Compositions and methods for treating lupus nephritis

Abstract

This provides methods for treating lupus nephritis. [Solution] This disclosure provides a method for treating lupus nephritis in an individual by administering an effective amount of type II anti-CD20 antibody to the individual having lupus. In other embodiments, this disclosure provides a similar method for treating membranous nephropathy.

Description

Cross-reference of related applications

[0001] This application claims priority rights to U.S. Patent Applications No. 62 / 899,706 filed September 12, 2019, No. 62 / 930,527 filed November 4, 2019, No. 62 / 931,032 filed November 5, 2019, and No. 63 / 005,071 filed April 3, 2020, each of which is incorporated herein by reference in its entirety.

[0002] Submission of sequence listings in ASCII text file The following submission in ASCII text file is incorporated herein by reference in its entirety: Sequence listing in computer-readable format (CRF) (filename: 146392048340 SEQLIST.TXT, date: August 17, 2020, size: 37KB) [Technical Field]

[0003] Field of Invention A method for treating lupus nephritis (LN) in an individual having lupus is provided herein by administering a type II anti-CD20 antibody. In another embodiment, a method for treating membranous nephropathy is provided herein. [Background technology]

[0004] background Proliferative lupus nephritis is the most common organ-threatening symptom of systemic lupus erythematosus. Glomerular damage and tubulointerstitial inflammation lead to proteinuria, hematuria, and progressive kidney damage. Treatment goals include reducing proteinuria, preventing kidney damage, and minimizing the toxicity of immunosuppressive therapy. (Hahn et al., Arthritis Care and Research 64:797-808, 2012; Fanouriakis et al., Ann. Rheum. Dis. 78:736-45, 2019.) Despite treatment, many patients have poor outcomes such as developing end-stage renal disease (ESRD), needing hemodialysis or kidney transplantation, or death, and the risk of ESRD has not substantially improved in the past 20 years. Hanly, et al., Rheumatology 55(2):252-62, 2016; Tektonidou et al., Arthritis Rheumatol 68(6):1432-1441, 2016). Currently, there are no approved treatments for lupus nephritis in the United States. Current unapproved standard treatments are associated with toxicity and low complete response rates.

[0005] Two anti-CD20 antibodies are being tested in clinical studies for their efficacy in treating lupus nephritis. Rituximab, a type I anti-CD20 antibody, depleted peripheral CD19+ B cells in 71 out of 72 patients and resulted in more responders and greater reductions in anti-dsDNA and C3 / C4 levels in a clinical trial (LUNAR). The dose regimen for the LUNAR study consisted of administering rituximab (1,000 mg) or placebo to patients with class III or class IV lupus nephritis (LN) on days 1, 15, 168, and 182 (weeks 0, 2, 24, and 26). However, rituximab therapy did not improve clinical outcomes at one year of treatment.

[0006] Another type I anti-CD20 antibody, ocrelizumab, was tested in a clinical trial (BELONG). Patients were randomized 1:1:1 to receive placebo, 400 mg of ocrelizumab, or 1,000 mg of ocrelizumab by intravenous infusion on days 1 and 15, followed by single infusions at week 16 and every 16 weeks thereafter, in addition to background glucocorticoids, combined with either mycophenolate mofetil (MMF) or the European Lupus Nephritis Trial (ELNT: Euro-Lupus Nephritis Trial) regimen (cyclophosphamide, followed by azathioprine). The study was terminated in part due to an imbalance in serious infection events (Mysler, E.F. et al. (2013) Arthritis Rheum. 65:2368-2379).

[0007] Therefore, there remains a need to test the effectiveness of other options in the treatment or prevention of LN in lupus patients.

[0008] All references cited in this specification, including patent applications and publications, are hereby incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0009] Summary In a particular embodiment, a method for treating lupus nephritis in an individual having lupus, comprising administering to the individual at least a first antibody exposure to type II anti-CD20 antibody, a second antibody exposure to type II anti-CD20 antibody, and a third antibody exposure to type II anti-CD20 antibody, wherein the second antibody exposure is not administered until approximately 18 to 26 weeks after the first antibody exposure, and the third antibody exposure is not administered until approximately 24 to 32 weeks after the second antibody exposure, and the first antibody exposure comprises one or two administrations of type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to 2200 mg of type II anti-CD20 antibody, and the second antibody A method is provided herein in which a body exposure comprises one or two doses of type II anti-CD20 antibody, a second antibody exposure comprising a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody, a third antibody exposure comprising one or two doses of type II anti-CD20 antibody, a third antibody exposure comprising a total exposure of approximately 800 mg to approximately 1200 mg of type II anti-CD20 antibody, wherein the type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6.A type II anti-CD20 antibody for use in a method of treating lupus nephritis in an individual, comprising administering to the individual a first antibody exposure to type II anti-CD20 antibody, a second antibody exposure to type II anti-CD20 antibody, and a third antibody exposure to type II anti-CD20 antibody, wherein the second antibody exposure is not administered until approximately 18 to 26 weeks after the first antibody exposure, and the third antibody exposure is not administered until approximately 24 to 32 weeks after the second antibody exposure, and the first antibody exposure comprises one or two administrations of type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to 2200 mg of type II anti-CD20 antibody, and the second antibody exposure comprises I Also provided herein is a type II anti-CD20 antibody comprising one or two doses of type I anti-CD20 antibody, wherein the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody, and the third antibody exposure comprises one or two doses of type II anti-CD20 antibody, wherein the third antibody exposure comprises a total exposure of approximately 800 mg to approximately 1200 mg of type II anti-CD20 antibody, and the type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6.

[0010] In some embodiments, the first antibody exposure includes a first dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the first antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the first antibody exposure of the second dose not being given until approximately 1.5 weeks to approximately 2.5 weeks after the first antibody exposure of the first dose. In some embodiments, the first antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the first antibody exposure of the second dose not being given until approximately 2 weeks after the first antibody exposure of the first dose. In some embodiments, the first dose of the first antibody exposure is approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the first dose of the second antibody exposure is approximately 1000 mg of type II anti-CD20 antibody.

[0011] In some embodiments, the second antibody exposure includes a first dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the second antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the second dose of the second antibody exposure not given until approximately 1.5 weeks to approximately 2.5 weeks after the second dose of the second antibody exposure. In some embodiments, the second antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the second dose of the second antibody exposure not given until approximately 2 weeks after the second dose of the second antibody exposure. In some embodiments, the first dose of the second antibody exposure is approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure is approximately 1000 mg of type II anti-CD20 antibody.

[0012] In some embodiments, the third antibody exposure includes a single dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the single dose of the third antibody exposure is approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the single dose of the third antibody exposure is not administered until approximately 52 weeks after the first dose of the first antibody exposure, or until approximately 28 weeks after the first dose of the second antibody exposure.

[0013] In some embodiments, the first antibody exposure, and / or the second antibody exposure, and / or the third antibody exposure, are administered intravenously.

[0014] In some embodiments, the individual has lupus nephritis. In some embodiments, the individual has class III or class IV lupus nephritis. In some embodiments, the individual is at risk of developing class III or class IV lupus nephritis. In some embodiments, the individual has class III(C) or class IV(C) lupus nephritis. In some embodiments, the individual has contingent class V lupus nephritis.

[0015] In some embodiments, the method further comprises administering an effective amount of an immunosuppressant to the individual. In some embodiments, the immunosuppressant comprises mycophenolic acid, its derivatives, or salts thereof. In some embodiments, the immunosuppressant comprises mycophenolate mofetil. In some embodiments, the method further comprises administering an effective amount of a glucocorticoid or corticosteroid to the individual. In some embodiments, the glucocorticoid or corticosteroid comprises methylprednisolone. In some embodiments, the glucocorticoid or corticosteroid comprises prednisone. In some embodiments, the method further comprises administering an effective amount of an antihistamine to the individual. In some embodiments, the antihistamine comprises diphenhydramine. In some embodiments, the method further comprises administering an effective amount of a nonsteroidal anti-inflammatory drug (NSAID) to the individual. In some embodiments, the NSAID comprises acetaminophen. In some embodiments, the method further comprises administering an effective amount of an antihypertensive agent to the individual. In some embodiments, the antihypertensive agent is an angiotensin-converting enzyme (ACE) inhibitor or an angiotensin receptor blocker. In some embodiments, the above method further includes administering a standard treatment to the individual. In some embodiments, the standard treatment includes treatment with one or more of angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, cyclophosphamide, mycophenolate mofetil, azathioprine, and glucocorticoids or corticosteroids.

[0016] In some embodiments, the above method results in a complete renal response (CRR) in the individual. In some embodiments, the above method results in a partial renal response (PRR) in the individual. In some embodiments, the above method results in depletion of circulating peripheral B cells in the individual. In some embodiments, the circulating peripheral B cells are CD19+ B cells. In some embodiments, the B cells are naive B cells (e.g., CD19+CD27- B cells), memory B cells (e.g., CD19+CD27+ B cells), or plasmablasts (e.g., CD19+CD27+CD38++ B cells). In some embodiments, the B cells are CD19+CD3-CD14-CD33-CD56- cells. In some embodiments, after administration of type II anti-CD20 antibody, B cells are depleted to a level where circulating peripheral B cells are present in the individual's peripheral blood at a rate of approximately 5 cells / μL or less. In some embodiments, B cells are depleted to a level where circulating peripheral B cells are present in the individual's peripheral blood at a rate of approximately 1 cell / μL or less. In some embodiments, B cells are depleted to a level where circulating peripheral B cells are present in the individual's peripheral blood at a rate of approximately 0.5 cells / μL or less. In some embodiments, B cells are depleted to a level where circulating peripheral B cells, depletion achieved after a first antibody exposure, are present in the individual's peripheral blood. In some embodiments, B cells are depleted to a level below the detection limit using HSFC. In some embodiments, HSFC has a limit of quantification (LLOQ) for B cells at approximately 1.0 cells / μL or less, approximately 0.8 cells / μL or less, approximately 0.6 cells / μL or less, approximately 0.5 cells / μL or less, or 0.441 cells / μL or less. In some embodiments, B cell depletion is sustained for at least 52 weeks after exposure to a first dose of the first antibody. In some embodiments, after administration of type II anti-CD20 antibody, circulating peripheral B cells in an individual are depleted by at least approximately 90% compared to the corresponding measurement in the same individual before administration of type II anti-CD20 antibody, or compared to the corresponding measurement in an individual not treated with type II anti-CD20 antibody.

[0017] In some embodiments, the individual is a human.

[0018] In some embodiments, the first antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 1 and 15 of treatment, the second antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 168 and 182 of treatment, and the third antibody exposure comprises one dose of 1000 mg of type II anti-CD20 antibody on day 364 of treatment, where the type II anti-CD20 antibody is obinutuzumab and the individual is human. In some embodiments, the first antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody at weeks 0 and 2 of treatment, the second antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody at weeks 24 and 26 of treatment, and the third antibody exposure comprises one dose of 1000 mg of type II anti-CD20 antibody at week 52 of treatment, the type II anti-CD20 antibody being obinutuzumab, the type II anti-CD20 antibody being administered intravenously, and the individual being human. In some embodiments, the first antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 1 and 15 of treatment, the first antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 168 and 182 of treatment, and the third antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 350 and 364 of treatment, the type II anti-CD20 antibody is obinutuzumab, and the individual is human. In some embodiments, the first antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody at weeks 0 and 2 of treatment, the second antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody at weeks 24 and 26 of treatment, and the third antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody at weeks 50 and 52 of treatment, the type II anti-CD20 antibody being obinutuzumab, and the individual being human.

[0019] In a particular embodiment, a method for depleting circulating peripheral B cells in an individual, comprising administering to the individual a first antibody exposure to type II anti-CD20 antibody, a second antibody exposure to type II anti-CD20 antibody, and a third antibody exposure to type II anti-CD20 antibody, wherein the second antibody exposure is not administered until approximately 18 to 26 weeks after the first antibody exposure, and the third antibody exposure is not administered until approximately 24 to 32 weeks after the second antibody exposure, and the first antibody exposure comprises one or two administrations of type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to 2200 mg of type II anti-CD20 antibody, and the second antibody exposure comprises one or two administrations of type II anti-CD20 antibody, and the second antibody exposure comprises I A method is provided herein comprising a total exposure of approximately 1800 mg to approximately 2200 mg of type I anti-CD20 antibody, a third antibody exposure comprising one or two doses of type II anti-CD20 antibody, the third antibody exposure comprising a total exposure of approximately 800 mg to approximately 1200 mg of type II anti-CD20 antibody, wherein the type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6, wherein after administration of the type II anti-CD20 antibody, B cells are depleted to a level in which circulating peripheral B cells are present in the individual's peripheral blood at a rate of approximately 5 cells / μL or less.Furthermore, a type II anti-CD20 antibody for use in a method for depleting peripheral circulating B cells in an individual, wherein the method comprises administering to the individual a first antibody exposure to type II anti-CD20 antibody, a second antibody exposure to type II anti-CD20 antibody, and a third antibody exposure to type II anti-CD20 antibody, wherein the second antibody exposure is not administered until approximately 18 to 26 weeks after the first antibody exposure, and the third antibody exposure is not administered until approximately 24 to 32 weeks after the second antibody exposure, and the first antibody exposure comprises one or two administrations of type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to 2200 mg of type II anti-CD20 antibody, and the second antibody exposure comprises one or two administrations of type II anti-CD20 antibody, and the second antibody A third antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody, and a third antibody exposure comprises one or two doses of type II anti-CD20 antibody, and a third antibody exposure comprises a total exposure of approximately 800 mg to approximately 1200 mg of type II anti-CD20 antibody, wherein the type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6, and after administration of the type II anti-CD20 antibody, B cells are depleted to a level in which circulating peripheral B cells are present in the individual's peripheral blood at a rate of approximately 5 cells / μL or less.

[0020] In some embodiments, the first antibody exposure includes a first dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the first antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the first antibody exposure of the second dose not being given until approximately 1.5 weeks to approximately 2.5 weeks after the first antibody exposure of the first dose. In some embodiments, the first antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the first antibody exposure of the second dose not being given until approximately 2 weeks after the first antibody exposure of the first dose. In some embodiments, the first dose of the first antibody exposure is approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the first dose of the second antibody exposure is approximately 1000 mg of type II anti-CD20 antibody.

[0021] In some embodiments, the second antibody exposure includes a first dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the second antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the second dose of the second antibody exposure not given until approximately 1.5 weeks to approximately 2.5 weeks after the second dose of the second antibody exposure. In some embodiments, the second antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the second dose of the second antibody exposure not given until approximately 2 weeks after the second dose of the second antibody exposure. In some embodiments, the first dose of the second antibody exposure is approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure is approximately 1000 mg of type II anti-CD20 antibody.

[0022] In some embodiments, the third antibody exposure includes a single dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the single dose of the third antibody exposure is approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the single dose of the third antibody exposure is not administered until approximately 52 weeks after the first dose of the first antibody exposure, or until approximately 28 weeks after the first dose of the second antibody exposure.

[0023] In some embodiments, the first antibody exposure, and / or the second antibody exposure, and / or the third antibody exposure, are administered intravenously.

[0024] In some embodiments, the individual has lupus nephritis. In some embodiments, the individual has class III or class IV lupus nephritis. In some embodiments, the individual is at risk of developing class III or class IV lupus nephritis. In some embodiments, the individual has class III(C) or class IV(C) lupus nephritis. In some embodiments, the individual has contingent class V lupus nephritis. In some embodiments, the individual has membranous nephropathy (MN), for example, primary membranous nephropathy (pMN). In some embodiments, the individual is at risk of developing membranous nephropathy (MN), for example, primary membranous nephropathy (pMN).

[0025] In some embodiments, circulating peripheral B cells are CD19+ B cells. In some embodiments, B cells are naive B cells (e.g., CD19+CD27- B cells), memory B cells (e.g., CD19+CD27+ B cells), or plasmablasts (e.g., CD19+CD27+CD38++ B cells). In some embodiments, B cells are CD19+CD3-CD14-CD33-CD56- cells. In some embodiments, B cells include CD19+CD20+ B cells, CD19+CD20- B cells, and CD19+CD22+ B cells. In some embodiments, B cells are depleted to a level where circulating peripheral B cells are present in individual peripheral blood at a rate of approximately 1 cell / μL or less. In some embodiments, B cells are depleted to a level where circulating peripheral B cells are present in individual peripheral blood at a rate of approximately 0.5 cells / μL or less. In some embodiments, B cells are depleted to below-detectable levels using HSFCs. In some embodiments, HSFCs have a limit of quantification (LLOQ) for B cells of approximately 1.0 cells / μL or less, approximately 0.8 cells / μL or less, approximately 0.6 cells / μL or less, approximately 0.5 cells / μL or less, or 0.441 cells / μL or less. In some embodiments, depletion is achieved after a first antibody exposure. In some embodiments, B cell depletion is sustained for at least 52 weeks after exposure to a first dose of the first antibody. In some embodiments, after administration of type II anti-CD20 antibody, circulating peripheral B cells in an individual are depleted by at least approximately 90% compared to a corresponding measurement in the same individual before administration of type II anti-CD20 antibody, or compared to a corresponding measurement in an individual not treated with type II anti-CD20 antibody. In some embodiments, an individual's serum B-cell activating factor (BAFF) level (e.g., BAFF level in a serum sample derived from the individual) increases after administration of the anti-CD20 type II antibody, for example, compared to a corresponding measurement in the same individual before administration of the anti-CD20 type II antibody, or compared to a corresponding measurement in an individual not treated with the anti-CD20 type II antibody.In some embodiments, an individual's serum B-cell activating factor (BAFF) level (e.g., BAFF level in a serum sample derived from the individual) increases within 6 weeks, 4 weeks, or 2 weeks after administration of anti-CD20 type II antibody, for example, compared to a corresponding measurement in the same individual before administration of anti-CD20 type II antibody, or compared to a corresponding measurement in an individual not treated with anti-CD20 type II antibody. In some embodiments, an individual's serum B-cell activating factor (BAFF) level (e.g., BAFF level in a serum sample derived from the individual) increases by at least 50%, at least 75%, at least 100%, at least 2-fold, or at least 3-fold after administration of anti-CD20 type II antibody, for example, compared to a corresponding measurement in the same individual before administration of anti-CD20 type II antibody, or compared to a corresponding measurement in an individual not treated with anti-CD20 type II antibody.

[0026] In some embodiments, the individual is a human.

[0027] In some embodiments, the first antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 1 and 15 of treatment, the second antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 168 and 182 of treatment, and the third antibody exposure comprises one dose of 1000 mg of type II anti-CD20 antibody on day 364 of treatment, where the type II anti-CD20 antibody is obinutuzumab and the individual is human. In some embodiments, the first antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody at weeks 0 and 2 of treatment, the second antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody at weeks 24 and 26 of treatment, and the third antibody exposure comprises one dose of 1000 mg of type II anti-CD20 antibody at week 52 of treatment, the type II anti-CD20 antibody being obinutuzumab, the type II anti-CD20 antibody being administered intravenously, and the individual being human. In some embodiments, the first antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 1 and 15 of treatment, the second antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 168 and 182 of treatment, and the third antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 350 and 364 of treatment, the type II anti-CD20 antibody is obinutuzumab, and the individual is human. In some embodiments, the first antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody at weeks 0 and 2 of treatment, the second antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody at weeks 24 and 26 of treatment, and the third antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody at weeks 50 and 52 of treatment, the type II anti-CD20 antibody being obinutuzumab, and the individual being human.

[0028] In a particular embodiment, a method for depleting peripheral circulating B cells in an individual, comprising administering to the individual a first antibody exposure to type II anti-CD20 antibody and a second antibody exposure to type II anti-CD20 antibody, wherein the second antibody exposure is not given until approximately 18 to 26 weeks after the first antibody exposure, the first antibody exposure comprises one or two administrations of type II anti-CD20 antibody, the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody, the second antibody exposure comprises one or two administrations of type II anti-CD20 antibody, and the second antibody exposure comprises type II anti-C A method is also provided herein, comprising a total exposure of approximately 1800 mg to approximately 2200 mg of D20 antibody, wherein the type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6, wherein after administration of the type II anti-CD20 antibody, B cells are depleted to a level in which circulating peripheral B cells are present in the individual's peripheral blood at a rate of approximately 5 cells / μL or less, and this state persists for at least 52 weeks after the first antibody exposure.Furthermore, a type II anti-CD20 antibody for use in a method for depleting peripheral circulating B cells in an individual, wherein the method comprises administering to the individual a first antibody exposure to type II anti-CD20 antibody and a second antibody exposure to type II anti-CD20 antibody, wherein the second antibody exposure is not given until approximately 18 weeks to approximately 26 weeks after the first antibody exposure, the first antibody exposure comprises one or two administrations of type II anti-CD20 antibody, the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody, and the second antibody exposure comprises one or two administrations of type II anti-CD20 antibody. Also provided herein is a type II anti-CD20 antibody in which body exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody, wherein the type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6, and after administration of the type II anti-CD20 antibody, B cells are depleted to a level in which circulating peripheral B cells are present in the individual's peripheral blood at a rate of approximately 5 cells / μL or less, and this is sustained for at least 52 weeks.

[0029] In some embodiments, the first antibody exposure includes a first dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the first antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the first antibody exposure of the second dose not being given until approximately 1.5 weeks to approximately 2.5 weeks after the first antibody exposure of the first dose. In some embodiments, the first antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the first antibody exposure of the second dose not being given until approximately 2 weeks after the first antibody exposure of the first dose. In some embodiments, the first antibody exposure of the first dose is approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the first dose of the second antibody exposure is approximately 1000 mg of type II anti-CD20 antibody.

[0030] In some embodiments, the second antibody exposure includes a first dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the second antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the second dose of the second antibody exposure not given until approximately 1.5 weeks to approximately 2.5 weeks after the second dose of the second antibody exposure. In some embodiments, the second antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the second dose of the second antibody exposure not given until approximately 2 weeks after the second dose of the second antibody exposure. In some embodiments, the first dose of the second antibody exposure is approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure is approximately 1000 mg of type II anti-CD20 antibody.

[0031] In some embodiments, the first antibody exposure and / or the second antibody exposure are administered intravenously.

[0032] In some embodiments, the individual has lupus nephritis. In some embodiments, the individual has class III or class IV lupus nephritis. In some embodiments, the individual is at risk of developing class III or class IV lupus nephritis. In some embodiments, the individual has class III(C) or class IV(C) lupus nephritis. In some embodiments, the individual has contingent class V lupus nephritis. In some embodiments, the individual has membranous nephropathy (MN), for example, primary membranous nephropathy (pMN). In some embodiments, the individual is at risk of developing membranous nephropathy (MN), for example, primary membranous nephropathy (pMN).

[0033] In some embodiments, circulating peripheral B cells are CD19+ B cells. In some embodiments, B cells are naive B cells (e.g., CD19+CD27- B cells), memory B cells (e.g., CD19+CD27+ B cells), or plasmablasts (e.g., CD19+CD27+CD38++ B cells). In some embodiments, B cells are CD19+CD3-CD14-CD33-CD56- cells. In some embodiments, B cells include CD19+CD20+ B cells, CD19+CD20- B cells, and CD19+CD22+ B cells. In some embodiments, B cells are depleted to a level where circulating peripheral B cells are present in individual peripheral blood at a rate of approximately 1 cell / μL or less. In some embodiments, B cells are depleted to a level where circulating peripheral B cells are present in individual peripheral blood at a rate of approximately 0.5 cells / μL or less. In some embodiments, B cells are depleted to below-detectable levels using HSFCs. In some embodiments, HSFCs have a limit of quantification (LLOQ) for B cells of approximately 1.0 cells / μL or less, approximately 0.8 cells / μL or less, approximately 0.6 cells / μL or less, approximately 0.5 cells / μL or less, or 0.441 cells / μL or less. In some embodiments, depletion is achieved after a first antibody exposure. In some embodiments, B cell depletion is sustained for at least 52 weeks after exposure to a first dose of the first antibody. In some embodiments, after administration of type II anti-CD20 antibody, circulating peripheral B cells in an individual are depleted by at least approximately 90% compared to a corresponding measurement in the same individual before administration of type II anti-CD20 antibody, or compared to a corresponding measurement in an individual not treated with type II anti-CD20 antibody.

[0034] In a particular embodiment, a method for treating an individual with rheumatoid arthritis, systemic lupus erythematosus (SLE), membranous nephropathy (MN), or extrarenal lupus (ERL) comprises administering to the individual a first antibody exposure to type II anti-CD20 antibody, a second antibody exposure to type II anti-CD20 antibody, and a third antibody exposure to type II anti-CD20 antibody, wherein the second antibody exposure is not administered until approximately 18 to 26 weeks after the first antibody exposure, and the third antibody exposure is not administered until approximately 24 to 32 weeks after the second antibody exposure, and the first antibody exposure comprises one or two administrations of type II anti-CD20 antibody, with the first antibody exposure comprising approximately 1800 mg to 2200 mg of type II anti-CD20 antibody. A method is provided herein comprising a total exposure to a type II anti-CD20 antibody, wherein the second antibody exposure comprises one or two doses of a type II anti-CD20 antibody, the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody, and the third antibody exposure comprises one or two doses of a type II anti-CD20 antibody, the third antibody exposure comprises a total exposure of approximately 800 mg to approximately 1200 mg of the type II anti-CD20 antibody, wherein the type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6.Furthermore, a type II anti-CD20 antibody for use in a method of treating rheumatoid arthritis, systemic lupus erythematosus (SLE), membranous nephropathy (MN), or extrarenal lupus (ERL) in an individual, wherein the method comprises administering to the individual a first antibody exposure to type II anti-CD20 antibody, a second antibody exposure to type II anti-CD20 antibody, and a third antibody exposure to type II anti-CD20 antibody, wherein the second antibody exposure is not administered until approximately 18 to 26 weeks after the first antibody exposure, and the third antibody exposure is not administered until approximately 24 to 32 weeks after the second antibody exposure, and the first antibody exposure comprises one or two administrations of type II anti-CD20 antibody, and the first antibody exposure comprises approximately 1800 mg to 22 Also provided herein are type II anti-CD20 antibodies comprising a total exposure of 00 mg, a second antibody exposure comprising one or two doses of type II anti-CD20 antibody, a second antibody exposure comprising a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody, a third antibody exposure comprising one or two doses of type II anti-CD20 antibody, a third antibody exposure comprising a total exposure of approximately 800 mg to approximately 1200 mg of type II anti-CD20 antibody, and a type II anti-CD20 antibody comprising a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6.

[0035] In some embodiments, the first antibody exposure includes a first dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the first antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the first antibody exposure of the second dose not being given until approximately 1.5 weeks to approximately 2.5 weeks after the first antibody exposure of the first dose. In some embodiments, the first antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the first antibody exposure of the second dose not being given until approximately 2 weeks after the first antibody exposure of the first dose. In some embodiments, the first antibody exposure of the first dose is approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the first dose of the second antibody exposure is approximately 1000 mg of type II anti-CD20 antibody.

[0036] In some embodiments, the second antibody exposure includes a first dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the second antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the second dose of the second antibody exposure not given until approximately 1.5 weeks to approximately 2.5 weeks after the second dose of the second antibody exposure. In some embodiments, the second antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the second dose of the second antibody exposure not given until approximately 2 weeks after the second dose of the second antibody exposure. In some embodiments, the first dose of the second antibody exposure is approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure is approximately 1000 mg of type II anti-CD20 antibody.

[0037] In some embodiments, the third antibody exposure includes a single dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the single dose of the third antibody exposure is approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the single dose of the third antibody exposure is not administered until approximately 52 weeks after the first dose of the first antibody exposure, or until approximately 28 weeks after the first dose of the second antibody exposure.

[0038] In some embodiments, the first antibody exposure, and / or the second antibody exposure, and / or the third antibody exposure, are administered intravenously.

[0039] In a particular embodiment, a method for treating lupus nephritis in an individual having lupus, or a method for depleting circulating peripheral B cells in an individual, comprising intravenous administration to the individual of first, second, and third antibody exposures to type II anti-CD20 antibody, wherein the first antibody exposure comprises two administrations of 1000 mg of type II anti-CD20 antibody at weeks 0 and 2 of treatment, and the second antibody exposure comprises 1000 mg of type II anti-CD20 antibody at weeks 24 and 26 of treatment. A method is provided herein comprising two administrations of an antibody, wherein a third antibody exposure comprises a single administration of 1000 mg of type II anti-CD20 antibody at week 52 of treatment, wherein the type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6, and the individual is a human. In some embodiments, the type II anti-CD20 antibody is obinutuzumab.

[0040] In a particular embodiment, a method for treating lupus nephritis in an individual having lupus, or a method for depleting circulating peripheral B cells in an individual, comprising intravenous administration to the individual of first, second and third antibody exposures to type II anti-CD20 antibody, wherein the first antibody exposure comprises two administrations of 1000 mg of type II anti-CD20 antibody at weeks 0 and 2 of treatment, and the second antibody exposure comprises 1000 mg of type II anti-CD20 antibody at weeks 24 and 26 of treatment. A method is provided herein comprising two doses, wherein a third antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody at weeks 50 and 52 of treatment, wherein the type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6, and the individual is a human. In some embodiments, the type II anti-CD20 antibody is obinutuzumab.

