Compositions and methods for treating idiopathic nephrotic syndrome in pediatric stage onset

Abstract

The present disclosure provides methods for treating or reducing the risk and / or frequency of relapse of Idiopathic Nephrotic Syndrome (INS) of a pediatric period onset in an individual greater than or equal to 2 years old and less than or equal to 25 years old, or of a pediatric period onset in the individual. In some embodiments, the methods comprise administering to the individual an effective amount of Ototuzumab.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority benefit of U.S. Provisional Patent Application Serial No. 63 / 423,767, filed on November 8, 2022, which is incorporated herein by reference in its entirety.

[0003] Reference to electronic sequence listing

[0004] The content of the electronic sequence listing (146392064240seqlist.xml; size: 41,773 bytes; and creation date: November 2, 2023) is incorporated herein by reference in its entirety. Technical field

[0005] Provided herein are methods for treating childhood - onset idiopathic nephrotic syndrome (INS) in an individual (e.g., an individual greater than or equal to 2 years old and less than or equal to 25 years old) by administering a type II anti - CD20 antibody, or for reducing the risk and / or frequency of relapse in an individual with childhood - onset INS. Background art

[0006] Childhood - onset idiopathic nephrotic syndrome (INS), also known as primary nephrotic syndrome (excluding secondary causes), encompasses minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). Although this disease is rare, the incidence of childhood - onset INS varies by race and region and is higher in certain ethnic groups (notably South Asians and African Americans), where the influence of genetic factors confers a higher risk, particularly for FSGS (Chanchlani and Parekh (2016) Front Pediatr 4:39). This disease is typically first diagnosed between 2 and 5 years of age (in 70% of patients with MCD), usually 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).

[0007] Patients with childhood-onset INS are initially treated with systemic oral corticosteroids. The frequency of relapses and response to corticosteroid treatment allow the disease to be classified into subtypes that reflect disease severity. These subtypes include "steroid-resistant nephrotic syndrome (underlying genetic cause)", "non / rarely relapsing steroid-sensitive nephrotic syndrome", "FRNS", and "SDNS" (Noone et al. (2018) Lancet 392:61-74). In the latter two subtypes, patients receive multiple courses of steroids during flares and often continue on steroid maintenance therapy to prevent further relapses. The optimal goal of treatment is to maximize steroid sparing while limiting the relapse rate, based on the patient's clinical response and drug-related adverse effects.

[0008] Childhood-onset INS relapses in more than 75% of patients, and nearly 50% of patients experience frequent relapses or steroid dependence (Abdel-Hafez et al. (2017) J. Nephropathol 6:180-186). In children in whom the disease is not well controlled with steroids, multiple steroid-sparing immunosuppressive agents (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).

[0009] The anti-CD20 monoclonal antibody rituximab was first described in 2004 for the treatment of childhood-onset INS (Benz et al. (2004) Pediatr Nephrol 19(7):794-797) and has since become a promising unapproved treatment option for both children and adults with FRNS or SDNS. Many investigator-initiated trials and case reports of its use have been described in the literature, with variable clinical response rates (50%-85% of patients achieving renal remission [no relapse] at 12 months), and rituximab has been shown to reduce the annual relapse rate in patients with FRNS and SDNS. However, patients who do not achieve complete remission at 12 months typically relapse between 8 and 9 months after rituximab treatment, and these relapses mostly occur 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).

[0010] Despite the tapering or discontinuation of concurrent immunosuppression, delayed reconstitution of the memory B cell repertoire is associated with prolonged remission (Colucci et al. (2019) Front Immunol 10:1653). Obinutuzumab is a humanized, glycoengineered type II anti-CD20 antibody with enhanced depletion of B cells in peripheral blood and tissues compared to type I antibodies such as rituximab and ofatumumab. It is administered by IV infusion. Consistent with its stronger B cell depletion, obinutuzumab is superior to rituximab when administered in combination with standard chemotherapy for the treatment of chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL) in adults, and obinutuzumab has currently been approved globally for these indications. Obinutuzumab is also indicated for the treatment of patients with FL who are refractory to treatment with rituximab or rituximab-containing regimens, or who progress during or after treatment with rituximab or rituximab-containing regimens. In addition, obinutuzumab is currently in clinical development for adult and pediatric autoimmune diseases (lupus nephritis [LN], membranous nephropathy, and systemic lupus erythematosus [SLE]).

[0011] Despite current treatments for childhood-onset INS, patients still face a significant risk of other notable side effects including growth impairment and steroid-related toxicities. Given the frequent clinical relapse rates following immunosuppressive therapy in childhood-onset INS and the associated potential risk of end-stage renal disease, there remains an unmet need for an approved steroid-sparing therapy with improved efficacy and better long-term outcomes.

[0012] All references (including patent applications and publications) cited herein are incorporated by reference in their entirety. SUMMARY OF THE INVENTION

[0013] In some aspects, the present disclosure provides a method for treating childhood-onset INS in an individual, the method comprising administering to the individual a first antibody exposure to at least a type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody; wherein the second antibody exposure is provided no earlier than about 18 weeks and no later than about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the first antibody exposure comprises: (a) a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (b) a total exposure of the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the individual weighs less than 45 kg; wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure comprises: (c) a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (d) a total exposure of the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the individual weighs less than 45 kg; wherein the type II anti-CD20 antibody comprises a heavy chain and a light chain, the heavy chain comprises 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, the light chain comprises the HVR-L1 sequence of SEQ ID NO:4, the HVR-L2 sequence of SEQ ID NO:5 and the HVR-L3 sequence of SEQ ID NO:6; and wherein the individual is a person who is greater than or equal to 2 years old and less than or equal to 25 years old.The present invention also provides a method for preventing recurrence, reducing the risk of recurrence, and / or reducing the frequency of recurrence in an individual with childhood-onset idiopathic nephrotic syndrome (INS), the method comprising exposing the individual to at least a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody; wherein the second antibody exposure is provided no earlier than about 18 weeks to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the first antibody exposure comprises: (a) a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (b) a total exposure of the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the individual weighs less than 45 kg; wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure comprises: (c) a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (d) a total exposure of the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the individual weighs less than 45 kg; wherein the type II anti-CD20 antibody comprises a heavy chain and a light chain, the heavy chain comprises 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, the light chain comprises the HVR-L1 sequence of SEQ ID NO:4, the HVR-L2 sequence of SEQ ID NO:5, and the HVR-L3 sequence of SEQ ID NO:6; and wherein the individual is a person who is greater than or equal to 2 years old and less than or equal to 25 years old.

[0014] In some embodiments, the individual weighs greater than or equal to 45 kg. In some embodiments, the first antibody exposure comprises a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg; the second antibody exposure comprises a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg; and wherein the individual weighs greater than or equal to 45 kg.

[0015] In some embodiments, the first antibody exposure comprises a first dose of the type II anti-CD20 antibody between about 900 mg and about 1100 mg, and a second dose of the 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 the type II anti-CD20 antibody between about 18 mg / kg and about 22 mg / kg, and a second dose of the type II anti-CD20 antibody between about 18 mg / kg and about 22 mg / kg, and wherein the individual weighs less than 45 kg. In some embodiments, 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 wherein the second dose of the first antibody exposure is provided until about 1.5 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 the type II anti-CD20 antibody and a second dose of the type II anti-CD20 antibody, and wherein the second dose of the first antibody exposure is provided until about 2 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 the type II anti-CD20 antibody. In some embodiments, the second dose of the first antibody exposure is about 1000 mg of the type II anti-CD20 antibody. In some embodiments, the first dose of the first antibody exposure is about 20 mg / kg of the type II anti-CD20 antibody, and wherein the individual weighs less than 45 kg. In some embodiments, the second dose of the first antibody exposure is about 20 mg / kg of the type II anti-CD20 antibody, and wherein the individual weighs less than 45 kg. In some embodiments (e.g., where one or more doses of the first antibody exposure are fixed doses), the individual weighs greater than or equal to 45 kg.

[0016] In some embodiments, the second antibody exposure comprises a first dose of the type II anti-CD20 antibody between about 18 mg / kg and about 22 mg / kg, and a second dose of the type II anti-CD20 antibody between about 18 mg / kg and about 22 mg / kg, and wherein the individual weighs less than 45 kg. In some embodiments, 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 wherein the second dose of the second antibody exposure is provided from 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 provided 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 the type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure is about 1000 mg of the type II anti-CD20 antibody. In some embodiments, the first dose of the second antibody exposure is about 20 mg / kg of the type II anti-CD20 antibody, and wherein the individual weighs less than 45 kg. In some embodiments, the second dose of the second antibody exposure is about 20 mg / kg of the type II anti-CD20 antibody, and wherein the individual weighs less than 45 kg. In some embodiments (e.g., where one or more doses of the second antibody exposure are fixed doses), the individual weighs greater than or equal to 45 kg.

[0017] In some embodiments, the first antibody exposure and the second antibody exposure are administered intravenously.

[0018] 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 (e.g., prior to administration of the first antibody exposure), the individual is in complete remission.

[0019] 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 (e.g., if the individual weighs less than 45 kg), 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 with 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, the acetaminophen is administered orally at a dose of 15 mg / kg (optionally with a maximum dose of 1000 mg).

[0020] In some embodiments, the method results in sustained complete remission in the individual at 1 year. In some embodiments, the method results in depletion of circulating peripheral B cells in the individual. In some embodiments, these circulating peripheral B cells are CD19+ B cells. In some embodiments, these 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, these B cells are CD19+CD3-CD14-CD33-CD56- cells. In some embodiments, following administration of the type II anti-CD20 antibody, B cells are depleted to a level such that circulating peripheral B cells are present in the peripheral blood from the individual at about 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 from the individual at about 1 cell / 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 from 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 from the individual at the level 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 lower limit of quantification (LLOQ) of B cells by HSFC is about 1.0 cell / μL or less, about 0.8 cell / μL or less, about 0.6 cell / μL or less, about 0.5 cell / μL or less, or 0.441 cell / 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, following administration of the type II anti-CD20 antibody, the circulating peripheral B cells of the individual are depleted by at least about 90% compared to the corresponding measurement in the same individual prior to administration of the type II anti-CD20 antibody, or compared to the corresponding measurement in an individual who has not received treatment with the type II anti-CD20 antibody.

[0021] In some embodiments, the first antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody on days 1 and 15 of treatment; the second antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody on days 168 and 182 of treatment; and the type II anti-CD20 antibody is ofatumumab. In some embodiments, the first antibody exposure comprises two doses of 20 mg / kg of the type II anti-CD20 antibody on days 1 and 15 of treatment; the second antibody exposure comprises two doses of 20 mg / kg of the type II anti-CD20 antibody on days 168 and 182 of treatment; the type II anti-CD20 antibody is ofatumumab; and the individual weighs less than 45 kg. In some embodiments, the first antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody at week 0 and week 2 of treatment; the second antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody at week 24 and week 26 of treatment; and the type II anti-CD20 antibody is ofatumumab. In some embodiments, the first antibody exposure comprises two doses of 20 mg / kg of the type II anti-CD20 antibody at week 0 and week 2 of treatment; the second antibody exposure comprises two doses of 20 mg / kg of the type II anti-CD20 antibody at week 24 and week 26 of treatment; the type II anti-CD20 antibody is ofatumumab; and the individual weighs less than 45 kg. In some embodiments (e.g., where the dose of antibody exposure is a fixed dose), the individual weighs greater than or equal to 45 kg.

[0022] In certain aspects, the present disclosure provides a method for treating childhood-onset INS in an individual, or reducing the risk and / or frequency of recurrence of childhood-onset INS in the individual, the method comprising intravenously administering to the individual a first antibody exposure and a second antibody exposure to a type II anti-CD20 antibody; wherein the first antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody at weeks 0 and 2 of treatment; wherein the second antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody at weeks 24 and 26 of treatment; wherein the type II anti-CD20 antibody comprises a heavy chain and a light chain, the 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 the 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; wherein the individual is a person who is greater than or equal to 2 years old and less than or equal to 25 years old; and wherein the individual weighs greater than or equal to 45 kg. In certain aspects, the present disclosure provides a method for treating childhood-onset INS in an individual, or reducing the risk and / or frequency of recurrence of childhood-onset INS in the individual, the method comprising intravenously administering to the individual a first antibody exposure and a second antibody exposure to a type II anti-CD20 antibody; wherein the first antibody exposure comprises two doses of 20 mg / kg of the type II anti-CD20 antibody at weeks 0 and 2 of treatment; wherein the second antibody exposure comprises two doses of 20 mg / kg of the type II anti-CD20 antibody at weeks 24 and 26 of treatment; wherein the type II anti-CD20 antibody comprises a heavy chain and a light chain, the 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 the 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; wherein the individual is a person who is greater than or equal to 2 years old and less than or equal to 25 years old; and wherein the individual weighs less than 45 kg.In some aspects, provided herein is a method for treating childhood-onset INS in an individual, or reducing the risk and / or frequency of recurrence of childhood-onset INS in the individual, the method comprising intravenously administering to the individual a first antibody exposure and a second antibody exposure to a type II anti-CD20 antibody; wherein the first antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody on day 1 and day 15 of treatment; wherein the second antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody on day 168 and day 182 of treatment; wherein the type II anti-CD20 antibody comprises a heavy chain and a light chain, the 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, the 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; wherein the individual is a person who is greater than or equal to 2 years old and less than or equal to 25 years old; and wherein the individual weighs greater than or equal to 45 kg. In some aspects, provided herein is a method for treating childhood-onset INS in an individual, or reducing the risk and / or frequency of recurrence of childhood-onset INS in the individual, the method comprising intravenously administering to the individual a first antibody exposure and a second antibody exposure to a type II anti-CD20 antibody; wherein the first antibody exposure comprises two doses of 20 mg / kg of the type II anti-CD20 antibody on day 1 and day 15 of treatment; wherein the second antibody exposure comprises two doses of 20 mg / kg of the type II anti-CD20 antibody on day 168 and day 182 of treatment; wherein the type II anti-CD20 antibody comprises a heavy chain and a light chain, the 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, the 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; wherein the individual is a person who is greater than or equal to 2 years old and less than or equal to 25 years old; and wherein the individual weighs less than 45 kg. In some embodiments, the type II anti-CD20 antibody is obinutuzumab.

[0023] 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 having 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 having the amino acid sequence of SEQ ID NO:8. In some embodiments, the type II anti-CD20 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and the 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 and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO:9; the light chain comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the type II anti-CD20 antibody is ofatumumab.

[0024] In some embodiments, the method further comprises administering prednisone (e.g., orally) to the individual. In some embodiments, oral prednisone is administered to the individual at a dose of 0.5 to 1 mg / kg / day (maximum 60 mg / day). In some embodiments, oral prednisone is administered to the individual at a dose of 0.5 to 1 mg / kg / day until week 2, and then tapered to a dose of 5 mg / day by week 24 of treatment. In some embodiments, oral prednisone is administered to the individual at a dose of 0.5 to 2 mg / kg / day (maximum 60 mg / day). In some embodiments, oral prednisone is administered to the individual at a dose of 0.5 to 2 mg / kg / day until week 2, and then tapered to a dose of 5 mg / day by week 24 of treatment. In some embodiments, the method further comprises administering methylprednisolone to the individual by intravenous (IV) infusion at weeks 0, 2, 24, 26, and 52 of treatment, e.g., prior to administration of the type II anti-CD20 antibody. In some embodiments, if the individual weighs greater than or equal to 45 kg, 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.

[0025] In some aspects, the present disclosure provides a kit for treating childhood-onset INS in an individual or reducing the risk and / or frequency of recurrence of childhood-onset INS, the kit comprising: a container comprising the type II anti-CD20 antibody, wherein the type II anti-CD20 antibody comprises a heavy chain and a light chain, the 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 the 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; a package insert having instructions for using the type II anti-CD20 in any of the above and herein-described methods.

[0026] In some aspects, the present disclosure provides a type II anti-CD20 antibody (e.g., ofatumumab) for use in any of the above and herein-described methods.

