TW202413404A - Anti-cd20 antibody compositions - Google Patents

Abstract

Provided herein are populations of anti-CD20 antibody proteins with specified ranges of post-translational modifications. Also provided are methods of using and methods of making such populations of anti-CD20 antibody proteins.

Description

Anti-CD20 antibody composition

1.    Priority Claim

This application claims the benefit of U.S. Provisional Application Serial No. 63/347,852 filed on June 1, 2022, U.S. Provisional Application Serial No. 63/421,078 filed on October 31, 2022, and U.S. Provisional Application Serial No. 63/445,082 filed on February 13, 2023. The entire contents of the foregoing are incorporated by reference. 2.    Reference to electronically submitted sequence listings

This application contains a sequence listing that has been submitted electronically as an XML file named "50581-0004WO1.XML". The XML file was created on May 22, 2023 and is 39,114 bytes in size. The data in the XML file is incorporated by reference in its entirety. 3.   Field of Invention

The present invention is in the field of recombinant anti-CD20 antibodies, methods of producing such antibodies and uses of such antibodies.

4. Invention Background

Therapeutic monoclonal antibodies (mAbs) produced in mammalian cells are heterogeneous due to post-translational modifications (PTMs). PTMs may occur during mAb production, purification, storage, and post-administration. PTMs are product quality attributes (PQAs) of therapeutic mAbs. Controlling PQAs within predefined acceptance criteria is critical to the biopharmaceutical industry as it ensures consistent product quality and reduces potential impacts on drug safety and efficacy (Xu, X. et al., Journal of Applied Bioanalysis 3 (2):21-5 (2017)).

The critical importance of antibody sequence variation is well established. Sequence diversity in antibody variable domains is critical for specific antigen recognition, while association with different constant domains results in different Fc-mediated effector activities. PTMs in these domains provide additional immune mechanisms by which antibody binding and activity can be modulated. PTMs vary by chain additions, such as N- and O-linked glycosylation, glycation, cysteinylation, and sulfation; chain trimming, such as C-terminal lysine clipping; amino acid modifications, such as cyclization (to N-terminal pyroglutamate), deamination, oxidation, isomerization, and carbamidomethylation; and disulfide scrambling of interchain disulfide bonds in hinge regions. Thus, each antibody can generate countless different antibody molecules with widely varying activities and potencies. Although post-translational modifications of antibodies have been observed and studied for decades, the full impact of microheterogeneity remains to be further investigated. PTMs may affect antibody functions, such as pharmacokinetic and pharmacodynamic properties, as well as clinical efficacy.

For a population of mAbs to have consistent product quality, clinical safety, and efficacy, having a defined range of post-translational modifications may be critical.

5. Invention content

Provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising about 20 to 40% fucosylated glycans and optionally about 10 to 20% galactosylated glycans.

In some embodiments, the N-glycan profile comprises 23% to 36% fucosylated glycans, preferably about 30% fucosylated glycans. In some embodiments, the N-glycan profile comprises 16% to 18% galactosylated glycans, preferably about 17% galactosylated glycans.

In some embodiments, the relative abundance of fucosylated glycans is the percentage of fucosylated glycans in all glycans in the N-glycan profile. In some embodiments, the relative abundance of galactosylated glycans is the percentage of galactosylated glycans in all glycans in the N-glycan profile.

In some embodiments, the N-glycan profile comprises 12% to 30% bisecting N-glycans, optionally about 18% bisecting N-glycans. In some embodiments, the bisecting N-glycans comprise one or more of G0B, G0FB, G1FB, G2FBS1, and G2FBS2. In some embodiments, the anti-CD20 antibody protein has an N-glycan profile comprising less than 5% sialylated glycans. In some embodiments, the N-glycan profile comprises less than 4%, 3%, 2.5%, 2%, 1%, or 0.5% sialylated glycans. In some embodiments, the N-glycan profile does not comprise detectable amounts of sialylated glycans.

In some embodiments, the anti-CD20 antibody protein has an N-glycan profile comprising 0.1% to 1.5% Man5 N-glycans. In some embodiments, the N-glycan profile comprises 0.4% to 0.7% Man5 N-glycans. In some embodiments, the N-glycan profile comprises about 0.6% Man5 N-glycans. In some embodiments, Man5 N-glycans are the only high mannose species in the N-glycan profile.

In some embodiments, the anti-CD20 antibody protein comprises 0.20 to 0.40 mol isoaspartic acid/mol protein. In some embodiments, the anti-CD20 antibody protein comprises 0.25 to 0.35 mol isoaspartic acid/mol protein.

In some embodiments, the glutamine at heavy chain position 1 is pyroglutamine and the glutamine at light chain position 1 is pyroglutamine.

In some embodiments, the anti-CD20 antibody protein has an N-glycan profile comprising N-glycans with a relative abundance ratio of G1 to G0 of 0.1 to 0.15. In some embodiments, the anti-CD20 antibody protein has an N-glycan profile comprising N-glycans with a relative abundance ratio of G1F to G1 of 0.5 to 0.9.

In some embodiments, the anti-CD20 antibody protein further comprises at least two N-glycans in the following relative abundance ranges: (a) 0.3% to 2% G0-GN; (b) 0.1% to 2% G0F-GN; (c) 30% to 60% G0; (d) 0.1% to 1% G1-GN; (e) 5% to 20% G0B; (f) 5% to 30% G0F; (g) 0.1% to 1.5% Man5; (h) 1% to 15% G0FB; (i) 1% to 13% G1; (j) 0.5% to 10% G1'; (k) 0.5% to 6% G1B; (l) 0.5% to 12% G1F; (m) 0.1% to 3% G1F'; (n) 0.1% to 3% G1FB; (o) 0.1% to 2% G2; and (p) 0.1% to 2% G2F.

In some embodiments, the anti-CD20 antibody protein further comprises at least two N-glycans within the following relative abundance ranges: (a) 0.8% to 1.1% G0-GN; (b) 0.5% to 1.1% G0F-GN; (c) 42.5% to 48.8% G0; (d) 0.3% to 0.6% G1-GN; (e) 9.5% to 14.1% G0B; (f) 12.8% to 19.7% G0F; (g) 0.4% to 0.7% Man5; (h) 5.1% to 7.0% G0FB; (i) 5.7% to 6.4% G1; (j) 2.7% to 3.3% G1'; (k) 1.4% to 2.0% G1B; (l) 2.6% to 4.2% G1F; (m) 1.1% to 1.6% G1F’; (n) 1.1% to 1.8% G1FB; (o) 0.5% to 0.7% G2; and (p) 0.3% to 0.5% G2F.

In some embodiments, the anti-CD20 antibody protein further comprises at least two N-glycans within the following relative abundance ranges: (a) 0.9% G0-GN; (b) 0.8% G0F-GN; (c) 46.1% G0; (d) 0.5% G1-GN; (e) 10.9% G0B; (f) 17.0% G0F; (g) 0.6% Man5; (h) 6.0% G0FB; (i) 6.1% G1; (j) 2.9% G1'; (k) 1.6% G1B; (l) 3.2% G1F; (m) 1.3% G1F'; (n) 1.3 G1FB; (o) 0.5% G2; and (p) 0.3% G2F.

In some embodiments, the anti-CD20 antibody protein further comprises at least three, four or five N-glycans in the following relative abundance ranges: (a) 0.3% to 2% G0-GN; (b) 0.1% to 2% G0F-GN; (c) 30% to 60% G0; (d) 0.1% to 1% G1-GN; (e) 5% to 20% G0B; (f) 5% to 30% G0F; (g) 0.1% to 1.5% Man5; (h) 1% to 15% G0FB; (i) 1% to 13% G1; (j) 0.5% to 10% G1'; (k) 0.5% to 6% G1B; (l) 0.5% to 12% G1F; (m) 0.1% to 3% G1F’; (n) 0.1% to 3% G1FB; (o) 0.1% to 2% G2; and (p) 0.1% to 2% G2F.

In some embodiments, the N-glycan profile of the anti-CD20 antibody protein is determined using a method comprising: (a) incubating the anti-CD20 antibody protein with an enzyme, wherein the enzyme catalyzes the release of N-glycans from the anti-CD20 antibody; (b) using one or more methods selected from chromatography, mass spectrometry, capillary electrophoresis, and combinations thereof to measure the relative abundance of the released N-glycans. In some embodiments, the method further comprises the following steps after step (a) and before step (b): (c) purifying the N-glycans; and (d) labeling the N-glycans with a fluorescent compound. In some embodiments, the enzyme is PNGase F. In some embodiments, the fluorescent compound is 2-aminobenzamide (2-AB).

In some embodiments, less than 10% of the anti-CD20 antibody proteins in the population are non-glycosylated. In some embodiments, less than 5% of the anti-CD20 antibody proteins in the population are non-glycosylated. In some embodiments, less than 1% of the anti-CD20 antibody proteins in the population are non-glycosylated.

In some embodiments, the anti-CD20 antibody protein comprises two or more of the following secondary structures as determined by circular dichroism at 205 nm to 260 nm: (a) 8.0% to 10.0% α-helix; (b) 32.0% to 36.0% antiparallel β-sheets; (c) 5.0% to 6.0% parallel β-sheets; (d) 16.0% to 18.0% β-turns; and (e) 35.0% to 36.0% random coils.

In some embodiments, the anti-CD20 antibody protein comprises the following secondary structure as determined by circular dichroism at 205 nm to 260 nm: (a) 8.0% to 10.0% α-helix; (b) 32.0% to 36.0% antiparallel β-pleats; (c) 5.0% to 6.0% parallel β-pleats; (d) 16.0% to 18.0% β-turns; and (e) 35.0% to 36.0% random coils.

In some embodiments, the anti-CD20 antibody protein comprises two or more of the following secondary structures as determined by circular dichroism at 205 nm to 260 nm: (a) about 9.0% α-helix; (b) about 33.0% antiparallel β-sheets; (c) about 5.6% parallel β-sheets; (d) about 17.5% β-turns; and (e) about 35.2% random coils.

In some embodiments, the population of anti-CD20 antibody proteins further comprises one or more of the following post-translational modifications in a specified abundance:

In some embodiments, one or more of the post-translational modifications are measured by peptide mapping using liquid chromatography-mass spectrometry (LC-MS).

In some embodiments, the population has a total protein amount of 25.5-25.8 mg/mL as measured by absorbance at 280 nm.

In some embodiments, the anti-CD20 antibody proteins in the population induce greater cytotoxicity in a cell-based antibody-dependent cytotoxicity (ADCC) assay compared to obinutuzumab, ofatumumab, rituximab, veltuzumab, ibritumomab tiuxetan and/or ocrelizumab.

In some embodiments, the population has a relative potency of 90% to 163% in a cell-based ADCC assay compared to a commercial reference standard. In some embodiments, the population has a relative potency of 78% to 116% or 73% to 128% in a cell-based complement-dependent cytotoxicity (CDC) assay compared to a commercial reference standard. In some embodiments, the population has a relative potency of 92% to 118% or 82% to 138% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In some embodiments, the population has a _KD value of 30 to 70 nM in an FcγRIIIa-158V binding assay as measured by surface plasmon resonance. In some embodiments, the population has a _KD value of 500 to 1000 nM in an FcγRIIIa 158F binding assay as measured by surface plasmon resonance. In some embodiments, the population has a significantly higher binding affinity to FcγRIIIa 158V or FcγRIIIa 158F than rituximab. In some embodiments, the population has a relative potency of 88% to 113% or 86% to 116% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In some embodiments, the population has a relative potency of 106% to 126% in a CD16 activity assay compared to a commercial reference standard.

In some embodiments, the population has 99.2% to 99.9% monomer as detected by size exclusion chromatography (SEC). In some embodiments, the population has 0.1% to 0.8% dimer as detected by SEC. In some embodiments, the population has no detectable amount of aggregates as detected by SEC; and/or the population has no detectable amount of fragments as detected by SEC.

In some embodiments, the population has 93.6 to 95.9% IgG after purification by non-reducing capillary gel electrophoresis (CGE). In some embodiments, the population has 0.1 to 0.3% high molecular weight species (HMWS) after purification by non-reducing CGE. In some embodiments, the population has 0.7 to 1.2% free light chain (LC) after purification by non-reducing CGE. In some embodiments, the population has 97.7 to 98.0% heavy chain plus light chain species (HC+LC) after purification by reducing CGE.

In some embodiments, the population has 20% to 25% acidic isoform as detected by imaged capillary isoelectric focusing (iCIEF). In some embodiments, the population has 50% to 60% predominant isoform as detected by iCIEF. In some embodiments, the population has 20% to 30% basic isoform as detected by iCIEF. In some embodiments, the population has an average molar ratio of free thiols to anti-CD20 antibody of about 2.0 to 2.2.

In some embodiments, the amino acid sequence of the anti-CD20 antibodies in the group comprises a deletion of the N-terminal residue. In some embodiments, the amino acid sequence of the anti-CD20 antibodies in the group comprises a deletion of up to 5 N-terminal residues. In some embodiments, the amino acid sequence of the anti-CD20 antibodies in the group comprises a deletion of up to 10 N-terminal residues. In some embodiments, the heavy chain terminal lysine residue of the anti-CD20 antibodies in the group is truncated.

Also provided herein is a pharmaceutical formulation comprising a composition described herein, wherein the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 10 mg/mL to 50 mg/mL. In some embodiments, the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 25 mg/mL.

In some embodiments, the pharmaceutical formulation further comprises one or more of the following: sodium chloride, trisodium citrate dehydrate, polysorbate 80, and hydrochloric acid. In some embodiments, the pharmaceutical formulation comprises about 9.0 mg/mL of sodium chloride, about 7.4 mg/mL of trisodium citrate dehydrate, about 0.7 mg/mL of polysorbate 80, and/or about 0.4 mg/mL of hydrochloric acid.

In some embodiments, the anti-CD20 antibody is present in a single dosage form.

Also provided herein is a pharmaceutical formulation comprising: (i) a composition as described herein, wherein the composition comprises a single dosage form of the group of anti-CD20 antibody proteins, wherein the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 25 mg/mL, (ii) about 9.0 mg/mL of sodium chloride, (iii) about 7.4 mg/mL of dehydrated trisodium citrate, (iv) about 0.7 mg/mL of polysorbate 80, and (v) about 0.4 mg/mL of hydrochloric acid.

Also provided herein is a single batch formulation of the population of anti-CD20 antibody proteins or pharmaceutical formulations described herein, wherein the single batch comprises at least 100 g, at least 120 g, or at least 150 g of anti-CD20 antibody protein.

Also provided herein are the anti-CD20 antibody proteins or pharmaceutical formulations described herein produced in a 15,000 L or 20,000 L bioreactor.

Also provided herein are methods of treating multiple sclerosis (MS) in a subject by administering to a subject in need thereof a therapeutically effective amount of a composition or pharmaceutical formulation described herein.

In some embodiments, the composition or pharmaceutical formulation is administered as follows: i) a first infusion at a dose of about 150 mg of the anti-CD20 antibody protein, ii) a second infusion two weeks later at a dose of about 450 mg of the anti-CD20 antibody protein, and iii) subsequent infusions every 24 weeks or six months at a dose of about 450 mg of the anti-CD20 antibody protein.

In some embodiments, administration of the composition or pharmaceutical formulation to a subject results in one or more of the following pharmacokinetic parameters: (a) AUC between 2,160 μg/mL and 3,840 μg/mL; (b) Cmax between 118,011 ng/mL and 159,989 ng/mL; (c) Cmin between 40 ng/mL and 375 ng/mL; and (d) Cavg between 6,437 ng/mL and 11,443 ng/mL.

In some embodiments, administration of the composition or pharmaceutical formulation to a subject results in one or more of the following pharmacokinetic parameters: (a) an AUC of about 3,000 μg/mL; (b) a Cmax of about 139,000 ng/mL; (c) a Cmin of about 139 ng/mL; and (d) a Cavg of about 8,940 ng/mL.

In some embodiments, the method comprises a treatment period of at least 96 weeks.

In some embodiments, the subject is pre-administered with a corticosteroid 30 to 60 minutes prior to administration of the composition or pharmaceutical formulation. In some embodiments, the corticosteroid is methylprednisone or dexamethasone. In some embodiments, methylprednisone is administered in an amount of about 100 mg and/or dexamethasone is administered in an amount of about 10 to 20 mg.

In some embodiments, the subject is pre-administered with an antihistamine 30 to 60 minutes prior to administration of the composition or pharmaceutical formulation. In some embodiments, the antihistamine is diphenhydramine HCl. In some embodiments, diphenhydramine HCl is administered in an amount of about 25 to 50 mg.

In some embodiments, the subject is pre-administered with an antipyretic 30 to 60 minutes prior to administration of the composition or pharmaceutical formulation. In some embodiments, the antipyretic is acetaminophen or an antipyretic bioequivalent thereto.

In some embodiments, the individual has an Expanded Disability Status Scale (EDSS) score of 0 to 5.5 before treatment.

Also provided herein are methods of treating multiple sclerosis (MS) in a subject by administering to a subject in need thereof a therapeutically effective amount of a composition or pharmaceutical formulation described herein, wherein administration of the composition or pharmaceutical formulation results in no evidence of disease activity (NEDA) in the subject 24 to 96 weeks after administration.

In some embodiments, administering the composition or pharmaceutical formulation results in NEDA in the subject after 24 weeks of administration.

Also provided herein are methods of treating multiple sclerosis (MS) in a subject by administering to a subject in need thereof a therapeutically effective amount of a composition or pharmaceutical formulation described herein, wherein administration of the composition or pharmaceutical formulation results in a temporary decrease in lymphocyte count in the subject.

In some embodiments, the lymphocyte count is normal before day 8 of administration.

In some embodiments, the MS is recurrent MS (RMS).

Also provided herein is a method of reducing the annualized relapse rate (ARR) of an individual suffering from a relapsing form of multiple sclerosis (MS) by administering to the individual an effective amount of a composition or pharmaceutical formulation described herein, the method comprising: administering the composition or pharmaceutical formulation for intravenous infusion in a multiple infusion dosage regimen, the dosage regimen comprising: a) a first infusion on day 1 comprising 150 mg of anti-CD20 antibody protein; b) a second infusion about 2 weeks after the first infusion comprising 450 mg of anti-CD20 antibody protein; c) a first subsequent infusion about 24 weeks or about six months from the first infusion comprising 450 mg of anti-CD20 antibody protein; and d) one or more subsequent infusions about 24 weeks or about six months from the previous infusion comprising 450 mg of anti-CD20 antibody protein.

In some embodiments, the effective amount of the composition or pharmaceutical formulation is sufficient to achieve an ARR of 0.091 or an ARR of 0.076.

In some embodiments, the second infusion, the first subsequent infusion, and the one or more subsequent infusions of the anti-CD20 antibody protein last for about one hour.

Also provided herein is a method for treating a relapsing form of multiple sclerosis (MS) in an individual by administering an effective amount of a composition or pharmaceutical formulation described herein to an individual in need thereof, the method comprising: administering a composition or pharmaceutical formulation for intravenous infusion in a multiple infusion dosage regimen, the dosage regimen comprising: a)  a first infusion on day 1, comprising 150 mg of anti-CD20 antibody protein; b) a second infusion about 2 weeks after the first infusion, comprising 450 mg of anti-CD20 antibody protein; c)  a first subsequent infusion about 24 weeks or about six months from the first infusion, comprising 450 mg of anti-CD20 antibody protein; and d) one or more subsequent infusions about 24 weeks or about six months from the previous infusion, comprising 450 mg of anti-CD20 antibody protein, The duration of the second infusion, the first subsequent infusion, and one or more subsequent infusions of the anti-CD20 antibody protein is about one hour.

In some embodiments, the method further comprises pre-administering a corticosteroid and an antihistamine to the individual 30 to 60 minutes prior to administering the composition or pharmaceutical formulation. In some embodiments, the corticosteroid is methylprednisolone or dexamethasone. In some embodiments, methylprednisolone is administered in an amount of about 100 mg and/or dexamethasone is administered in an amount of about 10 to 20 mg.

In some embodiments, the composition or pharmaceutical formulation for intravenous infusion is prepared in 250 mL of 0.9% Sodium Chloride Injection.

In some embodiments, the first subsequent infusion is about 24 weeks from the first infusion. In some embodiments, one or more subsequent infusions are about 24 weeks from the previous infusion. In some embodiments, the first subsequent infusion is about 6 months from the first infusion. In some embodiments, one or more subsequent infusions are about 6 months from the previous infusion.

In some embodiments, the duration of the first infusion of the anti-CD20 antibody protein is about four hours. In some embodiments, the first infusion of the anti-CD20 antibody protein is infused at the following rates: 10 mL per hour for the first 30 minutes; 20 mL per hour for the next 30 minutes; 35 mL per hour for the next hour; and 100 mL per hour for the remaining two hours.

In some embodiments, the second infusion, the first subsequent infusion, and the one or more subsequent infusions of the anti-CD20 antibody protein are infused at the following rate: 100 mL per hour for the first 30 minutes and 400 mL per hour for the remaining 30 minutes.

In some embodiments, a multiple infusion dosing regimen of an anti-CD20 antibody protein reduces or delays the progression of MS symptoms.

In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody protein achieved a reduction in the total number of gadolinium-enhancing T1 lesions based on MRI scans compared to subjects receiving 14 mg teriflunomide administered orally daily during the same treatment period.

In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody protein achieved a reduction in the total number of new and enlarging T2 hyperintense lesions based on MRI scans compared to subjects receiving 14 mg teriflunomide administered orally daily during the same treatment period.

In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody protein achieve an increase in the free disease activity (NEDA) status compared to subjects receiving daily oral administration of 14 mg teriflunomide during the same treatment period.

In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody protein achieved an increase in Confirmed Disability Improvement (CDI) compared to subjects receiving 14 mg teriflunomide orally daily during the same treatment period.

In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody protein achieved an increase in Multiple Sclerosis Functional Composite (MSFC) score compared to subjects receiving 14 mg teriflunomide orally daily during the same treatment period.

In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody protein achieved an improvement in a timed 25-Foot Walk (T25FW) score compared to subjects receiving 14 mg teriflunomide orally daily during the same treatment period.

In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody protein achieved an improvement in 9-Hole Peg test (9-HPT) scores compared to subjects receiving 14 mg teriflunomide orally daily during the same treatment period.

In some specific instances, subjects administered a multiple infusion dosing regimen of anti-CD20 antibody protein achieved a significant reduction in the volume and number of new T1 hypointense lesions based on MRI scans compared to subjects receiving 14 mg teriflunomide orally daily during the same treatment period.

In some embodiments, the multiple infusion dosing regimen of the anti-CD20 antibody protein results in a geometric mean steady-state AUC of 3000 mcg/mL per day (CV = 28%) and a mean maximum concentration of 139 mcg/mL (CV = 15%).

Also provided herein is a method for inactivating viruses or adventitious agents in rat myeloma cells expressing an anti-CD20 antibody protein recited in the compositions described herein, wherein the method maintains suitability for production of the antibody in a 15,000 L or 20,000 L bioreactor.

Also provided herein are methods for reducing the immunogenicity of an anti-CD20 antibody protein recited in the compositions described herein, wherein the method maintains suitability for production of the antibody in a 15,000 L or 20,000 L bioreactor.

Also provided herein are methods for treating multiple sclerosis, wherein the methods comprise administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical formulation comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein administration of the pharmaceutical formulation results in no disease activity (NEDA) in the subject 24 weeks after administration. In some embodiments, administration of the pharmaceutical formulation results in NEDA in the subject 24 to 96 weeks after administration.

Also disclosed herein are methods for treating multiple sclerosis, wherein the methods comprise administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical formulation comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein administration of the pharmaceutical formulation results in a temporary decrease in lymphocyte counts in the subject. In some embodiments, the lymphocyte counts are normal prior to day 8 of administration.

In some embodiments of any of the methods described herein, the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans.

In some embodiments of any of the methods described herein, multiple sclerosis (MS) is a relapsing form of MS (RMS). In certain embodiments, RMS comprises a clinically isolated syndrome (CIS); relapsing-remitting MS (RRMS); or active secondary progressive MS (SPMS). In some embodiments of any of the methods described herein, the individual is diagnosed with RMS according to the McDonald Criteria (2010) or by another suitable method known to those skilled in the art. In some embodiments of any of the methods described herein, the individual is a human.

Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10 to 20% galactosylated glycans.

Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 35% fucosylated glycans.

In some embodiments, the N-glycan profile comprises 28 to 33% fucosylated glycans. In some embodiments, the N-glycan profile comprises about 30% fucosylated glycans.

In some embodiments, the N-glycan profile comprises 16 to 18% galactosylated glycans. In some embodiments, the N-glycan profile comprises about 17% galactosylated glycans.

In some embodiments, the relative abundance of fucosylated glycans is the percentage of fucosylated glycans in all glycans in the N-glycan profile. In some embodiments, the relative abundance of galactosylated glycans is the percentage of galactosylated glycans in all glycans in the N-glycan profile.

Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising at least about 10% bisected N-glycans.

In some embodiments, the N-glycan profile comprises 12% to 30% aliquot N-glycans. In some embodiments, the N-glycan profile comprises about 18% aliquot N-glycans. In some embodiments, the aliquot N-glycans comprise one or more of G0B, GOFB, G1FB, G2FBS1, and G2FBS2.

Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody proteins in the population have an N-glycan profile comprising less than 5% sialylated glycans.

In some embodiments, the N-glycan profile comprises less than 4%, 3%, 2.5%, 2%, 1% or 0.5% sialylated glycans. In some embodiments, the N-glycan profile does not comprise detectable amounts of sialylated glycans.

Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising 5 to 15% G0B N-glycans.

In some embodiments, the N-glycan profile comprises 9 to 11% G0B N-glycans. In some embodiments, the N-glycan profile comprises about 10% G0B N-glycans.

Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising 0.1% to 1.5% Man5 N-glycans.

In some embodiments, the N-glycan profile comprises 0.4% to 0.7% Man5 N-glycans. In some embodiments, the N-glycan profile comprises about 0.6% Man5 N-glycans. In some embodiments, Man5 N-glycans are the only high mannose species in the N-glycan profile.

Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins comprises 0.20 to 0.40 mol isoaspartic acid/mol protein.

In some embodiments, the population of anti-CD20 antibody proteins comprises 0.25 to 0.35 mol isoaspartate/mol protein.

Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the glutamine at position 1 of the heavy chain is pyroglutamine, and wherein the glutamine at position 1 of the light chain is pyroglutamine.

Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising N-glycans with a relative abundance ratio of G1 to G0 of 0.1 to 0.15.

Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising a relative abundance ratio of G1F to G1 N-glycans of 0.5 to 0.9.

In some embodiments, the anti-CD20 antibody protein further comprises at least two N-glycans in the following relative abundance ranges: (a) 0.3% to 2% G0-GN; (b) 0.1% to 2% G0F-GN; (c) 0.1% to 1% G1-GN; (d) 5% to 20% G0B; (e) 5% to 30% G0F; (f) 0.1% to 1.5% Man5; (g) 1% to 15% G0FB; (h) 1% to 13% G1; (i) 0.5% to 10% G1'; (j) 0.5% to 6% G1B; (k) 0.5% to 12% G1F; (l) 0.1% to 3% G1F'; (m) 0.1% to 3% G1FB; (n) 0.1% to 2% G2; and (o) 0.1% to 2% G2F.

In some specific examples, the anti-CD20 antibody protein further comprises at least two N-glycans in the following relative abundance ranges: (a) 0.8% to 1.1% G0-GN; (b) 0.5% to 1.1% G0F-GN; (c) 0.3% to 0.6% G1-GN; (d) 9.5% to 14.1% G0B; (e) 12.8% to 19.7% G0F; (f) 0.4% to 0.7% Man5; (g) 5.1% to 7.0% G0FB; (h) 5.7% to 6.4% G1; (i) 2.7% to 3.3% G1'; (j) 1.4% to 2.0% G1B; (k) 2.6% to 4.2% G1F; (l) 1.1% to 1.6% G1F’; (m) 1.1% to 1.8% G1FB; (n) 0.5% to 0.7% G2; and (o) 0.3% to 0.5% G2F.

In some embodiments, the anti-CD20 antibody protein further comprises at least two N-glycans in the following relative abundances: (a) 0.9% G0-GN; (b) 0.8% G0F-GN; (c) 0.5% G1-GN; (d) 10.9% G0B; (e) 17.0% G0F; (f) 0.6% Man5; (g) 6.0% G0FB; (h) 6.1% G1; (i) 2.9% G1'; (j) 1.6% G1B; (k) 3.2% G1F; (l) 1.3% G1F'; (m) 1.3 G1FB; (n) 0.5% G2; and (o) 0.3% G2F.

In some embodiments, the population of anti-CD20 antibody proteins further comprises at least three, four or five N-glycans in a relative abundance or a relative abundance range as described herein.

In some embodiments, the N-glycan profile of the anti-CD20 antibody protein is determined using a method comprising: (a) incubating the anti-CD20 antibody protein with an enzyme, wherein the enzyme catalyzes the release of N-glycans from the anti-CD20 antibody; (b) using one or more methods selected from chromatography, mass spectrometry, capillary electrophoresis, and combinations thereof to measure the relative abundance of the released N-glycans. In some embodiments, the enzyme is PNGase F.

In some embodiments, the method further comprises the following steps after step (a) and before step (b): (c) purifying the N-glycans; and (d) labeling the N-glycans with a fluorescent compound. In some embodiments, the fluorescent compound is 2-aminobenzamide (2-AB).

In some embodiments, less than 10% of the anti-CD20 antibody proteins in the population are non-glycosylated. In some embodiments, less than 5% of the anti-CD20 antibody proteins in the population are non-glycosylated. In some embodiments, less than 1% of the anti-CD20 antibody proteins in the population are non-glycosylated.

