Compositions and methods for treating childhood-onset idiopathic nephrotic syndrome

Abstract

The present disclosure provides methods for treating childhood-onset idiopathic nephrotic syndrome (INS) or reducing the risk and / or frequency of relapse from childhood-onset INS in an individual aged 2 to 25. In some embodiments, the methods comprise administering to the individual an effective amount of obinutuzumab.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 423,767, filed November 8, 2022, which is incorporated herein by reference in its entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (146392064240seqlist.xml; size: 41,773 bytes; created on November 2, 2023) are incorporated herein by reference in their entirety.

[0003] FIELD OF THE INVENTION Provided herein are methods of treating childhood-onset idiopathic nephrotic syndrome (INS) in an individual (e.g., an individual aged 2 to 25 years) or reducing the risk and / or frequency of relapse in an individual with childhood-onset INS by administering a type II anti-CD20 antibody. [Background technology]

[0004] Childhood-onset idiopathic nephrotic syndrome (INS), also known as primary nephrotic syndrome (excluding secondary causes), encompasses minimal change disease (MCD) and focal and segmental glomerulosclerosis (FSGS). While the disease is rare, the incidence of childhood-onset INS varies by ethnicity and region, with higher rates among certain ethnic groups, particularly South Asians and African Americans, and genetic factors place them at higher risk for FSGS (Chanchlani and Parekh (2016) Front Pediatr 4:39). The disease is usually first diagnosed between the ages of 2 and 5 (in 70% of MCD cases), typically affects boys more than girls (2:1), and is defined by the presence of nephrotic-range proteinuria, edema, hyperlipidemia, and hypoalbuminemia (Noone et al. (2018) Lancet 392:61-74).

[0005] Patients with childhood-onset INS are initially treated with systemic oral corticosteroids. The frequency of relapses and response to corticosteroid treatment allow for classification of the disease into subtypes reflecting disease severity. These subtypes include "steroid-resistant nephrotic syndrome (underlying genetic causes)," "non- / rare relapsing steroid-sensitive nephrotic syndrome," "FRNS," and "SDNS" (Noone et al. (2018) Lancet 392:61-74). In the latter two subtypes, patients often receive multiple courses of steroids during an episode and continue on steroid maintenance therapy to prevent further relapses. The optimal goal of treatment is maximal steroid sparing while limiting the relapse rate based on the patient's clinical response and drug-related adverse effects.

[0006] Childhood-onset INS recurs in over 75% of patients, and nearly 50% of patients exhibit frequent relapses or steroid dependence (Abdel-Hafez et al. (2017) J. Nephropathol 6:180-186). Among children whose disease cannot be adequately controlled with steroids, several steroid-sparing immunosuppressants (e.g., cyclophosphamide, levamisole, cyclosporine A, tacrolimus, MMF, and rituximab) have been shown to reduce the risk of relapse. However, high-quality head-to-head data comparing these agents are limited (Mason et al. (2020) Clin. J. Am. Soc. Nephrol. 15:983-994).

[0007] The anti-CD20 monoclonal antibody rituximab was first described for the treatment of childhood-onset INS in 2004 (Benz et al. (2004) Pediatr Nephrol 19(7):794-797) and has since emerged as a promising unapproved treatment option in both children and adults with FRNS or SDNS. Numerous investigator-initiated clinical trials and case reports of its use have been described in the literature, with varying clinical response rates (50%–85% of patients in renal remission [relapse-free] at 12 months), and rituximab has been shown to reduce annual relapse rates in patients with FRNS and SDNS. However, patients who do not achieve complete remission at 12 months generally relapse between 8 and 9 months after rituximab treatment, and these relapses occur primarily in the context of B-cell recovery / reconstitution (Iijima et al. (2014) Lancet 384:1273-1281; Colucci et al. (2016) J. Am Soc Nephrol 27:1811-1822).

[0008] Delayed reconstitution of the memory B cell pool is associated with prolonged remission despite tapering or discontinuation of concomitant immunosuppression (Colucci et al. (2019) Front Immunol 10:1653). Obinutuzumab (GAZYVA®, GAZYVARO®) is a humanized, glycoengineered type II anti-CD20 antibody with enhanced depletion of B cells from peripheral blood and tissues compared with type I antibodies such as rituximab and ofatumumab. It is administered as an IV infusion. Consistent with its more potent B cell depletion, obinutuzumab is superior to rituximab for the treatment of adult chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL) when administered in combination with standard chemotherapy and is currently approved worldwide for these indications. Obinutuzumab is also indicated for the treatment of patients with FL who have failed to respond to, or progressed during or after, treatment with rituximab or rituximab-containing regimens. Additionally, obinutuzumab is currently in clinical development for adult and pediatric autoimmune diseases (lupus nephritis [LN], membranous nephropathy, and systemic lupus erythematosus [SLE]).

[0009] Despite current treatments for childhood-onset INS, patients remain at significant risk for growth failure and other significant side effects of steroid-related toxicity. Given the frequent clinical relapse rates after the use of immunosuppressive therapy and the associated potential risk of end-stage renal disease in childhood-onset INS, there remains an unmet need for approved steroid-sparing therapies with improved efficacy and better long-term outcomes.

[0010] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. Summary of the Invention

[0011] In certain aspects, provided herein are methods for treating childhood-onset INS in an individual, comprising administering to the individual at least a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to a type II anti-CD20 antibody, wherein the second antibody exposure is not provided for about 18 weeks to about 26 weeks after the first antibody exposure; the first antibody exposure comprises one or two doses of a type II anti-CD20 antibody, and the first antibody exposure comprises (a) a total exposure of between about 1800 mg and about 2200 mg of a type II anti-CD20 antibody, or (b) if the individual weighs less than 45 kg, a total exposure of between about 36 mg / kg and about 44 mg / kg of a type II anti-CD20 antibody. the second antibody exposure comprises one or two doses of a type II anti-CD20 antibody, and the second antibody exposure comprises (c) a total exposure of between about 1800 mg and about 2200 mg of a type II anti-CD20 antibody, or (d) if the individual's body weight is less than 45 kg, a total exposure of between about 36 mg / kg and about 44 mg / kg of a type II anti-CD20 antibody; the type II anti-CD20 antibody comprises a heavy chain comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3, and a light chain comprising an HVR-L1 sequence of SEQ ID NO: 4, an HVR-L2 sequence of SEQ ID NO: 5, and an HVR-L3 sequence of SEQ ID NO: 6; and the individual is a human between the ages of 2 and 25.Also provided herein is a method for preventing relapse, reducing the risk of relapse, and / or reducing the frequency of relapse in an individual with childhood-onset idiopathic nephrotic syndrome (INS), comprising administering to the individual at least a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to a type II anti-CD20 antibody, wherein the second antibody exposure is not provided for about 18 weeks to about 26 weeks after the first antibody exposure; the first antibody exposure comprises one or two doses of a type II anti-CD20 antibody, and the first antibody exposure comprises (a) a total exposure of between about 1800 mg and about 2200 mg of a type II anti-CD20 antibody, or (b) if the individual weighs less than 45 kg, about 36 mg / kg to about 44 mg / kg. the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, wherein the second antibody exposure comprises (c) a total exposure of between about 1800 mg and about 2200 mg of the type II anti-CD20 antibody, or (d) if the individual weighs less than 45 kg, a total exposure of between about 36 mg / kg and about 44 mg / kg of the type II anti-CD20 antibody; the type II anti-CD20 antibody comprises a heavy chain comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3, and a light chain comprising an HVR-L1 sequence of SEQ ID NO: 4, an HVR-L2 sequence of SEQ ID NO: 5, and an HVR-L3 sequence of SEQ ID NO: 6; and the individual is a human between the ages of 2 and 25.

[0012] In some embodiments, the individual weighs 45 kg or more. In some embodiments, the first antibody exposure comprises a total exposure of between about 1800 mg and about 2200 mg of type II anti-CD20 antibody; the second antibody exposure comprises a total exposure of between about 1800 mg and about 2200 mg of type II anti-CD20 antibody; and the individual weighs 45 kg or more.

[0013] In some embodiments, the first antibody exposure comprises a first dose of a type II anti-CD20 antibody between about 900 mg and about 1100 mg and a second dose of a type II anti-CD20 antibody between about 900 mg and about 1100 mg. In some embodiments, the first antibody exposure comprises a first dose of a type II anti-CD20 antibody between about 18 mg / kg and about 22 mg / kg and a second dose of a type II anti-CD20 antibody between about 18 mg / kg and about 22 mg / kg, and the individual weighs less than 45 kg. In some embodiments, the first 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 first antibody exposure is not provided until about 1.5 weeks to about 2.5 weeks after the first dose of the first antibody exposure. In some embodiments, the first antibody exposure comprises a first dose of a type II anti-CD20 antibody and a second dose of a type II anti-CD20 antibody, wherein the second dose of the first antibody exposure is not provided until about two weeks after the first dose of the first antibody exposure. In some embodiments, the first dose of the first antibody exposure is about 1000 mg of a type II anti-CD20 antibody. In some embodiments, the second dose of the first antibody exposure is about 1000 mg of a type II anti-CD20 antibody. In some embodiments, the first dose of the first antibody exposure is about 20 mg / kg of a type II anti-CD20 antibody, and the individual weighs less than 45 kg. In some embodiments, the second dose of the first antibody exposure is about 20 mg / kg of a type II anti-CD20 antibody, and the individual weighs less than 45 kg. In some embodiments (e.g., in embodiments where the dose(s) of the first antibody exposure are flat doses), the individual weighs 45 kg or more.

[0014] In some embodiments, the second antibody exposure comprises a first dose of a type II anti-CD20 antibody between about 18 mg / kg and about 22 mg / kg and a second dose of a type II anti-CD20 antibody between about 18 mg / kg and about 22 mg / kg, and the individual weighs less than 45 kg. 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 1.5 weeks 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. 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 dose of the second antibody exposure is about 20 mg / kg of a type II anti-CD20 antibody and the individual weighs less than 45 kg. In some embodiments, the second dose of the second antibody exposure is about 20 mg / kg of a type II anti-CD20 antibody and the individual weighs less than 45 kg. In some embodiments (e.g., in embodiments where the dose(s) of the second antibody exposure are flat doses), the individual weighs 45 kg or more.

[0015] In some embodiments, the first and / or second antibody exposure is administered intravenously.

[0016] In some embodiments, the individual has or has been diagnosed with childhood-onset INS. In some embodiments, the individual has frequently relapsing nephrotic syndrome (FRNS). In some embodiments, the childhood-onset INS is steroid-dependent nephrotic syndrome (SDNS). In some embodiments, the individual is in complete remission, for example, prior to administration of the first antibody exposure.

[0017] In some 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 methylprednisolone is administered intravenously to the individual at a dose of 80 mg. In some embodiments, for example, if the individual weighs less than 45 kg, the methylprednisolone is administered intravenously to the individual at a dose of 1.5 mg / kg. In some embodiments, the glucocorticoid or corticosteroid comprises prednisone. In some 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, the diphenhydramine is administered orally at a dose of 0.5 to 1 mg / kg, optionally up to a maximum dose of 50 mg. In some embodiments, the method further comprises administering to the individual an effective amount of acetaminophen. In some embodiments, acetaminophen is administered orally at a dose of 15 mg / kg, optionally up to a maximum dose of 1000 mg.

[0018] In some embodiments, the method results in a sustained complete remission in the individual at one year. In some embodiments, the method results in depletion of circulating peripheral B cells in the individual. In some embodiments, the circulating peripheral B cells are CD19+ B cells. In some embodiments, the B cells are naive B cells (e.g., CD19+CD27- B cells), memory B cells (e.g., CD19+CD27+ B cells), or plasmablasts (e.g., CD19+CD27+CD38++ B cells). In some embodiments, the B cells are CD19+CD3-CD14-CD33-CD56- cells. In some embodiments, after administration of a type II anti-CD20 antibody, B cells are depleted to a level such that no more than about 5 circulating peripheral B cells per μL are present in the peripheral blood from the individual. In some embodiments, B cells are depleted to a level such that no more than about 1 circulating peripheral B cell per μL is present in the peripheral blood from the individual. In some embodiments, B cells are depleted to a level such that circulating peripheral B cells are present in the peripheral blood of the individual at about 0.5 cells / μL or less. In some embodiments, B cells are depleted to a level such that circulating peripheral B cells are present in the peripheral blood of the individual, with depletion achieved after the first antibody exposure. In some embodiments, B cells are depleted to a level below the limit of detection using HSFC. In some embodiments, the HSFC has a lower limit of quantitation (LLOQ) of B cells of about 1.0 cells / μL or less, about 0.8 cells / μL or less, about 0.6 cells / μL or less, about 0.5 cells / μL or less, or 0.441 cells / μL or less. In some embodiments, B cell depletion persists for at least 52 weeks after the first dose of the first antibody exposure. In some embodiments, after administration of a type II anti-CD20 antibody, circulating peripheral B cells in an individual are depleted by at least about 90% compared to a corresponding measurement in the same individual before administration of the type II anti-CD20 antibody, or compared to a corresponding measurement in an individual not treated with a type II anti-CD20 antibody.

[0019] In some embodiments, the first antibody exposure comprises two doses of 1000 mg of a type II anti-CD20 antibody on days 1 and 15 of treatment; the second antibody exposure comprises two doses of 1000 mg of a type II anti-CD20 antibody on days 168 and 182 of treatment; and the type II anti-CD20 antibody is obinutuzumab. In some embodiments, the first antibody exposure comprises two doses of 20 mg / kg of a type II anti-CD20 antibody on days 1 and 15 of treatment; the second antibody exposure comprises two doses of 20 mg / kg of a type II anti-CD20 antibody on days 168 and 182 of treatment; the type II anti-CD20 antibody is obinutuzumab, and the individual weighs less than 45 kg. In some embodiments, the first antibody exposure comprises two doses of 1000 mg of a type II anti-CD20 antibody at weeks 0 and 2 of treatment; the second antibody exposure comprises two doses of 1000 mg of a type II anti-CD20 antibody at weeks 24 and 26 of treatment; the type II anti-CD20 antibody is obinutuzumab. In some embodiments, the first antibody exposure comprises two doses of 20 mg / kg of a type II anti-CD20 antibody at weeks 0 and 2 of treatment; the second antibody exposure comprises two doses of 20 mg / kg of a type II anti-CD20 antibody at weeks 24 and 26 of treatment; the type II anti-CD20 antibody is obinutuzumab, and the individual weighs less than 45 kg. In some embodiments (e.g., in embodiments where the antibody exposure doses are flat doses), the individual weighs 45 kg or more.