[0041] In some embodiments of the methods described herein, the type II anti-CD20 antibody is a humanized antibody. In some embodiments, the type II anti-CD20 antibody is defucosylated. In some embodiments, the heavy chain of the type II anti-CD20 antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7. In some embodiments, the light chain of the type II anti-CD20 antibody includes a light chain variable region containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the type II anti-CD20 antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the type II anti-CD20 antibody includes a heavy chain containing the amino acid sequence of SEQ ID NO: 9 and a light chain containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, the type II anti-CD20 antibody is obinutuzumab.

[0042] In some embodiments of the methods described herein, the method further comprises administering mycophenolate mofetil to an individual. In some embodiments, the method further comprises administering mycophenolate mofetil to an individual at a dose of 1500 mg / day on day 1 of treatment. In some embodiments, mycophenolate mofetil is administered to an individual at a dose of 1500 mg / day on day 1 of treatment and gradually increased to 2.0 g / day to 2.5 g / day at 500 mg / week by week 4 of treatment. In some embodiments, the method further comprises administering oral prednisone to an individual. In some embodiments, oral prednisone is administered to an individual at a dose of 0.5 mg / kg / day on day 2 of treatment. In some embodiments, oral prednisone is administered to an individual at a dose of 0.5 mg / kg / day on day 2 until week 2, and then gradually reduced to a dose of 5 mg / day until week 24 of treatment. In some embodiments, the method further comprises administering methylprednisolone to the individual by intravenous (IV) infusion at weeks 0, 2, 24, and 52 of treatment. In some embodiments, the method further comprises administering methylprednisolone to the individual by intravenous (IV) infusion at weeks 0, 2, 24, 26, and 52 of treatment. In some embodiments, the method further comprises orally administering 650 mg to 1000 mg of acetaminophen to the individual 30 to 60 minutes before one or more doses of type II anti-CD20 antibody. In some embodiments, the method further comprises orally administering 650 mg to 1000 mg of acetaminophen to the individual 30 to 60 minutes before each dose of type II anti-CD20 antibody. In some embodiments, the method further comprises orally administering 50 mg of diphenhydramine to the individual 30 to 60 minutes before one or more doses of type II anti-CD20 antibody. In some embodiments, the method further comprises orally administering 50 mg of diphenhydramine to the individual 30 to 60 minutes prior to each dose of type II anti-CD20 antibody.

[0043] In a particular embodiment, a kit for treating lupus nephritis in an individual having lupus is provided herein, comprising a container comprising a type II anti-CD20 antibody, wherein the type II anti-CD20 antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain comprising the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6, and comprising a package insert with instructions for using the type II anti-CD20 antibody in any of the methods described above and herein. In some embodiments, a package insert is provided that provides instructions for treating lupus nephritis in an individual, the instructions for administering a first antibody exposure to type II anti-CD20 antibody, a second antibody exposure to type II anti-CD20 antibody, and a third antibody exposure to type II anti-CD20 antibody to the individual, wherein the second antibody exposure is not given until approximately 18 to 26 weeks after the first antibody exposure, and the third antibody exposure is not given until approximately 24 to 32 weeks after the second antibody exposure, and the first antibody exposure is type II anti-CD20 The instructions indicate that the first antibody exposure includes one or two doses of the antibody, with the first antibody exposure including a total exposure of approximately 1800 mg to 2200 mg of type II anti-CD20 antibody; the second antibody exposure includes one or two doses of the antibody, with the second antibody exposure including a total exposure of approximately 1800 mg to 2200 mg of type II anti-CD20 antibody; and the third antibody exposure includes one or two doses of the antibody, with the third antibody exposure including a total exposure of approximately 800 mg to 1200 mg of type II anti-CD20 antibody.

[0044] In some embodiments, the third antibody exposure involves a single dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the first antibody exposure, and / or the second antibody exposure, and / or the third antibody exposure are administered intravenously.

[0045] In some embodiments, the kit further comprises a container containing a second pharmaceutical agent, the type II anti-CD20 antibody being the first pharmaceutical agent, and instructions relating to a package insert for administering the second pharmaceutical agent to a subject. In some embodiments, the second pharmaceutical agent is an immunosuppressant, glucocorticoid, corticosteroid, antimalarial agent, cytotoxic agent, integrin antagonist, cytokine antagonist, or hormone.

[0046] In a particular embodiment, a method for treating membranous nephropathy (MN) comprising administering to an individual in need a first antibody exposure to type II anti-CD20 antibody and a second antibody exposure to type II anti-CD20 antibody, wherein the second antibody exposure is not administered until approximately 18 to 26 weeks after the first antibody exposure, and the first antibody exposure comprises one or two administrations of type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody, and the second A method is also provided herein in which the antibody exposure comprises one or two doses of type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody, wherein the type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6. A type II anti-CD20 antibody for use in a method for treating membranous nephropathy (MN) in an individual, the method comprising administering to the individual a first antibody exposure to type II anti-CD20 antibody and a second antibody exposure to type II anti-CD20 antibody, wherein the second antibody exposure is not given until approximately 18 to 26 weeks after the first antibody exposure, the first antibody exposure comprises one or two doses of type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody. Also provided herein are type II anti-CD20 antibodies in which the second antibody exposure comprises one or two doses of type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody, wherein the type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6. In some embodiments, membranous nephropathy is primary membranous nephropathy (pMN).

[0047] In some embodiments, the first antibody exposure includes a first dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the first antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the first antibody exposure of the second dose not being given until approximately 1.5 weeks to approximately 2.5 weeks after the first antibody exposure of the first dose. In some embodiments, the first antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the first antibody exposure of the second dose not being given until approximately 2 weeks after the first antibody exposure of the first dose. In some embodiments, the first antibody exposure of the first dose is approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the first dose of the second antibody exposure is approximately 1000 mg of type II anti-CD20 antibody.

[0048] In some embodiments, the second antibody exposure includes a first dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the second antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the second dose of the second antibody exposure not given until approximately 1.5 weeks to approximately 2.5 weeks after the second dose of the second antibody exposure. In some embodiments, the second antibody exposure includes a first dose of type II anti-CD20 antibody and a second dose of type II anti-CD20 antibody, with the second dose of the second antibody exposure not given until approximately 2 weeks after the second dose of the second antibody exposure. In some embodiments, the first dose of the second antibody exposure is approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure is approximately 1000 mg of type II anti-CD20 antibody.

[0049] In some embodiments, the first and / or second antibody exposure is administered intravenously.

[0050] In some embodiments, the method further comprises administering an effective amount of glucocorticoid or corticosteroid to the individual. In some embodiments, the glucocorticoid or corticosteroid includes methylprednisolone. In some embodiments, 80 mg of methylprednisolone is administered intravenously to the individual 30 to 60 minutes before one or more doses of type II anti-CD20 antibody. In some embodiments, the method further comprises administering an effective amount of antihistamine to the individual. In some embodiments, the antihistamine includes diphenhydramine. In some embodiments, 50 mg of diphenhydramine is administered orally to the individual 30 to 60 minutes before one or more doses of type II anti-CD20 antibody. In some embodiments, the method further comprises administering an effective amount of nonsteroidal anti-inflammatory drug (NSAID) to the individual. In some embodiments, the NSAID includes acetaminophen. In some embodiments, 650 to 1000 mg of acetaminophen is orally administered to the individual 30 to 60 minutes before one or more doses of type II anti-CD20 antibody.

[0051] In some embodiments, the above method results in a complete renal response (CRR) in the individual. In some embodiments, the above method results in a partial renal response (PRR) in the individual. In some embodiments, the above method results in depletion of circulating peripheral B cells in the individual. In some embodiments, the circulating peripheral B cells are CD19+ B cells. In some embodiments, the B cells are naive B cells (e.g., CD19+CD27- B cells), memory B cells (e.g., CD19+CD27+ B cells), or plasmablasts (e.g., CD19+CD27+CD38++ B cells). In some embodiments, the B cells are CD19+CD3-CD14-CD33-CD56- cells. In some embodiments, after administration of type II anti-CD20 antibody, B cells are depleted to a level where circulating peripheral B cells are present in the individual's peripheral blood at a rate of approximately 5 cells / μL or less. In some embodiments, B cells are depleted to a level where circulating peripheral B cells are present in the individual's peripheral blood at a rate of approximately 1 cell / μL or less. In some embodiments, B cells are depleted to a level where circulating peripheral B cells are present in the individual's peripheral blood at a rate of approximately 0.5 cells / μL or less. In some embodiments, B cells are depleted to a level where circulating peripheral B cells, depletion achieved after a first antibody exposure, are present in the individual's peripheral blood. In some embodiments, B cells are depleted to a level below the detection limit using HSFC. In some embodiments, HSFC has a limit of quantification (LLOQ) for B cells at approximately 1.0 cells / μL or less, approximately 0.8 cells / μL or less, approximately 0.6 cells / μL or less, approximately 0.5 cells / μL or less, or 0.441 cells / μL or less. In some embodiments, B cell depletion is sustained for at least 52 weeks after exposure to a first dose of the first antibody. In some embodiments, after administration of type II anti-CD20 antibody, circulating peripheral B cells in an individual are depleted by at least approximately 90% compared to the corresponding measurement in the same individual before administration of type II anti-CD20 antibody, or compared to the corresponding measurement in an individual not treated with type II anti-CD20 antibody.

[0052] In some embodiments, the individual is a human.

[0053] In some embodiments, the first antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 1 and 15 of treatment, and the second antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 168 and 182 of treatment, the type II anti-CD20 antibody is obinutuzumab, and the individual is human. In some embodiments, the first antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on weeks 0 and 2 of treatment, and the second antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on weeks 24 and 26 of treatment, the type II anti-CD20 antibody is obinutuzumab, the type II anti-CD20 antibody is administered intravenously, and the individual is human. In some embodiments, the first antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 1 and 15 of treatment, and the second antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 168 and 182 of treatment, the type II anti-CD20 antibody is obinutuzumab, and the individual is human. In some embodiments, the first antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on weeks 0 and 2 of treatment, and the second antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on weeks 24 and 26 of treatment, the type II anti-CD20 antibody is obinutuzumab, and the individual is human.

[0054] In some embodiments of the methods described herein, the type II anti-CD20 antibody is a humanized antibody. In some embodiments, the type II anti-CD20 antibody is defucosylated. In some embodiments, the heavy chain of the type II anti-CD20 antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7. In some embodiments, the light chain of the type II anti-CD20 antibody includes a light chain variable region containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the type II anti-CD20 antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the type II anti-CD20 antibody includes a heavy chain containing the amino acid sequence of SEQ ID NO: 9 and a light chain containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, the type II anti-CD20 antibody is obinutuzumab.

[0055] In a particular embodiment, a kit for treating membranous nephropathy (MN) in an individual is provided herein, comprising a container containing a type II anti-CD20 antibody, wherein the type II anti-CD20 antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain comprising the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6, and the kit includes a package insert with instructions for using the type II anti-CD20 antibody in any of the methods described above and herein. In some embodiments, the accompanying documentation provides instructions for treating membranous nephropathy (MN) in an individual, and the instructions indicate that a first antibody exposure to type II anti-CD20 antibody and a second antibody exposure to type II anti-CD20 antibody are given, with the second antibody exposure not given until approximately 18 to 26 weeks after the first antibody exposure, and the first antibody exposure comprises one or two doses of type II anti-CD20 antibody, comprising a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody, and the second antibody exposure comprises one or two doses of type II anti-CD20 antibody, comprising a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody. In some embodiments, membranous nephropathy is primary membranous nephropathy (pMN). In some embodiments, the individual is a human.

[0056] It should be understood that one, some, or all of the characteristics of the various embodiments described herein can be combined to form other embodiments of the present invention. These and other embodiments of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following embodiments for carrying out the invention. [Brief explanation of the drawing]

[0057] [Figure 1A] Figure 1A shows the renal responses achieved at weeks 52 and 76 in the obinutuzumab treatment group and the placebo treatment group. CRR = complete renal response; PRR = partial renal response.

[0058] [Figure 1B] Figure 1B shows the renal response dynamics in the obinutuzumab treatment group and the placebo treatment group. CRR = complete renal response; PRR = partial renal response; ORR = total renal response.

[0059] [Figure 1C] Figure 1C shows a patient-level renal response heatmap. Rows represent individual patients; columns represent visits. UPCR = urinary protein to creatinine ratio; SCr = serum creatinine; ULN = upper limit of normal. Complete renal response required a urinary protein to creatinine ratio (UPCR) <0.5, serum creatinine below the upper limit of normal and not increasing by more than 15% from baseline, and urinary red blood cells (RBCs) <10 / hpf without RBC casts. Modified complete renal response required UPCR <0.5 with serum creatinine below the upper limit of normal. Partial renal response required a UPCR decrease of ≥50% to <1 from baseline (or <3 if baseline is ≥3), serum creatinine not increasing by more than 15% from baseline, and urinary RBCs <10 / hpf or not increasing by more than 50% from baseline.

[0060] [Figure 1D]Figure 1D shows the mean CD19+ peripheral B cells over time using high-sensitivity flow cytometry. Values ​​less than 0.441 were assigned to 0.441 for the calculation of the mean and SEM. SEM = standard error of the mean.

[0061] [Figure 2A] Figure 2A shows the changes from baseline in serological and laboratory parameters. The mean change from baseline was calculated by carrying over the last observation for missing data. In cases of treatment failure, the last measurement before treatment failure was used. SEM = standard error of the mean.

[0062] [Figure 2B] Figure 2B shows the complete response rate (CRR) over time for the obinutuzumab and placebo cohorts, based on acceptable serum creatinine criteria. CRR is defined as a UPCR of less than 0.5 and serum creatinine below the upper limit of normal.

[0063] [Figure 3A] Figure 3A shows the B-cell depletion dynamics, expressed as the percentage of patients with B-cell depletion using conventional high-sensitivity flow cytometry.

[0064] [Figure 3B] Figure 3B shows the mean B cell subset measurements over time using high-sensitivity flow cytometry. Values ​​less than 0.441 were assigned to 0.441 for the calculation of the mean and SEM. SEM = standard error of the mean.

[0065] [Figure 4]Figure 4 shows the response over time based on treatment group and peripheral B cell status. Persistent depletion was present if the peripheral B cell count was less than 0.441 at both week 24 and week 52. If week 24 data was missing, persistent depletion was present if the peripheral B cell count was less than 0.441 at week 12 and week 52 while obinutuzumab PK was high at week 24. Detectable B cells were present if either the peripheral B cell count at week 24 or week 52 was greater than 0.441. Obinutuzumab patients with insufficient data to determine their status were classified as having "insufficient data." All placebo-treated patients had detectable B cells at either week 24 or week 52. CRR = complete renal response; PRR = partial renal response; UPCR = urinary protein to creatinine ratio; SCr = serum creatinine; ULN = upper limit of normal.

[0066] [Figure 5] Figure 5 shows a visual predictive check (VPC) plot used to validate the pharmacokinetic (PK) model described in Example 2. The solid lines show the median, 5th percentile, and 95th percentile of the observed obinutuzumab concentration (μg / mL) at the indicated time (day). The shaded area shows the 90% confidence interval (90% CI) of the simulated median, 5th percentile, and 95th percentile obinutuzumab concentration (μg / mL) at the specified time (day), based on the PK model. Simulated concentrations were calculated from 1000 trials using the dosing, sampling, and covariate values ​​of the analytical dataset described in Example 2.

[0067] [Figure 6] Figure 6 shows the predicted obinutuzumab concentration profiles over time based on the PK model described in Example 2 for all patients following the drug regimen described in Example 1 (1000 mg of obinutuzumab on days 0, 14, 168, and 182). Concentration profiles were simulated using covariates and individual random effects for each patient. Residual variability was not included. The median, 5th percentile, and 95th percentile of the simulated obinutuzumab concentrations are plotted as shown.

[0068] [Figure 7] Figure 7 is a logistic regression analysis of the probability of occurrence of any grade of late SAE (severe adverse events occurring after dose 2) against the cumulative obinutuzumab exposure (AUC52) from the start of treatment to week 52. The circles indicate the observed responses (0 = no late SAE event; 1 = late SAE event). The circles are jittered vertically for better visualization. The logistic regression line is shown. The shaded area is the 90% confidence interval (CI) of the regression line. The p-value for the relationship of the probability of any grade of late SAE to obinutuzumab exposure is provided (p = 0.383). This logistic regression was performed based on late SAE data from only the placebo group patients.

[0069] [Figure 8] Figure 8 shows the observed B cell counts versus the individual predicted obinutuzumab concentrations from the study described in Example 1 (12 weeks after the first dose of obinutuzumab). The circles with a B cell count of 0.2 correspond to observations below the B cell quantification limit (BQL).

[0070] [Figure 9] Figure 9 shows the probability (B cells > BQL) of B cell counts rebounding above the BQL at week 52 compared to obinutuzumab exposure (AUC52) in patients administered obinutuzumab (1000 mg) at weeks 0, 2, 24, and 26. The circles indicate the observed B cell levels (1 = B cells > BQL; 0 = B cells < BQL). The logistic regression line is shown. The shaded area is the 90% confidence interval (CI) of the regression line. The p-value for the relationship of the probability (B cells > BQL) of B cell counts rebounding above the BQL at week 52 to obinutuzumab exposure is provided (p = 0.045).

[0071] [Figure 10]Figure 10 shows the baseline characteristics of patients in the obinutuzumab treatment group and the control treatment group. Patients in the obinutuzumab group were later further divided into those who showed persistent B cell depletion in response to obinutuzumab treatment and those who had detectable B cells in response to obinutuzumab treatment.

[0072] [Figure 11] Figure 11 shows the time course of (weekly) serum B-cell activating factor (BAFF) levels (pg / mL) in patients treated with obinutuzumab and MMF or placebo and MMF.

[0073] [Figure 12] Figure 12 shows the proportion of patients showing a response to treatment at week 76, stratified by baseline serum creatinine levels.

[0074] [Figure 13] Figure 13 shows the response rates between the obinutuzumab treatment group and the placebo treatment group at weeks 52 and 76, using the definition of surrogate response. OBI: obinutuzumab; PBO: placebo; UPCR: urine protein / creatinine ratio; SCr: serum creatinine; ULN: upper limit of normal. [Modes for carrying out the invention]

[0075] Detailed explanation Nonclinical data suggested the potential of treating lupus with both type I anti-CD20 antibodies (rituximab and ocrelizumab) and type II anti-CD20 antibody (obinutuzumab). However, unlike rituximab and ocrelizumab, obinutuzumab treatment met the primary and key secondary efficacy endpoints in the Phase II clinical trial (NOBILITY). In year one, obinutuzumab treatment, when added to mycophenolate and corticosteroids for the treatment of proliferative lupus nephritis, resulted in increased complete and partial renal responses compared to placebo. Furthermore, obinutuzumab was not associated with an increased rate of serious adverse events or serious infections. Pharmacokinetic (PK) and pharmacodynamic (PD) analyses from a Phase II clinical trial demonstrated that sustained peripheral B-cell depletion (<0.441 cells / μl) positively correlated with achieving complete renal response (CRR), and that obinutuzumab concentrations above 1 μg / mL were important for maintaining B-cell depletion. Simulations based on PK modeling of 1000 mg obinutuzumab dosing regimens at weeks 0, 2, 24, 26, and 52 showed that an additional single dose of 1000 mg obinutuzumab at week 52 was predicted to induce obinutuzumab concentrations above 1 μg / mL in the majority of patients at week 76. Therefore, an additional single dose of obinutuzumab at week 52 is predicted to maintain obinutuzumab concentrations above the critical level of 1 μg / mL, which is expected to maintain B-cell depletion and lead to better efficacy at week 76.

[0076] The previous obinutuzumab administration regimen was administered only at weeks 0, 2, 24, and 26 to allow for directional comparison with clinical trials of rituximab. It was also unclear whether obinutuzumab was effective, and there was no clear rationale for readministration without evidence of efficacy. The results shown in Examples 1 and 2 unexpectedly indicated that B-cell depletion was predicted to persist to week 76 with a 1000 mg obinutuzumab administration regimen at weeks 0, 2, 24, 26, and 52, without further adverse events. Maintaining B-cell depletion at week 76 is expected to lead to better efficacy (e.g., CRR). Subsequent additional doses every 24 weeks are expected to maintain efficacy. Currently, given the evidence of efficacy and the association of deeper B-cell depletion with obinutuzumab to greater response, continued administration may be considered to maintain B-cell depletion and clinical benefit.

[0077] In one embodiment, a method for treating lupus nephritis in an individual is provided herein, comprising administering to the individual a first antibody exposure to type II anti-CD20 antibody, a second antibody exposure to type II anti-CD20 antibody, and a third second antibody exposure to type II anti-CD20 antibody. In some embodiments, the individual has lupus. In some embodiments, the second antibody exposure is not administered until about 18 to 26 weeks after the first antibody exposure. In some embodiments, the third antibody exposure is not administered until about 24 to 32 weeks after the second antibody exposure. In some embodiments, the first antibody exposure comprises one or two administrations of type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of about 1800 mg to about 2200 mg of type II anti-CD20 antibody. In some embodiments, the second antibody exposure comprises one or two doses of type II anti-CD20 antibody, with the second antibody exposure comprising a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody. In some embodiments, the third antibody exposure comprises one or two doses of type II anti-CD20 antibody, with the third antibody exposure comprising a total exposure of approximately 800 mg to approximately 1200 mg of type II anti-CD20 antibody. In some embodiments, the antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6.

[0078] In another embodiment, a method for treating lupus nephritis in an individual is provided herein, comprising administering to the individual a first antibody exposure to at least type II anti-CD20 antibody and a second antibody exposure to type II anti-CD20 antibody. In some embodiments, the individual has lupus. In some embodiments, the second antibody exposure is not given until about 18 to about 26 weeks after the first antibody exposure. In some embodiments, the first antibody exposure comprises one or two administrations of type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of about 1800 mg to about 2200 mg of type II anti-CD20 antibody. In some embodiments, the second antibody exposure comprises one or two administrations of type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of about 1800 mg to about 2200 mg of type II anti-CD20 antibody. In some embodiments, the antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6.

[0079] In another embodiment, a method for treating lupus nephritis in an individual having lupus is provided herein, comprising administering an effective amount of type II anti-CD20 antibody to the individual. In some embodiments, the antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6. In some embodiments, the individual has class III or class IV lupus nephritis.

[0080] In another embodiment, a method for treating rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), membranous nephropathy (MN), or extrarenal lupus (ERL) in an individual is provided herein, comprising administering an effective amount of anti-CD20 antibody to the individual. In some embodiments, the antibody comprises a heavy chain variable region including the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain variable region including the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6.

[0081] I. General techniques The techniques and procedures described or referenced herein are generally well understood, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FMAusubel, et al. eds., (2003)); Methods in Enzymology series (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GRTaylor eds. (1995)), Harlow and Lane, eds. (1988); Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(JECellis,ed.,1998)Academic Press;Animal Cell Culture(RIFreshney),ed.,1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.,1993-8)J.Wiley and Sons;Handbook of Experimental Immunology(DMWeir and CCBlackwell, eds.);Gene Transfer Vectors for Mammalian Cells(JMMiller and MPCalos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D.Lane(Cold Spring Harbor Laboratory Press,1999);The Antibodies(M.Zanetti and Conventional methodologies, such as those described in JDCapra, eds., Harwood Academic Publishers, 1995, and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JBLippincott Company, 1993), are commonly used by those skilled in the art.

[0082] II. Definition The term "lupus nephritis (LN)" refers to the symptoms of lupus in the kidneys (e.g., systemic lupus erythematosus, drug-induced lupus, neonatal lupus, or discoid lupus).

[0083] The term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions having polyepitope specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., bispecific antibodies, diabodies, and single-chain molecules, and antibody fragments such as Fab, F(ab’)2, and Fv). The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

[0084] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of five basic heterotetrameric units together with an additional polypeptide called the J chain and contain ten antigen-binding sites, while IgA antibodies are composed of two to five basic four-chain units that can polymerize to form multivalent aggregates in combination with the J chain. In the case of IgG, the four-chain unit generally has a molecular weight of about 150,000 daltons. Each L chain is linked to the H chain by one disulfide covalent bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. The H and L chains also each have regularly spaced interchain disulfide bridges. Each H chain has a variable domain (V H ) at the N-terminus, followed by three constant domains (C H ) for each of the α and γ chains, and four C H domains for the μ and ε isotypes. Each L chain has a variable domain (V L ) at the N-terminus, followed by a constant domain at the opposite end. V L is aligned with V H , and C L is aligned with the first constant domain (C H 1) of the heavy chain. Certain amino acid residues are thought to form an interface between the light chain variable domain and the heavy chain variable domain. V H and V LWhen these two molecules pair together, a single antigen-binding site is formed. For the structure and properties of various classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6. Light chains derived from any vertebrate species can be assigned to one of two distinct types called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each having a heavy chain denoted as α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function. For example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0085] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain. The variable domains of the heavy and light chains are sometimes called "VH" and "VL," respectively. These domains are generally the most variable parts of the antibody (compared to other antibodies of the same class) and contain the antigen-binding site.

[0086] The term "variable" refers to the fact that specific segments of the variable domain differ extensively in sequence across antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody to its particular antigen. However, variability is not evenly distributed throughout the variable domain. Rather, it is concentrated in three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). The native heavy and light chain variable domains each contain four FR regions that largely adopt a beta-sheet configuration, connected by three HVRs that link beta-sheet structures and, in some cases, form loops that form part of the beta-sheet structure. The HVRs within each chain are closely linked to one another by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domain does not directly participate in antibody binding to antigens, but it exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cytotoxicity.

[0087] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population; that is, the individual antibodies in that population are identical except for any naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in trace amounts. Monoclonal antibodies are highly specific and directed to a single antigenic site. In contrast to polyclonal antibody preparations, which typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on an antigen. In addition to their specificity, monoclonal antibodies have the advantage of being synthesized by hybridoma culture and free from contamination with other immunoglobulins. The modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring the antibody to be produced in any particular way. For example, monoclonal antibodies used in accordance with the present invention are, for example, those produced by the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nded.1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, eg, US Patent No. 4,816,567), phage-display technologies (see, eg, Clackson et al. al.,Nature,352:624-628(1991);Marks et al.,J.Mol.Biol.222:581-597(1992);Sidhu et al.,J.Mol.Biol.338(2):299-310(2004);Lee et al. al., J.Mol.Biol.340(5):1073-1093(2004);Fellouse, Proc.Natl.Acad.Sci.USA101(34):12467-12472(2004);and Lee et al., J.Immunol.Methods 284(1-2):119-132(2004), and techniques for producing human or human-like antibodies in animals having some or all of the genes encoding the human immunoglobulin locus or human immunoglobulin sequence (e.g., International Publication No. 1998 / 24893, International Publication No. 1996 / 34096, International Publication No. 1996 / 33735, International Publication No. 1991 / 10741, Jakobovits et al., Proc.Natl.Acad.Sci.USA 90:2551(1993);Jakobovits et al.,Nature 362:255-258(1993);Bruggemann et al.,Year in Immunol.7:33 (1993), U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, Marks et al.,Bio / Technology 10:779-783(1992);Lonberg et al.,Nature 368:856-859(1994);Morrison,Nature 368:812-813(1994);Fishwild et al.It can be produced by a variety of techniques, including (see Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0088] The term "naked antibody" refers to an antibody that is not conjugated with a cytotoxic moiety or radiolabeling.

[0089] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, whole antibodies include those having heavy and light chains containing an Fc region. The constant domain may be the constant domain of the natural sequence (e.g., the constant domain of the human natural sequence) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

[0090] An "antibody fragment" is a portion of an intact antibody, preferably comprising the antigen-binding region and / or variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2; diabodies; linear antibodies (see Example 2 of U.S. Patent No. 5,641,870; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of the antibody yielded two identical antigen-binding fragments called "Fab" fragments and the remaining "Fc" fragment, denoted to reflect its readily crystallizable ability. The Fab fragment, along with the entire L chain, contains the variable region domain (V) of the H chain. H ), as well as the first constant domain of one heavy chain (C H1) consists of each Fab fragment, which is monovalent with respect to antigen binding, i.e., has a single antigen-binding site. Pepsin treatment of the antibody yields a single large F(ab')2 fragment, which is roughly equivalent to two Fab fragments with different antigen-binding activities disulfide-linked together, and is still capable of crosslinking to an antigen. The Fab' fragment contains one or more cysteines derived from the hinge region of the antibody, C H It differs from the Fab fragment in that it has several additional residues at the carboxyl terminus of one domain. Fab'-SH is the herein designation for Fab' fragments in which the cysteine ​​residue(s) of the constant domain have a free thiol group. The F(ab')2 antibody fragment was originally generated as a pair with a Fab' fragment having a hinged cysteine ​​between them. Other chemical couplings of antibody fragments are also known.

[0091] The Fc fragment contains the carboxyl ends of both H chains held together by a disulfide. The effector function of the antibody is determined by the sequence in the Fc region, which is also recognized by an Fc receptor (FcR) found in certain cell types.

[0092] "Fv" refers to the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer in which one heavy chain variable domain and one light chain variable domain are tightly bound noncovalently. The folding of these two domains creates six hypervariable loops (three from the H chain and three from the L chain) that provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to an antigen, albeit with lower affinity than the full binding site.