In some embodiments, the N-glycan spectrum of the anti-CD20 antibody protein is substantially as shown in Figure 2 .

Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins comprises two or more of the following secondary structures as determined by circular polarization dichroism at 205 nm to 260 nm: (a) 8.0% to 10.0% α-helix; (b) 32.0% to 36.0% antiparallel β-pleats; (c) 5.0% to 6.0% parallel β-pleats; (d) 16.0% to 18.0% β-turns; and (e) 35.0% to 36.0% random coils.

In some embodiments, the population of anti-CD20 antibody proteins comprises the following secondary structure as determined by circular dichroism at 205 nm to 260 nm: (a) 8.0% to 10.0% α-helix; (b) 32.0% to 36.0% antiparallel β-pleats; (c) 5.0% to 6.0% parallel β-pleats; (d) 16.0% to 18.0% β-turns; and (e) 35.0% to 36.0% random coil.

In some embodiments, the population of anti-CD20 antibody proteins comprises two or more of the following secondary structures as determined by circular dichroism at 205 nm to 260 nm: (a) about 9.0% α-helix; (b) about 33.0% antiparallel β-pleated sheets; (c) about 5.6% parallel β-pleated sheets; (d) about 17.5% β-turns; and (e) about 35.2% random coils.

Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain ("HC") comprising the amino acid sequence of SEQ ID NO: 1 and a light chain ("LC") comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins further comprises one or more of the following post-translational modifications in a specified abundance:

In some embodiments, the population of anti-CD20 antibody proteins comprises two, three, four, five or more post-translational modifications.

In some embodiments, the population of anti-CD20 antibody proteins comprises the following post-translational modifications in specified abundance:

In some embodiments, the population of anti-CD20 antibody proteins comprises the following post-translational modifications in specified abundance:

In some embodiments, one or more of the post-translational modifications are measured by peptide localization using liquid chromatography-mass spectrometry (LC-MS). In some embodiments, deamidation is measured by isoaspartate detection or peptide localization using LC-MS.

Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain ("HC") comprising the amino acid sequence of SEQ ID NO: 1 and a light chain ("LC") comprising the amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has the following properties:

In some embodiments, the population has a total protein amount of 25.5 to 25.8 mg/mL as measured by absorbance at 280 nm. In some embodiments, the population has a total protein amount of 25.6 mg/mL as measured by absorbance at 280 nm.

In some embodiments, the group induces higher cytotoxicity in a cell-based antibody-dependent cytotoxicity (ADCC) assay compared to obinutuzumab, ofatumumab, rituximab, veltuzumab, epilizumab and/or ocrelizumab. In some embodiments, the group has a relative potency of 90 to 163% in a cell-based ADCC assay compared to a commercial reference standard. In some embodiments, the group has a relative potency of 117% in a cell-based ADCC assay compared to a commercial reference standard. In some embodiments, the cell-based ADCC assay uses effector cells selected from CD16 effector cells and primary NK cells. In some embodiments, the population expresses more than 100% of a commercial reference standard in cell-based ADCC using CD16 effector cells.

In some embodiments, the population exhibits greater B cell depletion activity in a human whole blood B cell depletion assay compared to obinutuzumab, ofatumumab, rituximab, veltuzumab, epilizumab and/or ocrelizumab.

In some embodiments, the population has a relative potency of 78% to 116% or 73% to 128% in a cell-based complement-dependent cytotoxicity (CDC) assay compared to a commercial reference standard. In some embodiments, the population has a relative potency of about 91% in a cell-based CDC assay compared to a commercial reference standard.

In some embodiments, the population has a relative potency of 92 to 118% or 82 to 138% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In some embodiments, the population has a relative potency of about 109% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard.

In some embodiments, the population has a _K value of 30 to 70 nM in a FcγRIIIa-158V binding assay as measured by surface plasmon resonance. In some embodiments, the population has a _K value of about 59 nM in a FcγRIIIa-158V binding assay as measured by surface plasmon resonance.

In some embodiments, the population has a _K value of 500-1000 nM in a FcγRIIIa 158F binding assay as measured by surface plasmon resonance. In some embodiments, the population has a _K value of about 760 nM in a FcγRIIIa 158F binding assay as measured by surface plasmon resonance.

In some embodiments, the population has a significantly higher binding affinity for FcγRIIIa 158V or FcγRIIIa 158F than rituximab.

In some embodiments, the population has a relative potency of 88-113% or 86-116% in a C1q binding assay compared to a commercial reference standard as measured by ELISA. In some embodiments, the population has a relative potency of about 99% in a C1q binding assay compared to a commercial reference standard as measured by ELISA.

In some embodiments, the population has a relative potency of 106 to 126% in a CD16 activity assay compared to a commercial reference standard. In some embodiments, the population has a relative potency of about 115% in a CD16 activity assay compared to a commercial reference standard.

In some embodiments, the population of anti-CD20 antibody proteins has the following purification profile:

In some embodiments, the population has 99.2 to 99.9% monomers as detected by size exclusion chromatography (SEC). In some embodiments, the population has 0.1 to 0.8% dimers as detected by SEC. In some embodiments, the population has no detectable amount of aggregates as detected by SEC. In some embodiments, the population has no detectable amount of fragment content as detected by SEC.

In some embodiments, the population has 93.6 to 95.9% IgG after purification by non-reducing capillary gel electrophoresis (CGE). In some embodiments, the population has 0.1 to 0.3% high molecular weight species (HMWS) after purification by non-reducing CGE. In some embodiments, the population has 0.7 to 1.2% free light chains (LC) after purification by non-reducing CGE.

In some embodiments, the population has 97.7 to 98.0% heavy plus light chain species (HC+LC) after purification by reduced CGE.

In some embodiments, the population of anti-CD20 antibody proteins has the following distribution of charged isoforms:

In some embodiments, the population has 20 to 25% acidic isoforms as detected by imaged capillary isoelectric focusing (iCIEF). In some embodiments, the population has 50 to 60% major isoforms as detected by iCIEF. In some embodiments, the population has 20 to 30% basic isoforms as detected by iCIEF.

In some embodiments, the population has an average molar ratio of free thiol to anti-CD20 antibody of about 2.0 to 2.2.

In some embodiments, the amino acid sequence of the anti-CD20 antibodies in the population comprises a deletion of the N-terminal residue. In some embodiments, the amino acid sequence of the anti-CD20 antibodies in the population comprises a deletion of up to 5 N-terminal residues. In some embodiments, the amino acid sequence of the anti-CD20 antibodies in the population comprises a deletion of up to 10 N-terminal residues.

In some embodiments, the anti-CD20 antibodies in the population have a heavy chain terminal lysine residue truncated. In some embodiments, administration of the anti-CD20 antibody to a human patient results in one or more of the following pharmacokinetic parameters: (a) an AUC between 2,160 μg/mL and 3,840 μg/mL; (b) a Cmax between 118,011 ng/mL and 159,989 ng/mL; (c) a Cmin between 40 ng/mL and 375 ng/mL; and (d) a Cavg between 6,437 ng/mL and 11,443 ng/mL, and wherein the anti-CD20 antibody is administered as: i) a first infusion at a dose of about 150 mg, ii) a second infusion two weeks later at a dose of about 450 mg, and iii) subsequent infusions every six months at a dose of about 450 mg.

In some embodiments, administration of an anti-CD20 antibody to a human patient results in one or more of the following pharmacokinetic parameters: (a) an AUC of about 3,000 μg/mL; (b) a Cmax of about 139,000 ng/mL; (c) a Cmin of about 139 ng/mL; and (d) a Cavg of about 8,940 ng/mL.

In some embodiments, the anti-CD20 antibody is present in a single dosage form.

Also provided herein is a pharmaceutical formulation comprising the composition described herein, wherein the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 25 mg/mL.

Also provided herein is a pharmaceutical formulation comprising an anti-CD20 antibody described herein, wherein the anti-CD20 antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the pharmaceutical formulation comprises one or more of the following: sodium chloride, trisodium citrate dehydrate, polysorbate 80, and hydrochloric acid.

In some embodiments, the pharmaceutical formulation comprises about 9.0 mg/mL of sodium chloride. In some embodiments, the pharmaceutical formulation comprises about 7.4 mg/mL of trisodium citrate dehydrate. In some embodiments, the pharmaceutical formulation comprises about 0.7 mg/mL of polysorbate 80. In some embodiments, the pharmaceutical formulation comprises about 0.4 mg/mL of hydrochloric acid.

Also provided herein is a single batch preparation of a population of anti-CD20 antibody proteins described herein, wherein the anti-CD20 antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the single batch comprises at least 100 g of the anti-CD20 antibody protein.

Also provided herein is a single batch preparation of a population of anti-CD20 antibody proteins described herein, wherein the anti-CD20 antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the single batch comprises at least 120 g of the anti-CD20 antibody protein.

Also provided herein is a single batch preparation of a population of anti-CD20 antibody proteins described herein, wherein the anti-CD20 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the single batch comprises at least 150 g of the anti-CD20 antibody protein.

Also provided herein are the population of anti-CD20 antibody proteins described herein produced in a 15,000 L or 20,000 L bioreactor.

Also provided herein are methods of treating an autoimmune disease, wherein the method comprises administering to an individual in need thereof a composition as described herein, and wherein the autoimmune disease is selected from the group consisting of pityriasis, rheumatoid arthritis, vasculitis, inflammatory bowel disease, dermatitis, osteoarthritis, inflammatory muscle disease, allergic rhinitis, vaginitis, interstitial cystitis, scleroderma, osteoporosis, eczema, allogeneic or xenotransplantation, transplant rejection, Graft-versus-host disease, lupus erythematosus, inflammatory diseases, type 1 diabetes, pulmonary fibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis, myasthenia gravis, autoimmune hemolytic anemia, cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis, allergic conjunctivitis, atopic dermatitis, chronic obstructive pulmonary disease, glomerulonephritis, neuroinflammatory diseases, and uveitis.

Also provided herein are methods of treating multiple sclerosis, wherein the method comprises administering to a subject in need thereof a composition described herein.

In some embodiments, multiple sclerosis (MS) is a relapsing form of MS. In some embodiments, the relapsing form of MS is selected from clinically isolated syndrome (CIS), relapsing remitting MS (RRMS) and active secondary progressive MS (SPMS). In some embodiments, the relapsing form of MS is clinically isolated syndrome (CIS). In some embodiments, the relapsing form of MS is relapsing remitting multiple sclerosis (RRMS). In some embodiments, the relapsing form of MS is active secondary progressive multiple sclerosis (SPMS).

Also provided herein are methods of treating a neoplastic disease, wherein the method comprises administering to a subject in need thereof a composition described herein.

In some embodiments, the neoplastic disease is acute B-lymphoblastic leukemia, B-cell lymphoma, mature B-cell lymphoma, including B-type chronic lymphocytic leukemia (B-CLL), small B-cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmic lymphoma, mantle cell lymphoma, follicular lymphoma, marginal zone MALT-type lymphoma, marginal zone lymphoma of the lymph nodes with or without monocytoid B cells, marginal zone lymphoma of the spleen (with or without villous lymphocytes), tricholeucocytic leukaemia, diffuse large B-cell lymphoma, or Burkitt's lymphoma.

In some embodiments, the individual is a human being.

Also provided herein are methods for inactivating viruses or foreign substances in rat myeloma cells expressing an anti-CD20 antibody protein in a composition described herein, wherein the method maintains suitability for production of the antibody in a 15,000 L or 20,000 L bioreactor.

Also provided herein are methods for reducing the immunogenicity of an anti-CD20 antibody protein in a composition described herein, wherein the method maintains suitability for production of the antibody in a 15,000 L or 20,000 L bioreactor.

Also provided herein is a method for analyzing a TG-1101 (TG Therapeutics, Inc.) formulation, comprising: (i) providing an isolated N-glycan fraction from a TG-1101 (TG Therapeutics, Inc.) formulation; (ii) analyzing the N-glycan fraction to determine whether one or more N-glycans are the following N-glycans within the following relative abundance ranges: (a) 0.3% to 2% G0-GN; (b) 0.1% to 2% G0F-GN; (c) 0.1% to 1% G1-GN; (d) 5% to 20% G0B; (e) 5% to 30% G0F; (f) 0.1% to 1.5% Man5; (g) 1% to 15% G0FB; (h) 1% to 13% G1; (i) 0.5% to 10% G1’; (j) 0.5% to 6% G1B; (k) 0.5% to 12% G1F; (l) 0.1% to 3% G1F’; (m) 0.1% to 3% G1FB; (n) 0.1% to 2% G2; and (o) 0.1% to 2% G2F. In some embodiments, the method comprises analyzing the N-glycan portion to determine whether one or more N-glycans are the following N-glycans within the following relative abundance ranges: (a) 0.8% to 1.1% G0-GN; (b) 0.5% to 1.1% G0F-GN; (c) 0.3% to 0.6% G1-GN; (d) 9.5% to 14.1% G0B; (e) 12.8% to 19.7% G0F; (f) 0.4% to 0.7% Man5; (g) 5.1% to 7.0% G0FB; (h) 5.7% to 6.4% G1; (i) 2.7% to 3.3% G1'; (j) 1.4% to 2.0% G1B; (k) 2.6% to 4.2% G1F; (l) 1.1% to 1.6% G1F’; (m) 1.1% to 1.8% G1FB; (n) 0.5% to 0.7% G2; and (o) 0.3% to 0.5% G2F.

In some embodiments, the method comprises analyzing the N-glycan portion to determine whether one or more N-glycans are present in the following relative abundances: (a) 0.9% G0-GN; (b) 0.8% G0F-GN; (c) 0.5% G1-GN; (d) 10.9% G0B; (e) 17.0% G0F; (f) 0.6% Man5; (g) 6.0% G0FB; (h) 6.1% G1; (i) 2.9% G1'; (j) 1.6% G1B; (k) 3.2% G1F; (l) 1.3% G1F'; (m) 1.3 G1FB; (n) 0.5% G2; and (o) 0.3% G2F.

Also provided herein is a composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins further comprises at least two N-glycans in the following relative abundances: (a) 0.9% G0-GN; (b) 0.8% G0F-GN; (c) 0.5% G1-GN; (d) 10.9% G0B; (e) 17.0% G0F; (f) 0.6% Man5; (g) 6.0% G0FB; (h) 6.1% G1; (i) 2.9% G1′; (j) 1.6% G1B; (k) 3.2% G1F; (l) 1.3% G1F'; (m) 1.3 G1FB; (n) 0.5% G2; and (o) 0.3% G2F, and wherein the anti-CD20 antibody protein is produced in rat fusion tumor cells.

In some embodiments, the rat hybridoma cell is a YB2/0 cell.

7. Detailed description of the invention

Provided herein are anti-CD20 antibody protein groups with a specified range of post-translational modifications. The primary amino acid sequences of such antibodies are provided in Section 7.3. The types of such post-translational modifications and their corresponding abundance in the anti-CD20 antibody group are described in Section 7.4. The compositions (including single batch compositions) of such anti-CD20 antibody protein groups with a specific range of post-translational modifications and purity ranges are described in Section 7.5 (e). The quantitative analysis of such post-translational modifications in the anti-CD20 antibody protein group is described in Section 7.5. The analysis of the anti-CD20 antibody protein group is described in Section 7.5. The analysis used to demonstrate the biological and clinical significance of such post-translational modifications in a group of anti-CD20 antibody proteins is described in Section 7.7 (a). Methods of using these anti-CD20 antibody protein populations to treat and prevent medical conditions are described in Section 7.8. The pharmacokinetic and pharmacodynamic properties of the compositions provided herein in human patients are described in Section 7.9. Methods of making these anti-CD20 antibody protein populations are described in Section 7.10.

As used herein, "TG-1101" (TG Therapeutics, Inc.) (also known as ublituximab (UBX), UTX, TG-1101, TGTX-1101, Utuxin ^™ , LFB-R603, TG20, EMAB603) is the antibody source of the anti-CD20 antibodies described herein, which have a unique glycosylation profile produced by the disclosed methods.

The source antibody TG-1101 is a monoclonal antibody targeting epitopes on CD20, such as IRAHT (SEQ ID NO: 37) and EPAN (SEQ ID NO: 38). See Fox, E. et al., Mult. Scler. 27 : 420-429 (March 2021); Babiker et al., Expert Opin Investig Drugs 27 : 407-412 (2018); Cotchett, KR et al., Multiple Sclerosis and Related Disorders 49 : 102787 (2021); Miller et al., Blood 120 : Abstract No. 2756 (2012); Deng, C. et. al., J. Clin. Oncol. 31 : Abstract No. 8575 (2013). TG-1101 is also described in U.S. Patent Nos. 9,234,045 and 9,873,745. 7.1 Abbreviations and Conventions

The following abbreviations are used throughout this application. 7.2 Definition

As used herein, the term "anti-CD20 antibody protein population" refers to an anti-CD20 antibody protein composition being tested for the abundance of post-translational modifications. Individual anti-CD20 antibody proteins in a population may contain similar or different post-translational modifications. In some embodiments, a population of anti-CD20 antibody proteins refers to all anti-CD20 antibody proteins present in a single dosage form. In some embodiments, a population of anti-CD20 antibody proteins refers to all anti-CD20 antibody proteins present in a single batch. In some embodiments, the amount of a population of anti-CD20 antibody proteins is sufficient to determine whether the anti-CD20 antibody proteins in the batch meet or fail to reach a predetermined acceptable comparison value range compared to a reference standard.

As used herein, the term "single batch" in the context of anti-CD20 antibody protein refers to a composition derived from a single production or run of a single bioreactor of a specified volume. For example, an anti-CD20 antibody protein obtained from a single run of a 15,000 L bioreactor can be referred to as a single batch. In certain embodiments, the capacity of the bioreactor is at least 100; 200; 300; 400; 500; 750; 1,000; 2,000; 3,000; 4,000; 5,000; 7,500; 10,000; 15,000; 20,000; or at least 25,000 L.

As used herein, and unless otherwise indicated, the term "about" or "approximately" means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1 or 2 standard deviations. In certain embodiments, the term "about" or "approximately" means within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05 of a given value or range. 7.3 Primary Amino Acid Sequences of Anti-CD20 Antibodies

In some aspects, the anti-CD20 antibody proteins provided herein are represented by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2. The sequences are provided in the sequence listing below.

In some aspects, the anti-CD20 antibody proteins provided herein are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1, or amino acids that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to SEQ ID NO: 1, and a light chain comprising an amino acid sequence of SEQ ID NO: 2, or amino acids that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to SEQ ID NO: 2.

In some aspects, the anti-CD20 antibody proteins provided herein comprise the VH CDR1, CDR2 and CDR3 regions of SEQ ID NOs: 3, 4 and 5, and the VL CDR1, CDR2 and CDR3 regions of SEQ ID NOs: 8, 9 and 10.

In some aspects, the anti-CD20 antibody protein provided herein comprises the VH of SEQ ID NO:6 and the VL of SEQ ID NO:11.

In some aspects, the nucleic acid sequence encoding the heavy chain of the anti-CD20 antibody protein provided herein comprises the nucleic acid sequence of SEQ ID NO: 35. In some aspects, the nucleic acid sequence encoding the light chain of the anti-CD20 antibody protein provided herein comprises the nucleic acid sequence of SEQ ID NO: 36.

In some aspects, the anti-CD20 antibody proteins provided herein bind to the same epitope as TG-1101 (TG Therapeutics, Inc.).

In some aspects, the anti-CD20 antibody proteins provided herein are chimeric immunoglobulin G1 (IgG1) anti-CD20 monoclonal antibody proteins, each of which is assembled as a tetramer of two light chains (213 amino acids) and two heavy chains (448 amino acids).

In some aspects, the anti-CD20 antibody protein provided herein is represented by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, and comprises pyroglutamic acid instead of glutamic acid at position 1 of the light chain and/or the heavy chain, thereby obtaining a heavy chain of the amino acid sequence of SEQ ID NO: 13 and/or a light chain of the amino acid sequence of SEQ ID NO: 14.

In some aspects, the anti-CD20 antibody protein provided herein is represented by one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, and comprises a deletion at the C-terminal lysine of the heavy chain, thereby generating the amino acid sequence of SEQ ID NO: 15.

In some embodiments, provided herein are compositions or populations of anti-CD20 antibody proteins, wherein at least 50%, 60%, 70%, 80%, 90%, 95% or 98% comprise (i) pyroglutamine (instead of glutamine) at position 1 of the heavy chain, (ii) pyroglutamine (instead of glutamine) at position 1 of the light chain, and/or (iii) a deletion of the heavy chain C-terminal lysine.

In some embodiments, the anti-CD20 antibody is represented by one or more nucleic acid sequences encoding a light chain comprising an amino acid sequence of SEQ ID NO: 16. 7.4 Anti-CD20 Antibody Compositions

The anti-CD20 antibody compositions provided herein can be described by various post-translational modifications and/or by their three-dimensional configuration (see Section 7.5(e)). The corresponding amounts of various post-translational modifications can be quantified as described in Section 7.5. Without being limited by theory, these structural properties of the anti-CD20 antibody compositions provided herein lead to the biological and clinical properties described in Sections 7.7 and 7.9 below.

Anti-CD20 antibody compositions produced in vitro have various post-translational modifications. It should be understood that each individual anti-CD20 antibody protein may have its own unique post-translational modification pattern. In order to describe the properties of a group of multiple anti-CD20 antibody proteins, the overall presence of a specific post-translational modification can be quantified. Without being limited by theory, the extent of a specific post-translational modification in a group of anti-CD20 proteins can determine the biological and clinical properties of the composition (such as the dosage of a pharmaceutical formulation). Without being limited by theory, post-translational modifications are achieved by expression in rat fusion tumor cells (e.g., YB2/0 cells) in cell culture.

In some embodiments, the type of post-translational modification that can be used to describe the anti-CD20 antibody compositions provided herein is glycosylation. There are many known glycosylation. In one aspect, the glycosylation is N-glycosylation. The N-glycans that may be present may be any of the N-glycans shown in Figure 1. The N-glycosylation content is discussed below in Section 7.4 (a) and can be quantified using the analysis in Section 7.5.

In some embodiments, the type of post-translational modification that can be used to describe the anti-CD20 antibody compositions provided herein is deamination. Deamination is a chemical reaction in which an amide functional group in the side chain of the amino acids aspartic acid or glutamic acid is removed or converted to another functional group. Typically, aspartic acid is converted to aspartic acid or isoaspartic acid. The degree of deamination at a particular amino acid position in the anti-CD20 antibody compositions provided herein is described in Section 7.4(b) below and can be determined as described in Section 7.5 herein. (a)  N-Glycosylation

Various forms of N-glycosylation can be present in the anti-CD20 antibody compositions provided herein. Without being limited by theory, the relative distribution of various N-glycans of individual anti-CD20 antibody proteins or individual sugar residues present in those N-glycans in a population of anti-CD20 antibody proteins can determine the biological and clinical properties of the anti-CD20 antibody compositions provided herein (such as the biological and clinical properties discussed in Sections 7.7 and 7.9). The anti-CD20 antibody compositions provided herein can be described by any one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or all of the N-glycans or individual sugar residues described in the following subsections.

In some embodiments, the anti-CD20 antibody composition provided herein comprises at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14 or 15 N-glycans in the following relative abundance ranges: (a) 0.3% to 2% G0-GN; (b) 0.1% to 2% G0F-GN; (c) 30% to 60% G0; (d) 0.1% to 1% G1-GN; (e) 5% to 20% G0B; (f) 5% to 30% G0F; (g) 0.1% to 1.5% Man5; (h) 1% to 15% G0FB; (i) 1% to 13% G1; (j) 0.5% to 10% G1'; (k) 0.5% to 6% G1B; (l) 0.5% to 12% G1F; (m) 0.1% to 3% G1F’; (n) 0.1% to 3% G1FB; (o) 0.1% to 2% G2; and (p) 0.1% to 2% G2F.

In some embodiments, the anti-CD20 antibody composition provided herein comprises at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14 or 15 N-glycans in the following relative abundance ranges: (a) 0.8% to 1.1% G0-GN; (b) 0.5% to 1.1% G0F-GN; (c) 42.5% to 48.8% G0; (d) 0.3% to 0.6% G1-GN; (e) 9.5% to 14.1% G0B; (f) 12.8% to 19.7% G0F; (g) 0.4% to 0.7% Man5; (h) 5.1% to 7.0% G0FB; (i) 5.7% to 6.4% G1; (j) 2.7% to 3.3% G1’; (k) 1.4% to 2.0% G1B; (l) 2.6% to 4.2% G1F; (m) 1.1% to 1.6% G1F’; (n) 1.1% to 1.8% G1FB; (o) 0.5% to 0.7% G2; and (p) 0.3% to 0.5% G2F.

In some specific examples, the anti-CD20 antibody composition provided herein comprises at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14 or 15 N-glycans within the following relative abundance ranges: (a) 0.9% G0-GN; (b) 0.8% G0F-GN; (c) 46.1% G0; (d) 0.5% G1-GN; (e) 10.9% G0B; (f) 17.0% G0F; (g) 0.6% Man5; (h) 6.0% G0FB; (i) 6.1% G1; (j) 2.9% G1'; (k) 1.6% G1B; (l) 3.2% G1F; (m) 1.3% G1F’; (n) 1.3 G1FB; (o) 0.5% G2; and (p) 0.3% G2F.

In some embodiments, the anti-CD20 antibody protein of the population comprises an N-glycan profile comprising a relative abundance of about 0.3% to about 2% GO-GN, about 0.8% to about 1.1% GO-GN, or about 0.9% GO-GN. In some embodiments, the anti-CD20 antibody protein of the population comprises an N-glycan profile comprising a relative abundance of about 0.1% to about 2% GOF-GN, about 0.5% to about 1.1% GOF-GN, or about 0.8% GOF-GN. In some embodiments, the anti-CD20 antibody protein of the population comprises an N-glycan profile comprising a relative abundance of about 30% to 60% G0, about 42.5% to 48.8% G0, or about 46.1% G0. In some embodiments, the anti-CD20 antibody protein of the population comprises an N-glycan profile comprising a relative abundance of about 0.1% to about 1% G1-GN, about 0.3% to about 0.6% G1-GN, or about 0.5% G1-GN. In some embodiments, the anti-CD20 antibody protein of the population comprises an N-glycan profile comprising a relative abundance of about 5% to about 20% GOB, about 5% to about 15% GOB, about 9.5% to about 14.1% GOB, about 10.9% GOB, or about 10% GOB. In some embodiments, the anti-CD20 antibody protein of the population comprises an N-glycan profile comprising a relative abundance of about 5% to about 30% GOB, about 12.8% to about 19.7% GOB, or about 17.0% GOB. In some embodiments, the anti-CD20 antibody protein of the population comprises an N-glycan profile comprising a relative abundance of about 0.1% to about 1.5% Man5, about 0.4% to about 0.7% Man5, or about 0.6% Man5. In some embodiments, Man5 is the only high mannose N-glycan in the N-glycan profile. In some embodiments, the anti-CD20 antibody protein of the population comprises an N-glycan profile comprising a relative abundance of about 1% to about 15% GOFB, about 5.1% to about 7.0% GOFB, or about 6.0% GOFB. In some embodiments, the anti-CD20 antibody proteins of the population comprise an N-glycan profile comprising a relative abundance of about 1% to about 13% G1, about 5.7% to about 6.4% G1, or about 6.1% G1. In some embodiments, the anti-CD20 antibody proteins of the population comprise an N-glycan profile comprising a relative abundance of about 0.5% to about 10% G1', about 2.7% to about 3.3% G1', or about 2.9% G1'. In some embodiments, the anti-CD20 antibody proteins of the population comprise an N-glycan profile comprising a relative abundance of about 0.5% to about 6% G1B, about 1.4% to about 2.0% G1B, or about 1.6% G1B. In some embodiments, the anti-CD20 antibody protein of the population comprises an N-glycan profile comprising a relative abundance of about 0.5% to about 12% G1F, about 2.6% to about 4.2% G1F, or about 3.2% G1F. In some embodiments, the anti-CD20 antibody protein of the population comprises an N-glycan profile comprising a relative abundance of about 0.1% to about 3% G1F', about 1.1% to about 1.6% G1F', or about 1.3% G1F'. In some embodiments, the anti-CD20 antibody protein of the population comprises an N-glycan profile comprising a relative abundance of about 0.1% to about 3% G1FB, about 1.1% to about 1.8% G1FB, or about 1.3% G1FB. In certain embodiments, the anti-CD20 antibody protein of the population comprises an N-glycan profile comprising a relative abundance of about 0.1% to about 2% G2, about 0.5% to about 0.7% G2, or about 0.5% G2. In some embodiments, the anti-CD20 antibody protein of the population comprises an N-glycan profile comprising a relative abundance of about 0.1% to about 2% G2F, about 0.3% to about 0.5% G2F, or about 0.3% G2F.

In some embodiments, the anti-CD20 antibody protein comprises an N-glycan profile comprising about 0.3% to about 2% GO-GN, about 0.1% to about 2% GOF-GN, about 30% to 60% GO, about 0.1% to about 1% G1-GN, about 5% to about 20% G0B, about 5% to about 30% GOF, about 0.1% to about 1.5% Man5, about 1% to about 15% GOFB, about 1% to about 13% G1, about 0.5% to about 10% G1', about 0.5% to about 6% G1B, about 0.5% to about 12% G1F, about 0.1% to about 3% G1F', about 0.1% to about 3% G1FB, about 0.1% to about 2% G2, and about 0.1% to about 2% In some embodiments, Man5 is the only high mannose N-glycan in the N-glycan repertoire.