[0020] In certain aspects, provided herein is a method for treating childhood-onset INS in an individual or reducing the risk and / or frequency of its recurrence, comprising intravenously administering to the individual a first and second antibody exposure to a type II anti-CD20 antibody; the first antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody in weeks 0 and 2 of treatment; the second antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody in weeks 24 and 26 of treatment; the type II anti-CD20 antibody comprises a heavy chain comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3, and a light chain comprising an HVR-L1 sequence of SEQ ID NO: 4, an HVR-L2 sequence of SEQ ID NO: 5, and an HVR-L3 sequence of SEQ ID NO: 6; the individual is a human between the ages of 2 and 25; and the individual weighs 45 kg or more. In certain aspects, provided herein is a method for treating childhood-onset INS in an individual or reducing the risk and / or frequency of its recurrence, comprising intravenously administering to the individual a first and second antibody exposure to a type II anti-CD20 antibody; the first antibody exposure comprises two doses of 20 mg / kg of type II anti-CD20 antibody in weeks 0 and 2 of treatment; the second antibody exposure comprises two doses of 20 mg / kg of type II anti-CD20 antibody in weeks 24 and 26 of treatment; the type II anti-CD20 antibody comprises a heavy chain comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3, and a light chain comprising an HVR-L1 sequence of SEQ ID NO: 4, an HVR-L2 sequence of SEQ ID NO: 5, and an HVR-L3 sequence of SEQ ID NO: 6; the individual is a human between the ages of 2 and 25; and the individual weighs less than 45 kg.In certain aspects, provided herein is a method for treating childhood-onset INS in an individual or reducing the risk and / or frequency of its recurrence, comprising intravenously administering to the individual a first and a second antibody exposure to a type II anti-CD20 antibody; the first antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 1 and 15 of treatment; the second antibody exposure comprises two doses of 1000 mg of type II anti-CD20 antibody on days 168 and 182 of treatment; the type II anti-CD20 antibody comprises a heavy chain comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3, and a light chain comprising an HVR-L1 sequence of SEQ ID NO: 4, an HVR-L2 sequence of SEQ ID NO: 5, and an HVR-L3 sequence of SEQ ID NO: 6; the individual is a human between the ages of 2 and 25; and the individual weighs 45 kg or more. In certain aspects, provided herein is a method for treating childhood-onset INS in an individual or reducing the risk and / or frequency of its recurrence, comprising intravenously administering to the individual a first and second antibody exposure to a type II anti-CD20 antibody; the first antibody exposure comprises two doses of 20 mg / kg of type II anti-CD20 antibody on days 1 and 15 of treatment; the second antibody exposure comprises two doses of 20 mg / kg of type II anti-CD20 antibody on days 168 and 182 of treatment; the type II anti-CD20 antibody comprises a heavy chain comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3, and a light chain comprising an HVR-L1 sequence of SEQ ID NO: 4, an HVR-L2 sequence of SEQ ID NO: 5, and an HVR-L3 sequence of SEQ ID NO: 6; the individual is a human between the ages of 2 and 25; and the individual weighs less than 45 kg. In some embodiments, the type II anti-CD20 antibody is obinutuzumab.

[0021] In some embodiments of the methods described herein, the type II anti-CD20 antibody is a humanized antibody. In some embodiments, the type II anti-CD20 antibody is afucosylated. 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 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the type II 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. In some embodiments, the type II anti-CD20 antibody is obinutuzumab.

[0022] In some embodiments, the methods of the present invention further comprise administering (e.g., orally administering) prednisone to the individual. In some embodiments, oral prednisone is administered to the individual at a dose of 0.5-1 mg / kg / day, up to a maximum dose of 60 mg / day. In some embodiments, oral prednisone is administered to the individual at a dose of 0.5-1 mg / kg / day until the second week of treatment, tapering to a dose of 5 mg / day by the 24th week. In some embodiments, oral prednisone is administered to the individual at a dose of 0.5-2 mg / kg / day, up to a maximum dose of 60 mg / day. In some embodiments, oral prednisone is administered to the individual at a dose of 0.5-2 mg / kg / day until the second week of treatment, tapering to a dose of 5 mg / day by the 24th week. In some embodiments, the methods of the present invention further comprise administering methylprednisolone to the individual by intravenous (IV) infusion at weeks 0, 2, 24, 26, and 52 of treatment, e.g., prior to administration of the type II anti-CD20 antibody. In some embodiments, if the individual weighs 45 kg or more, 80 mg of methylprednisolone is administered to the individual. In some embodiments, if the individual weighs less than 45 kg, 1.5 mg / kg of methylprednisolone is administered to the individual.

[0023] In certain aspects, provided herein is a kit for treating childhood-onset INS or reducing the risk and / or frequency of its recurrence in an individual, the kit comprising: a container comprising a type II anti-CD20 antibody, wherein the type II anti-CD20 antibody comprises a heavy chain comprising the HVR-H1 sequence of SEQ ID NO: 1, the HVR-H2 sequence of SEQ ID NO: 2, and the HVR-H3 sequence of SEQ ID NO: 3, and a light chain comprising the HVR-L1 sequence of SEQ ID NO: 4, the HVR-L2 sequence of SEQ ID NO: 5, and the HVR-L3 sequence of SEQ ID NO: 6; and a package insert with instructions for using the type II anti-CD20 antibody in any of the methods described above and herein.

[0024] In certain aspects, provided herein is a type II anti-CD20 antibody (eg, obinutuzumab) for use in any of the methods described above and herein.

[0025] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows. [Brief explanation of the drawings]

[0026] [Figure 1]This provides a schematic diagram of a controlled study of the use of the type II anti-CD20 antibody obinutuzumab in the treatment of childhood-onset INS (e.g., frequently relapsing nephrotic syndrome or steroid-dependent nephrotic syndrome) in patients ≥2 to 25 years of age. BID = twice daily; CCOD = common end date of primary analysis; FRNS = frequently relapsing nephrotic syndrome; MMF = mycophenolate mofetil; PO = by mouth; SDNS = steroid-dependent nephrotic syndrome; SFU = safety follow-up; Wk = week. a) Administration of the first dose of study treatment (Day 1) should occur within 24 hours after baseline assessment. However, administration up to 72 hours is possible if necessary. The second infusion should occur on Day 15 ± 1. b) The primary efficacy endpoint of the proportion of participants with sustained complete remission at Year 1 will be measured at Week 52. DETAILED DESCRIPTION OF THE INVENTION

[0027] Childhood-onset idiopathic nephrotic syndrome (INS), also known as primary nephrotic syndrome (excluding secondary causes), encompasses minimal change disease (MCD) and focal and segmental glomerulosclerosis (FSGS). The disease is usually first diagnosed between the ages of 2 and 5 (in 70% of MCD patients), typically affects boys more than girls (2:1), and is defined by the presence of nephrotic-range proteinuria, edema, hyperlipidemia, and hypoalbuminemia (Noone et al. (2018) Lancet 392:61-74). Childhood-onset INS patients are initially treated with systemic oral corticosteroids. However, childhood-onset INS recurs in over 75% of patients, and nearly 50% of patients exhibit frequent relapses or steroid dependence (Abdel-Hafez et al. (2017) J. Nephropathol 6:180-186). Despite current treatments, patients remain at significant risk for growth failure and other significant side effects of steroid-related toxicity. Thus, there is a continuing need for safer and more effective treatments for childhood-onset INS (e.g., FRNS and / or SDNS).

[0028] In one aspect, provided herein is a method for treating childhood-onset INS in an individual, comprising administering to the individual at least a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to a type II anti-CD20 antibody, wherein the second antibody exposure is not provided for about 18 weeks to about 26 weeks after the first antibody exposure; the first antibody exposure comprises one or two doses of a type II anti-CD20 antibody, and the first antibody exposure comprises (a) a total exposure of between about 1800 mg and about 2200 mg of a type II anti-CD20 antibody, or (b) if the individual weighs less than 45 kg, a total exposure of between about 36 mg / kg and about 44 mg / kg of a type II anti-CD20 antibody. the second antibody exposure comprises one or two doses of a type II anti-CD20 antibody, and the second antibody exposure comprises (c) a total exposure of between about 1800 mg and about 2200 mg of a type II anti-CD20 antibody, or (d) if the individual's body weight is less than 45 kg, a total exposure of between about 36 mg / kg and about 44 mg / kg of a type II anti-CD20 antibody; the type II anti-CD20 antibody comprises a heavy chain comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3, and a light chain comprising an HVR-L1 sequence of SEQ ID NO: 4, an HVR-L2 sequence of SEQ ID NO: 5, and an HVR-L3 sequence of SEQ ID NO: 6; and the individual is a human between the ages of 2 and 25.In another aspect, provided herein is a method for reducing the risk and / or frequency of relapse in an individual with childhood-onset idiopathic nephrotic syndrome (INS), comprising administering to the individual at least a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to a type II anti-CD20 antibody, wherein the second antibody exposure is not provided for about 18 weeks to about 26 weeks after the first antibody exposure; the first antibody exposure comprises one or two doses of a type II anti-CD20 antibody, wherein the first antibody exposure is (a) a total exposure of between about 1800 mg and about 2200 mg of a type II anti-CD20 antibody, or (b) if the individual weighs less than 45 kg, between about 36 mg / kg and about 44 mg / kg. the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, wherein the second antibody exposure comprises (c) a total exposure of between about 1800 mg and about 2200 mg of the type II anti-CD20 antibody, or (d) if the individual weighs less than 45 kg, a total exposure of between about 36 mg / kg and about 44 mg / kg of the type II anti-CD20 antibody; the type II anti-CD20 antibody comprises a heavy chain comprising an HVR-H1 sequence of SEQ ID NO: 1, an HVR-H2 sequence of SEQ ID NO: 2, and an HVR-H3 sequence of SEQ ID NO: 3, and a light chain comprising an HVR-L1 sequence of SEQ ID NO: 4, an HVR-L2 sequence of SEQ ID NO: 5, and an HVR-L3 sequence of SEQ ID NO: 6; and the individual is a human between the ages of 2 and 25.

[0029] I. General Techniques The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, for example, widely used methodologies such as those described in: Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M.A.usubel, 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 (JECellis, ed., 1998) Academic Press; Animal Cell Culture (RIFreshney), ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells(JMMiller and MPCalos,eds.,1987);PCR: The Polymerase Chain Reaction,(Mullis et al.,eds.,1994);Current Protocols in Immunology(JEColigan et al.,eds.,1991);Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Press,2000);Using Antibodies:A Laboratory Manual(E.Harlow and D.Lane(Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JDCapra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VTDeVita et al., eds., JBLippincott Company, 1993). .

[0030] II. Definition The term "childhood-onset idiopathic nephrotic syndrome (INS)" refers to idiopathic nephrotic syndromes typically first diagnosed between the ages of 2 and 5 years, which encompass minimal change disease (MCD) and focal and segmental glomerulosclerosis (FSGS), also known as primary nephrotic syndrome (excluding secondary causes).

[0031] The term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab'), and Fv). The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

[0032] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of five basic heterotetrameric units, together with an additional polypeptide called a J chain, and contain ten antigen-binding sites, whereas IgA antibodies consist of two to five basic four-chain units, which can polymerize to form multivalent aggregates with the J chain. In the case of IgG, the four-chain unit is usually approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, whereas the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain contains a variable domain (V) at its N-terminus. H ), followed by three constant domains (C H ), and four C for μ and ε isotypes H Each L chain has a variable domain (V L ) followed by a constant domain at the opposite end. L is V H It is aligned with C L is the first constant domain of the heavy chain (C H 1). Particular 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 Land pair together to form a single antigen-binding site. For the structure and properties of various classes of antibodies, see, for example, page 71 and Chapter 6 of Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, CT, 1994. Light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain designated α, δ, ε, γ, and μ. The gamma and alpha classes are further divided into subclasses based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0033] 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 may be referred to as "VH" and "VL," respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen-binding site.

[0034] The term "variable" refers to the fact that certain segments of the variable domains vary extensively in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains. Rather, it is concentrated in three segments called hypervariable regions (HVRs) in both the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy- and light-chain variable domains each contain four FR regions that largely adopt a beta-sheet configuration, connected by three HVRs that form loops that connect, and in some cases form part of, the beta-sheet structure. The HVRs within each chain are held 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 antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, MD (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0035] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies within the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be produced by, for example, hybridoma techniques (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, 2002) nded.1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, eg, US Patent No. 4,816,567), phage-display technologies (see, eg, Clackson et al. al.,Nature,352:624-628(1991);Marks et al.,J.Mol.Biol.222:581-597(1992);Sidhu et al.,J.Mol.Biol.338(2):299-310(2004);Lee et al. al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. 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 that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., WO 1998 / 24893, WO 1996 / 34096, WO 1996 / 33735, WO 1991 / 10741, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551(1993);Jakobovits et al.,Nature 362:255-258(1993);Bruggemann et al.,Year in Immunol.7:33(1993), U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, Marks et al.,Bio / Technology 10:779-783(1992);Lonberg et al.,Nature 368:856-859(1994);Morrison,Nature 368:812-813(1994);Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0036] The term "naked antibody" refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0037] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. In particular, whole antibodies include those having heavy and light chains, including the Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody may have one or more effector functions.

[0038] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding and / or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10) :1057-1062

[1995] ); including single-chain antibody molecules and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, named for its ability to be readily crystallized. The Fab fragment contains the entire light chain and the variable region domain of the heavy chain (V H ), and the first constant domain of one heavy chain (C HEach 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 roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking antigen. The Fab' fragment consists of a C 3 fragment containing one or more cysteines from the antibody hinge region. H F(ab')2 antibody fragments differ from Fab fragments in that they have a few additional residues at the carboxy terminus of one domain. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) in the constant domains bear a free thiol group. F(ab')2 antibody fragments are originally produced as pairs of Fab' fragments, which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0039] The Fc fragment contains the carboxy-terminal portions of both heavy chains held together by disulfides. The effector functions of an antibody are determined by sequences within the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain cell types.

[0040] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, noncovalent association. The folding of these two domains generates six hypervariable loops (three loops each from the H and L chains) that contribute antigen-binding amino acid residues and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to antigen, albeit with lower affinity than the entire binding site.

[0041] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is a VFv consisting of V proteins joined together into a single polypeptide chain. H and V L Preferably, the sFv polypeptide is an antibody fragment containing the V H Domains and VL The sFv further comprises a polypeptide linker between the domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994).

[0042] "Functional fragments" of the antibodies of the present invention include portions of intact antibodies, generally including the antigen-binding or variable region of the intact antibody, or the Fc region of an antibody that retains or has altered FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules formed from antibody fragments, and multispecific antibodies.

[0043] The term "diabody" refers to a diabody that combines V domains to achieve inter-chain but not intra-chain V domain pairing, thereby resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. H Domains and V L This refers to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the domains. Bispecific diabodies are small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the domains. H Domains and V L Diabodies are heterodimers of two "crossover" sFv fragments in which the domains are present on different polypeptide chains. Diabodies are described, for example, in EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 9 0 :6444-6448 (1993).

[0044] Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). Chimeric antibodies of interest herein include PRIMATIZED® antibodies, in which the antigen-binding region of the antibody is derived from an antibody produced, for example, by immunizing macaque monkeys with an antigen of interest. As used herein, "humanized antibody" is used as a subset of "chimeric antibody."

[0045] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR (defined below) of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence and all or substantially all of the FR regions correspond to those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FRs will usually be no more than six in the H chain and no more than three in the L chain. Humanized antibodies optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that 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, e.g., 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.

[0046] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of 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). Human monoclonal antibodies can also be prepared using 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). van Dijk and van de Winkel,Curr.Opin.Pharmacol., 5 :368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomouse, which have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous gene loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 2001, 2002, 2003, 2004, regarding human antibodies generated via human B-cell hybridoma technology. 103 :3557-3562(2006).

[0047] As used herein, the terms "hypervariable region," "HVR," or "HV" refer to the region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops. Generally, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). In native antibodies, H3 and L3 exhibit the highest diversity of the six HVRs, and H3 in particular is thought to play a unique role in conferring superior specificity to antibodies. 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).

[0048] Several HVR delineations are used and encompassed herein. The Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia instead refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on an analysis of available complex crystal structures. Residues from each of these HVRs are shown below. TIFF2025537197000001.tif62170

[0049] HVRs may include the following "extended HVRs": 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. (supra) for each of these definitions.

[0050] The phrases "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering scheme used for heavy or light chain variable domains in the organization of antibodies in Kabat et al. (supra). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FRs or HVRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues 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 of the antibody's sequence with the "standard" Kabat-numbered sequence at the regions of homology.

[0051] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0052] A "human consensus framework" or "acceptor human framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is derived from a subgroup of variable domain sequences. Generally, these subgroups of sequences are described in Kabat et al., Sequences of Proteins of Immunological Interest, 5 thEd. Public Health Service, National Institutes of Health, Bethesda, MD (1991). For example, for VL, the subgroup can be subgroup kappa I, kappa II, kappa III, or kappa IV, as in Kabat et al. (supra). Additionally, for VH, the subgroup can be subgroup I, subgroup II, or subgroup III, as in Kabat et al. (supra). Alternatively, a human consensus framework may be derived from the above, such as when human framework residues are selected based on homology to the donor framework by aligning the donor framework sequence with a collection of different human framework sequences. An acceptor human framework "derived from" a human immunoglobulin framework or human consensus framework may comprise the same amino acid sequence or may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer.

