Compositions and methods of treating lupus nephritis

Abstract

To provide methods for treating or delaying progression of lupus nephritis in an individual that has lupus.SOLUTION: In some embodiments, the methods comprise administering to the individual an effective amount of a type II anti-CD20 antibody. The invention also provides methods for treating or delaying progression of rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) in an individual. In some embodiments, the methods comprise administering an effective amount of an anti-CD20 antibody.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 159,876, filed May 11, 2015, and U.S. Provisional Application No. 62 / 300,052, filed Feb. 25, 2016, each of which is hereby incorporated by reference in its entirety.

[0002] Submission of Sequence Listing in ASCII Text File The content of the following submission in ASCII text file is hereby incorporated by reference in its entirety: Sequence Listing in Computer - Readable Format (CRF) (filename: 146392032240SeqList.txt, date of record: May 5, 2016, size: 37 KB).

[0003] Provided herein are methods for treating or delaying the progression of lupus nephritis in an individual having lupus by administering a type II anti - CD20 antibody. Also provided herein are methods for treating or delaying the progression of rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) in an individual by administering an anti - CD20 antibody.

Background Art

[0004] Lupus is an autoimmune disease involving antibodies that attack connective tissue. This disease is estimated to affect nearly one million Americans, mainly women between the ages of 20 and 40. The main form of lupus is the systemic one (systemic lupus erythematosus, SLE). SLE has an incidence of about 1 in 700 women between the ages of 20 and 60. SLE can affect any organ system and can cause severe tissue damage. If lupus is not treated, it can progress from attacks on the skin and joints to attacks on internal organs including the lungs, heart, and kidneys, resulting in a kidney disease called lupus nephritis (LN), which is a major concern and can be fatal. Lupus mainly appears as a series of relapses with few or no disease symptoms in between.

[0005] LN is one of the most acute areas of damage related to the pathogenicity of SLE and is responsible for at least 50% of the mortality and morbidity of this disease. Currently, there is no curative treatment for patients diagnosed with SLE or LN. From a practical perspective, physicians generally use high-dose corticosteroids, such as prednisone, or some powerful immunosuppressive drugs such as azathioprine or cyclophosphamide, which are given during relapse periods and may be given continuously to those with frequent relapses. Even though these are effective treatments for reducing symptoms and extending lifespan, many of these drugs have potentially harmful side effects for the patients being treated. Therefore, there is still a need for more effective treatments for LN with fewer harmful side effects.

[0006] Two anti-CD20 antibodies have been tested in clinical studies for their efficacy in the treatment of lupus nephritis. Rituximab, a type I anti-CD20 antibody, did not meet the primary endpoint of overall response (weighted towards complete renal response, or CRR), but resulted in a 15.3% increase in partial renal response (PRR) (Rovin, B. H. et al. (2012) Arthritis Rheum. 64:1215 - 1226). Ocrelizumab, another type I anti-CD20 antibody, was partially terminated due to disproportionate serious infectious events (Mysler, E. F. et al. (2013) Arthritis Rheum. 65:2368 - 2379).

[0007] Obinutuzumab, a type II anti-CD20 antibody, showed superior B cell depletion compared to rituximab. Significantly more B cell depletion was observed with obinutuzumab treatment compared to rituximab treatment in cynomolgus monkeys (Mossner, E. et al. (2010) Blood 115:4393 - 4402). Therefore, it remains necessary to test the efficacy of type II anti-CD20 antibodies in the treatment or prevention of LN in patients with lupus.

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

Summary of the Invention

[0009] In certain embodiments, provided herein is a method for treating lupus nephritis or delaying its progression in an individual, comprising administering to the individual a type II anti-CD20 antibody at at least a first antibody exposure amount and a type II anti-CD20 antibody at a second antibody exposure amount. In some embodiments, the individual has lupus. In some embodiments, the second antibody exposure amount is not provided until about 18 to about 26 weeks after the first antibody exposure amount. In some embodiments, the second antibody exposure amount is not provided until about 4.5 to about 6.5 months after the first antibody exposure amount. In some embodiments, the first antibody exposure amount comprises one or two doses of a type II anti-CD20 antibody, and the first antibody exposure amount comprises a total exposure amount of about 1800 mg to about 2200 mg of the type II anti-CD20 antibody. In some embodiments, the second antibody exposure amount comprises one or two doses of a type II anti-CD20 antibody, and the second antibody exposure amount comprises a total exposure amount of about 1800 mg to about 2200 mg of the type II anti-CD20 antibody. In some embodiments, 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. In some embodiments, the individual is at risk of developing class III or class IV lupus nephritis. In some embodiments, the method relates to preventing lupus nephritis in an individual having lupus. In some embodiments, the method relates to preventing lupus nephritis in an individual having SLE. In some embodiments, the method relates to treating lupus nephritis or delaying its progression in an individual having SLE.

[0010] In some embodiments, the first antibody exposure amount comprises a first dose of a type II anti-CD20 antibody and a second dose of a type II anti-CD20 antibody, and the second dose of the first antibody exposure amount is not provided until about 1.5 weeks to about 2.5 weeks after the first dose of the first antibody exposure amount. In some embodiments, the first antibody exposure amount comprises a first dose of a type II anti-CD20 antibody and a second dose of a type II anti-CD20 antibody, and the second dose of the first antibody exposure amount is not provided until about 2 weeks after the first dose of the first antibody exposure amount. In some embodiments, the first antibody exposure amount comprises a first dose of a type II anti-CD20 antibody and a second dose of a type II anti-CD20 antibody, and the second dose of the first antibody exposure amount is not provided until about 10 days to about 17 days after the first dose of the first antibody exposure amount. In some embodiments, the first antibody exposure amount comprises a first dose of a type II anti-CD20 antibody and a second dose of a type II anti-CD20 antibody, and the second dose of the first antibody exposure amount is not provided until about 14 days after the first dose of the first antibody exposure amount. In some embodiments, the first dose of the first antibody exposure amount is about 1000 mg of a type II anti-CD20 antibody. In some embodiments, the second dose of the first antibody exposure amount is about 1000 mg of a type II anti-CD20 antibody. In some embodiments, the second antibody exposure amount comprises a first dose of about 900 mg to about 1100 mg of a type II anti-CD20 antibody and a second dose of about 900 mg to about 1100 mg of a type II anti-CD20 antibody. In some embodiments, the second antibody exposure amount comprises a first dose of a type II anti-CD20 antibody and a second dose of a type II anti-CD20 antibody, and the second dose of the second antibody exposure amount is not provided until about 1.5 weeks to about 2.5 weeks after the first dose of the second antibody exposure amount. In some embodiments, the second antibody exposure amount comprises a first dose of a type II anti-CD20 antibody and a second dose of a type II anti-CD20 antibody, and the second dose of the second antibody exposure amount is not provided until about 2 weeks after the first dose of the second antibody exposure amount. In some embodiments, the second antibody exposure amount comprises a first dose of a type II anti-CD20 antibody and a second dose of a type II anti-CD20 antibody, and the second dose of the second antibody exposure amount is not provided until about 10 days to about 17 days after the first dose of the second antibody exposure amount.In some embodiments, the second antibody exposure comprises a first dose of a type II anti-CD20 antibody and a second dose of a type II anti-CD20 antibody, and the second dose of the second antibody exposure is not provided until about 14 days after the first dose of the second antibody exposure. In some embodiments, the first dose of the second antibody exposure is about 1000 mg of a type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure is about 1000 mg of a type II anti-CD20 antibody. In some embodiments, the first antibody exposure and the second antibody exposure are administered intravenously. 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.

[0011] In certain aspects, provided herein is a method for treating or delaying the progression of lupus nephritis in an individual having lupus, comprising administering to the individual an effective amount of 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 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 method relates to preventing lupus nephritis in an individual having lupus. In some embodiments, the method relates to preventing lupus nephritis in an individual having SLE. In some embodiments, the method relates to treating or delaying the progression of lupus nephritis in an individual having SLE.

[0012] In certain embodiments, provided herein are methods for treating or delaying the progression of lupus nephritis in an individual having lupus, comprising administering to the individual a dose of about 1000 mg of 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 wherein the dose is administered to the individual once on days 1, 15, 168, and 182. In certain embodiments, provided herein are methods for treating or delaying the progression of lupus nephritis in an individual having lupus, comprising administering to the individual a dose of about 1000 mg of 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 wherein the dose is administered to the individual once at weeks 0, 2, 24, and 26. In some embodiments, week 0 corresponds to day 1. In some embodiments, the individual has class III or class IV lupus nephritis. In some embodiments, the type II anti-CD20 antibody is obinutuzumab.

[0013] In some embodiments of any of the above-described embodiments, the type II anti-CD20 antibody is administered intravenously. In some embodiments of any of the above-described embodiments, the individual does not have class III (C) or class IV (C) lupus nephritis. In some embodiments of any of the above-described embodiments, the individual has class V lupus nephritis. In some embodiments of any of the above-described embodiments, the method further comprises administering to the individual an effective amount of an immunosuppressive agent. In some embodiments, the immunosuppressive agent comprises mycophenolic acid, a derivative thereof, or a salt thereof. In some embodiments, the immunosuppressive agent comprises mycophenolate mofetil. In some embodiments of any of the above-described embodiments, the method further comprises administering to the individual an effective amount of a glucocorticoid or corticosteroid. In some embodiments, the glucocorticoid or corticosteroid comprises methylprednisolone. In some embodiments, the glucocorticoid or corticosteroid comprises prednisone. In some embodiments of any of the above-described embodiments, the method further comprises administering to the individual an effective amount of an antihistamine. In some embodiments, the antihistamine comprises diphenhydramine. In some embodiments of any of the above-described embodiments, the method further comprises administering to the individual an effective amount of a non-steroidal anti-inflammatory drug (NSAID). In some embodiments, the NSAID comprises acetaminophen. In some embodiments of any of the above-described embodiments, the method further comprises administering standard care treatment to the individual. In some embodiments, the standard care treatment comprises treatment with one or more of an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin receptor blocker, cyclophosphamide, mycophenolate mofetil, azathioprine, and a glucocorticoid or corticosteroid. In some embodiments, the standard care treatment is administered after the first antibody exposure to the type II anti-CD20 antibody and / or after the second antibody exposure to the type II anti-CD20 antibody. In some embodiments of any of the above-described embodiments, the method further comprises administering to the individual an effective amount of an antihypertensive agent.In some embodiments, the antihypertensive agent is an angiotensin-converting enzyme (ACE) inhibitor or an angiotensin receptor blocker. In some embodiments of any of the above embodiments, the method results in a complete renal response (CRR) in an individual. In some embodiments of any of the above embodiments, the method results in a reduction of circulating peripheral B cells in an individual. In some embodiments, the circulating peripheral B cells are CD19+ B cells. In some embodiments of any of the above embodiments, the type II anti-CD20 antibody is a humanized or human antibody. In some embodiments of any of the above embodiments, the type II anti-CD20 antibody is afucosylated. In some embodiments of any of the above embodiments, the type II anti-CD20 antibody is non-fucosylated (e.g., as described in U.S. Patent No. 8,883,980). In some embodiments of any of the above embodiments, the heavy chain of the type II anti-CD20 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments of any of the above embodiments, the light chain of the type II anti-CD20 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments of any of the above embodiments, the type II anti-CD20 antibody is obinutuzumab. In some embodiments of any of the above embodiments, the individual or patient is human.

[0014] In certain embodiments, a kit or product for treating lupus nephritis in an individual having lupus or delaying its progression is provided herein, comprising: (a) 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 (b) a package insert comprising instructions for use regarding treating lupus nephritis in an individual or delaying its progression, wherein the instructions for use indicate that at least a first antibody exposure amount of the type II anti-CD20 antibody and a second antibody exposure amount of the type II anti-CD20 antibody are administered to the individual, and the second antibody exposure amount is not provided until about 18 to about 26 weeks after the first antibody exposure amount, wherein the first antibody exposure amount comprises one or two doses of the type II anti-CD20 antibody, the first antibody exposure amount comprises a total exposure amount of about 1800 mg to about 2200 mg of the type II anti-CD20 antibody, the second antibody exposure amount comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure amount comprises a total exposure amount of about 1800 mg to about 2200 mg of the type II anti-CD20 antibody. In some embodiments, the kit or product further comprises: (c) a second drug, wherein the type II anti-CD20 antibody is the first drug; and (d) package insert instructions for use regarding administering the second drug to a subject. In some embodiments, the second drug is an immunosuppressant, glucocorticoid, corticosteroid, anti-malarial agent, cytotoxic agent, integrin antagonist, cytokine antagonist, or hormone. In some embodiments, the heavy chain of the type II anti-CD20 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7. In some embodiments, the light chain of the type II anti-CD20 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the type II anti-CD20 antibody is obinutuzumab. In some embodiments, the kit or product relates to preventing lupus nephritis in an individual having SLE.In some embodiments, the kit or product relates to treating or delaying the progression of lupus nephritis in an individual having SLE.

[0015] In certain aspects, provided herein is a method for treating or delaying the progression of rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) in an individual, comprising administering to the individual an effective amount of an anti-CD20 antibody, wherein the antibody comprises a heavy chain variable region 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 variable region 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. In some embodiments, the antibody is administered intravenously. In some embodiments, the method results in depletion of peripheral blood circulating B cells in the individual. In some embodiments, the peripheral blood circulating B cells are CD19+ B cells. In some embodiments, the antibody is a humanized antibody or a human antibody. In some embodiments, the antibody is afucosylated. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody is obinutuzumab. In some embodiments, the antibody comprises a modified Fc region. In some embodiments, the Fc region comprises modifications for attenuating effector function. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the human IgG1 Fc region is numbered according to the EU index and comprises the L234A, L235A, and P329G amino acid substitutions. In some embodiments of any of the above embodiments, the individual or patient is human.

[0016] In certain embodiments, provided herein is a composition for use in treating or delaying the progression of rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) in an individual, the composition comprising an anti-CD20 antibody, wherein the antibody comprises a heavy chain variable region 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 variable region 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. In some embodiments, the composition is administered intravenously. In some embodiments, administering the composition results in depletion of peripheral blood B cells in the individual. In some embodiments, the peripheral blood B cells are CD19+ B cells. In some embodiments, the antibody is a humanized antibody or a human antibody. In some embodiments, the antibody is afucosylated. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody is obinutuzumab. In some embodiments, the antibody comprises a modified Fc region. In some embodiments, the Fc region comprises modifications for attenuating effector function. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the human IgG1 Fc region is numbered according to the EU index and comprises L234A, L235A, and P329G amino acid substitutions.

[0017] Provided herein is the use of an anti-CD20 antibody for manufacturing a medicament for use in the treatment of rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) in an individual, wherein the antibody comprises a heavy chain variable region 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 variable region 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.

[0018] In certain embodiments, a kit or product for treating rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) in an individual or delaying the progression thereof is provided herein, comprising: (a) a container containing an anti-CD20 antibody, wherein the antibody comprises a heavy chain variable region 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 variable region 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 (b) a package insert comprising instructions for administering an effective amount of the anti-CD20 antibody to treat or delay the progression of RA or SLE in an individual. In some embodiments, the package insert comprises instructions for intravenous administration of the antibody. In some embodiments, the antibody is a humanized antibody or a human antibody. In some embodiments, the antibody is afucosylated. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody is obinutuzumab. In some embodiments, the antibody comprises a modified Fc region. In some embodiments, the Fc region comprises modifications for attenuating effector function. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the human IgG1 Fc region is numbered according to the EU index and comprises L234A, L235A, and P329G amino acid substitutions.

[0019] It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of the invention. These and other aspects of the invention will be apparent to those of ordinary skill in the art. These and other embodiments of the invention are further described in the following mode for carrying out the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020]

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Mode for Carrying Out the Invention

[0021] In one aspect, provided herein is a method for treating lupus nephritis or delaying its progression in an individual, comprising administering to the individual at least a type II anti-CD20 antibody with a first antibody exposure amount and a type II anti-CD20 antibody with a second antibody exposure amount. In some embodiments, the individual has lupus. In some embodiments, the second antibody exposure amount is not provided until about 18 to about 26 weeks after the first antibody exposure amount. In some embodiments, the first antibody exposure amount comprises one or two doses of the type II anti-CD20 antibody, and the first antibody exposure amount contains a total exposure amount of about 1800 mg to about 2200 mg of the type II anti-CD20 antibody. In some embodiments, the second antibody exposure amount comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure amount contains a total exposure amount of about 1800 mg to about 2200 mg of the type II anti-CD20 antibody. In some embodiments, the 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.

[0022] In another aspect, provided herein is a method for treating lupus nephritis or delaying its progression in an individual having lupus, comprising administering to the individual an effective amount of a 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. In some embodiments, the individual has class III or class IV lupus nephritis.

[0023] In another aspect, provided herein is a method for treating or delaying the progression of rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) in an individual, comprising administering to the individual an effective amount of an anti-CD20 antibody. In some embodiments, the antibody comprises a heavy chain variable region 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 variable region 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.

[0024] I. General Techniques The techniques and procedures described or referenced in this specification are generally well understood by those skilled in the art and are conventional methodologies, such as Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)), the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)), Oligonucleotide Synthesis (M.J. Gait, ed., 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press, Animal Cell Culture (R.I. Freshney), ed., 1987), Introduction to Cell and Tissue Culture (J.P. Mather and P.E.R. Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993 - 8) J.Wiley and Sons, Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.), Gene Transfer Vectors for Mammalian Cells (J.M.It is commonly used by those skilled in the art using a wide range of methodologies described in Miller and M.P.Calos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (J.E.Coligan et al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (C.A.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 J.D.Capra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (V.T.DeVita et al., eds., J.B.Lippincott Company, 1993), etc.

[0025] II. Definitions The term "lupus nephritis (LN)" refers to the signs of lupus (e.g., systemic lupus erythematosus, drug-induced lupus, neonatal lupus, or discoid lupus) in the kidney(s).

[0026] The term "antibody" includes monoclonal antibodies (including full-length antibodies having the immunoglobulin Fc region), antibody compositions having polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab')2, and Fv). The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody".

[0027] The basic four-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of five basic heterotetrameric units along 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 is generally 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 each also 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 and a constant domain at the other 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. Specific 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 LBy pairing them together, a single antigen-binding site is formed. For the structures and properties of different 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 from any vertebrate species can be assigned to one of two distinct types called kappa and lambda based on the amino acid sequences of their constant domains. Depending on the amino acid sequences of their heavy chain constant domains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequences and functions. For example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0028] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains can be referred to as "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.

[0029] The term "variable" refers to the fact that certain segments of the variable domains vary extensively in sequence among antibodies. The V domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domain. Rather, it is concentrated in three segments called hypervariable regions (HVRs) in both the variable domains of the light and heavy chains. The more highly conserved portions of the variable domains are called framework regions (FRs). The variable domains of the native heavy and light chains each adopt a largely beta-sheet conformation connected by three HVRs that also form loops connecting some of the beta-sheet structures and, in some cases, forming part of the beta-sheet structure, and contain four FR regions. The HVRs within each chain are held together in close proximity by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains do not directly participate in binding of the antibody to antigen but exhibit various effector functions such as the involvement of the antibody in antibody-dependent cell cytotoxicity.

[0030] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific and target 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 the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are free of contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be obtained, for example, 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 nd(ed. 1988), Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567), phage display techniques (see, for example, Clackson et 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., 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), and techniques for producing human or human-like antibodies in animals having a part or all of the human immunoglobulin locus or a gene encoding a human immunoglobulin sequence (see, for example, WO1998 / 24893, WO1996 / 34096, WO1996 / 33735, WO1991 / 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., Nature Biotechnol.See, e.g., 14:845-851(1996), Neuberger, Nature Biotechnol. 14:826(1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93(1995), and can be made by a variety of techniques.