In some embodiments, the anti-CD20 antibody protein comprises an N-glycan profile comprising about 0.8% to about 1.1% GO-GN, about 0.5% to about 1.1% GOF-GN, about 42.5% to 48.8% GO, about 0.3% to about 0.6% G1-GN, about 9.5% to about 14.1% G0B, about 12.8% to about 19.7% GOF, about 0.4% to about 0.7% Man5, about 5.1% to about 7.0% GOFB, about 5.7% to about 6.4% G1, about 2.7% to about 3.3% G1', about 1.4% to about 2.0% G1B, about 2.6% to about 4.2% G1F, about 1.1% to about 1.6% G1F', about 1.1% to about 1.8% In some embodiments, Man5 is the only high mannose N-glycan in the N-glycan repertoire.

In some embodiments, the anti-CD20 antibody protein comprises an N-glycan profile comprising about 0.9% GO-GN, about 0.8% GOF-GN, about 46.1% GO, about 0.5% G1-GN, about 10.9% G0B, about 17.0% GOF, about 0.6% Man5, about 6.0% GOFB, about 6.1% G1, about 2.9% G1', about 1.6% G1B, about 3.2% G1F, about 1.3% G1F', about 1.3% G1FB, about 0.5% G2, and about 0.3% G2F in relative abundance. In some embodiments, Man5 is the only high mannose N-glycan in the N-glycan profile.

In some embodiments, the anti-CD20 antibody proteins comprise an N-glycan profile comprising a relative abundance ratio of G1 to G0 N-glycans of about 0.1 to about 0.15. In some embodiments, the anti-CD20 antibody proteins comprise an N-glycan profile comprising a relative abundance ratio of G1F to G1 N-glycans of about 0.5 to about 0.9.

In some embodiments, the anti-CD20 antibody composition has an N-glycan profile as shown in FIG . 2 .

Specific ranges and values for various N-glycans are provided below. Compositions or populations of anti-CD20 antibody proteins provided herein can be described by any one of these N-glycan prevalences or by any one population or by all of these N-glycans. (i)   Galactosylation

In some specific examples, the anti-CD20 antibody compositions provided herein contain between 10 and 20% galactosylated glycans. Galactosylated glycans are those N-glycans with galactose residues (as shown by the hollow circles in FIG. 1 ) shown in FIG . 1 . Assays for determining the percentage of galactosylation (or galactosylated N-glycans) are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody proteins is enzymatically deglycosylated so that all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of galactosylated N-glycans is the percentage of galactosylated N-glycans in N-glycans cleaved using enzymatic digestion.

In some embodiments, the anti-CD20 antibody compositions provided herein contain between at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, or at least 18%, and at most 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or at most 20% galactosylated polysaccharides. In some embodiments, the anti-CD20 antibody compositions provided herein contain between 11% and 19%, 12% and 18%, 13% and 17%, or 14% and 16% galactosylated polysaccharides. For example, the anti-CD20 antibody compositions provided herein may contain about 17% galactosylated polysaccharides (where "about" means +/- 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%). (ii)  Fucosylation

In some embodiments, the anti-CD20 antibody compositions provided herein contain between 20% to 40% fucosylated glycans; between 23% to 36% fucosylated glycans; between 20% to 35% fucosylated glycans; between 28% to 33% fucosylated glycans; or about 33% fucosylated glycans (where "about" means +/- 1, 2, 3, 4, 5, 6, 7, 8 , 9, or 10%). Fucosylated glycans are those N-glycans with fucosylated residues shown in FIG1 (as indicated by the open triangles in FIG1). Assays for determining the percentage of fucosylation (or fucosylated N-glycans) are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is enzymatically deglycosylated to cleave all N-glycans from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of fucosylated N-glycans is the percentage of fucosylated N-glycans in the N-glycans cleaved using enzymatic digestion.

In some embodiments, the anti-CD20 antibody compositions provided herein contain between at least 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, or at least 33%, and at most 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 39%, 40%, 41%, or at most 42% fucosylated polysaccharides. (iii) Galactosylation to fucosylation ratio

In some embodiments, provided anti-CD20 antibody compositions are characterized by a specific ratio of total fucosylated glycans to total galactosylated glycans (or "fucose/galactose ratio"). This fucose/galactose ratio can be between 1.5 and 2.1; between 1.5 and 2; between 1.5 and 1.9; between 1.5 and 1.8; between 1.6 and 2.1; between 1.7 and 2.1; between 1.8 and 2.8; between 1.6 and 2.0; between 1.7 and 1.9; between 1.6 and 1.8; or the fucose/galactose ratio can be about 1.75 (where "about" means +/- 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%). Fucosylated glycans are those N-glycans shown in FIG . 1 with a fucose residue (as indicated by the open triangles in FIG. 1 ). Galactosylated glycans are those N-glycans shown in FIG . 1 with a galactose residue (as indicated by the open circles in FIG. 1 ). Assays for determining the percentage of fucosylation (or fucosylated N-glycans) are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is enzymatically deglycosylated so that all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of fucosylated N-glycans is the percentage of fucosylated N-glycans in the N-glycans cleaved using enzymatic digestion. (iv) Bisecting N-glycans

In some embodiments, the anti-CD20 antibody compositions provided herein comprise at least 10%, 15%, 20%, 25%, or at least 30% bisecting N-glycans; between 10% to 30%, between 12% to 30%, between 12% and 25%, between 12% and 20%, between 15% and 30%, between 15% and 25%, between 15% and 20%, between 18% and 30%, or between 18% and 25% bisecting N-glycans. Bisecting N-glycans are those N-glycans shown in FIG1 , whose third GlcNAc is attached to the mannose residue closest to the protein backbone (as shown by the open triangles in FIG1 ). Assays for determining the percentage of bisecting N-glycans are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is enzymatically deglycosylated to cleave all N-glycans from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of fucosylated N-glycans is the percentage of bisected N-glycans among the N-glycans cleaved using enzymatic digestion. (v) Sialylation

In some embodiments, the anti-CD20 antibody compositions provided herein contain less than 10%, 8%, 5%, 4%, 3%, 2.5%, 2%, 1% or 0.5% sialylated glycans. In some embodiments, the anti-CD20 antibody compositions provided herein contain between 10% and 0.5% sialylated glycans; between 10% and 5% sialylated glycans; between 5% and 0.5% sialylated glycans; between 4% and 0.5% sialylated glycans; between 2% and 0.5% sialylated glycans; or no detectable amount of sialylated glycans. Assays for determining the percentage of sialylation (or sialylated N-glycans) are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is enzymatically deglycosylated to cleave all N-glycans from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of sialylated N-glycans is the percentage of sialylated N-glycans in the N-glycans cleaved using enzymatic digestion.

In some embodiments, the anti-CD20 antibody compositions provided herein contain between at least undetectable amounts, 0.5%, 1%, 2%, 3%, 4%, or at least 5%, and at most 0.5%, 1%, 2%, 3%, 4%, 5%, or at most 10% sialylated glycans. In some embodiments, the anti-CD20 antibody compositions provided herein do not contain detectable amounts of sialylated glycans. (vi) G0B N-glycans

In some embodiments, the anti-CD20 antibody compositions provided herein include between 5% and 15% G0B N-glycans; between 9% and 11% G0B N-glycans; or about 10% G0B N-glycans (where "about" means +/- 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%). G0B N-glycans are shown in Figure 1. Assays for determining the percentage of G0B N-glycans are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody proteins is enzymatically deglycosylated so that all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of G0B N-glycans is the percentage of G0B N-glycans in the N-glycans cleaved using enzymatic digestion.

In some embodiments, the anti-CD20 antibody compositions provided herein contain between at least 5%, 6%, 7%, 8%, 9%, 10% or at least 11%, and at most 7%, 8%, 9%, 10%, 11%, 12% or at most 13% G0B N-glycans. (vii)       Man5 N-glycans

In some embodiments, the anti-CD20 antibody compositions provided herein comprise between 0.1% and 1.5% Man5 N-glycans; between 0.4% and 0.7% G0B N-glycans; or about 0.6% Man5 N-glycans (where "about" means +/- 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%). Man5 N-glycans are shown in FIG1 . Assays for determining the percentage of Man5 N-glycans are described in Section 7.5 . Briefly, a sample or population of anti-CD20 antibody proteins is enzymatically deglycosylated so that all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of Man5 N-glycans is the percentage of Man5 N-glycans in the N-glycans cleaved using enzymatic digestion.

In some embodiments, the anti-CD20 antibody compositions provided herein comprise between at least 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or at least 0.7%, and at most 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, or at most 0.9% Man5 N-glycans.

In some instances, Man5 N-glycans are the only high-mannose species in the N-glycan spectrum. (viii)      G0 N-glycans

In some embodiments, the anti-CD20 antibody compositions provided herein comprise between about 42% and about 52.8% G0 N-glycans (where "about" means +/- 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%). G0 N-glycans are shown in FIG1 . Assays for determining the percentage of G0 N-glycans are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody proteins is enzymatically deglycosylated so that all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of G0 N-glycans is the percentage of G0 N-glycans in the N-glycans cleaved using enzymatic digestion.

In some embodiments, the anti-CD20 antibody compositions provided herein contain between at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47% or at least 48%, and at most 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 52.8%, 53%, 54%, 55%, 56%, 57% or at most 58% G0 N-glycans. (ix) Ratio of G1 to G0 N-glycans

In some embodiments, anti-CD20 antibody compositions are provided that are characterized by a specified abundance ratio of G1 to G0-glycans. The abundance ratio of G1 to G0 N-glycans may be between 0.02 and 0.3; between 0.05 and 0.25; between 0.08 and 0.22; between 0.09 and 0.2; between 0.1 and 0.19; between 0.1 and 0.18; between 0.1 and 0.17; between 0.1 and 0.16; or the abundance ratio of G1 to G0 N-glycans may be between 0.1 and 0.15. G1 and G1 N-glycans are shown in FIG1 . Assays for determining the percentage of G1 or G0 glycans are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody protein is enzymatically deglycosylated to cleave all N-glycans from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of G1 or G0 glycans is the percentage of G1 or G0 glycans in the N-glycans cleaved using enzymatic digestion, respectively. (x) G1F to G1 N-glycan ratio

In some embodiments, anti-CD20 antibody compositions are provided that are characterized by a specified abundance ratio of G1F to G1 N-glycans. The abundance ratio of G1F to G1 N-glycans may be between 0.1 and 1.2; between 0.2 and 1.1; between 0.3 and 1; between 0.4 and 1; between 0.5 and 1; or the abundance ratio of G1 to G0 N-glycans may be between 0.5 and 0.9. G1F and G1 N-glycans are shown in Figure 1. Analytical methods for determining the percentage of G1F or G1 glycans are described in Section 7.5. Briefly, a sample or population of anti-CD20 antibody proteins is enzymatically deglycosylated so that all N-glycans are cleaved from the core. The resulting N-glycans can then be analyzed, for example, by mass spectrometry. The percentage of G1F or G1 glycans is the percentage of G1F or G1 glycans in the N-glycans cleaved by enzymatic digestion. (b) Deamination

In some embodiments, the anti-CD20 antibody proteins described herein comprise asparagine deamination at one or more asparagine residues present in the heavy chain. In some embodiments, the one or more deaminated aspartic acid residues present in the heavy chain are selected from Asn-33 (as shown in SEQ ID NO: 18), Asn-55 (as shown in SEQ ID NO: 19), Asn-61 (as shown in SEQ ID NO: 19), Asn-160 (as shown in SEQ ID NO: 22), Asn-202 (as shown in SEQ ID NO: 22), Asn-204 (as shown in SEQ ID NO: 22), Asn-277 (as shown in SEQ ID NO: 25), Asn-287 (as shown in SEQ ID NO: 25), Asn-362 (as shown in SEQ ID NO: 27), or Asn-385 (as shown in SEQ ID NO: 28). In some embodiments, the anti-CD20 antibody proteins described herein comprise asparagine deamination at one or more asparagine residues present in the light chain. In some embodiments, the one or more deaminated asparagine residues present in the light chain are selected from Asn-136 (as shown in SEQ ID NO: 31), Asn-137 (as shown in SEQ ID NO: 31), Asn-151 (as shown in SEQ ID NO: 32), or Asn-157 (as shown in SEQ ID NO: 32). (c) Oxidation

In some embodiments, the anti-CD20 antibody proteins comprise methionine oxidation at one or more methionine residues present in the heavy chain. In some embodiments, the one or more methionine residues present in the heavy chain are selected from Met-20, Met-34, Met-81, Met-253 or Met-428 as shown in SEQ ID NO: 17, 18, 21, 24 or 29, respectively. In some embodiments, the anti-CD20 antibody proteins comprise methionine oxidation at one or more methionine residues present in the light chain. In some embodiments, the one or more methionine residues present in the light chain are selected from Met-21 or Met-32 as shown in SEQ ID NO: 30. (d) Pyroglutamination

In some embodiments, the anti-CD20 antibody protein comprises pyroglutamination at an N-terminal glutamate residue in the heavy chain or the light chain. In some embodiments, the pyroglutamate at the N-terminal glutamate residue is present in the heavy chain, for example, as shown in pGlu-1 as SEQ ID NO: 13. In some embodiments, the pyroglutamate at the N-terminal glutamate residue is present in the light chain, for example, as shown in pGlu-1 as SEQ ID NO: 14. In some embodiments, the glutamate at position 1 of the heavy chain is pyroglutamate and the glutamate at position 1 of the light chain is pyroglutamate. (e) Lysine truncation

In some embodiments, the anti-CD20 antibody protein comprises a deletion of a C-terminal lysine amino acid residue present in the heavy chain or the light chain. In some embodiments, the C-terminal lysine amino acid residue of the heavy chain of the anti-CD20 antibody in the group is truncated. In some embodiments, the anti-CD20 antibody protein comprises a deletion of a C-terminal lysine at the heavy chain. In some embodiments, the heavy chain of the anti-CD20 antibody protein comprises the amino acid sequence of SEQ ID NO: 15. (f)   Configuration

The three-dimensional conformation or folding of the protein can be determined using the analysis described in Section 7.5(e).

In some embodiments, the composition or population of anti-CD20 antibody proteins provided herein (e.g., represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2) has a specified three-dimensional folding pattern or configuration. In some embodiments, the population of anti-CD20 antibody proteins comprises two or more of the following secondary structures as determined by circularly polarized dichroism (CD) spectroscopy at 205 nm to 260 nm: α-helices in the range of 3.0% to 15.0%; antiparallel β-sheets in the range of 25.0% to 40.0%; parallel β-sheets in the range of 0.5% to 12.0%; β-turns in the range of 10.0% to 25.0%; and random coils in the range of 30.0% to 42.0%.

In some embodiments, the population of anti-CD20 antibody proteins comprises two or more of the following secondary structures as determined by circular dichroism (CD) spectroscopy at 205 nm to 260 nm: α-helix in the range of 8.0% to 10.0%; antiparallel β-pleated sheets in the range of 32.0% to 36.0%; parallel β-pleated sheets in the range of 5.0% to 6.0%; β-turns in the range of 16.0% to 18.0%; and random coils in the range of 35.0% to 36.0%.

In some embodiments, the anti-CD20 antibody protein comprises two or more of the following secondary structures as determined by circular dichroism (CD) spectroscopy at 205 nm to 260 nm: about 9.0% α-helices; about 33.0% antiparallel β-sheets; about 5.6% parallel β-sheets; about 17.5% β-turns; and about 35.2% random coils, wherein the term "about" means ±5%. 7.5 Analysis of Quantitative Post-Translational Modifications

In some embodiments, the present invention provides a method for determining the amount of post-translational modification in a population of anti-CD20 antibody proteins. In some embodiments, the post-translational modification is selected from aspartate deamination, methionine oxidation, glycosylation, pyroglutamate formation, and lysine truncation. (a) Universal digestion

In some embodiments, the method for determining the amount of post-translational modification in a group of anti-CD20 antibody proteins comprises a step of digesting the group of anti-CD20 antibody proteins with an endoproteinase. In some embodiments, the group of anti-CD20 antibody proteins is reduced before digestion. In some embodiments, the group of anti-CD20 antibody proteins is alkylated before digestion. In some embodiments, the endoproteinase is selected from Asp-N, Lys-C or trypsin. In some embodiments, the digestion step is performed at 37°C for at least 8 hours, at least 12 hours, at least 16 hours, at least 20 hours, overnight, and/or less than 24 hours. In some embodiments, Asp-N or Lys-C is used to digest the group of anti-CD20 antibody proteins at 37°C overnight. In some embodiments, the anti-CD20 antibody protein is digested with trypsin at a ratio of 50:1 (w:w) at 37°C overnight. In some embodiments, the digested anti-CD20 antibody protein is purified from digestion reaction components and/or undigested anti-CD20 antibody protein. (b) Method for determining N-glycosylation

Illustrative analyses and their results are discussed in the examples in Sections 8.1 and 8.2 below.

In some embodiments, the method comprises a step of deglycosylation of a population of anti-CD20 antibody proteins to produce released N-glycans for labeling. In some embodiments, deglycosylation comprises cleaving glycosidic bonds between one or more or all N-glycans of a population of anti-CD20 antibody proteins. In some embodiments, deglycosylation comprises releasing some or most or substantially all N-glycans from a population of anti-CD20 antibody proteins. In some embodiments, deglycosylation releases greater than 50 percent, greater than 60 percent, greater than 70 percent, greater than 80 percent, greater than 90 percent, greater than 95 percent, greater than 97 percent, greater than 98 percent, greater than 99 percent, or 100 percent of N-glycans present on a population of anti-CD20 antibody proteins.

In some embodiments, deglycosylation comprises contacting the anti-CD20 antibody protein with one or more deglycosylation reagents that cleave N-glycans or N-linked oligosaccharides. In some embodiments, the deglycosylation reagent is a deglycosylation enzyme or chemical reagent. In some embodiments, the deglycosylation enzyme is PNGase F.

In some embodiments, deglycosylation comprises contacting the population of anti-CD20 antibody proteins with one or more deglycosylation enzymes or chemicals at a deglycosylation temperature of about 25°C to about 50°C, about 37°C to about 50°C, about 25°C, about 37°C, about 42°C, or about 50°C. In some embodiments, the deglycosylation temperature is 37°C. The deglycosylation rate can be increased by increasing the deglycosylation temperature. In some embodiments, deglycosylation comprises contacting a population of anti-CD20 antibody proteins with one or more deglycosylation enzymes or chemicals for a period of time sufficient to release some, most, or substantially all N-glycans from the population of anti-CD20 antibody proteins. In some embodiments, the deglycosylation comprises contacting a population of anti-CD20 antibody proteins with the one or more deglycosylation enzymes or chemicals for at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 20 hours, at least 24 hours, at least 30 hours, and/or less than 48 hours. In some embodiments, the deglycosylation comprises contacting a population of anti-CD20 antibody proteins with the one or more deglycosylation enzymes or chemicals for a period of about 30 minutes to about 2 hours, about 2 hours to about 4 hours, about 4 hours to about 8 hours, about 8 hours to about 12 hours, about 12 hours to about 20 hours, about 20 to about 30 hours, greater than about 30 hours, and/or less than 48 hours. In some embodiments, deglycosylation comprises contacting a population of anti-CD20 antibody proteins with one or more deglycosylation enzymes or chemicals for about 12 to about 20 hours. In some embodiments, deglycosylation comprises contacting a population of anti-CD20 antibody proteins with PNGase F at about 37° C. for about 12 to about 20 hours. In some embodiments, deglycosylation comprises contacting the anti-CD20 antibodies with PNGase F at 37° C. for a period of 12 to 20 hours. In some embodiments, deglycosylation using PNGase F occurs in the presence of a non-ionic detergent (i.e., NP-40).

In some embodiments, deglycosylation comprises denaturing the group of anti-CD20 antibody proteins before contacting the group of anti-CD20 antibody proteins with the one or more deglycosylation enzymes or chemicals. In some embodiments, denaturation comprises heat denaturation, chemical denaturation, or a combination of the two. In some embodiments, heat denaturation comprises incubating the group of anti-CD20 antibody proteins at a denaturation temperature and denaturation time sufficient to unfold some or most or all of the immunoglobulin fold domains of the anti-CD20 antibody proteins of the group. In some embodiments, the denaturation temperature is at least 50°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C, and/or less than about 100°C. In some embodiments, the denaturation temperature is about 50° C. to about 60° C., about 60° C. to about 70° C., about 70° C. to about 80° C., about 80° C. to about 90° C., or 90° C. to about 100° C. In some embodiments, the denaturation temperature is about 50° C., about 60° C., about 65° C., about 70° C., about 75° C., about 80° C., about 85° C., about 90° C., or about 100° C. In some embodiments, the denaturation temperature is 70° C. In some embodiments, the chemical denaturation comprises incubating a population of anti-CD20 antibody proteins at a denaturation temperature of at least 25° C., at least 30° C., at least 37° C., or at an elevated temperature (i.e., a thermal denaturation temperature). In some embodiments, the denaturation time is at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 10 hours, or at least 24 hours. In some embodiments, the denaturation time is about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 10 hours, or about 24 hours. In some embodiments, the denaturation time is 10 minutes. In some embodiments, chemical denaturation comprises contacting the population of anti-CD20 antibody proteins with one or more chemical denaturants. In some embodiments, one or more chemical denaturants are selected from ionic detergents, non-ionic detergents, zwitterionic detergents, ion-type detergents or reducing agents. In some embodiments, the chemical denaturant is selected from sodium dodecyl sulfate (SDS), urea or dithiothreitol (DDT). The effect of certain chemical denaturants may interfere with deglycosylation (i.e., enzymatic deglycosylation) and can be offset in the solution. In some embodiments, the chemical denaturant is offset by adding a non-ionic detergent to the solution. In some embodiments, the chemical denaturant is SDS and the additional non-ionic detergent is NP-40. In some embodiments, denaturing a population of anti-CD20 antibody proteins comprises heat denaturing at 70° C. for 10 minutes.

In some embodiments, the method comprises purifying N-glycans released from a population of anti-CD20 antibody proteins from a deglycosylation reaction mixture. In some embodiments, the released N-glycans are substantially free of deglycosylation reagents and deglycosylated or unreacted anti-CD20 antibodies. In some embodiments, the released N-glycans are substantially free of salts and/or detergents. In some embodiments, the released N-glycans are purified by hydrophilic interaction. In some embodiments, the released N-glycans are purified by chromatography comprising a hydrophilic stationary phase and a reverse phase solvent. In some embodiments, the released N-glycans are purified by hydrophilic interaction liquid chromatography (HILIC). In some embodiments, the released N-glycans are purified by Waters HILIC MassPrep μElution plates according to the manufacturer's protocol. In some embodiments, the released N-glycans are purified to a purity comparable to that obtained when purified by Waters HILIC MassPrep μElution plates according to the manufacturer's protocol.

In some embodiments, the method comprises a step of labeling a population of N-glycans released by an anti-CD20 antibody protein, thereby generating labeled N-glycans for detection. In some embodiments, labeling the released N-glycans comprises chemical derivatization, such as providing a detectable charge, UV activity, or fluorescent characteristic to the released N-glycans. In some embodiments, labeling the released N-glycans comprises reductive amination, hydrazide labeling, methylation, Michael addition, or permethylation. In some embodiments, labeling the released N-glycans comprises contacting the released N-glycans with a label selected from 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA), 2-aminopyridine (PA), 2-aminonaphthalene trisulfonic acid (ANTS), or 1-aminopyrene-3,6,8-trisulfonic acid (APTS). In some embodiments, the label is 2-AB. In some embodiments, labeling the released N-glycans by reductive amination comprises using a reducing agent. In some embodiments, the reducing agent is selected from sodium cyanoborohydride or 2-methylpyridine borane. In some embodiments, the reducing agent is cyanoborohydride. In some embodiments, labeling the released N-glycans by reductive amination comprises contacting the released N-glycans with a label suitable for reductive amination and a reducing agent at a reaction temperature and reaction time sufficient for labeling to occur. In some embodiments, the reaction temperature is about 25°C to about 40°C, about 40°C to about 50°C, about 50°C to about 60°C, about 60°C to about 70°C, or about 70°C to about 80°C. In certain embodiments, the reaction temperature is about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, or about 75°C. In some embodiments, the reaction temperature is 65°C. In some embodiments, the reaction time is at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, or at least 6 hours. In some embodiments, the reaction time is about 30 minutes to about 1 hour, about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, or about 4 hours to about 6 hours. In some embodiments, the reaction time is about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 6 hours. In some embodiments, the reaction time is 3 hours. In some embodiments, labeling a population of anti-CD20 antibody protein released N-glycans comprises reductive amination using 2-AB and cyanoborohydride at 65° C. for 3 hours to produce labeled N-glycans.

In some embodiments, the method comprises purifying a population of labeled N-glycans of anti-CD20 antibody proteins from a labeling reaction mixture. In some embodiments, the labeled N-glycans are substantially free of unreacted labels. In some embodiments, the labeled N-glycans are substantially free of unreacted released N-glycans. In some embodiments, the labeled N-glycans are substantially free of reducing agents (i.e., cyanoborohydrides). In some embodiments, the labeled N-glycans are purified by hydrophilic interactions. In some embodiments, the labeled N-glycans are purified by chromatography comprising a hydrophilic stationary phase and a reverse phase solvent. In some embodiments, the labeled N-glycans are purified by hydrophilic interaction liquid chromatography (HILIC). In some embodiments, the labeled N-glycans are purified using a Waters HILIC MassPrep μElution plate according to the manufacturer's protocol. In some embodiments, the labeled N-glycans are purified to a purity comparable to that obtained when purified using a Waters HILIC MassPrep μElution plate according to the manufacturer's protocol.

In some embodiments, the method comprises the steps of separating a population of labeled N-glycans of anti-CD20 antibody proteins and analyzing the N-glycan profile of the population. In some embodiments, separating the labeled N-glycans comprises separation based on hydrophilicity. In some embodiments, separating the labeled N-glycans comprises chromatography comprising a hydrophilic stationary phase and a reverse phase solvent. In a specific embodiment, the chromatography is hydrophilic interaction liquid chromatography (HILIC). In some embodiments of the method, separating the labeled N-glycans comprises chromatography comprising an amide stationary phase. In some embodiments, the amide stationary phase is a polysaccharide BEH amide column. In certain embodiments, the reverse phase solvent comprises one or more mobile phases, wherein the mobile phase comprises an acidic ammonium buffer and/or acetonitrile.

In some embodiments, the analysis is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time period, wherein the combination of parameters is sufficient to separate labeled N-glycans. In some embodiments, the analysis is performed using a chromatographic system (i.e., HPLC or UPLC system) that controls these parameters. In some embodiments, the chromatographic system is coupled to a detector for detecting labeled N-glycans of a group of anti-CD20 antibody proteins. In some embodiments, the detector is a fluorescent detector that detects fluorescence from a label (e.g., a label that contacts a group of released N-glycans of an anti-CD20 antibody protein in a labeling step). In some embodiments, the label is 2-AB. In some embodiments, detecting fluorescence comprises exciting with 360 nm wavelength light and observing fluorescence emission under 428 nm wavelength light. In some embodiments, the chromatography system is a HILIC-UPLC system. In some embodiments, separation and detection of labeled N-glycans are performed using a hydrophilic interactive stationary phase, a reverse phase solvent, and a Waters UPLC equipped with a fluorescence detector. In some embodiments, the hydrophilic interactive stationary phase comprises a polysaccharide BEH amide column (130Å, 1.7 μm, 2.1 mm X 150 mm). In some embodiments, the stationary phase (i.e., column) temperature is 50°C. In some embodiments, the flow rate is 0.50 mL/min. In some embodiments, the reverse phase eluent comprises one or more mobile phases, the mobile phases comprising a first mobile phase and a second mobile phase, the first mobile phase comprising about 250 mM ammonium formate, pH about 4.4, and the second mobile phase comprising acetonitrile. In some embodiments, the mobile phase gradient comprises the first mobile phase increasing from 22.0% to 44.1% in 38.5 minutes.

In some embodiments, detecting fluorescence comprises generating a chromatogram, wherein the dependent variable is selected from the volume of the mobile phase, the volume of the eluate passing through the chromatographic column, or time, and what can be observed is fluorescence. In certain embodiments, determining the N-glycan profile of a population of anti-CD20 antibody proteins comprises quantifying the relative amounts of labeled G0-GN, G0F-GN, G0, G1-GN, G0B, G0F, Man5, G0FB, G1, G1', G1B, G1F, G1F', G1FB, G2, and G2F N-glycans in the population. In certain embodiments, the analysis (i.e., HILIC-UPLC) is performed using flow rate, column temperature, mobile phase gradient, and time period parameters sufficient to separate GO-GN, GOF-GN, GO, G1-GN, G0B, GOF, Man5, GOFB, G1, G1', G1B, G1F, G1F', G1FB, G2, and G2F N-glycans for quantification. In certain embodiments, the amount of N-glycans is quantified by calculating the area under the curve comprising labeled GO-GN, GOF-GN, GO, G1-GN, G0B, GOF, Man5, GOFB, G1, G1', G1B, G1F, G1F', G1FB, G2, and G2F N-glycans in the chromatogram separating the N-glycans. In some embodiments, the relative abundance of N-glycans selected from G0-GN, G0F-GN, G0, G1-GN, G0B, G0F, Man5, G0FB, G1, G1 ', G1B, G1F, G1F', G1FB, G2 and G2F is quantified by calculating the peak area percentage of the N-glycans relative to the total peak area of G0-GN, G0F-GN, G0, G1-GN, G0B, G0F, Man5, G0FB, G1, G1 ', G1B, G1F, G1F', G1FB, G2 and G2F N-glycans in the chromatogram from which the N-glycans are separated. In some embodiments, the peak area of the N-glycans in the chromatogram is greater than or equal to 0.25%. In some embodiments, the peak area of N-glycans in the chromatogram has a signal-to-noise ratio greater than or equal to 3.0.

The N-glycan profile of a population of anti-CD20 antibody proteins can be determined by liquid chromatography-mass spectrometry (LC-MS) peptide mapping to determine the relative amount of glycosylation in the population, for example by summing the site-specific glycosylation of the digested population of anti-CD20 antibody proteins.