[0053] A "VH subgroup III consensus framework" comprises a consensus sequence obtained from the amino acid sequences in variable heavy chain subgroup III of Kabat et al. (supra). In one embodiment, the VH subgroup III consensus framework amino acid sequence comprises 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).

[0054] A "VL kappa I consensus framework" comprises a consensus sequence obtained from the amino acid sequences in variable light kappa subgroup I of Kabat et al. (supra). In one embodiment, the VH subgroup I consensus framework amino acid sequence comprises 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).

[0055] For example, an "amino acid modification" at a specified position in the Fc region refers to a substitution or deletion of the specified residue, or an insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent to" a particular residue means an insertion within 1 to 2 residues therefrom. The insertion may be on the N-terminal or C-terminal side of the particular residue. A preferred amino acid modification herein is a substitution.

[0056] An "affinity matured" antibody has one or more alterations in one or more HVRs thereof that result in improved affinity of the antibody for antigen, compared to a parent antibody lacking those alteration(s). In one embodiment, the affinity matured antibody has nanomolar or 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 shuffling VH and VL domains. Random mutagenesis of HVR and / or framework residues has been described, for example, by Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0057] As used herein, the terms "specifically binds to" or "specific for" refer to a measurable and reproducible interaction, e.g., binding between a target and an antibody, that determines the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can, but does not require, exclusive binding.

[0058] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain and includes native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined as stretching from the amino acid residue at Cys226, or from 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 recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, an intact antibody composition can include an antibody population in which all K447 residues have been removed, an antibody population in which the K447 residue has not been removed, and an antibody population having a mixture of antibodies with and without the K447 residue. Native-sequence Fc regions suitable for use in the antibodies of the present invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0059] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. A preferred FcR is a native-sequence human FcR. Additionally, a preferred FcR is one that binds IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., M. Daeron, Annu. Rev. Immunol. 15 :203-234 (1997). FcRs are described 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 also encompassed by the term "FcR" herein.

[0060] 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 (see, 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); WO 2004 / 92219 (Hinton et al.). The in vivo binding to FcRn and serum half-life of human FcRn high-affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides having variant Fc regions. WO 2004 / 42072 (Presta) describes antibody variants with improved or diminished binding to FcR. See, for example, Shields et al., J. Biol. Chem. 9 (2):6591-6604(2001).

[0061] The terms "substantially reduced" or "substantially different," as used herein, refer to a sufficiently high difference between two numerical values ​​(typically one associated with a molecule and the other associated with a reference / comparator molecule) such that one of skill in the art would consider the difference between the two values ​​to be statistically significant with respect to the biological characteristic measured by such values ​​(e.g., Kd values). The difference between the two values ​​is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than 40%, and / or greater than about 50%, depending on the value of the reference / comparator molecule.

[0062] The terms "substantially similar" or "substantially the same," as used herein, refer to a sufficiently high similarity between two numerical values ​​(typically one associated with a molecule and the other associated with a reference / comparator molecule) such that one of skill in the art would consider the difference between the two values ​​to have little or no biological and / or statistical significance with respect to the biological characteristic measured by such values ​​(e.g., Kd values). The difference between the 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%, depending on the reference / comparator value.

[0063] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the dosages and concentrations used.In many cases, physiologically acceptable carriers are pH-buffered aqueous solutions.Examples of physiologically acceptable carriers include buffers such as phosphate, 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™.

[0064] "Package insert" means instructions customarily included in commercial packaging of a drug containing information regarding indications, such as indications, usage, dosage, administration, contraindications, other drug products to be combined with the packaged product, and / or warnings regarding the use of such drug products.

[0065] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the treated individual or cell during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, reducing the rate of disease progression, improving or alleviating the disease state, remission or improved prognosis, and delaying disease progression. Delaying the progression of a disease (e.g., INS) means postponing, preventing, slowing, delaying, stabilizing, and / or postponing the onset of the disease. This delay can be of varying duration depending on the disease history and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can, in effect, include prevention, in that an individual, for example, an individual at risk of developing the disease, does not develop the disease.

[0066] As used herein, "sustained complete remission" refers to a response to treatment that includes a first morning void UPCR ≦0.2 g / g without relapse or any of the following specific intervening events (e.g., occurring after week 8): (1) relapse defined by any of the following events requiring systemic corticosteroid or other immunosuppressive treatment: (a) first morning void UPCR ≧2 g / g or (b) dipstick UA ≧3+ for 3 consecutive days with the most recent urine sample on those 3 days determining a UPCR >0.2 g / g, or (c) any 1 day dipstick UA protein ≧3+ with edema and a urine sample determining a UPCR >0.2 g / g; (2) any systemic corticosteroid use for >14 days in a 30-day period; (3) initiation of any rescue therapy for INS other than systemic corticosteroids; (4) treatment discontinuation due to lack of efficacy; or (5) death.

[0067] As used herein, "CD20" refers to the human B lymphocyte antigen CD20 (also known as CD20, B lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5; its sequence is characterized by SwissProt database entry P11836), a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B lymphocytes and mature B lymphocytes. (Valentine,MA,et al.,J.Biol.Chem.264(19)(1989 11282-11287;Tedder,TF,et al,Proc.Natl.Acad.Sci.USA85(1988)208-12;Stamenkovic,I.,et al.,J.Exp.Med.167(1988)1975-80;Einfeld,DA,et al.,EMBO J.7(1988)711-7;Tedder,TF,et al., J. Immunol. 142 (1989) 2560-8). The corresponding human gene is transmembrane 4 domain, subfamily A, member 1, also known as MS4A1. This gene encodes a member of the transmembrane 4A gene family. Members of this emerging protein family are characterized by common structural features and similar intron / exon splice boundaries and display unique expression patterns between hematopoietic cells and non-lymphoid tissues. This gene encodes a B lymphocyte surface molecule that plays a role in the development and differentiation of B cells into plasma cells. This family member is localized to 11q12 within a cluster of family members. Alternative splicing of this gene results in two transcript variants encoding the same protein.

[0068] The terms "CD20" and "CD20 antigen" are used interchangeably herein and include any variant, isoform, and species homolog of human CD20 that is naturally expressed by cells or expressed on cells transfected with the CD20 gene. Binding of the antibodies of the invention to the CD20 antigen mediates the killing of CD20-expressing cells (e.g., tumor cells) by inactivating CD20. The killing of CD20-expressing cells can occur by one or more of the following mechanisms: cell death / apoptosis induction, ADCC, and CDC.

[0069] Art-recognized synonyms of CD20 include B lymphocyte antigen CD20, B lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5.

[0070] The term "anti-CD20 antibody" according to the present invention refers to an antibody that specifically binds to the CD20 antigen. According to Cragg, MS, et al., Blood 103 (2004) 2738-2743; and Cragg, MS, et al., Blood 101 (2003) 1045-1052, anti-CD20 antibodies can be divided into two types (type I anti-CD20 antibodies and type II anti-CD20 antibodies) depending on their binding properties to the CD20 antigen and their biological activity. See Table 1 below. TIFF2025537197000002.tif66170

[0071] Examples of type II anti-CD20 antibodies include, for example, humanized B-LyI antibody IgG1 (a chimeric humanized IgG1 antibody as disclosed in WO 2005 / 044859), 11B8 IgG1 (as disclosed in WO 2004 / 035607), and AT80 IgG1. Typically, type II anti-CD20 antibodies of the IgG1 isotype exhibit characteristic CDC properties. Type II anti-CD20 antibodies have reduced CDC compared to type I antibodies of the IgG1 isotype (in the case of the IgG1 isotype).

[0072] Examples of Type I anti-CD20 antibodies include, for example, rituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (disclosed in WO 2005 / 103081), 2F2 IgG1 (disclosed in WO 2004 / 035607 and WO 2005 / 103081), and 2H7 IgG1 (disclosed in WO 2004 / 056312).

[0073] The afucosylated anti-CD20 antibody of the present invention is preferably a type II anti-CD20 antibody, more preferably an afucosylated humanized B-Ly1 antibody as described in WO 2005 / 044859 and WO 2007 / 031875.

[0074] 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 against the human CD20 antigen. However, this antibody is not glycoengineered or afucosylated, and therefore has a fucose content of at least 85%. This chimeric antibody contains a human gamma 1 constant domain and is identified under the name "C2B8" in U.S. Patent No. 5,736,137 (Andersen, et al.), issued April 17, 1998, and assigned to IDEC Pharmaceuticals, Inc. Rituximab is approved for the treatment of patients with relapsed or refractory low-grade or follicular CD20-positive B-cell non-Hodgkin's lymphoma. In vitro mechanism-of-action studies have previously shown that rituximab exhibits human complement-dependent cytotoxicity (CDC) (Reff, ME, et. al, Blood 83(2)(1994)435-445). In addition, it exhibits activity in assays measuring antibody-dependent cellular cytotoxicity (ADCC).

[0075] The term "GA101 antibody" as used herein refers to any one of the following antibodies that bind to human CD20: (1) an antibody comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and an 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 sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10; (4) the 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.

[0076] The term "humanized B-Ly1 antibody" refers to the humanized B-Ly1 antibody disclosed in WO 2005 / 044859 and WO 2007 / 031875, which was obtained from the mouse monoclonal anti-CD20 antibody B-Ly1 (variable region of mouse heavy chain (VH): SEQ ID NO: 11; variable region of mouse light chain (VL): SEQ ID NO: 12; see Poppema, S. and Visser, L., Biotest Bulletin 3 (1987) 131-139) by chimerization with a human constant domain derived from IgG1 and subsequent humanization (see WO 2005 / 044859 and WO 2007 / 031875). These "humanized B-Ly1 antibodies" are disclosed in detail in WO 2005 / 044859 and WO 2007 / 031875. Variable region (VH) of the heavy chain of mouse monoclonal anti-CD20 antibody B-Ly1 (SEQ ID NO: 11) TIFF2025537197000003.tif72170 Variable region (VL) of the light chain of mouse monoclonal anti-CD20 antibody B-Ly1 (SEQ ID NO: 12) TIFF2025537197000004.tif69170

[0077] In one embodiment, the "humanized B-LyI antibody" has a heavy chain variable region (VH) selected from the group of SEQ ID NOs: 7, 8, and 13 to 33 (corresponding in particular to B-HH2 to B-HH9 and B-HL8 to B-HL17 of WO 2005 / 044859 and WO 2007 / 031875). In a specific embodiment, such variable domains are 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 WO 2005 / 044859 and WO 2007 / 031875). In one specific embodiment, the "humanized B-Ly1 antibody" has a light chain variable region (VL) of SEQ ID NO: 8 (corresponding to B-KV1 in WO 2005 / 044859 and WO 2007 / 031875). In one specific embodiment, the "humanized B-Ly1 antibody" has a heavy chain variable region (VH) of SEQ ID NO: 7 (corresponding to B-HH6 in WO 2005 / 044859 and WO 2007 / 031875) and a light chain variable region (VL) of SEQ ID NO: 8 (corresponding to B-KV1 in WO 2005 / 044859 and WO 2007 / 031875). Furthermore, in one embodiment, the humanized B-Ly1 antibody is an IgG1 antibody. According to the present invention, such afucosylated humanized B-Ly1 antibodies are glycoengineered (GE) in the Fc region according to the procedures described in WO 2005 / 044859, WO 2004 / 065540, WO 2007 / 031875, Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180 and WO 99 / 154342. In one embodiment, the afucosylated glycoengineered 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.It replaces all previous versions (e.g., Vol. 25, No. 1, 2011, pp. 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). As used herein, obinutuzumab refers to Gazyva® and its biosimilar antibodies. In some embodiments, the humanized B-Ly1 antibody is an antibody or antigen-binding fragment thereof comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10. 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) TIFF2025537197000005.tif50170 Light chain (SEQ ID NO: 10) TIFF2025537197000006.tif30170

[0078] In some embodiments, the humanized B-Ly1 antibody is an afucosylated glycoengineered humanized B-Ly1. Such glycoengineered humanized B-Ly1 antibodies have an altered glycosylation pattern in the Fc region, preferably with a reduced level of fucose residues. Preferably, the amount of fucose is 60% or less of the total amount of oligosaccharides at Asn297 (in one embodiment, the amount of fucose is between 40% and 60%, in another embodiment, the amount of fucose is 50% or less, and in yet another embodiment, the amount of fucose is 30% or less). Furthermore, the oligosaccharides in the Fc region are bisected. These glycoengineered humanized B-Ly1 antibodies have increased ADCC.

[0079] The "ratio of binding ability of an anti-CD20 antibody to CD20 on Raji cells (ATCC-No. CCL-86) compared to rituximab" is determined by direct immunofluorescence measurement (mean fluorescence intensity (MFI) is measured) using the anti-CD20 antibody conjugated with Cy5 and rituximab conjugated with Cy5 in a FACSArray (Becton Dickinson) containing Raji cells (ATCC-No. CCL-86) as described in Example No. 2, and is calculated as follows: TIFF2025537197000007.tif26170

[0080] MFI is mean fluorescence intensity. As used herein, "Cy5 labeling ratio" refers to the number of Cy5-labeled molecules per antibody molecule.

[0081] Typically, the type II anti-CD20 antibody has a ratio of binding ability of the second anti-CD20 antibody to CD20 on Raji cells (ATCC-No. CCL-86) compared to rituximab of 0.3 to 0.6, in one embodiment 0.35 to 0.55, and in another embodiment 0.4 to 0.5.

[0082] In one embodiment, the type II anti-CD20 antibody, eg, the GA101 antibody, has increased antibody-dependent cellular cytotoxicity (ADCC).

[0083] By "antibody with increased antibody-dependent cellular cytotoxicity (ADCC)" is meant an antibody, as that term is defined herein, that has increased ADCC as determined by any suitable method known to those of skill in the art. One accepted in vitro ADCC assay is as follows: 1) The assay uses target cells known to express the target antigen recognized by the antigen-binding region of the antibody; 2) The assay uses human peripheral blood mononuclear cells (PBMCs) isolated from the blood of randomly selected healthy donors as effector cells; 3) The assay is performed according to the following protocol: i) Isolate PBMCs using standard density centrifugation procedures and collect 5x10 6 cells / ml suspended in RPMI cell medium; ii) Target cells are grown by standard tissue culture techniques, harvested from exponential growth phase where viability exceeds 90%, washed in RPMI cell culture medium, and incubated with 100 microcuries of PBS. 51 Label with Cr, wash twice with cell culture medium, and 5 Resuspend in cell culture medium at a density of 100 cells / ml; iii) transferring 100 microliters of the final target cell suspension described above to each well of a 96-well microtiter plate; iv) serially diluting the antibody in cell culture medium from 4000 ng / ml to 0.04 ng / ml and adding 50 microliters of the resulting antibody solution to target cells in a 96-well microtiter plate, testing in triplicate at various antibody concentrations covering the entire concentration range mentioned above; v) As a maximum release (MR) control, three additional wells in the plate containing labeled target cells receive 50 microliters of a 2% (VN) aqueous solution of non-ionic surfactant (Nonidet, Sigma, St. Louis) instead of the antibody solution (point iv above); vi) As a 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×g for 1 minute and incubated at 4° C. for 1 hour; viii) 50 microliters of PBMC suspension (point i above) is added to each well to obtain an effector:target cell ratio of 25:1, and the plate is placed in an incubator in a 5% CO2 atmosphere at 37°C for 4 hours; ix) Cell-free supernatant is collected from each well and experimentally released radioactivity (ER) is quantified using a gamma counter; x) The percentage of specific lysis is calculated for each antibody concentration according to the formula (ER-MR) / (MR-SR) x 100, 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 either an increase in the maximum percentage of specific lysis observed within the antibody concentration range tested above, and / or a decrease in the antibody concentration required to achieve half the maximum percentage of specific lysis observed within the antibody concentration range tested above. In one embodiment, the increase in ADCC is compared to the ADCC measured in the above assay mediated by the same antibody produced by the same host cell, using the same standard production, purification, formulation, and storage methods known to those of skill in the art, except that the comparison antibody (lacking increased ADCC) was not produced by host cells engineered to overexpress GnTIII and / or to reduce expression from the fucosyltransferase 8 (FUT8) gene (including, for example, those engineered for FUT8 knockout).