[0093] "Single-stranded Fv," also abbreviated as "sFv''" or "scFv," is a single polypeptide chain formed by the linking of V molecules. H and V L It is an antibody fragment containing an antibody domain. Preferably, the sFv polypeptide is V HDomain and V L The sFv further contains a polypeptide linker between the domain and the sFv, which allows the sFv to form a structure desirable for antigen binding. For an overview of sFv, see, for example, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0094] The "functional fragments" of the antibody of the present invention include a portion of an intact antibody, which generally includes the antigen-binding region or variable region of an intact antibody, or the Fc region of an antibody that retains or modifies FcR-binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0095] The term "diabody" refers to the process of achieving inter-chain V-domain pairing, rather than intra-chain pairing, thereby obtaining a bivalent fragment, i.e., a fragment having two antigen-binding sites. H Domain and V L This refers to a small antibody fragment prepared by constructing an sFv fragment (see previous paragraph) using a short linker (approximately 5-10 residues) between the domain and the sFv. A bispecificity diabody is a V of two antibodies. H Domain and V L It is a heterodimer of two “cross-over” sFv fragments whose domains are located on different polypeptide chains. The diabody is described in detail, for example, European Patent No. 404,097, International Publication No. 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0096] In this specification, monoclonal antibodies include, specifically, “chimeric” antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody belonging to a particular species or antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody belonging to a different species or antibody class or subclass, and fragments of such antibodies insofar as they exhibit the desired biological activity (U.S. Patent No. 4,816,567, Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In this specification, the chimeric antibody of interest includes PRIMATIZED® antibody, in which the antigen-binding region of the antibody is derived, for example, from an antibody produced by immunizing a macaque monkey with the antigen of interest. As used herein, “humanized antibody” is used as a subset of “chimeric antibody.”

[0097] Humanized non-human (e.g., mouse) antibodies are chimeric antibodies containing the smallest sequence derived from non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's HVR (defined below) are replaced with residues from the HVR of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having desired specificity, affinity, and / or capabilities. In some cases, framework ("FR") residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in either the recipient antibody or the donor antibody. These modifications may be made to further improve the antibody's performance, such as binding affinity. Generally, a humanized antibody includes at least one, typically substantially all, variable domains, where all or substantially all of the hypervariable loops correspond to a non-human immunoglobulin sequence, and all or substantially all of the FR region corresponds to a human immunoglobulin sequence, although the FR region may include one or more individual FR residue substitutions that improve antibody performance such as binding affinity, isomerization, and immunogenicity. The number of these amino acid substitutions in the FR is typically 6 or less in the H chain and 3 or less in the L chain. The humanized antibody also optionally includes the immunoglobulin constant region (Fc), typically at least a portion of the Fc of human immunoglobulin. For further details, see, for example, Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). For example, see Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0098] A “human antibody” is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or an antibody prepared using any of the techniques for preparing human antibodies disclosed herein. This definition of a human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, such as phage display libraries. Hoogenboom and Winter, J.Mol.Biol., 227:381 (1991); Marks et al., J.Mol.Biol., 222:581 (1991). The methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991) are also available for the preparation of human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigens to transgenic animals, such as immunized xenomouses, which have been modified to produce such antibodies in response to antigen administration, but whose endogenous gene loci are rendered inactive (see, for example, U.S. Patents 6,075,181 and 6,150,584 relating to XENOMOUSE® technology). See, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), relating to human antibodies produced via human B-cell hybridoma technology.

[0099] The terms “hypervariable region,” “HVR,” or “HV,” as used herein, refer to regions of antibody variable domains whose sequences are hypervariable and / or form structurally defined loops. Generally, antibodies contain six HVRs, three of which are located in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 exhibit the highest diversity among the six HVRs, and H3 in particular is thought to play a unique role in conferring excellent specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In practice, naturally occurring camel antibodies consisting only of heavy chains are functional and stable in the absence of light chains. For example, see Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0100] Several HVR descriptions are used and incorporated herein. Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia, on the other hand, refers to the location of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on the analysis of available complex crystal structures. The residues from each of these HVRs are listed below. TIFF2026090308000002.tif44170

[0101] HVR may include the following “extended HVRs”: in VL, 24–36 or 24–34 (L1), 46–56 or 50–56 (L2), and 89–97 or 89–96 (L3); and in VH, 26–35 (H1), 50–65 or 49–65 (H2), and 93–102, 94–102, or 95–102 (H3). Variable domain residues are numbered according to Kabat et al. (above) for each of these definitions.

[0102] The expressions "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat," and their variations, refer to the numbering scheme used for heavy chain variable domains or light chain variable domains in the antibody organization described above by Kabat et al. Even with this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of FR or HVR in the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after H2 residue 52 (residue 52a according to Kabat) and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). Kabat numbering of residues can be determined for a given antibody by alignment of the antibody sequence with the homologous region of the sequence numbered by "standard" Kabat.

[0103] A “framework” or “FR” residue is a variable domain residue other than an HVR residue as defined herein.

[0104] The "Human Consensus Framework" or "Acceptor Human Framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Generally, the subgroups of sequences are as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th These are subgroups in Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include those relating to VL, where subgroups may be subgroups Kappa I, Kappa II, Kappa III, or Kappa IV, as described in Kabat et al. (above). Furthermore, for VH, subgroups may be subgroups I, subgroup II, or subgroup III, as described in Kabat et al. (above). Alternatively, the human consensus framework may derive from the above, for example, when human framework residues are selected based on their homology to the donor framework by aligning specific residues, e.g., donor framework sequences, with a set of various human framework sequences. An acceptor human framework "derived" from a human immunoglobulin framework or human consensus framework may contain the same amino acid sequence, or it may contain existing amino acid sequence changes. In some embodiments, the number of existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0105] The "VH subgroup III consensus framework" includes a consensus sequence obtained from the amino acid sequences in variable heavy chain subgroup III by Kabat et al. (above). In one embodiment, the VH subgroup III consensus framework amino acid sequence includes at least part or all of each of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS(HC-FR1)(SEQ ID NO: 35), WVRQAPGKGLEWV(HC-FR2), (SEQ ID NO: 36), RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(HC-FR3, SEQ ID NO: 37), WGQGTLVTVSA(HC-FR4), (SEQ ID NO: 38).

[0106] The "VL Kappa I Consensus Framework" includes a consensus sequence obtained from the amino acid sequences of the variable light chain kappa subgroup I by Kabat et al. (above). In one embodiment, the VH subgroup I consensus framework amino acid sequence includes at least part or all of each of the following sequences: DIQMTQSPSSLSASVGDRVTITC(LC-FR1)(SEQ ID NO: 39), WYQQKPGKAPKLLIY(LC-FR2)(SEQ ID NO: 40), GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(LC-FR3)(SEQ ID NO: 41), and FGQGTKVEIKR(LC-FR4)(SEQ ID NO: 42).

[0107] For example, "amino acid modification" at a specified location in the Fc region refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent" to the specified residue means an insertion within one or two residues. The insertion may be on the N-terminal or C-terminal side of the specified residue. The preferred amino acid modification in this specification is substitution.

[0108] Affinity-matured antibodies have one or more changes in one or more HVRs, and these changes result in improved affinity of the antibody to the antigen compared to a parent antibody that does not have those changes. In one embodiment, affinity-matured antibodies have nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of HVR and / or framework residues has been described, for example, by Barbas et al. Proc Nat.Acad.Sci.USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J.Immunol.155:1994-2004 (1995); Jackson et al., J.Immunol.154(7):3310-9 (1995); and Hawkins et al., J.Mol.Biol.226:889-896 (1992).

[0109] As used herein, the terms “specifically bind to” or “specific to” refer to measurable and reproducible interactions, such as binding between a target and an antibody, that determine the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target more readily and / or for a longer duration with higher affinity, binding strength, than it would to other targets. In one embodiment, the extent to which an antibody binds to an unrelated target is less than about 10% of the antibody’s binding to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved between proteins of different species. In another embodiment, specific binding may include, but does not require, exclusive binding.

[0110] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. While the boundaries of the Fc region of an immunoglobulin heavy chain may differ, the Fc region of a human IgG heavy chain is typically defined as extending from an amino acid residue at position Cys226 or at position Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies may include antibody populations from which all K447 residues have been removed, antibody populations without removed K447 residues, and antibody populations having a mixture of antibodies with and without K447 residues. Suitable native sequence Fc regions for use in the antibodies of the present invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0111] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. The preferred FcR is the natural sequence human FcR. Furthermore, preferred FcRs are those that bind to IgG antibodies (gamma receptors), which include the FcγRI, FcγRII, and FcγRIII subclass receptors (including allele variants and, alternatively, splice forms of these receptors). The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, differing mainly in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor-activated tyrosine motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs have been reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.

[0112] The term "Fc receptor" or "FcR" also includes FcRn, a neonatal receptor 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)). Methods for measuring binding to FcRn are publicly known (see, for example, Ghetie and Ward, Immunol. Today 18:(12):592-8 (1997); Ghetie et al., Nature Biotechnology 15(7):637-40 (1997); Hinton et al., J.Biol.Chem. 279(8):6213-6 (2004); International Publication No. 2004 / 92219 (Hinton et al.)). The in vivo binding of human FcRn-high affinity binding polypeptides to FcRn and their serum half-lives can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides containing mutant Fc regions. International Publication No. 2004 / 42072 (Presta) describes antibody variants that enhance or reduce binding to FcR. See also, for example, Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0113] When used herein, the expressions “substantially reduced” or “substantially different” represent a sufficiently high difference between two numerical values ​​(generally one associated with a molecule and the other with a reference / comparison molecule), and as a result, a person skilled in the art would consider the difference between these two values ​​to be statistically significant with respect to the biological characteristic measured by such value (e.g., the Kd value). The difference between the two values ​​is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and / or greater than about 50% as a function of the value relative to the reference / comparison molecule.

[0114] When used herein, the terms “substantially similar” or “substantially identical” indicate a sufficiently high degree of similarity between two numerical values ​​(generally one associated with a nutrient and the other with a reference / comparison nutrient) to the extent that, as a result, a person skilled in the art would consider any difference between these two values ​​to be of little or no biological and / or statistical significance with respect to the biological characteristics measured by such values ​​(e.g., Kd values). The difference between the two values, as a function of the reference / comparison values, is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and / or less than about 10%.

[0115] As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals to which they are exposed at the doses and concentrations used. In many cases, physiologically acceptable carriers are pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphoric acid, citrate, and other organic acids; antioxidants, including ascorbic acid; 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, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0116] "Package insert" refers to instructions that are typically included in the commercial packaging of a pharmaceutical product and contain information regarding indications, dosage, administration, contraindications, other pharmaceuticals to be combined with the packaged product, and / or warnings regarding the use of such pharmaceuticals.

[0117] As used herein, the term “treatment” refers to a clinical intervention designed to alter the natural course of an individual or cell being treated during the course of clinicopathology. Desired effects of treatment include, but are not limited to, a reduction in the rate of disease progression, improvement or alleviation of the disease state, remission or improved prognosis, and delay in disease progression. For example, an individual is successfully “treated” if one or more symptoms associated with lupus nephritis, including, but not limited to, elevated serum creatinine, proteinuria, red blood cell casts, renal dysfunction, renal syndrome, granular casts, microhematuria, macrohematuria, hypertension, tubular abnormalities, hyperkalemia, rapidly progressive glomerulonephritis (RPGN), and acute renal failure (ARF), are alleviated or eliminated. Delaying the progression of a disease (e.g., lupus nephritis) means postponing, interfering with, delaying, delaying, stabilizing, and / or postponing the onset of the disease. This delay may be of varying duration depending on the medical history and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can effectively encompass prevention, in that an individual at risk of developing the disease does not develop it. For example, the progression of SLE in an individual prior to the onset of symptoms and / or pathology of LN can be delayed so as to postpone or prevent the onset of LN.

[0118] As used herein, “complete renal response (CRR)” refers to a response to treatment that includes normalization of serum creatinine, inactive urinary sediment, and a urinary protein-to-creatinine ratio of less than 0.5.

[0119] As used herein, “partial renal response (PRR)” refers to a response to treatment that is less than the CRR but still includes relief of one or more symptoms, including but not limited to a decrease in serum creatinine, a decrease in urinary sediment, and a decrease in proteinuria.

[0120] An "effective dose" is the minimum concentration necessary to achieve a measurable improvement or prevention of a particular disorder. The effective dose as used herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the individual's ability to induce the desired response with the antibody. An effective dose is also the dose at which the therapeutically beneficial effects outweigh any toxic or adverse effects of the treatment. In the case of prophylactic use, beneficial or desired outcomes include the elimination or reduction of the risk of disease, reduction of disease severity, or delay of disease onset, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that appear during the onset of the disease. In the case of therapeutic use, beneficial or desired outcomes include clinical outcomes such as the reduction of one or more symptoms caused by the disease, improvement of the quality of life of the person affected, reduction of the dose of other drugs required to treat the disease, enhancement of the effect of another drug (e.g., by targeting), delay of disease progression, and / or extension of survival. In the case of lupus nephritis, an effective dose of a drug may be effective in and / or alleviate to some extent one or more symptoms associated with the disorder. The effective dose may be a single dose or multiple doses. For the purposes of this invention, the effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in the clinical field, the effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, the “effective dose” may be considered in relation to the administration of one or more therapeutic agents, and a monotherapy may be considered to be given in an effective dose if, when combined with one or more other agents, the desired outcome can or does not occur.

[0121] 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; its sequence is characterized by SwissProt database entry P11836), a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B lymphocytes and mature B lymphocytes. (Valentine,MA,et al.,J.Biol.Chem.264(19)(1989 11282-11287;Tedder,TF,et al,Proc.Natl.Acad.Sci.USA85(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 a transmembrane 4-domain, subfamily A, member 1, also known as MS4A1. This gene encodes a member of the transmembrane 4A gene family. Members of this nascent protein family are characterized by common structural features and similar intron / exon splice boundaries, and exhibit unique expression patterns among hematopoietic cells and non-lymphoid tissues. This gene encodes a B lymphocyte surface molecule that plays a role in the development and differentiation of B cells into plasma cells. This family member is localized to 11q12 within the cluster of family members. Alternative splicing of this gene results in two transcriptional variants encoding the same protein.

[0122] 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 antibody of the present invention to the CD20 antigen mediates the death of CD20-expressing cells (e.g., tumor cells) by inactivating CD20. The death of CD20-expressing cells may occur by one or more mechanisms: cell death / apoptosis induction, ADCC, and CDC.

[0123] Synonyms for CD20 recognized in the art include B lymphocyte antigen CD20, B lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5.

[0124] The term "anti-CD20 antibody" according to the present invention refers to an antibody that specifically binds to the CD20 antigen. Depending on the binding characteristics and biological activity of the anti-CD20 antibody against the CD20 antigen, two types of anti-CD20 antibodies (type I and type II anti-CD20 antibodies) can be distinguished by Cragg, MS, et al., Blood 103 (2004) 2738-2743; and Cragg, MS, et al., Blood 101 (2003) 1045-1052 (see Table 1 below). [Table 1]

[0125] Examples of type II anti-CD20 antibodies include, for example, humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody as disclosed in International Publication No. 2005 / 044859), 11B8 IgG1 (as disclosed in International Publication No. 2004 / 035607), and AT80 IgG1. Typically, type II anti-CD20 antibodies of the IgG1 isotype exhibit characteristic CDC properties. Type II anti-CD20 antibodies have reduced CDC compared to type I antibodies of the IgG1 isotype (in the case of the IgG1 isotype).

[0126] Examples of type I anti-CD20 antibodies include, for example, rituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (disclosed in International Publication No. 2005 / 103081), 2F2 IgG1 (disclosed in International Publication No. 2004 / 035607 and International Publication No. 2005 / 103081), and 2H7 IgG1 (disclosed in International Publication No. 2004 / 056312).

[0127] The defucosylated anti-CD20 antibody according to the present invention is preferably a type II anti-CD20 antibody, and more preferably a defucosylated humanized B-Ly1 antibody described in International Publication No. 2005 / 044859 and International Publication No. 2007 / 031875.

[0128] Rituximab (reference antibody; example of a type I anti-CD20 antibody) is a genetically engineered chimeric human gamma 1 mouse constant domain containing a monoclonal antibody against the human CD20 antigen. However, this antibody is not glycoengineered, is not defucosylated, and therefore contains at least 85% fucose. This chimeric antibody, containing a human gamma 1 constant domain, is identified by the name "C2B8" in U.S. Patent No. 5,736,137 (Andersen, et al.), issued on April 17, 1998, and transferred 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 lymphoma. In vitro studies of its mechanism of action have shown that rituximab induces human complement-dependent cell-mediated cytotoxicity (CDC) (Reff, ME, et.al, Blood 83(2)(1994)435-445). Furthermore, it exhibits activity in assays measuring antibody-dependent cytotoxicity (ADCC).

[0129] As used herein, the term "GA101 antibody" refers to any one of the following antibodies that bind to human CD20: (1) an antibody comprising HVR-H1 containing the amino acid sequence of SEQ ID NO: 1, HVR-H2 containing the amino acid sequence of SEQ ID NO: 2, HVR-H3 containing the amino acid sequence of SEQ ID NO: 3, HVR-L1 containing the amino acid sequence of SEQ ID NO: 4, HVR-L2 containing the amino acid sequence of SEQ ID NO: 5, and HVR-L3 containing the amino acid sequence of SEQ ID NO: 6; (2) an antibody comprising a VH domain containing the amino acid sequence of SEQ ID NO: 7 and a VL domain containing the amino acid sequence of SEQ ID NO: 8; (3) an antibody comprising the amino acid sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10; (4) an antibody known as obinutuzumab; or (5) an antibody comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 9 and at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 10. In one embodiment, the GA101 antibody is an IgG1 isotype antibody. In some embodiments, the anti-CD20 antibody is a humanized B-Ly1 antibody.

[0130] The term "humanized B-Ly1 antibody" refers to the humanized B-Ly1 antibody disclosed in International Publication No. 2005 / 044859 and International Publication No. 2007 / 031875, which was obtained by chimerization with a human constant domain derived from IgG1 and subsequent humanization from mouse monoclonal anti-CD20 antibody B-Ly1 (variable region of mouse heavy chain (VH): SEQ ID NO. 11; variable region of mouse light chain (VL): SEQ ID NO. 12: - See Poppema, S. and Visser, L., Biotest Bulletin 3 (1987) 131-139) (see International Publication No. 2005 / 044859 and International Publication No. 2007 / 031875). These "humanized B-Ly1 antibodies" are disclosed in detail in International Publication No. 2005 / 044859 and International Publication No. 2007 / 031875. Variable region of mouse monoclonal anti-CD20 antibody B-Ly1 heavy chain (VH) (SEQ ID NO: 11) JPEG2026090308000004.jpg78170 Mouse monoclonal anti-CD20 antibody B-Ly1 light chain variable region (VL) (SEQ ID NO: 12) JPEG2026090308000005.jpg78170

[0131] In one embodiment, the "humanized B-Ly1 antibody" has a heavy chain variable region (VH) selected from the group of SEQ ID NOs. 7, 8, and 13-33 (in particular, corresponding to B-HH2 to B-HH9 and B-HL8 to B-HL17 in International Publication Nos. 2005 / 044859 and International Publication Nos. 2007 / 031875). In a specific embodiment, such a variable domain is selected from the group consisting of SEQ ID NOs. 14, 15, 7, 19, 25, 27, and 29 (corresponding to B-HH2, BHH-3, B-HH6, B-HH8, B-HL8, B-HL11, and B-HL13 in International Publication Nos. 2005 / 044859 and International Publication Nos. 2007 / 031875). In one specific embodiment, the "humanized B-Ly1 antibody" has a variable light chain region (VL) of SEQ ID NO: 8 (corresponding to B-KV1 in International Publication Nos. 2005 / 044859 and 2007 / 031875). In another specific embodiment, the "humanized B-Ly1 antibody" has a variable heavy chain region (VH) of SEQ ID NO: 7 (corresponding to B-HH6 in International Publication Nos. 2005 / 044859 and 2007 / 031875) and a variable light chain region (VL) of SEQ ID NO: 8 (corresponding to B-KV1 in International Publication Nos. 2005 / 044859 and 2007 / 031875). Furthermore, in one embodiment, the humanized B-Ly1 antibody is an IgG1 antibody. According to the present invention, such a defucosylated humanized B-Ly1 antibody has its Fc region glycosylated (GE) according to the procedure described in International Publication No. 2005 / 044859, International Publication No. 2004 / 065540, International Publication No. 2007 / 031875, Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180, and International Publication No. 99 / 154342. In one embodiment, the defucosylated, glycosylated humanized B-Ly1 is B-HH6-B-KV1 GE. In one embodiment, the anti-CD20 antibody is obinutuzumab (recommended by INN, WHO Drug Information, Vol. 26, No. 4, 2012, p. 453). As used herein, obinutuzumab is synonymous with GA101 or RO5072759.This replaces all previous versions (e.g., Vol.25, No.1, 2011, pp.75-76) and was previously known as aftuzumab (recommended by INN, WHO Drug Information, Vol.23, No.2, 2009, p.176; Vol.22, No.2, 2008, p.124). As used herein, references to obinutuzumab refer to GAZYVA® and its biosimilar antibodies. In some embodiments, the humanized B-Ly1 antibody is an antibody or antigen-binding fragment thereof comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 9 and a light chain containing the amino acid sequence of SEQ ID NO: 10. In some embodiments, the humanized B-Ly1 antibody comprises a heavy chain variable region containing three heavy chain CDRs of SEQ ID NO: 9 and a light chain variable region containing three light chain CDRs of SEQ ID NO: 10. JPEG2026090308000006.jpg88170

[0132] In some embodiments, the humanized B-Ly1 antibody is a defucosylated, glycoseptically engineered humanized B-Ly1. Such glycoseptically engineered humanized B-Ly1 antibodies have an altered glycosylation pattern in the Fc region, preferably with a reduced level of fucose residues. Preferably, the amount of fucose is 60% or less of the total amount of oligosaccharides in Asn297 (in one embodiment, the amount of fucose is 40% to 60%, in another embodiment, the amount of fucose is 50% or less, and in yet another embodiment, the amount of fucose is 30% or less). Furthermore, the oligosaccharides in the Fc region are preferably bisected. These glycoseptically engineered humanized B-Ly1 antibodies have increased ADCC.

[0133] The "ratio of the binding ability of anti-CD20 antibody to CD20 on Raji cells (ATCC-No.CCL-86) compared to rituximab" was determined by direct immunofluorescence measurement (mean fluorescence intensity (MFI) was measured) using the above anti-CD20 antibody conjugated with Cy5 and rituximab conjugated with Cy5 in a FACS array (Becton Dickinson) containing Raji cells (ATCC-No.CCL-86), as described in Example No. 2, and was calculated as follows. Ratio of CD20 binding ability on Raji cells (ATCC-No.CCL-86) = JPEG2026090308000007.jpg12170

[0134] MFI is the average fluorescence intensity. As used herein, "Cy5 labeling rate" refers to the number of Cy5-labeled molecules per antibody molecule.

[0135] Typically, the type II anti-CD20 antibody described above has a binding affinity ratio of 0.3 to 0.6 to rituximab for CD20 on Raji cells (ATCC-No.CCL-86), which is 0.35 to 0.55 in one embodiment and 0.4 to 0.5 in yet another embodiment.

[0136] In one embodiment, the above-mentioned type II anti-CD20 antibody, for example, GA101 antibody, exhibits enhanced antibody-dependent cell-mediated cytotoxicity (ADCC).

[0137] "An antibody having increased antibody-dependent cytotoxicity (ADCC)" means an antibody as defined herein that has an increase in ADCC, as determined by any suitable method known to those skilled in the art. One accepted in vitro ADCC assay is as follows: 1) The assay uses target cells known to express the target antigen recognized by the antigen-binding region of the antibody; 2) The assay uses human peripheral blood mononuclear cells (PBMCs) isolated from the blood of randomly selected healthy donors as effector cells; 3) The assay is performed according to the following protocol: i) Isolate PBMCs using a standard density centrifugation procedure, 5 × 10 6 Suspend cells in RPMI cell culture medium at a concentration of cells / ml; ii) Propagate target cells using standard tissue culture methods, harvest them from the exponential growth phase with a viability rate of more than 90%, wash them in RPMI cell culture medium, and dry them at 100 microcuries. 51 Labeled with Cr, washed twice with cell culture medium, 10 5 Resuspend the cells in the cell culture medium at a density of cells / ml; iii) Transfer 100 microliters of the above final target cell suspension to each well of a 96-well microtiter plate; iv) The antibody is sequentially diluted in cell culture medium from 4000 ng / ml to 0.04 ng / ml, and 50 microliters of the resulting antibody solution are added to target cells in a 96-well microtiter plate. A triple series of tests is performed to test various antibody concentrations covering the entire concentration range described above. As a maximum release (MR) control, add 50 microliters of 2% (VN) aqueous solution of a nonionic surfactant (Nonidet, Sigma, St. Louis) to three additional wells in a plate containing labeled target cells, instead of the antibody solution (see iv above); vi) As a spontaneous release (SR) control, place 50 microliters of RPMI cell culture medium in three additional wells of the plate containing labeled target cells instead of the antibody solution (as described in iv above); vii) Next, centrifuge the 96-well microtiter plate at 50 × g for 1 minute and incubate at 4°C for 1 hour; viii) Add 50 microliters of PBMC suspension (above i) to each well to obtain an effector:target cell ratio of 25:1, and place the plate in an incubator under a 5% CO2 atmosphere at 37°C for 4 hours; ix) Collect the cell-free supernatant from each well and quantify the experimentally released radioactivity (ER) using a gamma counter; x) The percentage of specific lysis is calculated for each antibody concentration according to the formula (ER-MR) / (MR-SR)×100, where ER is the quantified average radioactivity for that antibody concentration (see ix above), MR is the quantified average radioactivity for the MR control (see V above) (see ix above), and SR is the quantified average radioactivity for the SR control (see vi above) (see ix above); 4) “Increased ADCC” is defined as either 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 of the maximum percentage of specific lysis observed within the antibody concentration range tested above. In one embodiment, the increase in ADCC is compared to the ADCC measured in the above assay, mediated by the same antibody produced by the same type of host cells using the same standard production, purification, formulation and storage methods known to those skilled in the art, except that the comparative antibody (lacking increased ADCC) was not produced by host cells engineered to overexpress GnTIII and / or to reduce expression from the fucosyltransferase 8 (FUT8) gene (including those engineered for FUT8 knockout).

[0138] The "increased ADCC" mentioned above can be obtained, for example, by mutation and / or glycotechnology of the antibody. In one embodiment, the antibody is glycotechnologyly manipulated to have a branched oligosaccharide bound to the Fc region of a bisected antibody using GlcNAc, for example, in International Publication No. 2003 / 011878 (Jean-Mairet et al); U.S. Patent No. 6,602,684 (Umana et al.); U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.), P., et al., Nature Biotechnol. 17 (1999) 176-180). In another embodiment, the antibody is used in host cells lacking protein fucosylation (e.g., Lec13 CHO cells), or in cells lacking the alpha-1,6-fucosyltransferase gene (FUT8) or with knocked-down FUT gene expression (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al.). The antibody is glycotechnically engineered to lack fucose on the carbohydrate bound to the Fc region by expressing the antibody as described in *al., Biotechnol. Bioeng., 94(4):680-688(2006); and International Publication No. 2003 / 085107). In yet another embodiment, the antibody sequence is engineered to enhance ADCC in its Fc region (for example, in one embodiment, such an engineered antibody variant includes an Fc region having one or more amino acid substitutions at positions 298, 333 and / or 334 (EU numbering of residues) in the Fc region).

[0139] The term "complement-dependent cell-mediated cytotoxicity (CDC)" refers to the lysis of human tumor target cells by the antibody according to the present invention in the presence of complement. CDC can be measured by treating a preparation of CD20-expressing cells with the anti-CD20 antibody according to the present invention in the presence of complement. CDC is found when the antibody induces lysis (cell death) of 20% or more of tumor cells after 4 hours at a concentration of 100 nM. In one embodiment, the assay is performed: 51 This was performed using tumor cells labeled with Cr or Eu, and the released 51Cr or Eu levels are measured. The control includes incubation of tumor target cells containing complement but without antibodies.

[0140] The term “expression” of the “CD20” antigen is intended to indicate a significant level of expression of the CD20 antigen in cells, such as T cells or B cells. In one embodiment, a patient treated according to the method of the present invention expresses a significant level of CD20 on B cells. CD20 expression on B cells can be determined by standard assays known in the art, for example, CD20 antigen expression is measured by immunohistochemistry (IHC) detection, using FACS, or by PCR-based detection of the corresponding mRNA.

[0141] When used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple references unless otherwise explicitly indicated. Thus, for example, a reference to "a molecule" includes any combination of two or more such molecules.

[0142] As used herein, the term “approximately” refers to the normal margin of error for each value, as would be readily understood by those skilled in the art. References to values ​​or parameters referred to as “approximately” herein include (and describe) embodiments relating to the value or parameter itself.

[0143] The aspects and embodiments of the present invention described herein are understood to include the terms "comprising," "consisting," and "consisting essentially of."