[0027] 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 become apparent to those of ordinary skill in the art. These and other embodiments of the present invention are further described by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A schematic illustration of a controlled study using the type II anti-CD20 antibody ofatumumab to treat childhood-onset INS (e.g., frequently relapsing nephrotic syndrome or steroid-dependent nephrotic syndrome) in patients aged ≥2 years to 25 years is provided. BID = twice daily; CCOD = common cut-off date for primary analysis; FRNS = frequently relapsing nephrotic syndrome; MMF = mycophenolate mofetil; PO = by mouth; per os; 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 permitted if necessary. The second infusion should occur on Day 15 ± 1 day. b The primary efficacy endpoint of measuring the proportion of participants with sustained complete remission at one year is measured at Week 52. DETAILED DESCRIPTION

[0029] Childhood-onset idiopathic nephrotic syndrome (INS), also known as primary nephrotic syndrome (excluding secondary causes), encompasses minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). This disease is typically first diagnosed between the ages of 2 and 5 years (in 70% of patients with MCD), usually 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). Patients with childhood-onset INS are initially treated with systemic oral corticosteroids. However, childhood-onset INS recurs in more than 75% of patients, and nearly 50% of patients experience frequent relapses or steroid dependence (Abdel-Hafez et al. (2017) J. Nephropathol 6:180-186). Despite current treatments, patients still face a significant risk of other notable side effects, such as growth impairment and steroid-related toxicity. Therefore, there remains a need for safer and more effective treatments for childhood-onset INS (e.g., FRNS and / or SDNS).

[0030] In one aspect, the present disclosure provides a method for treating childhood-onset INS in an individual, the method comprising administering to the individual a first antibody exposure to at least a type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody; wherein the second antibody exposure is provided no sooner than about 18 to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the first antibody exposure comprises: (a) a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (b) a total exposure of the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the individual weighs less than 45 kg; wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure comprises: (c) a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (d) a total exposure of the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the individual weighs less than 45 kg; wherein the type II anti-CD20 antibody comprises a heavy chain and a light chain, the 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, the light chain comprising the HVR-L1 sequence of SEQ ID NO:4, the HVR-L2 sequence of SEQ ID NO:5 and the HVR-L3 sequence of SEQ ID NO:6; and wherein the individual is a person who is greater than or equal to 2 years old and less than or equal to 25 years old.In another aspect, the present disclosure provides methods for reducing the risk and / or frequency of relapse in an individual with childhood-onset idiopathic nephrotic syndrome (INS), the method comprising exposing the individual to at least a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody; wherein the second antibody exposure is provided no sooner than about 18 to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, the first antibody exposure comprising: (a) a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (b) a total exposure of the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the individual weighs less than 45 kg; wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, the second antibody exposure comprising: (c) a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (d) a total exposure of the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the individual weighs less than 45 kg; wherein the type II anti-CD20 antibody comprises a heavy chain and a light chain, the 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, the light chain comprising the HVR-L1 sequence of SEQ ID NO:4, the HVR-L2 sequence of SEQ ID NO:5, and the HVR-L3 sequence of SEQ ID NO:6; and wherein the individual is a person who is greater than or equal to 2 years old and less than or equal to 25 years old.

[0031] I. General Techniques

[0032] Those skilled in the art generally easily understand and typically use conventional methods to utilize the techniques and procedures described or referenced herein, such as, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (edited by F.M. Ausubel et al., (2003)); Methods in Enzymology series (Academic Press, Inc.): PCR 2: A Practical Approach (edited by M.J. MacPherson, B.D. Hames and G.R. Taylor (1995)), Harlow and Lane, editors (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (edited by R.I. Freshney (1987)); Oligonucleotide Synthesis (edited by M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by J.E. Cellis, 1998) Academic Press; Animal Cell Culture (edited by R.I. Freshney, 1987); Introduction to Cell and Tissue Culture (edited by J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, J.B. Griffiths and D.G. Newell, 1993 - 8) J.Wiley and Sons; Handbook of Experimental Immunology (edited by D.M. Weir and C.C. Blackwell); Gene Transfer Vectors for Mammalian Cells (edited by J.M. Miller and M.P.Methods widely described in Calos, Ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., Ed., 1994); Current Protocols in Immunology (J.E. Coligan et al., Ed., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty Ed., IRL Press, 1988 - 1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, Ed., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, Ed., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., Ed., J.B. Lippincott Company, 1993).

[0033] II. Definitions

[0034] The term "childhood - onset idiopathic nephrotic syndrome (INS)" refers to idiopathic nephrotic syndrome typically first diagnosed between 2 and 5 years of age, which encompasses minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) and is also referred to as primary nephrotic syndrome (excluding secondary causes).

[0035] The term "antibody" includes monoclonal antibodies (including full - length antibodies having an immunoglobulin Fc region), antibody compositions having multi - epitope specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies and single - chain molecules, and antibody fragments (e.g., Fab, F(ab') 2and Fv). The term "immunoglobulin" (Ig) is used interchangeably with antibody herein.

[0036] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units plus an additional polypeptide called the J chain, and contain ten antigen-binding sites, while IgA antibodies contain two to five basic four-chain units, which can associate with the J chain and polymerize to form multivalent assemblies. In the case of IgG, the four-chain unit is typically about 150,000 daltons. Each L chain is linked to an H chain by a covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the isotype of the H chain. Each H chain and L chain also has regularly spaced intra-chain disulfide bonds. Each H chain has a variable domain (V H ) at the N-terminus, followed by three constant domains (C H ) (for each α and γ chain) and four C H domains (for the μ and ε isotypes). Each L chain has a variable domain (V L ) at the N-terminus and a constant domain at the other end. V L is aligned with V H , and C L is aligned with the first constant domain (C H 1) of the heavy chain. It is believed that specific amino acid residues form an interface between the light and heavy chain variable domains. V H and V L pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, pp. 71 and Chapter 6. L chains derived from any vertebrate can be assigned to one of two distinct types based on the amino acid sequence of their constant domain, which are called kappa (κ) and lambda (λ), respectively. Immunoglobulins can be assigned to different classes or isotypes based on the amino acid sequence of their heavy chain constant domain (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains called α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function. For example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0037] 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 are sometimes referred to as "VH" and "VL", respectively. These domains are usually the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen-binding site.

[0038] The term "variable" refers to the fact that certain segments within the variable domains differ extensively between the sequences of antibodies. The V domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more conserved parts of the variable domains are called framework regions (FRs). The variable domains of the native heavy and light chains each contain four FR regions, which mainly adopt a β-sheet structure, connected by three HVRs, which form loops connecting the β-sheet structures and in some cases form part of the β-sheet structure. The HVRs within each chain are held tightly together by the FR regions and, together with the HVRs in the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda, MD (1991)). The constant domains do not directly participate in the binding of the antibody to the antigen but exhibit various effector functions, such as antibody-dependent cell-mediated cytotoxicity in which the antibody participates.

[0039] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor variations that may be present naturally and / or post-translational modifications (e.g., heteromerization, amidation). Monoclonal antibodies are highly specific for 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 an antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be made by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be prepared by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display techniques (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004)) and techniques for generating human or human-like antibodies in animals having a portion or all of the human immunoglobulin locus or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl.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 / Technology10:779-783(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)).

[0040] The term "naked antibody" refers to an antibody that is not conjugated to a cytotoxic moiety or a radiolabel.

[0041] The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably and refer to an antibody in its substantially intact form rather than an antibody fragment. Specifically, intact antibodies include those having a heavy chain and a light chain that include an Fc region. The constant domains may be the native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, intact antibodies may have one or more effector functions.

[0042] "Antibody fragment" encompasses a portion of an intact antibody, preferably including the antigen-binding region and / or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies (see U.S. Patent 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10) :1057-1062

[1995] ); single-chain antibody molecules and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments (referred to as "Fab" fragments) and a residual "Fc" fragment (the name reflecting its ability to crystallize readily). The Fab fragment consists of the entire L chain as well as the variable domain of the H chain (V H ) and the first constant domain of one heavy chain (C H1) Composition. Each Fab fragment is monovalent in antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody produces a single large F(ab') 2 fragment, which is roughly equivalent to two Fab fragments with different antigen-binding activities that are linked by a disulfide bond and are still capable of cross-linking antigens. The Fab' fragment differs from the Fab fragment in that the Fab' fragment has some additional residues added at the carboxyl terminus of the C H H1 domain, and these residues include one or more cysteines from the antibody hinge region. Fab'-SH is the nomenclature used herein for Fab' in which the cysteine residue of the constant domain bears a free thiol group. F(ab') 2 antibody fragments were initially produced as paired Fab' fragments with a hinge cysteine in between. Other chemical conjugations of antibody fragments are also known.

[0043] The Fc fragment contains the carboxyl-terminal portions of two heavy chains linked together by a disulfide bond. The effector functions of an antibody are determined by the sequences in the Fc region, which are also recognized by Fc receptors (FcR) present on certain types of cells.

[0044] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy-chain variable domain and a light-chain variable domain that are tightly and non-covalently associated. The folding of these two domains gives rise to six hypervariable loops (3 loops are produced by each of the H chain and the L chain), and these loops contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv that contains only three hypervariable regions (HVRs) specific for the antigen) has the ability to recognize and bind the antigen, although its affinity is lower than that of the complete binding site.

[0045] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment that contains the V H and V L antibody domains linked in a single polypeptide chain. Preferably, the sFv polypeptide further includes a polypeptide linker between the V H and V L domains to enable the sFv to form the required antigen-binding structure. For a review of sFv, see Pluckthun's The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Rosenberg and Moore, Springer-Verlag, New York, pages 269-315 (1994).

[0046] A "functional fragment" of an antibody of the invention comprises a portion of a whole antibody and generally includes the antigen-binding region or variable region of the whole antibody or the Fc region of an antibody that retains or has modified FcR-binding ability. Examples of antibody fragments include linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0047] The term diabody refers to small antibody fragments prepared by constructing sFv fragments (see previous paragraph), in which a short linker (about 5-10 residues) is present between the V H and V L domains, thereby enabling interchain pairing rather than intrachain pairing of the V domains, resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. A bispecific diabody is a heterodimer of two "crossed" sFv fragments, in which the V H and V L domains are on different polypeptide chains. Diabodies are described in more detail, for example, in EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90 :6444-6448 (1993).

[0048] Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of one or more chains is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of these antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). Chimeric antibodies for the purposes herein include antibodies in which the antigen-binding region of the antibody is derived from an antibody produced, for example, by immunizing macaques with the target antigen. As used herein, "humanized antibody" is used as a subset of "chimeric antibody".

[0049] A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains minimal sequences derived from non-human immunoglobulins. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient HVRs (as defined hereinafter) are replaced with residues from the HVRs 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 with the corresponding non-human residues. Additionally, a humanized antibody may contain residues not present in the recipient antibody or the donor antibody. These modifications can be made to further improve antibody properties, such as binding affinity. Generally, a humanized antibody will contain substantially all of at least one, usually two, variable domains in which all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of the human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions to enhance antibody properties, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FRs is usually no more than 6 in the H chain and no more than 3 in the L chain. A humanized antibody will also optionally contain at least a portion of the immunoglobulin constant region (Fc), which is usually a human immunoglobulin. For more 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 also, 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.

[0050] "Human antibody" refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human and / or produced using any of the techniques disclosed herein for the preparation of human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. A variety of techniques known in the art can be used to generate human antibodies, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Methods that can also be used to prepare human monoclonal antibodies are described, for example, 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). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5 :368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been inactivated, e.g., immunized xenomice (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 regarding the XENOMOUSE TM technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103 :3557-3562 (2006) regarding human antibodies produced by human B cell hybridoma technology.

[0051] As used herein, the terms "hypervariable region," "HVR," or "HV" refer to regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops. Generally, an antibody contains six HVRs; three in VH (H1, H2, H3), and three in VL (L1, L2, L3). In native antibodies, H3 and L3 exhibit the most diversity among the six HVRs, and in particular, H3 is thought to play a unique role in conferring fine specificity to the antibody. See, e.g.: Xu et al., Immunity 13 :37-45 (2000); Johnson and Wu, Methods in Molecular Biology 248: 1 - 25 (Lo, Editor, 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).

[0052] Many HVR descriptions are used and are included herein. Kabat complementarity - determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). In contrast, Chothia refers to the positions of structural loops (Chothia and Lesk, J. Mol. Biol. 196: 901 - 917 (1987)). AbM HVR represents a compromise between Kabat HVR and Chothia structural loops and is used by the AbM antibody modeling software of Oxford Molecular. "Contact" HVRs are based on the analysis of available complex crystal structures. The residues of each of these HVRs are described below.

[0053]

[0054] HVRs can 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. For each of these definitions, the variable domain residues are numbered according to the method of Kabat et al. described above.

[0055] The expression “Kabat-defined variable domain residue numbering” or “Kabat-defined amino acid position numbering” and variations thereof refer to the numbering system for the heavy-chain variable domain or the light-chain variable domain used in the compilation of antibodies in the Kabat et al. literature cited above. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to deletions or insertions in the FR or HVR of the variable domain. For example, the heavy-chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat numbering) and insertion residues after heavy-chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat numbering). The Kabat numbering of residues of a given antibody can be determined by aligning the antibody sequence with the homologous regions of the “standard” Kabat numbering sequence.

[0056] “Framework” or “FR” residues are those variable domain residues other than the HVR residues as defined herein.

[0057] “Human consensus framework” or “acceptor human framework” is a framework that represents the amino acid residues that are most frequently present in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include, for VL, the subgroup can be subgroup κI, κII, κIII, or κIV as described in Kabat et al. above. Additionally, for VH, the subgroup can be subgroup I, subgroup II, or subgroup III as described in Kabat et al. above. Alternatively, the human consensus framework can be derived from specific residues therein, such as when human framework residues are selected based on their homology to a donor framework sequence by aligning the donor framework sequence with a series of diverse human framework sequences. An acceptor human framework “derived” from a human immunoglobulin framework or a human consensus framework can contain the same amino acid sequence as the human immunoglobulin framework or the human consensus framework, or can contain pre-existing amino acid sequence variations. 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.

[0058] "VH subgroup III consensus framework" comprises a consensus sequence obtained from the amino acid sequences in the variable heavy 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).

[0059] "VLκI consensus framework" comprises a consensus sequence obtained from the amino acid sequences in the variable light κ 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).

[0060] "Amino acid modification" at a specific position, such as in the Fc region, refers to the substitution or deletion of a specific residue, or the insertion of at least one amino acid residue adjacent to a specific residue. An insertion "adjacent" to a specific residue refers to an insertion within one or two residues thereof. The insertion can be at the N-terminus or C-terminus of the specific residue. Preferred amino acid modifications herein are substitutions.

[0061] An "affinity matured" antibody is one in which one or more of the HVRs have one or more alterations that result in an improvement in the affinity of the antibody for the antigen as compared to the affinity of a parental antibody that does not have those alterations. In one embodiment, an affinity matured antibody has a nanomolar or even picomolar affinity for a target antigen. Affinity matured antibodies are produced by procedures well known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of HVRs and / or framework residues is described, for example, in: 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).

[0062] As used herein, the terms "specifically binds" or "is specific for" refer to a measurable and reproducible interaction, such as a binding between a target and an antibody, that identifies 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 can be an epitope) is one that binds that target with greater affinity, avidity, more readily, and / or for a longer duration than it binds other targets. In one embodiment, an antibody binds to an irrelevant target with a degree of binding that is less than about 10% of the binding of the antibody to the antigen, e.g., as measured by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of an antibody that specifically binds to a target is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved between proteins of different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0063] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which C-terminal region includes the native sequence Fc region and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is usually defined to extend from the amino acid residue at position Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of an antibody or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, a composition of a complete antibody can include a population of antibodies in which all K447 residues have been removed, a population of antibodies in which the K447 residues have not been removed, and a population of antibodies having a mixture of antibodies with and without the K447 residue. Suitable native sequence Fc regions for the antibodies of the invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0064] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. Preferred FcRs are native sequence human FcRs. In addition, preferred FcRs are receptors that bind IgG antibodies (γ receptors) and include the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See M. Annu.Rev.Immunol. 15 :203-234(1997)). FcRs are reviewed in Ravetch and Kinet, Annu.Rev.Immunol. 9 :457-92(1991); Capel et al., Immunomethods 4 :25-34(1994); and de Haas et al., J.Lab.Clin.Med. 126 :330-41(1995). The term "FcR" as used herein encompasses other FcRs, including those to be identified in the future.