[0084] Such "increased ADCC" can be obtained, for example, by mutation and / or glycoengineering of the antibody. In one embodiment, the antibody is glycoengineered to have biantennary oligosaccharides attached to the Fc region of the antibody, bisected by GlcNAc, e.g., in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); U.S. Pat. App. Pub. No. 2005 / 0123546 (Umana et al.), Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180). In another embodiment, the antibody is glycoengineered to delete fucose on the carbohydrate chains attached to the Fc region by expression in host cells deficient in protein fucosylation (e.g., Lec13 CHO cells or cells with an alpha-1,6-fucosyltransferase gene (FUT8) deletion 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 WO 2003 / 085107). In yet another embodiment, the antibody sequence has been engineered in its Fc region to enhance ADCC (e.g., in one embodiment, such an engineered antibody variant comprises an Fc region with one or more amino acid substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region).

[0085] The term "complement-dependent cytotoxicity (CDC)" refers to the lysis of human tumor target cells by an antibody of the invention in the presence of complement. CDC can be measured by treating a preparation of CD20-expressing cells with an anti-CD20 antibody according to the invention in the presence of complement. CDC is found when the antibody induces 20% or more lysis (cell death) of tumor cells at a concentration of 100 nM after 4 hours. In one embodiment, 51 Assays were performed using Cr- or Eu-labeled tumor cells, and the released 51Measurements of Cr or Eu are performed. Controls include incubation of tumor target cells with complement but no antibody.

[0086] The term "CD20 antigen expression" is intended to refer to a significant level of expression of the CD20 antigen on a cell, e.g., a T cell or a B cell. In one embodiment, a patient treated according to the methods of the present invention expresses a significant level of CD20 on B cells. CD20 expression on B cells can be determined by standard assays known in the art, for example, CD20 antigen expression is measured using immunohistochemical (IHC) detection, FACS, or via PCR-based detection of corresponding mRNA.

[0087] 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" includes any combination of two or more such molecules.

[0088] The term "about" as used herein refers to a normal error range for the respective value, which would be readily understood by one of ordinary skill in the art. Reference herein to a value or parameter with "about" includes (and describes) embodiments that are directed to the value or parameter itself.

[0089] It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0090] III. Method In one aspect, provided herein is a method for treating childhood-onset idiopathic nephrotic syndrome (INS) in an individual by administering an effective amount of a type II anti-CD20 antibody; the individual is a human between the ages of 2 and 25. In another aspect, provided herein is a method for reducing the risk and / or frequency of relapse in an individual having childhood-onset idiopathic nephrotic syndrome (INS) by administering an effective amount of a type II anti-CD20 antibody; the individual is a human between the ages of 2 and 25. In one aspect, provided herein is a method for treating childhood-onset INS in an individual or a method for depleting circulating peripheral B cells in an individual having childhood-onset INS by administering an effective amount of a type II anti-CD20 antibody; the individual is a human between the ages of 2 and 25.

[0091] In some embodiments, for example, when the individual weighs 45 kg or more, the method comprises administering to the individual a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to a type II anti-CD20 antibody, where the second antibody exposure is not provided for about 18 weeks to about 26 weeks after the first antibody exposure; the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, where the first antibody exposure comprises a total exposure of between about 1800 mg and about 2200 mg of the type II anti-CD20 antibody; and the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, where the second antibody exposure comprises a total exposure of between about 1800 mg and about 2200 mg of the type II anti-CD20 antibody. In some embodiments, for example, when the individual weighs less than 45 kg, the method comprises administering to the individual a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to a type II anti-CD20 antibody, where the second antibody exposure is not provided for about 18 weeks to about 26 weeks after the first antibody exposure; the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, where the first antibody exposure comprises a total exposure of between about 36 mg / kg and about 44 mg / kg of the type II anti-CD20 antibody; and the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, where the second antibody exposure comprises a total exposure of between about 36 mg / kg and about 44 mg / kg of the type II anti-CD20 antibody. As described herein, in some embodiments, an antibody of the invention 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 at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10.In some embodiments, the antibody is obinutuzumab.

[0092] Anti-CD20 antibody Certain aspects of the present disclosure relate to anti-CD20 antibodies, e.g., for use in the methods described herein, e.g., for treating childhood-onset INS (e.g., FRNS or SDNS) or reducing the risk and / or frequency of recurrence thereof. 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 a GA101 antibody.

[0093] Examples of type II anti-CD20 antibodies include, for example, humanized B-LyI antibody IgG1 (a chimeric humanized IgG1 antibody as disclosed in WO 2005 / 044859), 11B8 IgG1 (as disclosed in WO 2004 / 035607), and AT80 IgG1. Typically, type II anti-CD20 antibodies of the IgG1 isotype exhibit characteristic CDC properties. Type II anti-CD20 antibodies have reduced CDC compared to type I antibodies of the IgG1 isotype (in the case of the IgG1 isotype).

[0094] 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 bind to human CD20: (1) HVR-H1 comprising the amino acid sequence of GYAFSY (SEQ ID NO: 1), HVR-H2 comprising the amino acid sequence of FPGDGDTD (SEQ ID NO: 2), HVR-H3 comprising the amino acid sequence of NVFDGYWLVY (SEQ ID NO: 3), HVR-L1 comprising the amino acid sequence of RSSKSLLHSNGITYLY (SEQ ID NO: 4), HVR-L2 comprising the amino acid sequence of QMSNLVS (SEQ ID NO: 5), and HVR-L3 comprising the amino acid sequence of AQNLELPYT (SEQ ID NO: 6). -L3: (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 sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 10; (4) the antibody known as obinutuzumab, or (5) an antibody comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 9 and an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 10. In one embodiment, the GA101 antibody is an IgG1 isotype antibody. In some embodiments, the anti-CD20 antibody comprises HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3 of any of the antibodies described herein, e.g., three HVRs from SEQ ID NO: 7 and three HVRs from SEQ ID NO: 8, three HVRs from SEQ ID NO: 9 and three HVRs from SEQ ID NO: 10, or any of the HVRs of the amino acid sequences provided in Table 2.

[0095] 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. QVQLVQSGAEVKKPGSSVKVSCKAS GYAFSY SWINWVRQAPGQGLEWMGRI FPGDGDTD YNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR NVFDGYWLVYWGQGTLVTVSS (SEQ ID NO: 7) DIVMTQTPLSLPVTPGEPASISC RSSKSLLHSNGITYLY WYLQKPGQSPQLLIY QMSNLVS GVPDRFSGSGSGTDFTLKISRVEAEEDVGVYYC AQNLELPYT FGGGTKVEIKRTV (SEQ ID NO: 8).

[0096] 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. QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 9) DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10)

[0097] 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 SEQ ID NO: 9 and a light chain comprising SEQ ID NO: 10.

[0098] 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 a polypeptide sequence set forth in Table 2 below. TIFF2025537197000008.tif86170TIFF2025537197000009.tif214170TIFF2025537197000010.tif212170TIFF2025537197000011.tif48170

[0099] 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 glycosylation pattern in the Fc region, preferably with a reduced level of fucose residues. Preferably, the amount of fucose is 60% or less of the total amount of oligosaccharides at Asn297 (in one embodiment, the amount of fucose is between 40% and 60%, in another embodiment, the amount of fucose is 50% or less, and in yet another embodiment, the amount of fucose is 30% or less). Furthermore, the oligosaccharides of the Fc region are bisected. In some embodiments, a type II anti-CD20 antibody comprises an Fc region comprising biantennary oligosaccharides bisected by N-acetylglucosamine (GlcNAc). Such glycoengineered humanized anti-CD20 (e.g., B-Ly1) antibodies have increased ADCC.

[0100] The oligosaccharide constituents can significantly influence properties related to the efficacy of therapeutic glycoproteins, including physical stability, resistance to protease attack, interaction with the immune system, pharmacokinetics, and specific biological activity. Such properties may depend not only on the presence or absence of oligosaccharides but also on their specific structure. Some generalizations can be made between oligosaccharide structure and glycoprotein function. For example, certain oligosaccharide structures mediate rapid clearance of glycoproteins from the bloodstream through interactions with specific sugar-binding proteins, while other oligosaccharide structures may be bound by antibodies and elicit unwanted immune responses. (Jenkins, N., et al., Nature Biotechnol. 14 (1996) 975-81)

[0101] Mammalian cells are preferred hosts for the production of therapeutic glycoproteins due to their ability to glycosylate proteins in a manner most compatible with human application (Cumming, DA, et al., Glycobiology 1 (1991) 115-30; Jenkins, N., et al., Nature Biotechnol. 14 (1996) 975-81). Bacteria glycosylate proteins poorly and, like other common host types such as yeast, filamentous fungi, insect, and plant cells, produce glycosylation patterns associated with rapid clearance from the bloodstream, undesirable immune interactions, and, in some cases, reduced biological activity. Among mammalian cells, Chinese hamster ovary (CHO) cells have been the most commonly used over the past 20 years. In addition to providing appropriate glycosylation patterns, these cells allow for the consistent generation of genetically stable, highly productive clonal cell lines. These cells can be cultured to high densities in simple bioreactors using serum-free media, enabling the development of safe and reproducible bioprocesses. Other commonly used animal cells include baby hamster kidney (BHK) cells, NSO- and SP2 / 0 mouse myeloma cells. Recently, production from transgenic animals has also been tested (Jenkins, N., et al., Nature Biotechnol. 14 (1996) 975-981).

[0102] Antibodies contain glycan structures at conserved positions in the heavy chain constant region, and each isotype has a distinct array of N-linked glycan structures, which affect protein assembly, secretion, or functional activity in various ways (Wright, A., and Morrison, S.L., Trends Biotech. 15 (1997) 26-32). The structures of the attached N-linked glycans vary considerably depending on the degree of processing and can include high-mannose, multiantennary, and biantennary complex oligosaccharides (Wright, A., and Morrison, S.L., Trends Biotech. 15 (1997) 26-32). Typically, there is heterogeneous processing of the core oligosaccharide structure attached at a particular glycosylation site, and even monoclonal antibodies exist as multiple glycoforms. Similarly, significant differences in antibody glycosylation have been shown to occur between cell lines, and even subtle differences can be observed for specific cell lines grown under different culture conditions. (Lifely, MR, et al., Glycobiology 5(8)(1995)813-22).

[0103] One way to greatly increase efficacy while maintaining a simple production process and potentially avoiding serious undesirable 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 U.S. Patent No. 6,602,684. IgG1-type antibodies are the most commonly used antibodies in cancer immunotherapy and are glycoproteins with a conserved N-linked glycosylation site at Asn297 in each CH2 domain. Two complex biantennary oligosaccharides linked to Asn297 are buried between the CH2 domains and form extensive contacts with the polypeptide backbone, and their presence is essential for antibodies to mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC) (Lifely, MR, et al., Glycobiology 5 (1995) 813-822; Jefferis, R., et al., Immunol. Rev. 163 (1998) 59-76; Wright, A., and Morrison, SL, Trends Biotechnol. 15 (1997) 26-32).

[0104] It has previously been shown that overexpression of β(1,4)-N-acetylglucosaminyltransferase I11 ("GnTII17y"), a glycosyltransferase that catalyzes the formation of biantennary oligosaccharides, in Chinese hamster ovary (CHO) cells significantly increases the in vitro ADCC activity of an anti-neuroblastoma chimeric monoclonal antibody (chCE7) produced by the engineered CHO cells. (See Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180 and WO 99 / 154342, the entire contents of which are incorporated herein by reference.) The antibody chCE7 belongs to a large class of unconjugated monoclonal antibodies that have high tumor affinity and specificity but are clinically ineffective and have little efficacy when produced in standard industrial cell lines lacking the GnTIII enzyme (Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180). The study was the first to show that a significant increase in ADCC activity can be obtained by engineering antibody-producing cells to express GnTIII, which also leads to an increase in the proportion of constant region (Fc)-linked bisected oligosaccharides containing bisected, nonfucosylated oligosaccharides above the levels seen in native antibodies.

[0105] 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 modified N-linked oligosaccharides. 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 oligosaccharides are bisected complex oligosaccharides. In some embodiments, the N-linked oligosaccharides are modified to increase bisected nonfucosylated oligosaccharides. In some embodiments, the bisected nonfucosylated oligosaccharides are hybrid. In some embodiments, the bisected nonfucosylated oligosaccharides are complex. For a more detailed description, see, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); U.S. Pat. App. Pub. No. 2005 / 0123546 (Umana et al.); and U.S. Pat. No. 8,883,980 (Umana et al.).

[0106] In some embodiments, the type II anti-CD20 antibody is obinutuzumab.

[0107] Antibody preparation Antibodies according to any of the above embodiments (e.g., type II anti-CD20 antibodies of the present disclosure) can also incorporate any of the features, alone or in combination, as described in Sections 1-7 below.

[0108] 1. Antibody affinity In certain embodiments, the antibodies provided herein have a cytotoxicity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0109] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured in the presence of a titration series of unlabeled antigen at the lowest concentration ( 125 I) Fab is equilibrated with labeled antigen, followed by capturing the bound antigen on a plate coated with an anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / mL of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 [I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although incubation can be continued for a longer period (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes on a TOPCOUNT™ gamma counter (Packard). Concentrations of each Fab that result in 20% or less of maximal binding are selected for use in competitive binding assays.

[0110] According to another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, the assay using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C with an immobilized antigen CM5 chip at approximately 10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to reach approximately 10 response units (RU) of bound protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at a flow rate of approximately 25 μl / min at 25°C. The association rate (k) and dissociation rate (k) are calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (K) is calculated as the ratio of k / k. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).If the association rate by the surface plasmon resonance assay described above exceeds 10 M s, the association rate can be determined using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, with a 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen, as measured by a spectrometer such as a spectrophotometer equipped with a flow stop (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0111] 2. Antibody fragment 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 below. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a reference to scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a description of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0112] Diabodies are antibody fragments that have two antigen binding sites and can be bivalent or bispecific.See, for example, European Patent No. 404,097, International Publication No. 1993 / 01161, Hudson et al., Nat.Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al., Nat.Med. 9:129-134 (2003).

[0113] Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0114] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0115] 3. Chimeric and humanized antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. A chimeric antibody includes an antigen-binding fragment thereof.

[0116] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. Optionally, a humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues of a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0117] Humanized antibodies and methods for their production are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008) and further described below. Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing resurfacing); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach for FR shuffling).

[0118] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0119] 4. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0120] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace the endogenous immunoglobulin loci, or are extrachromosomally present or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., 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 KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VelociMouse® technology. The human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.

[0121] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines), and Ni, Xiandai Mianyixue, 26(4):265-268(2006) (which describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937(2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91(2005).

[0122] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. These variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0123] 5. Library-derived antibodies Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity(ies). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. 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).

[0124] In a specific phage display method, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding phage as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) without immunization to provide a single source of antibodies against a wide range of non-self antigens and also self antigens, as described by Griffiths et al., EMBO J., 12:725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells, using PCR primers containing random sequences to encode highly variable CDR3 regions, and achieving rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0125] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein.

[0126] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for CD20 and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of CD20. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing CD20. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0127] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983); WO 93 / 08829; and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004 A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 148(5):1547-1553 (1992)). al., J. Immunol., 152:5368 (1994)); and by preparation of trispecific antibodies, for example, as described in Tutt et al. J. Immunol. 147:60 (1991).

[0128] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006 / 0025576).

[0129] The antibodies or fragments herein also include "Dual Acting FAbs" or "DAFs" that contain antigen binding sites that bind to CD20 and another distinct antigen (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0130] 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 antibodies may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, as long as the final construct possesses the desired characteristics (e.g., antigen binding).