[0144] III. Method In one embodiment, a method is provided herein for treating lupus nephritis in an individual having lupus, or for depleting circulating peripheral B cells in an individual, by administering an effective amount of type II anti-CD20 antibody. In some embodiments, the individual has lupus nephritis or is at risk of developing lupus nephritis. In some embodiments, the lupus nephritis is class III or class IV lupus nephritis. In some embodiments, the individual has class III(C) or class IV(C) lupus nephritis. In some embodiments, the individual has contingent class V lupus nephritis. In some embodiments, the method comprises administering to an individual a first antibody exposure to type II anti-CD20 antibody, a second antibody exposure to type II anti-CD20 antibody, and a third antibody exposure to type II anti-CD20 antibody, wherein the second antibody exposure is not given from about 18 weeks to about 26 weeks after the first antibody exposure, the third antibody exposure is not given from about 24 weeks to about 32 weeks after the second antibody exposure, and the first antibody exposure comprises one or two administrations of type II anti-CD20 antibody. A method is provided herein in which an antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody, a second antibody exposure comprising one or two administrations of type II anti-CD20 antibody, a third antibody exposure comprising one or two administrations of type II anti-CD20 antibody, and a third antibody exposure comprising a total exposure of approximately 800 mg to approximately 1200 mg of type II anti-CD20 antibody. In some embodiments, the method comprises administering to an individual a first antibody exposure to type II anti-CD20 antibody and a second antibody exposure to type II anti-CD20 antibody, wherein the second antibody exposure is not given until approximately 18 to 26 weeks after the first antibody exposure, the first antibody exposure comprises one or two administrations of type II anti-CD20 antibody, the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody, and the second antibody exposure comprises one or two administrations of type II anti-CD20 antibody, the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of type II anti-CD20 antibody.As described herein, in some embodiments, the antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6. In some embodiments, the antibody comprises a VH domain containing the amino acid sequence of SEQ ID NO: 7 and a VL domain containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 9, and having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 10.

[0145] In another embodiment, a method for treating membranous nephropathy (MN) (e.g., primary membranous nephropathy, pMN) by administering an effective amount of anti-CD20 type II antibody is provided herein. In some embodiments, the method comprises administering to an individual a first antibody exposure to anti-CD20 type II antibody and a second antibody exposure to anti-CD20 type II antibody, wherein the second antibody exposure is not given until about 18 to 26 weeks after the first antibody exposure, and the first antibody exposure comprises one or two administrations of anti-CD20 type II antibody, comprising a total exposure of about 1800 mg to about 2200 mg of anti-CD20 type II antibody, and the second antibody exposure comprises one or two administrations of anti-CD20 type II antibody, comprising a total exposure of about 1800 mg to about 2200 mg of anti-CD20 type II antibody. As described herein, in some embodiments, the antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6. In some embodiments, the antibody comprises a VH domain containing the amino acid sequence of SEQ ID NO: 7 and a VL domain containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 9, and having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 10.

[0146] Anti-CD20 antibody Certain embodiments of this disclosure relate to anti-CD20 antibodies for use in the methods described herein, for example, for treating or preventing the progression of lupus nephritis, or for treating or preventing the progression of membranous nephropathy (e.g., pMN). In some embodiments, the anti-CD20 antibody is a type II antibody. In some embodiments, the anti-CD20 antibody is a human antibody or a humanized antibody. In some embodiments, the anti-CD20 antibody is defucosylated. In some embodiments, the anti-CD20 antibody is a GA 101 antibody.

[0147] Examples of type II anti-CD20 antibodies include, for example, humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody as disclosed in International Publication No. 2005 / 044859), 11B8 IgG1 (as disclosed in International Publication No. 2004 / 035607), and AT80 IgG1. Typically, type II anti-CD20 antibodies of the IgG1 isotype exhibit characteristic CDC properties. Type II anti-CD20 antibodies have reduced CDC compared to type I antibodies of the IgG1 isotype (in the case of the IgG1 isotype).

[0148] In some embodiments, the anti-CD20 antibody is the GA101 antibody described herein. In some embodiments, the anti-CD20 is one of the following antibodies that bind to human CD20: (1) an antibody comprising HVR-H1 containing the amino acid sequence GYAFSY (SEQ ID NO: 1), HVR-H2 containing the amino acid sequence FPGDGDTD (SEQ ID NO: 2), HVR-H3 containing the amino acid sequence NVFDGYWLVY (SEQ ID NO: 3), HVR-L1 containing the amino acid sequence RSSKSLLHSNGITYLY (SEQ ID NO: 4), HVR-L2 containing the amino acid sequence QMSNLVS (SEQ ID NO: 5), and HVR-L3 containing the amino acid sequence AQNLELPYT (SEQ ID NO: 6); (2) the amino acid sequence of SEQ ID NO: 7 (3) an antibody comprising a VH domain containing the amino acid sequence of SEQ ID NO: 8 and a VL domain containing the amino acid sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10; (4) an antibody known as obinutuzumab; or (5) an antibody comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 9 and an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 10. In one embodiment, the GA101 antibody is an IgG1 isotype antibody. In some embodiments, the anti-CD20 antibody comprises HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of any of the antibodies described herein, for example, three HVRs from SEQ ID NO: 7 and three HVRs from SEQ ID NO: 8; three HVRs from SEQ ID NO: 9 and three HVRs from SEQ ID NO: 10; or an HVR of any of the amino acid sequences provided in Table 2.

[0149] In some embodiments, the anti-CD20 antibody includes a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 7 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 8. JPEG2026090308000008.jpg41170

[0150] In some embodiments, the anti-CD20 antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 9 and a light chain containing the amino acid sequence of SEQ ID NO: 10. JPEG2026090308000009.jpg93170

[0151] In some embodiments, the anti-CD20 antibody is a humanized B-Ly1 antibody. In some embodiments, the humanized B-Ly1 antibody comprises a heavy chain variable region containing three heavy chain CDRs of SEQ ID NO: 9 and a light chain variable region containing three light chain CDRs of SEQ ID NO: 10. In some embodiments, the humanized B-Ly1 antibody comprises a heavy chain containing the sequence of SEQ ID NO: 9 and a light chain containing the sequence of SEQ ID NO: 10.

[0152] In some embodiments, the anti-CD20 antibody contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequences listed in Table 2 below. [Table 2] TIFF2026090308000011.tif235170TIFF2026090308000012.tif171170

[0153] In some embodiments, the anti-CD20 antibody (e.g., type II anti-CD20 antibody) is a defucosylated glycosylated antibody. Such glycoengineered antibodies have an altered glycosylation pattern in the Fc region, preferably with a reduced level of fucose residues. Preferably, the amount of fucose is 60% or less of the total amount of oligosaccharides in Asn297 (in one embodiment, the amount of fucose is 40% to 60%, in another embodiment, the amount of fucose is 50% or less, and in yet another embodiment, the amount of fucose is 30% or less). Furthermore, the oligosaccharides in the Fc region are preferably bisected. In some embodiments, the type II anti-CD20 antibody includes an Fc region containing bisected oligosaccharides bisected by N-acetylglucosamine (GlcNAc). These glycoengineered modified humanized anti-CD20 (e.g., B-Ly1) antibodies have increased ADCC.

[0154] Oligosaccharide components can significantly influence properties related to the efficacy of therapeutic glycoproteins, including physical stability, resistance to protease attack, interaction with the immune system, pharmacokinetics, and phenotypic activity. Such properties may depend not only on the presence or absence of oligosaccharides but also on their specific structure. Some generalizations can be made between oligosaccharide structure and glycoprotein function. For example, certain oligosaccharide structures mediate rapid clearance of glycoproteins from the bloodstream through interaction with specific carbohydrate-binding proteins, while others may be bound by antibodies, potentially triggering undesirable immune responses. (Jenkins, N., et al., Nature Biotechnol. 14(1996) 975-81).

[0155] Mammalian cells are preferred hosts for the production of therapeutic glycoproteins due to their ability to glycosylate proteins in the form most suitable for human application (Cumming, DA, et al., Glycobiology 1(1991)115-30; Jenkins, N., et al., Nature Biotechnol. 14(1996)975-81). Bacteria glycosylate proteins very rarely, and, as with other common host types such as yeast, filamentous fungi, insect and plant cells, produce glycosylation patterns associated with rapid clearance from the bloodstream, undesirable immune interactions, and, in some specific cases, reduced biological activity. Among mammalian cells, Chinese hamster ovary (CHO) cells have been the most commonly used over the past 20 years. In addition to providing a suitable glycosylation pattern, these cells enable the consistent generation of genetically stable and highly productive clonal cell lines. They can be cultured at high density in simple bioreactors using serum-free media, enabling the development of safe and reproducible bioprocesses. Other commonly used animal cells include baby hamster kidney (BHK) cells and NSO- and SP2 / 0- mouse myeloma cells. Recently, production from transgenic animals has also been investigated. (Jenkins, N., et al., Nature Biotechnol. 14(1996) 975-981).

[0156] Antibodies may contain carbohydrate structures at conserved positions in the heavy chain constant region, and each isotype has a different set of N-linked carbohydrate structures that variably affect protein assembly, secretion, or functional activity. (Wright, A., and Morrison, SL, Trends Biotech. 15(1997) 26-32). The structure of the bound N-linked carbohydrate varies considerably depending on the degree of processing and can include high-mannose, polybranched, and bibranched complex oligosaccharides. (Wright, A., and Morrison, SL, Trends Biotech. 15(1997) 26-32). Typically, heterogeneous processing of core oligosaccharide structures bound to specific glycosylation sites exists, even monoclonal antibodies, which exist as multiple glycoforms. Similarly, large differences in antibody glycosylation have been shown to occur between cell lines, and even slight differences can be observed in a given cell line grown under different culture conditions. (Lifely, MR, et al., Glycobiology 5(8)(1995)813-22).

[0157] One way to achieve a significant increase in potency while maintaining a simple manufacturing process and avoiding potentially significant undesirable side effects is to enhance the innate cell-mediated effector function of monoclonal antibodies by manipulating their oligosaccharide components, as described in Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180 and U.S. Patent No. 6,602,684. IgG1 antibodies are the most commonly used antibodies in cancer immunotherapy and are glycoproteins with conserved N-linked glycosylation sites at Asn297 of each CH2 domain. The two complex bifurcated oligosaccharides bound to Asn297 are embedded between the CH2 domains, forming extensive contact with the polypeptide backbone, and their presence is essential for antibodies to mediate effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) (Lifely, MR, et al., Glycobiology 5 (1995) 813-822; Jefferis, R., et al., Immunol. Rev. 163 (1998) 59-76; Wright, A., and Morrison, SL, Trends Biotechnol. 15 (1997) 26-32).

[0158] It has been previously shown that overexpression of β(1,4)-N-acetylglucosaminyltransferase I11 ("GnTII17y"), a glycosyltransferase that catalyzes the formation of bisected oligosaccharides, in Chinese hamster ovary (CHO) cells significantly increases the in vitro ADCC activity of the anti-neuroblastoma chimeric monoclonal antibody (chCE7) produced by engineered CHO cells. (Umana, P., et al., Nature Biotechnol. 17(1999) 176-180; and International Publication No. 99 / 154342 (the full contents thereof are incorporated herein by reference)) The antibody chCE7 belongs to a large class of non-conjugate monoclonal antibodies that possess high tumor affinity and specificity, but have insufficient potency to be clinically useful when produced in standard industrial cell lines lacking the GnTIII enzyme (Umana, P., et al., Nature Biotechnol. 17(1999) 176-180). This study was the first to demonstrate that a significant increase in ADCC activity could be obtained by manipulating antibody-producing cells to express GnTIII, which also increased the proportion of bisected oligosaccharides associated with the constant region (Fc) containing bisected non-fucosylated oligosaccharides beyond the levels found in naturally occurring antibodies.

[0159] In some embodiments, the anti-CD20 antibody (e.g., type II anti-CD20 antibody) includes a human Fc region (e.g., human IgG1 Fc region). In some embodiments, the Fc region includes a modified N-linked oligosaccharide. In some embodiments, the N-linked oligosaccharide in the Fc region has reduced fucose residues compared to antibodies with unmodified N-linked oligosaccharides. In some embodiments, the bisected oligosaccharide is a bisected complex oligosaccharide. In some embodiments, the N-linked oligosaccharide is modified to have an increased bisected non-fucosylated oligosaccharide. In some embodiments, the bisected non-fucosylated oligosaccharide is a hybrid type. In some embodiments, the bisected non-fucosylated oligosaccharide is a complex type. For a more detailed explanation, see, for example, International Publication No. 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.); and U.S. Patent No. 8,883,980 (Umana et al.).

[0160] In some embodiments, the type II anti-CD20 antibody is obinutuzumab.

[0161] Antibody preparation An antibody according to any of the above embodiments (for example, the type II anti-CD20 antibody of this disclosure) may incorporate any of the following features, either individually or in combination, as described in Sections 1 to 7 below.

[0162] antibody affinity In certain embodiments, the antibodies provided herein have concentrations of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (for example, 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 It has a dissociation constant (Kd) of M.

[0163] In one embodiment, Kd is measured by a radiolabeled antigen-binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of Fab to the antigen is measured in the presence of a titration series of the unlabeled antigen at a minimum concentration. 125 I) Fab is equilibrated with labeled antigen, and then measured by capturing the bound antigen with a plate coated with anti-Fab antibody (see, e.g., Chen et al., J.Mol.Biol.293:865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / mL of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2–5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc#269620), 100 pM or 26 pM [ 125 Mix the [I]-antigen with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight; however, incubation may be continued for a longer period (e.g., about 65 hours) to reach equilibrium. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). Then remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, add 150 μL / well of scintillant (MICROSCINT-20®, Packard) and count the plate on a TOPCOUNT® gamma counter (Packard) for 10 minutes. Select the concentration of each Fab that yields less than 20% of maximum binding for use in competitive binding assays.

[0164] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, assays using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) are performed at approximately 10 response units (RUs) at 25°C using an immobilized antigen CM5 chip. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE) is activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the instructions of those skilled in the art. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate at pH 4.8, and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RUs) of the coupled protein. After antigen injection, 1M ethanolamine is injected to block unreacted groups. For kinetic measurements, twofold serial dilutions of Fab (0.78 nM to 500 nM) (in PBS containing 0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST)) are injected at 25°C at a flow rate of approximately 25 μL / min. The association rate (kon) and dissociation rate (koff) are calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir coupling model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the koff / kon ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999).If the association rate by the surface plasmon resonance assay described above exceeds 10⁶ M⁻¹ s⁻¹, the association rate can be determined using a fluorescence quenching technique that measures the increase or decrease in the fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of a 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing antigen concentrations. This technique is measured using a spectrophotometer such as an Aviv Instruments spectrophotometer with stopped flow or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirring cuvette.

[0165] antibody fragment In certain embodiments, the antibodies provided herein are antibody fragments. Examples of 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 specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For references to scFv fragments, see, for example, Pluckthuen, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); also see International Publication No. 93 / 16185; and U.S. Patents Nos. 5,571,894 and 5,587,458. For a description of the Fab and F(ab')2 fragments, which contain salvage receptor-binding epitope residues and have a longer in vivo half-life, please refer to U.S. Patent No. 5,869,046.

[0166] A diabody is an antibody fragment having two antigen-binding sites, which may be bivalent or bispecific. See, for example, European Patent No. 404,097, International Publication No. 1993 / 01161, Hudson et al. Nat. Med. 9:129-134 (2003); and Hollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al. Nat. Med. 9:129-134 (2003).

[0167] A single-domain antibody is an antibody fragment that contains all or part of the heavy chain variable domains or all or part of the light chain variable domains of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc. (Massachusetts Waltham), U.S. Patent No. 6,248,516B1).

[0168] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., Escherichia coli or phages), as described herein.

[0169] Chimeric antibodies and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass is modified from those of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0170] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity against humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody contains one or more variable domains in which the HVR, e.g., CDR (or a portion thereof), is derived from a non-human antibody and the FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody optionally also contains at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which the HVR residues are derived) to restore or improve antibody specificity or affinity, for example.

[0171] Humanized antibodies and methods for their production are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described below: e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (Description of Slicity Determination Region (SDR) grafts); Padlan, Mol.Immunol.28:489-498 (1991) (Description of resurfacing); Dall'Acqua et al., Methods 36:43-60 (2005) (Description of FR shuffle); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br.J.Cancer,83:252-260 (2000) (Description of the "guided selection approach" to FR shuffle).

[0172] Human framework regions that may be used for humanization include, but are not limited to, the following: framework regions selected using the “best fit” method (see, e.g., Sims et al. J.Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain 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 maturation (somatic mutation) framework regions or human germ cell framework regions (see, e.g., Almagro and Fransson, Front.Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of the FR library (see, e.g., Baca et al. See al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0173] Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be prepared 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).

[0174] Human antibodies can be prepared by administering immunogens to transgenic animals modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain, or have extrachromosomal, or randomly integrated human immunoglobulin loci, replacing endogenous immunoglobulin loci. In such transgenic mice, 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 2007 / 0061900 describing VelociMouse® technology. Human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining them with different human constant regions.

[0175] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human xenomyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, 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 via human B-cell hybridoma technology have also been described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, U.S. Patent No. 7,189,826 (describes the production of monoclonal human IgM antibodies derived from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in 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).

[0176] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0177] Antibodies derived from the library The antibodies of the present invention can be isolated by screening a combinatorial library for one or more antibodies having desired activity. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies having desired binding properties. Such methods are outlined, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), 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. Further information is available in 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).

[0178] In certain phage display methods, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR), randomly recombined within a phage library, and then screened against antigen-binding phages, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). The phages typically display antibody fragments as either single-stranded Fv (scFv) fragments or Fab fragments. Libraries from immunogens provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) without immunization to provide a single source of antibodies against a wide range of non-self and also self-antigens, as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, natural libraries can also be synthetically constructed by cloning an unrearranged V gene segment from stem cells, encoding the highly variable CDR3 region using PCR primers containing random sequences, and performing rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Examples of patent publications describing human antibody phage libraries include U.S. Patent No. 5,750,373, and U.S. Patent Application Publications 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0179] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments in this specification.

[0180] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, for example, bispecific antibodies. A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites. In certain embodiments, one binding specificity is to CD20 and the other is to any other antigen. In certain embodiments, a bispecific antibody may bind to two different epitopes of CD20. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing CD20. Bispecific antibodies may be prepared as full-length antibodies or antibody fragments.

[0181] Techniques for producing multispecific antibodies include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), International Publication No. 93 / 08829, and Traunecker et al., EMBO J.10:3655 (1991)), and the "knob-into-hole" operation (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies also utilize techniques such as: manipulating the electrostatic steering effect to produce antibody Fc heterodimer molecules (International Publication No. 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J.Immunol., 148(5):1547-1553 (1992)); using "diabody" techniques to produce bispecific antibody fragments (see, e.g., Hollinger et al., Proc.Natl.Acad.Sci.USA, 90:6444-6448 (1993)); and using single-chain Fv(sFv) dimers (see, e.g., Gruber et al. See al., J.Immunol., 152:5368 (1994); and it can also be prepared by the preparation of a triplicate antibody as described, for example, Tutt et al. J.Immunol. 147:60 (1991).

[0182] This also includes antibodies designed to have three or more functional antigen-binding sites, such as "octopus antibodies." (See, for example, U.S. Patent Publication 2006 / 0025576A1)

[0183] The antibodies or fragments described herein also include "Dual Acting FAb" or "DAF" which include an antigen-binding site that binds to CD20 and another different antigen (see, for example, U.S. Patent Application Publication 2008 / 0069820).

[0184] 7. Antibody variants In certain embodiments, amino acid sequence variants of antibodies provided herein are intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from residues in the amino acid sequence of the antibody, and / or insertions into residues in the amino acid sequence of the antibody, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed so as to reach the final construct, insofar as the final construct has the desired characteristics (e.g., antigen binding).

[0185] a) Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for mutation introduction by substitution include HVR and FR. Conservative substitutions are shown in Table A under the heading "Preferred Substitutions." More substantial changes are shown in Table A under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions are introduced into the antibody of interest, and the product can be screened for desired activity, e.g., retention / improvement of antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC. [Table A]

[0186] Amino acids can be classified according to their general side-chain properties. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0187] Non-conservative substitution involves swapping one member of one of these classes with one of another.

[0188] A type of substitution mutant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting mutant selected for further study will have modifications (e.g., improvements) in specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or will substantially retain the specific biological properties of the parent antibody. Exemplary substitution mutants are affinity-matured antibodies, which can be readily generated using, for example, phage display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated, the mutant antibody is presented on a phage, and screened for specific biological activity (e.g., binding affinity).

[0189] Modifications (e.g., substitutions) may be made, for example, in HVR to improve antibody affinity. Such modifications may be made in HVR "hot spots," i.e., residues encoded by codons that are frequently mutated during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that come into contact with the antigen, and the resulting mutant VH or VL is tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described here. (For example, Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)) In some embodiments of affinity maturation, diversity is introduced into variable genes selected for maturation by one of several methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. This library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, which randomizes several HVR residues (e.g., 4-6 residues at a time). HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly often targeted.

[0190] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be made within an HVR. Such modifications may be, for example, outside the antigen-contact residue within the HVR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unmodified or contains one, two, or three or fewer amino acid substitutions.

[0191] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. This method identifies target residues or groups (e.g., charged residues such as arg, asp, his, lys, and glu) and substituted them with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex may be used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues may be targeted as candidates for substitution or removed. Mutants may be screened to determine whether they possess desired properties.

[0192] Amino acid insertions include amino-terminus and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. An example of terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include the fusion of the N-terminus or C-terminus of an antibody to an enzyme or polypeptide that increases the serum half-life of the antibody (for example, for ADEPT).

[0193] b) Glycosylated mutants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of glycosylation sites to an antibody can be conveniently achieved by modifying the amino acid sequence so that one or more glycosylation sites are created or removed.

[0194] If an antibody contains an Fc region, the carbohydrate bound to it may be modified. Natural antibodies produced by mammalian cells typically contain branched oligosaccharides that are commonly bound to Asn297 of the CH2 domain of the Fc region by an N-bond. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to the GlcNAc of the "stem" of the branched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibody of the present invention may be performed to create antibody variants having specific improved properties.

[0195] In one embodiment, an antibody variant is provided having a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. The amount of fucose in such an antibody may be, for example, 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycans at Asn297 relative to the total amount of all glycan structures attached to Asn297 (e.g., complex structures, hybrid structures, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, as described in, for example, International Publication No. 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (Eu numbering of Fc region residues) within the Fc region, although Asn297 may be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to slight sequence variations within the antibody. Such fucosylated mutants may possess improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.); and No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody mutants include: U.S. Patent Application Publication No. 2003 / 0157108; International Publication No. 2000 / 61739; International Publication No. 2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; International Publication No. 2002 / 0164328; International Publication No. 2004 / 0093621; International Publication No. 2004 / 01 Examples include publications No. 32140; No. 2004 / 0110704; No. 2004 / 0110282; No. 2004 / 0109865; International Publication No. 2003 / 085119; No. 2003 / 084570; No. 2005 / 035586; No. 2005 / 035778; No. 2005 / 053742; No. 2002 / 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 Publication No. 2003 / 0157108 A1, Presta, L; and International Publication No. 2004 / 056312 A1, Adams et al., particularly Example 11), and knockout cell lines, such as those containing the α-1,6-fucosyltransferase gene, FUT8, or 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 International Publication No. 2003 / 085107).

[0196] For example, antibody variants are further provided having a bisected oligosaccharide in which a bisected oligosaccharide bound 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 International Publication 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue of 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 International Publication No. 1997 / 30087 (Patel et al.); International Publication No. 1998 / 58964 (Raju, S.); and International Publication No. 1999 / 22764 (Raju, S.).

[0197] c) Fc region variant In certain embodiments, one or more amino acid modifications are introduced into the Fc region of the antibody provided herein, thereby creating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0198] In certain embodiments, the present invention aims to create antibody variants that, by possessing some, but not all, effector functions, are desirable candidate antibodies for applications where the in vivo half-life of the antibody is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only Fc(RIII), while monocytes express Fc(RI), Fc(RII, and Fc(RIII). FcR expression in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of target molecules include U.S. Patent No. 5,500,362 (e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al.). This is described in al., J.Exp.Med.166:1351-1361 (1987). Alternatively, non-radioactive assays may be used (see, for example, ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in animal models, such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J.Immunol.Methods 202:163(1996); Cragg, MS et al., Blood 101:1045-1052(2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743(2004)). FcRn binding and in vivo clearance / half-life can also be determined using methods known in the art (see, for example, Petkova, S B et al., Int'l.Immunol.18(12):1759-1769(2006)).

[0199] Antibodies with reduced effector function include antibodies having one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, and include the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0200] In certain embodiments, the Fc variants described herein further include one or more amino acid modifications to attenuate effector function (such as CDC and / or ADCC). In exemplary embodiments, the effector-attenuating modifications are modifications that do not alter the glycosylation pattern of the Fc region. In certain embodiments, the effector-attenuating modifications reduce or eliminate binding to human effector cells, binding to one or more Fc receptors, and / or binding to cells expressing Fc receptors. In exemplary embodiments, the Fc variants described herein include the following modifications, namely L234A, L235A, and P329G in the Fc region of human IgG1, which result in attenuated effector function. Substitutions L234A, L235A, and P329G (the L234A / L235A / P329G triple mutant is called LALAPG) have been previously shown to reduce binding to the Fc receptor and complement (see, for example, U.S. Patent Application Publication No. 2012 / 0251531).

[0201] In various embodiments, an Fc variant with reduced effector function refers to an Fc variant that reduces effector function (e.g., activity such as binding to CDC, ADCC, and / or FcR) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or more compared to the effector function achieved by a wild-type Fc region (e.g., an Fc region that may have other mutations but does not have mutations that reduce effector function). In certain embodiments, an Fc variant with reduced effector function refers to an Fc variant that eliminates all detectable effector function compared to a wild-type Fc region. Assays for measuring effector function are known in the art and are described below.

[0202] In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / absence of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity). NK cells, the main cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression in hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patent No. 5,500,362 (e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be employed (see, for example, ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in animal models, such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity.For example, see the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and 2005 / 100402. CDC assays can be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J.Immunol.Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)).

[0203] Specific antibody variants exhibiting improved or reduced binding to FcR have been described. (See, for example, U.S. Patent No. 6,737,056; International Publication No. 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001).)

[0204] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0205] In some embodiments, modifications are made in the Fc region that results in modified (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0206] Antibodies that have increased half-lives and improved binding to the embryonic Fc receptor (FcRn) and play a role in transferring maternal IgG to the fetus (Guyer et al., J.Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include variants having substitutions in 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, for example, a substitution in the Fc region residue 434 (U.S. Patent No. 7,371,826).

[0207] For other examples of variants in the Fc region, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and International Publication No. 94 / 29351.

[0208] d) Cysteine-modified antibody variants In certain embodiments, it may be desirable to create cysteine-modified artificial antibodies, e.g., “thioMAbs”, in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues are located in the accessible sites of the antibody. By substituting these residues with cysteine, the reactive thiol group is thereby positioned in the accessible sites of the antibody and can be used to create an immunoconjugate by conjugating the antibody to other sites, such as a drug site or a linker drug site, 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-modified antibodies can be produced, for example, as described in U.S. Patent No. 7,521,541.

[0209] e) Antibody derivative In certain embodiments, the antibodies provided herein can be further modified to include additional non-protein moieties known and readily available in the art. Suitable sites for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody varies, and if multiple polymers are attached, they may be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization is not limiting, but can be determined based on considerations such as the specific properties or functions of the antibody being improved, and whether the antibody derivative will be used for therapeutic purposes under defined conditions.

[0210] In another embodiment, a conjugate of an antibody and an unprotected site is provided that can be selectively heated by exposure to radiation. In one embodiment, the unprotected site is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation may be of any wavelength and may include, but is not limited to, wavelengths that do not harm normal cells but heat the unprotected site to a temperature that kills cells proximal to the antibody unprotected site.

[0211] A. Recombination method and composition The antibody may be produced, for example, using a recombinant method and composition described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding the anti-CD20 antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL of the antibody and / or the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In further embodiments, one or more vectors (e.g., an expression vector) comprising such nucleic acid are provided. In further embodiments, a host cell comprising such nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., transformed) a vector comprising: (1) a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a nucleic acid encoding an amino acid sequence comprising the VH of the antibody; or (2) a vector comprising a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, for example, a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method for producing an anti-CD20 antibody is provided, which includes culturing host cells containing nucleic acids encoding an antibody under conditions suitable for antibody expression, and optionally recovering the antibody from the host cells (or host cell culture medium).

[0212] For recombinant production of anti-CD20 antibodies, nucleic acids encoding the antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[0213] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, especially when glycosylation or Fc effector function is not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in Escherichia coli (E. coli).) After expression, antibodies may be isolated from bacterial cell paste in appropriate fractions and further purified.

[0214] In addition to prokaryotes, eukaryotes such as filamentous fungi and yeasts are suitable as cloning or expression hosts for antibody-encoding vectors. These include strains of fungi and yeast in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with partially or completely human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0215] Furthermore, suitable host cells for expressing glycosylated antibodies are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified and can be used in combination with insect cells, and are particularly suitable for transfection of Spodoptera frugiperda cells.