In some embodiments, LC-MS peptide mapping includes determining the molecular weight of peptides derived from the anti-CD20 antibody protein group digested by Lys-C to determine the presence and quantity of glycosylated residues (i.e., G0, G0F, G0B, G0FB, G1 or G1F). In some embodiments, the anti-CD20 antibody protein group is reduced and alkylated prior to digestion. In certain embodiments, LC is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time period, wherein the combination of parameters is sufficient to separate the peptides of the Lys-C digest. In certain embodiments, the relative abundance of the peptides of the Lys-C digest is calculated by integrating the area under the curve thereof compared to the total area.

In some embodiments, the observed molecular weight is compared to the theoretical mass of a glycosylated peptide selected from column 2 of Table 1. In some embodiments, the observed molecular weight is compared to the theoretical mass selected from column 4 of Table 1. Table 1 : Peptides used to determine glycosylation after Lys-C digestion

In some embodiments, the anti-CD20 antibody protein comprises glycosylation at residue Asn-298 of SEQ ID NO: 33 present in the heavy chain. (c) Methods for determining deamination (i) General deamination

Also provided herein is a method for determining the amount of aspartic acid deamination in a population of anti-CD20 antibody proteins. In some embodiments, the amount of aspartic acid deamination in a population of anti-CD20 antibody proteins is the amount of isoaspartic acid residues in the population. In some embodiments, the method comprises detecting the amount of isoaspartic acid residues in the digested population of anti-CD20 antibody proteins. (ii) Deamination according to Isoquant

The amount of aspartate deamination or isoaspartate in a population of anti-CD20 antibody proteins or a population of digested anti-CD20 antibody proteins can be determined enzymatically, for example, by adding a methyltransferase, protein isoaspartate methyltransferase (PIMT), which catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to isoaspartate. The amount of S-adenosylhomocysteine (SAH) produced by this PIMT-catalyzed reaction is directly proportional to the amount of isoaspartate present in the sample (~1:1 stoichiometry). SAH can be directly measured by isolating SAH (i.e., by ultra-high performance liquid chromatography (UPLC) or reversed-phase high performance liquid chromatography (RP-HPLC)), detecting it at 260 nm and quantifying the amount of SAH by comparison with a SAH standard curve (i.e., Trp-Ala-Gly-Gly-isoAsp-Ala-Ser-Gly-Glu peptide).

In some specific examples, the method comprises a derivatization step, which comprises contacting the group of anti-CD20 antibody proteins with PIMT and SAM. In some specific examples, the group of anti-CD20 antibody proteins is contacted with PIMT and SAM at a temperature sufficient to derivatize (i.e., methylate) substantially all aspartic acid residues present in the group and for a period of time. In some specific examples, the method optionally comprises a step of quenching the derivatization reaction. In certain specific examples, the method comprises a step of separating SAH based on hydrophobicity to quantify isoaspartic acid. In certain specific examples, separating SAH comprises chromatography, which comprises a hydrophobic interaction stationary phase and a reverse phase elution liquid. In certain specific examples, separating SAH comprises chromatography using a C18 stationary phase. In certain specific examples, the C18 stationary phase is a Hydro-RP column. In some embodiments, the reverse phase solvent comprises one or more mobile phases (i.e., weakly acidic phosphate buffer and/or methanol). In some embodiments, the analysis is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time period, wherein the combination of parameters is sufficient to separate SAH from the derivatization reaction components. In some embodiments, the analysis is performed using a chromatography system (i.e., HPLC or UPLC system) that controls these parameters. In some embodiments, the chromatography system is coupled to a detector for detecting the derivatized SAH (i.e., absorbance at 260 nm). In some embodiments, separation and detection of SAM is performed using a hydrophilic interactive stationary phase, a reverse phase elution solution, and an RP-HPLC or UPLC system equipped with a detector for detecting the absorbance of SAH at about 260 nm. In some embodiments, the hydrophobic interactive stationary phase comprises Synergi Hydro-RP (4.6 mm x 150 mm). In some embodiments, the stationary phase (i.e., column) temperature is about 25°C or room temperature. In a specific embodiment, the flow rate is 1 mL/min. In some embodiments, the reverse phase elution solution comprises one or more mobile phases, the mobile phase comprising a first mobile phase and a second mobile phase, the first mobile phase comprising about 50 mM potassium phosphate, pH about 6.2, and the second mobile phase comprising methanol. In some embodiments, the mobile phase gradient comprises a second mobile phase having a second mobile phase of 10.0% at 0 minutes, 40% at 7.5 minutes, 80% at 10.5 minutes, 80% at 12.5 minutes, 10% at 13.5 minutes, 10% at 20 minutes, and 10% at 25 minutes. In some embodiments, the amount of SAH is quantified by comparing its integrated curve area with a SAH standard curve. In some embodiments, the SAH standard curve is prepared using Trp-Ala-Gly-Gly-isoAsp-Ala-Ser-Gly-Glu. In some embodiments, the amount of aspartate deamination or isoaspartate in a panel of anti-CD20 antibody proteins is determined using the Promega Isoquant kit and Isoasp-DSIP standards according to the manufacturer's protocol. (iii) Deamination by LC-MS

The amount of aspartic acid deamination or isoaspartic acid in a population of anti-CD20 antibody proteins or a population of digested anti-CD20 antibody proteins can be determined by a liquid chromatography-mass spectrometry (LC-MS) peptide mapping method, for example, by summing the site-specific deamination results of the population of digested anti-CD20 antibody proteins.

In some embodiments, LC-MS peptide mapping comprises determining the molecular weight and/or relative abundance of peptides derived from a population of anti-CD20 antibody proteins digested with Lys-C to determine the presence and quantity of deamidated or isoaspartic acid residues. In some embodiments, the population of anti-CD20 antibody proteins is reduced and alkylated prior to digestion. In certain embodiments, LC is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time period, wherein the combination of parameters is sufficient to separate peptides of Lys-C digests. In certain embodiments, the relative abundance of peptides of Lys-C digests is calculated by integrating the area under the curve thereof compared to the total area.

In some embodiments, the observed molecular weight is compared to the theoretical mass of a deamidated peptide selected from column 2 of Table 2. In some embodiments, the observed molecular weight is compared to the theoretical mass selected from column 3 of Table 2. Table 2 : Peptides used to determine deamidation after Lys-C digestion

In some embodiments, LC-MS peptide mapping comprises determining the molecular weight and/or relative abundance of peptides derived from a population of anti-CD20 antibody proteins digested with Asp-N to determine the presence and quantity of deamidated or isoaspartic acid residues. In some embodiments, the population of anti-CD20 antibody proteins is reduced and alkylated prior to digestion. In certain embodiments, LC is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time period, wherein the combination of parameters is sufficient to separate peptides of the Asp-N digest. In certain embodiments, the relative abundance of peptides of the Asp-N digest is calculated by integrating the area under the curve thereof compared to the total area.

In some embodiments, the observed molecular weight is compared to the theoretical mass of a deamidated peptide selected from column 2 of Table 3. In some embodiments, the observed molecular weight is compared to the theoretical mass selected from column 3 of Table 3. Table 3 : Peptides used to determine deamidation after Asn-N digestion (d) Methods for determining oxidation (i) General oxidation

Also provided herein is a method for determining the extent of methionine oxidation in a population of anti-CD20 antibody proteins. In some embodiments, the amount of methionine oxidation in a population of anti-CD20 antibody proteins is the amount of Met sulfoxide (MetO) residues in the population. In some embodiments, the method comprises detecting the amount of Met sulfoxide (MetO) residues in the population. (ii) Oxidation by LC-MS

The amount of methionine oxidation or MetO residues in a population of anti-CD20 antibody proteins or a population of digested anti-CD20 antibody proteins can be determined by liquid chromatography-mass spectrometry (LC-MS) peptide mapping methods, for example, by summing the site-specific oxidation results of a population of digested CD20 antibody proteins.

In some embodiments, LC-MS peptide mapping comprises determining the molecular weight of peptides derived from a population of anti-CD20 antibody proteins digested with Lys-C to determine the presence and quantity of oxidation or MetO residues. In some embodiments, the population of anti-CD20 antibody proteins is reduced and alkylated prior to digestion. In some embodiments, LC is performed using parameters for flow rate, stationary phase (i.e., column) temperature, mobile phase gradient, and time period, wherein the combination of parameters is sufficient to separate peptides of Lys-C digests. In some embodiments, the relative abundance of peptides of Lys-C digests is calculated by integrating the area under the curve compared to the total area.

In some embodiments, the observed molecular weight is compared to the theoretical mass of an oxidized peptide selected from column 2 of Table 4. In some embodiments, the observed molecular weight is compared to the theoretical mass selected from column 3 of Table 4. Table 4 : Peptides used to measure oxidation after Lys-C digestion

In some embodiments, the anti-CD20 antibody protein comprises methionine oxidation at one or more methionine residues present in the heavy chain. In some embodiments, the one or more methionine residues present in the heavy chain are selected from Met-20, Met-34, Met-81, Met-253, or Met-428 as shown in SEQ ID NO: 17, 18, 21, 24 or 29, respectively. In some embodiments, the anti-CD20 antibody protein comprises methionine oxidation at one or more methionine residues present in the light chain. In some embodiments, the one or more methionine residues present in the light chain are selected from Met-21 or Met-32 as shown in SEQ ID NO: 30. (e) Analysis of protein conformation - circular dichroism

In some embodiments, circular dichroism (CD) spectroscopy is used to analyze the secondary structure of anti-CD20 antibody proteins by measuring the difference in absorption between left-circularly polarized light and right-circularly polarized light due to structural asymmetry. CD spectroscopy uses far-ultraviolet light between wavelengths of approximately 170 and 260 nm. At these wavelengths, different secondary structures commonly found in proteins can be analyzed, because alpha helices, parallel and antiparallel beta sheets, beta turns, and random coil conformations each produce a characteristic spectrum, and a given spectrum can be used to estimate its percentage of secondary structure. 7.6 Single Batch Composition

Provided herein are compositions or populations of scaled-up quantities of anti-CD20 antibody proteins described in Section 7.4. In certain embodiments, these scaled-up quantities are present in a single batch, i.e., a composition derived from a single run of a single bioreactor of a specified volume. For example, an anti-CD20 antibody protein obtained from a single run of a 15,000 L bioreactor can be referred to as a single batch. In certain embodiments, the capacity of the bioreactor is at least 100; 200; 300; 400; 500; 750; 1,000; 2,000; 3,000; 4,000; 5,000; 7,500; 10,000; 15,000; 20,000; or at least 25,000 L. In certain embodiments, the anti-CD20 antibody protein is present in such a single batch at a concentration of at least 10 mg/ml; 15 mg/mL; 20 mg/mL; 25 mg/mL; or at least 30 mg/ml as determined using the assay described in Section 7.6(a). In certain embodiments, the anti-CD20 antibody protein is present in such a single batch at a concentration of between 10 to 35 mg/ml; 10 to 30 mg/mL; 10 to 25 mg/mL; 10 to 20 mg/mL; 10 to 15 mg/mL; 15 to 35 mg/mL; 15 to 30 mg/mL; 15 to 25 mg/mL; 15 to 20 mg/mL; 20 to 35 mg/mL; 20 to 30 mg/mL; 20 to 25 mg/mL; 25 to 35 mg/mL; or 25 to 30 mg/mL as determined using the assay described in Section 7.6(a). In certain embodiments, the anti-CD20 antibody protein is present in such a single batch at a concentration of about 15 mg/ml; about 20 mg/mL; about 25 mg/ml; about 30 mg/mL; or about 35 mg/ml as determined using the assay described in Section 7.6(a) (where "about" means +/- 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%).

In some embodiments, the anti-CD20 antibody protein disclosed herein is produced on a commercial scale. In certain embodiments, the commercial scale is 10,000 to 25,000 L. (a) Total protein

In some embodiments, the amount of total protein is measured spectrophotometrically. In some embodiments, the amount of total protein is measured by absorbance at 280 nm.

In some specific examples, the following analytical procedure can be used to measure the amount of total protein. This method uses UV absorbance measurements at 280 nm and light scattering correction at 320 nm to determine the concentration of anti-CD20 antibody protein. The concentration calculation is based on the Beer-Lambert law. The extinction coefficient of the anti-CD20 antibody protein at 280 nm is 1.61 mL/mg/cm , as determined based on the amino acid composition. The extinction coefficient can be used. This method is applicable to in-process sample testing, release testing, and stability testing after protein A purification. Test samples are diluted to a target protein concentration of 0.4 mg/mL in triplicate using 0.9% sodium chloride by weight analysis. Saline is used as an instrument blank. Bovine serum albumin was used as a system suitability control and was tested before and after sample measurement. The test sample was loaded into a quartz cuvette with a 1 cm pathlength and the absorbance was measured at 280 and 320 nm. The test sample was tested in triplicate. In certain embodiments, the amount of total protein can be measured using the key materials and equipment listed in Table 5. In certain embodiments, the amount of total protein can be measured using the protein concentration system suitability standards listed in Table 6. Table 5 : Key Materials and Equipment Used in Protein Concentration Analysis UV = ultraviolet light; Vis = visible light Table 6 : Protein concentration system suitability criteria AU = absorbance unit; CV = coefficient of variation

In some embodiments, the anti-CD20 antibody protein provided herein has a total protein amount of 25.5-25.8 mg/mL. In certain embodiments, the population has a total protein amount of about 25.6 mg/mL. 7.7 Biological Properties

In some embodiments, the anti-CD20 antibody proteins provided herein have the biological properties described in detail in this section. In some embodiments, the biological properties can be measured using the assays described in Section 7.7(a). The biological properties of the anti-CD20 antibody protein compositions provided herein are described in Section 7.7(b). (a) Assays (i)   Cell-based antibody-dependent cytotoxicity (ADCC)

In some embodiments, the cell-based ADCC assay uses Raji cells as target cells. In some embodiments, Raji cells express CD20. In some embodiments, the cell-based ADCC assay uses effector cells selected from CD16 effector cells and primary NK cells. In some embodiments, the cell-based ADCC assay uses Raji cells as target cells and uses KILR CD16a effector cells as effector cells. In some embodiments, the cell-based ADCC assay uses Raji cells as target cells and uses primary NK cells as effector cells. In certain embodiments, the KILR CD16a effector cells are human CD8+ T lymphocytes derived from a single donor that have been engineered to express CD16/FcγRIII on their plasma membrane surface.

In some embodiments, the cell-based ADCC analysis uses target cell lysis as a readout. In some embodiments, the target cell lysis is quantified using CytoTox Glo ^™ (Promega). In some embodiments, the cell-based ADCC analysis shows the relative efficacy of the group relative to a commercial reference standard. In some embodiments, the cell-based ADCC analysis produces a dose response curve and an EC50 value. In some embodiments, the composition or population of the anti-CD20 antibody protein provided herein performs at 100% above the commercial reference standard RS-117808 in a cell-based ADCC using CD16 effector cells.

In some embodiments, ADCC activity can be measured using CD16 activity analysis. In some embodiments, CD16 activity analysis uses alternative readouts to assess ADCC activity. In some embodiments, CD16 activity analysis uses WIL2-S as a target cell. In some embodiments, the effector cell used is a stably transformed Jurkat cell strain expressing a chimeric molecule comprising an FcγRIIIa extracellular domain that is attached to a transmembrane domain and an intracellular domain of a mast cell/philosphere Fc receptor γ chain for IgE. In some embodiments, cells are combined and treated with serially diluted anti-CD20 antibodies in the presence of PMA (phorbol 12-myristate 13-acetate). In some embodiments, activation of effector cells induces the release of IL-2, which is measured by a commercial ELISA kit. In some embodiments, the potency is reported as a percentage relative to a reference standard.

In some embodiments, the population has a relative potency of 106% to 126% in a CD16 activity assay compared to a commercial reference standard. In some embodiments, the population has a relative potency of about 115% in a CD16 activity assay compared to a commercial reference standard. In some embodiments, the commercial reference standard is RS-117808.

In some embodiments, the cell-based ADCC assay uses the following assay procedure. An eight-point serial dilution of reference standards, QC reference controls, and test materials is used at a concentration range of 250.00 pg/mL to 0.04 pg/mL (250, 50, 16.7, 5.6, 1.9, 0.6, 0.2, 0.04 pg/mL). Two independent material preparations are prepared and analyzed on duplicate plates. Assay controls are prepared in triplicate and include: target cell alone control, target cell death control, effector cell alone control, and effector and target cell controls. KILR cells were obtained from Eurofins. They are from a master cell bank made from a unique human donor. The Master Cell Bank and Working Cell Bank system and qualification agreements allow suppliers to create thousands of vials in a reproducible manner, and TG maintains a secure supply of this critical reagent. Procedures such as the feed qualification process ensure that highly reproducible cells are used in each analysis. KILR cells are thawed and cultured at 1x10^6 cells/mL in medium supplemented with IL-2, left quiescent for at least 6 days before use, and not used more than 14 days after thawing. For the KILR ADCC assay, Raji cells, also managed through the Master Cell Bank and Working Cell Bank system, are seeded at 1x10 ⁵ cells/mL, and reference standards, internal analytical controls, and test sample dilutions are added to the Raji cells. KILR effector cells were seeded at ^5x105 cells/mL to achieve a final effector:target (E:T) ratio of 5:1. Cells were incubated at 36 ± 1°C, 5 ± 1% _CO2 for 18 to 22 hours. At the end of the incubation, CytoTox GLo™ preparation was added and the plates were incubated for 30 ± 10 minutes. Plates were read using a SpectraMax plate reader. SoftMax Pro was used to analyze the data using weighted nonlinear regression using a 4-parameter logical fit. The potency of the obtained data was evaluated relative to a reference standard using SoftMax Pro software. Representative reference standard and sample dose response curves are shown. Results are reported as a percentage of potency relative to a primary reference standard or its derivatives.

In some embodiments, the cell-based ADCC assay is used as a potency assay for batch release. In some embodiments, the cell-based ADCC assay is used as a potency assay for stability testing. In some embodiments, the cell-based ADCC assay is used as a potency assay for manufacturing quality control and process. In some embodiments, the cell-based ADCC assay is used for comparative studies (e.g., in research and development or clinical studies).

In some embodiments, the higher potency of the population in a cell-based ADCC assay is associated with a lower level of fucose in the N-glycan profile of the population. In some embodiments, the higher potency of the population in a cell-based ADCC assay is associated with a lower level of fucose in the N-glycan profile of the population. (ii) Antibody-dependent cellular phagocytosis (ADCP)

In some embodiments, antibody-dependent cellular phagocytosis (ADCP) activity can be measured using CD20-expressing Daudi cells as target cells (labeled with PKH26). In some embodiments, human monocytes are isolated from PBMCs and differentiated in vitro using GM-CSF to generate macrophages. In some embodiments, macrophages are cultured with PKH26-labeled target cells (previously incubated with serially diluted anti-CD20 antibody samples). In some embodiments, target cell phagocytosis is assessed by flow cytometry. In some embodiments, dose-response curves of test samples can be displayed along with calculated IC50. (iii) Complement-dependent cytotoxicity (CDC)

In some embodiments, the cell-based CDC assay uses Jeko-1 cells (as target cells) and a rabbit complement system. In some embodiments, the cell-based CDC assay uses Raji cells (as target cells) and a human complement system. In some embodiments, the cell-based CDC assay uses target cell lysis as a readout.

In some embodiments, the cell-based CDC assay uses the following assay procedure. A nine-point serial dilution of reference standards, QC reference controls, and test materials is used over a concentration range of 10,000 ng/mL to 10.42 ng/mL (10,000.00, 3333.33, 1666.67, 833.33, 416.67, 208.33, 104.17, 52.08, 10.42 ng/mL). Two independent preparations of material are prepared and analyzed in duplicate plates. Negative controls are prepared in triplicate and include target cell and complement controls as well as a separate target cell control set. In this assay, Jeko-1 cells (obtained from ATCC and maintained through a master bank system) are seeded at 3x10 ⁵ cells/mL and incubated for 60 to 90 minutes. Reference standards, internal analytical controls, and test samples diluted in duplicate in cell culture medium are added. Complements are then added to the wells and the plates are incubated at 36°C ± 1°C for approximately 2 hours, followed by 25 ± 5 minutes at room temperature. Control groups include target cells plus complement controls and target cells only controls to provide a baseline level of target cell viability during the assay. Cell Titer-Glo reagent is then added and incubated for an additional 30 ± 10 minutes at room temperature. At the end of the analysis, the plates were read using a SpectraMax M5 plate reader. SoftMax Pro was used to analyze the data using a 4-parameter logical fit with weighted nonlinear regression. The potency of the obtained data was assessed relative to a reference standard using Softmax Pro software. Results are reported as % potency relative to the reference standard. Representative reference standard and sample dose response curves are shown. (iv) CD20 binding activity

In some embodiments, the cell-based CD20 binding assay uses Jeko-1 cells and MSD analysis. In some embodiments, the cell-based CD20 binding assay generates a dose-dependent binding curve and an EC50 value.

In some embodiments, the cell-based CD20 binding assay uses the following assay procedure. An eight-point serial dilution of reference standards, QC reference controls, and test materials is prepared at a concentration range of 40,000.00 ug/mL to 0.23 ng/mL (40,000.00, 4,000.00, 1,000.00, 333.30, 111.10, 37.00, 4.60, 0.23 ng/mL). Two independent test material preparations are prepared for every 2 plates of test material evaluated. Assay controls included a no-cell control (reference standard/test material dilution + probe, cells omitted) and a cell-only control (cells + probe, reference standard/test material omitted). Jeko-1 cells were seeded at 3 x 10 ⁵ cells/mL in a final volume of 100 μL per well on MSD high-binding plates containing PBS and incubated at 35 to 37°C for 2 h ± 10 min. Unbound cells were removed by washing with PBS and the plates were blocked with 45% FBS. 50 μL of reference standard, QC reference control, or test material dilution was added and the plates were incubated at room temperature with shaking for 1 h ± 10 min. After incubation and three PBS washes, 50 μL of anti-human Fc detection antibody conjugated to STREP-SULFOTAG was added and incubated with shaking at room temperature for 1 hour ± 5 minutes. The plate was washed again with PBS and 150 μL of MesoScale reading buffer containing tripropylamine (TPA) was added as a co-reactant to generate light for electrochemical luminescence reading. The plate was read immediately on the MSD Reader using Workbench 4.0. The resulting data were evaluated using PLA software and analyzed using a restricted 4-parameter logic model to generate relative binding, 95% confidence intervals, and results relative to a reference standard. Binding activity results are reported as a percentage of potency relative to a reference standard. Relative potency test results were calculated from representative reference standard and test sample dose response curves. (v) FcγRIIIa 158V and FcγRIIIa 158F binding activity

In some embodiments, the FcγRIIIa binding assay uses surface plasmon resonance (SPR). In some embodiments, the FcγRIIIa binding assay produces a sensorgram showing dose-dependent binding, saturation, and dissociation. In some embodiments, the FcγRIIIa binding assay calculates the dissociation constant by association and dissociation rates and steady-state kinetics. In some embodiments, the binding affinity of the population to FcγRIIIa 158V is about one order of magnitude higher than the binding affinity of the population to FcγRIIIa 158F. In some embodiments, the binding affinity of the population to FcγRIIIa 158V is significantly higher than Rituxan. In some embodiments, the binding affinity of the population to FcγRIIIa 158F is significantly higher than Rituxan. In certain embodiments, the population has a significantly higher binding affinity to both FcγRIIIa 158V and FcγRIIIa 158F than Rituxan.

In some embodiments, the FcγRIIIa binding assay uses the following analytical procedure. FcγRIIIa 158V receptor (1.2 μg/ml) is immobilized on the chip surface using covalent amine coupling chemistry. Eight-point serial dilutions of reference standards, QC reference controls, and test materials are prepared at concentrations ranging from 2000 nM to 15.6 nM, with a dilution factor of 2. Independent replicates of sample dilutions are injected onto the chip and then regenerated between each cycle. Binding is measured in response units (RU). The kinetics of the binding reaction is determined by measuring the change in SPR due to the increase in mass close to the biosensor chip surface. The change in complex mass over time is visualized as a sensorgram. The equilibrium dissociation constant ( _KD ) and relative affinity of each sample for each receptor relative to the reference standard were determined. The rate of change of the SPR signal was analyzed using a 1:1 Langmuir model for the FcγRIIIa 158V variant to generate apparent rate constants for the association and dissociation phases of the reaction as well as the equilibrium dissociation constant. _KD was determined using the steady-state affinity of the FcγRIIIa 158F variant. The binding signal was exported to PLA to determine the relative binding response. The results are reported as % potency for each FcγRIIIa variant (158V and 158F) relative to the reference standard. (vi) C1q binding activity

In some embodiments, C1q binding activity can be measured using ELISA. In some embodiments, C1q binding activity can be measured using the following analytical procedure. Prepare seven-point serial dilutions of reference standards, QC reference controls, and test materials at concentrations ranging from 15.00 µg/mL to 0.12 µg/mL. Apply the reference standards, QC reference controls, and test material dilutions to ELISA plates and incubate the plates at room temperature for 1 hour ± 30 minutes (shaking 150 to 200 rpm). After coating, the plates are washed (3 times with PBS/0.05% Tween), blocked (with 1% BSA and incubated for 1 hour ± 10 minutes at room temperature with shaking at 150 to 200 rpm), and washed (3 times with PBS/0.05% Tween). Peroxidase-conjugated C1q is then added and the plates are incubated for 1.5 hours ± 30 minutes at room temperature with shaking at 150 to 200 rpm. After incubation and washing, tetramethylbenzidine (TMB) substrate solution is added and the plates are incubated for 7 minutes at room temperature (-1 minute/+30 seconds). This produces a colorimetric reaction that is proportional to the extent of C1q binding. The reaction was terminated by the addition of 1 M sulfuric acid and the color was measured at 450 nm using a Molecular Devices SpectraMax microplate reader. SoftMax Pro was used to analyze the data using a 4-parameter logic fit with weighted nonlinear regression. The potency of the resulting data was assessed relative to a reference standard using SoftMax Pro software. C1q binding activity results are reported as a percentage of potency relative to a reference standard. A representative dose-response curve for one assay can be displayed. (vii)       B cell depletion activity

In some embodiments, B cell depletion activity can be measured in a human whole blood B cell depletion assay. In some embodiments, B cell depletion activity can be measured in an autologous normal human whole blood B cell depletion assay. In some embodiments, B cell depletion can be measured by displaying cells in a CD45-positive lymphocyte gate and counting CD3-positive T cells, CD19-positive B cells, and CD20-positive B cells. In some embodiments, the percentage of B cell depletion (100-([100/B-/T-cell ratio in a sample without antibody] x [B-/T-cell ratio in a sample containing antibody])) can be calculated and plotted relative to sample concentration.

In some embodiments, B cell depletion activity can be measured using blood from three healthy donors. In some embodiments, B cell depletion can be measured by displaying cells in the CD45-positive lymphocyte gate and counting CD3-positive T cells, CD19-positive B cells, and CD20-positive B cells. (b)             Biological properties

In some embodiments, the biological properties of a composition or population of anti-CD20 antibody proteins provided herein can be measured and described in an assay described in Section 7.7(a) and using comparisons with a reference standard. In some embodiments, the reference standard is a commercial reference standard.

In some embodiments, the reference standard is an anti-CD20 antibody. In certain embodiments, the reference standard is GAZYVA (Obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan), or OCREVUS (ocrelizumab).

In some embodiments, the commercial reference standard is RS-117808. In certain embodiments, the anti-CD20 antibody protein groups provided herein have the biological properties shown in Table 7. RS-117808 ("antibody utuximab (TG-1101)") was deposited with the American Type Culture Collection (ATCC) at 10801 University Boulevard, Manassas, VA 20110, under the terms of the Budapest Treaty, and was received by ATCC on April 15, 2022 and assigned the informal patent deposit number PTA-127294. Table 7 : Biological Properties Compared to Reference Standards (i) Cell-based antibody-dependent cytotoxicity (ADCC)

In some embodiments, the compositions or populations of anti-CD20 antibody proteins provided herein exhibit a relative potency in cell-based ADCC using a CD16 effector cell assay (see Section 7.7(a)(i)) that is at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200% that of the commercial reference standard RS-117808. In certain embodiments, the compositions or populations of anti-CD20 antibody proteins provided herein exhibit a relative potency in a cell-based ADCC using a CD16 effector cell assay (see Section 7.7(a)(i)) that is at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 20-fold greater than the commercial reference standard, Rituxan® (Genentech/Biogen).

In some embodiments, the population induces cytotoxicity in a cell-based antibody-dependent cytotoxicity (ADCC) assay. In certain embodiments, the population induces greater cytotoxicity in an ADCC assay compared to a commercial reference standard.

In some embodiments, the group induces greater cytotoxicity in an ADCC assay compared to an anti-CD20 antibody. In some embodiments, the group induces greater cytotoxicity in an ADCC assay compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) and/or OCREVUS (ocrelizumab). In some embodiments, the group induces greater cytotoxicity in an ADCC assay compared to GAZYVA (obinutuzumab). In some embodiments, the group induces greater cytotoxicity in an ADCC assay compared to ARZERRA (ofatumumab). In certain embodiments, the group induces greater cytotoxicity in an ADCC assay compared to RITUXAN (rituximab). In certain embodiments, the group induces greater cytotoxicity in an ADCC assay compared to veltuzumab (IMMU-106). In certain embodiments, the group induces greater cytotoxicity in an ADCC assay compared to ZEVALIN (ibritumomab tiuxetan). In certain embodiments, the group induces greater cytotoxicity in an ADCC assay compared to OCREVUS (ocrelizumab).