[0131] a) Substitution, insertion and deletion variants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table A under the heading of "preferred substitutions." More substantial changes are shown in Table A under the heading of "exemplary substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the products are screened for the desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. TIFF2025537197000012.tif162170

[0132] Amino acids can be classified according to general side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0133] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0134] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study have altered (e.g., improved) certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques as described herein. Briefly, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0135] Alterations (e.g., substitutions) may be made, for example, in HVRs to improve antibody affinity. Such alterations may be made within HVR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact the antigen, and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by construction of and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0136] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the antibody's ability to bind to antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made within HVRs. Such changes may, for example, be outside the antigen contact residues within the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0137] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or groups of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact and adjacent residues can be targeted as candidates for substitution or removed. Variants can be screened to determine whether they contain the desired properties.

[0138] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., in the case of ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0139] b) Glycosylation variants In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0140] If the antibody contains an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are generally linked to Asn297 in the CH2 domain of the Fc region via an N-linkage. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention may be performed to generate antibody variants with specific improved properties.

[0141] In one embodiment, antibody variants are provided having a glycan structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures attached to Asn297 (e.g., complex structures, hybrid structures, and high-mannose structures) as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (Eu numbering of Fc region residues) within the Fc region; however, due to minor sequence variations within antibodies, Asn297 may be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, US Patent Application Publication No. 2003 / 0157108 (Presta, L.); US Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US Patent Application Publication No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US Patent Application Publication No. 2003 / 0115614; WO 2002 / 0164328; WO 2004 / 0093621; WO 2004 / 0093622; 0132140; 2004 / 0110704; 2004 / 0110282; 2004 / 0109865; WO 2003 / 085119; 2003 / 084570; 2005 / 035586; 2005 / 035778; 2005 / 053742; 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108, Presta, L; and WO 2004 / 056312, Adams et al., especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0142] Further provided are antibody variants having bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0143] c) Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody presented herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0144] In certain embodiments, the present invention contemplates antibody variants that possess some, but not all, effector functions, making them desirable candidates for applications in which in vivo antibody half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / absent CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express Fc(RIII) only, whereas monocytes express Fc(RI), Fc(RII, and Fc(RIII). FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest include those described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (Bruggemann, M. et al. al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA) and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody is unable to bind to C1q and lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0145] Antibodies with reduced effector function include antibodies with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0146] In certain embodiments, the Fc variants described herein further comprise one or more amino acid modifications to attenuate effector function (such as CDC and / or ADCC). In exemplary embodiments, the modification that attenuates effector function is a modification that does not alter the glycosylation pattern of the Fc region. In certain embodiments, the modification that attenuates effector function reduces or eliminates binding to human effector cells, binding to one or more Fc receptors, and / or binding to cells expressing Fc receptors. In exemplary embodiments, the Fc variants described herein comprise the following modifications: L234A, L235A, and P329G in the Fc region of human IgG1 (resulting in attenuated effector function). The substitutions L234A, L235A, P329G (the L234A / L235A / P329G triple variant is referred to as LALAPG) have previously been shown to reduce binding to Fc receptors and complement (see, e.g., U.S. Patent Application Publication No. 2012 / 0251531).

[0147] In various embodiments, an Fc variant with reduced effector function refers to an Fc variant that reduces effector function (e.g., activities such as CDC, ADCC, and / or FcR binding) 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 that reduce effector function, although it may have other mutations). In certain embodiments, an Fc variant with reduced effector function refers to an Fc variant that eliminates all detectable effector function compared to the wild-type Fc region. Assays for measuring effector function are known in the art and are described below.

[0148] To confirm the reduction / elimination of CDC and / or ADCC activity, in vitro and / or in vivo cytotoxicity assays can be performed. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the antibody lacks FcγR binding (and therefore likely lacks ADCC activity). NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA) and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody is unable to bind C1q and lacks CDC activity.See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)).

[0149] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0150] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0151] In some embodiments, modifications are made within the Fc region that result in altered (i.e., either improved or diminished) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0152] Antibodies with increased half-lives and improved binding to fetal Fc receptors (FcRn) that are responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826).

[0153] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351.

[0154] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine-engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with a cysteine ​​residue. In certain embodiments, the substituted residues are located at accessible sites on the antibody. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites on the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0155] e) Antibody derivative In certain embodiments, the antibodies provided herein may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Suitable sites for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous during manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when multiple polymers are attached, they can be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved and whether the antibody derivative will be used therapeutically under defined conditions.

[0156] In another embodiment, a conjugate of an antibody and a non-protective moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protective moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation may be of any wavelength, including but not limited to, a wavelength that does not harm normal cells but heats the non-protective moiety to a temperature that kills cells proximal to the antibody non-protective moiety.

[0157] A. Recombinant Methods and Compositions Antibodies may be produced using recombinant methods and compositions 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 may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of 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. In one embodiment, the host cell is eukaryotic, e.g., a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, or Sp20 cell). In one embodiment, a method of making an anti-CD20 antibody is provided, the method comprising culturing a host cell containing nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as described above, and optionally recovering the antibody from the host cell (or host cell medium).

[0158] For recombinant production of an anti-CD20 antibody, nucleic acid encoding the antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0159] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibodies of the present invention may be isolated in a soluble fraction from the bacterial cell paste or further purified.

[0160] In addition to prokaryotes, eukaryotic organisms such as filamentous fungi and yeast are suitable cloning or expression hosts for antibody-encoding vectors, including bacterial and yeast strains that have been "humanized" in their glycosylation pathways to produce antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0161] Additionally, suitable host cells for expressing glycosylated antibodies are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified and can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0162] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0163] 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 include the monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (e.g., 293 cells or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); see, e.g., Mather et al., Annals of NY Acad. Sci. 383:44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include DHFR - Examples include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0164] B. Assay The anti-CD20 antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art.

[0165] 1. Binding and Other Assays In one aspect, antibodies of the present invention are tested for their antigen-binding activity by known methods, such as 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 shown below. A CD20 antibody is iodinated, and a competition reaction mixture containing a fixed concentration of iodinated antibody and serially diluted, decreasing concentrations of unlabeled CD20 antibody is prepared. Cells expressing CD20 (e.g., BT474 cells stably transfected with human CD20) are added to this reaction mixture. After incubation, the cells are washed to separate free iodinated CD20 antibody from cell-bound CD20 antibody. The level of bound iodinated CD20 antibody is determined, for example, by measuring cell-bound radioactivity, and binding affinity is determined using standard methods. In another embodiment, the ability of a CD20 antibody to bind to surface-expressed CD20 (e.g., on B cell subsets) is assessed using flow cytometry. Peripheral white blood cells are obtained (e.g., from humans, cynomolgus monkeys, rats, or mice), and the cells are blocked with serum. 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). After sample incubation and washing, 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. An exemplary surface plasmon resonance method is provided in the Examples.

[0166] In another aspect, a competition assay can be used to identify antibodies that compete with any of the anti-CD20 antibodies disclosed herein for binding to CD20. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) bound by any of the anti-CD20 antibodies disclosed herein. Detailed exemplary methods for mapping antibody-binding epitopes are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ).

[0167] In an exemplary competitive assay, immobilized CD20 is incubated in a solution containing a first labeled antibody that binds to CD20 (e.g., rituximab, GA101 antibody, etc.) and a second unlabeled antibody being tested for its ability to compete with the first antibody for binding to CD20. The second antibody may be present in 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 allow binding of the first antibody to CD20, excess unbound antibody is removed and the amount of label associated with immobilized CD20 is measured. A substantial reduction in the amount of label associated with immobilized CD20 in the test sample compared to the control sample 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).

[0168] 2. Activity Assay 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 cellular cytotoxicity (ADCC) can be used, as described herein.

[0169] It will be understood that any of the above assays can be performed using an immunoconjugate of the invention in place of, or in addition to, an anti-CD20 antibody.

[0170] It will be understood that any of the above assays may be performed using an anti-CD20 antibody and an additional therapeutic agent.

[0171] Methods for administering type II anti-CD20 antibodies Provided herein is a method for treating childhood-onset idiopathic nephrotic syndrome (INS) in an individual, the method comprising administering to the individual a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to a type II anti-CD20 antibody. Also provided herein is a method for depleting circulating peripheral B cells in an individual, the method comprising administering to the individual a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to a type II anti-CD20 antibody, wherein after administration of the type II anti-CD20 antibody, B cells are depleted to a level such that no more than about 5 circulating peripheral B cells / μL are present in peripheral blood from the individual. Also provided herein are methods for depleting circulating peripheral B cells in an individual, comprising administering to the individual a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to a type II anti-CD20 antibody, wherein after administration of the type II anti-CD20 antibody, B cells are depleted to a level such that there are about 5 or fewer circulating peripheral B cells per μL in the peripheral blood from the individual, which depletion persists for at least 52 weeks after the first dose of the first antibody exposure. In some embodiments of the methods herein, the individual or patient is a human. In some embodiments, the individual or patient is a human between the ages of 2 and 25. In some embodiments, the individual or patient is a human between the ages of 2 and 25. In some embodiments, e.g., in embodiments using weight-based dosing of the type II anti-CD20 antibody, the individual weighs less than 45 kg. In some embodiments, e.g., in embodiments using fixed dosing of the type II anti-CD20 antibody, the individual weighs 45 kg or more.

[0172] In some embodiments, the individual or patient has been diagnosed with INS (e.g., FRNS or SDNS) before the age of 18. Guidelines for diagnosing childhood-onset INS (e.g., FRNS or SDNS) are known in the art and include, but are not limited to, those described in: Kidney Disease: Improving Global Outcomes Glomerular Diseases Work Group. KDIGO 2021 Clinical Practice Guideline for the Management of Glomerular Diseases (Kidney Int. 2021;100:S1-276).

[0173] In some embodiments, the individual or patient has childhood-onset frequently relapsing nephrotic syndrome (FRNS). In some embodiments, the individual or patient has experienced two or more relapses per 6 months within 6 months of disease onset, or four or more relapses per 12 months in any subsequent 12-month period.

[0174] In some embodiments, the individual or patient has childhood-onset steroid-dependent nephrotic syndrome (SDNS). In some embodiments, the individual or patient has experienced two consecutive relapses during treatment with prednisone or prednisolone (either full dose or tapering) or within 15 days of discontinuing prednisone or prednisolone.

[0175] In some embodiments, the individual or patient is in complete remission, e.g., prior to treatment with the methods of the present disclosure, in some embodiments, complete remission is defined as no edema, a UPCR < 0.2 g / g, and three consecutive daily urine dipstick readings that are trace or negative for protein.

[0176] In some embodiments, the individual or patient has experienced at least one relapse, for example, within six months, prior to treatment with the methods of the present disclosure.

[0177] In some embodiments, the individual or patient has received cyclophosphamide within six months, e.g., prior to treatment with the methods of the present disclosure, and has experienced at least one relapse after discontinuation of cyclophosphamide.

[0178] In some embodiments, the individual or patient has an estimated glomerular filtration rate (eGFR) within the normal range for their age.

[0179] In some embodiments, the methods of the present disclosure comprise administering to an individual a first antibody exposure to a type II anti-CD20 antibody of the present disclosure and a second antibody exposure to a type II anti-CD20 antibody of the present disclosure. In some embodiments, the second antibody exposure is not provided until about 18 weeks to about 26 weeks after the first antibody exposure. In some embodiments, the second antibody exposure is not provided until about 18 weeks after the first antibody exposure, about 19 weeks after the first antibody exposure, about 20 weeks after the first antibody exposure, about 21 weeks after the first antibody exposure, about 22 weeks after the first antibody exposure, about 23 weeks after the first antibody exposure, about 24 weeks after the first antibody exposure, about 25 weeks after the first antibody exposure, or about 26 weeks after the first antibody exposure. In some embodiments, the second antibody exposure is not provided less than about any of 26, 25, 24, 23, 22, 21, 20, or 19 weeks after the first antibody exposure. In some embodiments, the second antibody exposure is provided less than about any of 18, 19, 20, 21, 22, 23, 24, or 25 weeks after the first antibody exposure, i.e., the second antibody exposure is not administered until any of a range of weeks having an upper limit of 26, 25, 24, 23, 22, 21, 20, or 19 and an independently selected lower limit of 18, 19, 20, 21, 22, 23, 24, or 25, where the lower limit is less than the upper limit.

[0180] The dosing regimens described herein use a consistent system for tracking the time between doses, whereby the first dose is administered to the patient on day 1 or week 0. As described herein, the antibody exposures of the present disclosure can include one or two doses. When an antibody exposure contains one dose (as described herein), a reference to a second antibody exposure that is not provided until a certain period of time after the first antibody exposure refers to the time that elapses between the dose of the first antibody exposure (e.g., day 1 or week 0) and the dose of the second antibody exposure. When a first antibody exposure contains two doses, the first dose of the first antibody exposure is provided on day 1 or week 0. When an antibody exposure contains two doses (as described herein), a reference to a second antibody exposure that is not provided until a certain period of time after the first antibody exposure refers to the time that elapses between the first of the two doses of the first antibody exposure (e.g., day 1 or week 0) and the first of the two doses of the second antibody exposure. For example, if the methods of the disclosure include two doses of a first antibody exposure and two doses of a second antibody exposure, where the second antibody exposure is not provided until about 22 weeks after the first antibody exposure, then the interval between the first dose of the first antibody exposure and the first dose of the second antibody exposure is about 22 weeks.

[0181] In some embodiments, the first antibody exposure of the present disclosure comprises one or two doses of a type II anti-CD20 antibody of the present disclosure. In some embodiments, the first antibody exposure contains a total exposure of between about 1800 mg and about 2200 mg of a 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 a type II anti-CD20 antibody. In some embodiments, the individual weighs 45 kg or more.

[0182] In some embodiments, the first antibody exposure contains a total exposure of between about 36 mg / kg and about 44 mg / kg of type II anti-CD20 antibody. In some embodiments, the first antibody exposure contains a total exposure of about 36 mg / kg, about 38 mg / kg, about 40 mg / kg, about 42 mg / kg, or about 44 mg / kg of type II anti-CD20 antibody. In some embodiments, the individual weighs less than 45 kg.

[0183] In some embodiments, the first antibody exposure comprises two doses. In some embodiments, the first antibody exposure comprises a first dose of between about 900 mg and about 1100 mg of type II anti-CD20 antibody and a second dose of between about 900 mg and about 1100 mg of type II anti-CD20 antibody. In some embodiments, the first dose of the first antibody exposure contains about 1000 mg of type II anti-CD20 antibody. In some embodiments, the second dose of the first antibody exposure contains about 1000 mg of type II anti-CD20 antibody. In some embodiments, the individual weighs 45 kg or more.

[0184] In some embodiments, the first antibody exposure comprises two doses. In some embodiments, the first antibody exposure comprises a first dose of between about 18 mg / kg and about 22 mg / kg of type II anti-CD20 antibody and a second dose of between about 18 mg / kg and about 22 mg / kg of type II anti-CD20 antibody. In some embodiments, the first dose of the first antibody exposure contains about 20 mg / kg of type II anti-CD20 antibody. In some embodiments, the second dose of the first antibody exposure contains about 20 mg / kg of type II anti-CD20 antibody. In some embodiments, the individual weighs less than 45 kg.

[0185] In some embodiments, the second dose of the first antibody exposure is not provided until about 1.5 to about 2.5 weeks after the first dose of the first antibody exposure, hi some embodiments, the second dose of the first antibody exposure is not provided until about 2 weeks after the first dose of the first antibody exposure.

[0186] In some embodiments, the second antibody exposure of the present disclosure comprises one or two doses of a type II anti-CD20 antibody of the present disclosure. In some embodiments, the second antibody exposure contains a total exposure of between about 1800 mg and about 2200 mg of a type II anti-CD20 antibody. In some embodiments, the second antibody exposure contains a total exposure of about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, or about 2200 mg of a type II anti-CD20 antibody. In some embodiments, the individual weighs 45 kg or more.

[0187] In some embodiments, the second antibody exposure contains a total exposure of between about 36 mg / kg and about 44 mg / kg of type II anti-CD20 antibody. In some embodiments, the second antibody exposure contains a total exposure of about 36 mg / kg, about 38 mg / kg, about 40 mg / kg, about 42 mg / kg, or about 44 mg / kg of type II anti-CD20 antibody. In some embodiments, the individual weighs less than 45 kg.