[0065] 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.). In vivo binding to FcRn and the serum half - life of human FcRn - high affinity binding polypeptides can be determined, for example, in transgenic mice expressing human FcRn or transfected human cell lines or in primates administered polypeptides with variant Fc regions. WO 2004 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcR. See also, e.g., Shields et al., J. Biol. Chem. 9 (2):6591 - 6604(2001).

[0066] As used herein, the phrases “significantly reduced” or “significantly different” mean that there is a sufficiently high difference between two numerical values (usually one value is associated with a molecule and the other value is associated with a reference / control molecule) such that one of ordinary skill in the art would consider the difference between the two values to be statistically significant in the context of the biological property measured by the said value (e.g., Kd value). Depending on the value of the reference / control molecule, for example, the difference between the two values is greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40% and / or greater than about 50%.

[0067] As used herein, the terms “substantially similar” or “substantially identical” mean that there is a sufficiently high similarity between two numerical values (e.g., one value is associated with an antibody of the invention and the other value is associated with a reference / control antibody) such that one of ordinary skill in the art would consider the difference between the two values to have little biological and / or statistical significance in the context of the biological characteristic measured by the said value (e.g., Kd value). Depending on the value of the reference / control, for example, the difference between the two values is less than about 50%, less than about 40%, less than about 30%, less than about 20% and / or less than about 10%.

[0068] As used herein, "carrier" includes pharmaceutical carriers, excipients or stabilizers, which are non-toxic to cells or mammals exposed thereto at the doses and concentrations employed. Physiologically acceptable carriers are usually aqueous pH buffered solutions. Examples of physiologically acceptable carriers include: buffers such as phosphates, citrates 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 TM , polyethylene glycol (PEG) and PLURONICS TM .

[0069] "Package insert" means the instructions usually included in the commercial packaging of a drug, which contain information about indications (including indications, usage, dosage, mode of administration, contraindications, other drugs to be used in combination with the packaged product) and / or warnings etc. regarding the use of such drugs that are usually included in the commercial packaging of a drug.

[0070] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of an individual or cell being treated during a clinical pathological process. Desirable therapeutic effects include, but are not limited to, reducing the rate of disease progression, slowing or alleviating the disease state, relieving or improving the prognosis, and delaying disease progression. Delaying the progression of a disease (e.g., INS) means retarding, impeding, slowing, delaying, stabilizing, and / or postponing the development of the disease. Such delay can have different time lengths, depending on the medical history and / or the individual to be treated. It will be apparent to those skilled in the art that sufficient or significant delay can actually encompass prevention, since the individual (e.g., an individual at risk of developing the disease) does not develop the disease.

[0071] As used herein, "sustained complete remission" refers to a response to treatment that includes a first morning urine UPCR ≤ 0.2 g / g and no recurrence or any of certain concurrent events (e.g., occurring after week 8), such as (1) recurrence, defined as any one of the following events that require systemic corticosteroid or other immunosuppressive treatment: (a) first morning urine UPCR ≥ 2 g / g or (b) urine dipstick UA ≥ 3+ for 3 consecutive days, and the most recent urine sample during this 3-day period is determined to have a UPCR > 0.2 g / g or (c) urine dipstick UA protein ≥ 3+ on any day with edema, and the urine sample is determined to have a UPCR > 0.2 g / g; (2) use of any systemic corticosteroid for > 14 days within a 30-day period; (3) initiation of any INS rescue therapy other than systemic corticosteroid; (4) treatment discontinuation due to lack of efficacy; or (5) death.

[0072] As used herein, "CD20" refers to the human B-lymphocyte antigen CD20 (also known as CD20, B-lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5; the sequence is characterized by SwissProt database entry P11836), which is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B and mature B lymphocytes. (Valentine, M.A. et al., J. Biol. Chem. 264(19)(1989)11282-11287; Tedder, T.F. et al., Proc. Natl. Acad. Sci. U.S.A. 85(1988)208-12; Stamenkovic, I. et al., J. Exp. Med. 167(1988)1975-80; Einfeld, D.A. et al., EMBO J. 7(1988)711-7; Tedder, T.F. et al., J. Immunol. 142(1989)2560-8). The corresponding human gene is transmembrane 4 domain, subfamily A member 1, also known as MS4A1. This gene encodes a member of the transmembrane 4A gene family. Members of this nascent protein family are characterized by common structural features and similar intron / exon splicing boundaries and exhibit a unique expression pattern in hematopoietic cells and non-lymphoid tissues. This gene encodes a B-lymphocyte surface molecule that plays a role in B-cell development and differentiation into plasma cells. Members of this family are localized to 11q12 in a cluster of family members. Alternative splicing of this gene produces two transcript variants that encode the same protein.

[0073] The terms "CD20" and "CD20 antigen" are used interchangeably herein and include any variant, isotype, and species homolog of human CD20 that is naturally expressed by cells or expressed on cells transfected with the CD20 gene. The antibodies of the present invention mediate the killing of CD20-expressing cells (e.g., tumor cells) by binding to the CD20 antigen and 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.

[0074] As is well recognized in the art, aliases of CD20 include B lymphocyte antigen CD20, B lymphocyte surface antigen B1, Leu-16, Bp35, BM5, and LF5.

[0075] The term "anti-CD20 antibody" according to the present invention is an antibody that specifically binds to the CD20 antigen. According to the binding characteristics and biological activities of anti-CD20 antibodies to the CD20 antigen, two types of anti-CD20 antibodies (type I and type II anti-CD20 antibodies) can be distinguished according to the methods described by Cragg, M.S. et al., Blood 103 (2004) 2738-2743; and Cragg, M.S. et al., Blood 101 (2003) 1045-1052, see Table 1 below.

[0076] Table 1. Type I and Type II Anti-CD20 Antibodies

[0077] Type I anti-CD20 antibody Type II anti-CD20 antibody Type I CD20 epitope Type II CD20 epitope Localize CD20 to lipid rafts Do not localize CD20 to lipid rafts Enhance CDC (if IgG1 isotype) Reduce CDC (if IgG1 isotype) ADCC activity (if IgG1 isotype) ADCC activity (if IgG1 isotype) Full binding capacity Reduced binding capacity Homotypic polymerization Stronger homotypic polymerization Induce apoptosis after crosslinking Induce strong cell death without crosslinking

[0078] Examples of type II anti-CD20 antibodies include, for example, the humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody as disclosed in WO 2005 / 044859), 11B8 IgG1 (as disclosed in WO 2004 / 035607), and AT80 IgG1. Generally, type II anti-CD20 antibodies of the IgG1 isotype are characterized by characteristic CDC properties. Type II anti-CD20 antibodies have reduced CDC compared to type I antibodies of the IgG1 isotype (if of the IgG1 isotype).

[0079] Examples of type I anti-CD20 antibodies include, for example, rituximab, HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (as disclosed in WO 2005 / 103081), 2F2 IgG1 (as disclosed in WO 2004 / 035607 and WO 2005 / 103081), and 2H7 IgG1 (as disclosed in WO 2004 / 056312).

[0080] The afucosylated anti-CD20 antibody according to 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.

[0081] The "rituximab" antibody (reference antibody; an example of a type I anti-CD20 antibody) is a genetically engineered chimeric human γ1 murine constant domain that comprises a monoclonal antibody against the human CD20 antigen. However, the antibody is not glycoengineered and is not afucosylated, and thus the content of fucose is at least 85%. The chimeric antibody contains the human γ1 constant domain and is identified by the name "C2B8" in US 5,736,137 (Andersen et al.) granted to IDEC Pharmaceuticals Corporation on April 17, 1998. Rituximab is approved for the treatment of relapsed or refractory low-grade or follicular, CD20-positive B-cell non-Hodgkin's lymphoma. In vitro mechanism of action studies have shown that rituximab exhibits human complement-dependent cytotoxicity (CDC) (Reff, M.E. et al., Blood 83(2)(1994)435-445). In addition, it exhibits activity in assays measuring antibody-dependent cell cytotoxicity (ADCC).

[0082] As used herein, the term "GA101 antibody" refers to any of the following antibodies that bind to human CD20: (1) an antibody that comprises: HVR-H1, which comprises the amino acid sequence of SEQ ID NO:1; HVR-H2, which comprises the amino acid sequence of SEQ ID NO:2; HVR-H3, which comprises the amino acid sequence of SEQ ID NO:3; HVR-L1, which comprises the amino acid sequence of SEQ ID NO:4; HVR-L2, which comprises the amino acid sequence of SEQ ID NO:5; and HVR-L3, which comprises the amino acid sequence of SEQ ID NO:6; (2) an antibody that comprises: a VH domain that comprises the amino acid sequence of SEQ ID NO:7; and a VL domain that comprises the amino acid sequence of SEQ ID NO:8; (3) an antibody that comprises the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:10; (4) the antibody known as obinutuzumab; or (5) an antibody that 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 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: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.

[0083] The term "humanized B-Ly1 antibody" refers to the humanized B-Ly1 antibody as disclosed in WO 2005 / 044859 and WO 2007 / 031875, which is obtained by chimerizing the murine monoclonal anti-CD20 antibody B-Ly1 (murine heavy chain variable region (VH): SEQ ID NO:11; murine light chain variable region (VL): SEQ ID NO:12 - see Poppema, S. and Visser, L, Biotest Bulletin 3 (1987) 131 - 139) with human constant domains from IgG1 and then humanizing it (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.

[0084] Variable region of the murine monoclonal anti-CD20 antibody B-Ly1 heavy chain (VH) (SEQ ID NO:11)

[0085]

[0086] Variable region of the light chain (VL) of murine monoclonal anti-CD20 antibody B-Ly1 (SEQ ID NO:12)

[0087]

[0088] In one embodiment, the humanized B-Ly1 antibody has a heavy chain variable region (VH) selected from the group consisting of SEQ ID NOs: 7, 8, and 13 to 33 (specifically corresponding 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 a specific embodiment, the humanized B-Ly1 antibody has a light chain variable region (VL) of SEQ ID NO: 8 (corresponding to B-KV1 of WO2005 / 044859 and WO 2007 / 031875). In a specific embodiment, the humanized B-Ly1 antibody has a heavy chain variable region (VH) of SEQ ID NO: 7 (corresponding to B-HH6 of WO 2005 / 044859 and WO 2007 / 031875) and a light chain variable region (VL) of SEQ ID NO: 8 (corresponding to B-KV1 of WO 2005 / 044859 and WO 2007 / 031875). Further, in one embodiment, the humanized B-Ly1 antibody is an IgG1 antibody. According to the present invention, such afucosylated humanized B-Ly1 antibody is 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-KV1GE. In one embodiment, the anti-CD20 antibody is ofatumumab (proposed INN, WHO Drug Information, Vol. 26, No. 4, 2012, p. 453). As used herein, ofatumumab is synonymous with GA101 or RO5072759. It replaces all previous versions (e.g., Vol. 25, No. 1, 2011, pp. 75-76), and was originally called arfolitiximab (proposed INN, WHO Drug Information, Vol. 23, No. 2, 2009, p. 176; Vol. 22, No. 2, 2008, p. 124). As used herein, reference to ofatumumab refers to and its biosimilar antibodies. In some embodiments, the humanized B-Ly1 antibody is an antibody that comprises: a heavy chain that comprises the amino acid sequence of SEQ ID NO:9; and a light chain that comprises the amino acid sequence of SEQ ID NO:10; or an antigen-binding fragment thereof. In some embodiments, the humanized B-Ly1 antibody comprises: a heavy chain variable region that comprises the three heavy chain CDRs of SEQ ID NO:9; and a light chain variable region that comprises the three light chain CDRs of SEQ ID NO:10.

[0089] Heavy chain (SEQ ID NO:9)

[0090]

[0091]

[0092] Light chain (SEQ ID NO:10)

[0093]

[0094] In some embodiments, the humanized B-Ly1 antibody is a defucosylated 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 oligosaccharide 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). Additionally, the oligosaccharides in the Fc region are preferably quantitated. These glycoengineered humanized B-Ly1 antibodies have increased ADCC.

[0095] In a FACS assay (Becton Dickinson) using Raji cells (ATCC-No. CCL-86), the "binding ability ratio of the anti-CD20 antibody to CD20 on Raji cells (ATCC-No. CCL-86) compared to rituximab" was determined by direct immunofluorescence measurement (measuring the mean fluorescence intensity (MFI)) using the anti-CD20 antibody conjugated to Cy5 and rituximab conjugated to Cy5, as described in Example 2, and the calculation formula is as follows:

[0096] Binding ability ratio to CD20 on Raji cells (ATCC-No. CCL-86) =

[0097]

[0098] MFI is the mean fluorescence intensity. As used herein, "Cy5 labeling ratio" means the number of Cy5-labeled molecules per molecule of antibody.

[0099] Generally, the type II anti-CD20 antibody has a binding ability ratio to CD20 on Raji cells (ATCC-No. CCL-86) of 0.3 to 0.6, in one embodiment 0.35 to 0.55, and in yet another embodiment 0.4 to 0.5, compared to rituximab.

[0100] In one embodiment, the type II anti-CD20 antibody (e.g., the GA101 antibody) has enhanced antibody-dependent cell-mediated cytotoxicity (ADCC).

[0101] The term "antibody having increased antibody-dependent cell-mediated cytotoxicity (ADCC)" as defined herein means an antibody having increased ADCC measured by any suitable method known to those of ordinary skill in the art. One well-recognized in vitro ADCC assay is as follows:

[0102] 1) The assay uses target cells known to express the target antigen recognized by the antigen-binding region of the antibody;

[0103] 2) The assay uses human peripheral blood mononuclear cells (PBMCs) isolated from the blood of randomly selected healthy donors as effector cells;

[0104] 3) The assay is performed according to the following protocol:

[0105] i) PBMCs are isolated using standard density centrifugation procedures and suspended at a density of 5×10 6 cells / ml in RPMI cell culture medium;

[0106] ii) Target cells are grown by standard tissue culture methods, harvested from the exponential growth phase, with a cell viability greater than 90%, washed in RPMI cell culture medium, labeled with 100 microcuries of 51 Cr, washed twice with cell culture medium, and resuspended at a density of 10 5 cells / ml in cell culture medium;

[0107] iii) 100 microliters of the above final target cell suspension is transferred to each well of a 96-well microtiter plate;

[0108] iv) The antibody is serially diluted from 4000 ng / ml to 0.04 ng / ml in cell culture medium, and then 50 microliters of the resulting antibody solution is added to the target cells in the 96-well microtiter plate, and various antibody concentrations covering the entire above concentration range are assayed in triplicate;

[0109] v) For the maximum release (MR) control, 50 μl of an aqueous solution of 2% (VN) non-ionic detergent (Nonidet, Sigma, St.

[0110] Louis) is received in the other 3 wells of the plate containing the labeled target cells instead of the antibody solution (point iv above);

[0111] vi) For the spontaneous release (SR) control, 50 μl of RPMI cell culture medium is received in the other 3 wells of the plate containing the labeled target cells instead of the antibody solution (point iv above);

[0112] vii) The 96-well microtiter plate is then centrifuged at 50 × g for 1 minute and incubated at 4 °C for 1 hour;

[0113] viii) 50 μl of the 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 with a 5% CO2 atmosphere and at 37 °C for 4 hours;

[0114] ix) The cell-free supernatant is harvested from each well, and the radioactivity of the experimentally released (ER) is measured using a gamma counter;

[0115] x) The percentage of specific lysis at each antibody concentration is calculated according to the formula (ER - MR) / (MR - SR) × 100, where ER is the average radioactivity at that antibody concentration measured (see point ix above), MR is the average radioactivity of the MR control measured (see point V above) (see point ix above), and SR is the average radioactivity of the SR control measured (see point vi above) (see point ix above);

[0116] 4) "Increased ADCC" is defined as an increase in the maximum percentage of specific lysis observed within the range of antibody concentrations tested above, and / or a decrease in the antibody concentration required to reach half of the maximum percentage of specific lysis observed within the range of antibody concentrations tested above. In one embodiment, the increase in ADCC, relative to ADCC measured using the above assay, is mediated by the same antibody, produced by the same type of host cell, using the same standard production, purification, formulation, and storage methods known to those skilled in the art, except that the comparator antibody (lacking increased ADCC) is not produced by a host cell engineered to overexpress GnTIII and / or engineered to have reduced fucosyltransferase 8

[0117] (FUT8) gene expression (e.g., including designed for FUT8 knockout).