[0031] The term "naked antibody" refers to an antibody that is not conjugated to a cytotoxic moiety or a radioactive label.

[0032] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as contrasted with an antibody fragment. Specifically, whole antibodies include those having heavy and light chains containing an Fc region. The constant domains may be the constant domains of the native sequence (e.g., the constant domains of the human native sequence) or amino acid sequence variants thereof. In some cases, intact antibodies may have one or more effector functions.

[0033] An "antibody fragment" contains a portion of an intact antibody, preferably the antigen-binding region and / or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab´, F(ab´)2, and Fv fragments; diabodies; linear antibodies (see, e.g., U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, and the remaining "Fc" fragment (so-called because it is easily crystallizable). A Fab fragment consists of the entire L chain and the variable domain of the H chain (V H ), as well as the first constant domain of one heavy chain (C HIt consists of (1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab’)2 fragment, which approximately corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and can still crosslink antigens. The Fab’ fragment differs from the Fab fragment by having several additional residues containing one or more cysteines from the antibody hinge region at the carboxy terminus of the C H 1 domain. Fab´-SH is the name in this specification for a Fab´ in which the cysteine residue(s) of the constant domain have free thiol groups. F(ab´)2 antibody fragments were originally produced as pairs of Fab´ fragments having hinge cysteines in between. Other chemical couplings of antibody fragments are also known.

[0034] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of an antibody are determined by the sequences in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types.

[0035] "Fv" is the smallest antibody fragment that contains a complete antigen recognition site and antigen-binding site. This fragment consists of a dimer in which one heavy-chain variable region domain and one light-chain variable region domain are tightly bound non-covalently. The folding of these two domains gives rise to six hypervariable loops (three loops each from the H chain and the L chain), which provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only three HVRs specific for the antigen), although having a lower affinity than the whole binding site, has the ability to recognize and bind the antigen.

[0036] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment containing the V H and V L antibody domains connected to form a single polypeptide chain. Preferably, the sFv polypeptide has a VH and V L It further includes a polypeptide linker between the domain and V. For an overview of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0037] The "functional fragment" of the antibody of the present invention includes a part of the intact antibody, which generally includes the antigen-binding or variable region of the intact antibody, or the Fc region of the antibody that retains or has modified FcR-binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0038] The term "diabody" refers to a small antibody fragment prepared by constructing an sFv fragment (see the previous paragraph) using a short linker (about 5-10 residues) between the V H and V L domains to achieve inter-chain rather than intra-chain V domain pairing, thereby obtaining a bivalent fragment, i.e., a fragment having two antigen-binding sites. The diabody is a heterodimer of two "crossover" sFv fragments in which the V H and V L domains are present on different polypeptide chains. Diabodies are described in more detail, for example, in EP404,097, WO93 / 11161, Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

[0039] As used herein, monoclonal antibodies include “chimeric” antibodies (immunoglobulins) in which portions of the heavy and / or light chains are the same or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is derived from a different species or belongs to a different antibody class or subclass, and the corresponding sequences in fragments of such antibodies, provided that 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)). Chimeric antibodies of interest herein include PRIMATIZED™ antibodies, where the antigen-binding region of the antibody is derived from an antibody produced, for example, by immunizing macaques with the antigen of interest. As used herein, “humanized antibody” is used as a subset of “chimeric antibody”.

[0040] The "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains a minimal sequence derived from a non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's HVRs (defined below) have been replaced by residues from the HVRs of a non-human species such as mouse, rat, rabbit, or non-human primate (donor antibody) that have the desired specificity, affinity, and / or activity. In some instances, framework ("FR") residues of the human immunoglobulin are replaced by the corresponding non-human residues. Furthermore, a humanized antibody may contain residues that are not found in either the recipient antibody or the donor antibody. These modifications can be made to further improve the properties of the antibody, such as binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin sequence and all or substantially all of the FR regions correspond to those of the human immunoglobulin sequence, but the FR regions may include one or more individual FR residue substitutions that improve the properties of the antibody, such as binding affinity, isomerization, and immunogenicity. The number of these amino acid substitutions in the FRs is typically six or fewer for the H chain and three or fewer for the L chain. A humanized antibody optionally will also include the immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For further details, see, e.g., 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).See, for example, 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.

[0041] "Human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or an antibody produced using any of the techniques for producing a human antibody as disclosed herein. This definition of human antibody clearly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including 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, for example, by administering an antigen to a transgenic animal whose endogenous locus has been inactivated but which has been modified to produce such antibodies in response to antigen challenge, such as an immunized xenomouse (see, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) for human antibodies produced by human B cell hybridoma technology.

[0042] As used herein, the terms "hypervariable region", "HVR", or "HV" refer to the region of the antibody variable domain where the sequences are hypervariable and / or form structurally defined loops. Generally, an antibody contains six HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In native antibodies, H3 and L3 exhibit the greatest diversity among the six HVRs and H3 in particular is thought to play a unique role in conferring excellent specificity to the antibody. See, e.g., 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 fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993), Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0043] Several HVR descriptions are used herein and are included 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)). By contrast, Chothia refers to the positions of the structural loops (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 analysis of available complex crystal structures. The residues of each of these HVRs are described below. TIFF2025106262000002.tif98170

[0044] The HVR may include the following "extended HVR": 24 - 36 or 24 - 34 (L1), 46 - 56 or 50 - 56 (L2), and 89 - 97 or 89 - 96 (L3) in VL, and 26 - 35 (H1), 50 - 65 or 49 - 65 (H2), and 93 - 102, 94 - 102, or 95 - 102 (H3) in VH. The variable domain residues are numbered according to Kabat et al. (cited above) for each of these definitions.

[0045] The expressions "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat", and variations thereof, refer to the numbering system used for the heavy - chain variable domain or the light - chain variable domain in the construction of antibodies in Kabat et al. (cited above). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to deletions or insertions in the FR or HVR of the variable domain. For example, the heavy - chain variable domain may contain a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2, and residues inserted after heavy - chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat numbering of residues can be determined for a given antibody by alignment in the homologous regions between the antibody's sequence and the sequence numbered by "standard" Kabat.

[0046] "Framework" or "FR" residues are variable domain residues other than HVR residues as defined herein.

[0047] The "human consensus framework" or "acceptor human framework" is a framework that represents the amino acid residues that most commonly occur in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Generally, the subset of sequences is a subset such as that in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples, which include those for VL, the subset can be subset kappa I, kappa II, kappa III, or kappa IV as in Kabat et al. (supra). In addition, for VH, the subset can be subset I, subset II, or subset III as in Kabat et al. (supra). Alternatively, the human consensus framework can be derived from the above, such as when specific residues, e.g., human framework residues, are selected based on their homology to a donor framework sequence by aligning the donor framework sequence with a collection of various human framework sequences. The 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 variations. 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.

[0048] The "VH subgroup III consensus framework" includes a consensus sequence obtained from the amino acid sequences in the variable heavy chain subgroup III of Kabat et al. (referenced above). In one embodiment, the VH subgroup III consensus framework amino acid sequence includes at least a portion 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).

[0049] The "VL kappa I consensus framework" includes a consensus sequence obtained from the amino acid sequences in the variable light chain kappa subgroup I of Kabat et al. (referenced above). In one embodiment, the VH subgroup I consensus framework amino acid sequence includes at least a portion 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), FGQGTKVEIKR (LC-FR4) (SEQ ID NO: 42).

[0050] For example, "amino acid modification" at a defined position in the Fc region refers to substitution or deletion of a defined residue, or insertion of at least one amino acid residue adjacent to the defined residue. "Adjacent" insertion to a defined residue means insertion within one or two residues thereof. The insertion can be on the N-terminal side or the C-terminal side of the defined residue. Preferred amino acid modifications herein are substitutions.

[0051] An "affinity matured" antibody has one or more modifications in one or more of its HVRS, which result in an improvement in the affinity of the antibody for antigen as compared to the parental antibody that does not possess those modification(s). In one embodiment, the affinity matured antibody has a 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) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of HVRs and / or framework residues are 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).

[0052] The terms "specifically binds to" or "specific for", as used herein, refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the 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) binds to this target with higher affinity, binding strength, more readily, and / or for a longer duration than it binds to other targets. In one embodiment, the degree to which the antibody binds to an irrelevant target is, for example, less than about 10% of the antibody's binding to the target, as measured by a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved between proteins from different species. In another embodiment, specific binding may, but does not necessarily, include exclusive binding.

[0053] The term "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain and includes both native sequence Fc regions and variant Fc regions. The boundaries of the Fc region of an immunoglobulin heavy chain can vary, but the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification or by recombinant manipulation of the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include a population of antibodies in which all K447 residues have been removed, a population of antibodies without the removed K447 residues, and a population of antibodies that is a mixture of antibodies with and without the K447 residue. Suitable native sequence Fc regions for use in the antibodies of the present invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0054] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. Preferred FcRs are native sequence human FcRs. Further, preferred FcRs are those that bind to IgG antibodies (gamma receptors), and include receptors of the FcγRI, FcγRII, and FcγRIII subclasses (including allelic variants and alternatively spliced forms of these receptors). Among the FcγRII receptors, there are FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences but mainly differ in their cytoplasmic domain. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (M. Daeron, Annu. Rev. Immunol. 15:203 - 234 (1997)). FcRs are 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 included within the term "FcR" herein.

[0055] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus. Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994). Methods for measuring binding to FcRn are known (e.g., 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), WO2004 / 92219 (see Hinton et al.). The in vivo binding and serum half-life of a human FcRn high-affinity binding polypeptide can be assayed, for example, in transgenic mice expressing human FcRn or transfected human cell lines, or in primates administered a polypeptide having a mutated Fc region. WO2004 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcR. See also, e.g., Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0056] The expressions "substantially reduced" or "substantially different", as used herein, represent a sufficiently high degree of difference between two numerical values (generally, one related to a molecule and the other related to a reference / comparison molecule), such that one of ordinary skill in the art would consider the difference between the two values to be statistically significant in the context of the biological characteristic measured by the value (e.g., Kd value). The difference between such 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 of the reference / comparison molecule.

[0057] The terms "substantially similar" or "substantially the same", as used herein, represent a sufficiently high degree of similarity between two numerical values (e.g., one related to an antibody of the invention and the other related to a reference / comparative antibody), such that one of ordinary skill in the art would consider the difference between the two values to be of little or no biological and / or statistical significance in the context of the biological characteristic being measured by the value (e.g., the Kd value). The difference between such two 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% as a function of the reference / comparative value.

[0058] "Carriers", as used herein, include pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to the cells or mammals being exposed thereto at the dosages and concentrations employed. In many cases, the physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphoric acid, citric acid, 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™.

[0059] "Package insert" refers to the instructions customarily included in the commercial package of a drug, including information regarding indications, usage, dosage, administration, contraindications, information about other drugs to be combined with the packaged product, and / or warnings regarding the use of such drug, including information about the indications customarily included in the package insert.

[0060] As used herein, the term "treatment" refers to a clinical intervention designed to modify the natural course of an individual or cell being treated during the course of a clinical pathology. Desirable effects of treatment include slowing the rate of disease progression, restoring or alleviating the disease state, and improving remission or prognosis. For example, an individual has elevated serum creatinine, proteinuria, red blood cell casts, reduced renal function, nephrotic syndrome, granular casts, microscopic hematuria, gross hematuria, hypertension, tubular abnormalities, hyperkalemia, rapidly progressive glomerulonephritis (RPGN), and acute renal failure (ARF) Although included, but not limited to, one or more symptoms associated with lupus nephritis are reduced or eliminated, the "treatment" is successful.

[0061] As used herein, "delaying the progression" of a disease (e.g., lupus nephritis) means delaying, interfering with, slowing down, delaying, stabilizing, and / or extending the onset of the disease. This delay can be for different periods depending on the medical history and / or individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can virtually encompass prevention in that an individual, e.g., an individual at risk of developing the disease, does not develop the disease. For example, the progression of SLE in an individual prior to the onset of LN symptoms and / or pathology can be delayed to delay or prevent the onset of LN.

[0062] "Complete renal response (CRR)" as used herein refers to a response to treatment that includes normalization of serum creatinine, inactive urine sediment, and a urinary protein-to-creatinine ratio of less than 0.5.

[0063] As used herein, "partial renal response (PRR)" refers to a response to treatment that is less than CRR but still includes reduction of one or more symptoms including, but not limited to, reduction of serum creatinine, reduced urine sediment, and reduction of proteinuria.

[0064] "Effective amount" is at least the minimum concentration necessary to achieve a measurable improvement or prevention of a particular disorder. The effective amount herein can vary depending on factors such as the patient's disease state, age, gender, and weight, as well as the ability of the antibody to induce the desired response in the individual. The effective amount is also one in which the therapeutically beneficial effect exceeds any toxic or adverse effects of the treatment. In the case of prophylactic use, beneficial or desired results include elimination or reduction of the risk of disease, reduction in the severity of the disease, or delay in the onset of the disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that appear during the development of the disease. In the case of therapeutic use, beneficial or desired results include clinical outcomes such as reduction of one or more symptoms attributable to the disease, improvement in the quality of life of the affected person, reduction in the dosage of other medications required for treatment of the disease, enhancement of the effect of another medication (e.g., target-mediated), delay in the progression of the disease, and / or extension of the survival period. In the case of lupus nephritis, an effective amount of the drug may have an effect and / or some degree of reduction in one or more of the symptoms associated with the disorder. The effective amount can be administered in one or more doses. For purposes of the present invention, the effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a prophylactic or therapeutic treatment. As understood in the clinical art, the effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, "effective amount" can be considered in terms of administering one or more therapeutic agents, and a single agent, alone or in combination with one or more other agents, can be considered to be administered in an effective amount if the desired result is achieved or can be achieved.

[0065] "CD20", as used herein, refers to human B-lymphocyte antigen CD20 (also known as CD20, B-lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5, the sequence of which is characterized by SwissProt database entry P11836), a hydrophobic transmembrane protein having an approximate molecular weight of 35 kD located on pre-B and mature B lymphocytes. (Valentine, M.A., et al., J. Biol. Chem. 264(19)(1989 11282-11287, Tedder, T.F., et al, Proc. Natl. Acad. Sci. U.S.A. 85(1988)208-12, Stamenkovic, I., et al., J. Exp. Med. 167(1988)1975-80, Einfeld, D.A., et al., EMBO J. 7(1988)711-7, Tedder, T.F., et al., J. Immunol. 142(1989)2560-8). The corresponding human gene is transmembrane 4-domain, subfamily A, member 1, also known as MS4A1. This gene encodes a member of the transmembrane 4A gene family. Members of this family of developmental stage proteins are characterized by common structural features and similar intron / exon splice boundaries and display unique expression patterns in hematopoietic cells and non-lymphoid tissues. This gene encodes a B-lymphocyte surface molecule that plays a role in the development and differentiation of B cells into plasma cells. Members of this family are localized to 11q12 within the group of family members. Alternative splicing of this gene results in two transcript variants that encode the same protein.

[0066] The terms "CD20" and "CD20 antigen" are used interchangeably herein and include any variant form, isoform, and species homolog of human CD20 that is naturally expressed by a cell or expressed on a cell transfected with the CD20 gene. Binding of the antibodies of the present invention to the CD20 antigen mediates the death of cells expressing CD20 (e.g., tumor cells) by inactivating CD20. The death of cells expressing CD20 can occur by one or more of the following mechanisms: induction of cell death / apoptosis, ADCC, and CDC.

[0067] Synonyms of CD20 as recognized in the art include B-lymphocyte antigen CD20, B-lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5.

[0068] The term "anti-CD20 antibody" according to the present invention is an antibody that specifically binds to the CD20 antigen. Two types of anti-CD20 antibodies (type I and type II anti-CD20 antibodies) can be distinguished according to the binding characteristics and biological activities of the anti-CD20 antibodies to the CD20 antigen, according to Cragg, M.S., et al., Blood 103 (2004) 2738-2743, and Cragg, M.S., et al., Blood 101 (2003) 1045-1052. See Table 1 below. TIFF2025106262000003.tif83170

[0069] Examples of type II anti-CD20 antibodies include, for example, humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody as disclosed in WO2005 / 044859), 11B8 IgG1 (as disclosed in WO2004 / 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 (in the case of the IgG1 isotype) compared to type I antibodies of the IgG1 isotype.

[0070] Examples of type I anti-CD20 antibodies include, for example, rituximab, HI47IgG3 (ECACC, hybridoma), 2C6IgG1 (as disclosed in WO2005 / 103081), 2F2IgG1 (as disclosed and in WO2004 / 035607 and WO2005 / 103081), and 2H7IgG1 (as disclosed in WO2004 / 056312).

[0071] The afucosylated anti-CD20 antibody according to the present invention is preferably a type II anti-CD20 antibody as described in WO2005 / 044859 and WO2007 / 031875, more preferably an afucosylated humanized B-Ly1 antibody.

[0072] The "rituximab" antibody (reference antibody, an example of a type I anti-CD20 antibody) is a genetically engineered chimeric human gamma 1 mouse constant domain containing a monoclonal antibody directed against the human CD20 antigen. However, this antibody has not been glycosylated or afucosylated and thus has at least 85% of the amount of fucose. This chimeric antibody contains a human gamma 1 constant domain and is identified by the name "C2B8" in US5,736,137 (Andersen, et.al.), issued on April 17, 1998, and assigned to IDEC Pharmaceuticals Corporation. Rituximab is approved for the treatment of patients with CD20-positive, B-cell non-Hodgkin lymphoma that is low-grade or follicular and is relapsed or refractory. In vitro mechanistic studies have shown that rituximab exhibits human complement-dependent cytotoxicity (CDC) (Reff, M.E., et.al, Blood 83(2)(1994)435-445). In addition, it shows activity in assays measuring antibody-dependent cell-mediated cytotoxicity (ADCC).

[0073] The term "GA101 antibody", as used herein, refers to any one of the following antibodies that bind to human CD20: (1) an antibody comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; (2) an antibody comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 8; (3) an antibody comprising the amino acid sequences of SEQ ID NO: 9 and 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 to 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 to 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.