In some embodiments, the population has at least 1000%, 750%, 500%, 250%, 100%, 75%, 50% or at least 25% relative potency in a cell-based ADCC assay compared to a commercial reference standard. In certain embodiments, the population has at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold or at least 200-fold relative potency in a cell-based ADCC assay compared to a commercial reference standard. In certain embodiments, the population has at least greater than 100% relative potency in a cell-based ADCC assay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of greater than 100% in a cell-based ADCC assay compared to a commercial reference standard.

In some embodiments, the population has a relative potency in a cell-based ADCC assay of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater than a commercial reference standard. In certain embodiments, the population has a relative potency of 5% versus 50%, 50% versus 100%, 100% versus 500%, 500% versus 1000%, 10-fold versus 50-fold, 50-fold versus 100-fold, 100-fold and 150-fold, or 150-fold to 300-fold in a cell-based ADCC assay compared to a commercial reference standard.

In some embodiments, the commercial reference standard is an anti-CD20 antibody. In some embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) and/or OCREVUS (ocrezumab). In some embodiments, the group has a relative potency of about 38% to about 3 times greater in a cell-based ADCC assay compared to GAZYVA (obinutuzumab). In some embodiments, the group has a relative potency of about 10 times to about 31 times greater in a cell-based ADCC assay compared to ARZERRA (ofatumumab). In certain embodiments, the population has a relative potency of about 28-fold to about 2250-fold in a cell-based ADCC assay compared to RITUXAN (rituximab). In certain embodiments, the population has a relative potency of about 25-fold in a cell-based ADCC assay compared to OCREVUS (ocrelizumab).

In some embodiments, the population has a relative potency of 60 to 200%, 70 to 190%, 80% to 180%, 85 to 170%, or 90 to 163% in a cell-based ADCC assay compared to a commercial reference standard. In some embodiments, the population has a relative potency of 90 to 163% in a cell-based ADCC assay compared to a commercial reference standard. In some embodiments, the population has a relative potency of about 117% in a cell-based ADCC assay compared to a commercial reference standard. In some embodiments, the commercial reference standard is RS-117808.

In some embodiments, the EC50 value of the ADCC potency of the population as measured in a cell-based ADCC assay is between 2 and 6 pg/mL. In certain embodiments, the EC50 value of the ADCC potency of the population as measured in a cell-based ADCC assay is between 0.2 to 20 pg/mL, 0.3 to 18 pg/mL, 0.4 to 15 pg/mL, 0.5 to 12 pg/mL, 0.6 and 10 pg/mL, 0.7 and 9 pg/mL, 0.8 and 8 pg/mL, 0.9 and 7 pg/mL, or 1 and 6 pg/mL. In certain embodiments, the EC50 value for ADCC potency is the average EC50 value calculated from EC50 values obtained in two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, fifty, sixty or more replicates of a cell-based ADCC assay. In certain embodiments, the EC50 value for ADCC potency of the population is about 5.45 pg/mL as measured in a cell-based ADCC assay (e.g., using Eurofins/DiscoverX KILR CD16a effector cells). In certain embodiments, the EC50 value for ADCC potency of the population is about 2.42 pg/mL as measured in a cell-based ADCC assay (e.g., using Eurofins/DiscoverX KILR CD16a effector cells). (ii) Antibody-dependent cellular phagocytosis (ADCP)

In some embodiments, the population induces antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the population induces higher phagocytosis in an ADCP assay compared to a commercial reference standard. In some embodiments, the population induces higher phagocytosis in an ADCP assay compared to an anti-CD20 antibody.

In some embodiments, the group induces higher phagocytosis in an ADCP assay compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan) and/or OCREVUS (ocrelizumab). In certain embodiments, the group induces phagocytosis in an ADCP assay compared to GAZYVA (obinutuzumab). In certain embodiments, the group induces greater cytotoxicity in a CDC assay compared to ARZERRA (ofatumumab). In certain embodiments, the group induces higher phagocytosis in an ADCP assay compared to RITUXAN (rituximab). In certain embodiments, the group induces greater cytotoxicity in a CDC assay compared to veltuzumab (IMMU-106). In certain embodiments, the population induces higher phagocytosis in an ADCP assay compared to ZEVALIN (ibritumomab tiuxetan). In certain embodiments, the population induces higher phagocytosis in an ADCP assay compared to OCREVUS (ocrelizumab).

In some embodiments, the group has at least 1000%, 750%, 500%, 250%, 100%, 75%, 50% or at least 25% relative efficacy in the ADCP analysis compared to a commercial reference standard. In some embodiments, the group has at least 150%, 160%, 170%, 180%, 190%, 200%, 5 times, 10 times, 15 times, 20 times, 30 times, 50 times, 75 times, 100 times, 125 times, 150 times, or at least 200 times relative efficacy in the ADCP analysis compared to a commercial reference standard. In some embodiments, the group has at least more than 100% relative efficacy in the ADCP analysis compared to a commercial reference standard. In some embodiments, the group has more than 100% relative efficacy in the ADCP analysis compared to a commercial reference standard.

In some embodiments, the population has a relative potency in an ADCP assay of at least 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater than a commercial reference standard. In certain embodiments, the population has a relative potency in an ADCP assay of between 5% and 50%, 50% and 100%, 100% and 500%, 500% and 1000%, 10-fold and 50-fold, 50-fold and 100-fold, 100-fold and 150-fold, or 150-fold and 300-fold compared to a commercial reference standard.

In some embodiments, the commercial reference standard is an anti-CD20 antibody. In some embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) or OCREVUS (ocrezumab). In some embodiments, compared with ARZERRA (ofatumumab), the group has about 7 times the relative efficacy in ADCP analysis. In some embodiments, compared with RITUXAN (rituximab), the group has about 15 times the relative efficacy in ADCP analysis. In some embodiments, compared with GAZYVA (obinutuzumab), the group has about 23 times the relative efficacy in ADCP analysis.

In some embodiments, the EC50 value of the ADCP efficacy measured in the ADCP assay of the population is between 0.1 and 1 ng/mL. In some embodiments, the EC50 value of the ADCP efficacy measured in the ADCP assay of the population is between 1 and 10, 2 and 9, 3 and 8, 4 and 7, or 5 and 6 ng/mL. In some embodiments, the EC50 value of the ADCP efficacy measured in the ADCP assay of the population is between 0.05 and 20 ng/mL, 0.1 and 19 ng/mL, 0.15 and 18 ng/mL, 0.2 and 18 ng/mL, 0.25 and 15 ng/mL, 0.3 and 12 ng/mL, 0.3 and 10 ng/mL, 0.3 and 9 ng/mL, 0.3 and 8 ng/mL, 0.3 and 7 ng/mL, or 0.3 and 6 ng/mL. In certain embodiments, the EC50 value of ADCP potency is the average EC50 value calculated from the EC50 values obtained in two, three, four, five, six, seven, eight, nine, ten or more repeated ADCP assays. In certain embodiments, the EC50 value of ADCP potency of the population as measured in the ADCP assay is about 5.50 ng/mL. (iii)      Complement-dependent cytotoxicity (CDC)

In some embodiments, the population induces complement-dependent cytotoxicity (CDC). In some embodiments, the population induces greater cytotoxicity in a CDC assay compared to a commercial reference standard. In some embodiments, the population induces greater cytotoxicity in a CDC assay compared to an anti-CD20 antibody.

In some embodiments, the group induces greater cytotoxicity in a CDC assay compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan) and/or OCREVUS (ocrelizumab). In certain embodiments, the group induces greater cytotoxicity in a CDC assay compared to GAZYVA (obinutuzumab). In certain embodiments, the group induces greater cytotoxicity in a CDC assay compared to ARZERRA (ofatumumab). In certain embodiments, the group induces greater cytotoxicity in a CDC assay compared to RITUXAN (rituximab). In certain embodiments, the population induces greater cytotoxicity in a CDC assay compared to veltuzumab (IMMU-106). In certain embodiments, the population induces greater cytotoxicity in a CDC assay compared to ZEVALIN (ibritumomab tiuxetan). In certain embodiments, the population induces greater cytotoxicity in a CDC assay compared to OCREVUS (ocrelizumab).

In some embodiments, the population has a relative potency of at least 1000%, 750%, 500%, 250%, 100%, 75%, 50%, 25%, 12%, or at least 5% in a cell-based CDC assay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold in a cell-based CDC assay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of at least greater than 100% in a cell-based CDC assay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of greater than 100% in a cell-based CDC assay compared to a commercial reference standard.

In some embodiments, the population has a relative potency in a cell-based CDC assay of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater than a commercial reference standard. In certain embodiments, the population has a relative potency in a cell-based CDC assay of between 5% and 50%, 50% and 100%, 100% and 500%, 500% and 1000%, 10-fold and 50-fold, 50-fold and 100-fold, 100-fold and 150-fold, or 150-fold and 300-fold compared to a commercial reference standard.

In some embodiments, the commercial reference standard is an anti-CD20 antibody. In some embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) or OCREVUS (ocrezumab). In some embodiments, compared with ARZERRA (ofatumumab), the group has a relative efficacy of about 50% in a cell-based CDC analysis. In some embodiments, compared with RITUXAN (rituximab), the group has a relative efficacy of about 37% in a cell-based CDC analysis. In some embodiments, compared with OCREVUS (ocrezumab), the group has a relative efficacy of about 1.8 times in a cell-based CDC analysis.

In some embodiments, the group has a relative efficacy of 50 to 150%, 60 to 140%, 70 to 130%, 75 to 120% or 78 to 116% in a cell-based CDC assay compared to a commercial reference standard. In some embodiments, the group has a relative efficacy of 78 to 116% in a cell-based CDC assay compared to a commercial reference standard. In some embodiments, the group has a relative efficacy of 73 to 128% or 74 to 127% in a cell-based CDC assay compared to a commercial reference standard. In some embodiments, the group has a relative efficacy of about 91% in a cell-based CDC assay compared to a commercial reference standard. In some embodiments, the commercial reference standard is RS-117808.

In some embodiments, the EC50 value of the CDC potency of the population as measured in a cell-based CDC assay is between 0.4 and 0.7 μg/mL or between 0.4 and 0.6 μg/mL. In certain embodiments, the EC50 value of the CDC potency of the population as measured in a cell-based CDC assay is between 0.05 and 5 μg/mL, 0.1 and 4 μg/mL, 0.15 and 3 μg/mL, 0.2 and 2 μg/mL, 0.25 and 1 μg/mL, 0.3 and 0.9 μg/mL, 0.3 and 0.8 μg/mL, or 0.3 and 0.7 μg/mL. In certain embodiments, the EC50 value of CDC potency is the average EC50 value calculated from the EC50 values obtained in two, three, four, five, six, seven, eight, nine, ten or more replicates of a cell-based CDC assay. In certain embodiments, the EC50 value of CDC potency of the population as measured in a cell-based CDC assay is about 0.5 μg/mL. (iv) CD20 binding activity

In some embodiments, the population has CD20 binding activity in a cell-based CD20 binding assay. In some embodiments, the population has greater CD20 binding activity in a cell-based CD20 binding assay than a commercial reference standard.

In some embodiments, the population has greater CD20 binding activity in a cell-based CD20 binding assay compared to an anti-CD20 antibody. In some embodiments, the population has greater CD20 binding activity in a cell-based CD20 binding assay compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan) and/or OCREVUS (ocrelizumab). In some embodiments, the population has greater CD20 binding activity compared to GAZYVA (obinutuzumab). In some embodiments, the population has higher CD20 binding activity compared to ARZERRA (ofatumumab). In some embodiments, the population has greater CD20 binding activity compared to RITUXAN (rituximab). In some embodiments, the population has greater CD20 binding activity compared to veltuzumab (IMMU-106). In some embodiments, the population has greater CD20 binding activity compared to ZEVALIN (ibritumomab tiuxetan). In some embodiments, the population induces greater CD20 binding activity compared to OCREVUS (ocrelizumab).

In some embodiments, the population has a relative potency of at least 1000%, 750%, 500%, 250%, 100%, 75%, 50%, 25%, 12%, or at least 5% in a CD20 binding assay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold compared to a commercial reference standard. In certain embodiments, the population has a relative potency of at least greater than 100% in CD20 binding compared to a commercial reference standard. In certain embodiments, the population has a relative potency of greater than 100% in CD20 binding compared to a commercial reference standard.

In some embodiments, the population has a relative potency in a CD20 binding assay that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater than a commercial reference standard. In certain embodiments, the population has a relative potency in a CD20 binding assay of between 5% and 50%, 50% and 100%, 100% and 500%, 500% and 1000%, 10-fold and 50-fold, 50-fold and 100-fold, 100-fold and 150-fold, or 150-fold and 300-fold compared to a commercial reference standard.

In some embodiments, the commercial reference standard is an anti-CD20 antibody. In some embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan) or OCREVUS (ocrelizumab). In some embodiments, the group has a relative potency of about 1.6 times to about 5.8 times in the CD20 binding assay compared to GAZYVA (obinutuzumab). In some embodiments, the group has a relative potency of about 81% to about 4.3 times in the CD20 binding assay compared to ARZERRA (ofatumumab). In certain embodiments, the population has a relative potency of about 1.6-fold to about 4.1-fold in a CD20 binding assay compared to RITUXAN (rituximab).

In some embodiments, the population has a relative potency of 50 to 150%, 60 to 140%, 70 to 130%, 80 to 120%, 90 to 120%, or 92 to 118% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of 92 to 118% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of 82 to 138% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of about 109% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard. In some embodiments, the commercial reference standard is RS-117808.

In some embodiments, the EC50 value of CD20 binding potency of the population as measured in a cell-based CD20 binding activity bioassay is between 0.05 and 0.1 μg/mL. In certain embodiments, the EC50 value of CD20 binding potency of the population as measured in a cell-based CD20 binding activity bioassay is between 0.01 and 0.5 μg/mL, 0.02 and 0.4 μg/mL, 0.03 and 0.3 μg/mL, 0.04 and 0.2 μg/mL, or 0.05 and 0.1 μg/mL. In certain embodiments, the EC50 value of CD20 binding potency is the average EC50 value calculated from the EC50 values obtained in two, three, four, five, six, seven, eight, nine, ten or more replicates of the cell-based CDC binding activity bioassay. In certain embodiments, the population has an EC50 value of about 0.093 μg/mL for CD20 binding potency as measured in a cell-based CD20 binding activity bioassay. In certain embodiments, the population has an EC50 value of about 0.063 μg/mL for CD20 binding potency as measured in a cell-based CD20 binding activity bioassay. (v)         FcγRIIIa 158V and FcγRIIIa 158F binding activity

In some embodiments, the population has FcγRIIIa 158V binding activity in an FcγRIIIa binding assay. In some embodiments, the population has greater FcγRIIIa 158V binding activity in an FcγRIIIa binding assay than a commercial reference standard.

In some embodiments, the population has greater FcγRIIIa 158V binding activity in the FcγRIIIa binding fraction compared to an anti-CD20 antibody. In some embodiments, the population has greater CD20 binding activity in a cell-based CD20 binding assay compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan) and/or OCREVUS (ocrelizumab). In some embodiments, the population has greater FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to GAZYVA (obinutuzumab). In certain embodiments, the population has greater FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to ARZERRA (ofatumumab). In certain embodiments, the population has greater FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to RITUXAN (rituximab). In certain embodiments, the population has greater FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to veltuzumab (IMMU-106). In certain embodiments, the population has greater FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to ZEVALIN (ibrimumab tiuxetan). In certain embodiments, the population has greater FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to OCREVUS (ocrelizumab).

In some embodiments, the population has at least 1000%, 750%, 500%, 250%, 100%, 75%, or at least 50% relative FcγRIIIa 158V binding activity in an FcγRIIIa binding assay as compared to a commercial reference standard. In certain embodiments, the population has at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold relative FcγRIIIa 158V binding activity in an FcγRIIIa binding assay as compared to a commercial reference standard. In certain embodiments, the population has at least greater than 100% relative FcγRIIIa 158V binding activity in a cell-based ADCC assay compared to a commercial reference standard. In certain embodiments, the population has greater than 100% relative FcγRIIIa 158V binding activity in a FcγRIIIa binding assay compared to a commercial reference standard.

In some embodiments, the population has at least 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater relative FcγRIIIa 158V binding activity in an FcγRIIIa binding assay as compared to a commercial reference standard. In certain embodiments, the population has a relative FcγRIIIa 158V binding activity in an FcγRIIIa binding assay of at least 50% to 100%, 100% to 500%, 500% to 1000%, 10-fold to 50-fold, 50-fold to 100-fold, 100-fold to 150-fold, or 150-fold to 300-fold as compared to a commercial reference standard.

In some embodiments, the commercial reference standard is an anti-CD20 antibody. In some embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) or OCREVUS (ocrelizumab). In some embodiments, the group has about 3.8 times the relative FcγRIIIa 158V binding activity in the FcγRIIIa binding assay compared to GAZYVA (obinutuzumab). In some embodiments, the group has about 25.6 times the relative FcγRIIIa 158V binding activity in the FcγRIIIa binding assay compared to ARZERRA (ofatumumab). In certain embodiments, the population has about 18.7-fold greater relative FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to RITUXAN (rituximab). In certain embodiments, the population has about 16-fold greater relative FcγRIIIa 158V binding activity in an FcγRIIIa binding assay compared to OCREVUS (ocrelizumab).

In some embodiments, the population has FcγRIIIa 158F binding activity in an FcγRIIIa binding assay. In some embodiments, the population has higher FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to a commercial reference standard. In some embodiments, the population has greater FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to an anti-CD20 antibody. In some embodiments, the population has greater CD20 binding activity in a cell-based CD20 binding assay compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan) and/or OCREVUS (ocrelizumab). In certain embodiments, the population has greater FcγRIIIa 158F binding activity in the FcγRIIIa binding assay compared to GAZYVA (obinutuzumab). In certain embodiments, the population has greater FcγRIIIa 158F binding activity in the FcγRIIIa binding assay compared to ARZERRA (ofatumumab). In certain embodiments, the population has greater FcγRIIIa 158F binding activity in the FcγRIIIa binding assay compared to RITUXAN (rituximab). In certain embodiments, the population has greater FcγRIIIa 158F binding activity in the FcγRIIIa binding assay compared to veltuzumab (IMMU-106). In certain embodiments, the population has greater FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to ZEVALIN (ibritumomab tiuxetan). In certain embodiments, the population has greater FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to OCREVUS (ocrelizumab).

In some embodiments, the population has at least 1000%, 750%, 500%, 250%, 100%, 75%, or at least 50% relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay as compared to a commercial reference standard. In certain embodiments, the population has at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay as compared to a commercial reference standard. In some embodiments, the population has at least greater than 100% relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to a commercial reference standard. In certain embodiments, the population has greater than 100% relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to a commercial reference standard.

In some embodiments, the population has at least 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay as compared to a commercial reference standard. In certain embodiments, the population has a relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay of at least 5% to 50%, 50% to 100%, 100% to 500%, 500% to 1000%, 10-fold to 50-fold, 50-fold to 100-fold, 100-fold to 150-fold, or 150-fold to 300-fold as compared to a commercial reference standard.

In some embodiments, the commercial reference standard is an anti-CD20 antibody. In some embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) or OCREVUS (ocrelizumab). In some embodiments, the group has about 2.6 times the relative FcγRIIIa 158F binding activity in the FcγRIIIa binding assay compared to GAZYVA (obinutuzumab). In some embodiments, the group has about 21.8 times the relative FcγRIIIa 158F binding activity in the FcγRIIIa binding assay compared to ARZERRA (ofatumumab). In certain embodiments, the population has about 10.2 times the relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to RITUXAN (rituximab). In certain embodiments, the population has about 9.9 times the relative FcγRIIIa 158F binding activity in an FcγRIIIa binding assay compared to OCREVUS (ocrelizumab). In certain embodiments, the population has significantly higher binding affinity for FcγRIIIa 158V or FcγRIIIa 158F than rituximab.

In some embodiments, the population has a relative potency of 20 to 300%, 30 to 250%, 40% to 220%, or 50 to 200% in a cell-based FcγRIIIa binding assay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of 50 to 200% in a cell-based FcγRIIIa binding assay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of 76 to 130% or 82 to 130% in a cell-based FcγRIIIa 158V binding assay compared to a commercial reference standard. In certain embodiments, the commercial reference standard is RS-117808.

In some embodiments, the population has a _K value of 55 to 70 nM in a FcγRIIIa-158V binding assay as measured by surface plasmon resonance. In certain embodiments, the population has a K value of 10 to 100 nM, 15 to 90 nM, 10 to 80 nM, or 30 to 70 nM in a FcγRIIIa-158V binding assay as measured by surface plasmon resonance. In certain embodiments, the population has _{a K} value of 30 to 70 nM in a FcγRIIIa-158V binding assay as measured by surface plasmon resonance. In certain embodiments, the _K value is an average _{K value} calculated from K values obtained in two, three, four, five, six, seven, eight, nine, ten or more replicates of the FcγRIIIa-158V binding assay. In some embodiments, the population has a _K value of about 59 nM in an FcγRIIIa-158V binding assay as measured by surface plasmon resonance. In some embodiments, the population has a _K value of about 64.1 nM in an FcγRIIIa-158V binding assay as measured by surface plasmon resonance.

In some embodiments, the population has a _K value of 600 to 800 nM in a FcγRIIIa 158F binding assay as measured by surface plasmon resonance. In certain embodiments, the population has a K value of 100 to 2000 nM, 200 to 1800 nM, 300 to 1700 nM, 400 to 1600 nM, 500 to 1500 nM, 500 to 1200 nM, 600 to 1000 nM, or 600 to 800 nM in a FcγRIIIa 158F binding assay as measured by surface plasmon resonance. In certain embodiments, the population has a _{K value} of 500 to 1000 nM in a FcγRIIIa 158F binding assay as measured by surface plasmon resonance. In certain embodiments, the _KD value is an average _KD value calculated from KD values obtained in two, three, four, five, six, seven, eight, nine, ten or more replicates of the FcγRIIIa-158V binding assay. In certain embodiments, the population has a _KD value of 760 nM in the FcγRIIIa 158F binding assay as measured by surface plasmon resonance. In certain embodiments, the population has _{a KD} value of 680.3 nM in the FcγRIIIa 158F binding assay as measured by surface plasmon resonance. (vi) C1q binding activity

In some embodiments, the population has a Clq binding activity as measured by ELISA. In certain embodiments, the population has a Clq binding activity as measured by ELISA that is greater than a commercial reference standard.

In some embodiments, the population has greater C1q binding activity as measured by ELISA compared to an anti-CD20 antibody. In some embodiments, the population has greater C1q binding activity as measured by ELISA compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumomab tiuxetan) and/or OCREVUS (ocrelizumab). In some embodiments, compared to GAZYVA (obinutuzumab), there is greater C1q binding activity as measured by ELISA. In some embodiments, compared to ARZERRA (ofatumumab), there is greater C1q binding activity as measured by ELISA. In some embodiments, compared to RITUXAN (rituximab), there is greater C1q binding activity as measured by ELISA. In certain embodiments, the population has greater C1q binding activity as measured by ELISA compared to veltuzumab (IMMU-106). In certain embodiments, the population has greater C1q binding activity as measured by ELISA compared to ZEVALIN (ibritumomab tiuxetan). In certain embodiments, the population has greater C1q binding activity as measured by ELISA compared to OCREVUS (ocrelizumab).

In some embodiments, the population has a relative potency of at least 1000%, 750%, 500%, 250%, 100%, 75%, 50%, or at least 25% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In certain embodiments, the population has a relative potency of at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In certain embodiments, the population has a relative potency of at least greater than 100% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In certain embodiments, the population has a relative potency of greater than 100% in a C1q binding assay as measured by ELISA compared to a commercial reference standard.

In some embodiments, the population has a relative potency in a CIq binding assay as measured by ELISA of at least 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater than a commercial reference standard. In certain embodiments, the population has a relative potency between 5% and 50%, 50% and 100%, 100% and 500%, 500% and 1000%, 10-fold and 50-fold, 50-fold and 100-fold, 100-fold and 150-fold, or 150-fold and 300-fold in a C1q binding assay as measured by ELISA compared to a commercial reference standard.

In some embodiments, the commercial reference standard is an anti-CD20 antibody. In some embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan) or OCREVUS (ocrelizumab). In some embodiments, the group has a relative potency of about 142% as measured by ELISA in the C1q binding assay compared to ARZERRA (ofatumumab). In some embodiments, the group has a relative potency of about 123% as measured by ELISA in the C1q binding assay compared to RITUXAN (rituximab). In certain embodiments, the population has a relative potency of about 112% in a C1q binding assay as measured by ELISA compared to OCREVUS (ocrelizumab).

In some embodiments, the population has a relative potency of 30 to 180%, 40 to 170%, 50 to 160%, 60 to 150%, 70 to 140%, 80 to 130%, 85% to 120%, or 88 to 113% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In certain embodiments, the population has a relative potency of 88% to 113% or 86% to 117% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In certain embodiments, the population has a relative potency of 86 to 116% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In certain embodiments, the population has a relative potency of about 99% in a C1q binding assay as measured by ELISA compared to a commercial reference standard. In some embodiments, the commercial reference standard is RS-117808.

In some embodiments, the EC50 value of the C1q binding activity as measured by ELISA of the population is between 1.5 and 3 μg/mL. In certain embodiments, the EC50 value of the C1q binding activity as measured by ELISA of the population is between 0.2 and 9 μg/mL, 0.3 and 8 μg/mL, 0.4 and 7 μg/mL, 0.5 and 6 μg/mL, 0.6 and 5 μg/mL, 0.7 and 4 μg/mL, 0.8 and 3 μg/mL, 0.9 and 2.9 μg/mL, or 1 and 2.8 μg/mL. In certain embodiments, the EC50 value of the C1q binding activity is the average EC50 value calculated from the EC50 values obtained in two, three, four, five, six, seven, eight, nine, ten or more repeated ELISA experiments. In some embodiments, the EC50 value of the C1q binding activity of the population as measured by ELISA is about 1.92 μg/mL. In some embodiments, the EC50 value of the C1q binding activity of the population as measured by ELISA is about 2.6 μg/mL. (vii)     B cell depletion activity

In some embodiments, the population has B cell depletion activity as measured in a human whole blood B cell depletion assay. In certain embodiments, the population has greater B cell depletion activity as measured in a human whole blood B cell depletion assay compared to a commercial reference standard.

In some embodiments, the population has greater B cell depletion activity as measured in a human whole blood B cell depletion assay compared to an anti-CD20 antibody. In certain embodiments, the population has greater B cell depletion activity as measured in a human whole blood B cell depletion assay compared to GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan) and/or OCREVUS (ocrelizumab). In certain embodiments, the population has greater B cell depletion activity as measured in a human whole blood B cell depletion assay compared to GAZYVA (obinutuzumab). In certain embodiments, the population has greater B cell depletion activity as measured in a human whole blood B cell depletion assay compared to ARZERRA (ofatumumab). In certain embodiments, the population has greater B cell depletion activity as measured in a human whole blood B cell depletion assay compared to RITUXAN (rituximab). In certain embodiments, the population has greater B cell depletion activity as measured in a human whole blood B cell depletion assay compared to veltuzumab (IMMU-106). In certain embodiments, the population has greater B cell depletion activity as measured in a human whole blood B cell depletion assay compared to ZEVALIN (ibritumumab tiuxetan). In certain embodiments, the population has greater B cell depletion activity as measured in a human whole blood B cell depletion assay compared to OCREVUS (ocrelizumab).

In some embodiments, the population has a relative potency of at least 1000%, 750%, 500%, 250%, 100%, 75%, 50%, 25%, 12%, or at least 5% in a human whole blood B cell depletion assay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold in a human whole blood B cell depletion assay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of at least greater than 100% in a human whole blood B cell depletion assay compared to a commercial reference standard. In certain embodiments, the population has a relative potency of greater than 100% in a human whole blood B cell depletion assay compared to a commercial reference standard.

In some embodiments, the population has a relative potency in a human whole blood B cell depletion assay that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, or at least 200-fold, and at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 750%, 1000%, 15-fold, 20-fold, 30-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, or at most 300-fold greater than a commercial reference standard. In certain embodiments, the population has a relative potency in a human whole blood B cell depletion assay of between 5% and 50%, 50% and 100%, 100% and 500%, 500% and 1000%, 10-fold and 50-fold, 50-fold and 100-fold, 100-fold and 150-fold, or 150-fold and 300-fold compared to a commercial reference standard.

In certain embodiments, the commercial reference standard is an anti-CD20 antibody. In certain embodiments, the commercial reference standard is GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan), or OCREVUS (ocrelizumab). 7.8 Methods of Treating and Preventing Medical Conditions Methods of Treating Multiple Sclerosis

Provided herein is a method of treating a relapsing form of multiple sclerosis (RMS) in an individual by administering to an individual in need thereof an effective amount of an anti-CD20 antibody composition as described herein. In some embodiments, the relapsing form of multiple sclerosis (RMS) is selected from clinically isolated syndrome ("CIS"); relapsing remitting syndrome ("RRMS"); or active secondary progressive disease ("SPMS"). In some embodiments, RMS is CIS. In some embodiments, RMS is RRMS. In some embodiments, RMS is SPMS. In some embodiments, the individual is diagnosed as having RMS according to the McDonald Criteria (2010) or by another suitable method known to a person of ordinary skill.

In some embodiments, the method comprises administering an anti-CD20 antibody composition as described herein to an individual in a multiple dose regimen. In certain embodiments, the method comprises administering an anti-CD20 antibody composition as described herein to an individual by intravenous infusion. For example, the anti-CD20 antibody composition described herein can be administered to an individual by intravenous infusion in a multiple infusion dose regimen. The anti-CD20 antibody composition described herein can be administered to an individual by intravenous infusion in a multiple infusion dose regimen for 48 weeks. Additionally or alternatively, the anti-CD20 antibody composition described herein can be administered to an individual by intravenous infusion in a multiple infusion dose regimen for 96 weeks. In some embodiments, the multiple infusion dosing regimen comprises a first, second, and subsequent intravenous infusions of utuximab. In some embodiments, a "subsequent infusion" of utuximab is any number of infusions after the second infusion.