[0118] Said "increased ADCC" can be obtained, for example, by mutation of said antibody and / or glycoengineering. In one embodiment, the antibody is glycoengineered to have a bisected biantennary oligosaccharide linked to the antibody Fc region by GlcNAc, such as described in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); US2005 / 0123546 (Umana et al.), Umana, P. et al., Nature Biotechnol. 17(1999)176-180). In another embodiment, the antibody is glycoengineered by expressing the antibody in a host cell lacking protein fucosylation (e.g., Lec13 CHO cells or cells with deletion of the α-1,6-fucosyltransferase gene (FUT8) or knockdown of FUT gene expression) such that fucose is absent on the carbohydrate linked to the Fc region (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614(2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688(2006); and WO2003 / 085107). In yet another embodiment, the antibody sequence has been engineered in its Fc region to enhance ADCC (e.g., in one embodiment, such engineered antibody variants comprise an Fc region having one or more amino acid substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region).

[0119] The term "complement-dependent cytotoxicity (CDC)" refers to the lysis of human tumor target cells by an antibody according to 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 if the antibody at a concentration of 100 nM induces 20% or more tumor cell lysis (cell death) after 4 hours. In one embodiment, the assay uses 51 Cr- or Eu-labeled tumor cells and measures the released 51 Cr or Eu. Controls include co-incubating the tumor target cells with complement in the absence of antibody.

[0120] The term "expression of CD20 antigen" is intended to mean significant level of expression of the CD20 antigen in a cell (e.g., a T cell or a B cell). In one embodiment, a patient to be treated according to the method of the invention expresses a significant level of CD20 on B cells. CD20 expression on B cells can be determined by standard assays known in the art, e.g., measuring CD20 antigen expression using immunohistochemical (IHC) detection, FACS, or via PCR-based detection of the corresponding mRNA.

[0121] As used in this specification and the appended claims, the singular forms "a", "an", "the", and "said" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes combinations of two or more such molecules, and the like.

[0122] As used herein, the term "about" refers to the usual error range of the corresponding value that is readily known to those skilled in the art. Reference to "about" a value or parameter herein includes (and describes) embodiments that relate to that value or parameter itself.

[0123] It should be understood that aspects and embodiments of the invention described herein include those aspects and embodiments referred to by "comprising", "consisting of", and "consisting essentially of".

[0124] III. Methods

[0125] In one aspect, provided herein is a method of treating childhood-onset idiopathic nephrotic syndrome (INS) in an individual by administering an effective amount of a type II anti-CD20 antibody; wherein the individual is a person who is greater than or equal to 2 years old and less than or equal to 25 years old. In another aspect, provided herein is a method of treating the risk and / or frequency of recurrence of childhood-onset idiopathic nephrotic syndrome (INS) in an individual by administering an effective amount of a type II anti-CD20 antibody; wherein the individual is a person who is greater than or equal to 2 years old and less than or equal to 25 years old. In one aspect, provided herein is a method of treating childhood-onset INS in an individual or depleting the circulating peripheral B cells of an individual by administering an effective amount of a type II anti-CD20 antibody; wherein the individual is a person who is greater than or equal to 2 years old and less than or equal to 25 years old.

[0126] In some embodiments, such as 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 the type II anti-CD20 antibody, with the second antibody exposure not provided until about 18 to about 26 weeks after the first antibody exposure; wherein 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 to the type II anti-CD20 antibody of between about 1800 mg and about 2200 mg; and wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody and the second antibody exposure comprises a total exposure to the type II anti-CD20 antibody of between about 1800 mg and about 2200 mg. 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 the type II anti-CD20 antibody, with the second antibody exposure not provided until about 18 to about 26 weeks after the first antibody exposure, wherein 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 to the type II anti-CD20 antibody of between about 36 mg / kg and about 44 mg / kg; wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody and the second antibody exposure comprises a total exposure to the type II anti-CD20 antibody of between about 36 mg / kg and about 44 mg / kg. As described herein, the antibody comprises a heavy chain and a light chain, the 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; the 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 sequences of SEQ ID NO:9 and SEQ ID NO:10. In some embodiments, the antibody comprises an antibody having 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 having 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 some embodiments, the antibody is ofatumumab.

[0127] anti-CD20 antibody

[0128] 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 of childhood-onset INS. In some embodiments, the anti-CD20 antibody is a type II antibody. In some embodiments, the anti-CD20 antibody is human or humanized. In some embodiments, the anti-CD20 antibody is afucosylated. In some embodiments, the anti-CD20 antibody is the GA101 antibody.

[0129] Examples of type II anti-CD20 antibodies include, for example, the humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody as disclosed in WO 2005 / 044859), 11B8 IgG1 (as disclosed in WO 2004 / 035607), and AT80 IgG1. Generally, type II anti-CD20 antibodies of the IgG1 isotype are characterized by characteristic CDC properties. Type II anti-CD20 antibodies have reduced CDC compared to type I antibodies of the IgG1 isotype (if of the IgG1 isotype).

[0130] In some embodiments, the anti-CD20 antibody is the GA101 antibody described herein. In some embodiments, anti-CD20 refers to any one of the following antibodies that binds to human CD20: (1) an antibody that comprises: HVR-H1, which comprises the amino acid sequence of GYAFSY (SEQ ID NO:1); HVR-H2, which comprises the amino acid sequence of FPGDGDTD (SEQ ID NO:2); HVR-H3, which comprises the amino acid sequence of NVFDGYWLVY (SEQ ID NO:3); HVR-L1, which comprises the amino acid sequence of RSSKSLLHSNGITYLY (SEQ ID NO:4); HVR-L2, which comprises the amino acid sequence of QMSNLVS (SEQ ID NO:5); and HVR-L3, which comprises the amino acid sequence of AQNLELPYT (SEQ ID NO:6); (2) an antibody that comprises: a VH domain that comprises the amino acid sequence of SEQ ID NO:7; and a VL domain that comprises the amino acid sequence of SEQ ID NO:8; (3) an antibody that comprises the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:10; (4) the antibody known as obinutuzumab; or (5) an antibody that 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 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: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, such as 3 HVRs from SEQ ID NO:7 and 3 HVRs from SEQ ID NO:8, 3 HVRs from SEQ ID NO:9 and 3 HVRs from SEQ ID NO:10, or any HVRs of the amino acid sequences provided in Table 2.

[0131] In some embodiments, the anti-CD20 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7 and the light chain variable region comprising the amino acid sequence of SEQ ID NO:8.

[0132]

[0133] 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.

[0134]

[0135] In some embodiments, the anti-CD20 antibody is a humanized B-Ly1 antibody. In some embodiments, the humanized B-Ly1 antibody comprises: a heavy chain variable region comprising the three heavy chain CDRs of SEQ ID NO:9; and a light chain variable region comprising the three light chain CDRs of SEQ ID NO:10. In some embodiments, the humanized B-Ly1 antibody comprises: a heavy chain comprising the sequence of SEQ ID NO:9; and a light chain comprising the sequence of SEQ ID NO:10.

[0136] In some embodiments, the anti-CD20 antibody comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the polypeptide sequences listed in Table 2 below.

[0137] Table 2. Polypeptide sequences.

[0138]

[0139]

[0140]

[0141]

[0142]

[0143] In some embodiments, the anti-CD20 antibody (e.g., a type II anti-CD20 antibody) is a defucosylated 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 oligosaccharide 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). In addition, the oligosaccharides in the Fc region are preferably bisected. In some embodiments, the type II anti-CD20 antibody comprises an Fc region that comprises a biantennary oligosaccharide bisected with N-acetylglucosamine (GlcNAc). These glycoengineered humanized anti-CD20 (e.g., B-Ly1) antibodies have increased ADCC.

[0144] Oligosaccharide components can significantly affect properties related to the efficacy of therapeutic glycoproteins, including physical stability, resistance to protease attack, interaction with the immune system, pharmacokinetics, and specific biological activities. Such properties may depend not only on the presence or absence of oligosaccharides, but also on their specific structures. Some generalizations can be made between oligosaccharide structures and glycoprotein function. For example, certain oligosaccharide structures mediate the rapid clearance of glycoproteins from the bloodstream through interaction with specific carbohydrate-binding proteins, while other oligosaccharide structures can bind to antibodies and trigger unwanted immune responses. (Jenkins, N. et al., Nature Biotechnol. 14 (1996) 975 - 81).

[0145] Mammalian cells are the preferred hosts for the production of therapeutic glycoproteins because they are able to glycosylate proteins in a form that is most suitable for human applications. (Cumming, D.A. et al., Glycobiology 1 (1991) 115 - 30; Jenkins, N. et al., Nature Biotechnol. 14 (1996) 975 - 81). Bacteria rarely glycosylate proteins and, like other types of common hosts such as yeast, filamentous fungi, insects, and plant cells, produce glycosylation patterns associated with rapid clearance from the bloodstream, unwanted immune interactions, and reduced biological activity in certain specific cases. Among mammalian cells, Chinese hamster ovary (CHO) cells have been the most commonly used over the past two decades. In addition to providing a suitable glycosylation pattern, these cells also allow the continuous generation of genetically stable, high-yielding clonal cell lines. They can be cultured to high density in a simple bioreactor using serum-free media and allow 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. More recently, the production of transgenic animals has also been tested. (Jenkins, N. et al., Nature Biotechnol. 14 (1996) 975 - 981).

[0146] Antibodies can contain carbohydrate structures at conserved positions in the heavy-chain constant regions, where each isotype has a different array of N-linked carbohydrate structures that differentially affect protein assembly, secretion, or functional activity. (Wright, A. and Morrison, S.L., Trends Biotech. 15 (1997) 26-32). The structures of the attached N-linked carbohydrates vary widely depending on the degree of processing and can include high-mannose, multi-branched, and bi-antennary 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 specific glycosylation site such that even monoclonal antibodies exist as multiple glycoforms. Similarly, major differences in antibody glycosylation have been shown to occur between cell lines, and even minor differences are observed for a given cell line grown under different culture conditions. (Lifely, M.R. et al., Glycobiology 5(8) (1995) 813-22).

[0147] One approach to obtaining a substantial increase in potency while maintaining a simple production process and potentially avoiding significant adverse side effects is to enhance the natural, cell-mediated effector functions of monoclonal antibodies by engineering the oligosaccharide components of monoclonal antibodies, as described by Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180 and US 6,602,684. Antibodies of the IgG1 isotype, which are the most commonly used antibodies in cancer immunotherapy, are glycoproteins that have a conserved N-linked glycosylation site at Asn297 in each CH2 domain. The two complex bi-antennary oligosaccharides attached to Asn297 are buried between the CH2 domains, make extensive contacts with the polypeptide backbone, and their presence is essential for antibody-mediated effector functions such as antibody-dependent cell cytotoxicity (ADCC) (Lifely, M.R. et al., Glycobiology 5 (1995) 813-822; Jefferis, R. et al., Immunol. Rev. 163 (1998) 59-76; Wright, A., and Morrison, S.L., Trends Biotechnol. 15 (1997) 26-32).

[0148] Previous studies have shown that overexpression of β(1,4)-N-acetylglucosaminyltransferase I11 ("GnTII17y), a glycosyltransferase that catalyzes the formation of bisecting oligosaccharides, in Chinese hamster ovary (CHO) cells significantly increases the in vitro ADCC activity of an anti-neuroblastoma chimeric monoclonal antibody (chCE7) produced by engineered CHO cells. (See Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180; and WO 99 / 154342, the entire contents of these references are hereby incorporated by reference). The antibody chCE7 belongs to a large class of unconjugated monoclonal antibodies that have high tumor affinity and specificity, but are too low in potency to be used clinically when produced in standard industrial cell lines lacking the GnTIII enzyme (Umana, P. et al., Nature Biotechnol. 17 (1999) 176-180). This study was the first to show that engineering antibody-producing cells to express GnTIII can greatly enhance ADCC activity, which also results in an increased proportion of bisecting oligosaccharides (including bisecting non-fucosylated oligosaccharides) associated with the constant region (Fc) above the levels found in naturally occurring antibodies.

[0149] 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 bisecting oligosaccharide is a bisecting complex oligosaccharide. In some embodiments, the N-linked oligosaccharides have been modified to have increased bisecting non-fucosylated oligosaccharides. In some embodiments, the bisecting non-fucosylated oligosaccharide is of the hybrid type. In some embodiments, the bisecting non-fucosylated oligosaccharide is of the complex type. For a more detailed description, see, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); US2005 / 0123546 (Umana et al.); and U.S. Patent No. 8,883,980 (Umana et al.).

[0150] In some embodiments, the type II anti-CD20 antibody is ofatumumab.

[0151] Antibody Preparation

[0152] The antibody according to any of the above embodiments (e.g., the type II anti-CD20 antibody of the present disclosure) may incorporate any of the features, alone or in combination, as described in Sections 1-7 below:

[0153] 1. Antibody Affinity

[0154] In certain embodiments, provided herein is an antibody having a dissociation constant (Kd) 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 smaller, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M).

[0155] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed with a Fab form of the target antibody and its antigen. For example, by titrating the antigen with a minimum concentration ( 125 The solution binding affinity of Fab for antigen is measured by equilibrating Fab with 1) labeled antigen and then capturing bound antigen with a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To determine the conditions for the assay, 5 μg / ml capture anti-Fab antibody (CappelLabs) was coated in 50 mM sodium carbonate (pH 9.6). Multiwell plates (Thermo Scientific) were blocked overnight and then blocked with 2% (w / v) bovine serum albumin in PBS for two to five 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., following 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; however, the incubation can be continued for longer (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution is then removed and washed with 0.1% polysorbate 20 (TWEEN- ) Wash the plate eight times. When the plate has dried, add 150 μl / well of scintillant (MICROSCINT-20 TM ; Packard), and in TOPCOUNT TM The plates were counted for tens of minutes on a gamma counter (Packard).The concentration of each Fab that gave less than or equal to 20% of maximal binding was chosen for use in the competitive binding assay.

[0156] According to another embodiment, using Surface plasmon resonance assays are used to measure Kd. For example, assays are performed at 25 °C with a CM5 chip immobilized with antigen at ~10 response units (RU) using -2000 or -3000 (BIAcore, Inc., Piscataway, NJ). 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 (about 0.2 μM) in 10 mM sodium acetate pH 4.8 and then injected at a flow rate of 5 μl / min to obtain a coupled protein of approximately 10 response units (RU). After injecting the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, at 25 °C, a two-fold serial dilution (0.78 nM to 500 nM) of Fab in PBS containing 0.05% polysorbate 20 (TWEEN-20 TM ) surfactant (PBST) is injected at a flow rate of approximately 25 μl / min. The association rate (kon) and dissociation rate (koff) are calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model ( Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate exceeds 106 M-1 s-1 as determined by the above surface plasmon resonance assay, the association rate can be determined by using fluorescence quenching techniques, i.e., as measured in a spectrometer such as a stopped-flow equipped spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO TM spectrophotometer (ThermoSpectronic) using a stirred cuvette, by measuring the increase or decrease in the fluorescence emission intensity (excitation wavelength = 295 nm; emission wavelength = 340 nm, bandpass = 16 nm) of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25 °C in the presence of increasing concentrations of antigen.

[0157] 2. Antibody Fragments

[0158] 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, and other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., as described in The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Rosenberg and Moore, (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab fragments and F(ab') 2 fragments that contain salvage receptor binding epitope residues and have an extended in vivo half-life, see U.S. Patent No. 5,869,046.