[0074] The term "humanized B-Ly1 antibody" refers to humanized B-Ly1 antibodies as disclosed in WO2005 / 044859 and WO2007 / 031875, which were obtained from the murine monoclonal anti-CD20 antibody B-Ly1 (variable region of the murine heavy chain (VH): SEQ ID NO: 11; variable region of the murine light chain (VL): SEQ ID NO: 12 - see Poppema, S. and Visser, L., Biotest Bulletin 3 (1987) 131 - 139) by chimerization using human constant domains from IgG1 and subsequent humanization (see WO2005 / 044859 and WO2007 / 031875). Details of these "humanized B-Ly1 antibodies" are disclosed in WO2005 / 044859 and WO2007 / 031875. Variable region of the murine monoclonal anti-CD20 antibody B-Ly1 heavy chain (VH) (SEQ ID NO: 11) TIFF2025106262000004.tif Variable region of the B-Ly1 light chain (VL) (SEQ ID NO: 12) of the mouse monoclonal anti-CD20 antibody, 100170 TIFF2025106262000005.tif 97170

[0075] In one embodiment, the "humanized B-Ly1 antibody" has a variable heavy chain region (VH) selected from the group of SEQ ID NOs: 7, 8, and 13-33 (specifically corresponding to B-HH2-B-HH9 and B-HL8-B-HL17 of WO2005 / 044859 and WO2007 / 031875). In one specific embodiment, such 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 of WO2005 / 044859 and WO2007 / 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 of WO2005 / 044859 and WO2007 / 031875). In one specific embodiment, the "humanized B-Ly1 antibody" has a variable heavy chain region (VH) of SEQ ID NO: 7 (corresponding to B-HH6 of WO2005 / 044859 and WO2007 / 031875), and a variable light chain region (VL) of SEQ ID NO: 8 (corresponding to B-KV1 of WO2005 / 044859 and WO2007 / 031875). Further, in one embodiment, the humanized B-Ly1 antibody is an IgG1 antibody. According to the present invention, such afucosylated humanized B-Ly1 antibody is glycosylated in the Fc region according to the procedures described in WO2005 / 044859, WO2004 / 065540, WO2007 / 031875, Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180, and WO99 / 154342 (GE). In one embodiment, the afucosylated glycosylated humanized B-Ly1 is B-HH6-B-KV1 GE. In one embodiment, the anti-CD20 antibody is obinutuzumab (recommended 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, p. 75 - 76) and was previously known as afutuzumab (recommended INN, WHO Drug Information, vol. 23, No. 2, 2009, p. 176; Vol. 22, No. 2, 2008, p. 124). In some embodiments, the humanized B - Ly1 antibody is an antibody comprising a heavy chain having the amino acid sequence of SEQ ID NO: 9, a light chain having the amino acid sequence of SEQ ID NO: 10, or an antigen - binding fragment thereof. In some embodiments, the humanized B - Ly1 antibody comprises a heavy - chain variable region comprising the three heavy - chain CDRs of SEQ ID NO: 9 and a light - chain variable region comprising the three light - chain CDRs of SEQ ID NO: 10. Heavy chain (SEQ ID NO: 9) TIFF2025106262000006.tif44170 Light chain (SEQ ID NO: 10) TIFF2025106262000007.tif25170

[0076] In some embodiments, the humanized B - Ly1 antibody is afucosylated glyco - engineered humanized B - Ly1. Such glyco - engineered humanized B - Ly1 antibodies have an altered pattern of glycosylation within the Fc region and preferably have a reduced level of fucose residues. Preferably, the amount of fucose is 60% or less of the total amount of oligosaccharides at Asn297 (in one embodiment, the amount of fucose is 40% - 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). Further, the oligosaccharides in the Fc region are preferably bisected. These glyco - engineered humanized B - Ly1 antibodies have increased ADCC.

[0077] "Ratio of the binding ability of the anti-CD20 antibody to CD20 on Raji cells (ATCC number CCL-86) compared to rituximab" was determined by direct immunofluorescence measurement (where the mean fluorescence intensity (MFI) is measured) using the anti-CD20 antibody conjugated with Cy5 and rituximab conjugated with Cy5 in a FACSArray (Becton Dickinson) using Raji cells (ATCC number CCL-86) as described in Example 2, and is calculated as follows: Ratio of the binding ability to CD20 on Raji cells (ATCC number CCL-86) = TIFF2025106262000008.tif13170

[0078] MFI is the mean fluorescence intensity. "Cy5 labeling ratio", as used herein, means the number of Cy5-labeled molecules per molecule of antibody.

[0079] Typically, the type II anti-CD20 antibody has a ratio of binding ability to CD20 on Raji cells (ATCC number CCL-86) of the second anti-CD20 antibody compared to rituximab of 0.3 to 0.6, in one embodiment 0.35 to 0.55, and in yet another embodiment 0.4 to 0.5.

[0080] In one embodiment, the type II anti-CD20 antibody, for example, the GA101 antibody, has increased antibody-dependent cellular cytotoxicity (ADCC).

[0081] "Antibody having increased antibody-dependent cellular cytotoxicity (ADCC)" means an antibody such as that defined herein having increased ADCC as determined by any suitable method known to those skilled in the art. Those acceptable in an in vitro ADCC assay are 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) PBMCs are isolated using standard density centrifugation procedures and suspended at 5 x 10 6 cells / ml in RPMI cell culture medium, ii) Target cells are grown by standard tissue culture methods, harvested from the exponential growth phase with >90% viability, washed in RPMI cell culture medium, 51 labeled with 100 microcuries of 5 Cr, washed twice with cell culture medium, and resuspended in cell culture medium at a density of 10 iii) The above 100 microliter final target cell suspension is transferred to each well of a 96-well microtiter plate, iv) Antibodies are serially diluted in cell culture medium from 4000 ng / ml to 0.04 ng / ml, and 50 microliters of the resulting antibody solution is added to the target cells in the 96-well microtiter plate and tested at various antibody concentrations in triplicate covering all of the above concentration ranges, v) For the maximum release (MR) control, three additional wells in the plate containing labeled target cells receive 50 microliters of a 2% (v / v) aqueous solution of nonionic detergent (Nonidet, Sigma, St. Louis) instead of the antibody solution (point iv above), vi) For the spontaneous release (SR) control, three additional wells in the plate containing labeled target cells receive 50 microliters of RPMI cell culture medium instead of the antibody solution (point iv above), vii) The 96-well microtiter plate is then centrifuged at 50 x g for 1 minute and incubated at 4°C for 1 hour, viii) 50 microliters of the PBMC suspension (point i above) is added to each well, resulting in an effector to target cell ratio of 25:1, and the plate is placed in an incubator at 37°C in a 5% CO2 atmosphere for 4 hours, ix) Collect the cell-free supernatant from each well and quantify the experimentally released radioactivity (ER) using a gamma counter. x) For each antibody concentration, calculate the percentage of specific lysis according to the formula (ER - MR) / (MR - SR) x 100, where ER is the average radioactivity quantified for that antibody concentration (see point ix above), MR is the average radioactivity quantified for the MR control (see point V above) (see point ix above), and SR is the average radioactivity quantified for the SR control (see point vi above) (see point ix above). 4) "Increased ADCC" is defined as an increase in the maximum percentage of specific lysis observed within the antibody concentration range tested above and / or a reduction in the concentration of antibody 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 measured in the assay above, mediated by the same antibody, and against ADCC produced by the same type of host cell using the same standard production, purification, formulation, and storage methods known to those skilled in the art, except that the comparative antibody (lacking increased ADCC) is not produced by host cells engineered from the fucosyltransferase 8 (FUT8) gene (including those engineered for FUT8 knockout), engineered to overexpress GnTIII, and / or engineered to have reduced expression.

[0082] The "increased ADCC" can be obtained, for example, by mutation and / or glycosylation engineering of the antibody. In one embodiment, the antibody is glycosylation engineered, for example, to have a bisected oligosaccharide that binds to the Fc region of the antibody bisected by GlcNAc of WO2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), US2005 / 0123546 (Umana et al.), Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180). In another embodiment, the antibody is glycosylation engineered to lack fucose on the carbohydrate that binds to the Fc region by expressing the antibody in a host cell deficient in protein fucosylation (e.g., Lec13 CHO cells, or cells having a deleted alpha-1,6-fucosyltransferase gene (FUT8) or knockdown of FUT gene expression (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004), Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006), and WO2003 / 085107). In yet another embodiment, the antibody sequence is engineered within its Fc region to enhance ADCC (e.g., in one embodiment, such engineered antibody variants include an Fc region having one or more amino acid substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region).

[0083] The term "complement-dependent cytotoxicity (CDC)" refers to the lysis of human tumor target cells by the antibodies according to the invention in the presence of complement. CDC can be measured by treatment of a preparation of CD20-expressing cells with an anti-CD20 antibody according to the invention in the presence of complement. CDC is seen when the antibody induces lysis (cell death) of greater than 20% of the tumor cells after 4 hours at a concentration of 100 nM. In one embodiment, the assay is 51 performed using Cr or Eu-labeled tumor cells, and the released 51Measure Cr or Eu. As a control, incubation of tumor target cells using complement but not antibody is included.

[0084] 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 methods of the 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 immunohistochemical (IHC) detection, using FACS, or PCR-based detection of the corresponding mRNA.

[0085] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes combinations of two or more such molecules.

[0086] The term "about" as used herein refers to the normal error range of each value readily known to those of ordinary skill in the art. Reference to a value or parameter "about" in this specification includes (and describes) embodiments directed to that value or parameter itself.

[0087] It is understood that the aspects and embodiments of the invention described herein include aspects and embodiments "comprising", "consisting of", and "consisting essentially of".

[0088] III. Methods In one aspect, provided herein is a method for treating lupus nephritis or delaying its progression in an individual having lupus by administering an effective amount of a type II anti-CD20 antibody. In some embodiments, the individual has 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 method comprises administering to the individual at least a first antibody exposure amount of the type II anti-CD20 antibody and a second antibody exposure amount of the type II anti-CD20 antibody, wherein the second antibody exposure amount is not provided until about 18 to about 26 weeks after the first antibody exposure amount, wherein the first antibody exposure amount comprises one or two doses of the type II anti-CD20 antibody, the first antibody exposure amount comprises a total exposure amount of about 1800 mg to about 2200 mg of the type II anti-CD20 antibody, the second antibody exposure amount comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure amount comprises a total exposure amount of about 1800 mg to about 2200 mg of the type II anti-CD20 antibody. As described below, in some embodiments, the 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. In some embodiments, the antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 8. 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 to 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 to the amino acid sequence of SEQ ID NO: 10.

[0089] anti-CD20 antibody Certain aspects of the present disclosure relate to anti-CD20 antibodies for use, for example, in methods for treating or preventing the progression of lupus nephritis. In some embodiments, the anti-CD20 antibody is a type II antibody. In some embodiments, the anti-CD20 antibody is a human or humanized antibody. In some embodiments, the anti-CD20 antibody is afucosylated. In some embodiments, the anti-CD20 antibody is the GA101 antibody.

[0090] Examples of type II anti-CD20 antibodies include, for example, humanized B-Ly1 antibody IgG1 (chimeric humanized IgG1 antibody as disclosed in WO2005 / 044859), 11B8 IgG1 (as disclosed in WO2004 / 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 (in the case of the IgG1 isotype) compared to type I antibodies of the IgG1 isotype.

[0091] Examples of type I anti-CD20 antibodies include, for example, rituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (as disclosed in WO2005 / 103081), 2F2 IgG1 (as disclosed and in WO2004 / 035607 and WO2005 / 103081), and 2H7 IgG1 (as disclosed in WO2004 / 056312).

[0092] In some embodiments, the anti-CD20 antibody is the GA101 antibody described herein. In some embodiments, the anti-CD20 is any one of the following antibodies that binds to human CD20: (1) an antibody comprising an HVR-H1 comprising the amino acid sequence of GYAFSY (SEQ ID NO: 1), an HVR-H2 comprising the amino acid sequence of FPGDGDTD (SEQ ID NO: 2), an HVR-H3 comprising the amino acid sequence of NVFDGYWLVY (SEQ ID NO: 3), an HVR-L1 comprising the amino acid sequence of RSSKSLLHSNGITYLY (SEQ ID NO: 4), an HVR-L2 comprising the amino acid sequence of QMSNLVS (SEQ ID NO: 5), and an HVR-L3 comprising the amino acid sequence of AQNLELPYT (SEQ ID NO: 6); (2) an antibody comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 8; (3) an antibody comprising the amino acid sequences of SEQ ID NO: 9 and 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 to 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 to 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 any of the HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of any of the antibodies described herein, e.g., the 3HVR from SEQ ID NO: 7 and the 3HVR of SEQ ID NO: 8, the 3HVR from SEQ ID NO: 9 and the 3HVR from SEQ ID NO: 10, or any HVR of the amino acid sequences provided in Table 2.

[0093] In some embodiments, the anti-CD20 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8. TIFF2025106262000009.tif49170

[0094] In some embodiments, the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10. TIFF2025106262000010.tif101170

[0095] 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 comprising the three heavy chain CDRs of SEQ ID NO: 9 and a light chain variable region comprising the three light chain CDRs of SEQ ID NO: 10. In some embodiments, the humanized B-Ly1 antibody comprises a heavy chain comprising the sequence of SEQ ID NO: 9 and a light chain comprising the sequence of SEQ ID NO: 10.

[0096] In some embodiments, the anti-CD20 antibody comprises 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. TIFF2025106262000011.tif231170TIFF2025106262000012.tif226170TIFF2025106262000013.tif227170TIFF2025106262000014.tif220170

[0097] In some embodiments, the anti-CD20 antibody (e.g., a type II anti-CD20 antibody) is an afucosylated glycoengineered antibody. Such glycoengineered antibodies have an altered pattern of glycosylation within the Fc region and preferably have a reduced level of fucose residues. Preferably, the amount of fucose is 60% or less of the total amount of oligosaccharide at Asn297 (in one embodiment, the amount of fucose is 40% - 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). Further, the oligosaccharide of the Fc region is preferably bisected. These glycoengineered humanized anti-CD20 (e.g., B-Ly1) antibodies have increased ADCC.

[0098] Oligosaccharide constituents can significantly affect properties related to the efficacy of therapeutic glycoproteins, including physical stability, resistance to protease attack, interaction with the immune system, pharmacokinetics, and specific biological activities. Such properties can depend not only on the presence or absence of oligosaccharides but also on their specific structures. Some generalizations can be established between oligosaccharide structures and glycoprotein functions. For example, certain oligosaccharide structures mediate the rapid clearance of glycoproteins from the bloodstream through interaction with specific carbohydrate-binding proteins, while other oligosaccharide structures can be bound by antibodies and cause unwanted immune reactions. (Jenkins, N., et al., Nature Biotechnol. 14 (1996) 975-81).

[0099] Mammalian cells are preferred hosts for the production of therapeutic glycoproteins due to their ability to glycosylate proteins in the most highly compatible form for human application. (Cumming, D. A., et al., Glycobiology 1 (1991) 115 - 30, Jenkins, N., et al., Nature Biotechnol. 14 (1996) 975 - 81). Bacteria rarely glycosylate proteins, and other types of common hosts, such as yeast, filamentous fungi, insects, and plant cells, result in glycosylation patterns associated with rapid clearance from the bloodstream, unwanted immune interactions, and in some specific cases, reduced biological activity. Chinese hamster ovary (CHO) cells, which are mammalian cells, have been most commonly used in the past 20 years. In addition to providing a suitable glycosylation pattern, these cells enable the consistent generation of genetically stable, highly productive clonal cell lines. They can be cultured at high density in simple bioreactors using serum - free media, allowing the development of safe and reproducible biological processes. Other commonly used animal cells include baby hamster kidney (BHK) cells, NSO mouse myeloma cells, and SP2 / 0 mouse myeloma cells. Recently, production from transgenic animals has been tested. (Jenkins, N., et al., Nature Biotechnol. 14 (1996) 975 - 981).

[0100] All antibodies contain carbohydrate structures at conserved positions within the heavy chain constant region, and each isotype possesses a distinct array of N-linked carbohydrate structures, which variably influence protein composition, secretion, or functional activity. (Wright, A., and Morrison, S.L., Trends Biotech. 15 (1997) 26-32). The structure of the attached N-linked carbohydrates varies widely depending on the degree of processing, which can include high mannose, multi-branched, as well as bi-branched complex oligosaccharides. (Wright, A., and Morrison, S.L., Trends Biotech. 15 (1997) 26-32). Typically, there is a heterogeneous process of core oligosaccharide structures binding at specific glycosylation sites such that even monoclonal antibodies exist as multiple glycoforms. Similarly, major differences in antibody glycosylation occur between cell lines, and even minor differences have been shown to be seen for a given cell line growing under different culture conditions. (Lifely, M.R., et al., Glycobiology 5(8)(1995)813-22).

[0101] One way to obtain a substantial increase in efficacy while maintaining a simple production process and avoiding significant unwanted side effects is to enhance the natural cell-mediated effector functions of monoclonal antibodies by manipulating their oligosaccharide constituents, as described in Umana, P., et al., Nature Biotechnol. 17(1999)176-180 and US6,602,684. IgG1 antibodies, the most commonly used antibodies in cancer immunotherapy, are glycoproteins with a conserved N-linked glycosylation site at Asn297 within each CH2 domain. The two complex biantennary oligosaccharides that bind to Asn297 are buried between the CH2 domains, where they form extensive contacts with the polypeptide backbone, and their presence is essential for the antibody to mediate effector functions such as antibody-dependent cell cytotoxicity (ADCC) (Lifely, M.R., et al., Glycobiology 5(1995)813-822, Jefferis, R., et al., Immunol. Rev. 163(1998)59-76, Wright, A., and Morrison, S.L., Trends Biotechnol. 15(1997)26-32).

[0102] Overexpression of β(1,4)-N-acetylglucosaminyltransferase I11 (´´GnTII17y´´), a glycosyltransferase that catalyzes the formation of bisected oligosaccharides, in Chinese hamster ovary (CHO) cells has previously been shown to significantly increase the in vitro ADCC activity of an anti-neuroblastoma chimeric monoclonal antibody (chCE7) produced by engineered CHO cells. (See Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180; and WO99 / 154342, the entire contents of which are incorporated herein by reference). Antibody chCE7 has high tumor affinity and specificity but belongs to a large class of unconjugated monoclonal antibodies that have little clinically useful efficacy when produced in standard industrial cell lines lacking the GnTIII enzyme (Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180). That study first showed that a substantial increase in ADCC activity might be achievable by engineering antibody-producing cells to express GnTIII, which might also result in an increase in the proportion of bisected oligosaccharides associated with the constant region (Fc) that contain bisected, non-fucosylated oligosaccharides above levels seen in naturally occurring antibodies.

[0103] In some embodiments, the anti-CD20 antibody (e.g., a type II anti-CD20 antibody) comprises a human Fc region (e.g., a human IgG1 Fc region). In some embodiments, the Fc region comprises N-linked oligosaccharides that are modified. In some embodiments, the N-linked oligosaccharides of the Fc region have reduced fucose residues compared to an antibody having unmodified N-linked oligosaccharides. In some embodiments, the bisected oligosaccharide is a bisected complex oligosaccharide. In some embodiments, the N-linked oligosaccharides are modified to have increased bisected non-fucosylated oligosaccharides. In some embodiments, the bisected non-fucosylated oligosaccharide is of the hybrid type. In some embodiments, the bisected non-fucosylated oligosaccharide is of the complex type. For more detailed description, see, for example, WO2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), US2005 / 0123546 (Umana et al.), and U.S. Patent No. 8,883,980 (Umana et al.).

[0104] In some embodiments, the anti-CD20 antibody (e.g., a type II anti-CD20 antibody) is a multispecific antibody or a bispecific antibody.

[0105] Preparation of Antibodies An antibody according to any of the above embodiments (e.g., a type II anti-CD20 antibody of the present disclosure) may incorporate any of the features as described in items 1 to 7 below, alone or in combination.

[0106] 1. Antibody Affinity In certain embodiments, the antibodies provided herein have an affinity of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13has a dissociation constant (Kd) of (M).

[0107] In one embodiment, the Kd is measured by a radioimmunoassay (RIA). In one embodiment, the RIA is performed using the Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is determined in the presence of a series of titrations of unlabeled antigen by measuring the minimum concentration of ( 125 I)-labeled antigen that equilibrates and then binds to the antigen captured by a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the conditions for the assay, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23°C). In a non-adsorptive plate (Nunc #269620), 100 pM or 26 pM 125 I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although the incubation can be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to the capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, 150 μL / well of scintillant (MICROSCINT-20™ Packard) is added and the plate is counted on a TOPCOUNT® gamma counter (Packard) for 10 minutes. The concentration of each Fab that results in less than 20% of the maximum binding is selected for use in the competitive binding assay.