In some embodiments, the intravenous infusion comprises a multiple dose regimen (e.g., a multiple infusion dose regimen) comprising: a) a first infusion on day 1, comprising about 100 to about 200 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody proteins in the population have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 2 weeks after the first infusion, comprising about 400 to about 500 mg of mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody proteins in the population have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); c) a first subsequent infusion at about 24 weeks or about six months after the first infusion, comprising about 400 to about 500 mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody proteins in the population have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); NO:2, wherein the anti-CD20 antibody protein of the group has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and d) one or more subsequent infusions at about 24 weeks or about six months after the previous infusion, comprising about 400 to about 500 mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibody in the group consists of one or more heavy chains encoding an amino acid sequence comprising SEQ ID NO:1 and a light chain comprising SEQ ID NO:2, wherein the anti-CD20 antibody protein of the group has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and d) one or more subsequent infusions at about 24 weeks or about six months after the previous infusion, comprising about 400 to about 500 mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibody in the group consists of one or more heavy chains encoding an amino acid sequence comprising SEQ ID NO:1 and a light chain comprising SEQ ID NO:2, wherein the anti-CD20 antibody protein has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans).

In some embodiments, the intravenous infusion comprises a multiple dose regimen (e.g., a multiple infusion dose regimen) comprising: a) a first infusion comprising 150 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody proteins in the population have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 2 weeks after the first infusion comprising 450 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody proteins in the population have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody protein of the group has an N-glycan spectrum comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); c) a first subsequent infusion about 24 weeks or about six months after the first infusion, comprising 450 mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibody in the group is composed of one or more nucleic acid sequences encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody protein of the group has an N-glycan spectrum comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); NO:2, wherein the anti-CD20 antibody proteins of the group have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and d) one or more subsequent infusions at about 24 weeks or about six months after the previous infusion, comprising 450 mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibodies in the group are composed of one or more heavy chains encoding the amino acid sequence of SEQ ID NO:1 and a light chain encoding the amino acid sequence of SEQ ID NO:2, wherein the anti-CD20 antibody proteins of the group have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and d) one or more subsequent infusions at about 24 weeks or about six months after the previous infusion, comprising 450 mg of the anti-CD20 antibody composition described herein (i.e., the anti-CD20 antibodies in the group are composed of one or more heavy chains encoding the amino acid sequence of SEQ ID NO:1 and a light chain encoding the amino acid sequence of SEQ ID NO:2 NO:2, wherein the anti-CD20 antibody protein has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans). In some embodiments, the first infusion is administered within 4 hours; the second infusion is administered within 1 hour; and subsequent infusions (e.g., the first subsequent infusion and/or one or more subsequent infusions) are administered within 1 hour.

In some embodiments, the method comprises administering to the individual a first anti-CD20 antibody composition (e.g., GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan) or OCREVUS (ocrelizumab)) by intravenous infusion, and then administering to the individual a second anti-CD20 antibody as described herein (i.e., the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody proteins in the population have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans). In some embodiments, the intravenous infusion comprises a multiple infusion dosing regimen comprising: a) a first infusion on day 1 comprising the prescribed dose of an anti-CD20 antibody composition (e.g., GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan) or OCREVUS (ocrelizumab)); b) a second infusion about 2 weeks after the first infusion comprising about 400 to about 500 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibody in the population consists of one or more heavy chains encoding an amino acid sequence comprising SEQ ID NO: 1 and a heavy chain comprising SEQ ID NO: 2). NO:2, wherein the anti-CD20 antibody protein of the group has an N-glycan spectrum, the N-glycan spectrum comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); c) a first subsequent infusion at about 24 weeks or about six months after the first infusion, comprising about 400 to about 500 mg of the anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibody in the group consists of one or more heavy chains encoding the amino acid sequence of SEQ ID NO:1 and a light chain encoding the amino acid sequence of SEQ ID NO:2, wherein the anti-CD20 antibody protein of the group has an N-glycan spectrum, the N-glycan spectrum comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); NO:2, wherein the anti-CD20 antibody proteins of the group have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and d) one or more subsequent infusions at about 24 weeks or about six months after the previous infusion, comprising about 400 to about 500 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the group are composed of one or more heavy chains encoding an amino acid sequence comprising SEQ ID NO:1 and a light chain comprising SEQ ID NO:2, wherein the anti-CD20 antibody proteins of the group have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and d) one or more subsequent infusions at about 24 weeks or about six months after the previous infusion, comprising about 400 to about 500 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the group are composed of one or more heavy chains encoding an amino acid sequence comprising SEQ ID NO:1 and a light chain comprising SEQ ID NO:2, wherein the anti-CD20 antibody protein has N-glycans, and the N-glycans contain 10% to 20% galactosylated glycans and about 20 to 40% fucosylated glycans).

In some embodiments, the intravenous infusion comprises a multiple infusion dosing regimen comprising: a) a first infusion on day 1 comprising a prescribed dose of an anti-CD20 antibody composition (e.g., GAZYVA (obinutuzumab), ARZERRA (ofatumumab), RITUXAN (rituximab), veltuzumab (IMMU-106), ZEVALIN (ibritumumab tiuxetan), or OCREVUS (ocrelizumab)); b) a second infusion about 2 weeks after the first infusion comprising 450 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibody in the population consists of one or more heavy chains encoding an amino acid sequence comprising SEQ ID NO: 1 and a heavy chain comprising SEQ ID NO: 2). NO:2, wherein the anti-CD20 antibody protein of the group has an N-glycan spectrum, the N-glycan spectrum comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); c) about 24 weeks or about six months after the first infusion, the first subsequent infusion comprises 450 mg of the anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibody in the group consists of one or more heavy chains encoding the amino acid sequence of SEQ ID NO:1 and a light chain comprising SEQ ID NO:2, wherein the anti-CD20 antibody proteins of the group have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and d) one or more subsequent infusions at about 24 weeks or about six months after the previous infusion, comprising 450 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the group are composed of one or more heavy chains encoding an amino acid sequence comprising SEQ ID NO:1 and a light chain comprising SEQ ID NO:2, wherein the anti-CD20 antibody proteins of the group have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and d) one or more subsequent infusions at about 24 weeks or about six months after the previous infusion, comprising 450 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the group are composed of one or more heavy chains encoding an amino acid sequence comprising SEQ ID NO:1 and a light chain comprising SEQ ID NO:2, wherein the anti-CD20 antibody protein has an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans). In some embodiments, the first infusion is administered within 4 hours; the second infusion is administered within 1 hour; and subsequent infusions (e.g., the first subsequent infusion and/or one or more subsequent infusions) are administered within 1 hour.

In some embodiments, the method comprises a treatment period of at least 48 weeks. In some embodiments, the method comprises a treatment period of at least 96 weeks.

In some embodiments, the individual is a human. In some embodiments, the human individual is an adult. In some embodiments, the individual has experienced at least one relapse in the previous year before treatment or has experienced two relapses in the previous two years before treatment. In some embodiments, the individual has or had T1 gadolinium (Gd) enhancing lesions in the previous year before treatment with an anti-CD20 antibody composition as described herein. In some embodiments, before treatment with an anti-CD20 antibody composition as described herein, the individual had an Expanded Disability Status Scale (EDSS) score of 0 to 5.5.

In some embodiments of the method, the individual has not received nonsteroidal therapy for MS in the past five years prior to treatment with an anti-CD20 antibody composition as described herein. In some embodiments, the individual has not received treatment for MS. In some embodiments, the individual is negative for hepatitis B virus (HBV). In some embodiments, the individual is negative for hepatitis B virus surface antigen (HBsAg). In some embodiments, the individual is negative for anti-hepatitis B virus core antibodies. In some embodiments, the individual has not been immunized with a vaccine for at least 2 weeks or at least 4 weeks prior to treatment with an anti-CD20 antibody composition as described herein.

In some embodiments of the method, about 30 to about 60 minutes before administering an anti-CD20 antibody composition as described herein, a certain amount of a corticosteroid is pre-administered to the individual. In some embodiments, the corticosteroid is methylprednisolone or a bioequivalent corticosteroid thereof. In some embodiments, the amount of the corticosteroid is about 100 mg of methylprednisolone. In some embodiments, the corticosteroid is dexamethasone or a bioequivalent corticosteroid thereof. In some embodiments, the amount of the corticosteroid is about 10 to 20 mg of dexamethasone. In certain embodiments, about 30 to about 60 minutes before administering an anti-CD20 antibody composition as described herein, a certain amount of an antipyretic is pre-administered to the individual. In some embodiments, the antipyretic is acetaminophen or a bioequivalent antipyretic thereof. In some embodiments, an amount of an antihistamine is pre-administered to the subject about 30 to about 60 minutes prior to administration of an anti-CD20 antibody composition as described herein. In some embodiments, the antihistamine is diphenhydramine HCl or a bioequivalent antihistamine thereof. In some embodiments, the amount of antihistamine is about 25 to 50 mg diphenhydramine HCl. In some embodiments, an amount of a corticosteroid (as described above) and an amount of an antihistamine (as described above) are pre-administered to the subject about 30 to about 60 minutes prior to administration of an anti-CD20 antibody composition as described herein. In some embodiments, the corticosteroid and/or antihistamine are administered orally to the subject.

In some embodiments, the method reduces or delays the progression of one or more MS symptoms of an individual. In some embodiments, the method reduces the annualized relapse rate (ARR) of an individual after administering an anti-CD20 antibody (e.g., utuximab) composition as described herein. In some embodiments, the ARR is the sum of the total number of relapses of an individual divided by the duration of treatment (i.e., it is the sum of the sum of the RMS relapse counts of an individual divided by the sum of the duration of treatment of an individual (in years)). In some embodiments, the ARR is the number of times an individual confirms relapses by an independent relapse adjudication panel (IRAP) each year. In some embodiments, the reduction of the ARR is assessed at approximately 96 weeks after the first infusion. In some embodiments, individuals following administration of a multiple infusion dosing regimen of an anti-CD20 antibody composition described herein achieve a significant reduction in ARR compared to individuals receiving oral administration of 14 mg of teriflunomide daily during the same treatment period. For example, administration of an anti-CD20 antibody composition described herein can significantly reduce ARR and have a 59% relative reduction in relapse rate compared to administration of teriflunomide. Additionally or alternatively, administration of an anti-CD20 antibody composition described herein can significantly reduce ARR and have a 49% relative reduction in relapse rate compared to administration of teriflunomide. In some embodiments, treatment with the anti-CD20 antibody compositions described herein results in an ARR of about 0.100 to about 0.050; such as about 0.100 to about 0.090, about 0.090 to about 0.080, about 0.080 to about 0.070, about 0.070 to about 0.060, or about 0.060 to about 0.050. (e.g., about 0.099, 0.098, 0.097, 0.096, 0.095, 0.094, 0.093, 0.092, 0.091, 0.090, 0.089, 0.088, 0.087, 0.086, 0.085, 0.084, 0.083, 0.082, 0.081, 0.080, 0.079, 0.078, 0.077, 0.076, 0.075 , 0.074, 0.073, 0.072, 0.071, 0.070, 0.069, 0.068, 0.067, 0.066, 0.065, 0.064, 0.063, 0.062, 0.061, 0.060, 0.059, 0.058, 0.057, 0.056, 0.055, 0.054, 0.053, 0.052, 0.051, or 0.050).

In some embodiments, the method reduces the total number of T1 gadolinium (Gd) enhancing lesions in an individual after administration of an anti-CD20 antibody composition as described herein. In some embodiments, the reduction in Gd enhancing T1 lesions is assessed by MRI scan. In some embodiments, the reduction in Gd enhancing T1 lesions is assessed at about 96 weeks after the first infusion. In some embodiments, individuals administered a multiple infusion dosing regimen of an anti-CD20 antibody achieve a reduction in the total number of Gd enhancing T1 lesions as measured by MRI scan compared to individuals receiving 14 mg of teriflunomide orally daily during the same treatment period. In some embodiments, administration of an anti-CD20 antibody (e.g., utuximab) significantly reduces the total number of Gd-enhancing T1 lesions by about 97% relative reduction compared to administration of teriflunomide.

In some embodiments, the method reduces the number of new or enlarged T2 hyperintense lesions in an individual after administration of an anti-CD20 antibody composition as described herein. In some embodiments, the reduction in the number of new or enlarged T2 hyperintense lesions is assessed based on an MRI scan. In some embodiments, the reduction in the number of new or enlarged T2 hyperintense lesions is assessed at about 96 weeks after the first infusion. In some embodiments, individuals who are administered a multiple infusion dosing regimen of an anti-CD20 antibody (e.g., utuximab) achieve a reduction in the total number of new and enlarged T2 hyperintense lesions based on MRI scans compared to individuals who receive 14 mg of teriflunomide orally daily during the same treatment period. In some embodiments, administration of an anti-CD20 antibody (e.g., utuximab) reduces the total number of new or enlarging T2 hyperintense lesions by about 90 to 92% relative to administration of teriflunomide.

In some embodiments, the method achieves confirmed disability progression in the individual after administration of an anti-CD20 antibody composition as described herein. In some embodiments, confirming disability progression comprises an increase of greater than or equal to 1.0 points relative to the individual's baseline EDSS score attributable to MS, wherein the baseline EDSS score is 5.5 or less. In some embodiments, confirming disability progression comprises an increase of greater than or equal to 0.5 points relative to the individual's baseline EDSS score attributable to MS, wherein the baseline EDSS score is greater than 5.5.

In some embodiments, the method results in a subject having no disease activity (NEDA). In some instances, NEDA comprises one or more of: confirmed no relapse, no gadolinium-enhancing (Gd+) T1 lesions, no new and/or enlarged T2 lesions, and no 12-week confirmed disability progression. In some embodiments, the method of the invention results in NEDA in a subject at about 24 weeks after administration of a pharmaceutical formulation as described herein (e.g., an anti-CD20 antibody composition). For example, NEDA can be induced in an individual at about 24-96 weeks (e.g., 24-48 weeks, 24-72 weeks, 48-72 weeks, 72-96 weeks) or 48-96 weeks (e.g., 24 weeks, 36 weeks, 48 weeks, 60 weeks, 72 weeks, 84 weeks, 96 weeks, or any range therebetween) after administration. In some embodiments, an individual administered a multiple infusion dosing regimen of an anti-CD20 antibody (e.g., utuximab) achieves an increase in NEDA status compared to an individual who received daily oral administration of 14 mg teriflunomide during the same treatment period. In some embodiments, administration of an anti-CD20 antibody (e.g., utuximab) increases NEDA status by 197% compared to administration of teriflunomide. In some embodiments, administration of an anti-CD20 antibody (e.g., utuximab) increases NEDA status by 277% compared to administration of teriflunomide.

In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody (e.g., utuximab) achieved an increase in confirmed disability improvement (CDI) compared to subjects receiving 14 mg of teriflunomide orally daily during the same treatment period. In some embodiments, administration of an anti-CD20 antibody (e.g., utuximab) increased CDI by 116% at 12 weeks compared to teriflunomide. In some embodiments, administration of an anti-CD20 antibody (e.g., utuximab) increased CDI by 103% at 24 weeks compared to teriflunomide.

In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody (e.g., utuximab) achieved an increase in the Multiple Sclerosis Functional Composite (MSFC) score compared to subjects receiving 14 mg of teriflunomide orally daily during the same treatment period. In some embodiments, administration of an anti-CD20 antibody (e.g., utuximab) increased the MSFC score by about 76% to 90% compared to administration of teriflunomide.

In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody (e.g., utuximab) achieve an improvement in the Timed 25-Foot Walk (T25FW) score compared to subjects receiving 14 mg teriflunomide orally daily during the same treatment period. In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody (e.g., utuximab) achieve an improvement in the 9-Hole Peg Test (9-HPT) score compared to subjects receiving 14 mg teriflunomide orally daily during the same treatment period.

In some embodiments, subjects administered a multiple infusion dosing regimen of an anti-CD20 antibody (e.g., utuximab) achieved a significant reduction in the volume and number of new T1 hypointense lesions based on MRI scans compared to subjects receiving 14 mg of teriflunomide administered orally daily during the same treatment period.

In some embodiments, the treatment for MS (e.g., RMS) as disclosed herein further comprises administering an anti-CD20 antibody in combination with one or more additional therapeutic agents. For example, the anti-CD20 antibody in the methods of the present invention for treating RMS can be administered in combination with other compounds, drugs, and/or agents for treating RMS. Such compounds, drugs, and/or agents can include, for example, small molecule drugs, monoclonal antibodies, or other B cell depleting agents. In some embodiments, the methods described herein are used in combination with existing MS treatment care standards. In some cases, anti-CD20 antibodies are used in combination with Bruton's tyrosine kinase (BTK) inhibitors for the treatment of RMS, including, for example, fenebrutinib, evobrutinib, tolebrutinib, orelabrutinib BIIB091, AC0058, PRN473 (Dolgin, Nature Biotechnology 39 : 3-12 (2021), and those compounds described in U.S. Patent No. 9,951,077). In some cases, anti-CD20 antibodies are used in combination regimens that utilize a non-overlapping mechanism of utuximab to deplete B cells.

Anti-CD20 antibodies (e.g., Utuximab) can be combined with injectable, oral, or infused medications. Injectable medications for combination therapy with anti-CD20 antibodies can include interferons, including but not limited to AVONEX (interferon beta-1a), BETASERON (interferon beta-1b), EXTAYIA (interferon beta-1b), PLEGRIDY (pegylated interferon beta-1a), and REBIF (interferon beta-1a). Other injectable medications can include COPAXONE (glatiramer acetate). Oral medications combined with Utuximab can include AUBAGIO (teriflunomide), fumarate-based compositions (including BAFIERTAM), dimethyl fumarate, TECFIDERA, and VUMERITY. Additionally or in the alternative, an anti-CD20 antibody (e.g., utuximab) can be used in combination with the monoclonal antibody Tysabri (natalizumab). Orally administered agents that can be used in combination with anti-CD20 antibodies include GILENYI (fingolimod), MAYZENT (siponimod), ZEPOSIA (ozanimod), and PONVORY (ponesimod). Additional orally administered agents include MAVENCLAD (cladribine).

Anti-CD20 antibodies (such as utuximab) can also be used in combination with infusional medications, including LEMTRADE (alemtuzumab) and NOVANTRONE (mitoxantrone).

In some embodiments, an anti-CD20 antibody of the present invention (e.g., utuximab or an anti-CD20 antibody that binds to the same epitope as utuximab) can be administered to an individual (e.g., a patient) with known resistance to one or more anti-CD20 antibodies because utuximab targets a unique epitope region on the CD20 antigen that is not targeted by other anti-CD20 monoclonal antibodies.

In some embodiments, the additional therapeutic agent may include a B cell depleting agent other than an anti-CD20 antibody. In some instances, the B cell depleting agent is a PI3K inhibitor.

In some embodiments, the additional therapeutic agent comprises a B cell depleting agent, which is an anti-CD20 antibody like the anti-CD20 antibodies described herein (e.g., Utuximab). For example, Utuximab can be used in combination with an additional anti-CD20 antibody composition, such as OCREVUS® (ocrelizumab), KESIMPTA® (ofatumumab), and Rituxan® (rituximab).

Additional therapeutic agents may be given orally, parenterally, intravenously, or subcutaneously.

In some embodiments, the methods described herein may result in treatment-emergent adverse events (TEAEs). In some cases, a TEAE comprises cytopenia or a decrease in a subject's blood count. The blood count can be assessed by a blood test, such as a complete blood count (CBC). The blood count can be obtained by cell counting methods known in the art, including but not limited to manual methods (e.g., by using a blood cell counter) and automated methods (e.g., by using an automated cell counter). In some cases, cytopenia comprises a decrease or reduction in a subject's blood count of about 20 to 100% compared to a control or normal blood count. (e.g., about 20-30%, about 20-40%, about 30-40%, about 30-50%, about 40-50%, about 40-60%, about 50-60%, about 50-70%, about 60-70%, about 60-80%, about 70-80%, about 70-90%, about 80-90%, about 80-100%, about 90-100%, or any range therebetween (e.g., about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 100%, or any range therebetween)). A control or normal blood count may include the blood count of a healthy individual to whom the pharmaceutical formulation of the present invention has not been administered. Additionally or alternatively, the individual's blood count prior to treatment by the present method may serve as a control or normal blood count. The cytopenia or decreased blood count may include one or more of lymphopenia, neutropenia, leukopenia, and anemia.

In some cases, administration of a pharmaceutical formulation as described herein (e.g., an anti-CD20 antibody composition) results in cytopenia in a subject about 1 to 3 days (e.g., about 1 day, about 2 days, or about 3 days) after administration. For example, administration of a pharmaceutical formulation can result in lymphopenia in a subject about 2 days after administration. In certain instances, the cytopenia (e.g., lymphocytopenia, neutropenia, leukopenia, and/or anemia) is temporary, such that the individual's blood count normalizes (e.g., becomes the same or similar to a control or normal blood count) by about 15 days (e.g., by 14 days, by 13 days, by 12 days, by 11 days, by 10 days, by 9 days, by 8 days, by 7 days, by 6 days, by 5 days, or by 4 days) after administration of the pharmaceutical formulation. For example, the individual's lymphocyte count may be normalized by 8 days after administration of the pharmaceutical formulation. Methods of treating cancer

Also provided herein are methods of treating cancer in an individual in need thereof, comprising administering to the individual an effective amount of an anti-CD20 antibody composition as described herein. In some embodiments, the cancer is bladder cancer, breast cancer, cervical cancer, colorectal cancer, gynecological cancer (i.e., cervical cancer, ovarian cancer, uterine cancer, vaginal cancer, or vulvar cancer), head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, mesothelioma, myeloma, prostate cancer, skin cancer, or thyroid cancer.

Also provided herein are methods for treating a disease or condition associated with excessive B cell proliferation in an individual in need thereof, comprising administering to the individual an effective amount of an anti-CD20 antibody composition as described herein. In some embodiments, the disease or condition associated with excessive B cell proliferation is a blood cancer. In some embodiments, the blood cancer is a lymphoma, a leukemia, or a myeloma. In some embodiments, the blood cancer is selected from B-cell lymphoma, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), mantle cell lymphoma (MCL), follicular lymphoma (FL), Waldenstrom's macroglobulinemia (WM), diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma (MZL), hairy cell leukemia (HCL), Burkitt's lymphoma (BL), Reye's transformation, or primary central nervous system lymphoma (PCNSL). In various aspects of the method, the anti-CD20 antibody composition as described herein comprises an anti-CD20 antibody which is an IgG1 antibody comprising a heavy chain each comprising the amino acid sequence of SEQ ID NO: 1 and a light chain each comprising the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the blood cancer is B cell lymphoma. In some embodiments, the B cell lymphoma is relapsed or refractory. In some embodiments, the blood cancer is non-Hodgkin's lymphoma (NHL). In some embodiments, NHL is relapsed or refractory. In some embodiments, the blood cancer is Waldenstrom's macroglobulinemia (WM). In some embodiments, WM is relapsed or refractory. In some embodiments, the blood cancer is marginal zone lymphoma (MZL). In some embodiments, MZL is relapsed or refractory. In some embodiments, the blood cancer is chronic lymphocytic leukemia (CLL). In some embodiments, CLL is relapsed or refractory. In some embodiments, the blood cancer is small lymphocytic lymphoma (SLL). In some embodiments, SLL is relapsed or refractory. In some embodiments, the blood cancer is primary central nervous system lymphoma (PCNSL). In some embodiments, PCNSL is relapsed or refractory.

In some embodiments, the method of treating a blood cancer comprises administering an anti-CD20 antibody composition as described herein to a subject by intravenous infusion. In some embodiments, the intravenous infusion comprises: a) on days 1 and 2 of cycle 1 (divided into a 150 mg dose on day 1 and a 750 mg dose on day 2), day 8 and day 15 (each cycle is 28 days); on day 1 of cycles 2 to 6; and on day 1 of every 3 cycles after cycle 6 (e.g., cycle 9, 12, 15, etc.), comprising about 900 mg of an anti-CD20 antibody composition as described herein. In some embodiments, the blood cancer is CLL.

In some embodiments, the methods described herein may result in treatment-emergent adverse events (TEAEs). In some cases, a TEAE comprises cytopenia or a decrease in a subject's blood count. The blood count can be assessed by a blood test, such as a complete blood count (CBC). The blood count can be obtained by cell counting methods known in the art, including but not limited to manual methods (e.g., by using a blood cell counter) and automated methods (e.g., by using an automated cell counter). In some cases, cytopenia comprises a decrease or reduction in a subject's blood count of about 20 to 100% compared to a control or normal blood count. (e.g., about 20-30%, about 20-40%, about 30-40%, about 30-50%, about 40-50%, about 40-60%, about 50-60%, about 50-70%, about 60-70%, about 60-80%, about 70-80%, about 70-90%, about 80-90%, about 80-100%, about 90-100%, or any range therebetween (e.g., about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, about 100%, or any range therebetween)). A control or normal blood count may include the blood count of a healthy individual to whom the pharmaceutical formulation of the present invention has not been administered. Additionally or alternatively, the individual's blood count prior to treatment by the present method may serve as a control or normal blood count. Hemocytopenia or decreased blood count may include one or more of lymphopenia, neutropenia, leukopenia, and anemia. Multiple infusion doses

In any of the embodiments described herein, the intravenous infusion comprises a multiple infusion dosage regimen comprising: a) a first infusion on day 1 comprising about 400 to about 500 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody proteins of the population have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 1 week after the first infusion comprising about 500 to about 700 mg, about 800 to about 1000 mg, or about 1100 to about 1300 mg; c) a third infusion about 2 weeks after the first infusion, comprising about 500 to about 700 mg, about 800 to about 1000 mg, or about 1100 to about 1300 mg of the anti-CD20 antibody composition; d) a fourth infusion about 3 weeks after the first infusion, comprising about 500 to about 700 mg, about 800 to about 1000 mg, or about 1100 to about 1300 mg of the anti-CD20 antibody composition; and e) one or more subsequent infusions about one month after the previous infusion, comprising about 400 to about 500 mg, about 500 to about 700 mg, about 800 to about 1000 mg, or about 1100 to about 1300 mg of the anti-CD20 antibody composition. In some cases, the first infusion is administered over 4 hours; the second infusion is administered over 1 hour; the third infusion is administered over 1 hour; the fourth infusion is administered over 1 hour; and one or more subsequent infusions are administered over 1 hour.

In some embodiments, the intravenous infusion comprises a multiple infusion dosage regimen, comprising: a) a first infusion on day 1, comprising 450 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody proteins in the population have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 1 week after the first infusion, comprising 600, 900 or 1200 mg of the anti-CD20 antibody composition; c) a third infusion about 2 weeks after the first infusion, comprising 600, 900 or 1200 mg of the anti-CD20 antibody composition; mg of anti-CD20 antibody composition; d) a fourth infusion about 3 weeks after the first infusion, comprising a fourth infusion of 600, 900 or 1200 mg of the anti-CD20 antibody composition; and e) one or more subsequent infusions about one month after the previous infusion, comprising 450, 600, 900 or 1200 mg of the anti-CD20 antibody composition. In some cases, the first infusion is administered within 4 hours; the second infusion is administered within 1 hour; the third infusion is administered within 1 hour; the fourth infusion is administered within 1 hour; and the one or more subsequent infusions are administered within 1 hour.

In some embodiments, the intravenous infusion comprises a multiple infusion dosage regimen, comprising: a) a first infusion on day 1, comprising about 400 to about 500 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody proteins in the population have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 1 week after the first infusion, comprising about 500 to about 700 mg of mg of anti-CD20 antibody composition; c) a third infusion about 2 weeks after the first infusion, comprising about 800 to about 1000 mg of the anti-CD20 antibody composition; and d) one or more subsequent infusions about 1 month after the previous infusion, comprising about 400 to about 1000 mg of the anti-CD20 antibody composition. In some instances, the first infusion is administered within 4 hours; the second infusion is administered within 1 hour; the third infusion is administered within 1 hour; and the one or more subsequent infusions are administered within 1 hour.

In some embodiments, the intravenous infusion comprises a multiple infusion dosage regimen, comprising: a) a first infusion on day 1, comprising 450 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody proteins in the population have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 1 week after the first infusion, comprising 600 mg of the anti-CD20 antibody composition; c) a third infusion about 2 weeks after the first infusion, comprising 900 mg of the anti-CD20 antibody composition; mg of an anti-CD20 antibody composition; and d) one or more subsequent infusions about one month after the previous infusion, comprising 450, 600, or 900 mg of the anti-CD20 antibody composition. In some cases, the first infusion is administered within 4 hours; the second infusion is administered within 1 hour; the third infusion is administered within 1 hour; and the one or more subsequent infusions are administered within 1 hour.

In some embodiments, the intravenous infusion comprises a multiple infusion dosage regimen comprising: a) a first infusion on day 1, comprising about 5 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody proteins of the population have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and b) one or more subsequent infusions about one week after the previous infusion, comprising about 5 to about 450 mg of the anti-CD20 antibody composition, wherein the dose of each subsequent infusion is higher than the previous infusion. In some cases, the first infusion is administered over 4 hours; and one or more subsequent infusions are administered over 1 hour.

In some embodiments, the intravenous infusion comprises a multiple infusion dosage regimen comprising: a) a first infusion on day 1 comprising about 100 to about 200 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody proteins of the population have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); and b) one or more subsequent infusions about one week after the previous infusion comprising about 400 to about 500 mg of the anti-CD20 antibody composition. In some cases, the first infusion is administered over 4 hours; and one or more subsequent infusions are administered over 1 hour.