[0159] Diabodies are antibody fragments that have two antigen-binding sites and can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetra-bodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0160] 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, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0161] Antibody fragments can be prepared 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.

[0162] 3. Chimeric and Humanized Antibodies

[0163] In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody 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 another example, a chimeric antibody is a "class-switch" antibody in which the class or subclass has been changed from that of the parental antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0164] In certain embodiments, the chimeric antibodies are humanized antibodies. Generally, non-human antibodies are humanized to reduce their immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. Typically, a humanized antibody comprises one or more variable domains in which the HVRs, such as CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. A humanized antibody optionally also will comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), such as to restore or improve antibody specificity or affinity.

[0165] Humanized antibodies and methods of making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specific determinant region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing "guided selection" methods for FR shuffling).

[0166] Human framework regions useful 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 from consensus sequences of human antibodies from specific subgroups of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); human mature (somatic mutation) 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)).

[0167] 4. Human Antibodies

[0168] In certain embodiments, the antibodies provided herein are human antibodies. A variety of techniques known in the art can be used to generate human antibodies. 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).

[0169] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce a complete human antibody or a complete antibody having human variable regions in response to antigenic stimulation. Such animals typically contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus, or is present extrachromosomally or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically 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 the XENOMOUSE TM technology; U.S. Patent No. 5,770,429, which describes technology; U.S. Patent No. 7,041,870, which describes K-M technology, and U.S. Patent No. 7,041,870, which describes The human variable regions of the antibodies can be further modified from those produced by such animals, for example, by combining with different human constant regions.

[0170] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines have been described for the production of human monoclonal antibodies. (See, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (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).

[0171] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0172] 5. Antibodies from Libraries

[0173] The antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies having one or more desired activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies having desired binding characteristics. Such methods are reviewed in, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001) and further described in the following references: for example, 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, Methods in Molecular Biology 248:161-175 (Lo ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34); 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0174] In certain phage display methods, all components of the VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, from which antigen-binding phages can then be selected, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies against the immunogen without the need to construct hybridomas. Alternatively, all native components (e.g., all native components from a human) can be cloned to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, a naive library can also be generated by cloning unrearranged V gene segments from stem cells; and using PCR primers containing random sequences to encode the highly variable CDR3 region and complete in vitro rearrangement, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373, and U.S. Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0175] Antibodies or antibody fragments isolated from a human antibody library herein are considered human antibodies or human antibody fragments.

[0176] 6. Multispecific Antibodies

[0177] In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for CD20 and the other is for any other antigen. In certain embodiments, the bispecific antibody 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 made as full-length antibodies or antibody fragments.

[0178] Techniques for preparing 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 "stuffer" engineering (see, e.g., U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic manipulation effects to prepare antibody Fc-heterodimer molecules (WO 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to prepare 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., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al., J. Immunol. 147:60 (1991).

[0179] Engineered antibodies having three or more functional antigen-binding sites, including "octopus antibodies", are also included herein (see, e.g., US2006 / 0025576A1).

[0180] The antibodies or fragments herein also include "dual action FAb" or "DAF", which contain antigen-binding sites that bind to CD20 as well as other different antigens (see, e.g., US 2008 / 0069820).

[0181] 7. Antibody Variants

[0182] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions, and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to achieve the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

[0183] a) Substitutional, Insertional, and Deletional Variants

[0184] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitution mutations include HVRs and FRs. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table A. More substantial changes are provided under the heading "Exemplary Substitutions" in Table A 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 screened for the desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0185] Table A

[0186]

[0187] Amino acids can be grouped according to common side-chain properties:

[0188] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0189] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0190] (3) Acidic: Asp, Glu;

[0191] (4) Basic: His, Lys, Arg;

[0192] (5) Residues that influence chain orientation: Gly, Pro;

[0193] (6) Aromatic: Trp, Tyr, Phe.

[0194] Non-conservative substitutions will entail exchanging a member of one of these classes for another.

[0195] One type of substitution variant involves substituting one or more hypervariable region residues of a parental antibody (e.g., a humanized antibody or a human antibody). Generally, one or more of the resulting variants selected for further study will have an alteration (e.g., improvement) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parental antibody and / or will substantially retain certain biological properties of the parental antibody. Exemplary substitution variants are affinity matured antibodies, which can be readily generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0196] For example, the HVRs can be altered (e.g., substituted) to improve antibody affinity. Such alterations can be made in HVR “hotspots,” i.e., residues encoded by codons that undergo high frequency mutation during somatic maturation (see, e.g., Chowdhury, Methods Mol.Biol. 207:179-196 (2008)) and / or residues that contact the antigen, and the binding affinity of the resulting variant VH or VL is tested. Affinity maturation by construction and reselection from secondary libraries has been described, e.g., by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al. eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation using any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is subsequently screened to identify any antibody variants having the desired affinity. Another method of introducing diversity involves HVR-directed methods, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. The HVR residues involved in antigen binding can be specifically identified, e.g., using alanine-scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0197] In certain embodiments, substitutions, insertions, or deletions can occur within one or more HVRs, so long as such alterations do not substantially reduce the antigen-binding ability of the antibody. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in the HVRs. Such alterations can be outside of the antigen-contact residues of the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR remains unchanged or contains no more than one, two, or three amino acid substitutions.

[0198] A method that can be used to identify 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, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the points of contact between the antibody and the antigen. Such contact residues and adjacent residues that are candidates for substitution can be targeted or eliminated. Variants can be screened to determine whether they possess the desired properties.

[0199] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to polypeptides containing one hundred or more residues, as well as in-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N-terminal or C-terminal of the antibody with an enzyme (e.g., for ADEPT) or polypeptide that increases the serum half-life of the antibody.

[0200] b) Glycosylation variants

[0201] In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. Addition or deletion of glycosylation sites to the antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0202] When the antibody contains an Fc region, the carbohydrates attached thereto can be altered. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides that are generally attached by an N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention can be modified to produce antibody variants having certain improved properties.

[0203] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose that is linked (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose at Asn297 in the sugar chain relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn 297 as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue at approximately position 297 in the Fc region (Eu numbering of Fc region residues); however, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants can have improved ADCC function. See, for example, U.S. Patent Publication No. US2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibody variants that are "defucosylated" or "fucose-deficient" include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US 2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing afucosylated antibodies include the protein fucosylation-deficient Lec13 CHO cells (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US2003 / 0157108 A1, Presta, L.; and WO 2004 / 056312 A1, Adams et al., particularly 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 WO2003 / 085107).

[0204] Antibody variants having a bisected oligosaccharide are further provided, e.g., where the bisected oligosaccharide linked to the Fc region of the antibody is bisected with GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0205] c) Fc region variants

[0206] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. The Fc region variants may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that comprises an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0207] In certain embodiments, the present invention contemplates antibody variants having some but not all effector functions, making them ideal candidates for applications where the in vivo half-life of the antibody is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus may lack ADCC activity), but retains the ability to bind FcRn. Primary cells, NK cells, which mediate ADCC, express only Fc(RIII, while monocytes express Fc(RI, Fc(RII, and Fc(RIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Other non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom 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 can be used (see, e.g., ACTI for flow cytometry TM Non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated 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 can also be performed to confirm that the antibody does not bind C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example, Petkova, S.B. et al., Int’l. Immunol. 18(12):1759-1769 (2006)).

[0208] Antibodies with reduced effector function include those having substitutions in one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0209] In certain embodiments, the Fc variants described herein further comprise one or more amino acid modifications for attenuating effector functions (e.g., CDC and / or ADCC). In an exemplary embodiment, the modification for attenuating effector functions is a modification that does not alter the glycosylation pattern of the Fc region. In certain embodiments, the modification for attenuating effector functions reduces or eliminates binding to human effector cells, binding to one or more Fc receptors, and / or binding to cells expressing Fc receptors. In one exemplary embodiment, the Fc variant described herein comprises the following modifications: L234A, L235A, and P329G in the human IgG1 Fc region result in attenuated effector functions. Substitution of L234A, L235A, and P329G (the L234A / L235A / P329G triple variant is referred to as LALAPG) has previously been shown to reduce binding to Fc receptors and complement (see, e.g., U.S. Publication No. 2012 / 0251531).

[0210] In various embodiments, an Fc variant with reduced effector function refers to an Fc variant that reduces effector functions (e.g., activities such as CDC, ADCC, and / or binding to FcR) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more compared to the effector functions achieved by a wild-type Fc region (e.g., an Fc region without mutations that reduce effector functions, 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 functions compared to a wild-type Fc region. Assays for measuring effector functions are known in the art and are described below.

[0211] In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus may lack ADCC activity). The primary cells NK cells that mediate ADCC only express FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Other non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom 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 can be used (see, for example, ACTI TM for flow cytometry; and CytoTox non-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA); and CytoTox

[0212] 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, the ADCC activity of the molecule of interest can be evaluated 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). C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)).

[0212] Certain antibody variants are described that have improved or reduced binding to FcR. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0213] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0214] In some embodiments, changes are made in the Fc region, resulting in altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0215] Antibodies having an extended half-life and improved neonatal Fc receptor (FcRn) binding are described in US2005 / 0014934A1 (Hinton et al.), where the neonatal Fc receptor 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)). Those antibodies comprise an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include Fc variants having substitutions 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).

[0216] For other examples of Fc region variants, also see: Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0217] d) Antibody variants engineered with cysteine

[0218] In certain embodiments, it may be desirable to generate cysteine-engineered antibodies, such as "thioMAbs", in which one or more residues of the antibody are replaced with cysteine residues. In particular embodiments, the replaced residues are present at accessible sites of the antibody. As further described herein, by replacing those residues with cysteine, reactive thiol groups are positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as a drug moiety or a linker-drug moiety, to produce an immunoconjugate. In certain embodiments, any one or more of the following residues may be replaced 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 as described, for example, in U.S. Patent No. 7,521,541.

[0219] e) Antibody derivatives

[0220] In certain embodiments, the antibodies provided herein can be further modified to include additional non-protein moieties known in the art and readily available. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propanediol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, poly(propylene oxide / ethylene oxide) copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacture due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, and the like.

[0221] In another embodiment, conjugates of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can have any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-protein moiety to a temperature at which cells proximal to the antibody-non-protein moiety are killed.

[0222] A. Recombinant methods and compositions

[0223] Antibodies can be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-CD20 antibody as described herein is provided. Such nucleic acids can 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 and a second vector, the first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and the second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, e.g., a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cells). In one embodiment, a method of preparing an anti-CD20 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or the host cell culture medium).

[0224] For recombinant production of an anti-CD20 antibody, a 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 a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the antibody).

[0225] Suitable host cells for cloning or expressing vectors encoding antibodies include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not 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 (edited by B.K.C. Lo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). Antibodies can be isolated from the bacterial cell paste in the soluble fraction after expression and can be further purified.

[0226] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains in which the glycosylation pathway has been "humanized" such that antibodies with a partially or fully human glycosylation pattern are produced. See Gerngross, Nat. Biotech. 22:1409-1414 (2004); and Li et al., Nat. Biotech. 24:210-215 (2006).

[0227] Suitable host cells for the expression of glycosylated antibodies also derive from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains that can be used with insect cells have been identified, particularly for transfection of Spodoptera frugiperda cells.

[0228] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing the PLANTIBODIES TM technology) for the production of antibodies in transgenic plants.

[0229] Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed by SV40 (COS-7); the human embryonic kidney line (293 or 293 cells, as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells (as described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, which include DHFR -CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0230] B. Assays

[0231] The physical / chemical properties and / or biological activities of the anti-CD20 antibodies provided herein can be identified, screened, or characterized by a variety of assays known in the art.

[0232] 1. Binding Assays and Other Assays

[0233] In one aspect, the antigen-binding activity of the antibodies of the invention is tested, for example, by known methods such as ELISA, Western blotting, etc. Methods known in the art can be used to determine CD20 binding and exemplary methods are disclosed herein. In one embodiment, radioimmunoassay is used to measure binding. An exemplary radioimmunoassay is provided below. The CD20 antibody is iodinated and a competitive reaction mixture is prepared that contains a fixed concentration of the iodinated antibody and serially diluted, unlabeled CD20 antibody at decreasing concentrations. Cells expressing CD20 (e.g., BT474 cells stably transfected with human CD20) are added to the reaction mixture. After incubation, the cells are washed to separate free iodinated CD20 antibody from CD20 antibody bound to the cells. For example, the level of bound iodinated CD20 antibody is determined by counting the radioactivity associated with the cells and using standard methods to determine the binding affinity. In another embodiment, flow cytometry is used to evaluate the ability of the CD20 antibody to bind to surface-expressed CD20 (e.g., on B cell subsets). Peripheral white blood cells (e.g., from human, cynomolgus monkey, rat, or mouse) are obtained and the cells are blocked with serum. Labeled CD20 antibody is added in serial dilutions and the T cells are also stained to identify T cell subsets (using methods known in the art). After incubating and washing the samples, the cells are sorted using a flow cytometer and the data are analyzed using methods well known in the art. In another embodiment, surface plasmon resonance can be used to analyze CD20 binding. Exemplary surface plasmon resonance methods are illustrated in the examples.

[0234] In another aspect, competitive assays 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., linear or conformational epitope) bound by any of the anti-CD20 antibodies disclosed herein. Exemplary methods for mapping the epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols" in Methods in Molecular Biology, Volume 66 (Humana Press, Totowa, NJ).

[0235] 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 whose ability to compete with the first antibody for binding to CD20 is being tested. The second antibody can be present in a hybridoma supernatant. As a control, immobilized CD20 is incubated in a solution containing the first labeled antibody but not containing the second unlabeled antibody. After incubation under conditions that permit the first antibody to bind to CD20, the excess unbound antibody is removed, and the amount of label associated with the immobilized CD20 is measured. If the amount of label associated with the immobilized CD20 is substantially reduced in the test sample relative to the control sample, it indicates that the second antibody competes with the first antibody for binding to CD20. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0236] 2. Activity Assays

[0237] The anti-CD20 antibodies of the present disclosure (e.g., type II antibodies) can be identified and / or characterized by one or more activity assays known in the art. For example, complement-dependent cytotoxicity (CDC) and / or antibody-dependent cell cytotoxicity (ADCC) can be used as described herein.

[0238] It should be understood that any of the above assays can be performed using the immunoconjugates of the present invention in place of or in addition to the anti-CD20 antibodies.

[0239] It should be understood that any of the above assays can be performed using the anti-CD20 antibodies and additional therapeutic agents.

[0240] Methods of Administering Type II Anti-CD20 Antibodies

[0241] The present disclosure provides methods for treating childhood-onset idiopathic nephrotic syndrome (INS) in an individual, wherein the methods comprise administering to the individual a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody. The present disclosure also provides methods for depleting circulating peripheral B cells in an individual, wherein the methods comprise administering to the individual a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody, and wherein after administering the type II anti-CD20 antibody, the B cells are depleted to a level such that circulating peripheral B cells are present at about 5 cells / μL or less in peripheral blood from the individual. The present disclosure also provides methods for depleting circulating peripheral B cells in an individual, wherein the methods comprise administering to the individual a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody, and wherein after administering the type II anti-CD20 antibody, the B cells are depleted to a level such that circulating peripheral B cells are present at about 5 cells / μL or less in peripheral blood from the individual, and this level 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 who is greater than or equal to 2 years old and less than or equal to 25 years old. In some embodiments, the individual or patient is a human who is greater than 2 years old and less than 25 years old. In some embodiments (e.g., embodiments of weight-based dosing using a type II anti-CD20 antibody), the weight of the individual is less than 45 kg. In some embodiments (e.g., embodiments of fixed dosing using a type II anti-CD20 antibody), the weight of the individual is greater than or equal to 45 kg.

[0242] In some embodiments, the individual or patient has been diagnosed with INS (e.g., FRNS or SDNS) before 18 years of age. 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 the following documents: 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).

[0243] In some embodiments, the individual or patient has frequently relapsing nephrotic syndrome (FRNS) with childhood onset. In some embodiments, the individual or patient has ≥2 relapses every 6 months within 6 months of disease onset, or ≥4 relapses every 12 months within any subsequent 12-month period.