[0108] According to another embodiment, Kd can be measured using a BIACORE® surface plasmon resonance assay. For example, an assay using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25° C. with an immobilized antigen CM5 chip at approximately 10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated using N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. After diluting the antigen with 10 mM sodium acetate (pH 4.8) to 5 μg / mL (about 0.2 μM), it is injected at a flow rate of 5 μL / min to achieve a coupled protein of approximately 10 response units (RU). After injecting the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected at 25° C. in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at a flow rate of approximately 25 μL / min. The association rate (k on ) and dissociation rate (k off ) are calculated by fitting the association sensorgram and dissociation sensorgram simultaneously using a simple 1:1 Langmuir binding model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is calculated as k off / k onIt is calculated as a ratio. See, for example, Chen et al., J. Mol. Biol. 293:865 - 881 (1999). When the on - rate by the above - described surface plasmon resonance assay exceeds 106 M−1s−1, the on - rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in the fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm band - pass) of 20 nM anti - antigen antibody (Fab - type) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen, measured in a spectrometer such as a stopped - flow - equipped spectrophotometer (Aviv Instruments) with a stirred cuvette or an 8000 series SLM - AMINCO (trademark) spectrophotometer (ThermoSpectronic).

[0109] 2. Antibody fragments In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab’, Fab’ - SH, F(ab’)2, Fv, and scFv fragments, as well as other fragments described hereinafter. For an overview of certain antibody fragments, see Hudson et al. Nat. Med. 9:129 - 134 (2003). For an overview of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., (Springer - Verlag, New York), pp. 269 - 315 (1994), WO93 / 16185, as well as U.S. Patent Nos. 5,571,894 and 5,587,458. See also U.S. Patent No. 5,869,046 for discussion of Fab and F(ab´)2 fragments that contain salvage receptor - binding epitope residues and have an increased in vivo half - life.

[0110] A diabody is an antibody fragment having two antigen-binding sites that can be bivalent or bispecific. See, for example, EP404,097, WO1993 / 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 tetra-bodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0111] A single-domain antibody is an antibody fragment that includes all or part of the heavy-chain variable domain of an antibody, or all or part of the light-chain variable domain. In certain embodiments, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, MA, for example, U.S. Patent No. 6,248,516 B1).

[0112] Antibody fragments can be made by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies as described herein and production by recombinant host cells (e.g., E. coli or phage).

[0113] 3. Chimeric 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 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 mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switch" antibody in which the class or subclass has changed from that of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0114] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody includes one or more variable domains, wherein the HVRs, such as CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody will optionally also include at least a portion of a 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., the antibody from which the HVR residues are derived) so as to, for example, restore or improve antibody specificity or affinity.

[0115] Humanized antibodies and methods of making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat’l Acad. Sci. USA 86:10029-10033 (1989), U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al., Methods 36:25-34 (2005) (describing specific determinant region (SDR) grafting), Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”), Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing “guided selection” approaches to FR shuffling).

[0116] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)), framework regions derived from consensus sequences of certain subsets of human antibodies of the light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992), and Presta et al. J. Immunol., 151:2623 (1993)), human mature (somatic mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions resulting from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0117] 4. Human Antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of 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).

[0118] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies having human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, is present episomally, or is randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For an overview 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, which describe XENOMOUSE™ technology, U.S. Patent No. 5,770,429, which describes HUMAB® technology, U.S. Patent No. 7,041,870, which describes K-M MOUSE® technology, and U.S. Patent Application Publication No. US2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions derived from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0119] Human antibodies can also be made by methods based on hybridomas. Human myelomas and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133:3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies generated by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (triooma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0120] Human antibodies can also be generated 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.

[0121] 5. Library-Derived Antibodies The antibodies of the present invention can be isolated by screening a combinatorial library for antibodies having the desired activity(ies). For example, methods for generating phage display libraries and screening such libraries for antibodies having the desired binding characteristics are known in the art. Such methods are reviewed, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, 2001), and are further described, for example, in the McCafferty et al., Nature 348:552-554, Clackson et al., Nature 352:624-628 (1991), Marks et al., J. Mol. Biol. 222:581-597 (1992), Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003), Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004), Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004), Fellouse, Proc. Natl. Acad. Sci. USA101(34):12467-12472 (2004), and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0122] In certain phage display methods, the repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR), randomly recombined within a phage library, and then selected for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries derived from immunization sources provide high-affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, as described by Griffiths et al., EMBO J, 12:725-734 (1993), naive repertoires can be cloned (e.g., from humans) and provide a single source of antibodies to a wide range of non-self or self antigens without any immunization. Finally, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992), naive libraries can also be synthetically generated by cloning V gene segments that have not been rearranged from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro. Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, as well as U.S. Patent Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0123] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0124] 6. Multispecific Antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies having binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for CD20 and the other is for any other antigen. In certain embodiments, the bispecific antibody can bind to two different epitopes of CD20. The bispecific antibody can be used to localize a cytotoxic agent to cells expressing CD20. The bispecific antibody can be prepared as a full-length antibody or an antibody fragment.

[0125] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-chain-light-chain pairs having different specificities (see Milstein and Cuello, Nature 305:537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and the "knob-in-hole" maneuver (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can be prepared by manipulation of the electrostatic steering effect for making antibody Fc-heterodimer molecules (WO2009 / 089004A1), cross-linking of two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)), use of leucine zippers for producing bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)), use of the "diabody" technique for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)), and can also be made by the preparation of trispecific antibodies such as, for example, described in Tutt et al. J. Immunol. 147:60 (1991).

[0126] Engineered antibodies having three or more functional antigen-binding sites, including "Octopus antibodies", are also included herein (see, e.g., US2006 / 0025576A1).

[0127] Antibodies or fragments thereof as used herein also include CD20, as well as "Dual Acting FAb" or "DAF" that includes antigen-binding sites that bind to another different antigen (see, for example, US2008 / 0069820).

[0128] 7. Antibody variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions of residues from the amino acid sequence of the antibody and / or insertions of residues into the amino acid sequence and / or substitutions of residues in the amino acid sequence. Any combination of deletions, insertions, and substitutions can be made to achieve the final construct, provided that the final construct retains the desired characteristics, such as antigen-binding ability.

[0129] a) Substitution, insertion, and deletion variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table A under the heading "Preferred substitutions". More substantial changes are provided 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 the desired activity, such as retention / improvement of antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC. TIFF2025106262000015.tif184170

[0130] Amino acids can be grouped according to common side-chain characteristics. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues affecting chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe

[0131] Non-conservative substitutions will involve an exchange of a member of one of these classes with a member of another class.

[0132] Certain types of substitution variants are involved in substituting one or more hypervariable region residues of a parental antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variant(s) selected for further study will have a modification (e.g., improvement) of a particular biological property (e.g., increased affinity, reduced immunogenicity) relative to the parental antibody and / or will have a particular biological property of the parental antibody that is substantially retained. Exemplary substitution variants are affinity matured antibodies that are conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated, the variant antibodies are displayed on phage, and selected for a particular biological activity (e.g., binding affinity).

[0133] Modifications (e.g., substitutions) can be made in the HVRs to, for example, improve antibody affinity. Such modifications can be made to residues encoded by codons that mutate frequently during the somatic maturation process, i.e., HVR “hot spots” (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or to residues that contact the antigen, and the resulting mutant VH or VL is tested for binding affinity. Affinity maturation by constructing a secondary library and rescreening therefrom is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, N.J., (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods, such as error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis. A secondary library is then created. The library is then screened to identify any antibody mutants having the desired affinity. Another method for introducing diversity involves a method directed to the HVRs in which multiple HVR residues (e.g., 4-6 residues at a time) are randomized. The HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0134] In certain embodiments, substitutions, insertions, or deletions can occur within one or more HVRs so long as such modifications do not substantially reduce the ability of the antibody to bind the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in the HVRs. Such modifications can be, for example, at locations other than antigen contact residues within the HVRs. In certain embodiments of the mutant VH and VL sequences provided above, each HVR either is not modified or contains no more than one, two, or three amino acid substitutions.

[0135] A method useful for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" and is described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or groups of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the first substitution. Alternatively, or in addition, the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or excluded as candidates for substitution. Mutant forms can be selected to determine whether they contain the desired properties.

[0136] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions in the range of polypeptides containing from 1 residue to 100 or more residues, as well as in-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion mutants of the antibody molecule include fusions of the antibody to an enzyme (e.g., for ADEPT) or polypeptide that increase the serum half-life of the antibody at the N-terminus or C-terminus of the antibody.

[0137] b) Glycosylation mutants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Addition or deletion of glycosylation sites to the antibody can be conveniently achieved by modifying the amino acid sequence such that one or more glycosylation sites are created or removed.

[0138] When the antibody contains an Fc region, the carbohydrates binding thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides that are generally bound by N-linkage to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose that binds to GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibodies of the present invention can be performed to create antibody variants with improved certain properties.

[0139] In one embodiment, an antibody variant having a carbohydrate structure lacking fucose (either directly or indirectly) linked to the Fc region is provided. For example, the amount of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined, for example, as described in WO2008 / 077546, by calculating the average amount of fucose within the sugar chain at Asn297 relative to the total of all sugar structures (e.g., complex, hybrid, and high-mannose structures) that bind to Asn297 when measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue located at approximately position 297 within the Fc region (Eu numbering of Fc region residues), but Asn297 can also be located at approximately ±3 amino acids upstream or downstream of position 297, i.e., positions 294 to 300, due to minor sequence variations in the antibody. Such fucosylation variants can have improved ADCC function. See, for example, U.S. Patent Publication Nos. US2003 / 0157108 (Presta, L.), US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications regarding "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, US2004 / 0132140, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing afucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986), US Patent Application No. US2003 / 0157108 A1, Presta, L., and WO2004 / 056312 A1, Adams et al., particularly Example 11), and knockout cell lines such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004), Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006), and WO2003 / 085107).

[0140] For example, there is further provided an antibody variant having a bisected oligosaccharide in which the 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 WO2003 / 011878 (Jean-Mairet et al.), US Patent No. 6,602,684 (Umana et al.), and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide bound to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.), WO1998 / 58964 (Raju, S.), and WO1999 / 22764 (Raju, S.).

[0141] c) Fc region variants In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. The Fc region variants can include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0142] In certain embodiments, the invention contemplates antibody variants that are desirable candidates for applications where the in vivo half-life of an antibody is important but where certain effector functions (such as complement and ADCC) are unnecessary or harmful, by possessing some but not all effector functions. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / abrogation of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and thus is likely to lack ADCC activity), but retains FcRn binding ability. NK cells, which are 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 a molecule of interest are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985), U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods are used (see, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA, and the CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo in an animal model such as those disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody is unable to bind to C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. A CDC assay can be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), Cragg, M. S. et al., Blood 101:1045-1052 (2003), and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, S. B. et al., Int’l. Immunol. 18(12):1759-1769 (2006)).

[0143] Antibodies having reduced effector function include those having one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant having substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0144] In certain embodiments, the Fc variants described herein further comprise one or more amino acid modifications to attenuate effector functions (such as CDC and / or ADCC). In exemplary embodiments, the modifications to attenuate effector functions are modifications that do not alter the glycosylation pattern of the Fc region. In certain embodiments, the modifications to attenuate effector functions reduce or eliminate binding to human effector cells, binding to one or more Fc receptors, and / or binding to cells expressing Fc receptors. In an exemplary embodiment, the Fc variants described herein comprise the following modifications: L234A, L235A, and P329G within the Fc region of human IgG1, which result in attenuated effector functions. The substitutions L234A, L235A, and P329G (the L234A / L235A / P329G triple variant is referred to as LALAPG) have been previously shown to reduce binding to Fc receptors and complement (see, e.g., US Publication No. 2012 / 0251531).

[0145] In various embodiments, an Fc variant having reduced effector function refers to an Fc variant that reduces effector function (such as activities such as CDC, ADCC, and / or binding to 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 does not have mutations to reduce effector function but may have other mutations). In certain embodiments, an Fc variant having reduced effector function refers to an Fc variant that eliminates all detectable effector functions compared to a wild-type Fc region. Assays for measuring effector function are known in the art and are described below.

[0146] In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / impairment of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that an antibody lacks FcγR binding (and thus is likely to lack ADCC activity). While NK cells, which are primary cells for mediating ADCC, express only FcγRIII, 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 molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985), U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods are used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for flow cytometry (Cell Technology, Inc. Mountain View, CA and CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of a molecule of interest can be evaluated in vivo, for example, in an animal model such as those disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that an antibody is unable to bind C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 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, M. S. et al., Blood 101:1045-1052 (2003), and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)).

[0147] Certain antibody variants with improved or decreased binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056, WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0148] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (EU numbering of residues) in the Fc region.

[0149] In some embodiments, modifications that result in altered (i.e., improved or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC), such as those described in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000), can be made within the Fc region.

[0150] Antibodies having an increased half-life and improved binding to the neonatal Fc receptor (FcRn) that are responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976), and Kim et al., J. Immunol. 24:249 (1994)) are described in US2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include substitutions at one or more of the Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, for example, those having a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0151] See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO94 / 29351 for other examples of Fc region variants.

[0152] d) Cysteine-engineered antibody variants In certain embodiments, it may be desirable to generate a cysteine-engineered antibody, e.g., a "thioMAb", in which one or more residues of the antibody are replaced with cysteine residues. In certain embodiments, the replacement residues occur at accessible sites of the antibody. By replacing with residues having cysteine, a reactive thiol group is thereby positioned at an accessible site of the antibody and the antibody can be conjugated to other moieties such as a drug moiety or a linker-drug moiety to generate an immunoconjugate as further described herein. In certain embodiments, any one or more of the following residues may be replaced with cysteine: V205 of the light chain (Kabat numbering), A118 of the heavy chain (EU numbering), and S400 of the heavy chain Fc region (EU numbering). The cysteine-engineered antibody can be generated, for example, as described in U.S. Patent No. 7,521,541.

[0153] e) Antibody derivatives In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties that are known in the art and are readily available. Moieties suitable for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / prolylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homo- or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof, but are not limited thereto. Polyethylene glycol propionaldehyde may be advantageous for manufacture due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary and, if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the improved antibody, whether the antibody derivative is to be used in therapy under a given set of conditions, and the like.

[0154] In another embodiment, conjugates of antibodies and non-proteinaceous moieties that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not damage normal cells but heat the non-proteinaceous moiety to a temperature at which cells proximal to the antibody-non-proteinaceous moiety die.

[0155] A. Recombinant Methods and Compositions Antibodies can be produced using recombinant methods and compositions such as those described, for example, in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-CD20 antibody described herein is provided. Such a nucleic acid can encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) 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 (e.g., transformed therewith). In one embodiment, the host cell is eukaryotic, e.g., Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method of making an anti-CD20 antibody is provided, the method comprising culturing a host cell comprising a nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody and optionally recovering the antibody from the host cell (or the host cell culture medium).

[0156] With respect to the recombinant production of anti-CD20 antibodies, for example, a nucleic acid encoding an antibody as described above is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such a nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody).

[0157] Host cells suitable for the cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste in the soluble fraction and further purified.

[0158] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized," which result in the production of antibodies with a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0159] Host cells suitable for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used, particularly for the transfection of Spodoptera frugiperda cells, in conjunction with insect cells.

[0160] Plant cell cultures can also be used as hosts. See U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0161] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines are SV40 (COS-7), human embryonic kidney cell lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), dog kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT060562), and monkey kidney CV1 cells transformed by TRI cells, MRC5 cells, and FS4 cells as described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982). Other useful mammalian host cell lines include DHFR - Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), as well as myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0162] B. Assays The anti-CD20 antibodies provided herein can be identified, selected, or characterized with respect to their physical / chemical properties and / or biological activities by a variety of assays known in the art.

[0163] 1. Conjugation Assays and Other Assays In one aspect, the antibodies of the invention are tested for their antigen binding activity by known methods such as, for example, ELISA, Western blot, etc. CD20 binding can be determined using methods known in the art, and exemplary methods are disclosed herein. In one embodiment, binding is measured using a radioimmunoassay. An exemplary radioimmunoassay is provided below. The CD20 antibody is iodinated and a competitive reaction mixture containing a fixed concentration of the iodinated antibody and decreasing concentrations of serially diluted unlabeled CD20 antibody is prepared. Cells expressing CD20 (e.g., BT474 cells stably transfected with human CD20) are added to the reaction mixture. Following incubation, the cells are washed to separate free iodinated CD20 antibody from the CD20 antibody bound to the cells. For example, the level of bound iodinated CD20 antibody is determined by counting the radioactivity associated with the cells, and the binding affinity is determined using standard methods. In another embodiment, the ability of the CD20 antibody to bind surface-expressed CD20 (e.g., on a subset of B cells) is evaluated using flow cytometry. Peripheral white blood cells are obtained (e.g., from human, cynomolgus monkey, rat, or mouse), and the cells are blocked with serum. The labeled CD20 antibody is added in serial dilutions and T cells are also stained to identify T cell subsets (using methods known in the art). Following incubation and washing of the sample, the cells are sorted using a flow cytometer and the data are analyzed using methods well known in the art. In another embodiment, CD20 binding can be analyzed using surface plasmon resonance. Exemplary surface plasmon resonance methods are illustrated in the Examples.

[0164] In another aspect, the competition assay can be used to identify an antibody that competes with any of the anti-CD20 antibodies disclosed herein with respect to binding to CD20. In certain embodiments, such a competing antibody binds to the same epitope (e.g., linear or conformational epitope) that is bound by any of the anti-CD20 antibodies disclosed herein. Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0165] 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 that is being tested for its ability to compete with the first antibody with respect to binding to CD20. The second antibody can be present in a hybridoma supernatant. As a control, immobilized CD20 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that permit binding of the first antibody 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 relative to the control sample, this indicates that the second antibody competes 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).

[0166] 2. Activity Assay The anti-CD20 antibodies (e.g., type II antibodies) of the present disclosure can be identified and / or characterized by one or more activity assays known in the art. For example, complement-dependent cytotoxicity (CDC) and / or antibody-dependent cell-mediated cytotoxicity (ADCC) can be used as described herein.

[0167] It is understood that the immunoconjugates of the present invention can be used in place of or in addition to the anti-CD20 antibodies to perform any of the above assays.

[0168] It is understood that any of the above assays can be performed using an anti-CD20 antibody and an additional therapeutic agent.

[0169] C. Immunoconjugate The present invention also provides an immunoconjugate comprising an anti-CD20 antibody herein conjugated to one or more cytotoxic agents such as a chemotherapeutic agent or chemotherapeutic drug, a growth inhibitor, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope.

[0170] In one embodiment, the immunoconjugate is a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent EP0 425 235 B1); an auristatin such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatin; calicheamicin or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296, Hinman et al., Cancer Res. 53:3336-3342 (1993), and Lode et al., Cancer Res. 58:2925-2928 (1998)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med.Chem. 13:477-523 (2006), Jeffrey et al., Bioorganic & Med.Chem.Letters 16:358-362 (2006), Torgov et al., Bioconj.Chem. 16:717-721 (2005), Nagy et al., Proc.Natl.Acad.Sci.USA 97:829-834 (2000), Dubowchik et al., Bioorg. & Med.Chem.Letters 12:1529-1532 (2002), King et al., J.Med.Chem. 45:4336-4343 (2002), and U.S. Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecin; and an antibody-drug conjugate (ADC) in which an antibody, including but not limited to these, is conjugated to one or more drugs.

[0171] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, saporin, dianthin, American pokeweed proteins (PAPI, PAPII, and PAP-S), cucurbitacin inhibitor, curcin, crocin, bryodin inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichothecene.