In some embodiments, the intravenous infusion comprises a multiple infusion dosing regimen comprising: a) a first infusion on day 1 comprising 150 mg of the anti-CD20 antibody composition; and b) one or more subsequent infusions about one week after the previous infusion comprising 450 mg of the anti-CD20 antibody composition. In some embodiments, the method comprises seven or more subsequent infusions. In some embodiments, the method comprises seven subsequent infusions. In some cases, the first infusion is administered over 4 hours; and the one or more subsequent infusions are administered over 1 hour.

In some embodiments, the intravenous infusion comprises a multiple infusion dosage regimen, comprising: a) a first infusion on day 1, comprising about 500 to about 1000 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody proteins in the population have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 1 week after the first infusion, comprising about 500 to about 1000 mg of the anti-CD20 antibody; c) a third infusion about 2 weeks after the first infusion, comprising about 500 to about 1000 mg of the anti-CD20 antibody; mg of anti-CD20 antibody composition; and d) one or more subsequent infusions about one month after the previous infusion, comprising about 500 to about 1000 mg of anti-CD20 antibody composition. In certain embodiments, one or more subsequent infusions each comprise 600 mg of an anti-CD20 antibody composition as described herein. In certain embodiments, the first infusion, the second infusion, the third infusion, and one or more subsequent infusions each comprise 600 mg of an anti-CD20 antibody composition as described herein. In some embodiments, the first infusion, the second infusion, the third infusion, and one or more subsequent infusions each comprise 900 mg of an anti-CD20 antibody composition as described herein. In some cases, the first infusion is administered within 4 hours; the second infusion is administered within 1 hour; the third infusion is administered within 1 hour; and the one or more subsequent infusions are administered within 1 hour.

In some embodiments, the intravenous infusion comprises a multiple infusion dosage regimen, which includes: a) a first infusion on day 1, comprising about 100 to about 200 mg of an anti-CD20 antibody composition as described herein (i.e., the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the anti-CD20 antibody proteins in the population have an N-glycan profile comprising 10 to 20% galactosylated glycans and about 20 to 40% fucosylated glycans); b) a second infusion about 1 week after the first infusion, comprising about 700 to about 800 mg of mg of the anti-CD20 antibody composition; c) a third infusion about 2 weeks after the first infusion, comprising about 850 to about 950 mg of the anti-CD20 antibody composition; and d) one or more subsequent infusions about 1 month after the previous infusion, comprising about 850 to about 950 mg of the anti-CD20 antibody composition. In some embodiments, the first infusion comprises 150 mg of the anti-CD20 antibody composition, the second infusion comprises 750 mg of the anti-CD20 antibody composition, and the third and one or more subsequent infusions comprise 900 mg of the anti-CD20 antibody composition. In some cases, the first infusion is administered within 4 hours; the second infusion is administered within 1 hour; the third infusion is administered within 1 hour; and the one or more subsequent infusions are administered within 1 hour. Individual

In some embodiments, the individual is a human. In some embodiments, the human individual is an adult. In some embodiments, the individual has relapsed or refractory B-cell lymphoma. In some embodiments, the individual has previously been treated with at least one prior course of rituximab or a rituximab-based therapy. In some embodiments, the individual has previously been treated with at least one prior course of fludarabine or a fludarabine-based therapy. In some embodiments, the individual has not received prior treatment for B-cell lymphoma. In some embodiments, the individual is eligible for high-dose or combination chemotherapy and/or stem cell transplantation. In some embodiments, prior to treatment with an anti-CD20 antibody composition as described herein, the individual has an Eastern Cooperative Oncology (ECOG) score of 0 to 2. In some embodiments, prior to treatment with an anti-CD20 antibody composition as described herein, the individual has a peripheral blood lymphocyte count greater than 5,000/μL.

In some specific examples, the anti-CD20 antibody compositions provided herein can be used to treat and/or prevent chronic inflammatory demyelinating polyneuropathy (CIDP); myositis; lupus nephritis; other forms of MS-PPMS, SPMS; myasthenia gravis (MG); antiphospholipid syndrome; thrombotic thrombocytopenic purpura (TTP); ulcerative colitis; minimal change nephropathy syndrome (MCNS); aplastic anemia; autoimmune glomerulopathy; rheumatoid arthritis (RA); interstitial lung disease; myasthenia gravis ( MG); subepidermal autoimmune buccal disease; infectious lung disease; acquired hemophilia; refractory mixed cryoglobulinemia; primary immune thrombocytopenia; graft-versus-host disease (GVHD); autoimmune buccal disease; anti-myelin-associated glycoprotein (MAG) polyneuropathy; granulomatosis with polyangiitis (GPA); neuromyelitis optica; systemic lupus erythematosus; ulcer; post-transplantation lymphoproliferative disease; autoimmune hemolytic anemia; cerebral vasculitis; microscopic polyangiitis (MPA); or idiopathic nephrotic syndrome. 7.9  Pharmacokinetic properties

Also provided herein are methods of treating a human patient suffering from a disease (e.g., an autoimmune disease) comprising administering to the patient an anti-CD20 antibody provided herein.

In some embodiments, the anti-CD20 antibody is administered as follows: i) a first infusion at a dose of about 150 mg, ii) a second infusion two weeks later at a dose of about 450 mg, and iii) subsequent infusions at a dose of about 450 mg every six months.

In some embodiments, administration of the anti-CD20 antibody results in an area under the curve (AUC) between about 2,160 μg/mL and about 3,840 μg/mL. In some embodiments, the AUC is about 3,000 μg/mL. In some embodiments, the AUC is a steady-state AUC.

In some embodiments, administration of the anti-CD20 antibody produces a Cmax between about 118,011 ng/mL and about 159,989 ng/mL. In some embodiments, the Cmax is about 139,000 ng/mL. In some embodiments, the Cmax is a steady-state Cmax.

In some embodiments, administration of the anti-CD20 antibody produces a Cmin of about 0 ng/mL and about 375 ng/mL. In some embodiments, the Cmin is about 139 ng/mL. In some embodiments, the Cmin is a steady-state Cmin.

In some embodiments, administration of an anti-CD20 antibody produces a Cavg between about 6,437 ng/mL and about 11,443 ng/mL. In some embodiments, the Cavg is about 8,940 ng/mL. In some embodiments, the Cavg is a steady-state Cavg.

In some embodiments, the autoimmune disease is selected from multiple sclerosis, tinea pedis, rheumatoid arthritis, vasculitis, inflammatory bowel disease, dermatitis, osteoarthritis, inflammatory muscle disease, allergic rhinitis, vaginitis, interstitial cystitis, scleroderma, osteoporosis, eczema, allogeneic or xenotransplantation (organ, bone marrow, stem cells and other cells and tissues), transplant rejection, graft-versus-host disease, lupus erythematosus, inflammatory disease, type 1 diabetes, pulmonary fibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis (e.g., Hashimoto's thyroiditis and autoimmune thyroiditis), myasthenia gravis, autoimmune hemolytic anemia, cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis, allergic conjunctivitis, atopic dermatitis, chronic obstructive pulmonary disease, glomerulonephritis, neuroinflammatory diseases, and uveitis.

In some embodiments, the autoimmune disease is multiple sclerosis. In some embodiments, multiple sclerosis is a relapsing form of multiple sclerosis. In some embodiments, the relapsing form of multiple sclerosis is clinically isolated syndrome (CIS); relapsing remitting MS (RRMS); active secondary progressive MS (SPMS); or primary progressive MS (PPMS). In some embodiments, the relapsing form of MS is clinically isolated syndrome (CIS). In some embodiments, the relapsing form of MS is relapsing remitting multiple sclerosis (RRMS). In some embodiments, the relapsing form of MS is active secondary progressive multiple sclerosis (SPMS). In some embodiments, the relapsing form of MS is primary progressive MS (PPMS).

In certain embodiments, the anti-CD20 antibody is administered intravenously. 7.10      Manufacturing methods

Also provided herein are methods for producing the anti-CD20 antibody proteins provided herein with the specified range of post-translational modifications. Example 15 provides an exemplary method for producing the anti-CD20 antibody proteins provided herein.

In some embodiments, the method of producing the anti-CD20 antibody protein group having a post-translational modification as described above in a specified range comprises: i) culturing rat fusion tumor cells at a first culture pH of about 7.0 to about 7.55 for 0 to 3 days, ii) culturing rat fusion tumor cells at a second culture pH of about 6.5 to about 6.99 on day 3, iii) maintaining the culture pH at a second culture pH of about 6.5 to about 6.99 from culture day 3 to day 14 of the cell culture, and iv) controlling the culture pCO ₂ content to less than about 200 mmHg during the entire culture period. In some embodiments, the second culture pH is about 6.60 to about 6.96 (e.g., the second culture pH is 6.8).

In some embodiments, the second culture pH results in a higher integrated viable cell density (IVCD) at harvest and a higher titer.

In some embodiments, the second culture pH results in a lower fucosylation percentage.

In some embodiments, rat hybridoma cells expressing the recombinant protein are cultured in chemically defined basal medium and animal derived component-free (ADCF) medium.

In some embodiments, the basal medium is supplemented with a feed medium.

In some embodiments, the method further comprises an initial temperature set point of about 37°C, wherein the initial temperature set point is set on culture day 0 to culture day 1.

In some aspects, the method further comprises a second temperature set point of about 35°C, wherein the second temperature set point is set at the end of culture day 1 to culture day 3.

In some embodiments, the method further comprises a third temperature set point of about 32°C to about 33°C, wherein the third temperature set point is set on culture day 3 and maintained during the harvesting period. The term "harvesting" refers to the time point during the mammalian cell culture process when the cells containing the recombinant protein are separated and removed from the cell culture medium and subjected to additional processing (such as, for example, centrifugation, filtration or purification).

In some embodiments, cells are harvested on day 12, 13, or 14 of the cell culture process, or when cell viability drops below 20%, whichever occurs first.

In some embodiments, the method further comprises collecting the recombinant protein produced by the rat hybridoma cells.

In some embodiments, the method further comprises purifying the recombinant protein by affinity analysis and/or ion exchange analysis. In some embodiments, the affinity analysis comprises protein A purification.

In some embodiments, the method increases the yield of the recombinant protein. For example, the recombinant protein is increased by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, or at least about 150% relative to the recombinant protein produced by a culture method that does not use the culture conditions cited above. Sequence Listing 7.11 Preparation of Utuximab for Intravenous Infusion in the Disclosed Treatment Methods

In some embodiments, the utuximab infusion is prepared in 250 mL of 0.9% sodium chloride injection. For example, the utuximab infusion can be prepared in an infusion bag containing 0.9% sodium chloride injection.

In some embodiments, the first infusion of 150 mg of utuximab is prepared as follows: (1) inspect one vial of 150 mg/6 mL utuximab solution for any particulate matter or discoloration and do not use the solution if the solution contains loose foreign particulate matter; (2) if the vial is free of particulate matter, use one vial of 150 mg/6 mL utuximab solution to prepare a 250 mL infusion bag for the first infusion; (3) withdraw 6 mL of 0.9% sodium injection from the infusion bag and discard; (4) withdraw 6 mL of utuximab from the vial; (5) dilute 6 mL (150 mg) of utuximab into the 250 mL bag containing 0.9% sodium injection; mL infusion bag for immediate administration; and (6) mix the infusion bag by gently inverting it without shaking.

In some embodiments, a second or subsequent infusion of 450 mg of utuximab is prepared by: (1) inspecting three vials of 150 mg/6 mL utuximab solution for any particulate matter or discoloration and not using the solution if it contains loose foreign particulate matter; (2) if the vials are free of particulate matter, using the three vials of 150 mg/6 mL utuximab solution to prepare a 250 mL infusion bag for the second or subsequent infusion; (3) withdrawing 18 mL of 0.9% sodium injection from the infusion bag and discarding it; (4) removing 18 mL of utuximab from the vial; and (5) diluting 18 mL (450 mg) of utuximab into the 250 mL of 0.9% sodium injection. mL infusion bag for immediate administration; and (6) mix the infusion bag by gently inverting it without shaking.

In some embodiments, the contents of the infusion bag are at room temperature prior to initiating the intravenous infusion of utuximab. 7.12    Administering utuximab in the treatment methods disclosed herein

In some embodiments, the individual is pre-screened for hepatitis B virus (HBV) prior to initiating administration of utuximab. If the individual tests positive for hepatitis B surface antigen (HbsAg), utuximab should not be administered. In some embodiments, the individual administered utuximab infusion is negative for hepatitis B surface antigen (HBsAg).

In some embodiments, the subject is pre-administered with a corticosteroid and an antihistamine, both of which can be administered orally or intravenously, 30 to 60 minutes prior to administration of utuximab or an anti-CD20 antibody that binds to the same epitope as utuximab. In some embodiments, the corticosteroid and/or antihistamine is administered orally to the subject. In some instances, the pre-treatment dose of the corticosteroid is about 100 mg of methylprednisolone, 10 to 20 mg of dexamethasone, or an equivalent corticosteroid.

In some embodiments, utuximab is administered in a multiple dose regimen. In certain instances, utuximab is administered by intravenous infusion. For example, utuximab may be administered by intravenous infusion in a multiple infusion dose regimen.

In some embodiments, utuximab is administered to patients with RMS by intravenous infusion for 48 weeks in a multiple infusion dosing schedule. In some embodiments, utuximab is administered to patients with RMS by intravenous infusion for 96 weeks in a multiple infusion dosing schedule.

The multiple infusion dosing regimen may include a first infusion, a second infusion, and a subsequent intravenous infusion of Utuximab. In some embodiments, a "subsequent infusion" of Utuximab may be any number of infusions after the first infusion and the second infusion.

In some embodiments, the multiple infusion dosing regimen comprises a first infusion and a second infusion of 150 mg utuximab intravenously (first infusion), followed two weeks later by 450 mg utuximab intravenously (second infusion). In some embodiments, the multiple infusion dosing regimen further comprises subsequent infusions of 450 mg utuximab intravenously every 6 months.

In some embodiments, the duration of the first infusion of utuximab or an anti-CD20 antibody that binds to the same epitope as utuximab is about four hours. For example, the infusion rate of the first infusion of utuximab or an anti-CD20 antibody that binds to the same epitope as utuximab may be 10 mL per hour for the first 30 minutes; 20 mL per hour for the next 30 minutes; 35 mL per hour for the next hour; and 100 mL per hour for the remaining two hours. In some embodiments, the duration of the second and subsequent infusions of utuximab is about one hour. For example, the infusion rate of the second and/or subsequent infusions of utuximab may be 100 mL per hour for the first 30 minutes and 400 mL per hour for the remaining 30 minutes. If the infusion is interrupted or slowed, the infusion may last longer.

In some embodiments, individuals are monitored for at least one hour after the completion of the first two infusions of Utuximab. Post-infusion monitoring is not required for subsequent infusions unless an infusion-related reaction (IRR) and/or allergic reaction is observed.

In some embodiments, if the individual has symptoms of a life-threatening infusion-related reaction, the intravenous infusion of utuximab is stopped and permanently discontinued. In some embodiments, if the individual has symptoms of a severe infusion-related reaction, the intravenous infusion of utuximab is discontinued and restarted once the individual's infusion-related reaction symptoms disappear. In some embodiments, the intravenous infusion of utuximab is restarted at half the infusion rate at the onset of the infusion-related reaction. If the individual tolerates half the infusion rate, the infusion rate of utuximab can be increased to the original infusion rate. In some embodiments, if the individual has symptoms of a mild to moderate infusion-related reaction, the infusion rate of utuximab is reduced by half, wherein the half infusion rate is maintained for at least 30 minutes. If the individual tolerates half the infusion rate, the utuximab infusion rate can be increased to the original infusion rate.

In some embodiments, utuximab is administered by intravenous infusion such that the geometric mean steady-state AUC is 3000 mcg/mL (CV=28%) per day and the mean maximum concentration is 139 mcg/mL (CV=15%).

In some embodiments, utuximab may be administered by a route other than intravenous infusion (e.g., subcutaneous injection, intramuscular injection, oral, epidermal, spinal, or inhalation). 7.13      Pharmaceutical compositions

A "pharmaceutical composition" may refer to a composition that is acceptable for pharmaceutical administration, such as to humans. Such a composition may include substances in amounts no greater than those acceptable for pharmaceutical administration, including the absence of such impurities, and may include, in addition to any active agent, pharmaceutically acceptable excipients, vehicles, carriers, stabilizers, and other inactive ingredients, for example, to prepare such a composition for administration.

The present invention provides a pharmaceutical composition comprising utuximab or an antibody that binds to the same epitope as utuximab for use in treating a subject suffering from a relapsing form of multiple sclerosis according to any of the methods disclosed herein.

In some embodiments, the pharmaceutical composition comprises utuximab. In some embodiments, the pharmaceutical composition comprising utuximab is formulated with a pharmaceutical carrier. Suitable pharmaceutical carriers are those known to those of ordinary skill (Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA (1990)).

Pharmaceutical compositions may contain any number of excipients. Excipients that may be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients are taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy , 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

The pharmaceutical compositions described herein may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound may be coated in a material to protect it from the action of acids and other natural conditions that may inactivate it. As used herein, "parenteral administration" may refer to modes of administration other than enteral and topical administration (usually by injection), and may include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Additionally or alternatively, the pharmaceutical compositions described herein can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration (e.g., intranasal, oral, vaginal, rectal, sublingual or topical).

In some embodiments, the pharmaceutical composition comprising utuximab is for administration to a subject via intravenous infusion.

The following examples are provided for purposes of illustration and not limitation. 8. Examples 8.1 Example 1 - Glycosylation Spectra

The glycosylation profile of the anti-CD20 antibody protein samples provided herein is determined by measuring fluorescently labeled N-glycans (the fluorescent label is 2-aminobenzamide), which are enzymatically cleaved from the anti-CD20 antibody using PNGase F. The labeled glycans are resolved using a hydrophilic interaction column equipped. After separation, the glycans are passed through a fluorescent detector. Peak identification in the chromatogram of the test sample is based on retention time and relative to the peaks in the glycan standard that have been confirmed by mass spectrometry. The relative percentage of each N-glycan is calculated based on the N-glycan peak area divided by the total peak area of all N-glycans. The glycosylation spectrum is shown in Figure 2. 8.2 Example - Intact Mass Method

The glycosylation spectrum of the anti-CD20 antibody protein provided herein was evaluated under non-reducing conditions by intact mass analysis (LC-MS). During the chromatographic step using SEC and a mobile phase containing TFA, acetonitrile and water, the anti-CD20 antibody protein sample provided herein was first exchanged into an appropriate MS buffer. The sample was then introduced into ESI-QTOF for intact mass analysis. The mass spectrum was deconvoluted and the peaks were assigned according to mass. The relative abundance of each anti-CD20 antibody protein containing N-glycans provided herein was calculated by obtaining the abundance of N-glycans and dividing it by the total abundance of all identified peaks. The results are provided in the table below and in Figure 3. Table 8 : Intact molecular weight of anti -CD20 antibody protein sample according to LC-MS Abbreviation: ND = Not Detected. 8.3 Example 3 - Cell-based ADCC Assay (a) Materials and Methods

Antibody-dependent cytotoxicity (ADCC) is mediated by the binding of the Fc portion of TG-1101 (TG Therapeutics, Inc.) to FcγIIIA receptors on effector cells. The assay used for this analysis employed Eurofins-DiscoverX's "KILR CD16a Effector Cells", which are human CD8+ T lymphocytes derived from a single donor that have been engineered to express CD16 (FcγRIII) on their plasma membrane surface. These cytotoxic T cells reduce background killing and improve accuracy and reproducibility compared to PMBC preparations isolated from fresh blood. Raji cells were used as target cells, and ADCC activity was measured by lysis of target cells.

KILR cells were obtained from Eurofins, and Raji cells were obtained from ATCC. Reagent quality was ensured using a master cell bank and working cell bank system. Raji cells were seeded at 1x10 ⁵ cells/mL, KILR effector cells were seeded at 5x10 ⁵ cells/mL, and the final effector: target (E:T) ratio was 5:1. Eight-point serial dilutions of samples were used with a concentration range of 250.00 pg/ml to 0.04 pg/ml (250, 50, 16.7, 5.6, 1.9, 0.6, 0.2, 0.04 pg/ml). Cell mixtures and test samples were incubated at 36 ± 1°C, 5 ± 1% CO ₂ for 18 to 22 hours. At the end of the incubation, CytoTox GLo™ preparation was added and the plates were incubated for 30 ± 10 minutes. Plates were read using a SpectraMax plate reader. Two independent preparations of material were prepared and analyzed on duplicate plates. Assay controls were prepared in triplicate and included: target cell alone control, target cell death control, effector cell alone control, and effector and target cell controls. SoftMax Pro was used to analyze the data using a 4-parameter logical fit using weighted nonlinear regression. Results are reported as percent ADCC activity compared to the Ublituximan reference standard. EC50s for each test sample were generated based on the 4-parameter logical fit curve to obtain more information. This test method is a validated analytical method CTSOP482, which was used for release and stability testing of TG-1101 (TG Therapeutics, Inc.) drug substance and drug product. (b) Results

The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, Ocrevus, and Arzerra. The commercial reference standard RS-117808 (117808) of TG-1101 (TG Therapeutics, Inc.) was used as a control. As shown in Figure 4 , all anti-CD20 samples showed dose-dependent ADCC activity, and TG-1101 (TG Therapeutics, Inc.) had the highest ADCC activity compared to other anti-CD20s. Compared to the TG-1101 (TG Therapeutics, Inc.) reference standard, the ADCC activity expressed as a percentage of the TG-1101 (TG Therapeutics, Inc.) reference standard is shown in Table 9. Gazava has relatively similar ADCC activity to TG-1101 (TG Therapeutics, Inc.), while the ADCC activities of Rituxan, Ocrevus and Arzerra are significantly lower. The ED50 of each anti-CD20 is also shown in Table 9. Overall, TG-1101 (TG Therapeutics, Inc.) and Gazyva have lower EC50s than Arzerra, Rituxan and Ocrevus. The EC50 of TG-1101 (TG Therapeutics, Inc.) is approximately 25 times lower than that of Ocrevus. Table 9 : ADCC activity and EC50 8.4 Example 4 - Cell-based ADCC assay using primary NK cells (a) Materials and methods

This ADCC assay was performed using CD20 expressing Raji cells as target cells, primary NK cells as effector cells, and LDH as target cell lysis readout. Raji cells (ATCC, Cat# CCL-86TM) were plated at 1x10 ⁵ cells/well. Primary NK cells were isolated from human donor PBMC using the Miltenyibiotec kit (Cat# 130-092-657). NK92/CD16a cells and primary NK cells with an E/T ratio of 5:1 were used in this assay. Eight-point serial dilutions of samples were used in triplicates ranging from 0.01 μg/ml to 0 ug/ml with a dilution factor of 10. The target cells and the test sample dilution were incubated in a 37°C incubator for 30 minutes. The effector cells were added to the target cell culture and then incubated for 6 hours, after which the supernatant was collected. The OD492 nm data minus the background (OD650 nm) was used to calculate LDH release. The percentage of cell lysis was calculated according to the following formula: Cell lysis % = 100 * (OD sample data - OD tumor cells plus effector cells) / (OD maximum release - OD minimum release). (b) Results

The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, and Arzerra. As shown in Figure 5 , all anti-CD20 samples showed dose-dependent ADCC activity. The calculated EC50 values are summarized in Table 10. TG-1101 (TG Therapeutics, Inc.) and Gazyva showed higher ADCC activity and lower EC50 than Rituxan and Arzerra. Table 10 : EC50 of ADCC analysis using primary NK cells 8.5 Example 5 - Cell-based ADCP Assay (a) Materials and Methods

Antibody-dependent cellular phagocytosis (ADCP) is another potential mechanism of action (MOA) of anti-CD20. ADCP activity was assessed using an assay using CD20-expressing Daudi cells as target cells (ATCC, Cat# CCL-213, labeled with PKH26). Human monocytes were isolated from PBMCs of 20 human donors (using the Human Pan Monocyte Isolation Kit, MiltenyiBiotec, Cat# 130-096-537) and differentiated in vitro using GM-CSF to generate macrophages. An E/T ratio of 5:1 was used; and ADCP was assessed by flow cytometry in this assay. Eight serial dilutions of samples ranging from 100 μg/ml to 0 ug/ml (dilution factor 10) were incubated with PKH26-labeled target cells in triplicate. Macrophages were then co-cultured with PKH26-labeled target cells for 22 hours. Target cell phagocytosis was assessed by flow cytometry. Controls in the analysis included a target cell control of Daudi cells stained only with PKH26; and an effector cell control of MDM stained only with PKH67.

Effector cells and target control and non-specific IgG1 antibody; effector cells and target cell control (background control). ADCP was determined by FACS as the percentage of PKH26/PKH67 double positive cells/PKH26. (b) Results

The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, and Arzerra. As shown in Figure 6 , all anti-CD20 samples exhibited dose-dependent ADCP activity. The calculated EC50 values are summarized in Table 11. The EC50 was in the ng/ml range, and the ADCP activity of all samples was considered similar considering the nature of the assay. Table 11 : EC50 of ADCP Assay Blind code: 50K068:70T:2003 = Gazyva; 52W243:70T:2003 = ARZERRA; 54A157:70T:2003 = Rituxan 8.6 Example 6 - Cell-based CDC assay (a) Materials and methods

Complement-dependent cytotoxicity (CDC) is mediated by binding of the Fc portion of TG-1101 (TG Therapeutics, Inc.) to the C1q receptor in the complement system. The CDC activity assay used in this analysis is a cell-based assay using the CD20-expressing human mantle cell lymphoma cell line Jeko-1 and rabbit serum as a complement source. CDC-mediated cell lysis was measured by Cell Titer-Glo™ Assay (Promega). Nine-point serial dilutions of samples were used with a concentration range of 10,000 ng/ml to 10.42 ng/ml (10,000.00, 3333.33, 1666.67, 833.33, 416.67, 208.33, 104.17, 52.08, 10.42 ng/ml). Two independent preparations were prepared for each sample and analyzed on duplicate plates. Negative controls were prepared and analyzed in triplicate and included target cell and complement controls as well as target cell alone controls.

Jeko-1 cells obtained from ATCC and maintained through a master bank system were inoculated at 3x10 ⁵ cells/mL and incubated for 60 to 90 minutes. Sample dilutions were added, followed by complements, and the plates were incubated for approximately 1 hour at 37°C and 25 minutes at room temperature. Controls with only target cells plus complements and only target cells provided a baseline level of target cell viability during the analysis. Cell Titer-Glo reagent was then added and incubated for an additional 30 minutes at room temperature. At the end of the analysis, the plates were read using a SpectraMax M5 plate reader. SoftMax Pro was used to analyze the data using a 4-parameter logical fit using weighted nonlinear regression. The concurrency and potency of the obtained data were evaluated relative to a reference standard using PLA software. Results are reported as % potency relative to the reference standard. EC50 for each test sample was generated based on a 4-parameter logical fit to obtain additional information. This test method is a validated assay CTSOP463 used for TG-1101 (TG Therapeutics, Inc.) drug substance and drug product release and stability testing. (b) Results

The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, Ocrevus, and Arzerra. The commercial reference standard RS-117808 (117808) of TG-1101 (TG Therapeutics, Inc.) was used as a control. As shown in Figure 7 , all samples, except Gazyva (known to have reduced CDC activity), showed dose-dependent CDC activity. Rituxan and Arzerra had comparable CDC activity; Ubli and Ocrevus had comparable CDC activity. The CDC activity expressed as a percentage of the TG-1101 (TG Therapeutics, Inc.) reference standard is shown in Table 12. Gazava has relatively similar ADCC activity to TG-1101 (TG Therapeutics, Inc.), while Rituxan, Ocrevus, and Arzerra have significantly lower ADCC activity compared to TG-1101 (TG Therapeutics, Inc.). The ED50 of each anti-CD20 is also shown in Table 12, which shows similar comparison results with the degree of CDC activity. Table 12 : CDC Activity and EC50 8.7 Example 7 - Cell-based CD20 binding assay (a) Materials and methods

The CD20 binding used for this analysis is a cell-based binding assay using the CD20 expressing human mantle cell lymphoma cell line Jeko-1 and the MSD (MesoScale Discovery) assay format. Jeko-1 target cells are seeded onto the MSD plates and the test samples are incubated and allowed to bind to the Jeko-1 cells using an anti-human Fc detection antibody conjugated to streptavidin-SULFOTAG™ to emit an electrochemiluminescent signal. An eight-point serial dilution of the test sample was used over a concentration range of 40,000.00 µg/ml to 0.23 ng/ml (40,000.00, 4,000.00, 1,000.00, 333.30, 111.10, 37.00, 4.60, 0.23 ng/ml). Two independent test material preparations were prepared for every 2 plates evaluated. Analytical controls included a no-cell control (reference standard/test material dilution + probe, cells omitted) and a cell-only control (cells + probe, reference standard/test material omitted).