[0216] 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 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0217] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspensions may be useful. Other examples of useful mammalian host cell lines include the CV1 monkey kidney cell line transformed with SV40 (COS-7); human embryonic kidney cells (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells described in Mather et al., Annals NYAcad. Sci. 383:44-68 (1982); MRC These are 5 cells and FS4 cells. Other useful mammalian host cell lines include DHFR - Examples include Chinese hamster ovary (CHO) cells, including 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 specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0218] B. Assay The anti-CD20 antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0219] Binding assays and other assays In one embodiment, the antibody of the present invention is tested for its antigen-binding activity by known methods, such as ELISA or Western blotting. CD20 binding can be determined using methods known in the art, exemplary methods of which are disclosed herein. In one embodiment, binding is measured using radioimmunoassay. An exemplary radioimmunoassay is provided below. The CD20 antibody is iodized, and a competitive reaction mixture is prepared containing a constant concentration of the iodized antibody and a decreased concentration of serially diluted unlabeled CD20 antibody. CD20-expressing cells (e.g., BT474 cells stably transfected with human CD20) are added to the reaction mixture. After incubation, the cells are washed to separate the free iodized CD20 antibody from the CD20 antibody bound to the cells. The level of bound iodized CD20 antibody is determined, for example, by counting the radioactivity associated with the cells, and the binding affinity is determined using standard methods. In another embodiment, flow cytometry is used to evaluate the ability of the CD20 antibody to bind to surface-expressed CD20 (e.g., on a B cell subset). Peripheral leukocytes are obtained (e.g., from humans, cynomolgus monkeys, rats, or mice), and the cells are blocked with serum. Labeled CD20 antibody is added to serial dilutions, and T cells are also stained (using methods known in the art) to identify T cell subsets. After incubation and washing of the samples, cells are sorted using flow cytometry, and the data are analyzed using methods known in the art. In another embodiment, CD20 binding may be analyzed using surface plasmon resonance. Exemplary surface plasmon resonance methods are illustrated in the examples.

[0220] In another embodiment, a competitive assay can be used to identify antibodies that compete with any of the anti-CD20 antibodies disclosed herein for binding to CD20. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) to which any of the anti-CD20 antibodies disclosed herein bind. Detailed illustrative methods for mapping the epitopes to which antibodies bind are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0221] In an exemplary competition assay, immobilized CD20 is incubated in a solution containing a first labeled antibody that binds to CD20 (e.g., rituximab, GA101 antibody, etc.) and a second unlabeled antibody being tested for its ability to compete with the first antibody for binding to CD20. The second antibody may be present in the hybridoma supernatant. As a control, immobilized CD20 is incubated in a solution containing the first labeled antibody but not in a solution containing the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to CD20, excess unbound antibody is removed and the amount of label associated with the immobilized CD20 is measured. If the amount of label associated with the immobilized CD20 is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to CD20. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0222] Activity assay The anti-CD20 antibodies of this disclosure (e.g., type II antibodies) may be identified and / or characterized by one or more activity assays known in the art. For example, complement-dependent cell-mediated cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) may be used, as described herein.

[0223] It is understood that any of the above assays can be performed by using the immunoconjugate of the present invention in place of or in addition to the anti-CD20 antibody.

[0224] It is understood that all of the above assays can be performed using anti-CD20 antibodies and additional therapeutic agents.

[0225] Method of administering type II anti-CD20 antibody A method for treating lupus nephritis (LN) in an individual having lupus is provided herein, comprising administering to the individual a first antibody exposure to type II anti-CD20 antibody, a second antibody exposure to type II anti-CD20 antibody, and a third antibody exposure to type II anti-CD20 antibody. A method for depleting the circulating peripheral B cells of an individual is also provided herein, comprising administering to the individual a first antibody exposure to type II anti-CD20 antibody, a second antibody exposure to type II anti-CD20 antibody, and a third antibody exposure to type II anti-CD20 antibody, wherein, after administration of type II anti-CD20 antibody, the B cells are depleted to a level such that circulating peripheral B cells are present in the peripheral blood of the individual at a rate of approximately 5 cells / μL or less. A method for depleting the circulating peripheral B cells of an individual is also provided herein, comprising administering to the individual a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to a type II anti-CD20 antibody, wherein, after administration of the type II anti-CD20 antibody, the B cells are depleted to a level in which circulating peripheral B cells are present in the peripheral blood of the individual at a rate of approximately 5 cells / μL or less, and this effect lasts for at least 52 weeks after a first dose of the first antibody exposure. In some embodiments of the methods herein, the individual or patient is human.

[0226] LN is known in the art as a sign of lupus in the kidneys (e.g., systemic lupus erythematosus, drug-induced lupus, neonatal lupus, or discoid lupus). The most common type of lupus that manifests in the kidneys is systemic lupus erythematosus (SLE). 25–50% of SLE patients have abnormal urine and / or renal function early in the course of the disease, and it is estimated that up to 60% of adults and 80% of children eventually develop LN (see Cameron, JS (1999) J.Am.Soc.Nephrol. 10:413–424 for further details). LN is thought to account for at least 50% of SLE-related morbidity and mortality.

[0227] Furthermore, renal symptoms have also been observed in other types of lupus, such as discoid lupus (Roujeau, J.C. et al. (1984) Acta Derm. Venereol. 64:160-163) and drug-induced lupus (Smith, PR. et al. (1999) Rheumatology (Oxford) 38:1017-1018). In some embodiments, individuals have SLE, discoid lupus, or drug-induced lupus.

[0228] The diagnosis of SLE can follow current American College of Rheumatology (ACR) criteria. Active disease can be defined by one British Isles Lupus Activity Group (BILAG) "A" criterion or two BILAG "B" criteria; the SLE Disease Activity Index (SLEDAI); or the Systemic Lupus Erythematosus (SLE) Responder Index (SRI), as described in the following examples and explained in Furie et al., Arthritis Rheum. 61(9):1143-51 (2009). Tan et al. "The Revised Criteria for the Classification of SLE" Arthritis Rheum Several signs, symptoms, or other indicators used to diagnose SLE, as applied from 25 (1982), include: butterfly rash, discoid rash, or raised rash on the cheeks; photosensitivity, such as a reaction to sunlight causing the onset or increase of skin rashes; oral ulcers, such as ulcers of the nose or mouth; usually painless arthritis, such as non-erosive arthritis (arthritis in which the bone around the joint is not destroyed), involving two or more peripheral joints; serositis, pleurisy, or pericarditis; excessive protein in the urine (more than 0.5 g / day or 3+ on a test stick); and / or cellular casts (urinary and / or leukemia). These may include renal impairment (such as abnormal elements derived from globules and / or tubular cells), neurological signs, symptoms, or other indicators, seizures (convulsions), and / or psychosis in the absence of drugs or metabolic disorders known to cause such effects, as well as hematological signs, symptoms, or other indicators such as hemolytic anemia or leukopenia (white blood cell count less than 4,000 cells / cubic millimeter), lymphopenia (lymphocyte count less than 1,500 lymphocytes / cubic millimeter), or thrombocytopenia (platelet count less than 100,000 platelets / cubic millimeter). Leukopenia and lymphopenia must be detected at least twice. Thrombocytopenia must be detected in the absence of drugs known to induce it. The present invention is not limited to these signs, symptoms, or other indicators of lupus.

[0229] The presence of autoantibodies can be tested as an indicator of lupus. Autoantibodies may include, but are not limited to, anti-dsDNA antibodies, anti-complement antibodies, and antinuclear antibodies (e.g., ENA panels). ENAs are extractable nuclear antigens, i.e., McNeilage et al., J., Clin. Lab. Immunol. 15:1-17 (1984); Whittingham, Ann. Acad. Med. 17(2):195-200 (1988); Wallace and Hahn, dubois' lupus erythematosus, 7 th This refers to a group of nuclear antigens including RNP, Ro / SS-A, La / SS-B, Sm, SCL-70, and Jo-1, as described in Lippincott (2007); Tang et al., Medicine 89(1):62-67 (2010). Antibodies against ENA correlate with lupus. McNeilage et al., 1984; Whittingham 1988; Asherson et al., Medicine 68(6):366-374 (1989); and Tang et al., 2010. Decreased complement activity can also be associated with lupus, as measured, for example, by C3 level, C4 level and / or CH50 assays.

[0230] As described above regarding SLE, it is known in the art that lupus (LN) often develops progressively in patients with lupus (e.g., systemic lupus erythematosus, drug-induced lupus, neonatal lupus, or discoid lupus). That is, a patient may be diagnosed with lupus without the presence of one or more clinical or pathological symptoms of LN. Nevertheless, a patient may still be considered at risk of developing LN, given the high frequency of lupus patients who eventually develop LN. Therefore, in some embodiments, the methods of the present disclosure may find applications in delaying the progression of LN or preventing LN in patients with lupus. In some embodiments, the methods of the present disclosure may be used to delay or prevent the development of LN in patients with lupus (e.g., a form of lupus lacking renal expression).

[0231] LN pathology can be classified according to the International Society of Nephrology / Renal Pathology (ISN / RPS) 2003 classification system, as shown in the table below (for further explanation and definition of terms, see Markowitz GS, D'Agati VD (2007) Kidney Int 71:491-495 and Weening, JJ (2004) Kidney Int 65:521-530). [Table 3]

[0232] In some embodiments, the patient has a Class III or Class IV LN. In some embodiments, the patient has a Class III LN. For example, in some embodiments, the patient has a Class III(A) or Class III(A / C) LN. In some embodiments, the patient has a Class IV LN. For example, in some embodiments, the patient has a Class IV-S(A), IV-G(A), IV-S(A / C), or IV-G(A / C) LN. As shown in Table 3 above, Class V LN may also occur concurrently with Class III or Class IV LN. In some embodiments, the methods of this disclosure are used to treat patients having a Class III or Class IV LN and a contingent Class V LN.

[0233] As described above, patients with a high incidence of lupus (e.g., SLE) eventually develop LN. In some embodiments, patients are at risk of developing LN. In some embodiments, patients are at risk of developing Class III or Class IV LN. In some embodiments, patients are at risk of developing Class III or Class IV LN along with contingent Class V LN.

[0234] In some embodiments, the patient does not have a class III(C)LN (e.g., as listed in Table 3 above). In some embodiments, the patient does not have a class IV(C)LN such as a class IV-S(C) or IV-G(C)LN (e.g., as listed in Table 3 above).

[0235] In some embodiments, the patient has a urine-to-protein-creatinine ratio (UPCR) greater than 1 prior to treatment, for example, during 24-hour urine collection. In some embodiments, the patient has received at least one pulsed dose of methylprednisolone (e.g., 500-1000 mg IV) prior to treatment. In some embodiments, the patient has been administered a stable dose of an ACE inhibitor or angiotensin receptor blocker (ARB) at least 10 days prior to treatment.

[0236] In some embodiments, the patient does not have severe renal impairment and does not require dialysis or kidney transplantation prior to treatment as described herein. In some embodiments, the patient does not have sclerosis in more than 50% of glomeruli on renal biopsy prior to treatment as described herein. In some embodiments, the patient does not have active central nervous system SLE prior to treatment as described herein. In some embodiments, the patient does not have a history of progressive multifocal leukoencephalopathy (PML) prior to treatment as described herein. In some embodiments, the patient does not have a positive hepatitis C serology, hemoglobin <7 g / dL (unless caused by autoimmune hemolytic anemia due to SLE), platelet count <20,000 / uL, or positive serum human chorionic gonadotropin prior to treatment as described herein. In some embodiments, the patient does not have a known HIV infection prior to treatment as described herein. In some embodiments, the patient has not been treated with one or more of the following prior to the treatment described herein (for example, three months prior to the treatment described herein): cyclophosphamide, calcineurin inhibitor, JAK inhibitor, BTK inhibitor, TYK2 inhibitor, or IV antibiotic.

[0237] Using some laboratory tests known in the art, the presence, progression and / or efficacy against treatment in lupus nephritis can be diagnosed and / or monitored. In some embodiments, serum creatinine can be measured. In some embodiments, the normal range of serum creatinine can be from about 0.6 to about 1.3 mg / dL, with some variation among age, between male and female, and among laboratories. In some embodiments, the presence of urinary sediment and / or casts can be measured, for example, by microscopic examination of urine. For example, the number of red blood cells in a urine sample can be assayed by microscopic examination. In some embodiments, the normal value of urinary sediment can be about 4 or fewer red blood cells (RBC) per high power field (HPF). Urinary casts can include, but are not limited to, red blood cell casts, white blood cell casts, renal tubular epithelial cell casts, waxy casts, hyaline casts, granular casts, and fatty casts. In some embodiments, the urine protein / creatinine ratio (UPCR) can be measured. The presence of protein in urine (proteinuria) can also be assayed by tests including, but not limited to, the urine albumin / creatinine ratio (UACR) and dipstick urine test. Other tests and / or measurements that may be useful for examining renal function can include, but are not limited to, renal panel, creatinine clearance, sodium, potassium, chloride, bicarbonate, phosphorus, calcium, albumin, blood urea nitrogen (BUN), creatinine, glucose, estimated glomerular filtration rate (eGFR), BUN / creatinine ratio, and anion gap, and, where appropriate, measurement of the above parameters in blood and / or urine may be included. For more details, see, for example, the American College of Rheumatology Guidelines for Screening, Case Definition, Treatment and Management of Lupus Nephritis (Hahn, B. et al. (2012) Arthritis Care Res. 64:797-808).

[0238] A method for treating membranous nephropathy (MN), such as primary membranous nephropathy (pMN), which comprises administering to an individual a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to a type II anti-CD20 antibody, is further provided herein.

[0239] In some embodiments, pMN is confirmed by renal biopsy, for example, prior to treatment with a type II anti-CD20 antibody. For example, in some embodiments, pMN is diagnosed based on light microscopy, immunofluorescence microscopy, and / or electron microscopy.

[0240] In some embodiments, the individual has been treated with, or is being treated simultaneously with, blockade of the renin-angiotensin system, such as an angiotensin-converting enzyme (ACE) inhibitor and / or an angiotensin receptor blocker (ARB). In some embodiments, the individual has a urinary protein-to-creatinine ratio (UPCR) of 5 g or more (e.g., from a 24-hour urine collection) despite treatment with a renin-angiotensin system blocker, such as an angiotensin-converting enzyme (ACE) inhibitor and / or an angiotensin receptor blocker (ARB), for at least 3 months prior to treatment with a type II anti-CD20 antibody, or a UPCR of 4 g or more (e.g., from a 24-hour urine collection) despite treatment with a renin-angiotensin system blocker, such as an angiotensin-converting enzyme (ACE) inhibitor and / or an angiotensin receptor blocker (ARB), for at least 6 months prior to treatment with a type II anti-CD20 antibody. In some embodiments, the individual has an estimated glomerular filtration rate (eGFR) ≧ 40 mL / min / 1.73m 2 or an endogenous creatinine clearance ≧ 40 L / min (e.g., based on a 24-hour urine collection). In some embodiments, the eGFR is calculated using the CKD-EPI equation.

[0241] In some embodiments, the individual does not have secondary neuropathy (MN). In some embodiments, the individual is not hypertensive or has uncontrolled blood pressure for at least three months prior to treatment with, for example, type II anti-CD20 antibody. In some embodiments, the individual has a systolic blood pressure of ≤140 mmHg and a diastolic blood pressure of ≤90 mmHg prior to treatment with, for example, type II anti-CD20 antibody. In some embodiments, the individual has not been treated with calcineurin inhibitors (CNIs) (e.g., cyclosporine A or mTOR inhibitors) or alkylating agents for at least six months prior to treatment with type II anti-CD20 antibody. In some embodiments, the individual has not been treated with rituximab for at least six months prior to treatment with type II anti-CD20 antibody. In some embodiments, the individual has not been treated with renal replacement therapy (e.g., kidney transplantation, chronic dialysis) prior to treatment with type II anti-CD20 antibody. In some embodiments, the individual does not have type 1 or type 2 diabetes. In some embodiments, the individual has no history of active infection, severe recurrent or chronic infection, HIV infection, or TB infection. In some embodiments, the individual has no history of cancer or PML. In some embodiments, the individual is not positive for HBV, HCV, or serum human chorionic gonadotropin. In some embodiments, the individual does not have any or all of the following (for example, prior to treatment with type II anti-CD20 antibody): AST or ALT greater than 2.5 times the upper limit of normal (ULN), amylase or lipase greater than 2 times the ULN, or neutrophils greater than 1.5 × 10⁶. 3 Less than 5 cells / μL, less than 5 CD19+ B cells / μL, less than 9 g / dL hemoglobin, or less than 75,000 platelets / μL.

[0242] In some embodiments, the method of the present disclosure includes administering to an individual a first antibody exposure to the type II anti-CD20 antibody of the present disclosure, a second antibody exposure to the type II anti-CD20 antibody of the present disclosure, and a third antibody exposure to the type II anti-CD20 antibody of the present disclosure. In some embodiments, the second antibody exposure is not given until about 18 to 26 weeks after the first antibody exposure. In some embodiments, the second antibody exposure is not given until about 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, or 26 weeks after the first antibody exposure. In some embodiments, the second antibody exposure is not administered until approximately 26, 25, 24, 23, 22, 21, 20, or 19 weeks after the first antibody exposure. In some embodiments, the second antibody exposure is not administered until approximately 18, 19, 20, 21, 22, 23, 24, or 25 weeks after the first antibody exposure. That is, the second antibody exposure is not administered until any range of weeks having an upper limit of 26, 25, 24, 23, 22, 21, 20, or 19 and an independently selected lower limit of 18, 19, 20, 21, 22, 23, 24, or 25, where the lower limit is less than the upper limit. In some embodiments, the third antibody exposure is not administered until approximately 24 to 32 weeks after the second antibody exposure. In some embodiments, the third antibody exposure is not administered until approximately 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, or 32 weeks after the second antibody exposure. In some embodiments, the third antibody exposure is not administered until approximately 32, 31, 30, 29, 28, 27, 26, or 25 weeks after the second antibody exposure. In some embodiments, the third antibody exposure is not administered until approximately 24, 25, 26, 27, 28, 29, 30, or 31 weeks after the second antibody exposure.In other words, the third antibody exposure is not given up to any of the weeks in which the upper limit is 32, 31, 30, 29, 28, 27, 26, or 25 and the independently selected lower limit is 24, 25, 26, 27, 28, 29, 30, or 31, with the lower limit being less than the upper limit.

[0243] In some embodiments, the method of the present disclosure includes administering to an individual a first antibody exposure to the type II anti-CD20 antibody of the present disclosure and a second antibody exposure to the type II anti-CD20 antibody of the present disclosure. In some embodiments, the second antibody exposure is not given until about 18 to 26 weeks after the first antibody exposure. In some embodiments, the second antibody exposure is not given until about 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, or 26 weeks after the first antibody exposure. In some embodiments, the second antibody exposure is not administered until less than approximately 26, 25, 24, 23, 22, 21, 20, or 19 weeks after the first antibody exposure. In some embodiments, the second antibody exposure is not administered until more than approximately 18, 19, 20, 21, 22, 23, 24, or 25 weeks after the first antibody exposure. That is, the second antibody exposure is not administered until any range of weeks having an upper limit of 26, 25, 24, 23, 22, 21, 20, or 19 and an independently selected lower limit of 18, 19, 20, 21, 22, 23, 24, or 25, where the lower limit is less than the upper limit.

[0244] The drug regimens described herein use a consistent system for tracking the time between doses, so that the first dose is administered to the patient on day 1 or week 0. As described herein, the antibody exposure in this disclosure may consist of one or two doses. If the antibody exposure consists of one dose, a reference to a second antibody exposure that is not given until a certain period has elapsed after the first antibody exposure (as described herein) refers to the amount of time that has elapsed between the dose of the first antibody exposure (e.g., on day 1 or week 0) and the dose of the second antibody exposure. If the first antibody exposure consists of two doses, the first dose of the first antibody exposure is provided on day 1 or week 0. If the antibody exposure consists of two doses, a reference to a second antibody exposure that is not given until a certain period has elapsed after the first antibody exposure (as described herein) refers to the amount of time that has elapsed between the first of the two doses of the first antibody exposure (e.g., on day 1 or week 0) and the first of the two doses of the second antibody exposure. For example, if the method of the present disclosure includes a first antibody exposure by two doses and a second antibody exposure by two doses, and the second antibody exposure is not administered until approximately 22 weeks after the first antibody exposure, then the interval between the first dose of the first antibody exposure and the second dose of the first antibody exposure is approximately 22 weeks.

[0245] In some embodiments, the first antibody exposure of the Disclosure comprises one or two doses of the type II anti-CD20 antibody of the Disclosure. In some embodiments, the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. In some embodiments, the first antibody exposure comprises a total exposure of approximately 1800 mg, approximately 1900 mg, approximately 2000 mg, approximately 2100 mg, or approximately 2200 mg of the type II anti-CD20 antibody.

[0246] In some embodiments, the first antibody exposure comprises two doses. In some embodiments, the first antibody exposure comprises a first dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the first dose of the first antibody exposure comprises approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the second dose of the first antibody exposure comprises approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the second dose of the first antibody exposure is not administered from approximately 1.5 weeks to approximately 2.5 weeks after the first dose of the first antibody exposure. In some embodiments, the second dose of the first antibody exposure is not administered from approximately 2 weeks after the first dose of the first antibody exposure.

[0247] In some embodiments, the second antibody exposure of the Disclosure comprises one or two doses of the type II anti-CD20 antibody of the Disclosure. In some embodiments, the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. In some embodiments, the second antibody exposure comprises a total exposure of approximately 1800 mg, approximately 1900 mg, approximately 2000 mg, approximately 2100 mg, or approximately 2200 mg of the type II anti-CD20 antibody.

[0248] In some embodiments, the second antibody exposure comprises two doses. In some embodiments, the second antibody exposure comprises a first dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the second antibody exposure of the first dose comprises approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the second antibody exposure of the second dose comprises approximately 1000 mg of type II anti-CD20 antibody. In some embodiments, the second antibody exposure of the second dose is not administered from approximately 1.5 weeks to approximately 2.5 weeks after the second antibody exposure of the first dose. In some embodiments, the second antibody exposure of the second dose is not administered from approximately 2 weeks after the second antibody exposure of the first dose.

[0249] In some embodiments, the third antibody exposure of the Disclosure comprises one or two doses of the type II anti-CD20 antibody of the Disclosure. In some embodiments, the third antibody exposure comprises a total exposure of about 800 mg to about 1200 mg of the type II anti-CD20 antibody. In some embodiments, the third antibody exposure comprises a total exposure of about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, or about 1200 mg of the type II anti-CD20 antibody.

[0250] In some embodiments, the third antibody exposure comprises a single dose. In some embodiments, the third antibody exposure comprises a single dose of approximately 900 mg to approximately 1100 mg of type II anti-CD20 antibody. In some embodiments, the single dose of the third antibody exposure comprises approximately 1000 mg of type II anti-CD20 antibody.

[0251] In some embodiments, the type II anti-CD20 antibody of this disclosure is administered intravenously (e.g., by IV infusion).

[0252] In some embodiments, the methods of the present disclosure further include administering an effective amount of an immunosuppressant (for example, in combination with a type II anti-CD20 antibody as described herein). Several classes of immunosuppressants are known in the art, including but not limited to cell proliferation inhibitors (e.g., cytotoxic agents such as antibiotics, alkylating agents (e.g., cyclophosphamide (also known as cytophosphan)), antimetabolites such as inosine monophosphate dehydrogenase inhibitors and protein synthesis inhibitors, folic acid analogs, purine analogs, pyrimidine analogs, etc.), immunosuppressive antibodies, glucocorticoids, immunophilin-targeting drugs (e.g., tacrolimus, sirolimus, rapamycin and its analogs, cyclosporine, etc.), mTOR active site inhibitors, mycophenolic acids and their derivatives or salts, TNF-binding proteins, interferons, opioids, and other small molecules (e.g., fingolimod). In some embodiments, the immunosuppressant comprises mycophenolic acid, a derivative of mycophenolic acid, or a salt of mycophenolic acid. In some embodiments, the immunosuppressant is mycophenolate mofetil. In some embodiments, the immunosuppressant comprises CellCept® (Roche). In some embodiments, the immunosuppressant comprises Myfortic® (Novartis). The effective doses of the immunosuppressants of this disclosure are known in the art and can be readily confirmed by standard assays. For example, mycophenolate mofetil may be administered at a dose of 2.0–2.5 g / day. In some embodiments, mycophenolate mofetil may be administered starting at 1000 mg / day in divided doses (twice / day) and gradually increased to a maximum of 2.0–2.5 g / day in divided doses (twice / day) by week 4.

[0253] In some embodiments, immunosuppressants may be administered before, during, or after administration of the type II anti-CD20 antibody of this disclosure, for example, as a treatment for lupus. In some embodiments, immunosuppressants may be administered throughout the period of treatment with the type II anti-CD20 antibody of this disclosure. In some embodiments, mycophenolate mofetil may be administered as described above throughout the period of treatment with the type II anti-CD20 antibody.

[0254] In some embodiments, the methods of the present disclosure further include administering an effective dose of a glucocorticoid or corticosteroid (in combination with, for example, a type II anti-CD20 antibody as described herein). A variety of natural and synthetic glucocorticoids / corticosteroids are known in the art, including but not limited to beclomethasone, triamcinolone, dexamethasone, betamethasone, prednisone, methylprednisolone, prednisolone, cortisone, and cortisol. In some embodiments, the glucocorticoid / corticosteroid includes methylprednisolone. In some embodiments, the glucocorticoid / corticosteroid includes prednisone. The effective doses of the glucocorticoids / corticosteroids of the present disclosure are known in the art and can be readily confirmed by standard assays. For example, methylprednisolone may be administered once daily in doses of 750 to 1000 mg by IV. Alternatively, prednisone may be administered orally at a dose of 0.5 mg / kg, and then gradually reduced to 7.5 mg / day as needed.

[0255] In some embodiments, glucocorticoids may be administered before, during, or after administration of the type II anti-CD20 antibody of this disclosure, for example, to treat LN clinical activity. In some embodiments, glucocorticoids may be administered before administration of the type II anti-CD20 antibody of this disclosure, for example, 30 to 60 minutes prior to administration of the type II anti-CD20 antibody. In some embodiments, 80 mg of methylprednisolone may be administered IV 30 to 60 minutes prior to administration of the type II anti-CD20 antibody of this disclosure. In some embodiments, prednisone (e.g., orally) and / or methylprednisolone (e.g., IV administration) may be administered in conjunction with treatment, followed by maintenance therapy (e.g., mycophenolate mofetil or cyclophosphamide).

[0256] In some embodiments, the method of the Disclosure further comprises administering an effective dose of an antihistamine (for example, in combination with a type II anti-CD20 antibody as described herein). Antihistamines known in the Art and currently used clinically include histamine H1 receptor and histamine H2 receptor antagonists or inverse agonists. In some embodiments, the antihistamine includes diphenhydramine. The effective dose of the antihistamines of the Disclosure is known in the Art and can be readily confirmed by standard assays. For example, diphenhydramine may be administered in an oral dose of 50 mg.

[0257] In some embodiments, an antihistamine may be administered before, during, or after administration of the type II anti-CD20 antibody of the Disclosure, for example, as prophylactic treatment. In some embodiments, an antihistamine may be administered before administration of the type II anti-CD20 antibody of the Disclosure, for example, 30 to 60 minutes prior to administration of the type II anti-CD20 antibody. In some embodiments, 50 mg of diphenhydramine may be administered orally 30 to 60 minutes prior to administration of the type II anti-CD20 antibody of the Disclosure.

[0258] In some embodiments, the methods of the present disclosure further include administering an effective amount of a nonsteroidal anti-inflammatory drug (NSAID) (for example, in combination with a type II anti-CD20 antibody as described herein). NSAIDs known in the art include acetic acid derivatives, propionic acid derivatives, salicylates, enolic acid derivatives, anthranilic acid derivatives, selective COX-2 inhibitors, and sulfonanilides. In some embodiments, the NSAID includes acetaminophen. The effective amount of the NSAID of the present disclosure is known in the art and can be readily confirmed by standard assays. For example, acetaminophen may be administered in oral doses of 650 to 1000 mg.

[0259] In some embodiments, the NSAID can be administered, for example as prophylactic treatment, before, during, or after administration of the type II anti-CD20 antibody of the present disclosure. In some embodiments, the NSAID can be administered before administration of the type II anti-CD20 antibody of the present disclosure, for example, 30 to 60 minutes before the type II anti-CD20 antibody. In some embodiments, 650 to 1000 mg of acetaminophen can be orally administered 30 to 60 minutes before administration of the type II anti-CD20 antibody of the present disclosure.

[0260] In some embodiments, the method of the present disclosure further comprises administering an effective amount of an antimalarial drug (e.g., in combination with the type II anti-CD20 antibody described herein). Examples of antimalarial drugs that can be used include, but are not limited to, hydroxychloroquine, chloroquine, and quinacrine. In some embodiments, the antimalarial drug can be administered before, during, or after administration of the type II anti-CD20 antibody of the present disclosure, for example, as treatment for one or more symptoms of lupus.

[0261] In some embodiments, the method of the present disclosure further comprises administering an effective amount of an integrin antagonist (e.g., in combination with a type II anti-CD20 antibody as described herein). Examples of integrin antagonists that can be used include, but are not limited to, LFA-1 antibodies, such as efalizumab (RAPTIVA (registered trademark)) commercially available from Genentech, or alpha4 integrin antibodies, such as natalizumab (ANTEGREN (registered trademark)) available from Biogen, or diazacyclic phenylalanine derivatives, phenylalanine derivatives, phenylpropionic acid derivatives, enamine derivatives, propanoic acid derivatives, alkanoic acid derivatives, substituted phenyl derivatives, aromatic amine derivatives, ADAM disintegrin domain polypeptides, antibodies against alphabeta3 integrin, and aza-bridged bicyclic amino acid derivatives. In some embodiments, the integrin antagonist can be administered before, during, or after administration of the type II anti-CD20 antibody of the present disclosure, for example, as treatment for one or more symptoms of lupus.