[0172] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioactive conjugate. A variety of radioisotopes are available for the production of radioactive conjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioisotopes of Lu. When a radioactive conjugate is used for detection, it may include radioactive atoms for scintigraphy studies, e.g., tc99m or I123, or again spin labels for nuclear magnetic resonance (NMR) imaging methods (also known as magnetic resonance imaging, mri) such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0173] Antibodies and cytotoxic conjugates can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies. See WO94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug intracellularly. For example, acid-labile linkers, peptidase-sensitive linkers, photosensitive linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992), U.S. Patent No. 5,208,020) can be used.

[0174] The immunoconjugates or ADCs in this specification clearly contemplate, but are not limited to, such conjugates prepared using crosslinking reagents including BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as commercially available SVSB (succinimidyl-(4-vinylsulfone)benzoate) (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

[0175] A method for treating or delaying the progression of lupus nephritis Certain embodiments of the present disclosure relate to a method for treating or delaying the progression of lupus nephritis (LN) in an individual having lupus. In some embodiments, the individual or patient is human.

[0176] LN is known in the art as a manifestation of lupus (e.g., systemic lupus erythematosus, drug-induced lupus, neonatal lupus, or discoid lupus) in the kidney(s). The most common form of lupus that manifests in the kidney is systemic lupus erythematosus (SLE). 25-50% of SLE patients have abnormalities in urine and / or renal function early in the course of the disease, and it is thought that up to 60% of adults and 80% of children will ultimately develop LN (see, for more details, Cameron, J.S. (1999) J. Am. Soc. Nephrol. 10:413-424). LN is thought to be responsible for at least 50% of the morbidity and mortality associated with SLE.

[0177] In addition, kidney manifestations have also been described in other forms of lupus such as discoid (Roujeau, J.C. et al. (1984) Acta Derm. Venereol. 64:160-163) and drug-induced lupus (Smith, P.R. et al. (1999) Rheumatology (Oxford) 38:1017-1018). In some embodiments, the individual has SLE, discoid lupus, or drug-induced lupus.

[0178] The diagnosis of SLE can be made according to the 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 Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), or the Systemic Lupus Erythematosus (SLE) Responder Index (SRI) as described in the examples below and as described in Furie et al., Arthritis Rheum 61(9):1143-51(2009). Some signs, symptoms, or other indicators used to diagnose SLE adapted from Tan et al. "The Revised Criteria for the Classification of SLE" Arthritis Rheum 25(1982) include malar rash such as a rash across the cheeks, discoid rash, or butterfly rash such as a red, raised patch; photosensitivity such as a reaction to sunlight that causes the onset or increase of a skin rash; oral ulcers such as ulcers in the nose or mouth; arthritis that is usually painless such as non-erosive arthritis involving two or more peripheral joints (arthritis where the bone around the joint is not destroyed), serositis, pleurisy, or pericarditis; kidney impairment such as excessive protein in the urine (more than 0.5 gm / day or 3+ on a test stick) and / or cellular casts (abnormal elements derived from urine and / or white blood cells and / or renal tubular cells); neurological signs, symptoms, or other indicators; seizures; and / or psychosis in the absence of drugs or known metabolic disorders that 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 per cubic millimeter) or lymphopenia (less than 1,500 lymphocytes per cubic millimeter) or thrombocytopenia (less than 100,000 platelets per cubic millimeter). Leukopenia and lymphopenia need to be detected on two or more occasions. Thrombocytopenia needs to be detected in the absence of known drugs that induce it. The present invention is not limited to these signs, symptoms, or other indicators of lupus.

[0179] The presence of autoantibodies can be tested as an indicator for lupus. Autoantibodies can include, but are not limited to, anti-dsDNA antibodies, anti-complement antibodies, and anti-nuclear antibodies (e.g., ENA panel). ENA refers to extractable nuclear antigens, i.e., a group of nuclear antigens including, for example, RNP, Ro / SS-A, La / SS-B, Sm, SCL-70, Jo-1 as described in 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 ED. LIPPINCOTT (2007), Tang et al., Medicine 89(1):62-67 (2010). Antibodies to ENA are correlated with lupus. McNeilage et al., 1984, Whittingham 1988, Asherson et al., Medicine 68(6):366-374 (1989), and Tang et al., 2010. For example, reduced complement activity as measured by C3 level, C4 level, and / or CH50 assay is also associated with lupus.

[0180] As described above in the context of SLE, it is known in the art that LN often gradually presents as a symptom in patients with lupus (such as systemic lupus erythematosus, drug-induced lupus, neonatal lupus, or discoid lupus). That is, a patient can be diagnosed with lupus without clinical or pathological signs of one or more LN symptoms. Nevertheless, the patient can still be considered at risk of developing LN because lupus patients ultimately develop LN at a high frequency. Thus, in some embodiments, it can be found that the methods of the present disclosure are used in delaying the progression of LN or preventing LN in patients with lupus. In some embodiments, it can be found that the methods of the present disclosure are used in delaying or preventing the occurrence of LN in patients with lupus (such as the form of lupus lacking symptoms in the kidney(s)).

[0181] LN pathology can be classified according to the classification system of the International Society of Nephrology / Renal Pathology Society (ISN / RPS) 2003, as shown in the following table (for further explanations and definitions of terms, see Markowitz GS, D’Agati VD (2007) Kidney Int 71:491-495 and Weening, JJ (2004) Kidney Int 65:521-530). TIFF2025106262000016.tif255170TIFF2025106262000017.tif63170

[0182] In some embodiments, the patient has Class III or Class IV LN. In some embodiments, the patient has Class III LN. For example, in some embodiments, the patient has Class III(A) or Class III(A / C) LN. In some embodiments, the patient has Class IV LN. For example, in some embodiments, the patient has 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 can also occur concurrently with Class III or Class IV LN. In some embodiments, the methods of the present disclosure are used to treat patients having Class III or Class IV LN, and concomitant Class V LN.

[0183] As discussed above, patients with lupus (e.g., SLE) will ultimately develop LN with high frequency. In some embodiments, the patient is at risk of developing LN. In some embodiments, the patient is at risk of developing Class III or Class IV LN. In some embodiments, the patient is at risk of developing Class III or Class IV LN, along with concomitant Class V LN.

[0184] In some embodiments, the patient does not have Class III(C) LN (e.g., as described in Table 3 above). In some embodiments, the patient does not have Class IV(C) LN, e.g., Class IV-S(C) or IV-G(C) LN (e.g., as described in Table 3 above).

[0185] Multiple laboratory tests known in the art can be used to diagnose and / or monitor the presence, progression, and / or response to treatment of lupus nephritis. 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, although there are some differences depending on age, gender, and laboratory. 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 to creatinine ratio (UPCR) can be measured. The presence of protein in the urine (proteinuria) can also be assayed by tests including, but not limited to, the urine albumin to creatinine ratio (UACR) and dipstick urine analysis. Other tests and / or measurements that may be useful in investigating renal function can include, but are not limited to, a 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. For more detailed description, see, for example, 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).

[0186] In some embodiments, the method of the present disclosure comprises administering to a subject a type II anti-CD20 antibody having at least a first antibody exposure amount of the present disclosure and a type II anti-CD20 antibody having a second antibody exposure amount. Any of the type II anti-CD20 antibodies described herein, such as the GA101 antibody such as obinutuzumab, can be used. In some embodiments, the second antibody exposure amount is not provided until about 18 weeks to about 26 weeks after the first antibody exposure amount. In some embodiments, the second antibody exposure amount is not provided until about 18 weeks after the first antibody exposure amount, about 19 weeks after the first antibody exposure amount, about 20 weeks after the first antibody exposure amount, about 21 weeks after the first antibody exposure amount, about 22 weeks after the first antibody exposure amount, about 23 weeks after the first antibody exposure amount, about 24 weeks after the first antibody exposure amount, about 25 weeks after the first antibody exposure amount, or about 26 weeks after the first antibody exposure amount. In some embodiments, the second antibody exposure amount is not provided until any of less than about 26, 25, 24, 23, 22, 21, 20, or 19 weeks after the first antibody exposure amount. In some embodiments, the second antibody exposure amount is not provided until any of greater than about 18, 19, 20, 21, 22, 23, 24, or 25 weeks after the first antibody exposure amount. That is, the second antibody exposure amount is not provided until any of a range of weeks having an upper limit of 26, 25, 24, 23, 22, 21, 20, or 19 weeks and an independently selected lower limit of 18, 19, 20, 21, 22, 23, 24, or 25 weeks, where the lower limit is less than the upper limit.

[0187] The dosing regimens described herein use a consistent system for observing and recording the time between doses, whereby a first dose is administered to a patient on day 1. As described herein, the antibody exposure of the present disclosure may include one or two doses. In cases where the antibody exposure contains one dose, the reference (as described herein) to a second antibody exposure that is not provided until a period of time has elapsed after the first antibody exposure refers to the amount of time elapsed between the dose of the first antibody exposure (e.g., on day 1) and the dose of the second antibody exposure. When the first antibody exposure includes two doses, the first dose of the first antibody exposure is provided on day 1. In cases where the antibody exposure contains two doses, the reference (as described herein) to a second antibody exposure that is not provided until a period of time has elapsed after the first antibody exposure refers to the amount of time elapsed between the first dose of the two doses of the first antibody exposure (e.g., on day 1) and the first dose of the two doses of the second antibody exposure. For example, if the method of the present disclosure includes a first antibody exposure having two doses and a second antibody exposure having two doses, and the second antibody exposure is not provided until about 22 weeks after the first antibody exposure, the interval between the first dose of the first antibody exposure and the first dose of the second antibody exposure is about 22 weeks.

[0188] In some embodiments, the first antibody exposure of the present disclosure includes one or two doses of the type II anti-CD20 antibody of the present disclosure. In some embodiments, the first antibody exposure contains a total exposure of about 1800 mg to about 2200 mg of the type II anti-CD20 antibody. In some embodiments, the first antibody exposure contains a total exposure of about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, or about 2200 mg of the type II anti-CD20 antibody.

[0189] In some embodiments, the first antibody exposure amount includes a dose for two administrations. In some embodiments, the first antibody exposure amount includes a first dose of a type II anti-CD20 antibody of about 900 mg to about 1100 mg and a second dose of a type II anti-CD20 antibody of about 900 mg to about 1100 mg. In some embodiments, the first dose of the first antibody exposure amount contains about 1000 mg of the type II anti-CD20 antibody. In some embodiments, the second dose of the first antibody exposure amount contains about 1000 mg of the type II anti-CD20 antibody. In some embodiments, the second dose of the first antibody exposure amount is not provided until about 1.5 weeks to about 2.5 weeks after the first dose of the first antibody exposure amount. In some embodiments, the second dose of the first antibody exposure amount is not provided until about 2 weeks after the first dose of the first antibody exposure amount.

[0190] In some embodiments, the second antibody exposure amount of the present disclosure includes a dose of the type II anti-CD20 antibody of the present disclosure for one or two administrations. In some embodiments, the second antibody exposure amount contains a total exposure amount of the type II anti-CD20 antibody of about 1800 mg to about 2200 mg. In some embodiments, the second antibody exposure amount contains a total exposure amount of the type II anti-CD20 antibody of about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, or about 2200 mg.

[0191] In some embodiments, the second antibody exposure comprises two doses. In some embodiments, the second antibody exposure comprises a first dose of a type II anti-CD20 antibody of about 900 mg to about 1100 mg and a second dose of a type II anti-CD20 antibody of about 900 mg to about 1100 mg. In some embodiments, the first dose of the second antibody exposure contains about 1000 mg of the type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure contains about 1000 mg of the type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure is not provided until about 1.5 to about 2.5 weeks after the first dose of the second antibody exposure. In some embodiments, the second dose of the second antibody exposure is not provided until about 2 weeks after the first dose of the second antibody exposure.

[0192] In some embodiments, the type II anti-CD20 antibody of the present disclosure is administered intravenously (e.g., by IV infusion).

[0193] In some embodiments, the methods of the present disclosure further comprise administering an effective amount of an immunosuppressant (e.g., in combination with a type II anti-CD20 antibody as described herein). Multiple classes of immunosuppressants are known in the art, including, but not limited to, cytotoxic agents such as cell growth inhibitors (e.g., antibiotics, alkylating agents (e.g., cyclophosphamide, also known as cytophosphane)), inosine monophosphate dehydrogenase inhibitors, antimetabolites such as protein synthesis inhibitors, folic acid analogs, purine analogs, pyrimidine analogs, etc.), immunosuppressive antibodies, glucocorticoids, drugs targeting immunophilins (e.g., tacrolimus, sirolimus, rapamycin, and their analogs, cyclosporine, etc.), mTOR active site inhibitors, mycophenolic acid and its 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 comprises mycophenolate mofetil. In some embodiments, the immunosuppressant comprises CellCept® (Roche). In some embodiments, the immunosuppressant comprises Myfortic® (Novartis). An effective amount of the immunosuppressant of the present disclosure is known in the art and can be readily confirmed by standard assays. For example, mycophenolate mofetil can be administered at 2.0 - 2.5 g / day as illustrated in FIG. 1. In some embodiments, mycophenolate mofetil can be started at a divided dose of 1000 mg / day (twice daily) and titrated to a maximum divided dose of 2.0 - 2.5 g / day (twice daily) by the fourth week.

[0194] In some embodiments, the immunosuppressant can be administered before, during, or after administration of the type II anti-CD20 antibody of the present disclosure, for example, as a treatment for lupus. In some embodiments, the immunosuppressant can be administered throughout the period of treatment with the type II anti-CD20 antibody of the present disclosure. In some embodiments, mycophenolate mofetil can be administered as described above throughout the period of treatment with the type II anti-CD20 antibody.

[0195] In some embodiments, the methods of the present disclosure further comprise administering an effective amount of a glucocorticoid or corticosteroid (e.g., in combination with a type II anti-CD20 antibody as described herein). A variety of naturally occurring 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 comprises methylprednisolone. In some embodiments, the glucocorticoid / corticosteroid comprises prednisone. An effective amount of the glucocorticoid / corticosteroid of the present disclosure is known in the art and can be readily confirmed by standard assays. For example, methylprednisolone can be administered IV once daily at a dose of 750-1000 mg. As another example, prednisone can be administered orally at 0.5 mg / kg and optionally tapered to 7.5 mg / day.

[0196] In some embodiments, the glucocorticoid can be administered before, during, or after administration of the type II anti-CD20 antibody of the present disclosure, for example, to treat LN clinical activity. In some embodiments, the glucocorticoid 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, 80 mg of methylprednisolone can be administered IV 30 to 60 minutes before administration of the type II anti-CD20 antibody of the present disclosure. In some embodiments, prednisone (e.g., administered orally) and / or methylprednisolone (e.g., administered IV) can be administered by treatment followed by maintenance therapy (e.g., mycophenolate mofetil or cyclophosphamide).

[0197] In some embodiments, the methods of the present disclosure further comprise administering an effective amount of an antihistamine agent (e.g., in combination with a type II anti-CD20 antibody as described herein). Antihistamine agents known in the art and currently in clinical use include histamine H1-receptor and histamine H2-receptor antagonists, or inverse agonists. In some embodiments, the antihistamine agent comprises diphenhydramine. The effective amount of the antihistamine agent of the present disclosure is known in the art and can be readily confirmed by standard assays. For example, diphenhydramine can be administered at an oral dose of 50 mg.

[0198] In some embodiments, the antihistamine agent can be administered before, during, or after administration of the type II anti-CD20 antibody of the present disclosure, for example, as prophylactic treatment. In some embodiments, the antihistamine agent 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, 50 mg of diphenhydramine can be administered orally 30 to 60 minutes before administration of the type II anti-CD20 antibody of the present disclosure.

[0199] In some embodiments, the methods of the disclosure further comprise administering an effective amount of a non-steroidal anti-inflammatory drug or NSAID (e.g., 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, sulfonanilides, and the like. In some embodiments, the NSAID comprises acetaminophen. The effective amount of the NSAID of the disclosure is known in the art and can be readily confirmed by standard assays. For example, acetaminophen can be administered at an oral dosage of 650 - 1000 mg.

[0200] In some embodiments, the NSAID can be administered, for example, as a prophylactic treatment, before, during, or after administration of the type II anti-CD20 antibody of the disclosure. In some embodiments, the NSAID can be administered before administration of the type II anti-CD20 antibody of the disclosure, for example, 30 - 60 minutes before the type II anti-CD20 antibody. In some embodiments, 650 - 1000 mg of acetaminophen can be orally administered 30 - 60 minutes before administration of the type II anti-CD20 antibody of the disclosure.

[0201] In some embodiments, the methods of the disclosure further comprise administering an effective amount of an antimalarial agent (e.g., in combination with a type II anti-CD20 antibody as described herein). Examples of antimalarial agents that can be used include, but are not limited to, hydroxychloroquine, chloroquine, and quinacrine. In some embodiments, the antimalarial agent can be administered before, during, or after administration of the type II anti-CD20 antibody of the disclosure, for example, as a treatment for one or more symptoms of lupus.

[0202] In some embodiments, the methods of the disclosure further comprise 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 may be used include LFA-1 antibodies such as efalizumab (RAPTIVA®) commercially available from Genentech or alpha4 integrin antibodies such as natalizumab (ANTEGREN®) available from Biogen, or diazacyclic phenylalanine derivatives, phenylalanine derivatives, phenylpropionic acid derivatives, enamine derivatives, propanoic acid derivatives, alkanonic acid derivatives, substituted phenyl derivatives, aromatic amine derivatives, ADAM disintegrin domain polypeptides, antibodies against alphavbeta3 integrin, aza-bridged bicyclic amino acid derivatives, and the like, but are not limited thereto. In some embodiments, the integrin antagonist may be administered before, during, or after administration of the type II anti-CD20 antibody of the disclosure, for example, as a treatment for one or more symptoms of lupus.

[0203] In some embodiments, the methods of the disclosure further comprise administering an effective amount of a cytokine antagonist (e.g., in combination with a type II anti-CD20 antibody as described herein). Examples of cytokine antagonists that may be used include antagonists for IL-1, IL-lα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15 (e.g., antagonist antibodies); tumor necrosis factors such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit ligand (KL), but are not limited thereto. In some embodiments, the cytokine antagonist may be administered before, during, or after administration of the type II anti-CD20 antibody of the disclosure, for example, as a treatment for one or more symptoms of lupus.

[0204] In some embodiments, the methods of the disclosure further comprise administering an effective amount of a hormone (e.g., in combination with a type II anti-CD20 antibody as described herein). In some embodiments, the hormone (e.g., for hormone replacement therapy) can be administered, for example, for medical treatment in women with lupus, before, during, or after administration of the type II anti-CD20 antibody of the disclosure.

[0205] In some embodiments, the methods of the disclosure further comprise administering standard of care treatment (e.g., in combination with a type II anti-CD20 antibody as described herein). In some embodiments, the standard of care treatment can be administered, for example, to treat or prevent one or more symptoms of lupus, before, during, or after administration of the type II anti-CD20 antibody of the disclosure. In certain embodiments, the standard of care treatment can be administered after a second antibody exposure of the disclosure. For example, the type II anti-CD20 antibody of the disclosure can be administered to a patient as described herein as induction therapy, and then the patient can be treated according to care as standard maintenance therapy. Standard of care treatment for lupus is well known in the art and includes, but is not limited to, angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor blockers, cyclophosphamide, mycophenolate mofetil (e.g., at a dosage such as 2.0 - 2.5 g / day as described herein), azathioprine, and glucocorticoids or corticosteroids (e.g., prednisone such as prednisone taper).