[0244] In some embodiments, the individual or patient has childhood-onset steroid-dependent nephrotic syndrome (SDNS). In some embodiments, the individual or patient has had two consecutive relapses during treatment with prednisone or prednisolone (at full dose or during tapering) or within 15 days after discontinuation of prednisone or prednisolone.

[0245] In some embodiments, the individual or patient (e.g., prior to treatment using the methods of the present disclosure) is in a state of complete remission. In some embodiments, complete remission is defined as the absence of edema, UPCR ≤ 0.2 g / g, and a urine dipstick reading of trace or negative for protein for three consecutive days.

[0246] In some embodiments, the individual or patient has had at least 1 relapse within 6 months (e.g., prior to treatment according to the methods of the present disclosure).

[0247] In some embodiments, the individual or patient has received cyclophosphamide treatment within 6 months (e.g., prior to treatment according to the methods of the present disclosure) and has had at least 1 relapse after discontinuation of cyclophosphamide.

[0248] In some embodiments, the estimated glomerular filtration rate (eGFR) of the individual or patient is within the normal range for their age.

[0249] In some embodiments, the methods of the present disclosure include administering to the 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 to about 26 weeks after the first antibody exposure. In some embodiments, the second antibody exposure is not provided until about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, or about 26 weeks after the first antibody exposure. In some embodiments, the second antibody exposure is not provided until less than about any of the following weeks after the first antibody exposure: 26, 25, 24, 23, 22, 21, 20, or 19. In some embodiments, the second antibody exposure is not provided until greater than about any of the following weeks after the first antibody exposure: 18, 19, 20, 21, 22, 23, 24, or 25. That is, the second antibody exposure is not provided until any of the week ranges with an independently selected lower limit of 18, 19, 20, 21, 22, 23, 24, or 25 and an upper limit of 26, 25, 24, 23, 22, 21, 20, or 19, wherein the lower limit is less than the upper limit.

[0250] The dosing regimens described herein use a consistent system to track the time between doses, where a first dose is administered to a patient on Day 1 or Week 0. As described herein, antibody exposure of the present disclosure can include one or two doses. In the case where the antibody exposure contains one dose, referring to a period of time passing after the first antibody exposure (as described herein) before a second antibody exposure is provided refers to the amount of time passing between the dose of the first antibody exposure (e.g., Day 1 or Week 0) and the dose of the second antibody exposure. If the first antibody exposure includes two doses, the first dose of the first antibody exposure is provided on Day 1 or Week 0. In the case where the antibody exposure contains two doses, referring to a period of time passing after the first antibody exposure (as described herein) before a second antibody exposure is provided refers to the amount of time passing between the first dose of the first antibody exposure (e.g., Day 1 or Week 0) of the two doses and the first dose of the second antibody exposure of the two doses. For example, if the method of the present disclosure includes a first antibody exposure having two doses and a second antibody exposure having two doses, and the second antibody exposure is not provided until about 22 weeks after the first antibody exposure, the interval between the first dose of the first antibody exposure and the first dose of the second antibody exposure is about 22 weeks.

[0251] In some embodiments, the first antibody exposure of the present disclosure includes one or two doses of the type II anti-CD20 antibody of the present disclosure. In some embodiments, the first antibody exposure contains a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg. In some embodiments, the first antibody exposure contains a total exposure of about 1800 mg, about 1900 mg, about 2000 mg, about 2100 mg, or about 2200 mg of the type II anti-CD20 antibody. In some embodiments, the individual's body weight is greater than or equal to 45 kg.

[0252] In some embodiments, the first antibody exposure contains a total exposure of the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg. 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 the type II anti-CD20 antibody. In some embodiments, the individual's body weight is less than 45 kg.

[0253] In some embodiments, the first antibody exposure comprises two doses. 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 dose of the first antibody exposure contains about 1000 mg of the type II anti-CD20 antibody. In some embodiments, the second dose of the first antibody exposure contains about 1000 mg of the type II anti-CD20 antibody. In some embodiments, the individual weighs greater than or equal to 45 kg.

[0254] In some embodiments, the first antibody exposure comprises two doses. 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. In some embodiments, the first dose of the first antibody exposure contains about 20 mg / kg of the type II anti-CD20 antibody. In some embodiments, the second dose of the first antibody exposure contains about 20 mg / kg of the type II anti-CD20 antibody. In some embodiments, the individual weighs less than 45 kg.

[0255] In some embodiments, the second dose of the first antibody exposure is provided until about 1.5 to about 2.5 weeks after the first dose of the first antibody exposure. In some embodiments, the second dose of the first antibody exposure is provided until about 2 weeks after the first dose of the first antibody exposure.

[0256] In some embodiments, the second antibody exposure of the present disclosure comprises one or two doses of the type II anti-CD20 antibody of the present disclosure. In some embodiments, the second antibody exposure contains a total exposure of a type II anti-CD20 antibody between about 1800 mg and about 2200 mg. 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 the type II anti-CD20 antibody. In some embodiments, the individual weighs greater than or equal to 45 kg.

[0257] In some embodiments, the second antibody exposure contains a total exposure of a type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg. 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 the type II anti-CD20 antibody. In some embodiments, the individual weighs less than 45 kg.

[0258] In some embodiments, the second antibody exposure comprises two doses. In some embodiments, the second antibody exposure comprises a first dose of a type II anti-CD20 antibody 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 dose of the second antibody exposure contains about 1000 mg of the type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure contains about 1000 mg of the type II anti-CD20 antibody. In some embodiments, the individual has a body weight greater than or equal to 45 kg.

[0259] In some embodiments, the second antibody exposure comprises two doses. In some embodiments, the second antibody exposure comprises a first dose of a type II anti-CD20 antibody 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. In some embodiments, the first dose of the second antibody exposure contains about 20 mg / kg of the type II anti-CD20 antibody. In some embodiments, the second dose of the second antibody exposure contains about 20 mg / kg of the type II anti-CD20 antibody. In some embodiments, the individual has a body weight less than 45 kg.

[0260] In some embodiments, the second dose of the second antibody exposure is provided until about 1.5 to about 2.5 weeks after the first dose of the second antibody exposure. In some embodiments, the second dose of the second antibody exposure is provided until about 2 weeks after the first dose of the second antibody exposure.

[0261] In some embodiments, the type II anti-CD20 antibody of the present disclosure is administered intravenously (e.g., by IV infusion).

[0262] In some embodiments, the methods of the present disclosure further comprise administering an effective amount of a glucocorticoid or corticosteroid (e.g., in combination with a type II anti-CD20 antibody as described herein). A variety of naturally occurring and synthetic glucocorticoids / corticosteroids are known in the art and include, but are not limited to, beclomethasone, triamcinolone, dexamethasone, betamethasone, prednisone, methylprednisolone, prednisolone, cortisone, and cortisol. In some embodiments, the glucocorticoid / corticosteroid comprises methylprednisolone. In some embodiments, the glucocorticoid / corticosteroid comprises prednisone. An effective amount of the glucocorticoid / corticosteroid of the present disclosure is known in the art and can be readily determined by standard assays. For example, methylprednisolone can be administered IV once daily at a dose of 750 - 1000 mg. As another example, prednisone can be administered orally at 0.5 mg / kg and optionally tapered to 7.5 mg / day. In some embodiments, methylprednisolone can be administered prior to each anti-CD20 antibody infusion. In some embodiments, methylprednisolone can be administered intravenously at 80 mg (e.g., if the individual weighs greater than or equal to 45 kg) or 1.5 mg / kg (e.g., if the individual weighs less than 45 kg). In some embodiments, oral prednisone or equivalent can be administered at a dose of 0.5 - 1 mg / kg / day (maximum 60 mg / day). In some embodiments, oral prednisone or equivalent can 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 can be administered at a dose of 0.5 to 2 mg / kg / day (maximum 60 mg / day). In some embodiments, oral prednisone or equivalent can be administered at a dose of 0.5 to 2 mg / kg / day (maximum 60 mg / day) and tapered to a target of 5 mg / day.

[0263] In some embodiments, the glucocorticoid can be administered before, during, or after administration of the type II anti-CD20 antibody of the present disclosure. In some embodiments, the glucocorticoid can be administered before administration of the type II anti-CD20 antibody of the present disclosure, e.g., the glucocorticoid can be administered 30 - 60 minutes before administration of the type II anti-CD20 antibody. In some embodiments, 80 mg of methylprednisolone can be administered IV 30 - 60 minutes before administration of the type II anti-CD20 antibody of the present disclosure. In some embodiments, prednisone (e.g., orally administered) and / or methylprednisolone (e.g., IV administered) can be administered in conjunction with the treatment, followed by maintenance therapy (e.g., mycophenolate mofetil or cyclophosphamide).

[0264] 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 as described herein). Antihistamines known in the art and currently in clinical use include histamine H 1-receptor and histamine H 2 -receptor antagonists or inverse agonists. In some embodiments, the antihistamine includes diphenhydramine. An effective amount of the antihistamines of the present disclosure is known in the art and can be readily determined by standard assays. For example, diphenhydramine can be administered at an oral dose of 0.5 - 1 mg / kg (rounded to the nearest available pill formulation) up to a maximum dose of 50 mg.

[0265] In some embodiments, the antihistamine can be administered before, during, or after administration of the type II anti-CD20 antibody of the present disclosure, for example, as a prophylactic treatment. In some embodiments, the antihistamine can be administered before administration of the type II anti-CD20 antibody of the present disclosure, for example, the antihistamine is administered 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 can be orally administered 30 - 60 minutes before administration of the type II anti-CD20 antibody of the present disclosure.

[0266] In some embodiments, the methods of the present disclosure further include administering an effective amount of acetaminophen. For example, acetaminophen can be administered at an oral dose of 15 mg / kg up to a maximum dose of 1000 mg.

[0267] In some embodiments, acetaminophen can be administered before, during, or after administration of the type II anti-CD20 antibody of the present disclosure, for example, as a prophylactic treatment. In some embodiments, acetaminophen can be administered before administration of the type II anti-CD20 antibody of the present disclosure, for example, acetaminophen is administered 30 - 60 minutes before administration of the type II anti-CD20 antibody. In some embodiments, 15 mg / kg (rounded to the nearest available pill formulation) or up to 1000 mg of acetaminophen can be orally administered 30 - 60 minutes before administration of the type II anti-CD20 antibody of the present disclosure.

[0268] In some embodiments, the methods of the present disclosure further include administering standard of care treatment (e.g., in combination with the type II anti-CD20 antibody described herein). In some embodiments, the standard of care treatment can be administered before, during, or after administration of the type II anti-CD20 antibody of the present disclosure, for example, for treating or preventing one or more symptoms of INS.

[0269] In some embodiments, the methods of the present disclosure result in complete remission in an individual. In some embodiments, the individual is in complete remission at 1 year after initiation of treatment (e.g., at week 52, as described herein). In some embodiments, complete remission refers to a state where the first morning urine UPCR ≤ 0.2 g / g and no concurrent events (occurring after week 8) have occurred, e.g., after completion of steroid taper, as described herein. In some embodiments, (e.g., at 1 year after initiation of treatment, e.g., at week 52, as described herein) there is sustained complete remission. In some embodiments, sustained complete remission includes a first morning urine UPCR ≤ 0.2 g / g and no relapse or any of certain concurrent events have occurred: (1) relapse (occurring after week 8, e.g.), defined as any one of the following events requiring systemic corticosteroid or other immunosuppressive therapy: (a) first morning urine UPCR ≥ 2 g / g or (b) urine dipstick UA ≥ 3+ for 3 consecutive days and the most recent urine sample from this 3-day period is determined to have a UPCR > 0.2 g / g or (c) urine dipstick UA protein ≥ 3+ on any day with edema and the urine sample is determined to have a UPCR > 0.2 g / g; (2) use of any systemic corticosteroid for > 14 days within a 30-day period (occurring after week 8, e.g.); (3) initiation of any INS rescue therapy other than systemic corticosteroids (occurring at any time, e.g.); (4) treatment discontinuation due to lack of efficacy (occurring at any time, e.g.); or (5) death (occurring at any time, e.g.). In some embodiments, the individual has sustained complete remission from week 8 to week 52 of treatment and the individual has not experienced (1) relapse, defined as any one of the following events requiring systemic corticosteroid or other immunosuppressive therapy: (a) first morning urine UPCR ≥ 2 g / g or (b) urine dipstick UA ≥ 3+ for 3 consecutive days and the most recent urine sample from this 3-day period is determined to have a UPCR > 0.2 g / g or (c) urine dipstick UA protein ≥ 3+ on any day with edema and the urine sample is determined to have a UPCR > 0.2 g / g; or (2) use of any systemic corticosteroid for > 14 days within a 30-day period. In some embodiments, the individual's complete remission persists until week 52 of treatment and no INS rescue therapy other than systemic corticosteroids has been initiated.

[0270] In some embodiments, the methods of the present disclosure result in the depletion of circulating peripheral B cells in an individual. In some embodiments, these 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), the circulating peripheral B cells present in peripheral blood 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 some embodiments, the level of circulating peripheral B cells is measured using high-sensitivity flow cytometry (HSFC) as described herein. In some embodiments, the B cells are depleted to a level below the limit of detection using HSFC. In some embodiments, the lower limit of quantification (LLOQ) of HSFC for B cells is about 1.0 cell / μL or less, about 0.8 cell / μL or less, about 0.6 cell / μL or less, about 0.5 cell / μL or less, or 0.441 cell / μL or less. In some embodiments, the circulating peripheral B cells of an 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, after exposure to the first dose of the first antibody, the depletion of circulating peripheral B cells persists for at least 52 weeks. In some embodiments, after exposure to the first dose of the first antibody, the 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.In some embodiments, depletion of circulating peripheral B cells refers to a measurement of circulating peripheral B cells obtained 3 months after treatment (e.g., after receiving the first and / or second antibody exposure as described herein), 6 months after treatment (e.g., after receiving the first and / or second antibody exposure as described herein), 9 months after treatment (e.g., after receiving the first and / or second antibody exposure as described herein), or 12 months after treatment (e.g., after receiving the first and / or second antibody exposure as described herein), for example, compared to a corresponding measurement of the same individual before treatment or compared to a corresponding measurement of a control individual (e.g., an individual not receiving treatment), after a first antibody exposure (e.g., including 1 or 2 doses of an anti-CD20 antibody as described herein) and after a second antibody exposure (e.g., including 1 or 2 doses of an anti-CD20 antibody as described herein).

[0271] Methods for determining depletion of circulating peripheral B cells in an individual are known in the art, such as flow cytometry using one or more antibodies that recognize B cell markers. In some embodiments, high-sensitivity flow cytometry (HSFC) can be used to determine depletion of circulating peripheral B cells (see, for example, Vital, E.M. et al. (2011) Arthritis Rheum. 63:3038-3047 and Example 1). In some embodiments, the B cells are CD19+ B cells. In some embodiments, these B cells are naïve 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, these 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, such as from a peripheral blood sample.

[0272] In some embodiments, the level of circulating peripheral B cells present in a peripheral blood sample is measured (e.g., by HSFC measurement) as follows. Lymphocytes in the sample are identified 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 scatter 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 parental CD45+ lymphocyte gate (e.g., by plotting CD19 versus CD3 / CD14 and gating on CD19+CD3-CD14- cells), and CD19+CD33-CD56- B cells can be identified from the parental CD19+CD3-CD14- cells (e.g., by plotting CD19 versus CD33 / CD56 and gating on CD19+CD33-CD56- cells). The B cell count 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, and the B cell count can then be determined, for example, by calculating (CD19+ events x bead count) / (bead count x sample volume).

[0273] In some embodiments, after administration of a type II anti-CD20 antibody of the present disclosure (e.g., according to any method described herein), the circulating peripheral B cells present in peripheral blood 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, such as 5 cells / μL or less. In some embodiments, B cells are depleted to a level below the limit of detection using HSFC. In some embodiments, the lower limit of quantification (LLOQ) for B cells by HSFC is 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.