[0262] In some embodiments, the method of the present disclosure further comprises administering an effective amount of a cytokine antagonist (for example, in combination with a type II anti-CD20 antibody as described herein). Examples of cytokine antagonists that may be used include, but are not limited to, antagonists against IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, and IL-15 (e.g., antagonist antibodies); tumor necrosis factors such as TNF-α and TNF-β; and other polypeptide factors including LIF and kit ligand (KL). In some embodiments, the cytokine antagonist may be administered before, during, or after administration of the type II anti-CD20 antibody of the present disclosure, for example, as treatment for one or more symptoms of lupus.

[0263] In some embodiments, the method of the Disclosure further comprises administering an effective amount of hormone (for example, in combination with the type II anti-CD20 antibody described herein). In some embodiments, the hormone (for example, for hormone replacement therapy) may be administered before, during, or after administration of the type II anti-CD20 antibody of the Disclosure, for example, for medical treatment in women with lupus.

[0264] In some embodiments, the methods of the present disclosure further include administering standard treatment (e.g., in conjunction with a type II anti-CD20 antibody as described herein). In some embodiments, standard treatment may be administered before, during, or after administration of the type II anti-CD20 antibody of the present disclosure to treat or prevent one or more symptoms of lupus, for example. In certain embodiments, standard treatment may be administered after exposure to a second antibody of the present disclosure. In certain embodiments, standard treatment may be administered after exposure to a third antibody of the present disclosure. For example, the type II anti-CD20 antibody of the present disclosure may be administered to a patient as induction therapy, as described herein, and the patient may then be treated according to standard treatment as maintenance therapy. Standard treatments for lupus are well known in the art and include, but are not limited to, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, cyclophosphamide, mycophenolate mofetil (e.g., in the doses specified herein, such as 2.0-2.5 g / day), azathioprine, and glucocorticoids or corticosteroids (e.g., prednisone, e.g., tapering prednisone).

[0265] In some embodiments, the method of the present disclosure further includes administering an antihypertensive agent (for example, in combination with a type II anti-CD20 antibody as described herein). In some embodiments, the antihypertensive agent may be administered, for example, before, during, or after administration of the type II anti-CD20 antibody of the present disclosure to treat or prevent hypertension. In some embodiments, the antihypertensive agent includes, but is not limited to, ACE inhibitors and angiotensin receptor blockers.

[0266] In some embodiments, the methods of this disclosure result in complete renal response (CRR) in the individual. In some embodiments, CRR includes all of the following: normalization of serum creatinine, inactive urinary sediment, and a urinary protein-to-creatinine ratio of less than 0.5. In some embodiments, normalization of serum creatinine is characterized by serum creatinine below the upper limit of the normal range (ULN) of the central laboratory value, and / or ≤15% above baseline and below the ULN range of the central laboratory value if baseline (e.g., day 1) serum creatinine is within the normal range of the central laboratory value. In some embodiments, inactive urinary sediment is characterized by <10 RBCs / high magnification field (HPF) and / or the absence of erythrocyte casts. For a more detailed discussion of CRR and partial renal response (PRR) in LN, see, for example, Chen, YE et al. (2008) Clin. J. Am. Soc. Nephrol. 3:46-53.

[0267] In some embodiments, the methods of the present disclosure result in a complete renal response (CRR) or partial renal response (PRR) in an individual. In some embodiments, PRR includes one or more of the following: normalization of serum creatinine, inactive urinary sediment, and a urinary protein-to-creatinine ratio of less than 0.5. In some embodiments, PRR includes one or more of the alleviation of one or more symptoms, including but not limited to a decrease in serum creatinine, a decrease in urinary sediment, a decrease in proteinuria, and any other improvement in renal function. In some embodiments, CRR or PRR includes a decrease in one or more biomarkers of lupus activity, including but not limited to anti-dsDNA antibodies, antinuclear antibodies / ENA, anti-complement antibodies, decreased levels of complement C3 and / or C4, and decreased complement activity (as measured by, for example, the CH50 assay).

[0268] In some embodiments, the method of the present disclosure results in the depletion of circulating peripheral B cells in an individual. In some embodiments, the circulating peripheral B cells are CD19+ B cells. In some embodiments, the circulating peripheral B cells are naive B cells. In some embodiments, the circulating peripheral B cells are memory B cells. In some embodiments, the circulating peripheral B cells are plasmablasts or plasma cells. In some embodiments, after administration of the type II anti-CD20 antibody of the present disclosure (e.g., by any of the methods described herein), circulating peripheral B cells are present in peripheral blood at concentrations of approximately 7 cells / μL or less, approximately 6 cells / μL or less, approximately 5 cells / μL or less, approximately 4 cells / μL or less, approximately 3 cells / μL or less, approximately 2 cells / μL or less, approximately 1 cell / μL or less, or approximately 0.5 cells / μL or less. In some embodiments, the level of circulating peripheral B cells is measured using high-sensitivity flow cytometry (HSFC) as described herein. In some embodiments, B cells are depleted to subdetectable levels using HSFC. In some embodiments, HSFCs have a limit of quantification (LLOQ) for B cells of approximately 1.0 cells / μL or less, approximately 0.8 cells / μL or less, approximately 0.6 cells / μL or less, approximately 0.5 cells / μL or less, or 0.441 cells / μL or less. In some embodiments, circulating peripheral B cells in the individual are depleted by at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, or approximately 100%. In some embodiments, the depletion of circulating peripheral B cells persists for at least 52 weeks after the first dose of the first antibody exposure.In some embodiments, depletion of circulating peripheral B cells persists for at least 51 weeks, at least 50 weeks, at least 49 weeks, at least 48 weeks, at least 47 weeks, at least 46 weeks, at least 45 weeks, at least 44 weeks, at least 43 weeks, at least 42 weeks, at least 41 weeks, at least 40 weeks, at least 39 weeks, at least 38 weeks, at least 37 weeks, at least 36 weeks, at least 35 weeks, at least 34 weeks, at least 33 weeks, at least 32 weeks, at least 31 weeks, at least 30 weeks, at least 29 weeks, at least 28 weeks, at least 27 weeks, at least 26 weeks, at least 25 weeks, or at least 24 weeks after exposure to the first dose of the first antibody. In some embodiments, circulating peripheral B cell depletion is measured, for example, after a first antibody exposure (e.g., including one or two doses of the anti-CD20 antibody described herein), after a second antibody exposure (e.g., including one or two doses of the anti-CD20 antibody described herein), after a third antibody exposure (e.g., including one or two doses of the anti-CD20 antibody described herein), and 3 months after treatment (e.g., according to the Specified Specified Specified Specified Specified), compared to the corresponding measurement in the same individual before treatment or in a control individual (e.g., an untreated individual). This refers to measurements of circulating peripheral B cells performed after receiving the first antibody exposure and / or the second antibody exposure and / or the third antibody exposure described herein), 6 months after treatment (for example, after receiving the first antibody exposure and / or the second antibody exposure and / or the third antibody exposure described herein), 9 months after treatment (for example, after receiving the first antibody exposure and / or the second antibody exposure and / or the third antibody exposure described herein), or 12 months after treatment (for example, after receiving the first antibody exposure and / or the second antibody exposure and / or the third antibody exposure described herein).

[0269] Methods for assaying depletion of circulating peripheral B cells in an organism, such as flow cytometry using one or more antibodies that recognize B cell markers, are known in the art. In some embodiments, high-sensitivity flow cytometry (HSFC) can be used to assay depletion of circulating peripheral B cells (see, e.g., Vital, E.M. et al. (2011) Arthritis Rheum. 63:3038-3047 and Example 1). In some embodiments, the B cells are CD19+ B cells. In some embodiments, the B cells are naive B cells (e.g., CD19+CD27- B cells), memory B cells (e.g., CD19+CD27+ B cells), or plasmablasts (e.g., CD19+CD27+CD38++ B cells). In some embodiments, the B cells are CD19+CD3-CD14- cells and / or CD19+CD33-CD56- cells. In some embodiments, the B cells are CD19+CD3-CD14-CD33-CD56-cells. In some embodiments, the B cells include CD19+CD20+B cells, CD19+CD20-B cells, and CD19+CD22+B cells. In some embodiments, the B cells are, for example, circulating peripheral B cells from a peripheral blood sample.

[0270] In some embodiments, the level of circulating peripheral B cells present in a peripheral blood sample is measured as follows (e.g., HSFC). Lymphocytes are identified in the sample by flow cytometry (e.g., by plotting CD45 versus side scattering and gating CD45+ cells). In some embodiments, doublets are excluded from the analysis prior to this step (e.g., by gating single cells and excluding forward-scattering and / or side-scattering doublets). CD19+ B cells are then identified by excluding T cells, NK cells, and monocytes. For example, CD19+CD3-CD14- cells can be identified from parental CD45+ lymphocyte gates (e.g., by plotting CD19 versus CD3 / CD14 and gating CD19+CD3-CD14- cells), and CD19+CD33-CD56-B cells can be identified from parental CD19+CD3-CD14- cells (e.g., by plotting CD19 versus CD33 / CD56 and gating CD19+CD33-CD56- cells). The number of B cells can then be determined, for example, by dividing the number of detected CD19+ B cells (e.g., CD19+CD3-CD14-CD33-CD56- cells) by the sample volume. In some embodiments, the number of beads or other QC controls can also be quantified, and then the number of B cells can be determined, for example, by calculating (CD19+ events × number of beads) / (number of beads × sample volume).

[0271] In some embodiments, after administration of the type II anti-CD20 antibody of this disclosure (for example, by any of the methods described herein), circulating peripheral B cells are present in peripheral blood at concentrations of approximately 7 cells / μL or less, approximately 6 cells / μL or less, approximately 5 cells / μL or less, approximately 4 cells / μL or less, approximately 3 cells / μL or less, approximately 2 cells / μL or less, approximately 1 cell / μL or less, or approximately 0.5 cells / μL or less, for example, 5 cells / μL or less. In some embodiments, B cells are depleted to subdetectable levels using HSFCs. In some embodiments, HSFCs have a limit of quantification (LLOQ) for B cells of approximately 1.0 cells / μL or less, approximately 0.8 cells / μL or less, approximately 0.6 cells / μL or less, approximately 0.5 cells / μL or less, or 0.441 cells / μL or less.

[0272] IV. Manufactured goods or kits In another embodiment, a manufactured article or kit is provided containing the type II anti-CD20 antibody of this disclosure, which is useful in any of the methods described herein (e.g., for the treatment, prevention and / or diagnosis of the disorders described herein). The manufactured article or kit comprises a container and a label or package insert inserted into or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, etc. The container may be formed from a variety of materials, such as glass or plastic. The container may hold a composition used alone or in combination with another composition effective for treating, preventing and / or diagnosing a condition or for depleting circulating peripheral B cells, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous needle). At least one active agent in the composition is the antibody described herein (e.g., the type II anti-CD20 antibody of this disclosure). The label or package insert indicates that the composition is used to treat a selected condition or to deplete circulating peripheral B cells, according to any of the methods described herein. Alternatively, or in addition, the product or kit may further include a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, etc.

[0273] In some embodiments, a manufactured product or kit is provided herein that includes a container comprising the type II anti-CD20 antibody of the present disclosure and an optional pharmaceutically acceptable carrier, and a package insert optionally containing instructions for treating lupus nephritis or depleting circulating peripheral B cells in an individual, for example, the instructions for administering a first antibody exposure to the type II anti-CD20 antibody, a second antibody exposure to the type II anti-CD20 antibody, and a third antibody exposure to the type II anti-CD20 antibody to an individual, wherein the second antibody exposure is not given until approximately 18 to 26 weeks after the first antibody exposure, and the third antibody exposure is given until approximately 26 weeks after the second antibody exposure. It is indicated that the first antibody exposure is not given from approximately 24 weeks to approximately 32 weeks after the first antibody exposure, and that the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, with a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody; the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, with a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody; and the third antibody exposure comprises one or two doses of the type II anti-CD20 antibody, with a total exposure of approximately 800 mg to approximately 1200 mg of the type II anti-CD20 antibody. In some embodiments, kits are provided herein that include a container comprising the type II anti-CD20 antibody of the present disclosure and an optional pharmaceutically acceptable carrier, and optionally a package insert containing instructions for treating class III or class IV lupus nephritis in an individual. In some embodiments of any of the above embodiments, the type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6. In some embodiments of any of the above embodiments, the type II anti-CD20 antibody is obinutuzumab. In some embodiments, the individual is human.

[0274] The product or kit may further include a second or third container containing a second pharmaceutical, the anti-CD20 antibody (e.g., the type II anti-CD20 antibody of this disclosure) being the first pharmaceutical, and the product further includes a package insert describing how to treat a subject with the second pharmaceutical. Examples of the second pharmaceutical include chemotherapeutic agents, immunosuppressants, antimalarial agents, cytotoxic agents, integrin antagonists, cytokine antagonists, hormones, and any treatments that may be used in conjunction with the type II anti-CD20 antibody described herein. The products in these embodiments may further include a package insert indicating that the composition may be used to treat a particular condition.

[0275] This specification is deemed sufficient to enable those skilled in the art to practice the invention. In addition to the modifications shown and described herein, various modifications of the invention will be apparent to those skilled in the art from the foregoing description and are within the scope of the appended claims. All publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety for all purposes. [Examples]

[0276] The present invention will be better understood by referring to the following embodiments. However, these embodiments should not be construed as limiting the scope of the invention. It will be understood that the embodiments and examples described herein are for illustrative purposes only, and that various modifications or changes taking them into account will be proposed to those skilled in the art and should be included within the spirit and scope of this application and the appended claims.

[0277] Example 1: Obinutuzumab, mycophenolic acid, and corticosteroids for the treatment of proliferative lupus nephritis B cells are central to the pathogenesis of lupus nephritis, but randomized controlled trials of type I anti-CD20 antibodies have failed to demonstrate superiority over standard treatment alone. Obinutuzumab is a glycoengineered type II anti-CD20 monoclonal antibody that induces greater B cell depletion than type I anti-CD20 antibodies. We compared obinutuzumab treatment versus placebo treatment in patients with proliferative lupus nephritis treated with mycophenolate and corticosteroids.

[0278] The results of the Phase 2, multicenter, randomized, double-blind trial (NOBILITY) comparing obinutuzumab with placebo in patients with proliferative lupus nephritis treated with mycophenolate and corticosteroids are shown below.

[0279] Materials and methods 126 patients were enrolled at 43 sites in North America, South America, Europe, and Israel. After a 4-week screening period, patients were randomly assigned in a 1:1 ratio via an interactive Web response system to receive either intravenous obinutuzumab 1000 mg or placebo infusion on days 1, 15, 168, and 182 of the study. To reduce the risk of infusion-related reactions, patients randomized to obinutuzumab or placebo received either blinded intravenous (IV) methylprednisolone 80 mg or placebo, respectively, prior to administration of the study drug. All patients received mycophenolate (mycophenolate mofetil, target dose of 2–2.5 grams per day or an equivalent dose of mycophenolate) and a standardized corticosteroid tapering agent (prednisone 0.5 mg / kg / day, starting at a maximum of 60 mg / day and tapering to 7.5 mg / day up to 12 weeks). Patients were followed in a blinded manner until week 104, and those with persistent B-cell depletion were followed for safety and B-cell evaluation. See also the protocol published in International Publication No. 2016 / 183104.

[0280] Clinical trial visits were scheduled at weeks 4, 12, 24, 36, 52, 76, and 104 to assess safety, urinary protein excretion (measured by 24-hour urine collection and / or random UPCR, preferably UPCR from early morning urination), serum creatinine, autoantibody and serum complement component levels, and clinical disease activity. Peripheral blood B cells were quantified using high-sensitivity flow cytometry (HSFC) at baseline (week 0) and at weeks 2, 4, 12, 24, 52, and 104 (data obtained from some patients at week 76 for investigational findings). Optional repeat renal biopsies were offered to all patients at week 52 and performed according to local clinical practice.

[0281] patient Patients were aged 18 to 75 years, had systemic lupus erythematosus (SLE) as defined by the American College of Rheumatology criteria, had renal biopsy findings of International Society of Nephrology / Renal Pathology 2003 class III or IV within 6 months of randomization (contingent class V was acceptable), had a urinary protein-to-creatinine ratio (UPCR) > 1 in 24-hour urine collection, and an estimated glomerular filtration rate (eGFR) ≥ 30 mL / min / 1.73 m² without rapidly progressive decline in renal function. 2 They were eligible if they met the following criteria. All patients provided written informed consent.

[0282] Evaluation items The primary endpoints were the proportion of patients who achieved complete renal response at week 52, defined as UPCR < 0.5, serum creatinine below the upper limit of normal and ≤ 15% above baseline, and fewer than 10 red blood cells per high-magnification field of view (HPF) without red blood cell casts in urinalysis. Key secondary endpoints included partial renal response, defined as a 50% or greater reduction in UPCR from baseline to less than 1 (or less than 3 if baseline was UPCR ≥ 3), no increase of more than 15% in serum creatinine from baseline, and no increase of less than 10 or more than 50% in urinary erythrocytes from baseline; total renal response, defined as achieving a complete or partial response; modified complete renal response, defined as a complete renal response excluding urinary sediment criteria; a second modified complete renal response, enabling serum creatinine to be below the upper limit of normal or not increase by more than 15% from baseline; changes from baseline in biomarkers of lupus nephritis disease activity, including dsDNA antibody levels, complement component 3 (C3), and complement component 4 (C4); and safety. Patients who received rescue from pulsed-dose methylprednisolone (≥500 mg), cyclophosphamide, rituximab, or other novel immunosuppressive therapies after baseline, or who withdrew from the study early, were attributed as non-responders for all response endpoints.

[0283] High-sensitivity flow cytometry (HSFC) To provide the absolute number of peripheral B cells, a minimal residual B cell (MRB 1.1) panel containing CD19, CD20, and CD22 markers was assayed by HSFC. CD19 appears early in B cell ontogeny and remains expressed on all B lineage cells, but is downregulated on plasma cells. CD20 is expressed on all normal B cells except very early progenitor cells and terminally differentiated plasma cells. CD22 is mainly found on mature B cells.

[0284] The HSFC assay used two tubes each for quality control (QC) and test samples: a fluorescence-minus-one (FMO) tube for control and gating, and a laboratory tube. Both were analyzed using 405 nm, 488 nm, and 633 nm lasers and a FACSCanto® II flow cytometer (Becton Dickinson) with BD FACSDiva® flow cytometry analysis software (Becton Dickinson). Briefly, whole blood was collected from patients. 300 μL of QC or whole blood was pipetteed to the bottom of each staining tube using reverse pipetting. 50 μL of mAb cocktail was mixed and added to each tube. The following FMO tubes were used: 5 μL anti-CD3:FITC, 5 μL anti-CD14:FITC, 5 μL anti-CD33:PerCP-Cy5.5, 5 μL anti-CD56:PerCP-Cy5.5, 15 μL anti-CD45:APC-H7, and 15 μL PBS. The following laboratory tubes were used: 5 μL anti-CD19:BV 421, 5 μL anti-CD3:FITC, 5 μL anti-CD14:FITC, 5 μL anti-CD22:PE, 5 μL anti-CD33:PerCP-Cy5.5, 5 μL anti-CD56:PerCP-Cy5.5, 5 μL anti-CD20:APC, and 15 μL anti-CD45:APC-H7. The tubes were thoroughly mixed (vortexed) and allowed to cool at room temperature (18-26°C). o C) The samples were incubated in the dark for 15 minutes. Next, 1.5 mL of BD FACSLysing solution was added to each tube. The tubes were thoroughly mixed (vortexed) and allowed to stand at room temperature in the dark for 30 minutes. The tubes were vortexed again and then analyzed.

[0285] Samples were acquired using a FACSCanto® II flow cytometer (Becton Dickinson) with a saved FACSDiva® MRB panel-1.1 acquisition template. The threshold was set to 1,000 with parameter 780 / 60(633)(CD45 APC-H7). Before acquisition, the SSC and FSC voltages and thresholds were confirmed to be properly set in the first sample. A minimum of 20,000 events in the lymph gate was set as the stop gate.

[0286] The following gating strategies were used. First, a dot plot of time (x-axis) against CD3 / CD14 FITC-A (y-axis) was used to monitor acquisition quality (Plot 1). Events collected during system failures were negatively selected from the analysis using the "time" gate. Single cells were gated and side-scatter doublets were excluded using a bivariate dot plot of SSC-A (x-axis) against SSC-H (y-axis) (Plot 2). Single cells were gated and forward-scatter doublets were excluded using a bivariate dot plot of FSC-A (x-axis) against FSC-H (y-axis) from the previous gate (Plot 3). CD45+ lymphocytes were gated using a bi-exponential bivariate dot plot of CD45 APC-H7-A (x-axis) against SSC-A (y-axis) from the previous gate (Plot 4). The arrangement of lymphocyte gates from plot 4 was verified using a bivariate dot plot of FSC-A (x-axis) vs. SSC-A (Y-axis) (plot 5). CD3+ T cells and CD14+ monocytes were excluded using a bivariate dot plot of lymphocyte gates from plot 4, CD3+ BV421-A (x-axis) vs. CD3 / CD14 FITC-A (Y-axis). - CD14 - CD19 + Cells were gated (plot 6). CD3 - CD14 - CD19 + Using a bivariate dot plot of gated CD19 BV421-A (x axis) versus CD33 / CD56 PerCP-C5.5-A (Y axis), we excluded CD33+ monocytes and T cells or NK cells expressing CD56, and CD19+ B cells (CD33+- CD56 - CD19 + The results for the following were reported (Plot 7). Bead events were gated to calculate the absolute number using a bivariate dot plot of CD33 / CD56 PerCP-Cy5.5-A (x-axis) vs. CD22 PE-A (Y-axis) with time gates from Plot 1 (Plot 8). The absolute number was determined as follows: CD19 B cells: cells / μL = (CD19 + event × number of beads) / (number of beads × 300 μL of blood volume used for staining). The assay was validated to a limit of quantification (LLOQ) of 0.441 cells / μL.

[0287] statistical analysis The estimates, using the Cochrane-Mantel-Harensel test with a two-sided α=0.2, showed that enrolling 60 patients in each treatment group could provide 83% power to detect a 20% difference in achieving complete renal response (CRR) between the obinutuzumab group (50% response rate) and the control group (30% response rate). The assumptions were based on response rates observed in recent randomized clinical trials, including patients with proliferative lupus nephritis. To control for type I errors in the primary analysis, hypothesis tests against the study endpoint were performed sequentially, starting from the primary endpoint.

[0288] The safety analysis population consisted of all patients who received at least one dose of obinutuzumab or placebo. Safety was assessed using descriptive statistics.

[0289] result 126 patients were randomized. One patient was randomized but discontinued the study due to pregnancy before the first blinded infusion, leaving the remaining 125 patients to receive at least one dose of their assigned intervention and be included in the modified treatment intention population. 115 patients (92%) completed 52 weeks of treatment. Four patients (6%) in the obinutuzumab group and seven patients (11%) in the control group required rescue immunosuppression before week 52.

[0290] Most patients (85%) were female, with a mean age of 33 years (Table 4). 73% were identified as Hispanic or Latino, and 43% were Caucasian. A total of 78% had class IV lupus nephritis, with the remainder having class III lupus nephritis. Concurrent class V lupus nephritis was present in 29%. The mean (+ / -SD) UPCR at baseline was 3.12±2.56, the mean serum creatinine at baseline was 0.84±0.77, and the mean eGFR at baseline was 102.0±31.7. Patient disease characteristics at baseline were similar in both treatment groups. A further classification of the obinutuzumab group, later divided into patients with persistent B-cell depletion after obinutuzumab treatment vs. patients with detectable B cells after obinutuzumab treatment, is shown in Figure 10. [Table 4]

[0291] Clinical results Complete renal response (primary endpoint) at week 52 was achieved in 22 patients (35%) in the obinutuzumab group and 14 patients (23%) in the control group (risk difference, 12 percent points; 80% CI, 2-22; P=0.115) (Figure 1A). Overall response at week 52 was achieved in 35 patients (56%) in the obinutuzumab group and 22 patients (36%) in the control group (risk difference, 20 percent points; 80% CI, 9-31; P=0.025). Modified complete renal response at week 52 was achieved in 25 patients (40%) in the obinutuzumab group and 16 patients (26%) in the control group (risk difference, 14 percent points; 80% CI, 3-25; P=0.09). The primary and secondary efficacy endpoints are shown in Table 5A.

[0292] In the exploratory analysis at week 76, complete renal response was achieved in 25 patients (40%) in the obinutuzumab group and 11 patients (18%) in the control group (risk difference, 22 percent points; 80% CI, 12–32). The pre-specified surrogate definition of complete renal response was an increase in the response rate in both groups while maintaining the therapeutic benefit of obinutuzumab at week 76. Renal response over time is shown in Figure 1B. The results of the exploratory analysis at week 76 are shown in Tables 5A and 5B. Further efficacy data are shown in Figures 1C and 1D.

[0293] Six patients (10%) in the obinutuzumab group and twelve patients (19%) in the placebo group received salvage therapy up to week 76. Of these, two patients in the obinutuzumab group and six patients in the placebo group received cyclophosphamide salvage therapy.

[0294] Obinutuzumab was associated with significant improvements in C3, C4, anti-dsDNA antibody, and UPCR compared to placebo (Table 5A). At weeks 52 and 76, the adjusted mean differences in UPCR reduction from baseline between the treatment groups were 0.57 (80% CI, 0.2–1.0) and 0.72 (80% CI, 0.4–1.1), respectively. The changes from baseline for each of these measurements are shown in Figure 2A. Obinutuzumab was associated with increased achievement of CRR (40% vs. 18%, P=0.007) and ORR (51% vs. 29%, P=0.015) at week 76. The definition of alternative response showed an increased response rate in both treatment groups (Table 5A and Figure 13). [Table 5A]

[0295] Recent studies suggest that a 25% threshold for serum creatinine increase may be appropriate for patients with normal serum creatinine levels. Therefore, we applied new modified complete renal response (mCRR) and modified partial renal response (mPRR) criteria to the data. Under the new criteria, mCRR required all of the following: UPCR < 0.5; serum creatinine below the upper limit of normal; and serum creatinine did not increase by more than 25% from baseline; and mPRR required all of the following: a decrease of 50% or more in UPCR to less than 1 (or less than 3 if baseline was 3 or higher); and serum creatinine did not increase by more than 25% from baseline. Using these criteria, obinutuzumab was associated with an increase in mCRR compared to placebo at week 52 (43% vs. 29%, a 14% difference, p<0.2) and week 76 (54% vs. 31%, a 23% difference, p<0.01), and obinutuzumab was also associated with an increase in mPRR at week 52 (68% vs. 45%, a 23% difference, p<0.05) and week 76 (68% vs. 50%, an 18% difference, p<0.05). The time-course CRR (CRR definition: UPCR <0.5 and serum creatinine below the upper limit of normal) for both cohorts is shown in Figure 2B.

[0296] At week 4, 98% and 89% of patients in the obinutuzumab group had peripheral CD19+ B cell depletion below the lower limit of quantification using conventional flow cytometry (<5 cells / μL) and high-sensitivity flow cytometry (<0.441 cells / μL), respectively. The proportion of patients with depletion by high-sensitivity flow cytometry at weeks 24 and 52 was 73% and 80%, respectively. At week 4, 98% and 89% of patients in the obinutuzumab group had peripheral CD19+ B cell depletion below the lower limit of quantification using conventional flow cytometry (<5 cells / μL) and high-sensitivity flow cytometry (<0.441 cells / μL), respectively. The proportion of patients with depletion by high-sensitivity flow cytometry at weeks 24 and 52 was 73% and 80%, respectively (Figure 3A). Memory and naive B cells and plasmablasts were rapidly depleted, and evidence of naive B cell and plasmablast regrowth was obtained at week 24, prior to the third infusion (Figure 3B). Mean serum B-cell activator (BAFF) levels increased from baseline 4,585 pg / mL to 14,601 pg / mL at week 52 in the obinutuzumab group, compared to a 36% increase from baseline 5,341 to 7,278 pg / mL in the placebo group at week 52 (Figure 11). In the obinutuzumab group, BAFF levels began to rise within two weeks.

[0297] In the obinutuzumab group, 32 patients (51%) remained subdetectable depleted using high-sensitivity flow cytometry at both weeks 24 and 52. These patients had numerically higher CRR (50%) and ORR (66%) rates at week 76 compared to patients who had detectable B cells at any point in time, with CRR and ORR rates of 35% and 45%, respectively (Figure 4 and Table 5B). Among patients treated with obinutuzumab, achieving sustained B cell depletion was associated with a greater renal response benefit at week 76 (Table 5B), but patients who achieved sustained B cell depletion also had lower baseline proteinuria and serum creatinine. Sustained B cell depletion was achieved in 32 / 52 (62%) of patients with complete data. [Table 5B]

[0298] The response rate was low among patients with baseline SCr <0.65 mg / dL (n=45), due to the requirement that SCr not increase by more than 15% from baseline (Figure 12). Increasing this threshold to 25% increased the response rate to a level similar to that of the other groups.