[0206] In some embodiments, the methods of the disclosure further comprise administering an antihypertensive agent (e.g., in combination with a type II anti-CD20 antibody as described herein). In some embodiments, the antihypertensive agent can be administered before, during, or after administration of the type II anti-CD20 antibody of the disclosure, for example, to treat or prevent hypertension. In some embodiments, antihypertensive agents include, but are not limited to, ACE inhibitors and angiotensin receptor blockers. In some embodiments, the antihypertensive agents listed in Table 5 are administered, for example, at doses within the ranges described in Table 5.

[0207] In some embodiments, the methods of the disclosure result in a complete renal response (CRR) in an individual. In some embodiments, CRR includes all of the following: normalization of serum creatinine, inactive urinary sediment, and a urine protein-to-creatinine ratio of <0.5. In some embodiments, normalization of serum creatinine is characterized by serum creatinine below the upper limit of normal (ULN) of the central laboratory value and / or, if the baseline (e.g., day 1) serum creatinine is within the normal range of the central laboratory value, serum creatinine that is ≤15% above baseline and below the ULN of the central laboratory value range. In some embodiments, inactive urinary sediment is characterized by <10 RBCs / high power field (HPF) and / or the absence of red blood cell casts. For a more detailed discussion of CRR and partial renal response (PRR) in LN, see, for example, Chen, Y.E. et al. (2008) Clin. J. Am. Soc. Nephrol. 3:46-53.

[0208] In some embodiments, the methods of the disclosure result in a complete renal response (CRR) or a partial renal response (PRR) in an individual. In some embodiments, the PRR includes one or more of the following: normalization of serum creatinine, inactive urine sediment, and a urine protein-to-creatinine ratio of <0.5. In some embodiments, the PRR includes one or more of the following: reduction of serum creatinine, reduced urine sediment, reduction of one or more symptoms including, but not limited to, reduction of proteinuria, and any other improvement in renal function. In some embodiments, the CRR or PRR includes reduction of one or more biomarkers of lupus activity including, but not limited to, reduced levels of anti-dsDNA antibody, anti-nuclear antibody / ENA, anti-complement antibody, complement C3 and / or C4, and reduced complement activity (e.g., as measured by the CH50 assay).

[0209] In some embodiments, the methods of the disclosure result in the depletion of circulating peripheral B cells in an individual. In some embodiments, after administration of the type II anti-CD20 antibody of the disclosure (e.g., according to any of the methods described herein), the circulating peripheral B cells are present in peripheral blood at about 10 cells / μL or less, about 9 cells / μL or less, about 8 cells / μL or less, about 7 cells / μL or less, about 6 cells / μL or less, about 5 cells / μL or less, about 4 cells / μL or less, about 3 cells / μL or less, about 2 cells / μL or less, or about 1 cell / μL or less. In some embodiments, the circulating peripheral B cells in an individual are depleted by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%. In some embodiments, the depletion of circulating peripheral B cells is, for example, compared to a pre-treatment corresponding measurement in the same individual, or compared to a corresponding measurement in a control individual (e.g., an untreated individual), after a first antibody exposure (e.g., comprising 1 or 2 doses of an anti-CD20 antibody as described herein), after a second antibody exposure (e.g., comprising 1 or 2 doses of an anti-CD20 antibody as described herein), 3 months after treatment (e.g., after receiving the first and / or second antibody exposure as described herein), 6 months after treatment (e.g., after receiving the first and / or second antibody exposure as described herein), 9 months after treatment (e.g., after receiving the first and / or second antibody exposure as described herein), or 12 months after treatment (e.g., after receiving the first and / or second antibody exposure as described herein), and refers to a measurement of circulating peripheral B cells taken at that time.

[0210] Methods for assaying for depletion of peripheral blood circulating B cells in an individual, 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 for depletion of peripheral blood circulating B cells (see, e.g., Vital, E.M. et al. (2011) Arthritis Rheum. 63:3038-3047). 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).

[0211] IV. Product or Kit In another aspect of the present invention, there is provided a product or kit comprising a material useful for the treatment, prevention, and / or diagnosis of the disorders described above. The product or kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials such as glass or plastic. The container can hold a composition effective for the treatment, prevention, and / or diagnosis of a medical condition, either by itself or in combination with another composition, and can have a sterile access port (e.g., the container can be a vial having a stopper penetrable by an intravenous injection solution bag or a hypodermic needle). At least one active agent in the composition is an antibody described herein (e.g., a type II anti-CD20 antibody of the present disclosure). The label or package insert indicates that the composition is to be used for treating a selected medical condition, for example, according to any of the methods described herein. Alternatively, or in addition, the product or kit can further comprise 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 can further comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0212] In some embodiments, a kit is provided herein that includes a container comprising a type II anti-CD20 antibody of the present disclosure and any pharmaceutically acceptable carrier, and optionally, a package insert including instructions for use regarding treating or delaying the progression of lupus nephritis in an individual, wherein, for example, the instructions indicate that a type II anti-CD20 antibody of at least a first antibody exposure amount and a type II anti-CD20 antibody of a second antibody exposure amount are administered to the individual, and the second antibody exposure amount is not provided until about 18 to about 26 weeks after the first antibody exposure amount, wherein the first antibody exposure amount comprises one or two doses of the type II anti-CD20 antibody, the first antibody exposure amount comprises a total exposure amount of about 1800 mg to about 2200 mg of the type II anti-CD20 antibody, the second antibody exposure amount comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure amount comprises a total exposure amount of about 1800 mg to about 2200 mg of the type II anti-CD20 antibody. In some embodiments, a kit is provided herein that includes a container comprising a type II anti-CD20 antibody of the present disclosure and any pharmaceutically acceptable carrier, and optionally, a package insert including instructions for use regarding treating or delaying the progression of 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 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. In some embodiments of any of the above embodiments, the type II anti-CD20 antibody is obinutuzumab.

[0213] The product may still further include a second or third container containing a second drug, wherein the anti-CD20 antibody (e.g., a type II anti-CD20 antibody of the present disclosure) is the first drug, and the article further includes a package insert regarding the use of the second drug to treat a subject. Exemplary second drugs include chemotherapeutic agents, immunosuppressive agents, antimalarial agents, cytotoxic agents, integrin antagonists, cytokine antagonists, hormones, and any of the therapeutic agents that can be used in combination with a type II anti-CD20 antibody as described herein. The products in these embodiments may further include a package insert indicating that the composition can be used to treat a particular medical condition.

[0214] It is understood that any of the above products may include the immunoconjugate of the present invention instead of, or in addition to, the anti-CD20 antibody.

[0215] This specification is considered to be sufficient to enable those skilled in the art to practice the present invention. In addition to the modifications shown and described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description, and those are within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Examples

[0216] The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the present invention. The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes taking them into account have been proposed to those skilled in the art, and it is understood that they should be included within the spirit and scope of this specification and the scope of the appended claims.

[0217] Example 1: Pharmacological study of obinutuzumab administered with mycophenolate mofetil in patients with class III / IV lupus nephritis Study design This Phase II study was designed to evaluate the safety and efficacy of obinutuzumab (i.e., a type II anti-CD20 antibody) as add-on therapy to mycophenolate mofetil (MMF) in patients with active ISN / RPS class III / IV lupus nephritis (LN). The Phase II study is a parallel-group, double-blind, placebo-controlled study that compares the efficacy and safety of obinutuzumab plus MMF with the efficacy and safety of placebo plus MMF in patients with class III and IV proliferative LN (Figure 1).

[0218] The study is also a prospective, multi-center study. In some embodiments, including the diagnosis of SLE according to current ACR criteria (at least four criteria must be present, one of which must be a positive antinuclear antibody), patients diagnosed with ISN / RPS class III or IV LN are enrolled at sites worldwide. The study includes standard care therapy using an angiotensin-converting enzyme (ACE) inhibitor / angiotensin II receptor blocker, MMF (dosed at 2.0 - 2.5 g / day), and prednisone taper.

[0219] As described in more detail below, patients are 18 - 75 years old and have ISN / RPS 2003 class III or IV proliferative LN based on a renal biopsy performed within six months prior to screening (see Weening, JJ (2004) J. Am. Soc. Nephrol. 15:241 - 250) and may have concomitant class V disease (e.g., class III / V or class IV / V). Patients with class III (C) or class IV (C) disease are excluded due to a lower likelihood of response in these classifications.

[0220] The inclusion criteria for study subjects include the following. (a) Signed informed consent form (b) 18 - 75 years of age, (c) Ability to comply with the study protocol, (d) Diagnosis of systemic lupus erythematosus (SLE) according to current ACR criteria (at least 4 criteria must be present, one of which must be a positive antinuclear antibody), (e) Diagnosis of ISN / RPS 2003 class III or IV LN based on a renal biopsy performed within 6 months prior to screening (the patient may also show class V disease simultaneously in addition to class III or class IV disease), (f) Presence of active urinary sediment based on ≥10 RBC / HPF or erythrocyte casts, and (g) Proteinuria (>1.0 urinary protein-to-creatinine ratio based on 24-hour urine collection).

[0221] Important exclusion criteria include the following. (a) Currently active retinitis, seizure disorder that is not fully controlled, acute confusional state, myelitis, stroke or stroke syndrome, cerebellar ataxia, or dementia due to SLE, (b) Presence of rapidly progressive glomerulonephritis (presence of crescents in ≥50% of glomeruli evaluated by renal biopsy, or defined by a doubling of serum creatinine within 12 weeks prior to screening), (c) Estimated GFR <30 mL / min, or severe renal dysfunction defined by the need for dialysis or renal transplantation, (d) Sclerosis involving more than 50% of glomeruli in renal biopsy, (e) Treatment with cyclophosphamide or calcineurin inhibitor within 3 months prior to randomization, and (f) Unstable diseases with thrombocytopenia or at risk of developing clinically significant bleeding or organ failure requiring treatment such as plasma exchange, or acute blood or platelet transfusion.

[0222] Patients receive an initial 1000 mg of methylprednisolone intravenous (IV) injection before or during screening and may receive up to 3000 mg of methylprednisolone IV before randomization for severe clinical activity according to the guidelines for daily care for these patients. Patients receive 80 mg of methylprednisolone (or methylprednisolone placebo) IV on the day of obinutuzumab / placebo infusion to reduce infusion-related events. The oral prednisone taper is 0.5 mg / kg and is tapered over 12 weeks. This modified taper is initiated recognizing that prednisone doses above 10 mg / day are associated with significant adverse events including an increased risk of cardiovascular events (Bichile, T. and Petri, M. (2014) Presse Med. 43:e187-195). Experience with rituximab in the past has shown that complete and partial renal responses may be possible in the absence of oral prednisone or with prednisone taper, thus allowing the use of lower doses of corticosteroids (Condon, M.B. et al. (2013) Ann. Rheum. Dis. 72:1280-1286).

[0223] Patients are observed for 12 months until the primary endpoint is assessed, and at 6 months, an interim analysis is performed to assess the initial differences in CRR. All patients have a central reading of renal biopsy histopathology and repeat renal biopsies if available based on clinical status and local practice. All patients are assessed by high-sensitivity flow cytometry (HSFC) to assess the ability of obinutuzumab to deplete circulating peripheral B cells and an interim PD analysis is performed to evaluate whether patients have completely depleted peripheral CD19+ B cells as expected.

[0224] Medication and Non-Study Medicinal Products The dosing regimen for the study is obinutuzumab administered by IV infusion at a dose of 1000 mg on days 1, 15, 168, and 182 (test group); or an obinutuzumab placebo (e.g., saline IV corresponding to a 1000 mg dose of obinutuzumab) administered by IV infusion on days 1, 15, 168, and 182. Obinutuzumab / placebo is administered in a hospital or clinical setting where a complete resuscitation facility is immediately available and under the close supervision of the investigator or a designated person. At the end of the infusion, if necessary, the IV line is left in place for at least 1 hour to allow for the administration of IV medications. During this period, the IV line may be removed if no adverse events occur.

[0225] After screening, patients who have not yet received MMF receive MMF at a dose of 1500 mg / day in divided doses (2 - 3 times / day) and, if tolerated, the dose is titrated to a target dose of up to 2.0 - 2.5 g / day in divided doses (2 - 3 times / day) by week 4 for all patients. If the dose needs to be reduced, it may be decreased in decrements of 250 - 500 mg. During screening or randomization, if clinically indicated, patients may receive methylprednisolone IV at a dose of 750 - 1000 mg once daily for up to 3 days to treat underlying LN clinical activity. Patients receive oral prednisone at a dose of 0.5 mg / kg during screening or randomization, and the tapering of this prednisone dose is initiated on day 16 according to the protocol and the prednisone dose is reduced to 7.5 mg / day by week 12. These treatments are further detailed below.

[0226] Combination Therapy and Clinical Practice Patients who have not yet taken vitamin D (400 IU / day) and calcium supplements (1200 mg / day of calcium citrate or 1500 mg / day of calcium carbonate) start taking these supplements in the randomization. All patients take an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker that is titrated to achieve adequate blood pressure control as recommended by the National Kidney Foundation for chronic kidney disease. Other agents that affect proteinuria, including but not limited to nondihydropyridine calcium antagonists, dihydropyridine calcium antagonists, aldosterone antagonists, and direct renin antagonists, are not initiated during the study.

[0227] Mycophenolate mofetil (MMF) All patients continue or initiate the use of MMF during screening or at the latest on day 1. The initial oral dose is 1500 mg / day, given as a two- or three-divided dose and titrated upward to 2.0 - 2.5 g / day as a divided dose by week 4. MMF may be increased upward to a maximum dose of 2.5 g / day in 500 mg / week increments if tolerated. Reduction is possible due to adverse effects.

[0228] Newly diagnosed LN patients with no prior exposure to MMF are recommended to initiate an induction agent (MMF or cyclophosphamide) and then re-evaluate eligibility. Some patients initially treated with MMF or cyclophosphamide achieve a complete renal response (CRR) and thus require minimal additional immunosuppression (Dall’Era, M. et al. (2011) Arthritis Care Res. 63:351 - 357).

[0229] For patients enrolled in the study who are already receiving a dose of MMF above 1500 mg / day, titrate MMF upward to a target of 2.5 g / day as a divided dose by week 4 if tolerated. Give the patient's current MMF dose as a two- or three-divided dose and increase by 500 mg / week if tolerated.

[0230] Corticosteroid Administration All patients receive a combination of IV and oral corticosteroids as part of their initial LN therapy. Methylprednisolone (e.g., Solu-Medrol®) is administered for two purposes: as part of routine care for patients with class III or IV active LN and to reduce infusion-related reactions (IRR) on the days of obinutuzumab / placebo infusions. Based on the investigator's judgment and local practice, up to three doses of 1000 mg of IV methylprednisolone are given. Up to three infusions of 1000 mg may be initiated before screening or between screening.

[0231] On days 1, 15, 168, and 186, patients receive 80 mg of IV methylprednisolone or placebo 30 to 60 minutes before the study drug infusion to prevent IRR. In addition, oral prednisone may be initiated before screening or between screening, and tapering is started on day 2. Oral prednisone is given at a dose of 0.50 mg / kg / day (maximum dose of 60 mg) from day 2 to day 16, excluding the days of IV methylprednisolone / placebo infusions, and continued until day 16. After day 16, tapering of prednisone is started.

[0232] All patients receive the planned corticosteroid taper starting on day 16. Patients gradually reduce their prednisone dose over 12 weeks until the dose reaches 7.5 mg / day by week 12. After a 14-week taper, patients continue prednisone at 7.5 mg / day or less. In patients in whom the disease is too clinically active for them to proceed to the first step of their prednisone taper, such as based on active urine sediment, rising serum creatinine, or moderate to severe additional renal symptoms, these patients may continue to receive their initial prednisone dose for up to 28 days.

[0233] To maintain consistency in the treatment of renal erythema, retreatment with higher doses of corticosteroids is permitted if a clinically appropriate judgment is made by the investigator and the patient meets the criteria for renal erythema. Patients may be treated with prednisone (maximum 0.5 mg / kg, not exceeding 60 mg / day) for 2 weeks. Prednisone is then tapered to achieve 10 mg / day within 6 weeks after the initial corticosteroid increase. Patients may receive corticosteroids or undergo surgery for urgent illnesses (mental trauma, severe asthma) if clinically warranted, and if possible, limit the use of corticosteroids to a total of ≤7 days. The investigator may then increase the prednisone dose to ≤2.5 mg / day to treat symptoms of adrenal insufficiency or corticosteroid withdrawal after the patient's dose has been tapered to 10 mg / day.

[0234] Patients experiencing severe further renal SLE erythema may receive treatment with additional oral corticosteroids if a clinically appropriate judgment is made by the investigator. Based on the severity of the disease and organ system complications, these patients may be retreated with prednisone (maximum 1.0 mg / kg) for up to 2 weeks, and the dose is tapered to 7.5 mg / day. Patients experiencing mild or moderate further renal erythema may temporarily increase their prednisone dose up to a maximum of 20 mg per day, and if a clinically appropriate judgment is made by the investigator, this dose may be tapered over 4 weeks. If gastrointestinal complications temporarily preclude treatment with oral corticosteroids, equivalent doses of IV corticosteroids are possible.

[0235] Antimalarial agents Patients taking antimalarial agents in the study registration maintain a constant dose throughout the study. Patients who have not taken antimalarial agents in the past may be registered in the study, but should not initiate antimalarial agents if they are experiencing disease erythema that is unresponsive to corticosteroids. Table 4 lists the antimalarial agents and dose ranges expected to be used during the study. TIFF2025106262000018.tif34170

[0236] Hypotensive therapy All patients who are not currently taking an ACE inhibitor or an angiotensin receptor blocker should start on Day 1 of screening. Patients should take an ACE inhibitor or an angiotensin receptor blocker for at least 10 days before randomization. Combination therapy using the two drugs is not possible.

[0237] During screening, make every effort to adequately manage the patient's blood pressure. Titrate the dose of the ACE inhibitor or angiotensin receptor blocker up to the maximum dose recommended in the current prescribing information to achieve adequate blood pressure control as recommended by the Eighth Report of the Joint National Committee on the Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (James, P.A. et al. (2014) JAMA 311:507 - 520). If adequate blood pressure control is not achieved, the patient may start an additional antihypertensive agent, but should not start an agent that affects proteinuria (e.g., non - dihydropyridine calcium channel blockers, aldosterone antagonists, direct renin antagonists). Do not start additional agents that specifically target the renin - angiotensin system during the study. The presented dosage ranges for specific ACE inhibitors and angiotensin receptor blockers are listed in Table 5. If patients cannot tolerate ACE inhibitors and angiotensin receptor blockers, they may use a direct renin inhibitor or an aldosterone antagonist, but not in combination. TIFF2025106262000019.tif131170

[0238] Study objectives and evaluation items The primary objective of this proof-of-concept study is to measure the complete renal response (CRR) at week 52 following obinutuzumab administration. The ability of obinutuzumab plus MMF to achieve CRR at week 52 is compared to placebo plus MMF and evaluated by improvement in renal function, urine sediment, and proteinuria. The second objective includes assessing the safety of obinutuzumab in this patient population, the ability of obinutuzumab to induce an overall response (CRR + PRR) at week 52, the ability of obinutuzumab to improve the time to response (CRR + PRR) over 52 weeks, and the ability of obinutuzumab to improve biomarkers of LN disease activity (e.g., see Tew, G.W. et al. (2010) Lupus 19:146-157 for reduced anti-dsDNA antibody levels, increased C3 and C4 levels).