[0188] In some embodiments, the second antibody exposure comprises two doses. In some embodiments, the second antibody exposure comprises a first dose of between about 900 mg and about 1100 mg of type II anti-CD20 antibody and a second dose of between about 900 mg and about 1100 mg of type II anti-CD20 antibody. In some embodiments, the first dose of the second antibody exposure contains about 1000 mg of type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure contains about 1000 mg of type II anti-CD20 antibody. In some embodiments, the individual weighs 45 kg or more.

[0189] In some embodiments, the second antibody exposure comprises two doses. In some embodiments, the second antibody exposure comprises a first dose of between about 18 mg / kg and about 22 mg / kg of type II anti-CD20 antibody and a second dose of between about 18 mg / kg and about 22 mg / kg of type II anti-CD20 antibody. In some embodiments, the first dose of the second antibody exposure contains about 20 mg / kg of type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure contains about 20 mg / kg of type II anti-CD20 antibody. In some embodiments, the individual weighs less than 45 kg.

[0190] 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, hi 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.

[0191] In some embodiments, a Type II anti-CD20 antibody of the present disclosure is administered intravenously (eg, IV infusion).

[0192] 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 described herein). Various natural and synthetic glucocorticoids / corticosteroids are known in the art, including, but not limited to, beclomethasone, triamcinolone, dexamethasone, betamethasone, prednisone, methylprednisolone, prednisolone, cortisone, and cortisol. In some embodiments, the glucocorticoid / corticosteroid comprises methylprednisolone. In some embodiments, the glucocorticoid / corticosteroid comprises prednisone. Effective amounts of glucocorticoids / corticosteroids of the present disclosure are known in the art and readily ascertainable by standard assays. For example, methylprednisolone may be administered intravenously at a dose of 750-1000 mg once daily. As another example, prednisone may be administered orally at 0.5 mg / kg and optionally tapered to 7.5 mg / day. In some embodiments, methylprednisolone may be administered before each anti-CD20 antibody infusion. In some embodiments, methylprednisolone may be administered intravenously at 80 mg (e.g., if the individual weighs 45 kg or more) or 1.5 mg / kg (e.g., if the individual weighs less than 45 kg). In some embodiments, oral prednisone or equivalent may be administered at a dose of 0.5-1 mg / kg / day (maximum 60 mg / day). In some embodiments, oral prednisone or equivalent may be administered at a dose of 0.5-1 mg / kg / day (maximum 60 mg / day) and tapered to a target of 5 mg / day. In some embodiments, oral prednisone or equivalent may be administered at a dose of 0.5-2 mg / kg / day (maximum 60 mg / day). In some embodiments, oral prednisone or equivalent may be administered at a dose of 0.5-2 mg / kg / day (maximum 60 mg / day) and tapered to a target of 5 mg / day.

[0193] In some embodiments, a glucocorticoid may be administered before, during, or after administration of a type II anti-CD20 antibody of the present disclosure. In some embodiments, a glucocorticoid may be administered before administration of a type II anti-CD20 antibody of the present disclosure, for example, 30 to 60 minutes before administration of the type II anti-CD20 antibody. In some embodiments, 80 mg of methylprednisolone may be administered intravenously 30 to 60 minutes before administration of a type II anti-CD20 antibody of the present disclosure. In some embodiments, prednisone (e.g., orally) and / or methylprednisolone (e.g., IV) may be administered in conjunction with treatment, followed by maintenance treatment (e.g., mycophenolate mofetil or cyclophosphamide).

[0194] In some embodiments, the methods of the present disclosure further comprise administering an effective amount of an antihistamine (e.g., in combination with a type II anti-CD20 antibody described herein). Antihistamines 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 comprises diphenhydramine. Effective amounts of antihistamines of the present disclosure are known in the art and readily ascertainable by standard assays. For example, diphenhydramine may be administered at an oral dose of 0.5-1 mg / kg (nearest round available tablet formulation), up to a maximum dose of 50 mg.

[0195] In some embodiments, an antihistamine may be administered before, during, or after administration of a type II anti-CD20 antibody of the present disclosure, e.g., as a prophylactic treatment. In some embodiments, an antihistamine may be administered before administration of a type II anti-CD20 antibody of the present disclosure, e.g., 30-60 minutes before administration of the type II anti-CD20 antibody. In some embodiments, 0.5-1 mg / kg or up to 50 mg of diphenhydramine may be administered orally 30-60 minutes before administration of a type II anti-CD20 antibody of the present disclosure.

[0196] In some embodiments, the methods of the present disclosure further comprise administering an effective amount of acetaminophen, for example, acetaminophen may be administered at an oral dose of 15 mg / kg, up to a maximum dose of 1000 mg.

[0197] In some embodiments, acetaminophen may be administered before, during, or after administration of a type II anti-CD20 antibody of the present disclosure, e.g., as a prophylactic treatment. In some embodiments, acetaminophen may be administered before administration of a type II anti-CD20 antibody of the present disclosure, e.g., 30-60 minutes before administration of a type II anti-CD20 antibody. In some embodiments, 15 mg / kg (nearest round available tablet formulation) or up to 1000 mg of acetaminophen may be administered orally 30-60 minutes before administration of a type II anti-CD20 antibody of the present disclosure.

[0198] In some embodiments, the methods of the present disclosure further comprise administering a standard of care (e.g., in combination with a type II anti-CD20 antibody described herein), which may be administered before, during, or after administration of a type II anti-CD20 antibody of the present disclosure, e.g., for the treatment or prevention of one or more symptoms of INS.

[0199] In some embodiments, the method of the present disclosure results in complete remission in an individual. In some embodiments, the individual is in complete remission one year after initiation of treatment, for example, at week 52 as described herein. In some embodiments, complete remission refers to a state in which, after completion of steroid tapering, for example, as described herein, the first morning void UPCR is ≦0.2 g / g without the occurrence of an intervening event (occurring after week 8). In some embodiments, complete remission persists, for example, one year after initiation of treatment, for example, at week 52 as described herein. In some embodiments, sustained complete remission includes a first morning void UPCR≦0.2 g / g without the occurrence of relapse or any of the following specific intervening events: (1) relapse (e.g., occurring after week 8) defined by any of the following events requiring systemic corticosteroid or other immunosuppressive treatment: (a) first morning void UPCR≧2 g / g or (b) dipstick UA≧3+ for 3 consecutive days with the most recent urine sample on those 3 days determining a UPCR>0.2 g / g, or (c) dipstick UA protein≧3+ on any 1 day with edema and a urine sample determining a UPCR>0.2 g / g; (2) any systemic corticosteroid use for >14 days in a 30-day period (e.g., occurring after week 8); (3) initiation of any rescue therapy for INS other than systemic corticosteroids (e.g., occurring at any time); (4) treatment discontinuation due to lack of efficacy (e.g., occurring at any time); or (5) death (e.g., occurring at any time). In some embodiments, complete remission is sustained in an individual from week 8 through week 52 of treatment, and the individual does not experience: (1) a relapse defined by any of the following events requiring systemic corticosteroid or other immunosuppressive treatment: (a) first morning void UPCR ≧2g / g or (b) dipstick UA ≧3+ for 3 consecutive days with the most recent urine sample during those 3 days determining UPCR>0.2g / g, or (c) any daily dipstick UA protein ≧3+ with edema and a urine sample determining UPCR>0.2g / g; or (2) any systemic corticosteroid use for >14 days in a 30-day period.In some embodiments, complete remission is maintained in the individual through week 52 of treatment without initiating any salvage therapy for INS other than systemic corticosteroids.

[0200] In some embodiments, the methods of the present disclosure result in depletion of circulating peripheral B cells in an individual. In some embodiments, the circulating peripheral B cells are CD19+ B cells. In some embodiments, the circulating peripheral B cells are naive B cells. In some embodiments, the circulating peripheral B cells are memory B cells. In some embodiments, the circulating peripheral B cells are plasmablasts or plasma cells. In some embodiments, after administration of a type II anti-CD20 antibody of the present disclosure (e.g., according to any of the methods described herein), there are 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, about 1 cell / μL or less, or about 0.5 cells / μL or less in the peripheral blood. In some embodiments, the level of circulating peripheral B cells is measured using high-sensitivity flow cytometry (HSFC) as described herein. In some embodiments, B cells are depleted to levels below the limit of detection using HSFC. In some embodiments, the HSFC has a lower limit of quantitation (LLOQ) of B cells of about 1.0 cells / μL or less, about 0.8 cells / μL or less, about 0.6 cells / μL or less, about 0.5 cells / μL or less, or 0.441 cells / μL or less. In some embodiments, circulating peripheral B cells in the individual are depleted by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%. In some embodiments, depletion of circulating peripheral B cells persists for at least 52 weeks after the first dose of the first antibody exposure. In some embodiments, depletion of circulating peripheral B cells persists for at least 51 weeks, at least 50 weeks, at least 49 weeks, at least 48 weeks, at least 47 weeks, at least 46 weeks, at least 45 weeks, at least 44 weeks, at least 43 weeks, at least 42 weeks, at least 41 weeks, at least 40 weeks, at least 39 weeks, at least 38 weeks, at least 37 weeks, at least 36 weeks, at least 35 weeks, at least 34 weeks, at least 33 weeks, at least 32 weeks, at least 31 weeks, at least 30 weeks, at least 29 weeks, at least 28 weeks, at least 27 weeks, at least 26 weeks, at least 25 weeks, or at least 24 weeks after the first dose of the first antibody exposure.In some embodiments, depletion of circulating peripheral B cells refers to a measurement of circulating peripheral B cells taken after a first antibody exposure (e.g., comprising one or two doses of an anti-CD20 antibody as described herein), after a second antibody exposure (e.g., comprising one or two doses of an anti-CD20 antibody as described herein), three months after treatment (e.g., after receiving the first and / or second antibody exposure as described herein), six months after treatment (e.g., after receiving the first and / or second antibody exposure as described herein), nine months after treatment (e.g., after receiving the first and / or second antibody exposure as described herein), or twelve months after treatment (e.g., after receiving the first and / or second antibody exposure as described herein), e.g., compared to a corresponding measurement in the same individual before treatment or compared to a corresponding measurement in a control individual (e.g., an individual not receiving treatment).

[0201] Methods for assaying depletion of circulating peripheral 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 depletion of circulating peripheral B cells (see, e.g., Vital, EM et al. (2011) Arthritis Rheum. 63:3038-3047 and Example 1). In some embodiments, the B cells are CD19+ B cells. In some embodiments, the B cells are naive B cells (e.g., CD19+CD27- B cells), memory B cells (e.g., CD19+CD27+ B cells), or plasmablasts (e.g., CD19+CD27+CD38++ B cells). In some embodiments, the B cells are CD19+CD3-CD14- cells and / or CD19+CD33-CD56- cells. In some embodiments, the B cells are CD19+CD3-CD14-CD33-CD56- cells. In some embodiments, the B cells include CD19+CD20+ B cells, CD19+CD20- B cells, and CD19+CD22+ B cells. In some embodiments, the B cells are circulating peripheral B cells, e.g., from a peripheral blood sample.

[0202] In some embodiments, the level of circulating peripheral B cells present in a peripheral blood sample is measured (e.g., by HSFC) as follows: Lymphocytes are identified in the sample by flow cytometry (e.g., by plotting CD45 versus side scatter and gating on CD45+ cells). In some embodiments, doublets are excluded from the analysis prior to this step (e.g., by gating on single cells and excluding forward and / or side scatter doublets). CD19+ B cells are then identified by excluding T cells, NK cells, and monocytes. For example, CD19+CD3-CD14- cells can be identified from the parent CD45+ lymphocyte gate (e.g., by plotting CD19 vs. CD3 / CD14 and gating on CD19+CD3-CD14- cells), and CD19+CD33-CD56- B cells can be identified from the parent CD19+CD3-CD14- cells (e.g., by plotting CD19 vs. CD33 / CD56 and gating on CD19+CD33-CD56- cells). The number of B cells can then be determined, for example, by dividing the number of detected CD19+ B cells (e.g., CD19+CD3-CD14-CD33-CD56- cells) by the sample volume. In some embodiments, the number of beads or other QC controls is also quantified, after which the number of B cells can be determined, for example, by calculating (CD19+ events x number of beads) / (number of beads x sample volume).

[0203] In some embodiments, after administration of a type II anti-CD20 antibody of the present disclosure (e.g., according to any of the methods described herein), circulating peripheral B cells are present in the peripheral blood at 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, about 1 cell / μL or less, or about 0.5 cells / μL or less, e.g., 5 cells / μL or less. In some embodiments, B cells are depleted to levels below the limit of detection using HSFC. In some embodiments, the HSFC has a lower limit of quantitation (LLOQ) of about 1.0 cells / μL or less, about 0.8 cells / μL or less, about 0.6 cells / μL or less, about 0.5 cells / μL or less, or 0.441 cells / μL or less.

[0204] IV. Manufactured Articles or Kits In another aspect, an article of manufacture or kit containing a type II anti-CD20 antibody of the present disclosure useful in any of the methods described herein (e.g., for the treatment, prevention, and / or diagnosis of a disorder described herein) is provided. The article of manufacture or kit of the present invention comprises a container and a label or package insert affixed to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition, alone or in combination with another composition, that is effective for the treatment, prevention, and / or diagnosis of a condition or for depleting circulating peripheral B cells, and may have a sterile access port (e.g., the container may be an IV solution bag or vial with a stopper pierceable with a hypodermic needle). At least one active agent in the composition of the present invention 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 used in accordance with any of the methods described herein to treat a selected condition or to deplete circulating peripheral B cells. Alternatively, or additionally, the article of manufacture or kit of the invention may 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. The kit or article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0205] In some embodiments, provided herein is an article of manufacture or kit comprising a container containing a type II anti-CD20 antibody of the present disclosure and optionally a pharmaceutically acceptable carrier, and optionally a package insert comprising instructions for treating childhood-onset INS in an individual or reducing the risk and / or frequency of recurrence of childhood-onset INS in the individual, e.g., the instructions indicating that a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to a type II anti-CD20 antibody are administered to the individual, wherein the second antibody exposure is not provided for about 18 weeks to about 26 weeks after the first antibody exposure; the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of between about 1800 mg and about 2200 mg of the type II anti-CD20 antibody; and the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of between about 1800 mg and about 2200 mg of the type II anti-CD20 antibody. In some embodiments, the instructions indicate the individual is between 2 and 25 years of age. In some embodiments, the instructions indicate the individual weighs 45 kg or more. In some embodiments, the antibody is obinutuzumab.

[0206] In some embodiments, provided herein is an article of manufacture or kit that includes a container containing a type II anti-CD20 antibody of the present disclosure and optionally a pharmaceutically acceptable carrier, and optionally a package insert that includes instructions for treating childhood-onset INS in an individual or for reducing the risk and / or frequency of recurrence of childhood-onset INS in an individual, e.g., the instructions include a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to a type II anti-CD20 antibody. and the second antibody exposure is administered to the body, and the second antibody exposure is not provided for about 18 weeks to about 26 weeks after the first antibody exposure; the first antibody exposure includes one or two doses of a type II anti-CD20 antibody, and the first antibody exposure contains a total exposure of about 36 mg / kg to about 44 mg / kg of the type II anti-CD20 antibody; and the second antibody exposure includes one or two doses of a type II anti-CD20 antibody, and the second antibody exposure contains a total exposure of about 36 mg / kg to about 44 mg / kg of the type II anti-CD20 antibody. In some embodiments, the description indicates the individual is between 2 and 25 years old. In some embodiments, the description indicates the individual weighs less than 45 kg. In some embodiments, the antibody is obinutuzumab.

[0207] The article of manufacture or kit of the invention may further comprise a second or third container containing a second medicament, wherein the anti-CD20 antibody (e.g., a type II anti-CD20 antibody of the present disclosure) is the first medicament, and the article of manufacture further comprises package insert instructions for treating a subject with the second medicament. The article of manufacture in these embodiments may further comprise a package insert indicating that the composition of the invention can be used to treat a particular condition.