[0299] The B-cell depletion achieved in this Phase II clinical trial (NOBILITY) investigating obinutuzumab was compared to the B-cell depletion achieved in a previous clinical trial (LUNAR) with rituximab, a type I anti-CD20 antibody. Both antibodies were administered at 1000 mg at weeks 0, 2, 24, and 26. As shown in Tables 5C and 5D, obinutuzumab treatment achieved superior B-cell depletion, down to less than 5 cells / μL as measured using conventional methodology (Table 5C), or down to 0 cells / μL as measured by the HSFC described herein (Table 5D). [Table 5C] [Table 5D]

[0300] Adverse events: Table 6 summarizes the safety data. The median follow-up period at the data cutoff date was 78 weeks (range, 5–104 weeks). One patient randomized to placebo inadvertently received two obinutuzumab infusions during the first cycle and was included in the obinutuzumab group for safety analysis. The incidence of serious adverse events was 23% in the obinutuzumab group and 30% in the placebo group, and the incidence of serious infections was 6% in the obinutuzumab group and 18% in the placebo group, respectively. One patient in the obinutuzumab group and three patients in the placebo group discontinued blinded obinutuzumab infusions due to adverse events. Infusion-related reactions occurred in 10 patients (16%) in the obinutuzumab group and 6 patients (10%) in the placebo group, all of which were non-serious and resolved with supportive care. The most frequent adverse events associated with obinutuzumab were bronchitis and infusion-related reactions.

[0301] At the cutoff date, there were five deaths: one in the obinutuzumab group and four in the placebo group. One fatal case of progressive multifocal leukoencephalopathy occurred in the placebo group in a patient who had received cyclophosphamide rescue approximately six months prior to diagnosis. [Table 6] As shown above, at weeks 52 and 76, obinutuzumab resulted in a higher CRR than placebo. The results at week 104 are shown in Table 7 below. [Table 7]

[0302] Similar to the results at weeks 52 and 76, at week 104, obinutuzumab showed a higher CRR than placebo (41% vs. 23%, P=0.026). At week 104, patients treated with obinutuzumab had an eGFR of +6.5 vs. -3.2 mL / min / 1.73m². 2 Greater improvements were observed in UPCR, anti-dsDNA, C3, and C4 (P=0.018). Serious adverse events (OBI 25% vs. PBO 30%), serious infections (8% vs. 18%), and death (1 vs. 4) were not increased in OBI.

[0303] In summary, this trial demonstrated the sustained benefits of obinutuzumab up to week 104, approximately 18 months after the final obinutuzumab infusion.

[0304] Conclusion and Discussion Nonclinical data suggested the potential of treating lupus with the anti-CD20 antibodies rituximab, ocrelizumab, and obinutuzumab. However, clinical trials of rituximab and ocrelizumab failed to meet their primary or key secondary endpoints for treatment. Unlike rituximab and ocrelizumab, the data presented herein demonstrate that clinical trials of obinutuzumab met both the primary and key secondary efficacy endpoints. At year one, patients treated with obinutuzumab, in addition to mycophenolate mofetil and corticosteroids for the treatment of proliferative lupus nephritis, showed increased complete and partial renal responses compared to patients treated with placebo. Furthermore, obinutuzumab was not associated with an increased rate of serious adverse events or serious infections.

[0305] Obinutuzumab treatment resulted in rapid and complete depletion of peripheral CD19+ B cells, memory and naive B cell subsets compared to ocrelizumab treatment, and plasmablast formation in the majority of patients with a lower incidence of safety events. At week 4, 89% of patients in the obinutuzumab group had peripheral CD19+ B cell depletion below the lower limit of quantification (<0.441 cells / μL) using high-sensitivity flow cytometry (HSFC). Furthermore, improved clinical efficacy was observed compared to the placebo group.

[0306] Of the 63 patients in the obinutuzumab / mycophenolate mofetil group, 36 showed persistent depletion of ≤5 cells / uL from day 28 to week 52, 6 had at least one measurement above 5 cells / uL (no persistent depletion), and 21 could not be evaluated in this analysis (missing one or more data points). Therefore, the majority of patients in this group had persistent B-cell depletion up to week 52.

[0307] The trial results showed that obinutuzumab, when administered in combination with mycophenolate and corticosteroids, was superior to placebo in achieving complete and overall response at week 52 for the treatment of proliferative lupus nephritis. Exploratory analysis at week 76 demonstrated a higher efficacy benefit compared to the control group. Furthermore, patients receiving obinutuzumab showed greater improvements in anti-dsDNA antibody levels, C3, C4, and UPCR compared to the control group.

[0308] The inventors hypothesized that residual B cells in peripheral blood and renal tissue explained the lack of efficacy of type I anti-CD20 monoclonal antibodies in previous lupus nephritis studies. They also hypothesized that enhanced B cell depletion with obinutuzumab significantly improved renal response compared to controls. The results of this study suggest that B cells play a crucial role in the pathogenesis of lupus nephritis, and that achieving complete depletion is associated with clinical benefit.

[0309] Obinutuzumab has been associated with improved achievement of complete and partial renal response at one year, which are linked to improved long-term outcomes in lupus nephritis, respectively (Chen, YE. et al. (2008) Clin J Am Soc Nephrol 3:46-53; Davidson, J. et al. (2018) The Journal of Rheumatology 45:5). Because complete response is rare in the first year of treatment, the European League Against Rheumatism (EULAR) guidelines now recommend partial renal response as the initial treatment goal in the first year (Fanouriakis, A. et al. (2019) Ann Rheum Dis. Jun;78(6):736-745). Exploratory results from week 76 suggest that the benefit of obinutuzumab for total renal response compared to controls precedes a similarly large benefit for complete renal response at 18 months by one year.

[0310] Infusion-related reactions were more common in patients treated with obinutuzumab than in the control group. In CLL, the incidence and severity of infusion-related reactions with obinutuzumab appeared to be greater than those observed with rituximab and were associated with the increased release of pro-inflammatory cytokines, particularly IL-6 and IL-8 (Freeman, CL. et al. (2015) Blood 126:2646-2649). Enhanced cross-linking between CD20-expressing leukemia cells and FcγRIIIA-carrying effector cells has been proposed as a mechanism (Freeman, CL. et al. (2016) Leukemia 30:1763-1766). We hypothesize that the relatively low rate of infusion-related reactions and the absence of severe infusion-related reactions observed in this study may be due to the corticosteroid regimens administered for the treatment of lupus nephritis and differences in CD20 expression between oncology patients and lupus nephritis patients. As in oncology, the incidence of infusion-related reactions was highest with the first obinutuzumab infusion and decreased with subsequent infusions.

[0311] The emergence of immunosuppressive therapies for the treatment of lupus nephritis has been associated with improvements in short-term and long-term outcomes. However, the use of current unapproved standard treatments has not been associated with improvements in the incidence of ESRD in recent decades, and there are still no approved treatments for lupus nephritis in the United States.

[0312] Example 2: Modified obinutuzumab dosing regimen for the treatment of proliferative lupus nephritis in combination with mycophenolic acid and corticosteroids. The study described in Example 1 demonstrated that a dosing regimen of 1000 mg obinutuzumab infusions at weeks 0, 2, 24, and 26, combined with standard immunosuppression, demonstrated efficacy and tolerable safety at weeks 52 and 76 in patients with lupus nephritis (LN). This example describes how a modeling approach was used to predict expected obinutuzumab PK after a 1000 mg dosing regimen at weeks 0, 2, 24, 26, and 52 in combination with mycophenolate mofetil and corticosteroids.

[0313] Population pharmacokinetic models Based on the data presented in Example 1, a population pharmacokinetic (PK) model was developed. The analytical dataset used to develop the PK model included 658 post-administration serum concentration values ​​from 63 patients treated with 1000 mg obinutuzumab infusions at weeks 0, 2, 24, and 26, in combination with standard immunosuppression, as described in Example 1.

[0314] The PK of obinutuzumab was well explained by a two-compartment linear population model in which clearance is the sum of two elimination pathways: a) damping coefficient (k des ) accompanied by a decrease in time-varying clearance (CL) over time T0 a) likely associated with a decrease in CD20 targets over time and improvement in proteinuria; and b) time-independent clearance (CL) related to the endogenous catabolic process of IgG. INF The covariates found to affect obinutuzumab PK parameters were body weight (BW), baseline serum albumin (ALB), and baseline serum IgG levels.

[0315] The final model was the CL INF CL T0 This included the allometric dependence of Q (intercompartmental clearance) on body weight (force coefficient 0.66), the dependence of the central and peripheral volumes of the distribution on body weight (force coefficient 0.600), and the dependence of the peripheral volume of the distribution on body weight (force coefficient 1). CL T0 and CL INF These decreased by powers of 2.8 and 0.685, respectively, with increasing albumin levels. INF The value decreased exponentially with increasing IgG concentration, in a power of 0.4 increment. No significant effect of anti-drug antibodies was observed. The estimated model parameters for the final model are shown in Table 8. [Table 8]

[0316] Table 9 shows the effects of covariates on the model parameters. [Table 9]

[0317] The final model was validated using goodness-of-fit plots, plots of random effects and between-individual parameters, and prediction check procedures such as visual prediction checks (VPC).

[0318] PK model validation Model validation demonstrated that the final PK model could be used to predict obinutuzumab exposure. For example, the visual predictive test (VPC) plot showed good agreement between observed obinutuzumab concentrations and data simulated using the final PK model (Figure 5).

[0319] The time-course obinutuzumab concentration profile was simulated using the final PK model validated for all patients according to the dosing regimen described in Example 1 (1000 mg at weeks 0, 2, 24, 26, and 52). The predicted time-course obinutuzumab concentration profile is shown in Figure 6.

[0320] Safety and efficacy exposure analysis Exploratory graphs and logistic regression analyses of exposure-safety relationships did not reveal a clear relationship with the adverse events (AEs) analyzed. This suggests that a favorable therapeutic concentration range may exist and that efficacy can be improved by increasing the dose without adversely affecting the safety profile. Logistic regression analyses of the probability of adverse events versus obinutuzumab exposure evaluated three types of events (late SAEs after initial administration, infection and invasiveness, and infusion-related reactions) and showed no correlation between exposure to obinutuzumab and the probability of events. For example, as shown in Figure 7, the probability of late SAE occurrence and cumulative exposure (AUC) from the start of treatment to week 52 were not observed. 52 There was no statistically significant relationship between the two (p=0.383).

[0321] Exploratory graphical analysis of the B-cell-efficacy relationship from the studies described in Example 1 suggested that a greater proportion of obinutuzumab-treated patients who achieved sustained peripheral B-cell depletion achieved complete renal response (CRR) compared to those who did not. For example, as shown in Table 10, 73.7% of patients who achieved CRR had B-cell levels below the limit of B-cell quantification (BQL = 0.441 cells / μl), while 65.8% of patients who did not achieve CRR had B-cell levels below BQL. Furthermore, pharmacokinetic and pharmacodynamic analyses showed that patients with higher obinutuzumab exposure were more likely to have sustained peripheral B-cell depletion at weeks 24 and 52 compared to patients with lower exposure. For example, as shown in Table C, 82.8% of patients with high obinutuzumab exposure (cumulative AUC at week 52 above the median) had B cells below the limit of quantification, compared to 53.6% of patients in the low-exposure group (cumulative AUC at week 52 below the median). [Table 10]

[0322] Furthermore, B cell depletion appeared to persist longer in patients with higher exposure. For example, as shown in Figure 8, after week 12, B cell counts fell below BQL and remained low as long as the obinutuzumab concentration remained above 1 μg / mL. Moreover, as shown in Figure 9, the probability of B cell counts rebounding above BQL at week 52 (B cells > BQL) decreased with increasing exposure (AUCC). 52 (p=0.045). Model-based simulation of the proposed additional dose of 1000 mg of obinutuzumab at week 52.

[0323] To maintain B-cell depletion and potentially increase efficacy at week 76, an additional infusion of 1000 mg of obinutuzumab administered at week 52 was proposed (1000 mg of obinutuzumab at weeks 0, 2, 24, 26, and 52). The proportion of patients with trough obinutuzumab concentrations exceeding 1 μg / mL at week 76 after the additional dose at week 52 was evaluated by simulation using the population PK model described above.

[0324] As shown in Table 11, simulations of exposure parameters for five different obinutuzumab dosing regimens showed that dosing at weeks 0, 2, 24, 26, and 52 resulted in 45.9% of subjects being predicted to have trough obinutuzumab concentrations above 1 μg / mL at week 76. In contrast, only 1.6% of subjects administered at weeks 0, 2, 24, and 26, or at weeks 0, 2, 12, 24, and 26, were predicted to have trough obinutuzumab concentrations above 1 μg / mL at week 76. Similarly, only 3.3% of subjects administered at weeks 0, 2, 16, 18, 32, and 34 were predicted to have trough obinutuzumab concentrations above 1 μg / mL at week 76. [Table 11]

[0325] As shown in Figure 8, from week 12 onward, the B cell count fell below the BQL level and remained low as long as the obinutuzumab concentration remained above 1 μg / mL. Therefore, the additional dose at week 52 is expected to maintain B cell depletion and lead to better efficacy at week 76.

[0326] Furthermore, based on the overall knowledge regarding the safety of obinutuzumab, the lack of a statistically significant exposure-safety relationship, and the 76-week safety data collected in the study described in Example 1 (see Table 6 in Example 1), an additional dose at 52 weeks would still result in an acceptable safety profile.

[0327] To account for the risk of infusion-related reactions, 80 mg of methylprednisolone should be administered intravenously before each obinutuzumab administration.

[0328] In summary, based on estimated exposures from simulations of the drug regimen and evaluation of available safety data, a drug regimen similar to that used in the study described in Example 1 (i.e., intravenous administration of obinutuzumab 1000 mg at weeks 0, 2, 24, and 26) is proposed, followed by additional doses of 1000 mg intravenously every six months, starting at week 52.

[0329] The data from Example 1, as well as the modeling and simulation results presented in this example, suggest that the proposed drug regimen (i.e., dosing at weeks 0, 2, 24, 26, and 52) induces rapid and prolonged B-cell depletion and is effective in treating LN. This drug regimen (including the additional dose at w52) is expected to provide a safety profile similar to that observed in the study described in Example 1.

[0330] Example 3: Testing of a modified obinutuzumab dosing regimen for the treatment of proliferative lupus nephritis in combination with mycophenolic acid and corticosteroids. Using the modeling approach described in Example 2, PK exposure for 1000 mg obinutuzumab dosing regimens at weeks 0, 2, 24, 26, and 52 in combination with mycophenolate mofetil and corticosteroids was predicted. This example describes the administration of 1000 mg obinutuzumab dosing regimens at weeks 0, 2, 24, 26, and 52.

[0331] dosage Patients will be randomly assigned to one of two groups: the obinutuzumab group or the placebo group. Patients in the obinutuzumab group will receive 1000 mg of obinutuzumab intravenously (IV) at baseline and at weeks 2, 24, 26, and 52 of the study, along with premedication. Patients in the obinutuzumab group will be divided into two subgroups. Both subgroups will receive 1000 mg of obinutuzumab intravenously (IV). One subgroup will receive infusions at baseline and at weeks 2, 24, 26, and 52 of the study, along with premedication, while the other subgroup will receive infusions at baseline and at weeks 2, 24, 26, 50, and 52 of the study, along with premedication. Mycophenolate mofetil (MMF) will be administered on day 1, with dose escalation to the target dose by week 4, and the target dose will be maintained until week 80. MMF is administered in divided doses starting at a dose of 1500 mg / day (or equivalent), gradually increasing from 500 mg / week to 2.0-2.5 g / day by week 4, and then maintained at the target dose until week 80. Oral prednisone is started at 0.5 mg / kg / day from day 2 until the visit in week 2. Prednisone is gradually tapered starting on day 16, reaching the target dose of 5 mg / day by week 24. Prednisone is maintained at 5 mg / day from weeks 24 to 80.

[0332] Patients in the placebo group will receive a placebo equivalent to obinutuzumab IV infusion at baseline and at weeks 2, 24, 26, and 52, along with premedication. MMF will be administered on day 1, with dose escalation to the target dose by week 4, and the target dose will be maintained until week 80. MMF will be administered in divided doses starting at 1500 mg / day (or equivalent), with dose escalation from 500 mg / week to 2.0-2.5 g / day by week 4, and then maintained at the target dose until week 80. Oral prednisone will be started at 0.5 mg / kg / day from day 2 until the visit at week 2. Prednisone tapering will begin on day 16, reaching the target dose of 5 mg / day by week 24. Prednisone will remain at 5 mg / day from weeks 24 to 80.

[0333] For premedication, methylprednisolone 80 mg is administered by IV infusion at weeks 0, 2, 24, 26, and 52. Acetaminophen is administered orally at a dose of 650-1000 mg 30-60 minutes before obinutuzumab or placebo infusion. Diphenhydramine is administered orally at a dose of 50 mg 30-60 minutes before obinutuzumab or placebo infusion.

[0334] Patients will be followed in a blinded manner, and those with persistent B-cell depletion will be followed for safety and B-cell assessment. See also the protocol published in International Publication No. 2016 / 183104.

[0335] patient Patients are eligible if they are between 18 and 75 years of age, have systemic lupus erythematosus (SLE) as established by current American College of Rheumatology criteria, have a diagnosis of International Society of Nephrology / Nephrology 2003 Class III or IV (may show a Class III or Class IV disease in addition to an associated Class V disease) as confirmed by a renal biopsy performed 6 months prior to or during screening, have a urinary protein-to-creatinine ratio (UPCR) greater than 1 on a 24-hour urine collection, have received at least one dose of pulsed methylprednisolone IV (500-1000 mg) or an equivalent dose for the treatment of a current episode of active LN in the 6 months prior to or during screening, and have received a stable dose of an ACE inhibitor or angiotensin receptor blocker (ARB) at least 10 days prior to randomization.

[0336] Exclusion criteria include: pregnancy, breastfeeding, or intending to become pregnant during the study or within 18 months after the last dose of the study drug; severe renal impairment or need for dialysis or kidney transplantation; glomerulosclerosis of more than 50% of the kidneys on a renal biopsy; presence of rapidly progressive glomerulonephritis; having received exclusion therapy (any anti-CD20 therapy in the 12 months prior to randomization, cyclophosphamide, tacrolimus, cyclosporine, or voclosporine in the 2 months prior to randomization, any biological therapy other than anti-CD20 in the 3 months prior to randomization, an oral inhibitor of Janus-related kinase (JAK), Bruton's tyrosine kinase (BTK), or tyrosine kinase 2 (TYK2) in the 3 months prior to randomization, or any live vaccine in the 2 months prior to randomization); severe active central nervous system SLE; high risk of clinically significant bleeding or organ failure due to thrombocytopenia, anemia, and / or coagulation disorders, or plasma apheresis, venous In cases requiring transfusion of internal immunoglobulin or acute blood products; known HIV infection; known active infection of any kind except fungal infections of the nail bed; any major episode of infection requiring treatment with IV antibiotics or anti-infective agents for 3 months prior to hospitalization or randomization, or treatment with oral antibiotics or anti-infective agents for 6 weeks prior to randomization; a history of progressive multifocal leukoencephalopathy (PML); a history of cancer other than non-melanoma carcinoma of the skin that has been treated or excised and resolved; and intolerance or contraindications to the study therapy, e.g., positive hepatitis C serology, hemoglobin <7 g / dL (unless caused by autoimmune hemolytic anemia due to SLE), platelet count <25,000 / uL, positive serum human chorionic gonadotropin measured before the first obinutuzumab infusion, AST or ALT >2.5 × upper limit of normal (ULN), amylase or lipase >2 × ULN, neutrophils <1.5 × 10⁻¹⁰ 3 This includes a blood glucose level of / uL and positivity for hepatitis B surface antigen (HBsAg).

[0337] Evaluation items Peripheral blood B cells, safety, urinary protein excretion, serum creatinine, autoantibody and serum complement component levels, and clinical disease activity are evaluated as described in Example 1. Evaluation items such as the percentage of patients achieving CRR, the percentage of patients achieving modified CRR, and the percentage of patients achieving PRR are measured as described in Example 1.

[0338] The primary endpoint is the percentage of participants with a complete renal response (CRR) at week 76.

[0339] Secondary endpoints include the proportion of participants achieving overall renal response (ORR), defined as achieving either a chronic renal response (CRR) or partial renal response (PRR); changes in anti-dsDNA titer; changes in complement C3; time to the first CRR; proportion of participants achieving a CRR including urinary sediment (CRR sediment); proportion of participants achieving an ORR including urinary sediment (ORR sediment); changes in the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2k); changes in the Fatigue Scale (FACIT-F); changes in the HRQoL (SF-36) scale; changes in estimated glomerular filtration rate (eGFR); proportion of participants achieving a CRR by eGFR criteria; proportion of participants experiencing adverse events; maximum serum concentration of obinutuzumab; proportion of participants with baseline and post-treatment anti-drug antibodies (ADA); and changes from baseline in total peripheral B cell count.

[0340] Example 4: Phase III randomized, open-label, active-controlled, multicenter study to evaluate the efficacy and safety of obinutuzumab in patients with primary membranous nephropathy. This study will evaluate the efficacy, safety, pharmacodynamics, and pharmacokinetics of obinutuzumab compared to tacrolimus in patients with primary membranous nephropathy (pMN). This is a phase III randomized, parallel-group, effective-controlled, open-label trial evaluating the efficacy and safety of obinutuzumab compared to tacrolimus in patients with pMN.

[0341] Membranous nephropathy (MN) is classified as either primary or secondary MN depending on its etiology. Idiopathic or primary MN (pMN) is autoimmune in nature, caused by autoantibodies targeting podocyte membranes. Secondary MN can be caused by underlying conditions such as cancer, infections, autoimmune diseases such as systemic lupus erythematosus, or treatment with certain drugs, such as gold / penicillamine. pMN is a kidney-specific autoimmune glomerular disease that presents with increased urinary protein associated with a characteristic pattern of glomerular damage. Most pMNs are mediated by other antibodies, such as antibodies against the M-type phospholipase A2 receptor (anti-PLA2R) (70-85%), antibodies against the thrombospondin type 1 domain containing 7A (THSD7A) (3-5%), or unidentified anti-podocyte autoantibodies (10%) (Couser WG. Clin J Am Soc Nephrol 2017;12(6):983-97). These autoantibodies target the podocyte membrane, leading to subepithelial deposition of immune complexes and widespread loss of podocyte foot processes, resulting in impaired filtration and proteinuria. These autoantibodies may originate from dysregulated B cells, and patients with persistent proteinuria show improvement with treatment with immunosuppressants (KDIGO Clinical Practice Guideline for Glomerulonephritis - Chapter 7: Idiopathic membranous nephropathy).

[0342] pMN is the most common cause of idiopathic nephropathy syndrome in non-diabetic adults worldwide, accounting for 20–37% of cases, and rising to as high as 40% in adults over 60 years of age (Couser WG. Clin J Am Soc Nephrol 2017;12(6):983-97). Signs and symptoms of renal syndrome include hypoalbuminemia, edema, weight gain, hyperlipidemia, fatigue, and loss of appetite. Thromboembolism, infection, hypothyroidism, hypertension, anemia, and coronary artery disease are common complications (de Seigneux S, Martin PY. Swiss Med Wkly 2009;139(29-30):416-22). The natural course of pMN varies; one-third of patients achieve spontaneous remission, another third develop chronic subnephrotic proteinuria, and the remaining third progress to end-stage renal disease (ESRD) over 5-10 years. Clinically, 80% of pMN patients exhibit renal syndrome, and 20% exhibit non-renal proteinuria (Couser WG. Clin J Am Soc Nephrol 2017;12(6):983-97).Complete remission of nephrotic proteinuria predicts superior long-term kidney and patient survival, and achieving partial remission to subnephrotic proteinuria also significantly reduces the risk of progression to ESRD requiring dialysis or kidney transplantation (Cattran DC, Brenchley PE. Kidney Int 91 2017;566-574; Troyanov S, et al., and the Toronto Glomerulonephritis Registry Group. Kidney Int 2004;66(3):1199-205; Fervenza FC, Sethi S, Specks U. Clin J Am Soc Nephrol 2008;3(3):905-19; Hladunewich MA, et al., and the Metropolitan Toronto Glomerulonephritis Registry. Clin J Am Soc Nephrol 2009;4(9):1417-22; Polanco N, et al., and the Grupo de Estudio de las Enfermedades Glomerulares de la Sociedad Espanola de Nefrologia.J Am Soc Nephrol 2010;21(4):697-704).

[0343] Currently, there are no FDA-approved treatments for pMN, and current treatment options are controversial. Because the natural course of the disease varies, the KDIGO guidelines suggest that initial treatment for MN patients should be optimized supportive care (including renin-angiotensin system [RAS] blockade and blood pressure control), and that immunosuppressive therapy is recommended for patients with persistent renal syndrome. Alternating regimens of glucocorticoids and alkylating agents, such as chlorambucil (Italian Ponticelli protocol) or cyclophosphamide (modified Ponticelli protocol), are effective in achieving some form of remission in 60–70% of patients, but are associated with clinically significant toxicity and adverse effects, including hyperglycemia, myelosuppression, infection, infertility, and cancer (Waldman M, Austin HA 3rd. J Am Soc Nephrol 2012;23(10):1617-30). Calcineurin inhibitors (CNIs), including cyclosporine, are effective and are the preferred treatment for neuropathy (MN) in the United States and Canada. However, these drugs are associated with a high incidence of relapse after discontinuation and frequent side effects, including hypertension, hyperlipidemia, and nephrotoxicity (Rojas-Rivera JE, Carriazo S, Ortiz A. Clin Kidney J 2019;12(5):629-38; Fervenza FC, Appel GB, Barbour SJ, et al., and the MENTOR Investigators. N Engl J Med 2019,381(1):36-46). Given the high relapse rate and serious adverse effects, there is a significant unmet need for effective treatment options for pMN.

[0344] Since B cells play a crucial role in autoantibody production and the development of such pMN pathology, rituximab, a type I anti-CD20 antibody, has been used in several studies, resulting in B cell depletion and remission of renal syndrome (Fervenza FC, et al., and the MENTOR Investigators. N Engl J Med 2019;381(1):36-46; Dahan K, et al. Kidney Int Rep 2018;3(2):498-501; Ruggenenti P, et al. J Am Soc Nephrol 2015;26(10):2545-58). A recent MENTOR trial, a randomized controlled clinical trial comparing rituximab with cyclosporine A (CsA) in 130 patients with pMN, showed that rituximab demonstrated superior overall renal response compared to the active-controlled CsA at 24 months, with reduced relapses and serious adverse events (Fervenza FC, et al., and the MENTOR Investigators. N Engl J Med 2019;381(1):36-46). However, the use of rituximab highlights that approximately 40% of patients either fail treatment or show no response of any kind (neither complete nor partial) at 24 months, leaving an unmet medical need (Fervenza FC, et al., and the MENTOR Investigators. N Engl J Med 2019;381(1):36-46).

[0345] the purpose The primary efficacy objective is to evaluate the efficacy of obinutuzumab compared to tacrolimus based on the proportion of patients achieving complete response (CR) at week 104. CR is defined as a stable estimated glomerular filtration rate (eGFR) with a urinary protein / creatinine ratio (UPCR) of ≤0.3 (24-hour collection), eGFR ≤15% of baseline, and no occurrence of intermediate events (escape therapy, treatment failure, or early study discontinuation). eGFR is calculated using the Chronic Kidney Disease Epidemiological Collaborative Study (CKD-EPI) formula (Levey, ASet al. (2009) Ann. Intern. Med. 150:604-12).

[0346] The secondary efficacy objective is to evaluate the efficacy of obinutuzumab compared to tacrolimus based on the following endpoints. ● The percentage of patients who achieved complete remission (OR) (CR ...

Claims

1. A method for treating lupus nephritis in an individual having lupus, comprising administering to the individual a first antibody exposure to type II anti-CD20 antibody, a second antibody exposure to the type II anti-CD20 antibody, and a third antibody exposure to the type II anti-CD20 antibody, The second antibody exposure was not administered from approximately 18 weeks to approximately 26 weeks after the first antibody exposure. The third antibody exposure was not administered from approximately 24 weeks to approximately 32 weeks after the second antibody exposure. The first antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The second antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The third antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the third antibody exposure comprises a total exposure of approximately 800 mg to approximately 1200 mg of the type II anti-CD20 antibody. A method comprising a type II anti-CD20 antibody comprising a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO:

6.

2. The method according to claim 1, wherein the first antibody exposure comprises a first dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody.

3. The method according to claim 1 or claim 2, wherein the first antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the exposure of the second dose of the first antibody is not given from about 1.5 weeks to about 2.5 weeks after the exposure of the first dose of the first antibody.

4. The method according to claim 3, wherein the first antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the first antibody exposure of the second dose is not administered until approximately two weeks after the first antibody exposure of the first dose.

5. The method according to any one of claims 2 to 4, wherein the first dose of the first antibody exposure is approximately 1000 mg of the type II anti-CD20 antibody.

6. The method according to any one of claims 2 to 5, wherein the second dose of the first antibody exposure is approximately 1000 mg of the type II anti-CD20 antibody.

7. The method according to any one of claims 1 to 6, wherein the second antibody exposure comprises a first dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody.

8. The method according to any one of claims 1 to 7, wherein the second antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the second dose of the second antibody exposure is not administered from about 1.5 weeks to about 2.5 weeks after the first dose of the second antibody exposure.

9. The method according to claim 8, wherein the second antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the second dose of the second antibody exposure is not administered until approximately two weeks after the first dose of the second antibody exposure.

10. The method according to any one of claims 7 to 9, wherein the first dose of the second antibody exposure is approximately 1000 mg of the type II anti-CD20 antibody.

11. The method according to any one of claims 7 to 10, wherein the second dose of the second antibody exposure is about 1000 mg of the type II anti-CD20 antibody.

12. The method according to any one of claims 1 to 11, wherein the third antibody exposure comprises a single dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody.

13. The method according to claim 12, wherein the single dose of the third antibody exposure is approximately 1000 mg of the type II anti-CD20 antibody.