[0239] The primary efficacy endpoint is the proportion of subjects who achieve CRR as assessed at week 52. In this study, CRR is defined by the attainment of all of the following: (a) Normalization of serum creatinine based on the following: (i) If the baseline (day 1) is not within the normal range of the central laboratory values, serum creatinine below the upper limit of normal (ULN) of the central laboratory values (ii) If the serum creatinine at baseline (day 1) is within the normal range of the central laboratory values, serum creatinine below 15% of baseline and below the ULN of the central laboratory values (b) Inactive urine sediment (based on <10 RBC / high power field (HPF) and / or absence of red blood cell casts), and (c) A urine protein-to-creatinine ratio of <0.5.

[0240] Any patient who switches to rescue medication at 52 weeks is considered a non-responder. The proportion of patients achieving CRR within the treatment group is compared using the Cochran-Mantel-Haenszel (CMH) test, with race (African Caribbean / African American vs. other races) and region (USA vs. non-USA) as stratifying factors. If the test results are favorable for the obinutuzumab group at the α < 0.1-level (one-sided), it is concluded as a change towards a better renal response associated with the obinutuzumab group.

[0241] The secondary efficacy evaluation items are as follows: (a) Analysis of the proportion of patients achieving overall response (CRR + PRR) at week 52, (b) Time to overall response (CRR + PRR) during 52 weeks, (c) Percent reduction or increase from baseline, and mean and median evaluations of biomarkers of LN disease activity (e.g., reduction in anti-dsDNA antibody levels, increased C3 and C4 levels), (d) Proportion of patients achieving PRR at week 52, as defined by all of the following: (i) Serum creatinine of ≤ 15% above baseline level (ii) Absence of RBC / HPF and erythrocyte casts of ≤ 50% above baseline (iii) 50% improvement in the urine protein-to-creatinine ratio and meeting one of the following conditions: (A) Urine protein-to-creatinine ratio of < 1.0 if the baseline urine protein-to-creatinine ratio is ≤ 3.0, (B) Urine protein-to-creatinine ratio of < 3.0 if the baseline protein-to-creatinine ratio is > 3.0, (e) Proportion of patients achieving CRR at week 24, (f) Time to CRR during 52 weeks, (g) Proportion of patients achieving modified CRR (mCRR1) at week 52, using the definition of the primary efficacy measurement and removing the urine sediment analysis criteria, mCRR1 refers to the achievement of normalization of serum creatinine based on the following: (i) Serum creatinine below the ULN of the central laboratory value range (ii) If the serum creatinine at baseline (day 1) is within the normal range of the central laboratory value, serum creatinine > 15% above baseline and below the ULN of the central laboratory value range (iii) Urine protein - to - creatinine ratio < 0.5 (h) The proportion of patients achieving a second CRR (mCRR2) at week 52, as defined by the following attainment (i) Normalization of serum creatinine based on the following (A) Serum creatinine ≤ ULN of the central laboratory value range (B) If the serum creatinine at baseline (day 1) exceeds the normal range of the central laboratory value, serum creatinine > 15% above baseline, or if the serum creatinine at baseline (day 1) is within the normal range of the central laboratory value, S ≤ ULN of the central laboratory value range (ii) Inactive urine sediment (based on < 10 RBC / HPF and absence of red blood cell casts) (iii) Urine protein - to - creatinine ratio < 0.5

[0242] The pharmacodynamic (PD) objective is to compare the changes in CD19 + B cells in peripheral blood after treatment with obinutuzumab versus placebo. The levels of circulating CD19 + B cells are measured at screening and on days 15, 28, 84, 168, 364, and 728.

[0243] The pharmacokinetic (PK) objectives are to characterize the pharmacokinetics of obinutuzumab in the LN population and to evaluate potential PK interactions between obinutuzumab and concomitant medications including mycophenolate mofetil (MMF). The dose-concentration-time data of obinutuzumab are analyzed using a non-linear mixed effects model (by the software NONMEM). The PK model is further developed in main parameters (e.g., clearance) using PK profile data including the effects of main covariates (e.g., gender, race / ethnicity, weight, baseline biochemical and hematological parameters, degree of underlying disease). The derivation of individual measures of exposure, e.g., the observed concentration-time curve (AUC) and the area under the maximum concentration (C max ) depends on the final PK model used. Serum obinutuzumab is summarized (mean, minimum, maximum, SD, and geometric mean) and reported.

[0244] The exploratory objectives of the study include the assessment of exploratory biomarkers at the pre-dose level (including but not limited to B cell subsets and levels of proteins and / or mRNAs in serum, blood, and urine) and their potential association with outcomes, the assessment of changes in exploratory biomarkers (including but not limited to B cell subsets and levels of proteins and / or mRNAs in serum, blood, and urine) over time in patients dosed with obinutuzumab versus placebo, the assessment of the occurrence of further renal erythema, the assessment of the impact of the therapy on outcomes reported by patients and physicians, and the evaluation of renal biopsy histopathology (e.g., the presence of CD19+ B cells at screening and / or subsequent biopsies). The exploratory assessment items include the following: (a) Levels of circulating B cell subsets at screening and on days 15, 28, 84, 168, 364, and 728, (b) Levels of exploratory biomarkers (including but not limited to B cell subsets and levels of proteins and / or mRNAs in serum, blood, and urine) at screening and on days 1, 15, 28, 84, 168, 252, 364, 532, and 728, (c) Percentage of patients experiencing flushing in the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)-2K, (d) Percentage of patients experiencing renal flushing over a period of 52 to 104 weeks, (e) Percentage of patients achieving CRR, mCRR1, and mCRR2 at additional time points (including week 12 and week 36), (f) General assessment by the physician (visual analog scale captured at screening, at the baseline visit, and at multiple time points during the study execution), and (g) Renal biopsy findings.

[0245] Laboratory, Biomarkers, and Other Biological Samples The following laboratory evaluations are recorded during the study: (a) Hematology: Hemoglobin, Hematocrit, RBC, Mean Corpuscular Volume, Mean Corpuscular Hemoglobin, WBC (absolute and differential), and quantitative platelet count, (b) Blood Chemistry: AST / SGOT, ALT / SGPT, Alkaline Phosphatase, Amylase, Lipase, Total Protein, Albumin, Cholesterol, Total Bilirubin, Urea, Uric Acid, Creatinine, Random Glucose, Potassium, Sodium, Chloride, Calcium, Phosphorus, Lactate Dehydrogenase, CPK, and Triglyceride, (c) Urinalysis: Dipsticks for blood, nitrates, protein, and glucose, as well as urine microscopy, (d) 24-hour urine collection performed at randomization and at months 3, 6, 9, and 12 (analysis regarding total protein, total creatinine, and creatinine clearance), (e) Flow Cytometry: B cells (including CD19, CD27, CD38, and IgD), T cells (CD3, CD4, CD8), and NK cells (CD16, CD56), (f) Autoantibody Profile: Antinuclear Antibody (ANA), Anti-dsDNA, Anti-Sm, Anti-RNP, Anti-Ro, Anti-La, and Anti-C1q, (g) Anti-dsDNA Antibody: Measured by ELISA at all visits as part of the SLEDAI-2K assessment, (h) Quantitative immunoglobulins: Total Ig levels including IgG, IgM, and IgA isotypes. (i) Complement: C3, C4, and CH50. (j) Antibody titers: Antibody titers against common antigens (rubella, tetanus, influenza, pneumococcus), and (k) Pregnancy test: Urine pregnancy tests performed at screening and before each study drug infusion. Infusions are not administered if the test is not negative. At all other time points, urine pregnancy tests are performed based on menstrual history and risk of pregnancy.

[0246] Send the following samples for analysis: Cells from blood and urine for B cells and lupus-related biomarkers (including, but not limited to, CD19+ B cells and mRNA related to B cell activity), serum and urine for B cells and lupus-related biomarkers (including, but not limited to, B cell activating factor or BAFF), and renal biopsy slides for immunopathological evaluation.

[0247] Infusion Before each infusion of the study drug or placebo, patients receive prophylactic treatment with oral acetaminophen (650 – 1000 mg) and diphenhydramine (50 mg, or equivalent dose of a similar agent), which are given 30 – 60 minutes before the start of the infusion period. Patients receiving obinutuzumab receive 80 mg of methylprednisolone IV, and patients receiving placebo receive placebo-methylprednisolone IV, which are given 30 – 60 minutes before the start of the obinutuzumab / placebo infusion. If a patient experiences a low-grade infusion-related reaction (IRR) that is considered clinically significant by the investigator, the infusion rate should be reduced to half of the initial infusion rate (adhering to the non-Hodgkin lymphoma protocol infusion rate and schedule). After the reaction has resolved, the infusion should maintain the reduced rate for an additional 30 minutes. If tolerated at the reduced rate, the infusion rate may be increased to the next rate in the infusion schedule. Patients experiencing a severe IRR should immediately interrupt their infusion and receive aggressive symptomatic treatment. The infusion should not be restarted until all symptoms have resolved. At the restart of the infusion, the rate should be half of the rate that induced the reaction. Instructions regarding the administration of obinutuzumab infusions are provided in Table 6 below. TIFF2025106262000020.tif128170

[0248] All renal biopsies and reports obtained as part of the study registration were microscopically imaged and sent to an online central reading portal to avoid histological evaluation oversight by local renal pathologists. Specialist researchers evaluated these biopsies and made the final determination. Every effort was made to screen patients to complete this process, but completion of the screening activities was not mandatory. All new biopsies obtained during screening or the study were processed in a manner that allowed immunohistochemical staining of the tubulointerstitium for the presence of B cells. The study encourages, but does not require, repeat renal biopsies for patients who have not achieved a CRR, and calls for the strengthening of study sites that perform repeat renal biopsies.

[0249] Example 2: Obinutuzumab is superior to rituximab in inducing B cell cytotoxicity in samples from patients with rheumatoid arthritis and systemic lupus erythematosus by Fc gamma receptor-dependent and independent effector mechanisms. Some patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) treated with standard-dose rituximab (RTX) exhibit inefficient B cell depletion and poor clinical responses that can be increased by delivering higher doses, indicating that standard-dose RTX is a suboptimal therapy in these patients. To investigate whether better responses could be achieved with other anti-CD20 mAbs, RTX was compared with obinutuzumab (OBZ), a next-generation glycoengineered type II anti-CD20 mAb, in a series of in vitro assays measuring B cell cytotoxicity in SLE and RA samples. In an in vitro whole blood assay, OBZ was found to be at least two-fold more efficient than RTX in inducing B cell cytotoxicity. By analyzing this difference in detail, it was found that RTX induced more potent complement-dependent cytotoxicity (CDC) than OBZ. In contrast, OBZ was more effective in triggering Fc gamma receptor (FcγR)-mediated effector mechanisms, including activation of NK cells and neutrophils. OBZ was also more efficient in inducing direct cell death. This was true overall for all CD19+ B cells and naïve (IgD+CD27−), and specifically for switched (IgD−CD27+) memory B cells, whose higher frequency is associated with poor clinical responses after RTX.

[0250] Materials and methods Patients All participants in this study provided consent in accordance with the Declaration of Helsinki, which was approved by the local research ethics committee. All patients with RA fulfilled the American College of Rheumatology (ACR) / European League Against Rheumatism (EULAR) classification criteria (Aletaha D. et al. 2010 Ann Rheum Dis. 2010;69(9):1580 - 8), and all patients with SLE met the ACR classification criteria (Petri M. et al. Arthritis Rheum. 2012;64(8):2677 - 86).

[0251] Antibodies and reagents The anti - CD20 mAbs used in the study included rituximab (RTX), ofatumumab (OBZ), and unglycosylated wild - type glycosylated OBZ (OBZ Gly ), and in some experiments, OBZ with a mutated Fc portion (P329G LALA) that does not engage any Fc - related effector functions (Herter S. et al. Cancer Research. 2015;75(15 Supplement):2460), OBZ - PG LALA. Roche Innovation Center Zurich, Switzerland generated all anti - CD20 mAbs except RTX, which was a gift from the Pharmacy Department of University College Hospital, U.K. The FcγRII antagonist (Greenman J. et al. Mol Immunol. 1991;28(11):1243 - 54) was produced in - house.

[0252] Flow cytometry and B - cell isolation mAbs conjugated with fluorescent dyes were procured from Becton Dickinson biosciences or Biolegend, U.K.): CD3 (phycoerythrin [PE]-Cy7), CD15 (fluorescein isothiocyanate, FITC), CD16 (allophycocyanin, APC), CD19 (Alexa Fluor 700), CD45 (PE), CD56 (PE), CD107a (Brilliant Violet 421), CD11b (PE), CD62L (APC), propidium iodide and annexin V (FITC). Flow cytometry was performed using a Becton Dickinson LSR Fortessa cell analyzer. Lymphocytes were identified based on forward and side scatter characteristics. B cells were identified as CD19+ or CD20+, T cells as CD3+, and NK cells as CD3−56+. Neutrophils were identified based on forward and side scatter characteristics and positivity for CD15. The mean fluorescence intensity (MFI) of CD11b and CD62L in samples incubated with mAbs was compared with the MFI in samples incubated without the antibody.

[0253] In all experiments, peripheral blood mononuclear cells (PBMCs) were separated from whole blood samples by Ficoll-Hypaque density gradient, and B cells were isolated from PBMCs using the EasySep™ Human B Cell Enrichment Kit (Cambridge, U.K.).

[0254] Whole blood B cell depletion assay Briefly, 300 μl of freshly drawn whole blood anticoagulated with heparin was incubated at 37 °C and 5% CO2 for 24 hours with or without 1 μg / mL of mAb. Then, as described above, the samples were stained with anti-CD3, anti-CD19, and anti-CD45, after which the red blood cells were lysed and analyzed by flow cytometry (Reddy V. et al. Arthritis & rheumatology. 2015;67(8):2046 - 55). As described above, the B cell depletion % was calculated from the ratio of B cells remaining after treatment to T cells and defined as the cytotoxicity index (CTI) (Mossner E. et al. Blood. 2010;115(22):4393 - 402, and Reddy V. et al. Arthritis & rheumatology. 2015;67(8):2046 - 55).

[0255] Surface fluorescence quenching assay As described above, a surface fluorescence quenching assay was performed (Beers S.A. et al. Blood. 2008;112(10):4170 - 7, and Reddy V. et al. Arthritis & rheumatology. 2015;67(8):2046 - 55) to evaluate the internalization of mAb by B cells. Isolated B cells were incubated for 6 hours with 5 μg / mL of mAb conjugated with Alexa - 488 and then analyzed by flow cytometry.

[0256] Complement - dependent cytotoxicity assay As described above, the CDC assay was performed (Cragg M.S. et al. Blood. 2004;103(7):2738-43). Isolated B cells were incubated with mAb at a concentration of 10 μg / mL for 30 minutes at 37°C and 5% CO2. Samples were stained with fluorescent-conjugated anti-CD19 antibody, annexin V (Av), and propidium iodide (PI), and the frequency of CD19+Av+PI+ cells was evaluated by flow cytometry. Freshly collected healthy human serum was used as a source of complement. To define the activity regarding complement, a portion of the serum was heat-inactivated (HIS) at 56°C for 30 minutes. The ability of the mAb to activate complement and lyse target cells was evaluated by the relative frequency of CD19+Av+PI+ cells in samples incubated with healthy serum or HIS.

[0257] Direct cell death Isolated B cells were incubated in RPMI supplemented with 10% heat-inactivated fetal bovine serum, with or without mAb at a concentration of 10 μg / mL for 6 hours at 37°C and 5% CO2. The frequency of CD19+Av+ cells in samples with mAb compared to that in samples without mAb represented the ability of the mAb to induce direct cell death.

[0258] NK cell degranulation assay By measuring the expression of CD107a or LAMP-1 (lysosome-associated membrane protein 1), samples from a whole blood B cell depletion assay were used to evaluate NK cell degranulation, which is upregulated in NK cell activation and correlates with NK cell-mediated ADCC (Alter G. et al. J Immunol Methods. 2004;294(1-2):15-22, and Aktas E. et al. Cell Immunol. 2009;254(2):149-54). Thus, the frequency of CD3-56+107a+ NK cells in samples using mAb was compared with the frequency of CD3-56+107a+ NK cells in samples incubated without using mAb. NK cell activation is associated with increased activity of metalloproteinases, which cleave CD16 whose expression is reduced in NK cell activation (Romee R. et al. Blood. 2013;121(18):3599-608). Thus, the degree of CD16 loss was also used as an indirect measure of NK cell activation (Grzywacz B. et al. Leukemia. 2007;21(2):356-9; author reply9, and Bowles J.A. et al. Blood. 2006;108(8):2648-54).

[0259] Neutrophil activation assay Neutrophil activation was evaluated in a whole blood assay by measuring the increase in CD11b or the decrease in CD62L on CD15+ neutrophils by flow cytometry (Golay J. et al. Blood. 2013;122(20):3482-91, and Wittmann S. et al. Cytometry A. 2004;57(1):53-62). The ability of mAb to induce neutrophil activation was evaluated by comparing the mean fluorescence intensity (MFI) of CD11b and CD62L on CD15+ neutrophils in samples incubated with or without using mAb.

[0260] Statistical analysis Data were analyzed using GraphPad Prism Software version 5.0. Comparisons between groups were made using the Mann-Whitney test or Wilcoxon paired signed-rank as appropriate. The Spearman correlation coefficient was used to analyze correlations.

[0261] Results Type II mAb is more efficient than type I in inducing B cell cytotoxicity. To evaluate the effects of type I and type II mAbs on B cell cytotoxicity in RA and SLE samples, whole blood B cell depletion assays were performed as described above (Reddy V. et al. Arthritis & rheumatology. 2015;67(8):2046 - 55). OBZ is >2-fold more efficient than RTX in depleting B cells from patients with RA (n = 31) and SLE (n = 34), and unglycosylated OBZ Gly and both OBZ were more efficient than RTX in all samples tested (Figure 2A). In both RA and SLE, the median CTI of OBZ was significantly higher than the CTIs of OBZ Gly and RTX, and the CTI of OBZ Gly was significantly higher than the CTI of RTX in both RA and SLE. In RA, the median (interquartile range) CTIs of RTX, OBZ Gly , and OBZ were 29 (13 - 50), 60 (47 - 70), and 67 (60 - 77), respectively, and in SLE were 19 (11 - 39), 40 (31 - 53), and 59 (52 - 70), respectively. Thus, in both RA and SLE, there was a hierarchy of mAb-induced B cell depletion: RTX < OBZ Gly < OBZ. Noticeable variability between samples, particularly with RTX, was also described in B cell depletion. The superior efficiency of OBZ Gly (having an Fc similar to RTX that is not glycosylated) is due to its type II nature being the cause of the difference between the two types of mAbs in the efficiency of B cell depletion in the whole blood assay, while the increased efficiency of OBZ compared to OBZ Gly presents as being attributable to afucosylation of the Fc portion.

[0262] B cells internalize RTX more rapidly than OBZ. Next, the superior efficiency of type II mAbs in B cell depletion was investigated to determine whether it was consistent with their type II nature, and thereby to evaluate whether B cells from patients with RA and SLE internalized RTX to a greater extent than OBZ. The median (range) percentage of surface-accessible RTX to OBZ, 55 (51–57) vs 83 (81–84) respectively in RA (n = 5), and 60 (49–77) vs 76 (70–80) respectively in SLE (n = 8), revealed that RTX internalized more extensively than OBZ after 6 h incubation (Figure 2B). To evaluate the role of FcγRIIb in this internalization, experiments were performed in the presence of the FcγR-blocking mAb AT10 as previously observed to partially inhibit the internalization of RTX and to a lesser extent for OBZ, using a non-glycosylated type II antibody variant of OBZ (Figure 2B) (Reddy V. et al. Arthritis & rheumatology. 2015;67(8):2046–55).