[0208] The specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description, and fall 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. [Example]

[0209] The present invention will be more fully understood by reference to the following examples, which, however, should not be construed as limiting the scope of the present invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the scope of the appended claims.

[0210] Example 1: A Phase III, Multicenter, Randomized, Open-Label Study to Evaluate the Efficacy and Safety of Obinutuzumab Versus MMF in Patients With Childhood-Onset Idiopathic Nephrotic Syndrome The following is a phase III, randomized, open-label, multicenter, active-comparison study to evaluate the efficacy, safety, and pharmacokinetics (PK) / pharmacodynamics (PD) of obinutuzumab versus MMF in maintaining remission in participants with childhood-onset frequently relapsing nephrotic syndrome (FRNS) or steroid-dependent nephrotic syndrome (SDNS) who achieved complete remission at study entry and were considered at high risk for relapse.

[0211] Patients with childhood-onset FRNS / SDNS are at risk for short- and long-term steroid toxicity, serious infections, edema, thromboembolic events, and acute kidney injury during relapses. Reduction of these risks depends on identifying effective treatments and maintaining clinical remission with sustained reduction in proteinuria. Current standard treatment remains limited to a combination of systemic corticosteroids and immunosuppressive therapy, although most available regimens result in complete renal response and remission in fewer than half of treated patients.

[0212] Objectives and evaluation items This study will evaluate the efficacy, safety, pharmacokinetics, and pharmacodynamics of obinutuzumab compared to MMF in participants aged 2 years or older and up to 25 years with childhood-onset FRNS or SDNS. The specific primary and secondary objectives of the study and corresponding endpoints are outlined below. In this protocol, "study treatment" refers to the treatment assigned to participants as part of this study (i.e., obinutuzumab or MMF).

[0213] The primary objective of the study was to evaluate the efficacy of obinutuzumab compared with MMF in participants aged 2 to 25 years with childhood-onset FRNS or SDNS. The primary endpoint was the proportion of participants achieving sustained complete remission at 1 year, defined as no relapse or any of the following intercurrent events and a first morning void urinary protein-to-creatinine ratio (UPCR) ≤ 0.2 g / g at week 52: Intercurrent events occurring after Week 8 included: (1) relapse defined by any of the following events: (a) first morning void UPCR ≥ 2 g / g, (b) dipstick urinalysis (UA) ≥ 3+ for 3 consecutive days (home monitoring) with the most recent urine sample during those 3 days measured by a central laboratory as UPCR > 0.2 g / g, or (c) any daily dipstick UA protein ≥ 3+ with edema and a urine sample measured by a central laboratory as UPCR > 0.2 g / g; and (2) any systemic corticosteroid use for > 14 days in a 30-day period. Intercurrent events occurring after randomization included: (1) initiation of any rescue therapy for idiopathic nephrotic syndrome (INS) other than systemic corticosteroids, as determined by the investigator's best medical judgment; (2) treatment discontinuation due to lack of efficacy; and / or (3) death.

[0214] Secondary objectives are to evaluate the efficacy of obinutuzumab compared with MMF. Corresponding secondary endpoints are: (1) overall recurrence-free survival (RFS); (2) probability of RFS at 52 weeks; (3) cumulative corticosteroid dose; (4) number of relapses in randomized study treatment; (5) proportion of participants experiencing edema-related relapses during the 52-week treatment period; and (6) proportion of patients with sustained complete remission at 76 weeks.

[0215] Another secondary objective was to assess change in fatigue in participants treated with obinutuzumab compared with MMF. The corresponding secondary endpoint was the mean change from baseline to week 52 in the "General Fatigue" domain of the Pediatric Quality of Life Inventory (PedsQL) Multidimensional Fatigue scale total score.

[0216] The third secondary objective was to assess the change in quality of life in participants treated with obinutuzumab compared with MMF. The corresponding secondary endpoint was the mean change from baseline to week 52 in the "Physical Functioning" domain of the PedsQL Quality of Life Inventory.

[0217] The fourth secondary objective is to assess edema over time. The corresponding secondary endpoint is the mean change in the Cure Glomerulonephropathy (CureGN) edema scale over time from baseline to week 52.

[0218] The fifth secondary objective is to evaluate the safety of obinutuzumab compared with MMF. Corresponding secondary endpoints are: (1) the incidence, nature, and severity of adverse events (AEs), with AE intensity (mild, moderate, severe, life-threatening) and severity determined according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) grading, from baseline to Week 52, if applicable; and (2) the incidence of laboratory findings or vital sign abnormalities from baseline to Week 52.

[0219] The sixth secondary endpoint is to characterize the PK profile of obinutuzumab. The corresponding secondary endpoint is serum concentrations of obinutuzumab at specific time points.

[0220] The seventh secondary objective is to characterize PD changes induced by obinutuzumab. Corresponding secondary endpoints are: (1) the proportion of participants achieving B-cell depletion (e.g., using high-sensitivity flow cytometry (HSFC)) at specified time points; and (2) total peripheral B-cell and B-cell subset (e.g., memory B-cell) counts and changes from baseline at specified time points.

[0221] The study objective was to evaluate the efficacy of obinutuzumab compared with MMF. Corresponding study endpoints were: (1) change in UPCR from baseline to week 52; (2) change in estimated glomerular filtration rate (eGFR) from baseline to week 52; (3) proportion of participants achieving sustained peripheral B-cell depletion at weeks 24, 52, and 76; (4) proportion of participants achieving RFS at week 24; (5) proportion of participants in sustained complete remission at week 76; (6) change in Physician's Global Assessment of Disease Activity (PGA) from baseline to weeks 24 and 52; (7) change in Subject Global Assessment of Disease Activity (SGA) from baseline to weeks 24 and 52; and (8) proportion of participants free of relapse before completion of steroid tapering.

[0222] The secondary study objective was to investigate the relationship between exposure-effectiveness and exposure-safety. Corresponding study endpoints were: (1) change in obinutuzumab exposure and selected efficacy endpoints (including sustainable complete response (SCR) and RFS) from baseline to Week 52 and over time; and (2) change in obinutuzumab exposure and the incidence, nature, and severity of selected adverse events from baseline to Week 52 and over time.

[0223] The third investigational objective is to evaluate the potential relationship between drug exposure and B-cell depletion. Corresponding investigational endpoints are: (1) obinutuzumab exposure and change from baseline over time in circulating CD19+ B-cell counts; and (2) total peripheral B-cell and B-cell subset (e.g., memory B-cell) counts before / at relapse.

[0224] The fourth study objective is to evaluate the immune response to obinutuzumab. The corresponding study endpoints are the proportion of participants with anti-drug antibodies (ADAs) at baseline and the incidence of ADAs after treatment during the study.

[0225] The fifth study objective is to evaluate the potential effect of ADA. The corresponding study endpoint is the relationship between ADA status and efficacy, safety, PD, or PK endpoints.

[0226] The sixth research objective is to identify and / or evaluate biomarkers that provide evidence of obinutuzumab activity (i.e., pharmacodynamic biomarkers) or increase knowledge and understanding of disease biology and drug safety. Corresponding research endpoints are the relationship between blood biomarkers and efficacy, safety, PK, immunogenicity, or other biomarker endpoints.

[0227] Inclusion and Exclusion Criteria The study inclusion criteria included: (1) participants were between 2 and 25 years of age at the time of randomization; (2) participants were included in accordance with international guidelines [e.g., KDIGO 2021, IPNA 2023 (Trautmann et al. (2023) Pediatric Nephrol 38:877-919] before age 18 years; (3) complete remission, defined by the absence of edema, with UPCR ≤ 0.2 g / g at screening and three consecutive daily urine dipstick readings that were trace or negative for protein within the week before randomization; (4) having had at least one relapse in the 6 months before screening after or while receiving oral corticosteroids and / or immunosuppressive therapy (e.g., oral cyclophosphamide, levamisole, mizoribine, MMF, or CNI) to prevent relapse; (5) patients who received cyclophosphamide 6 months before randomization must have experienced at least one relapse after discontinuation of cyclophosphamide; and (6) an estimated glomerular filtration rate (eGFR) within the normal range for age (according to the modified Schwartz formula if under 18 years of age or the Chronic Kidney Disease Epidemiology Association if 18 years of age or older). (Using the formula from the CKD-EPI Collaboration).Frequently relapsing nephrotic syndrome (FRNS) is defined as: 2 or more relapses per 6 months within 6 months of disease onset, or 4 or more relapses per 12 months in any subsequent 12 months.Steroid-dependent nephrotic syndrome (SDNS) is defined as: 2 consecutive relapses during treatment with prednisone or prednisolone (either full dose or during tapering) or within 15 days of discontinuing prednisone or prednisolone.

[0228] Exclusion criteria included the following: (1) secondary nephrotic syndrome (i.e., reflux nephropathy, IgA nephropathy, lupus nephritis, etc.); (2) history of steroid-resistant nephrotic syndrome; (3) history of genetic defects known to directly cause nephrotic syndrome (i.e., NPHS2 [podocin], NPHS1 [nephrin], PLCE1, WT1, or other known genetic causes); (4) treatment with other immunosuppressants to prevent relapse other than MMF or oral corticosteroids within 2 months prior to randomization; (5) history of organ or bone marrow transplantation; (6) participation in another therapeutic trial within 30 days of enrollment or within 5 half-lives of the investigational drug (whichever is longer); (7) intolerance or contraindication to the investigational therapy, including any of the following: (a) history of a severe allergic or anaphylactic reaction to a monoclonal antibody or known hypersensitivity to any component of the obinutuzumab infusion, (b) lack of peripheral venous access, (c) intolerance or contraindication to oral or IV corticosteroids, or (d) intolerance or contraindication to MMF; (8) MMF for a duration of at least 6 months (9) participants who, in the investigator's judgment, are likely to require systemic corticosteroids during the study for reasons other than idiopathic nephrotic syndrome; (10) have received any of the exclusionary treatments (see below); (11) have had any major episode of active infection of any kind (excluding fungal infection of the nail bed) or infection requiring hospitalization or treatment with IV anti-infectives within 4 weeks prior to screening, or oral anti-infectives within 2 weeks prior to randomization (12) Completion of anti-infective medications; (13) Evidence of active tuberculosis (TB) infection; (14) History of currently active primary or secondary immunodeficiency, including known history of HIV infection and other severe immunodeficiency hematologic disorders; (15) History of severe recurrent or chronic infection; (16) History of cancer, including solid tumors, hematologic malignancies, and carcinoma in situ (excluding excised and cured basal cell carcinoma and squamous cell carcinoma of the skin) within the past 5 years; (17) Major surgery requiring hospitalization within the 4 weeks prior to or during screening;(17) High risk of clinically significant bleeding or any condition requiring plasmapheresis, intravenous immunoglobulin, or acute blood product transfusion; (18) Evidence of any significant or uncontrolled concomitant illness (including, but not limited to, neurological, respiratory, cardiac, hepatic, endocrine, malignant, or gastrointestinal disorders) that, in the investigator's judgment, would prevent the participant from participating; (19) Current active alcohol or drug abuse, or a history of alcohol or drug abuse; (20) Any of the following laboratory parameters at screening: AST or ALT > 2.5 x upper limit of normal (ULN) (for age and sex) (cannot be attributed to underlying nephrotic syndrome) Amylase or lipase >2×ULN Absolute neutrophil count <1.5 x 10 3 / μL Hemoglobin <8g / dL Platelet count <110,000 / μL for participants under 12 years of age and platelet count <50,000 / μL for patients 12 years of age or older -Positive hepatitis B surface antigen - Positive Hepatitis B core antibody -Positive hepatitis C antibody Positive serum human chorionic gonadotropin measured at screening Excluded treatments include: - Cyclophosphamide, levamisole, mizoribine, tacrolimus, cyclosporine or voclosporin in the 2 months prior to screening or during screening. Any biologic B-cell depleting therapy (e.g., anti-CD19, anti-CD20, anti-CD22), such as but not limited to, rituximab, ocrelizumab, or ofatumumab, within 9 months prior to the Day 1 baseline visit Any biologic therapy (other than anti-CD19, anti-CD20, anti-CD22) in the 2 months prior to or during screening, including but not limited to belimumab, daratumumab, ustekinumab, anifrolumab, secukinumab, or atacicept - Oral inhibitors of Janus-associated kinase (JAK), Bruton's tyrosine kinase (BTK), or tyrosine kinase 2 (TYK2) (including baricitinib, tofacitinib, upadacitinib, filgotinib, ibrutinib, or fenebrutinib or any investigational agent) in the 2 months prior to screening or during screening Any live vaccine in the 28 days prior to or during screening

[0229] research treatment The study consisted of four periods: a screening period of up to 28 days, a 52-week initial open-label treatment period, a 52-week extension period, and a minimum 12-month safety follow-up (SFU) period beginning upon completion or discontinuation of study treatment. The study schema is shown in Figure 1.

[0230] Approximately 80 participants aged ≥2-25 years will be randomized in a 1:1 ratio to one of two open-label treatment arms: Arm A (obinutuzumab) or Arm B (MMF). Randomization will be stratified by participant's disease type (FRNS vs. SDNS) and use of immunosuppressive treatment other than corticosteroids for INS prior to study entry (MMF / other immunosuppressants vs. no MMF / other immunosuppressants).

[0231] The investigational product for this study is obinutuzumab.

[0232] After a 28-day (+ / - 7-day) screening period, randomized participants enter a 52-week treatment period. During the treatment period, participants receive either a 1000 mg IV infusion of obinutuzumab on days 1, 15, 168 (week 24), and 182 (week 26) during the first 52 weeks of treatment, or initiate or continue daily oral MMF (either tablet, capsule, or liquid formulation) on day 1 according to Figure 1. Participants weighing 45 kg or greater receive a 1000 mg obinutuzumab dose. Participants weighing less than 45 kg receive a weight-adjusted dose of 20 mg / kg for obinutuzumab infusion. Methylprednisolone is administered at 80 mg IV (or 1.5 mg / kg if weighing 45 kg or less) as a premedication before the infusion. Acetaminophen / paracetamol is administered at 15 mg / kg (maximum dose 1000 mg) PO as a premedication before the infusion. Diphenhydramine hydrochloride is administered 0.5 to 1 mg / kg (maximum dose 50 mg) PO or IV as a premedication before infusion.

[0233] Participants randomized to MMF received 1200 mg / m in divided doses. 2 Participants receiving daily oral prednisone (or prednisolone equivalent) at randomization will be tapered to a target dose of 0 mg / day by Week 8 after randomization (or sooner, e.g., by Study Weeks 4-6, if appropriate) and will continue without prednisone for the remainder of the study. Participants who experience disease recurrence will be tapered to a maximum of 2 mg / kg / day (60 mg / m) until 3 or more consecutive days of negative / trace urine protein dipstick (or UPCR ≤ 0.2 g / g). 21 x 1000 mg / day to a maximum dose of 60 mg / day) prednisone or prednisolone, followed by a tapering of oral corticosteroids within 4 weeks while continuing the study treatment regimen. If participants in the MMF arm meet the criteria for salvage therapy, they are considered to have met the definition of an intercurrent event and will receive alternative therapy of obinutuzumab (2 x 1000 mg 14 days apart, or 20 mg / kg if <45 kg) or INS. If participants in the obinutuzumab arm meet the criteria for salvage therapy, they are considered to have met the definition of an intercurrent event and will receive MMF (600 mg / m in divided doses) every 4 weeks to treat recurrence according to the investigator's discretion. 2 BID [Target 1200mg / m 2 ], up to 2 g / day) or receive alternative therapy. If obinutuzumab is used, discontinue MMF.

[0234] The primary endpoint of the proportion of participants with sustained complete remission (UPCR ≤ 0.2 g / g without relapse or occurrence of other intercurrent events) at 1 year will be assessed when the last randomized participant completes Week 52.