14. The method according to claim 12 or claim 13, wherein the single-dose exposure to the third antibody is not administered until approximately 52 weeks after the first dose exposure to the first antibody, or until approximately 28 weeks after the first dose exposure to the second antibody.

15. The method according to any one of claims 1 to 14, wherein the individual has lupus nephritis.

16. The method according to any one of claims 1 to 14, wherein the individual has class III or class IV lupus nephritis.

17. The method according to any one of claims 1 to 14, wherein the individual is at risk of developing class III or class IV lupus nephritis.

18. The method according to any one of claims 1 to 14, wherein the individual has class III(C) or class IV(C) lupus nephritis.

19. The method according to any one of claims 1 to 14, wherein the individual has congenital class V lupus nephritis.

20. The method according to any one of claims 1 to 19, further comprising administering an effective amount of immunosuppressant to the individual.

21. The method according to claim 20, wherein the immunosuppressant comprises mycophenolic acid, a derivative thereof, or a salt thereof.

22. The method according to claim 21, wherein the immunosuppressant comprises mycophenolate mofetil.

23. The method according to any one of claims 1 to 22, further comprising administering an effective amount of glucocorticoid or corticosteroid to the individual.

24. The method according to claim 23, wherein the glucocorticoid or corticosteroid comprises methylprednisolone.

25. The method according to claim 23, wherein the glucocorticoid or corticosteroid comprises prednisone.

26. The method according to any one of claims 1 to 25, further comprising administering an effective amount of an antihistamine to the individual.

27. The method according to claim 26, wherein the antihistamine comprises diphenhydramine.

28. The method according to any one of claims 1 to 27, further comprising administering an effective amount of a nonsteroidal anti-inflammatory drug (NSAID) to the individual.

29. The method according to claim 28, wherein the NSAID comprises acetaminophen.

30. The method according to any one of claims 1 to 29, further comprising administering an effective amount of antihypertensive agent to the individual.

31. The method according to claim 30, wherein the antihypertensive agent is an angiotensin-converting enzyme (ACE) inhibitor or an angiotensin receptor blocker.

32. The method according to any one of claims 1 to 31, further comprising administering standard treatment to the individual.

33. The method according to claim 32, wherein the standard treatment comprises treatment with one or more of angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, cyclophosphamide, mycophenolate mofetil, azathioprine, and glucocorticoids or corticosteroids.

34. The method according to any one of claims 1 to 33, which produces a complete renal response (CRR) in the aforementioned individual.

35. The method according to any one of claims 1 to 33, which produces a partial renal response (PRR) in the aforementioned individual.

36. The method according to any one of claims 1 to 35, which causes depletion of circulating peripheral B cells in the said individual.

37. The method according to claim 36, wherein the circulating peripheral B cells are CD19+ B cells.

38. The method according to any one of claims 1 to 37, wherein, after administration of the type II anti-CD20 antibody, B cells are depleted to a level in which circulating peripheral B cells are present in the peripheral blood of the individual at a concentration of approximately 5 cells / μL or less.

39. The method according to claim 38, wherein B cells are depleted to a level in which circulating peripheral B cells are present in the peripheral blood of the individual at a rate of about 1 cell / μL or less or about 0.5 cells / μL or less.

40. The method according to any one of claims 36 to 39, wherein the depletion is achieved after exposure to the first antibody.

41. The method according to any one of claims 36 to 40, wherein B cell depletion is sustained for at least 52 weeks after exposure to the first dose of the first antibody.

42. The method according to any one of claims 1 to 41, wherein, after administration of the type II anti-CD20 antibody, the circulating peripheral B cells in the individual are depleted by at least about 90% compared to a corresponding measurement in the same individual before administration of the type II anti-CD20 antibody, or compared to a corresponding measurement in an individual not treated with the type II anti-CD20 antibody.

43. The method according to any one of claims 1 to 42, wherein the individual is a human.

44. A method for depleting circulating peripheral B cells in an individual, comprising administering to the individual a first antibody exposure to type II anti-CD20 antibody, a second antibody exposure to the type II anti-CD20 antibody, and a third antibody exposure to the type II anti-CD20 antibody, The second antibody exposure was not administered from approximately 18 weeks to approximately 26 weeks after the first antibody exposure. The third antibody exposure was not administered from approximately 24 weeks to approximately 32 weeks after the second antibody exposure. The first antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The second antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The third antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the third antibody exposure comprises a total exposure of approximately 800 mg to approximately 1200 mg of the type II anti-CD20 antibody. The type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO:

6. A method comprising administering the type II anti-CD20 antibody, wherein B cells are depleted to a level where circulating peripheral B cells are present in the peripheral blood of the individual at a concentration of approximately 5 cells / μL or less.

45. The method according to claim 44, wherein the first antibody exposure comprises a first dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody.

46. The method according to claim 44 or claim 45, wherein the first antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the exposure of the second dose of the first antibody is not given from about 1.5 weeks to about 2.5 weeks after the exposure of the first dose of the first antibody.

47. The method according to claim 46, wherein the first antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the first antibody exposure of the second dose is not administered until approximately two weeks after the first antibody exposure of the first dose.

48. The method according to any one of claims 45 to 47, wherein the first dose of the first antibody exposure is about 1000 mg of the type II anti-CD20 antibody.

49. The method according to any one of claims 45 to 48, wherein the second dose of the first antibody exposure is about 1000 mg of the type II anti-CD20 antibody.

50. The method according to any one of claims 44 to 49, wherein the second antibody exposure comprises a first dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody.

51. The method according to any one of claims 44 to 50, wherein the second antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the second dose of the second antibody exposure is not given from about 1.5 weeks to about 2.5 weeks after the first dose of the second antibody exposure.

52. The method according to claim 51, wherein the second antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the second dose of the second antibody exposure is not administered until approximately two weeks after the first dose of the second antibody exposure.

53. The method according to any one of claims 50 to 52, wherein the first dose of the second antibody exposure is approximately 1,000 mg of the type II anti-CD20 antibody.

54. The method according to any one of claims 50 to 53, wherein the second dose of the second antibody exposure is approximately 1,000 mg of the type II anti-CD20 antibody.

55. The method according to any one of claims 44 to 54, wherein the third antibody exposure comprises a single dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody.

56. The method according to claim 55, wherein the single dose of the third antibody exposure is approximately 1000 mg of the type II anti-CD20 antibody.

57. The method according to claim 55 or claim 56, wherein the single-dose exposure to the third antibody is not administered until approximately 52 weeks after the first dose exposure to the first antibody, or until approximately 28 weeks after the first dose exposure to the second antibody.

58. The method according to any one of claims 44 to 57, wherein the individual has lupus nephritis.

59. The method according to any one of claims 44 to 57, wherein the individual has class III or class IV lupus nephritis.

60. The method according to any one of claims 44 to 57, wherein the individual is at risk of developing class III or class IV lupus nephritis.

61. The method according to any one of claims 44 to 57, wherein the individual has class III(C) or class IV(C) lupus nephritis.

62. The method according to any one of claims 44 to 57, wherein the individual has congenital class V lupus nephritis.

63. The method according to any one of claims 44 to 62, wherein the circulating peripheral B cells are CD19+ B cells.

64. The method according to any one of claims 44 to 63, wherein B cells are depleted to a level in which circulating peripheral B cells are present in the peripheral blood of the individual at a rate of about 1 cell / μL or less or about 0.5 cells / μL or less.

65. The method according to any one of claims 44 to 64, wherein the depletion is achieved after the first antibody exposure.

66. The method according to any one of claims 44 to 65, wherein B cell depletion is sustained for at least 52 weeks after exposure to the first dose of the first antibody.

67. The method according to any one of claims 44 to 66, wherein, after administration of the type II anti-CD20 antibody, the circulating peripheral B cells in the individual are depleted by at least about 90% compared to a corresponding measurement in the same individual before administration of the type II anti-CD20 antibody, or compared to a corresponding measurement in an individual not treated with the type II anti-CD20 antibody.

68. The method according to any one of claims 44 to 67, wherein the individual is a human.

69. The method according to any one of claims 1 to 68, wherein the first antibody exposure and / or the second antibody exposure and / or the third antibody exposure are administered intravenously.

70. The method according to any one of claims 1 to 69, wherein the antibody is humanized.

71. The method according to any one of claims 1 to 70, wherein the antibody is defucosylated.

72. The method according to any one of claims 1 to 71, wherein the heavy chain of the type II anti-CD20 antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO:

7.

73. The method according to any one of claims 1 to 72, wherein the light chain of the type II anti-CD20 antibody includes a light chain variable region containing the amino acid sequence of SEQ ID NO:

8.

74. The method according to any one of claims 1 to 73, wherein the heavy chain variable region of the type II anti-CD20 antibody comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region of the type II anti-CD20 antibody comprises the amino acid sequence of SEQ ID NO:

8.

75. The method according to any one of claims 1 to 74, wherein the heavy chain of the type II anti-CD20 antibody comprises the amino acid sequence of SEQ ID NO: 9, and the light chain of the type II anti-CD20 antibody comprises the amino acid sequence of SEQ ID NO:

10.

76. The method according to any one of claims 1 to 69, wherein the type II anti-CD20 antibody is obinutuzumab.

77. The method according to claim 1 or claim 44, wherein the first antibody exposure comprises two administrations of 1,000 mg of the type II anti-CD20 antibody on day 1 and day 15 of treatment, the second antibody exposure comprises two administrations of 1,000 mg of the type II anti-CD20 antibody on day 168 and day 182 of treatment, the third antibody exposure comprises one administration of 1,000 mg of the type II anti-CD20 antibody on day 364 of treatment, the type II anti-CD20 antibody is obinutuzumab, and the individual is a human.

78. The method according to claim 1 or claim 44, wherein the first antibody exposure comprises two administrations of 1,000 mg of the type II anti-CD20 antibody on day 1 and day 15 of treatment, the second antibody exposure comprises two administrations of 1,000 mg of the type II anti-CD20 antibody on day 168 and day 182 of treatment, the third antibody exposure comprises two administrations of 1,000 mg of the type II anti-CD20 antibody on day 350 and day 364 of treatment, the type II anti-CD20 antibody is obinutuzumab, and the individual is a human.

79. The method according to claim 1 or claim 44, wherein the first antibody exposure comprises two administrations of 1,000 mg of the type II anti-CD20 antibody at weeks 0 and 2 of treatment, the second antibody exposure comprises two administrations of 1,000 mg of the type II anti-CD20 antibody at weeks 24 and 26 of treatment, the third antibody exposure comprises one administration of 1,000 mg of the type II anti-CD20 antibody at week 52 of treatment, the type II anti-CD20 antibody is obinutuzumab, and the individual is human.

80. The method according to claim 1 or claim 44, wherein the first antibody exposure comprises two administrations of 1,000 mg of the type II anti-CD20 antibody at weeks 0 and 2 of treatment, the second antibody exposure comprises two administrations of 1,000 mg of the type II anti-CD20 antibody at weeks 24 and 26 of treatment, the third antibody exposure comprises two administrations of 1,000 mg of the type II anti-CD20 antibody at weeks 50 and 52 of treatment, the type II anti-CD20 antibody is obinutuzumab, and the individual is human.

81. A method for depleting circulating peripheral B cells in an individual, comprising administering to the individual a first antibody exposure to a type II anti-CD20 antibody, and a second antibody exposure to the type II anti-CD20 antibody, The second antibody exposure was not administered from approximately 18 weeks to approximately 26 weeks after the first antibody exposure. The first antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The second antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO:

6. A method comprising administering the type II anti-CD20 antibody, wherein B cells are depleted to a level where circulating peripheral B cells are present in the peripheral blood of the individual at a concentration of approximately 5 cells / μL or less, and this state persists for at least 52 weeks after exposure to the first dose of the first antibody.

82. The method according to claim 81, wherein the first antibody exposure comprises a first dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody.

83. The method according to claim 81 or claim 82, wherein the first antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the exposure of the second dose of the first antibody is not given from about 1.5 weeks to about 2.5 weeks after the exposure of the first dose of the first antibody.

84. The method according to claim 83, wherein the first antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the first antibody exposure of the second dose is not administered until approximately two weeks after the first antibody exposure of the first dose.

85. The method according to any one of claims 82 to 84, wherein the first dose of the first antibody exposure is about 1000 mg of the type II anti-CD20 antibody.

86. The method according to any one of claims 82 to 85, wherein the second dose of the first antibody exposure is approximately 1000 mg of the type II anti-CD20 antibody.

87. The method according to any one of claims 81 to 86, wherein the second antibody exposure comprises a first dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody.

88. The method according to any one of claims 81 to 87, wherein the second antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the second dose of the second antibody exposure is not given from about 1.5 weeks to about 2.5 weeks after the first dose of the second antibody exposure.

89. The method according to claim 88, wherein the second antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the second dose of the second antibody exposure is not administered until approximately two weeks after the first dose of the second antibody exposure.

90. The method according to any one of claims 87 to 89, wherein the first dose of the second antibody exposure is approximately 1000 mg of the type II anti-CD20 antibody.

91. The method according to any one of claims 87 to 90, wherein the second dose of the second antibody exposure is approximately 1000 mg of the type II anti-CD20 antibody.

92. The method according to any one of claims 81 to 91, wherein the individual has lupus nephritis.

93. The method according to any one of claims 81 to 91, wherein the individual has class III or class IV lupus nephritis.

94. The method according to any one of claims 81 to 91, wherein the individual is at risk of developing class III or class IV lupus nephritis.

95. The method according to any one of claims 81 to 91, wherein the individual has class III(C) or class IV(C) lupus nephritis.

96. The method according to any one of claims 81 to 91, wherein the individual has congenital class V lupus nephritis.

97. The method according to any one of claims 81 to 91, wherein the individual has membranous nephropathy (MN).

98. The method according to any one of claims 81 to 91, wherein the individual is at risk of developing membranous nephropathy (MN).

99. The method according to claim 97 or claim 98, wherein the membranous nephropathy is primary membranous nephropathy (pMN).

100. The method according to any one of claims 81 to 99, wherein the circulating peripheral B cells are CD19+ B cells.

101. The method according to any one of claims 81 to 100, wherein B cells are depleted to a level in which circulating peripheral B cells are present in the peripheral blood of the individual at a rate of about 1 cell / μL or less or about 0.5 cells / μL or less.

102. The method according to any one of claims 81 to 101, wherein the depletion is achieved after the first antibody exposure.

103. The method according to any one of claims 81 to 102, wherein B cell depletion is sustained for at least 52 weeks after exposure to the first dose of the first antibody.

104. The method according to any one of claims 81 to 103, wherein, after administration of the type II anti-CD20 antibody, the circulating peripheral B cells in the individual are depleted by at least about 90% compared to a corresponding measurement in the same individual before administration of the type II anti-CD20 antibody, or compared to a corresponding measurement in an individual not treated with the type II anti-CD20 antibody.

105. The method according to any one of claims 81 to 104, wherein the first antibody exposure and / or the second antibody exposure are administered intravenously.

106. The method according to any one of claims 81 to 105, wherein the type II anti-CD20 antibody is a humanized antibody.

107. The method according to any one of claims 81 to 106, wherein the type II anti-CD20 antibody is defucosylated.

108. The method according to any one of claims 81 to 107, wherein the heavy chain variable region of the type II anti-CD20 antibody includes the amino acid sequence of SEQ ID NO:

7.

109. The method according to any one of claims 81 to 108, wherein the light chain variable region of the type II anti-CD20 antibody includes the amino acid sequence of SEQ ID NO:

8.

110. The method according to any one of claims 81 to 107, wherein the heavy chain variable region of the type II anti-CD20 antibody comprises the amino acid sequence of SEQ ID NO: 7, and the light chain variable region of the type II anti-CD20 antibody comprises the amino acid sequence of SEQ ID NO:

8.

111. The method according to any one of claims 81 to 110, wherein the heavy chain of the type II anti-CD20 antibody comprises the amino acid sequence of SEQ ID NO: 9, and the light chain of the type II anti-CD20 antibody comprises the amino acid sequence of SEQ ID NO:

10.

112. The method according to any one of claims 81 to 105, wherein the type II anti-CD20 antibody is obinutuzumab.

113. The method according to any one of claims 81 to 112, wherein the individual is a human.

114. A method for treating lupus nephritis in an individual having lupus, comprising intravenously administering to the individual a first antibody exposure, a second antibody exposure, and a third antibody exposure against type II anti-CD20 antibody, The first antibody exposure comprises two administrations of 1000 mg of the type II anti-CD20 antibody during weeks 0 and 2 of treatment. The second antibody exposure comprises two administrations of 1000 mg of the type II anti-CD20 antibody at weeks 24 and 26 of treatment. The third antibody exposure comprises a single dose of 1000 mg of the type II anti-CD20 antibody at week 52 of treatment. The type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO:

6. A method for determining that the aforementioned individual is human.

115. A method for treating lupus nephritis in an individual having lupus, comprising intravenously administering to the individual a first antibody exposure, a second antibody exposure, and a third antibody exposure against type II anti-CD20 antibody, The first antibody exposure comprises two administrations of 1000 mg of the type II anti-CD20 antibody during weeks 0 and 2 of treatment. The second antibody exposure comprises two administrations of 1000 mg of the type II anti-CD20 antibody at weeks 24 and 26 of treatment. The third antibody exposure includes two administrations of 1000 mg of the type II anti-CD20 antibody at weeks 50 and 52 of treatment. The type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO:

6. A method for determining that the aforementioned individual is human.

116. A method for depleting circulating peripheral B cells in an individual, comprising intravenously administering to the individual a first antibody exposure, a second antibody exposure, and a third antibody exposure against type II anti-CD20 antibody, The first antibody exposure comprises two administrations of 1000 mg of the type II anti-CD20 antibody during weeks 0 and 2 of treatment. The second antibody exposure comprises two administrations of 1000 mg of the type II anti-CD20 antibody at weeks 24 and 26 of treatment. The third antibody exposure comprises a single dose of 1000 mg of the type II anti-CD20 antibody at week 52 of treatment. The type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO:

6. A method for determining that the aforementioned individual is human.

117. A method for depleting circulating peripheral B cells in an individual, comprising intravenously administering to the individual a first antibody exposure, a second antibody exposure, and a third antibody exposure against type II anti-CD20 antibody, The first antibody exposure comprises two administrations of 1000 mg of the type II anti-CD20 antibody during weeks 0 and 2 of treatment. The second antibody exposure comprises two administrations of 1000 mg of the type II anti-CD20 antibody at weeks 24 and 26 of treatment. The third antibody exposure includes two administrations of 1000 mg of the type II anti-CD20 antibody at weeks 50 and 52 of treatment. The type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO:

6. A method for determining that the aforementioned individual is human.

118. The method according to any one of claims 114 to 117, further comprising administering mycophenolate mofetil to the individual.

119. The method according to claim 118, wherein mycophenolate mofetil is administered to the individual at a dose of 1500 mg / day on the first day of treatment.

120. The method according to claim 118 or claim 119, wherein mycophenolate mofetil is administered to the individual at a dose of 1500 mg / day on the first day of treatment, and the dose is gradually increased to 2.0 g / day to 2.5 g / day at a rate of 500 mg / week by the fourth week of treatment.

121. The method according to any one of claims 114 to 120, further comprising administering oral prednisone to the individual.

122. The method according to claim 121, wherein oral prednisone is administered to the individual at a dose of 0.5 mg / kg / day on the second day of treatment.

123. The method according to claim 122, wherein oral prednisone is administered to the individual at a dose of 0.5 mg / kg / day on the second day until the second week, and then gradually reduced to a dose of 5 mg / day until the 24th week of treatment.

124. The method according to any one of claims 114 to 123, further comprising administering methylprednisolone to the individual by intravenous (IV) infusion at weeks 0, 2, 24, and 52 of treatment.

125. The method according to claim 124, further comprising administering methylprednisolone to the individual by intravenous (IV) infusion at the 26th week of treatment.

126. The method according to any one of claims 114 to 125, further comprising orally administering acetaminophen to the individual in an amount of 650 mg to 1000 mg 30 to 60 minutes before one or more doses of the type II anti-CD20 antibody.

127. The method according to any one of claims 114 to 126, further comprising orally administering 50 mg of diphenhydramine to the individual 30 to 60 minutes before one or more doses of the type II anti-CD20 antibody.

128. A kit for treating lupus nephritis in individuals with lupus, (a) A container comprising a type II anti-CD20 antibody, wherein the type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6, (b) A package insert containing instructions for treating lupus nephritis in an individual, wherein the instructions indicate that a first antibody exposure to the type II anti-CD20 antibody, a second antibody exposure to the type II anti-CD20 antibody, and a third antibody exposure to the type II anti-CD20 antibody are administered to the individual, and the second antibody exposure is not administered from approximately 18 weeks to approximately 26 weeks after the first antibody exposure, and the third antibody exposure is not administered from approximately 24 weeks to approximately 32 weeks after the second antibody exposure, Equipped with, The first antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The second antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. A kit wherein the third antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the third antibody exposure comprises a total exposure of approximately 800 mg to approximately 1200 mg of the type II anti-CD20 antibody.

129. (c) A second pharmaceutical product wherein the type II anti-CD20 antibody is the first pharmaceutical product, and (d) Instructions for the package insert for administering the second drug to the subject. The kit according to claim 128, further comprising a container having the following:

130. The kit according to claim 128 or claim 129, wherein the third antibody exposure comprises a single dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody.

131. The kit according to claim 129 or claim 130, wherein the second pharmaceutical product is an immunosuppressant, a glucocorticoid, a corticosteroid, an antimalarial agent, a cytotoxic agent, an integrin antagonist, a cytokine antagonist, or a hormone.

132. A type II anti-CD20 antibody for use in a method for treating lupus nephritis in an individual, wherein the method comprises administering to the individual a first antibody exposure to the type II anti-CD20 antibody, a second antibody exposure to the type II anti-CD20 antibody, and a third antibody exposure to the type II anti-CD20 antibody, The second antibody exposure was not administered from approximately 18 weeks to approximately 26 weeks after the first antibody exposure. The third antibody exposure was not administered from approximately 24 weeks to approximately 32 weeks after the second antibody exposure. The first antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The second antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The third antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the third antibody exposure comprises a total exposure of approximately 800 mg to approximately 1200 mg of the type II anti-CD20 antibody. The type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO:

6.

133. A type II anti-CD20 antibody for use in a method for depleting circulating peripheral B cells in an individual, wherein the method comprises administering to the individual a first antibody exposure to the type II anti-CD20 antibody, a second antibody exposure to the type II anti-CD20 antibody, and a third antibody exposure to the type II anti-CD20 antibody, The second antibody exposure was not administered from approximately 18 weeks to approximately 26 weeks after the first antibody exposure. The third antibody exposure was not administered from approximately 24 weeks to approximately 32 weeks after the second antibody exposure. The first antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The second antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The third antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the third antibody exposure comprises a total exposure of approximately 800 mg to approximately 1200 mg of the type II anti-CD20 antibody. The type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO:

6. A type II anti-CD20 antibody that, after administration, depletes B cells to a level where circulating peripheral B cells are present in the peripheral blood of the individual at a concentration of approximately 5 cells / μL or less.

134. A type II anti-CD20 antibody for use in a method for depleting circulating peripheral B cells in an individual, comprising administering to the individual a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody, The second antibody exposure was not administered from approximately 18 weeks to approximately 26 weeks after the first antibody exposure. The first antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The second antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO:

6. A type II anti-CD20 antibody, wherein, after administration, B cells are depleted to a level where circulating peripheral B cells are present in the peripheral blood of the individual at a concentration of approximately 5 cells / μL or less, and this state persists for at least 52 weeks.

135. A type II anti-CD20 antibody for use in the method according to any one of claims 1 to 127.

136. A method for treating membranous nephropathy (MN), comprising administering a first antibody exposure to type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody to an individual in need thereof, The second antibody exposure was not administered from approximately 18 weeks to approximately 26 weeks after the first antibody exposure. The first antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The second antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. A method comprising a type II anti-CD20 antibody comprising a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO:

6.

137. The method according to claim 136, wherein the first antibody exposure comprises a first dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody.

138. The method according to claim 136 or claim 137, wherein the first antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the first antibody exposure of the second dose is not administered from about 1.5 weeks to about 2.5 weeks after the first antibody exposure of the first dose.

139. The method according to claim 137, wherein the first antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the first antibody exposure of the second dose is not administered until approximately two weeks after the first antibody exposure of the first dose.

140. The method according to any one of claims 136 to 139, wherein the first dose of the first antibody exposure is about 1000 mg of the type II anti-CD20 antibody.

141. The method according to any one of claims 136 to 140, wherein the second dose of the first antibody exposure is about 1000 mg of the type II anti-CD20 antibody.

142. The method according to any one of claims 136 to 141, wherein the second antibody exposure comprises a first dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody and a second dose of approximately 900 mg to approximately 1100 mg of the type II anti-CD20 antibody.

143. The method according to any one of claims 136 to 142, wherein the second antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the second dose of the second antibody exposure is not given from about 1.5 weeks to about 2.5 weeks after the first dose of the second antibody exposure.

144. The method according to claim 143, wherein the second antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the second dose of the second antibody exposure is not administered until approximately two weeks after the first dose of the second antibody exposure.

145. The method according to any one of claims 142 to 144, wherein the first dose of the second antibody exposure is about 1000 mg of the type II anti-CD20 antibody.

146. The method according to any one of claims 142 to 145, wherein the second dose of the second antibody exposure is approximately 1,000 mg of the type II anti-CD20 antibody.

147. The method according to any one of claims 136 to 146, wherein the individual has primary membranous nephropathy (pMN).

148. The method according to any one of claims 136 to 147, further comprising administering an effective amount of glucocorticoid or corticosteroid to the individual.

149. The method according to claim 148, wherein the glucocorticoid or corticosteroid comprises methylprednisolone.

150. The method according to claim 149, wherein 80 mg of methylprednisolone is administered intravenously to the individual 30 to 60 minutes before one or more doses of the type II anti-CD20 antibody.

151. The method according to any one of claims 136 to 150, further comprising administering an effective amount of an antihistamine to the individual.

152. The method according to claim 151, wherein the antihistamine comprises diphenhydramine.

153. The method according to claim 152, wherein 50 mg of diphenhydramine is orally administered to the individual 30 to 60 minutes before one or more doses of the type II anti-CD20 antibody.

154. The method according to any one of claims 136 to 153, further comprising administering an effective amount of a nonsteroidal anti-inflammatory drug (NSAID) to the individual.

155. The method according to claim 154, wherein the NSAID comprises acetaminophen.

156. The method according to claim 155, wherein 650 to 1000 mg of acetaminophen is orally administered to the individual 30 to 60 minutes before one or more doses of the type II anti-CD20 antibody.

157. The method according to any one of claims 136 to 156, which produces a complete response (CR) in the aforementioned individual.

158. The method according to any one of claims 136 to 156, which produces a partial response (PR) in the aforementioned individual.

159. The method according to any one of claims 136 to 158, which causes depletion of circulating peripheral B cells in the said individual.

160. The method according to claim 159, wherein the circulating peripheral B cells are CD19+ B cells.

161. The method according to any one of claims 136 to 160, wherein, after administration of the type II anti-CD20 antibody, B cells are depleted to a level in which circulating peripheral B cells are present in the peripheral blood of the individual at a concentration of approximately 5 cells / μL or less.

162. The method according to claim 161, wherein B cells are depleted to a level in which circulating peripheral B cells are present in the peripheral blood of the individual at a concentration of approximately 1 cell / μL or less or approximately 0.5 cells / μL or less.

163. The method according to any one of claims 159 to 162, wherein the depletion is achieved after the first antibody exposure.

164. The method according to any one of claims 159 to 163, wherein B cell depletion is sustained for at least 52 weeks after exposure to the first dose of the first antibody.

165. The method according to any one of claims 136 to 164, wherein, after administration of the type II anti-CD20 antibody, the circulating peripheral B cells in the individual are depleted by at least about 90% compared to a corresponding measurement in the same individual before administration of the type II anti-CD20 antibody, or compared to a corresponding measurement in an individual not treated with the type II anti-CD20 antibody.

166. The method according to any one of claims 136 to 165, wherein the individual is a human.

167. A method for treating primary membranous nephropathy (pMN) in an individual, comprising intravenously administering to the individual a first antibody exposure to type II anti-CD20 antibody and a second antibody exposure to type II antibody, The first antibody exposure comprises two administrations of 1000 mg of the type II anti-CD20 antibody during weeks 0 and 2 of treatment. The second antibody exposure comprises two administrations of 1000 mg of the type II anti-CD20 antibody at weeks 24 and 26 of treatment. The type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO:

6. A method for determining that the aforementioned individual is human.

168. A kit for treating primary membranous nephropathy (pMN) in an individual, (a) A container comprising a type II anti-CD20 antibody, wherein the type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6, (b) A package insert containing instructions for treating pMN in an individual, wherein the instructions indicate that a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody are administered to the individual, and the second antibody exposure is not given from about 18 weeks to about 26 weeks after the first antibody exposure, The first antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. A kit wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody.

169. A type II anti-CD20 antibody for use in a method for treating primary membranous nephropathy (pMN) in an individual, comprising administering to the individual a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody, The second antibody exposure was not administered from approximately 18 weeks to approximately 26 weeks after the first antibody exposure. The first antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The second antibody exposure comprises one or two administrations of the type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of approximately 1800 mg to approximately 2200 mg of the type II anti-CD20 antibody. The type II anti-CD20 antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain containing the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO:

6.

170. A type II anti-CD20 antibody for use in the method according to any one of claims 136 to 167.

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