[0263] RTX is more efficient than OBZ in inducing complement-dependent cytotoxicity. The ability of these mAbs to induce CDC was also investigated. The frequency of lysed B cells (CD19+Av+PI+) was significantly greater in samples incubated with RTX in the presence of normal human serum (NHS) compared to heat-inactivated serum (HIS), with a median (range) difference of 10.9% (8.1–21), while the difference for samples incubated with OBZ was found to be 4.8% (0.9–6.5) (Figure 2C). The mean ± SD fold increase in lysed cells in samples incubated with NHS vs HIS was 1.9 ± 0.5 and 1.2 ± 0.2 for RTX and OBZ, respectively (Figure 2D). Thus, the data demonstrate that RTX was superior to OBZ in causing CDC.

[0264] OBZ is more efficient than RTX in activating NK cells. These CDC results were consistent with the nature of the mAbs of type I and II, but did not match the excellent efficiency of the type II mAbs in the whole blood assay. Next, the ability of the mAbs to induce FcγR-mediated effector mechanisms was investigated; first, NK activation in the whole blood B cell depletion assay was evaluated. Gating as shown in Figure 3E enabled the assessment of NK degranulation associated with CD16 expression (increase in CD107a). The highest percentage of CD107a+ NK (CD3-CD56+) cells was seen in the CD56+CD16- fraction (Figures 3A-3G), which demonstrated that NK cell degranulation downregulated CD16, as previously reported (Grzywacz B. et al. Leukemia. 2007;21(2):356-9; author reply 9).

[0265] By establishing these parameters, a comparable assay was performed to compare RTX and OBZ. After 24 hours of incubation in the absence of mAb, there were no significant differences in the frequencies of NK cells, CD107a+ NK cells, CD16+ NK cells, or B cells between patients with RA (n = 18) and SLE (n = 23) (Figure 4A). However, the median (range) frequencies of CD3−CD56+CD107a+ activated NK cells were significantly higher in samples incubated with OBZ compared to RTX, 5.1% (1.9–22) vs. 2.8% (0.3–14) and 5.5% (0.6–12) vs. 4.3% (1.2–8.9), respectively, in both RA and SLE, there were significantly lower median (range) frequencies of CD16+ NK cells, 69 (36–94) vs. 89 (83–97) and 66 (42–91) vs. 84 (61–95), respectively (Figure 4B). Furthermore, in SLE, the frequency of CD3−CD56+CD107a+ NK cells in samples incubated with OBZ was significantly higher and doubled compared to the frequency of CD3−CD56+CD107a+ NK cells incubated with RTX (Figure 4B). Additionally, NK cell activation as evaluated by acquisition of CD107a or loss of CD16, or the doubling rate of the frequency of CD3−CD56+CD107a+ NK cells was found to be greater in RA compared to SLE (Figure 4B). NK cell activation as evaluated by the frequency of CD3−CD56+CD107a+ NK cells by RTX and OBZ was significantly correlated with the frequency of CD3−CD56+CD107a+ NK cells in samples incubated without using mAb, r 2 = 0.89, p < 0.05; r 2 = 0.78, p < 0.05 (Figure 4C), and in SLE, r 2 = 0.52, p < 0.05; r 2=0.36, p < 0.05 (Figure 4D). However, there was a stronger correlation in RA compared to SLE. Collectively, these data suggest that disease-related defects in NK cell activation in SLE may contribute to the inefficient B cell depletion described in the total blood depletion assay (Figure 2A), and that the baseline activation state of NK cells can affect the response to mAb-mediated activation in both RA and SLE (Figures 4B and 4C).

[0266] Next, we investigated whether the different activation of NK cells by RTX and OBZ was due to type I and type II characteristics and / or due to the use of Fc engineering with OBZ Gly ) which may have wild-type glycosylation or completely lack FcγR engagement and consequently have reduced efficiency / ineffectiveness in inducing ADCC and CDC (OBZ-PG LALA). In both RA (n = 6) and SLE (n = 12), no significant differences were seen in the frequency of CD3-CD56+CD107a+ or CD3-CD56+CD16+ NK cells in samples incubated without mAb compared to samples incubated with OBZ-PG LALA, indicating, as expected, that FcγR engagement is essential. In these samples, furthermore, OBZ was more efficient than OBZ Gly and RTX in depleting B cells in the total blood assay (Figure 5A), but the increasing hierarchy in the frequency and fold increase of CD3-CD56+CD107a+ NK cells was described as follows: no mAb = OBZ-PG LALA < RTX < OBZ (Figures 5B and 5C). The frequency of CD3-CD56+CD16+ NK cells in samples incubated with OBZ was significantly lower compared to other samples (Figure 5D). The frequency of CD3-CD56+CD16+ NK cells in RA was also lower in samples incubated with OBZ Gly compared to RTX, but not in SLE (Figure 5D).

[0267] Thus, compared to samples incubated without mAb, the differences in mean fold change in samples incubated with RTX, OBZ-PG LALA, OBZ Gly , and OBZ were 1.2, 1.5, 1.9, and 3.1 in RA and 1.5, 0.8, 1.4, and 1.8 in SLE, respectively, such that the ability of mAb to upregulate CD107a expression on CD3−CD56+ NK cells was greater in RA than in SLE (Figure 5C). The pattern of B cell depletion achieved by mAb in RA and SLE (Figure 5A) was similar to the pattern of NK cell activation by mAb (Figures 5B and 5D), and there was no direct correlation between the percentage of B cell depletion achieved by mAb in individual samples and the frequency of CD3−CD56+CD107a+ NK cells (data not shown).

[0268] OBZ is more efficient than RTX in activating neutrophils. In addition to NK cells, neutrophils have also been presented as mAb effector cells (Golay J. et al. Blood. 2013;122(20):3482-91). Thus, next, the ability of mAb to induce neutrophil activation was evaluated by measuring the expression of CD11b and CD62L as described above (Wittmann S. et al. Cytometry A. 2004;57(1):53-62), and is shown in Figure 9. CD11b forms part of the β-integrin (Mac-1) complex, and multiple genetic variants of this complex are associated with lupus-related phagocyte defects (Bologna L. et al. J Immunol. 2011;186(6):3762-9). In neutrophil activation, while the surface expression of CD11b is upregulated, the expression of the adhesion molecule CD62L is downregulated (Golay J. et al. Blood. 2013;122(20):3482-91, and Wittmann S. et al. Cytometry A. 2004;57(1):53-62). When compared to samples incubated without mAb, it was found that the MFI of CD11b in samples incubated with mAb was significantly higher in both RA (n = 10) and SLE (n = 22) (Figure 6A). In both RA and SLE, a significant correlation was described between the MFI of CD11b in samples incubated in the absence of mAb and the MFI of CD11b in samples incubated with RTX (r 2 = 0.81, 0.82), while a significant correlation regarding OBZ was described in SLE (r 2 = 0.81) but not in RA (Figure 6B). A hierarchy was also described in the ability of mAb to upregulate CD11b such that the MFI of CD11b was lower in the order of samples incubated with RTX < OBZ Gly < OBZ. Furthermore, the MFI of CD62L was greater in the order of samples incubated with RTX > OBZ Gly > OBZ (Figure 6C). In both RA and SLE, the MFI of CD62L in samples incubated in the absence of mAb and RTX (r2 = 0.93, 0.91) and OBZ (r values of 2 = 0.64, 0.71) with the MFI of CD62L in the samples incubated with them was described (Figure 6D). Thus, the hierarchy of mAbs in the ability to activate neutrophils was OBZ > OBZ Gly > RTX. In summary, these data presented that type II mAbs were superior to RTX in activating neutrophils in whole blood assays for both RA and SLE samples. OBZ-PG LALA did not induce significant changes to the markers in both RA (n = 7) and SLE (n = 12) compared to samples incubated in the absence of mAbs.

[0269] OBZ is more efficient than RTX in inducing direct cell death. Next, as shown in Figure 10, the annexin V assay was used to evaluate direct cell death (DCD). The ability of OBZ to induce direct cell death was greater than that of RTX with respect to both the total CD19+ cells and B cell subsets in Figure 7A, namely IgD+CD27− naive cells and IgD−CD27+ switched memory cells (RA, n = 5 and SLE, n = 4). Even in samples incubated without mAbs, the proportion of annexin V+ cells was in the order of DN cells > IgD+CD27+ non-switched memory cells > IgD−CD27+ switched memory cells > IgD+CD27− naive cells from the highest. Nevertheless, OBZ was superior to RTX in inducing DCD.

[0270] B cell subsets: Expression of CD20, FcγRIIb and internalization of mAbs Next, we investigated whether differences between B cell subsets and / or their ability to internalize mAbs in the expression of CD20 and FcγRIIb provided an explanation for the different sensitivities to mAb-induced DCD. B cell subsets displayed different abilities to internalize mAbs such that IgD−CD27+ switched memory cells internalized less mAb than other B cell subsets, and IgD+CD27+ non-switched memory cells internalized mAb to a greater extent than other B cell subsets. Antagonizing the effect of FcγRIIb with AT10 significantly reduced internalization in both cases. Compared to naive and IgD−CD27+ switched memory cells, IgD+CD27+ non-switched memory cells had significantly higher expression of CD20 and FcγRIIb and displayed a significantly greater ability to internalize mAbs, while naive and IgD−CD27+ switched memory cells had significantly lower expression of CD20 and FcγRIIb and displayed significantly lower levels of internalization. DN cells had variable levels of CD20 and FcγRIIb expression worthy of note but internalized RTX to a significantly greater extent than IgD−CD27+ switched memory cells. B cells from both RA and SLE samples consistently displayed low levels of internalization of OBZ. Collectively, these data showed no clear relationship between the susceptibility of B cell subsets to mAb-induced DCD and their ability to internalize mAbs or express CD20 or FcγRIIb.

[0271] Here, obinutuzumab, a type II anti-CD20 mAb with a glycosylated Fc, has been shown to demonstrate at least two-fold greater efficacy in depleting B cells from whole blood samples of patients with both RA and SLE, compared to RTX. This increased activity of OBZ was mainly affected by the Fc gamma receptor (FcγR)-mediated effector mechanism and DCD. In contrast, RTX more efficiently restored complement against CDC but was rapidly internalized and significantly less efficient in causing ADCC and DCD. Subsequent analysis revealed that the expression of the CD20 target molecule was lower in IgD-CD27+ switched memory and DN cells, perhaps causing their relative resistance to depletion by RTX.

Claims

**Claim 1** A method for treating lupus nephritis or delaying its progression in an individual having lupus, comprising administering to the individual at least a first antibody exposure amount of a type II anti-CD20 antibody and a second antibody exposure amount of the type II anti-CD20 antibody, wherein the second antibody exposure amount is not provided until about 18 weeks to about 26 weeks after the first antibody exposure amount, the first antibody exposure amount comprises one or two doses of the type II anti-CD20 antibody, and the first antibody exposure amount comprises a total exposure amount of about 1800 mg to about 2200 mg of the type II anti-CD20 antibody, the second antibody exposure amount comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure amount comprises a total exposure amount of about 1800 mg to about 2200 mg of the type II anti-CD20 antibody, 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, the method. **Claim 2** The method according to claim 1, wherein the first antibody exposure amount comprises a first dose of about 900 mg to about 1100 mg of the type II anti-CD20 antibody and a second dose of about 900 mg to about 1100 mg of the type II anti-CD20 antibody. **Claim 3** The method according to claim 1 or claim 2, wherein the first antibody exposure amount 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 first antibody exposure amount is not provided until about 1.5 weeks to about 2.5 weeks after the first dose of the first antibody exposure amount. **Claim 4** The method according to claim 3, wherein the first antibody exposure amount 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 first antibody exposure amount is not provided until about 2 weeks after the first dose of the first antibody exposure amount. **Claim 5** The method according to claim 3 or claim 4, wherein the first dose of the first antibody exposure amount is about 1000 mg of the type II anti-CD20 antibody. **Claim 6** The method according to any one of claims 3 to 5, wherein the second dose of the first antibody exposure amount is about 1000 mg of the type II anti-CD20 antibody. **Claim 7** The method according to any one of claims 1 to 6, wherein the second antibody exposure amount comprises a first dose of the type II anti-CD20 antibody of about 900 mg to about 1100 mg and a second dose of the type II anti-CD20 antibody of about 900 mg to about 1100 mg.

8. The method according to any one of claims 1 to 7, wherein the second antibody exposure amount 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 amount is not provided until about 1.5 weeks to about 2.5 weeks after the first dose of the second antibody exposure amount.

9. The method according to claim 8, wherein the second antibody exposure amount 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 amount is not provided until about 2 weeks after the first dose of the second antibody exposure amount.

10. The method according to claim 8 or claim 9, wherein the first dose of the second antibody exposure amount is the type II anti-CD20 antibody of about 1000 mg.

11. The method according to any one of claims 8 to 10, wherein the second dose of the second antibody exposure amount is the type II anti-CD20 antibody of about 1000 mg.

12. The method according to any one of claims 1 to 11, wherein the first antibody exposure amount and the second antibody exposure amount are administered intravenously.

13. The method according to any one of claims 1 to 12, wherein the individual has class III or class IV lupus nephritis.

14. The method according to any one of claims 1 to 12, wherein the individual is at risk of developing class III or class IV lupus nephritis.

15. A method for treating lupus nephritis or delaying its progression in an individual having lupus, comprising administering to the individual an effective amount of 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 individual has class III or class IV lupus nephritis, said method.

16. The method according to claim 14, wherein the type II anti-CD20 antibody is administered intravenously.

17. The method according to any one of claims 1 to 16, wherein the individual does not have class III (C) or class IV (C) lupus nephritis.

18. The method according to any one of claims 1 to 17, wherein the individual has class V lupus nephritis.

19. The method according to any one of claims 1 to 18, further comprising administering to the individual an effective amount of an immunosuppressive agent.

20. The method according to claim 19, wherein the immunosuppressive agent comprises mycophenolic acid, a derivative thereof, or a salt thereof.

21. The method according to claim 20, wherein the immunosuppressive agent comprises mycophenolate mofetil.

22. The method according to any one of claims 1 to 21, further comprising administering to the individual an effective amount of a glucocorticoid or corticosteroid.

23. The method according to claim 22, wherein the glucocorticoid or corticosteroid comprises methylprednisolone.

24. The method according to claim 22, wherein the glucocorticoid or corticosteroid comprises prednisone.

25. The method according to any one of claims 1 to 24, further comprising administering to the individual an effective amount of an antihistamine.

26. The method according to claim 25, wherein the antihistamine comprises diphenhydramine.

27. The method according to any one of claims 1 to 26, further comprising administering to the individual an effective amount of a non-steroidal anti-inflammatory drug (NSAID).

28. The method according to claim 27, wherein the NSAID comprises acetaminophen.

29. The method according to any one of claims 1 to 28, further comprising administering to the individual an effective amount of an antihypertensive agent.

30. The method according to claim 29, wherein the antihypertensive agent is an angiotensin-converting enzyme (ACE) inhibitor or an angiotensin receptor blocker.

31. The method according to any one of claims 1 to 30, further comprising subjecting the individual to standard care treatment.

32. The method according to claim 31, wherein the standard care treatment comprises treatment using one or more of an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin receptor blocker, cyclophosphamide, mycophenolate mofetil, azathioprine, and a glucocorticoid or corticosteroid.

33. The method according to any one of claims 1 to 32, wherein the method results in a complete renal response (CRR) in the individual.

34. The method according to any one of claims 1 to 33, wherein the method results in a reduction of circulating peripheral B cells in the individual.

35. The method according to claim 34, wherein the circulating peripheral B cells are CD19+ B cells.

36. The method according to any one of claims 1 to 35, wherein the type II anti-CD20 antibody is a humanized or human antibody.

37. The method according to any one of claims 1 to 36, wherein the type II anti-CD20 antibody is afucosylated.

38. The method according to any one of claims 1 to 37, wherein the heavy chain of the type II anti-CD20 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

7.

39. The method according to any one of claims 1 to 38, wherein the light chain of the type II anti-CD20 antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:

8.

40. The method according to any one of claims 1 to 39, wherein the type II anti-CD20 antibody is obinutuzumab.

41. The method according to any one of claims 1 to 40, wherein the individual is human.

42. A kit for treating lupus nephritis or delaying its progression in an individual having lupus, comprising: (a) 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, said container; An attached document containing instructions for use regarding treating or delaying the progression of lupus nephritis in an individual, wherein the instructions for use indicate that at least a type II anti-CD20 antibody with a first antibody exposure amount and the type II anti-CD20 antibody with a second antibody exposure amount are administered to the individual, and the second antibody exposure amount is not provided until about 18 to about 26 weeks after the first antibody exposure amount, the attached document, wherein the first antibody exposure amount includes one or two doses of the type II anti-CD20 antibody, the first antibody exposure amount includes a total exposure amount of the type II anti-CD20 antibody of about 1800 mg to about 2200 mg, the second antibody exposure amount includes one or two doses of the type II anti-CD20 antibody, and the second antibody exposure amount includes a total exposure amount of the type II anti-CD20 antibody of about 1800 mg to about 2200 mg, the kit.

43. (c)A second drug, wherein the type II anti-CD20 antibody is a first drug, the second drug, and (d)The attached document instructions for use regarding administering the second drug to a subject, further comprising a container according to claim 42.

44. The kit according to claim 43, wherein the second drug is an immunosuppressive agent, glucocorticoid, corticosteroid, antimalarial agent, cytotoxic agent, integrin antagonist, cytokine antagonist, or hormone.

45. A kit for treating or delaying the progression of lupus nephritis in an individual having lupus, (a)A container containing a type II anti-CD20 antibody, wherein the type II anti-CD20 antibody includes 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, the container, and (b)An attached document containing instructions for use regarding treating or delaying the progression of class III or class IV lupus nephritis in an individual, the kit.

46. (c)A second drug, wherein the type II anti-CD20 antibody is a first drug, the second drug, and (d)The attached document instructions for use regarding administering the second drug to a subject, further comprising a container according to claim 45.

47. The kit according to claim 46, wherein the second drug is an immunosuppressant, a glucocorticoid, a corticosteroid, an antimalarial agent, a cytotoxic agent, an integrin antagonist, a cytokine antagonist, or a hormone.

48. A method for treating rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) in an individual or delaying its progression, comprising administering to the individual an effective amount of an anti-CD20 antibody, wherein the antibody comprises a heavy chain variable region 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 variable region 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.

49. The method according to claim 47, wherein the antibody is administered intravenously.

50. The method according to claim 47 or claim 48, wherein the method results in depletion of circulating peripheral B cells in the individual.

51. The method according to claim 49, wherein the circulating peripheral B cells are CD19+ B cells.

52. The method according to any one of claims 47 to 50, wherein the antibody is a humanized or human antibody.

53. The method according to any one of claims 47 to 51, wherein the antibody is afucosylated.

54. The method according to any one of claims 47 to 52, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

7.

55. The method according to any one of claims 47 to 53, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

8.

56. The method according to any one of claims 47 to 54, wherein the antibody is obinutuzumab.

57. The method according to any one of claims 47 to 54, wherein the antibody comprises a modified Fc region.

58. The method according to claim 56, wherein the Fc region comprises a modification for attenuating effector function.

59. The method according to claim 56, wherein the Fc region is a human IgG1 Fc region.

60. The method according to claim 58, wherein the Fc region is numbered according to the EU index and comprises the L234A, L235A, and P329G amino acid substitutions.

61. The method according to any one of claims 48 to 60, wherein the individual is human.

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