[0235] After the Week 52 assessment, participants could continue obinutuzumab in the treatment extension period until the common end date (CCOD) or enter directly into safety follow-up (SFU). Participants who relapse after Week 52, whether in the MMF or obinutuzumab arm, will be treated according to the investigator's best judgment, which may include administering an initial or booster dose of obinutuzumab. Patients who do not meet the criteria for relapse after Week 52 are not eligible for obinutuzumab treatment in the extension period but will be followed every 3 months at study visits until CCOD. Participants with peripheral B-cell depletion will be followed in the SFU every 12 weeks for 6 months until the end of the study, and then every 6 months thereafter until peripheral CD19 B cells return to pretreatment values ​​or within the central laboratory normal range, whichever is lower for this patient population.

[0236] The first SFU visit will be scheduled during the treatment period or approximately 12 weeks after the last study visit during CCOD, whichever occurs first. No obinutuzumab infusions or MMF will be provided during the SFU. Standard of care therapy will be provided at the investigator's discretion. Patients not receiving obinutuzumab will be seen at only one SFU visit, with the assessment specified at SFU Visit 1. Patients receiving obinutuzumab will be followed at the SFU until such patients meet both of the following criteria: (1) peripheral B cells have returned to pre-obinutuzumab baseline levels or within the normal range for the population, whichever is lower; and (2) the last obinutuzumab infusion was at least 12 months prior.

[0237] At each SFU visit, absolute CD19+ B-cell counts will be measured. For participants with persistent B-cell depletion, defined as an absolute CD19+ B-cell count below the lowest pretreatment value and below the lower limit of normal (LLN) for this population, and who have not received additional therapy associated with peripheral B-cell depletion, SFU will continue every 6 months until one of the following occurs: (1) peripheral CD19+ B cells return to the lowest pretreatment value or the age-specific LLN for the patient population, whichever is lower; (2) additional therapy associated with peripheral B-cell depletion (e.g., belimumab, rituximab, or cyclophosphamide, or use of obinutuzumab outside of the study protocol); or (3) the study ends.

[0238] A participant completes the study if: (1) the SFU request is completed; (2) the last study visit of treatment at or after completion of CCOD, when the investigator intends to treat the participant for nephrotic syndrome outside the study protocol without completing the required SFU; or (3) the study is terminated. Continued access to the Roche Investigational Medicinal Product (IMP; obinutuzumab) will be provided to eligible participants by the study sponsor upon completion of the study.

[0239] Length of research period The minimum duration of study participation for each individual is expected to be approximately 1.5-2 years (the SFU is a minimum of 12 months after the last obinutuzumab infusion for all patients receiving obinutuzumab). Participants may continue to participate in the study and receive follow-up or repeat treatment according to the activity schedule until the last participant recruited has been in the study for at least 18 months.

[0240] The maximum duration of study participation is estimated to be approximately 3 years, or longer if peripheral B cells remain below LLN at the clinical cutoff, in which case participants will be required to return for SFU visits every 12 weeks for 6 months, and then every 6 months thereafter until B cells return to pre-obinutuzumab dose baseline or normal central laboratory LLN for the participant population, or until the end of the study.

Claims

1. A pharmaceutical agent for treating idiopathic nephrotic syndrome (INS) in an individual, comprising a type II anti-CD20 antibody, wherein a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody are applied to the individual, The second antibody exposure is not provided until approximately 18 to 26 weeks have elapsed since the first antibody exposure; The first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, (a) Total exposure to the type II anti-CD20 antibody between approximately 1800 mg and approximately 2200 mg, or (b) If the body weight of the individual is less than 45 kg, the total exposure to the type II anti-CD20 antibody is between approximately 36 mg / kg and approximately 44 mg / kg; The second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure is (c) Total exposure to the type II anti-CD20 antibody between approximately 1800 mg and approximately 2200 mg, or (d) If the body weight of the individual is less than 45 kg, the total exposure to the type II anti-CD20 antibody is between approximately 36 mg / kg and approximately 44 mg / kg; The aforementioned type II anti-CD20 antibody is obinutuzumab; and The aforementioned individual is a human being between 2 and 25 years of age, and is a pharmaceutical product.

2. A pharmaceutical agent comprising a type II anti-CD20 antibody for reducing the risk and / or frequency of relapse in an individual having idiopathic nephrotic syndrome (INS), wherein a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody are applied to the individual, The second antibody exposure is not provided until approximately 18 to 26 weeks have elapsed since the first antibody exposure; The first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, (a) Total exposure to the type II anti-CD20 antibody between approximately 1800 mg and approximately 2200 mg, or (b) If the body weight of the individual is less than 45 kg, the total exposure to the type II anti-CD20 antibody is between approximately 36 mg / kg and approximately 44 mg / kg; The second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure is (c) Total exposure to the type II anti-CD20 antibody between approximately 1800 mg and approximately 2200 mg, or (d) If the body weight of the individual is less than 45 kg, the total exposure to the type II anti-CD20 antibody is between approximately 36 mg / kg and approximately 44 mg / kg; The aforementioned type II anti-CD20 antibody is obinutuzumab; and The aforementioned individual is a human being between 2 and 25 years of age, and is a pharmaceutical product.

3. The pharmaceutical product according to claim 1 or 2, wherein the first antibody exposure comprises a total exposure of the type II anti-CD20 antibody between approximately 1800 mg and approximately 2200 mg; the second antibody exposure comprises a total exposure of the type II anti-CD20 antibody between approximately 1800 mg and approximately 2200 mg; and the body weight of the individual is 45 kg or more.

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

5. The pharmaceutical product according to claim 1 or 2, wherein the first antibody exposure comprises a first dose of the type II anti-CD20 antibody between approximately 18 mg / kg and approximately 22 mg / kg, and a second dose of the type II anti-CD20 antibody between approximately 18 mg / kg and approximately 22 mg / kg, and the body weight of the individual is less than 45 kg.

6. The pharmaceutical product according to claim 1 or 2, wherein the first antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the second dose of the first antibody exposure is not provided until approximately 1.5 weeks to approximately 2.5 weeks have elapsed after the first dose of the first antibody exposure.

7. The pharmaceutical product according to claim 6, wherein the first antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the second dose of the first antibody exposure is not provided until approximately two weeks have elapsed after the first dose of the first antibody exposure.

8. The pharmaceutical product according to claim 4, wherein the first dose of the first antibody exposure is approximately 1000 mg of the type II anti-CD20 antibody.

9. The pharmaceutical product according to claim 4, wherein the second dose of the first antibody exposure is approximately 1000 mg of the type II anti-CD20 antibody.

10. The pharmaceutical product according to claim 5, wherein the first dose of the first antibody exposure is the type II anti-CD20 antibody at approximately 20 mg / kg, and the body weight of the individual is less than 45 kg.

11. The pharmaceutical product according to claim 5, wherein the second dose of the first antibody exposure is the type II anti-CD20 antibody at approximately 20 mg / kg, and the body weight of the individual is less than 45 kg.

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

13. The pharmaceutical product according to claim 1 or 2, wherein the second antibody exposure comprises a first dose of the type II anti-CD20 antibody between approximately 18 mg / kg and approximately 22 mg / kg, and a second dose of the type II anti-CD20 antibody between approximately 18 mg / kg and approximately 22 mg / kg, and the body weight of the individual is less than 45 kg.

14. The pharmaceutical product according to claim 1 or 2, wherein the second antibody exposure comprises a first dose of the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and the second dose of the second antibody exposure is not provided until approximately 1.5 weeks to approximately 2.5 weeks have elapsed after the first dose of the second antibody exposure.

15. The pharmaceutical product according to claim 14, wherein the second dose of the second antibody exposure is not provided until approximately two weeks have elapsed after the first dose of the second antibody exposure.

16. The pharmaceutical product according to claim 12, wherein the first dose of the second antibody exposure is approximately 1000 mg of the type II anti-CD20 antibody.

17. The pharmaceutical product according to claim 12, wherein the second dose of the second antibody exposure is approximately 1,000 mg of the type II anti-CD20 antibody.

18. The pharmaceutical product according to claim 13, wherein the first dose of the second antibody exposure is approximately 20 mg / kg of the type II anti-CD20 antibody, and the body weight of the individual is less than 45 kg.

19. The pharmaceutical product according to claim 13, wherein the second dose of the second antibody exposure is approximately 20 mg / kg of the type II anti-CD20 antibody, and the body weight of the individual is less than 45 kg.

20. The pharmaceutical product according to claim 1 or 2, wherein the individual has or has been diagnosed with having INS.

21. The pharmaceutical product according to claim 1 or 2, wherein the INS is frequently relapsing nephrotic syndrome (FRNS).

22. The pharmaceutical product according to claim 1 or 2, wherein the INS is steroid-dependent nephrotic syndrome (SDNS).

23. The pharmaceutical product according to claim 1 or 2, wherein the individual is in complete remission before administration.

24. The pharmaceutical product according to claim 1 or 2, wherein an effective amount of glucocorticoid or corticosteroid is further administered to the individual.

25. The pharmaceutical product according to claim 24, wherein the glucocorticoid or corticosteroid comprises methylprednisolone.

26. The pharmaceutical product according to claim 25, wherein methylprednisolone is administered intravenously to the individual at a dose of 80 mg.

27. The pharmaceutical product according to claim 25, wherein methylprednisolone is administered intravenously to the individual at a dose of 1.5 mg / kg, and the individual's body weight is less than 45 kg.

28. The pharmaceutical product according to claim 24, wherein the glucocorticoid or corticosteroid comprises prednisone.

29. The pharmaceutical product according to claim 1 or 2, wherein an effective amount of antihistamine is further administered to the individual.

30. The pharmaceutical product according to claim 29, wherein the antihistamine comprises diphenhydramine.

31. The pharmaceutical product according to claim 30, wherein diphenhydramine hydrochloride is administered orally or intravenously to the individual at a dose of 0.5 to 1 mg / kg.

32. The pharmaceutical product according to claim 1 or 2, wherein an effective amount of acetaminophen is further administered to the individual.

33. The pharmaceutical product according to claim 32, wherein the acetaminophen is administered orally at a dose of 15 mg / kg, with a maximum dose of 1000 mg.

34. The pharmaceutical agent according to claim 1 or 2, which causes sustained complete remission in the individual within one year.

35. The pharmaceutical product according to claim 1 or 2, wherein the first antibody exposure comprises two doses of 1,000 mg of the type II anti-CD20 antibody on day 1 and day 15 of treatment; and the second antibody exposure comprises two doses of 1,000 mg of the type II anti-CD20 antibody on day 168 and day 182 of treatment.

36. The pharmaceutical product according to claim 1 or 2, wherein the first antibody exposure comprises two doses of the type II anti-CD20 antibody at 20 mg / kg on day 1 and day 15 of treatment; the second antibody exposure comprises two doses of the type II anti-CD20 antibody at 20 mg / kg on day 168 and day 182 of treatment; and the body weight of the individual is less than 45 kg.

37. The pharmaceutical product according to claim 1 or 2, wherein the first antibody exposure comprises two doses of 1,000 mg of the type II anti-CD20 antibody during week 0 and week 2 of treatment; and the second antibody exposure comprises two doses of 1,000 mg of the type II anti-CD20 antibody during week 24 and week 26 of treatment.

38. The pharmaceutical product according to claim 1 or 2, wherein the first antibody exposure comprises two doses of the type II anti-CD20 antibody at 20 mg / kg during weeks 0 and 2 of treatment; the second antibody exposure comprises two doses of the type II anti-CD20 antibody at weeks 24 and 26 of treatment; and the body weight of the individual is less than 45 kg.

39. A pharmaceutical agent comprising obinutuzumab for treating INS in an individual or for reducing the risk and / or frequency thereof, wherein first and second antibody exposure to obinutuzumab is administered intravenously to the individual. The first antibody exposure comprises two doses of 1000 mg of obinutuzumab during week 0 and week 2 of treatment; The second antibody exposure comprises two doses of 1000 mg of obinutuzumab at weeks 24 and 26 of treatment; The aforementioned individual is a human being between 2 and 25 years of age; and A pharmaceutical product in which the weight of the aforementioned individual is 45 kg or more.

40. A pharmaceutical agent comprising obinutuzumab for treating INS in an individual or for reducing the risk and / or frequency thereof, wherein a first antibody exposure to obinutuzumab and a second antibody exposure are administered intravenously to the individual. The first antibody exposure described above comprises two doses of obinutuzumab at 20 mg / kg during week 0 and week 2 of treatment; The second antibody exposure comprises two doses of obinutuzumab at 20 mg / kg during weeks 24 and 26 of treatment; The aforementioned individual is a human being between 2 and 25 years of age; and A pharmaceutical product in which the weight of the aforementioned individual is less than 45 kg.

41. A pharmaceutical agent comprising obinutuzumab for treating INS in an individual or for reducing the risk and / or frequency thereof, wherein a first antibody exposure to obinutuzumab and a second antibody exposure are administered intravenously to the individual. The first antibody exposure comprises two doses of 1000 mg of obinutuzumab on day 1 and day 15 of treatment; The second antibody exposure comprises two doses of 1000 mg of obinutuzumab on days 168 and 182 of treatment; The aforementioned individual is a human being between 2 and 25 years of age; and A pharmaceutical product in which the weight of the aforementioned individual is 45 kg or more.

42. A pharmaceutical agent comprising obinutuzumab for treating INS in an individual or reducing the risk and / or frequency thereof, wherein a first antibody exposure to obinutuzumab and a second antibody exposure are administered intravenously to the individual. The first antibody exposure comprises two doses of obinutuzumab at 20 mg / kg on day 1 and day 15 of treatment; The second antibody exposure described above comprises two doses of obinutuzumab at 20 mg / kg on days 168 and 182 of treatment; The aforementioned individual is a human being between 2 and 25 years of age; and A pharmaceutical product in which the weight of the aforementioned individual is less than 45 kg.

43. The pharmacopoeia according to claim 39 or 40, wherein methylprednisolone is further administered to the individual by intravenous (IV) infusion during weeks 0, 2, 24, and 26 of treatment, prior to the administration of obinutuzumab.

44. The pharmacopoeia according to claim 41 or 42, wherein methylprednisolone is further administered to the individual by intravenous (IV) infusion on days 1, 15, 168, and 182 of treatment prior to the administration of obinutuzumab.

45. (a) If the body weight of the individual is 45 kg or more, 80 mg of methylprednisolone is administered to the individual; or (b) If the body weight of the individual is less than 45 kg, 1.5 mg / kg of methylprednisolone is administered to the individual. The pharmaceutical product according to claim 43.

46. (a) If the body weight of the individual is 45 kg or more, 80 mg of methylprednisolone is administered to the individual; or (b) If the body weight of the individual is less than 45 kg, 1.5 mg / kg of methylprednisolone is administered to the individual. The pharmaceutical product according to claim 44.

47. A kit for treating INS in individuals, (a) A container comprising a type II anti-CD20 antibody, wherein the type II anti-CD20 antibody is obinutuzumab; (b) a package insert with instructions for treating INS in an individual, wherein the instructions indicate that the individual is a human being between 2 and 25 years of age; and the instructions indicate that a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody are applied to the individual, and the second antibody exposure is not provided until approximately 18 to 26 weeks after the first antibody exposure; The first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of the type II anti-CD20 antibody between approximately 1800 mg and approximately 2200 mg; The accompanying document further indicates that the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and that the second antibody exposure comprises a total exposure of the type II anti-CD20 antibody between approximately 1800 mg and approximately 2200 mg. A kit that includes this.

48. A kit for treating INS in individuals, (a) A container comprising a type II anti-CD20 antibody, wherein the type II anti-CD20 antibody is obinutuzumab; (b) a package insert with instructions for treating INS in an individual, wherein the instructions indicate that the individual is a human being between 2 and 25 years of age and weighing less than 45 kg; and the instructions indicate that a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody are applied to the individual, and the second antibody exposure is not provided until approximately 18 to 26 weeks after the first antibody exposure; The first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the first antibody exposure comprises a total exposure of the type II anti-CD20 antibody between approximately 36 mg / kg and approximately 44 mg / kg; The accompanying document further indicates that the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and that the second antibody exposure comprises a total exposure of the type II anti-CD20 antibody between approximately 36 mg / kg and approximately 44 mg / kg. A kit that includes this.

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