[0274] IV. Articles or Kits

[0275] On the other hand, provided are articles or kits containing the type II anti-CD20 antibodies of the present disclosure, which can be used in any of the methods described herein (e.g., for treating, preventing, and / or diagnosing the conditions described herein). The article or kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition that, either alone or in combination with another composition, is capable of effectively treating, preventing, and / or diagnosing a condition or for depleting circulating peripheral B cells, and the container can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a subcutaneous injection needle). At least one active agent in the composition is the antibody described herein (e.g., the type II anti-CD20 antibody of the present disclosure). The label or package insert indicates that the composition is for treating a selected condition or for depleting circulating peripheral B cells according to any of the methods described herein. Alternatively or additionally, the article or kit can further contain a second (or third) container that contains a pharmaceutical buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. The article can also include other substances required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0276] In some embodiments, provided herein is an article or kit that includes a container containing the type II anti-CD20 antibody of the present disclosure and an optional pharmaceutical carrier, and an optional package insert containing instructions for treating childhood-onset INS in an individual or reducing the risk and / or frequency of recurrence of childhood-onset INS in an individual, e.g., wherein the instructions indicate administering a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody to the individual, and the second antibody exposure is provided until about 18 to about 26 weeks after the first antibody exposure; wherein the first antibody exposure includes one or two doses of the type II anti-CD20 antibody, and the first antibody exposure includes a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg; wherein the second antibody exposure includes one or two doses of the type II anti-CD20 antibody, and the second antibody exposure includes a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg. In some embodiments, the instructions indicate that the individual is greater than or equal to 2 years old and less than or equal to 25 years old. In some embodiments, the instructions indicate that the individual weighs greater than or equal to 45 kg. In some embodiments, the antibody is ofatumumab.

[0277] In some embodiments, provided herein is an article or a kit that includes a container comprising a type II anti-CD20 antibody of the present disclosure and an optional pharmaceutically acceptable carrier, and an optional 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 an individual. For example, the instructions indicate administering a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody to the individual, wherein the second antibody exposure is not provided until about 18 to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the first antibody exposure has a total exposure of the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg; wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure has a total exposure of the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg. In some embodiments, the instructions indicate that the individual is greater than or equal to 2 years old and less than or equal to 25 years old. In some embodiments, the instructions indicate that the individual weighs less than 45 kg. In some embodiments, the antibody is ofatumumab.

[0278] The article or kit may further include a second or third container comprising a second drug, wherein the anti-CD20 antibody (e.g., the type II anti-CD20 antibody of the present disclosure) is the first drug, and the article further includes instructions on treating the subject with the second drug on the package insert. The article in these embodiments may further include a package insert that indicates that the composition can be used to treat a specific condition.

[0279] This specification is considered to be sufficient to enable those skilled in the art to practice the invention. Various modifications of the invention will become apparent to those skilled in the art from the foregoing specification 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.

[0280] Examples

[0281] The invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes will be suggested to those skilled in the art and will be included within the spirit and scope of this application and the scope of the appended claims.

[0282] Example 1: A Phase III, multicenter, randomized, open-label study to evaluate the efficacy and safety of ofatumumab versus MMF in patients with childhood-onset idiopathic nephrotic syndrome

[0283] The following presents a Phase III, randomized, open-label, multicenter, active-controlled 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 have achieved complete remission at study entry and are considered to be at high risk of relapse.

[0284] Patients with childhood-onset FRNS / SDNS are at risk of short- and long-term steroid toxicity, serious infections, edema, thromboembolic events, and acute kidney injury during relapses. Reduction of these risks depends on identification of effective therapies and maintenance of clinical remission via sustained reduction of proteinuria. Current standard of care remains limited to combinations of systemic corticosteroids and immunosuppressive therapies, but most available regimens result in complete renal response and remission in less than half of treated patients.

[0285] Objectives and Endpoints

[0286] This study evaluated the efficacy, safety, pharmacokinetics, and pharmacodynamics of obinutuzumab versus MMF in participants aged ≥2 years and ≤25 years with childhood-onset FRNS or SDNS. The specific important objectives and secondary objectives of the study, and the corresponding endpoints, are outlined below. In this protocol, "study treatment" refers to the treatment assigned to a participant as part of this study (i.e., obinutuzumab or MMF).

[0287] The primary objective of the study was to evaluate the efficacy of obinutuzumab compared to MMF in participants aged ≥2 years and ≤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 a urinary protein-to-creatinine ratio (UPCR) ≤0.2 g / g in the first morning urine at week 52 and no recurrence or any of the following concurrent events. Concurrent events occurring after week 8 included: (1) recurrence, defined as any of the following events: (a) first morning urine UPCR ≥2 g / g or (b) urine dipstick urinalysis (UA) ≥3+ for 3 consecutive days (home monitoring) and the most recent urine sample during this 3-day period was determined to have a UPCR >0.2 g / g, as measured by the central laboratory or (c) urine dipstick UA protein ≥3+ on any day with edema and the urine sample was determined to have a UPCR >0.2 g / g, as measured by the central laboratory; and (2) use of any systemic corticosteroid for >14 days within a 30-day period. Concurrent 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.

[0288] The secondary objectives were to evaluate the efficacy of obinutuzumab compared to MMF. The corresponding secondary endpoints were: (1) overall relapse-free survival (RFS); (2) probability of RFS at week 52; (3) cumulative corticosteroid dose; (4) number of relapses at the time of randomization to 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 week 76.

[0289] Another secondary objective was to evaluate the change in fatigue in participants treated with obinutuzumab compared to 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 Total Score.

[0290] The third secondary objective was to evaluate the change in quality of life in participants treated with obinutuzumab compared to 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.

[0291] The fourth secondary objective was to evaluate edema over time. The corresponding secondary endpoint was the mean change from baseline over time to week 52 in the CureGN Edema Scale.

[0292] The fifth secondary objective is to evaluate the safety of obinutuzumab compared to MMF. The corresponding secondary endpoints are: (1) the incidence, nature, and severity of adverse events (AEs), where severity is determined from baseline to week 52 according to AE intensity (mild, moderate, severe, life-threatening) and graded using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) (if applicable); and (2) the incidence of laboratory or vital sign abnormalities from baseline to week 52.

[0293] The sixth secondary objective is to characterize the obinutuzumab PK profile. The corresponding secondary endpoint is the serum concentration of obinutuzumab at specified time points.

[0294] The seventh secondary objective is to characterize the PD changes induced by obinutuzumab. The corresponding secondary endpoints are: (1) the proportion of participants achieving B cell depletion at specific time points (e.g., using high-sensitivity flow cytometry (HSFC)); and (2) total peripheral B cells and B cell subsets (e.g., memory B cell counts) and changes relative to baseline at specific time points.

[0295] The exploratory objectives are to evaluate the efficacy of obinutuzumab compared to MMF. The corresponding exploratory endpoints are: (1) the change in UPCR from baseline to week 52; (2) the change in estimated glomerular filtration rate (eGFR) from baseline to week 52; (3) the proportion of participants achieving sustained peripheral B cell depletion at weeks 24, 52, and 76; (4) the proportion of participants achieving RFS at week 24; (5) the proportion of participants in sustained complete remission at week 76; (6) the change in the physician's overall disease activity assessment (PGA) from baseline to weeks 24 and 52; (7) the change in the subject's overall disease activity assessment (SGA) from baseline to weeks 24 and 52; and (8) the proportion of participants who do not experience a relapse before completion of steroid taper.

[0296] The second exploratory objective is to explore the relationship between exposure-efficacy and exposure-safety. The corresponding exploratory endpoints are: (1) the change in obinutuzumab exposure and selected efficacy endpoints (including sustained complete remission (SCR) and RFS) from baseline to week 52 and over time; and (2) the change in the incidence, nature, and severity of obinutuzumab exposure and selected adverse events from baseline to week 52 and over time.

[0297] The third exploratory objective is to evaluate the potential relationship between drug exposure and B cell depletion. The corresponding exploratory endpoints are: (1) the change in obinutuzumab exposure and circulating CD19+ B cell counts relative to baseline over time; and (2) total peripheral B cells and B cell subsets (e.g., memory B cells) counts before / at relapse.

[0298] The fourth exploratory objective is to evaluate the immune response to obinutuzumab. The corresponding exploratory endpoints are the proportion of participants with anti-drug antibodies (ADA) at baseline and the incidence of ADA after treatment during the study.

[0299] The fifth exploratory objective is to evaluate the potential impact of ADA. The corresponding exploratory endpoint is the relationship between ADA status and efficacy, safety, PD, or PK endpoints.

[0300] The sixth exploratory objective is to identify and / or evaluate biomarkers that provide evidence of obinutuzumab activity (i.e., pharmacodynamic biomarkers) or to enhance the knowledge and understanding of disease biology and drug safety. The corresponding exploratory endpoints are the relationships between biomarkers in the blood and efficacy, safety, PK, immunogenicity, or other biomarker endpoints.

[0301] Inclusion and exclusion criteria

[0302] The inclusion criteria for the study include: (1) participants being between ≥2 years and ≤25 years of age at randomization; (2) diagnosis of FRNS or SDNS before the age of 18 according to international guidelines [e.g., KDIGO 2021, IPNA 2023, see Trautmann et al. (2023) Pediatr Nephrol 38:877-919]; (3) being in complete remission, defined as the absence of edema at screening, UPCR ≤0.2 g / g, and having three consecutive daily urine dipstick readings of trace or negative for protein within weeks before randomization; (4) having had at least one relapse within 6 months before screening, after stopping oral corticosteroids and / or immunosuppressive therapy (e.g., oral cyclophosphamide, levamisole, mizoribine, MMF, or CNI), or while receiving oral corticosteroids and / or immunosuppressive therapy to prevent relapse; (5) patients who received cyclophosphamide within 6 months before randomization must have experienced at least 1 relapse after cyclophosphamide withdrawal; and (6) estimated glomerular filtration rate (eGFR) being within the normal range for age (using the modified Schwartz formula if less than 18 years old, or the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation if 18 years old or older). Frequent relapsing nephrotic syndrome (FRNS) is defined as ≥2 relapses every 6 months within 6 months of disease onset, or ≥4 relapses every 12 months in any subsequent 12 months. Steroid-dependent nephrotic syndrome (SDNS) is defined as two consecutive relapses during treatment with prednisone or prednisolone (at full dose or during taper) or within 15 days after prednisone or prednisolone withdrawal.

[0303] Exclusion criteria included: (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 immunosuppressive drugs other than MMF or oral corticosteroids for preventing recurrence within 2 months before randomization; (5) history of organ or bone marrow transplantation; (6) participation in another treatment trial within 30 days of enrollment or within 5 half-lives (whichever is longer) of the study drug; (7) intolerance or contraindications to the study therapy, including any of the following: (a) history of severe allergy or anaphylactic shock reaction to monoclonal antibodies, or known hypersensitivity to any component of ocrelizumab infusion, (b) lack of peripheral venous access, (c) intolerance or contraindications to oral or intravenous corticosteroids, or (d) intolerance or contraindications to MMF; (8) previous treatment failure of the patient to MMF, defined as two or more relapses within any 6-month period while receiving MMF for at least 6 months; (9) in the judgment of the investigator, the participant may need to use systemic corticosteroids for reasons other than idiopathic nephrotic syndrome during the study; (10) receiving exclusion therapy (see below); (11) any type of active infection (excluding onychomycosis), or any severe infection episode that required hospitalization or treatment with intravenous anti-infective drugs within 4 weeks before screening, or completion of oral anti-infective drugs within 2 weeks before randomization; (12) evidence of active tuberculosis (TB) infection; (13) current history of active primary or secondary immunodeficiency, including history of known HIV infection and other severe immunodeficiency blood disorders; (14) history of severe recurrent or chronic infections; (15) history of progressive multifocal leukoencephalopathy; (15) history of having cancer (including solid tumors, hematological malignancies, and carcinoma in situ (except for excised and cured basal cell carcinoma and squamous cell carcinoma of the skin)) within the past 5 years or currently having these cancers; (16) major surgery that required hospitalization during the 4-week period before screening or during screening; (17) clinical significant bleeding or high risk of any condition that requires plasma exchange, intravenous immunoglobulin, or acute blood product transfusion; (18) evidence of any significant or uncontrolled concomitant disease that, in the judgment of the investigator, would prevent the participant from participating, including but not limited to neurological, respiratory, cardiac, hepatic, endocrine, malignant, or gastrointestinal disorders; (19) current active alcohol or drug abuse, or history of alcohol or drug abuse; and (20) any of the following laboratory parameters at screening:

[0304] —AST or ALT > 2.5 × upper limit of normal (ULN) (age- and sex-adjusted) not attributable to underlying nephrotic syndrome

[0305] —Amylase or lipase > 2 × ULN

[0306] —Absolute neutrophil count < 1.5 × 10 3 / μL

[0307] —Hemoglobin < 8 g / dL

[0308] —For participants < 12 years of age, platelet count < 110,000 / μL, and for patients ≥ 12 years of age, platelet count < 50,000 / μL

[0309] —Positive hepatitis B surface antigen

[0310] —Positive hepatitis B core antibody

[0311] —Positive hepatitis C antibody

[0312] —Positive serum human chorionic gonadotropin measured at screening

[0313] Excluded treatments include:

[0314] —Cyclophosphamide, levamisole, mizoribine, tacrolimus, cyclosporine, or voclosporin taken during the 2 months prior to screening or during screening

[0315] —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 Day 1 baseline visit

[0316] —Any biologic therapy (other than anti-CD19, anti-CD20, anti-CD22), such as but not limited to belimumab, daratumumab, ustekinumab, anifrolumab, secukinumab, or abatacept, during the 2 months prior to screening or during screening

[0317] —Oral inhibitors of Janus-associated kinase (JAK), Bruton tyrosine kinase (BTK), or tyrosine kinase 2 (TYK2), including baricitinib, tofacitinib, upadacitinib, filgotinib, ibrutinib, or fenebrutinib, or any investigational drug, taken during the 2 months prior to screening or during screening

[0318] investigational drug

[0319] —Any live vaccine administered during the 28 days prior to screening or during screening

[0320] Study treatment

[0321] The study consisted of four periods: a 28-day screening period, a 52-week initial open-label treatment period, an extension period after 52 weeks, and a safety follow-up (SFU) period of at least 12 months starting from the completion or discontinuation of study treatment. Figure 1 The study protocol is provided in

[0322] Approximately 80 participants aged ≥2 years to 25 years were randomized in a 1:1 ratio to one of the following two open-label treatment groups: Group A (obinutuzumab) or Group B (MMF). Randomization was stratified according to the participant's disease type (FRNS compared to SDNS) and the use of immunosuppressive therapy (except for corticosteroids used for INS prior to study entry) (MMF / other immunosuppressants compared to no MMF / other immunosuppressants).

[0323] The study product used in the study was obinutuzumab.

[0324] After a 28-day (+ / -7 days) screening period, randomized participants entered a 52-week treatment period. According to Figure 1 , during the treatment period, participants received obinutuzumab 1000 mg IV infusion on Days 1, 15, 168 (Week 24), and 182 (Week 26) during the initial 52-week treatment period, or started or continued oral MMF (tablet, capsule, or liquid formulation) daily starting on Day 1. Participants weighing 45 kg or more received a 1000 mg obinutuzumab dose. Participants weighing less than 45 kg received a weight-adjusted 20 mg / kg dose of obinutuzumab infusion. Methylprednisolone 80 mg IV (or 1.5 mg / kg if ≤45 kg) was administered as premedication before the infusion. Acetaminophen / paracetamol 15 mg / kg (maximum dose 1000 mg) PO was administered as premedication before the infusion. Diphenhydramine hydrochloride 0.5 - 1 mg / kg (maximum dose 50 mg) PO or IV was administered as premedication before the infusion.

[0325] Participants randomized to MMF took the target dose of 1200 mg / m 2 / day (maximum 2.5 g / day) in divided doses. Participants who were receiving oral prednisone (or prednisone equivalent) daily at randomization were tapered to reach a target of 0 mg / day by Week 8 after randomization (or earlier, e.g., by Week 4 - 6 of the study if applicable), and prednisone was continued to be withheld for the remainder of the study. Participants who experienced disease relapse received up to 2 mg / kg / day (60 mg / m 2Prednisone or prednisolone (at a maximum dose of 60 mg / day) per day until the urine protein urine dipstick test is negative / trace (or UPCR ≤ 0.2 g / g) for 3 consecutive days or longer, then gradually reduce the oral corticosteroid over 4 weeks while continuing the study treatment regimen. If a participant in the MMF group meets the criteria for rescue therapy, the patient is considered to meet the definition of a concurrent event and receives an alternative therapy of obinutuzumab (2 × 1000 mg, 14 days apart; or 20 mg / kg if < 45 kg) or INS. If a participant in the obinutuzumab group meets the criteria for rescue therapy, the patient is considered to meet the definition of a concurrent event and receives MMF (600 mg / m 2 BID [target 1200 mg / m 2 , in divided doses, maximum 2 g / day) or alternative therapy to treat the recurrence. When obinutuzumab is used, MMF is discontinued.

[0326] When the last randomized participant completes week 52, the primary endpoint will be evaluated: the proportion of participants with a sustained complete remission (PCR ≤ 0.2 g / g and no recurrence or other concurrent events) at 1 year.

[0327] After the week 52 assessment, participants can continue to receive obinutuzumab until the Common Completion Date (CCOD) during the treatment extension period, or proceed directly to Safety Follow-up (SFU). Participants who experience a recurrence after week 52 (regardless of whether in the MMF group or the obinutuzumab group) will be treated at the investigator's best judgment, which includes administering an initial or additional dose of obinutuzumab. Patients who do not meet the recurrence criteria at week 52 or later are not eligible for obinutuzumab treatment during the extension period, but are followed up every 3 months at study visits until the CCOD. Participants with peripheral B cell depletion are followed up every 12 weeks for 6 months in the SFU, and then every 6 months thereafter until the peripheral CD19 B cells return to pre-treatment values or within the central laboratory normal values for this patient population (whichever is lower), until the end of the study.

[0328] The first SFU visit is scheduled approximately 12 weeks after the last study visit during the treatment period or at CCOD, whichever comes first. Obinutuzumab infusion or MMF is not provided during SFU. Standard care therapy is provided at the discretion of the investigator. Patients who do not receive obinutuzumab only need to have one SFU visit and undergo the assessments specified in SFU Visit 1. Patients who receive obinutuzumab are followed up during SFU until such patients meet two of the following criteria: (1) Peripheral B cells have recovered to the pre-obinutuzumab baseline level or within the normal range of the population (whichever is lower); and (2) The last infusion of obinutuzumab was at least 12 months ago.

[0329] At each SFU visit, the absolute CD19+ B cell count is measured. For participants with persistent B cell depletion (defined as an absolute CD19+ B cell count below the lowest pre-treatment value and below the lower limit of normal (LLN) for this population) and who have not received additional therapy related to peripheral B cell reduction, SFU will continue every 6 months until any of the following occurs: (1) Peripheral CD19+ B cells recover to the lowest pre-treatment value or the age-specific LLN of the patient population (whichever is lower); (2) Receive additional therapy related to peripheral B cell reduction (e.g., belimumab, rituximab, or cyclophosphamide, or use of obinutuzumab outside the study protocol); or (3) The study ends.

[0330] Participants complete the study when: (1) The SFU requirements are completed; (2) The last treatment study visit is completed at or after CCOD and the investigator intends to treat the subject for nephrotic syndrome outside the study protocol without completing the required SFU; (3) Or the study ends. After the study is completed, the study sponsor will provide eligible participants with continued access to the Roche investigational medicinal product (IMP; obinutuzumab).

[0331] Duration of the study period

[0332] The expected minimum duration of participation in the study for each individual is approximately 1.5 - 2 years (for all patients who have received obinutuzumab, SFU will be at least 12 months after the last obinutuzumab infusion). Participants may continue to participate in the study and be followed up according to the activity schedule or receive repeat treatment until the last recruited participant has been in the study for at least 18 months.

[0333] The maximum expected study participation time is approximately 3 years, or longer if peripheral B cells are still below the LLN at clinical cutoff. In such cases, participants will be asked to return for SFU visits every 12 weeks for 6 months, then every 6 months until B cells return to the pre-ofatumumab-dose baseline or the central laboratory normal LLN for the participant population, or until the end of the study.

Claims

1. 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 the type II anti-CD20 antibody; wherein the second antibody exposure is provided no earlier than about 18 weeks to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the first antibody exposure comprises: (a) a total exposure to the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (b) a total exposure to the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the individual weighs less than 45 kg; wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure comprises: (c) a total exposure to the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (d) a total exposure to the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the individual weighs less than 45 kg; wherein the type II anti-CD20 antibody is ofatumumab; and wherein the individual is a person greater than or equal to 2 years old and less than or equal to 25 years old.

2. A method for reducing the risk and / or frequency of relapse in an individual with childhood-onset idiopathic nephrotic syndrome (INS), the method comprising administering to the individual a first antibody exposure to a type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody; wherein the second antibody exposure is provided no earlier than about 18 weeks to about 26 weeks after the first antibody exposure; wherein the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the first antibody exposure comprises: (a) a total exposure to the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (b) a total exposure to the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the individual weighs less than 45 kg; wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure comprises: (c) a total exposure to the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (d) a total exposure to the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the individual weighs less than 45 kg; wherein the type II anti-CD20 antibody is ofatumumab; and wherein the individual is a person greater than or equal to 2 years old and less than or equal to 25 years old.

3. The method according to claim 1 or claim 2, wherein the first antibody exposure comprises a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg; wherein the second antibody exposure comprises a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg; and wherein the individual weighs greater than or equal to 45 kg.

4. The method according to any one of claims 1 to 3, wherein the first antibody exposure comprises: a first dose of the type II anti-CD20 antibody between about 900 mg and about 1100 mg, and a second dose of the type II anti-CD20 antibody between about 900 mg and about 1100 mg.

5. The method according to any one of claims 1 to 3, wherein the first antibody exposure comprises: a first dose of the type II anti-CD20 antibody between about 18 mg / kg and about 22 mg / kg, and a second dose of the type II anti-CD20 antibody between about 18 mg / kg and about 22 mg / kg, and wherein the individual weighs less than 45 kg.

6. The method according to any one of claims 1 to 5, 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 wherein the second dose of the first antibody exposure is provided until about 1.5 to about 2.5 weeks after the first dose of the first antibody exposure.

7. The method 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 wherein the second dose of the first antibody exposure is provided until about 2 weeks after the first dose of the first antibody exposure.

8. The method according to any one of claims 1 to 4, 6 and 7, wherein the first dose of the first antibody exposure is about 1000 mg of the type II anti-CD20 antibody.

9. The method according to any one of claims 1 to 4 and 6 to 8, wherein the second dose of the first antibody exposure is about 1000 mg of the type II anti-CD20 antibody.

10. The method according to any one of claims 1 to 3 and 5 to 7, wherein the first dose of the first antibody exposure is about 20 mg / kg of the type II anti-CD20 antibody, and wherein the individual weighs less than 45 kg.

11. The method according to any one of claims 1 to 3, 5 to 7 and 10, wherein the second dose of the first antibody exposure is about 20 mg / kg of the type II anti-CD20 antibody, and wherein the individual weighs less than 45 kg.

12. The method according to any one of claims 1 to 4 and 6 to 9, wherein the second antibody exposure comprises: A first dose of the type II anti-CD20 antibody between about 900 mg and about 1100 mg, and a second dose of the type II anti-CD20 antibody between about 900 mg and about 1100 mg.

13. The method according to any one of claims 1 to 3, 5 to 7, 10 and 11, wherein the second antibody exposure comprises: A first dose of the type II anti-CD20 antibody between about 18 mg / kg and about 22 mg / kg, and a second dose of the type II anti-CD20 antibody between about 18 mg / kg and about 22 mg / kg, and wherein the body weight of the individual is less than 45 kg.

14. The method according to any one of claims 1 to 13, 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 wherein the second dose of the second antibody exposure is provided until about 1.5 to about 2.5 weeks after the first dose of the second antibody exposure.

15. The method according to claim 14, wherein the second dose of the second antibody exposure is provided until about 2 weeks after the first dose of the second antibody exposure.

16. The method according to any one of claims 1 to 4, 6 to 9, 12, 14 and 15, wherein the first dose of the second antibody exposure is about 1000 mg of the type II anti-CD20 antibody.

17. The method according to any one of claims 1 to 4, 6 to 9, 12 and 14 to 16, wherein the second dose of the second antibody exposure is about 1000 mg of the type II anti-CD20 antibody.

18. The method according to any one of claims 1 to 3, 5 to 7, 10, 11 and 13 to 15, wherein the first dose of the second antibody exposure is about 20 mg / kg of the type II anti-CD20 antibody, and wherein the body weight of the individual is less than 45 kg.

19. The method according to any one of claims 1 to 3, 5 to 7, 10, 11, 13 to 15 and 18, wherein the second dose of the second antibody exposure is about 20 mg / kg of the type II anti-CD20 antibody, and wherein the body weight of the individual is less than 45 kg.

20. The method according to any one of claims 1 to 19, wherein the individual has or has been diagnosed with childhood-onset INS.

21. The method according to any one of claims 1 to 20, wherein the childhood-onset INS is frequently relapsing nephrotic syndrome (FRNS).

22. The method according to any one of claims 1 to 20, wherein the childhood-onset INS is steroid-dependent nephrotic syndrome (SDNS).

23. The method according to any one of claims 1 to 22, wherein the individual is in complete remission before the administration.

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

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

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

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

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

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

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

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

32. The method according to any one of claims 1 to 31, further comprising administering to the individual an effective amount of acetaminophen.

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

34. The method according to any one of claims 1 to 33, wherein the method induces sustained complete remission in the individual at 1 year.

35. The method according to claim 1 or claim 2, wherein the first antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody on day 1 and day 15 of treatment; and wherein the second antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody on day 168 and day 182 of treatment.

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

37. The method according to claim 1 or claim 2, wherein the first antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody in week 0 and week 2 of treatment; and wherein the second antibody exposure comprises two doses of 1000 mg of the type II anti-CD20 antibody in week 24 and week 26 of treatment.

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

39. A method for treating childhood-onset INS in an individual or reducing the risk and / or frequency of recurrence of childhood-onset INS in the individual, the method comprising intravenously administering to the individual a first antibody exposure and a second antibody exposure to obinutuzumab; wherein the first antibody exposure comprises two doses of 1000 mg of obinutuzumab at week 0 and week 2 of treatment; wherein the second antibody exposure comprises two doses of 1000 mg of obinutuzumab at week 24 and week 26 of treatment; wherein the individual is a person aged greater than or equal to 2 years and less than or equal to 25 years; and wherein the body weight of the individual is greater than or equal to 45 kg.

40. A method for treating childhood-onset INS in an individual or reducing the risk and / or frequency of recurrence of childhood-onset INS in the individual, the method comprising intravenously administering to the individual a first antibody exposure and a second antibody exposure to obinutuzumab; wherein the first antibody exposure comprises two doses of 20 mg / kg of obinutuzumab at week 0 and week 2 of treatment; wherein the second antibody exposure comprises two doses of 20 mg / kg of obinutuzumab at week 24 and week 26 of treatment; wherein the individual is a person aged greater than or equal to 2 years and less than or equal to 25 years; and wherein the body weight of the individual is less than 45 kg.

41. A method for treating childhood-onset INS in an individual or reducing the risk and / or frequency of recurrence of childhood-onset INS in the individual, the method comprising intravenously administering to the individual a first antibody exposure and a second antibody exposure to obinutuzumab; wherein the first antibody exposure comprises two doses of 1000 mg of obinutuzumab at day 1 and day 15 of treatment; wherein the second antibody exposure comprises two doses of 1000 mg of obinutuzumab at day 168 and day 182 of treatment; wherein the individual is a person aged greater than or equal to 2 years and less than or equal to 25 years; and wherein the body weight of the individual is greater than or equal to 45 kg.

42. A method for treating childhood-onset INS in an individual or reducing the risk and / or frequency of recurrence of childhood-onset INS in the individual, the method comprising intravenously administering to the individual a first antibody exposure and a second antibody exposure to obinutuzumab; wherein the first antibody exposure comprises two doses of 20 mg / kg of obinutuzumab at day 1 and day 15 of treatment; wherein the second antibody exposure comprises two doses of 20 mg / kg of obinutuzumab at day 168 and day 182 of treatment; wherein the individual is a person who is greater than or equal to 2 years old and less than or equal to 25 years old; and wherein the weight of the individual is less than 45 kg.

43. The method according to claim 39 or claim 40, further comprising administering methylprednisolone to the individual by intravenous (IV) infusion prior to administering obinutuzumab at week 0, week 2, week 24, and week 26 of the treatment.

44. The method according to claim 41 or claim 42, further comprising administering methylprednisolone to the individual by intravenous (IV) infusion prior to administering obinutuzumab on day 1, day 15, day 168, and day 182 of the treatment.

45. The method according to claim 43 or claim 44, wherein: (a) if the weight of the individual is greater than or equal to 45 kg, then 80 mg of methylprednisolone is administered to the individual; or (b) if the weight of the individual is less than 45 kg, then 1.5 mg / kg of methylprednisolone is administered to the individual.

46. A kit for treating childhood-onset INS in an individual, the kit comprising: (a) a container containing a type II anti-CD20 antibody, wherein the type II anti-CD20 antibody is obinutuzumab; (b) a package insert having instructions for treating childhood-onset INS in an individual, wherein the instructions indicate that the individual is a person who is greater than or equal to 2 years old and less than or equal to 25 years old; and wherein the instructions further indicate administering a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody to the individual, and providing the second antibody exposure until about 18 to about 26 weeks after the first antibody exposure; wherein 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 about 1800 mg and about 2200 mg; wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg.

47. A kit for treating childhood-onset INS in an individual, the kit comprising: (a) a container containing a type II anti-CD20 antibody, wherein the type II anti-CD20 antibody is obinutuzumab; (b) a package insert having instructions for treating childhood-onset INS in an individual, wherein the instructions indicate that the individual is a person who is greater than or equal to 2 years old and less than or equal to 25 years old and has a weight less than 45 kg; and wherein the instructions further indicate administering a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody to the individual, and providing the second antibody exposure until about 18 to about 26 weeks after the first antibody exposure; Wherein 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 about 36 mg / kg and about 44 mg / kg; Wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure comprises a total exposure of the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg.

48. A type II anti-CD20 antibody for use in a method of treating childhood-onset INS in an individual in need thereof, wherein the method comprises administering to the individual a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody; Wherein the second antibody exposure is provided no sooner than about 18 weeks to about 26 weeks after the first antibody exposure; Wherein the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the first antibody exposure comprises: (a) a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (b) a total exposure of the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the individual weighs less than 45 kg; Wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure comprises: (c) a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (d) a total exposure of the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the individual weighs less than 45 kg; Wherein the type II anti-CD20 antibody is obinutuzumab; and Wherein the individual is a person greater than or equal to 2 years old and less than or equal to 25 years old.

49. A type II anti-CD20 antibody for use in a method of reducing the risk and / or frequency of relapse in an individual with childhood-onset INS in need thereof, wherein the method comprises administering to the individual a first antibody exposure to the type II anti-CD20 antibody and a second antibody exposure to the type II anti-CD20 antibody; Wherein the second antibody exposure is provided no sooner than about 18 weeks to about 26 weeks after the first antibody exposure; Wherein the first antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the first antibody exposure comprises: (a) a total exposure of the type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (b) a total exposure of the type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the individual weighs less than 45 kg; Wherein the second antibody exposure comprises one or two doses of the type II anti-CD20 antibody, and the second antibody exposure comprises: (c) The total exposure of said type II anti-CD20 antibody between about 1800 mg and about 2200 mg, or (d) The total exposure of said type II anti-CD20 antibody between about 36 mg / kg and about 44 mg / kg if the body weight of said individual is less than 45 kg; wherein said type II anti-CD20 antibody is ofatumumab; and wherein said individual is a person who is greater than or equal to 2 years old and less than or equal to 25 years old.

50. A type II anti-CD20 antibody for use in the method according to any one of claims 1 to 45.

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