Jeko-1 cells obtained from ATCC and maintained by the master bank system were seeded at 3x10 ⁵ cells/mL into MSD high binding plates containing PBS (final volume of 100 µL per well) and incubated at 35 to 37°C for 2 hours ± 10 minutes. Unbound cells were removed by washing with PBS, and the plates were blocked and then washed. 50 µL of sample dilution was added and the plates were incubated at room temperature with shaking for 1 hour ± 10 minutes. After incubation and 3 washes, 50 µL of anti-human Fc detection antibody conjugated with STREP-SULFOTAG was added and incubated at room temperature with shaking for 1 hour ± 10 minutes. The plate was washed again and 150 µL of MesoScale reading buffer containing tripropylamine (TPA) was added as a co-reactant to generate light for the electrochemical luminescence readout. The plate was immediately read on an MSD Reader using Workbench 4.0. The resulting data were evaluated using PLA software and analyzed using a constrained 4-parameter logic model. Binding activity results are reported as percent potency relative to a reference standard. An EC50 for each test sample was generated based on the 4-parameter logic fit curve to obtain additional information. This test method is a validated assay CTSOP466 used for TG-1101 (TG Therapeutics, Inc.) drug substance and drug product release and stability testing. (b) Results

The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, Ocrevus, and Arzerra. The TG-1101 (TG Therapeutics, Inc.) commercial reference standard RS-117808 (117808) was used as a control. As shown in Figure 8 , all samples exhibited dose-dependent CD20 binding. Except for Gazyva (a type II anti-CD20 known to have a target occupancy of approximately 50%), Utuximab, Rituxan, Ocrevus, and Arzerra also had similar maximum binding. The CD20 binding activity expressed as a percentage of the TG-1101 (TG Therapeutics, Inc.) reference standard and the binding EC50 are shown in Table 13. The CD20 binding affinity of the four anti-CD20s is similar. Table 13 : CD20 binding activity and EC50

EC50 values listed are negative values, logarithmic transformation from PLA software converts concentrations to base 2. The actual ED50 (in µg/ml) is ²ⁿ , where n = the number listed. 8.8 Example 8 - Cell Surface CD20 Binding by FACS (a) Materials and Methods

Binding of TG-1101 (TG Therapeutics, Inc.) to CD20 on the surface of Raji and Daudi cells was characterized by FACS analysis at LakePharma. Six serial dilutions of the sample were used in duplicate with a concentration range of 40 μg/ml to 0 ug/ml, with a dilution factor of 5. Cells were incubated with sample dilutions; binding was detected using PE-conjugated anti-human secondary antibodies. (b) Results

The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Arzerra, and Rituxan. As shown in Figure 9, all anti-CD20 samples exhibited dose-dependent CD20 binding to Daudi cells and Raji cells. The binding characteristics as assessed by FACS analysis were similar to those assessed by MSD analysis. With the exception of Gazyva (a type II anti-CD20 known to have a target occupancy of approximately 50%), Utuximab, Rituxan, and Arzerra also had similar maximum binding. The calculated EC50 values are summarized in Table 14. The CD20 binding affinities of the four anti-CD20 antibodies were similar. Table 14 : EC50 of cell surface CD20 binding by FACS 8.9 Example 9 - FcγRIIIA binding analysis (a) Materials and methods

The assay used in this analysis is a surface plasmon resonance (SPR) based approach that measures binding to both FcγRIIIa 158V and FcγRIIIa 158F receptors. The method follows a direct binding assay approach where the FcγRIIIa receptor is immobilized directly to a flow cell on the surface of a sensor chip and samples are injected onto the chip to assess binding. Either the FcγRIIIa 158V receptor (3 μg/ml) or the FcγRIIIa 158F receptor (6 μg/ml) was immobilized on the chip surface using covalent amine coupling chemistry. Eight point serial dilutions of the test samples were made at concentrations ranging from 1000 nM to 15.6 nM with a dilution factor of 2. The sample dilutions were injected onto the chip in independent duplicates and the surface was regenerated between each cycle. Binding is measured in response units (RU). The kinetics of the binding reaction are determined by measuring the change in SPR due to the increase in mass close to the biosensor chip surface. The change in complex mass over time is visualized as a sensorgram.

The equilibrium dissociation constant ( _KD ) was determined for each sample for each receptor. The rate of change of the SPR signal was analyzed using a 1:1 Langmuir model for the FcγRIIIa 158V variant to generate apparent rate constants for the association and dissociation phases of the reaction, as well as the equilibrium dissociation constant. The _KD was determined for the FcγRIIIa 158F variant using steady-state affinity. The binding signal was exported to PLA to determine the relative binding reactivity, relative affinity, and relative binding of the samples and reported relative to the TG-1101 (TG Therapeutics, Inc.) reference standard. This assay is a validated assay, CTSOP477, used for TG-1101 (TG Therapeutics, Inc.) drug substance release testing. (b) Results

The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, Ocrevus, and Arzerra. As shown in Table 15, among the anti-CD20 antibodies tested, TG-1101 (TG Therapeutics, Inc.) had the highest binding affinity for both FcγRIIIa 158V and FcγRIIIa 158F receptors. Gazyva ranked second in terms of binding affinity. For the high-affinity receptor FcγRIIIa158V, the affinity of TG-1101 (TG Therapeutics, Inc.) was approximately 15 times higher than that of Ocrevus; for the low-affinity receptor FcγRIIIa158F, the affinity of TG-1101 (TG Therapeutics, Inc.) was approximately 10 times higher than that of Ocrevus. Table 16 shows the relative affinity and relative binding values using TG-1101 (TG Therapeutics, Inc.) reference standard as a reference. The results demonstrate that TG-1101 (TG Therapeutics, Inc.) has higher relative binding and relative affinity than all other anti-CD20s. Table 15 : KD for FcγRIIIa 158V and FcγRIIIa 158V based on SPR binding *: Average of 4 values Table 16 : Summary of relative affinity and relative binding results 8.10 Example 10 - Fc receptor binding based on Octet ( a) Materials and methods

This analysis was performed by LakePharma. Binding characterization was performed on an Octet HTX instrument at 25°C. Human Fc receptor groups were loaded onto Anti-PentaHis (H1S1K) biosensors. The loaded sensors were immersed in serial dilutions of the test samples (starting at 300 nM, 1:3 dilution, 7 points). Kinetic constants were calculated using a monovalent (1:1) binding model. (b) Results

The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, Ocrevus, and Arzerra. As shown in Table 17, among the anti-CD20 antibodies tested, TG-1101 (TG Therapeutics, Inc.) had the highest binding affinity for both FcγRIIIa 158V and FcγRIIIa 158F receptors, confirming the SPR data. The binding affinity to FCRN was similar among all anti-CD20s, which will affect the PK. TG-1101 (TG Therapeutics, Inc.) also had higher affinity for FcγRIIA and FcγRIIIB compared to Ocrevus. Table 17 : Summary of _KD Results for Fc Receptors 8.11 Example 11-C1q binding analysis (a) Materials and methods

The C1q binding assay used for this analysis is an ELISA assay. The sample is coated on an ELISA plate and human C1q conjugated to HRP is incubated with the sample on the plate. In the presence of the substrate TMB, the bound HRP produces a colorimetric signal. A 7-point serial dilution of the test material is prepared at a concentration range of 15.00 ug/ml to 0.12 ug/ml with a dilution factor of 2. The sample dilutions are coated on the ELISA plate and the plate is incubated at room temperature for 1 hour ± 30 minutes. After coating, the plate is washed, blocked, and washed again. Peroxidase-conjugated C1q is added and the plate is incubated at room temperature for 1.5 hours ± 30 minutes. After incubation and washing, tetramethylbenzidine (TMB) substrate solution was added and the plates were incubated at room temperature for 7 minutes (-1 minute/+30 seconds). This produces a colorimetric reaction that is proportional to the extent of C1q binding. The reaction was stopped by adding 1M sulfuric acid and the color was measured at 450 nm using a Molecular Devices SpectraMax microplate reader. SoftMax Pro was used to analyze the data using weighted nonlinear regression using a 4-parameter logic fit. Binding activity results are reported as a percentage of potency relative to the TG-1101 (TG Therapeutics, Inc.) reference standard. EC50s for each test sample were generated based on the 4-parameter logic fit curve to obtain additional information. This test method is a validated assay CTSOP455 for TG-1101 (TG Therapeutics, Inc.) drug substance and drug product release and stability testing. (b) Results

The samples tested included TG-1101 (TG Therapeutics, Inc.), Gazyva, Rituxan, Ocrevus, and Arzerra. The TG-1101 (TG Therapeutics, Inc.) commercial reference standard RS-117808 (117808) was used as a control. As shown in Figure 10 , Gazyva had minimal C1q binding (as expected), while the other four anti-CD20s exhibited dose-dependent C1q binding. The maximum binding of TG-1101 (TG Therapeutics, Inc.) was slightly higher. The C1q binding activity expressed as a percentage of the TG-1101 (TG Therapeutics, Inc.) reference standard is shown in Table 18. The ED50 of each anti-CD20 is also shown in Table 18. The EC50 of TG-1101 (TG Therapeutics, Inc.) is slightly lower than all anti-CD20, indicating that TG-1101 (TG Therapeutics, Inc.) has a higher affinity for C1q. Table 18 : C1q binding activity and EC50 8.12 Example 12 - Human whole blood B cell depletion analysis (a) Materials and methods

TG-1101 (TG Therapeutics, Inc.) was characterized in an autologous normal human whole blood B cell depletion assay and compared to Gazyva, Rituxan, Ocrevus, and Arzerra. Whole blood from three human donors was used, and donors with the FCGR3A_SNP target (rs396991) 158V/158V genotype were selected. Sample dilutions ranging from 0.000001 to 100 µg/ml were added to whole blood and incubated at 37°C in a humidified cell incubator for 24 hours. Blood aliquots were stained for markers including CD45 (lymphocyte population), CD3 (T cells), CD19 (B cells), and CD20 (B cells). B-cell depletion was assessed by displaying cells in the CD45-positive lymphocyte gate and counting CD3-positive T cells, CD19-positive B cells, and CD20-positive B cells. The percentage of B-cell depletion was calculated (100-([100/B-/T-cell ratio in samples without antibody] x [B-/T-cell ratio in samples with antibody])) and plotted against sample concentration. (b) Results

The samples tested included TG-1101 (TG Therapeutics, Inc.) commercial reference standard RS-117808 (117808) (API batch produced by commercial process (C2) at Samsung Biologics (PPQ1), Gazyva, Rituxan, Ocrevus, and Arzerra. As shown in Figures 11A to C , all samples exhibited dose-dependent B cell depletion activity, although there were some minor differences between donors. Overall, TG-1101 (TG Therapeutics, Inc.) and Gazyva had higher B cell depletion activity than Arzerra, Rituxan, and Ocrevus. Three anti-CD20 antibodies and one anti-CD19 antibody were used in this experiment. The calculated ED50s for B cell depletion using various antibodies for B cell markers are summarized in Table 19. Overall, the EC50s for TG-1101 (TG Therapeutics, Inc.) and Gazyva were also lower than those for Arzerra, Rituxan, and Ocrevus. On average, the EC50 for TG-1101 (TG Therapeutics, Inc.) was more than ten-fold lower than that for Ocrevus. Table 19 : Summary of ED50s for B cell depletion for multiple B cell markers 8.13 Example 13 - Calculation of Pharmacokinetic (PK) Values

Relevant steady-state PK parameters are calculated according to methods known in the art. Steady state is achieved when the amount of drug eliminated per unit time is equal to the amount of drug reaching the systemic circulation per unit time. Therefore, the half-life represents the time required for the plasma concentration of the drug to decrease by 50% to reach steady state. Where; t = time, Vd = volume of distribution, Cl = clearance. The half-life is calculated using the following formula:

AUC represents the total dose of drug exposed over time. AUC is used as a measure when determining equivalent doses of a formulation and the resulting tissue or plasma exposure. AUC is equivalent to the average concentration over a time interval. Where t = time and Cpt = the last measured drug concentration relative to time. AUC is calculated using the following formula:

After the absorption phase ends and the elimination phase begins, Cmax is obtained by measuring the highest point of drug concentration during the observation period. After the absorption phase ends and the elimination phase begins, Cmin is obtained by measuring the highest point of drug concentration during the observation period. 8.14 Example 14 - Method for Determining Population Pharmacokinetic (PPK) Values of TG-1101 for the Treatment of Autoimmune Diseases

Pop PK analysis of TG-1101 was performed by pooling TG-1101 serum concentration-time, dose, demographic, and covariate data from one Phase 2 study (TG1101-RMS201) and two Phase 3 studies (TG1101-RMS301 and TG1101-RMS302) in RMS subjects. The data sets were combined with previous TG-1101 data sets in subjects with hematological malignancies.

All subjects who received at least one dose of TG-1101 in studies TG1101-RMS201, TG1101-RMS301, and TG1101-RMS302 were included in the dataset for PK analysis. Subjects who did not have at least one quantifiable post-dose TG-1101 concentration were included in the dataset and flagged. Exposures for these subjects were determined based on typical population PK parameters.

In the Phase 2a clinical trial TG1101-RMS201, TG-1101 was administered as a single agent and compared to placebo to investigate the extent of B cell depletion by TG-1101 and to determine the optimal dose and infusion schedule of TG-1101 in RMS subjects. Based on the results of this study, a dose of 150 mg (infused over 4 hours) on Day 1, Week 1, followed by a dose of 450 mg (infused over one hour) on Day 15, Week 3, achieved a median >99% B cell depletion at Week 4 that was sustained through Week 24. The dosing regimen was well tolerated by subjects, with infusion-related reactions (Grades 1 and 2) being the most commonly reported adverse events. Two Phase 3 studies in RMS subjects, TG1101-RMS301 (also known as ULTIMATE I) and TG1101-RMS302 (ULTIMATE II), have been completed as randomized, double-blind, double-dummy, active-controlled studies comparing TG-1101 with oral teriflunomide to evaluate the ARR, safety, and tolerability in RMS subjects.

Overall, the dataset for PopPK analysis included a total of 8672 PK samples collected from 931 subjects. Predose samples accounted for 10.02% of the PK samples, while records with missing information or outliers (>10 standard deviations from the mean TG-1101 concentration at the nominal dosing time) accounted for 0.20% and were excluded. Postdose samples from BLQ accounted for 3.47% of the data. The exclusion resulted in 36 subjects without quantifiable postdose PK samples. Therefore, the PK analysis dataset included 7485 PK samples from 895 subjects, of which 5624 PK samples were from 591 RMS subjects.

The final PopPK parameter estimates are presented in Table 20. The PK parameter estimates for a typical subject (defined as a male subject from North America or Western Europe, weighing 73 kg, and ADA-negative) were as follows: CL was estimated to be 11.6 mL/h, IIV was 38.1%; Ve was estimated to be 3.18 L (IIV=15.0%); Vp was estimated to be 3.60 L (IIV=21.3%); and Q was estimated to be 11.6 mL/h.

Body weight and ADA were found to be statistically significant predictors of TG-1101 CL. ADA-positive subjects had a modest increase in TG-1101 CL of 14% compared to subjects without quantifiable ADA. For the RMS subpopulation with a wide range of weights (45.1 to 154 kg), CL ranged from a low of 22% to a high of 48% relative to a typical subject weighing 73 kg. Additionally, at the late stage (417 days after treatment initiation), the median CL reduction was 12.5%.

Weight, sex, and region were found to be statistically significant predictors of Vc. Subjects from Eastern Europe had slightly higher Vc than Western Europeans and North Americans (10%), and females had slightly lower Vc than males (7%). For the RMS subgroup with a wide range of weights (45.1 to 154 kg), Vc ranged from a low of 19% to a high of 38% for a typical subject weighing 73 kg.

After incorporating body weight into the model, age, hemoglobin concentration, platelet count, white blood cell count, renal impairment, or liver impairment had no effect on TG-1101 PK. Table 20 : Parameter estimates for the final TG-1101 PK model Abbreviations: CL-clearance; CV%-coefficient of variation percentage; ETA-individual-specific random effect; IIV-interindividual variability; Q-intercompartmental clearance; RSE-relative standard error; SD-standard deviation; SE-standard error; U2-TG-1101 + umbralisib; Vc-volume of the central compartment; Vp-volume of the peripheral compartment. ^a RSE for parameter estimates was calculated as 100 x (SE/typical value); RSE for IIV magnitude was calculated as 100 x (SE/estimated variability). ^b Estimates of random effect and IIV are presented as CV% and are based on estimates calculated as √variability x 100. ^c Contraction (%) was calculated as 100 x (1-SD[ETA]/√[variance]). ^d The correlation coefficient between CL and Vc was estimated to be 0.434.

In estimating the final TG-1101 PopPK model, standard errors were obtained and are presented along with the parameter estimates in Table 20. Model parameters were accurately estimated with RSE < 20% for structural and covariate model parameters and RSE < 30% for random effect estimates. The shrinkage for CL and Vc was acceptable (5.32% and 32.6%, respectively), whereas the shrinkage for Vp was larger (40%).

The key goodness of fit (GOF) diagnostics for the final PopPK model for TG-1101 showed satisfactory fit over time with minimal residual bias between predicted concentration values, and good agreement between predicted and observed concentrations. Relative to the base model, ETA-covariate relationships were resolved, and there were no significant further trends between ETA (for CL and Vc) and covariates in the RMS subpopulation, indicating that the model adequately captured significant covariate relationships. Overall, the prediction-corrected visual prediction test (pcVPC) plot indicated that the model adequately predicted the central tendency of observed TG-1101 concentrations and adequately represented the range of the data.

The relative importance of the effects of the covariates included in the final PoPK model was assessed using forest plots of relative changes in exposure (Cmax,ss, Cmin,ss, and AUCss) when the covariates were varied one at a time (i.e., univariate analysis). The effects of these covariates (including changes in body weight, sex, region, ADA, and late CL fraction) on TG-1101 exposure ranged from 0.8 to 1.25 compared to the reference exposure (defined as exposure in male subjects from North America/Western Europe weighing 73 kg, ADA negative, and who had been treated for <416 days). Therefore, none of the covariates were considered clinically relevant. In addition, the covariates did not significantly affect the magnitude of the IIV of CL or Vc. The combined effect of weight and ADA reduced IIV by only 2.8%, from 39.2% in the baseline model to 38.1% in the final model (Table 20). Weight, sex, and region also reduced IIV in Vc by 18.5%, from 18.4% in the baseline model to 15.0% in the final model.

Figure 12 shows the goodness of fit (GOF) diagnosis of the final model for TG-1101. Figure 13 shows the pcVPC of the final PK model for TG-1101 based on the study. Figure 14 shows the forest plot of the covariate effects on TG-1101 drug exposure.

The final PopPK model was reestimated using the data set including the exclusion of outliers. All structural parameters were accurately estimated, with no relevant changes in those estimates, whereas the IIV term was inflated by the inclusion of outliers. Therefore, it was expected that the exclusion of outliers during the model development process would eliminate the effects of these outliers and minimize spurious results in the covariate analysis. The final PopPK model including only the RMS subpopulation was also reestimated. The fit of the model to the RMS data yielded similar structural parameters and random effects, with the exception of a slightly lower effect of CL on IIV; the precision of the parameter estimates was also similar.

Conclusion: TG-1101 PPK Analysis

The final model for TG-1101 was used to obtain individual post hoc estimates of PK parameters. For each subject enrolled in studies TG1101-RMS201, TG1101-RMS301, and TG1101-RMS302, PK parameters (AUCss, Cavg,ss, Cmax,ss, and Cmin,ss) were estimated at Week 48 based on the post hoc PK parameters.

The geometric mean 1½ (90% confidence interval [CI]) was calculated to be 21.8 days (21.4, 22.1 days). The median time to steady state was determined to be 15.5 weeks. Thus, there was no accumulation in per-protocol subjects who received 150 mg TG-1101 on Day 1, followed by 450 mg TG-1101 on Day 15, Week 24, and Week 48. The ratio of the median Cmax at Week 24 to the Cmax at Day 1 was 3.04 (range, 3.00 to 3.42), consistent with a 3-fold increase in the number of doses and indicating no accumulation. Similarly, the ratio of the Cmax at Week 48 to Week 24 was 1, indicating no accumulation.

The model predicted geometric mean values of AUCss, Cavgss, Cmaxss, and Cminss were 3000 ug·d/mL (±28%), 8940 ng/mL (±28%), 139000 (±15.1%), and 139 (±170%), respectively. 8.15 Example 15 - Commercial-scale manufacturing method for producing TG-1101 in YB2/0 rat fusion tumor cells

In this example, the production method of TG-1101 expressed in YB2/0 rat fusion tumor cells at 15,000 μL is described. The flow chart in Table 21 illustrates an overview of the production method of TG-1101. Table 21 : Flow chart of the production method of TG-1101

In summary, the production of each batch of TG-1101 begins with thawing of a working cell bank (WCB) vial, as further described below. The culture is expanded through a series of shake flasks and inoculation bioreactors to meet the inoculum requirements of a 15,000 L production bioreactor, which is operated in fed-batch mode. The bioreactor is harvested and clarified by centrifugation followed by deep filtration. The clarified harvest is purified through three chromatographic steps, including protein A, cation exchange, and anion exchange, which are designed to purify TG-1101 and reduce process impurities (such as host cell proteins and residual DNA). The purification process (as shown in Example 4) contains steps to ensure viral safety, including viral inactivation (solvent/detergent) and viral filtration steps. The final UFDF and formulation steps are used to concentrate TG-1101 and buffer exchange it into the formulation buffer and achieve the desired product concentration. The API to be filled is formulated at a concentration of 25.0 mg/mL in 25 mM sodium citrate, 154 mM sodium chloride, 0.07% polysorbate 80, pH 6.5 to obtain TG-1101. After filling, the TG-1101 API is frozen at ≤-60°C and then stored frozen at ≤-35°C.

Additional descriptions of upstream and downstream process operations are provided below. (a) Expression vectors, production cell lines, and cell banks

The host cell line used to generate the TG-1101 production cell line is the rat cell line YB2/0. The production cell line R603-12D11 was developed by transfecting the expression vector HK463-25 (containing the immunoglobulin heavy and light chain cDNA sequences of TG-1101) into the YB2/0 host cell line. Figure 15 depicts the expression vector diagram of HK463-25 producing TG-1101 in a 15,000 L bioreactor. Expression vector

The expression vector HK463-25 incorporates various elements optimized for stable expression in the YB2/0 host cell line. The Rous sarcoma virus long terminal repeat (RSV LTR) promoter is used for constitutive expression of heavy and light chain cDNAs. This promoter corresponds to the long terminal repeat sequence of the RSV genome, contains enhancer elements in the 5' region, and has strong transcriptional activity in the YB2/0 cell line. Transcriptional termination and polyadenylation of both heavy and light chain cDNAs are provided by the human growth hormone polyadenylation sequence (hGH polyA). Chimeric introns are introduced 5' to the cDNA sequence of each antibody chain to enhance expression. This intron is optimized for splicing and consists of a 5' donor sequence from human β-globin and a 3' acceptor sequence from an Ig heavy chain variable gene. The β-lactamase gene confers resistance to ampicillin (AmpR) and is used to produce plasmids in E. coli. The enzyme neomycin-phosphotransferase II (NeoR) is under the control of the SV40 promoter and confers resistance to the antibiotic G418 to transfected cell lines, thereby serving as a selectable marker. Dihydrofolate reductase (Dhfr) is under the control of the SV40 promoter, confers resistance to methotrexate (MTX), and can also serve as a selectable and amplifiable marker in transfected cell lines.

The HK463-25 expression vector ( Figure 15 ) is 11.1 kb in size and contains five open reading frames for the antibody heavy chain, light chain, Dhfr, NeoR, and AmpR genes in the same orientation. The restriction sites indicated in the figure were used for Southern blot analysis of the integration construct. A unique NotI restriction site located 3' of the NeoR gene was used to linearize the vector prior to transfection. Production cell line R603-12D11

Transfection of host cell lines, selection and screening of transfectants, and generation of production cell line R603-12D11 after limiting dilution selection. Screening of clones, selection of production cell line R603-12D11, and adaptation to serum-free medium. A pre-seeded stock solution (PSS) cell bank was prepared. An overview of the steps involved in the generation of production cell line R603-12D11 is shown in Table 22. Table 22 : Generation of production cell line R603-12D11

A frozen tube of YB2/0 cell bank (YB2/0-301 04/147) was thawed and the cells were grown by diluting to a cell density of 1×10 ⁵ cells/mL with fresh medium (EMS medium with 5% FCS) every 3 to 4 days. The cells were inoculated at a density of 2×10 ⁵ cells/mL on the day before transfection to reach the exponential phase before transfection. 44.5 μg of NotI-linearized expression vector HK463-25 ( FIG. 15 ) was transfected into 5×10 ⁶ cells by electroporation using animal component-free Optimix reagent (Equibio). The cells were diluted in medium and inoculated in 96-well plates at 100 cells/well. Three days after electroporation, screening was started with 1 g/L G418 in the culture medium.

After selection in G418 medium, transformants were initially screened for titer by ELISA. Over 3000 transformants were screened and over 200 of the best producing wells were selected for further testing and continued to be passaged. A second titer screen was performed to further reduce the number of clones. This screen was followed by additional screening of the antibodies for fucose content by ELISA. To provide an expected degree of CD16 activation of the antibody, a fucose content of less than 40% was considered ideal; CD16 activation is inversely proportional to fucose content. Further screening using a cell-based CD16 activation assay, which also assessed IL-2 secretion in addition to the free kappa/IgG ratio (<0.2 to facilitate purification), narrowed the results to a total of five cell lines for further development.

Five candidate cell lines were selected by limiting dilution at 0.4 cells/well in EMS medium with 5% FCS. The clones were screened for IgG productivity, fucose content, CD16 activation, and free kappa/IgG ratio to identify the production cell line R603-12D11. As the selected cell line was expanded from the selection step, it also underwent a switch to serum-free medium and was rescreened to ensure the desired phenotype before preparing a mini-cell bank. The mini-cell bank was then thawed and expanded to generate the R603-12D11 pre-stocked cell bank (PSS). The R603-12D11 PSS cell bank has been certified to be free of mold, adventitious viruses, and microbial contamination prior to MCB generation.

Master Cell Bank (MCP) Preparation and Testing

MCB was manufactured at Henogen (later acquired by NovaSep). MCB batch G071/MCB/070208 was prepared by thawing and expanding a vial of the production cell line R603-12D11 pre-seeded stock in serum-free medium EM-SF2 P500 H4 (EMS minimal medium supplemented with 2-hydroxyethanol, ethanolamine, NaHCO ₃ , ferric citrate, pluronic acid, HEPES, and recombinant human insulin). The cells were expanded for eleven days in T-flasks and roller flasks. The cell suspension was concentrated by centrifugation and aliquoted into 13 individual portions. Each fraction was centrifuged and resuspended in cryomedia (90% EM-SF2 P500 H4 + 10% DMSO) to a target cell density of 10×10 ⁶ cells/mL. The resuspended fractions were then aliquoted into 18 cryovials per fraction, for a total of 234 MCB cryovials. The cryovials were placed on dry ice and then placed in a cryovial box, which was placed in a -80°C freezer for 21 hours. The cryovials were transferred to liquid nitrogen tanks for long-term storage on February 19, 2007 and are currently stored at multiple locations. The cell passage number from PSS to MCB for the production cell line is 10.4.

Following the generation of MCB lot G071/MCB/070208, the MCB was tested directly and further characterized by testing the WCB derived from it. The identity of the MCB was tested; the test results confirmed the rat origin of the MCB. Performance qualification was confirmed by thawing a tube of MCB and monitoring cell viability. Copy number, restriction endonuclease spectrum, number of integration sites, integrity of RNA coding sequences, and RNA quantification were evaluated. The number of heavy and light chain copies integrated into the genome was estimated to be 1.13 and 2.14, respectively, based on quantitative polymerase chain reaction (Q-PCR). Southern blot evaluation of plastid integration sites indicated equivalent hybridization patterns for MCB and PSS. The number of integration sites was measured by Southern blotting in both MCB and WCB, and was 1 for both cell pools. In addition, intrachromosomal integration of the HK463-25 expression plasmid insertion at a single locus on the isochromosome was demonstrated by fluorescence in situ hybridization (FISH) analysis. For MCB, the integrity of the RNA coding sequence was consistent with the reference sequence, and the RNA quantification of the heavy and light chains was consistent with the PSS cell pool.

The MCB was subjected to next generation sequencing (NGS) (Cergentis, Utrecht, Netherlands) using targeted locus amplification (TLA) to confirm that the expressed TG-1101 antibody had the correct amino acid sequence and to check for the presence of low-level sequence variants. The MCB was tested for adventitious viruses, fungi, and microorganisms and met the acceptance criteria established for MCB release, including the acceptance criteria for adventitious viruses, fungi, and microorganisms. The overall testing results of the MCB confirmed the suitability of the MCB for establishment. Working Cell Bank Lot G140/R603/WCB001

The first WCB was made at NovaSep (after the acquisition of Henogen). To prepare the WCB, one tube of MCB G071/MCB/070208 was thawed and expanded in serum-free medium EM-SF2 P500 H4 in flasks and roller flasks over eleven days. The expanded cell suspension was concentrated by centrifugation and aliquoted into 22 equal parts. Each part was centrifuged and resuspended in freezing medium (90% EM-SF2 P500 H4 + 10% DMSO) to a target cell density of 12.1×10 ⁶ cells/mL. The suspended parts were then aliquoted into 18 cryovials each, resulting in a total of 396 WCB cryovials. The batch was designated G140/R603/WCB001. The cryovials were placed on dry ice and then in a freezer box, which was placed in a -80°C freezer for 24 hours. The cryovials were transferred to a liquid nitrogen tank for long-term storage on September 22, 2009. The WCB was stored in at least two different storage locations, including a small number of vials that were stored at the manufacturer for a shorter period of time. The cell generation number from the production cell line PSS to WCB was 21.4.

WCB lot G140/R603/WCB001 was tested, including identity testing. Test results confirmed the rat origin of WCB. Performance qualification was confirmed by thawing a tube of WCB and monitoring cell viability, doubling time, and IgG productivity. Copy number, restriction endonuclease spectrum, and the number of integration sites were evaluated. The number of heavy and light chain copies integrated into the genome was estimated to be 1.2 and 2.5, respectively, by quantitative polymerase chain reaction (Q-PCR). These results are consistent with the results of MCB. Southern blot evaluation of plastid integration sites demonstrated that the hybridization patterns of WCB and MCB are equivalent. The number of integration sites of WCB was determined to be 1 by Southern blot.

The MCB was subjected to next generation sequencing (NGS) (Cergentis, Utrecht, Netherlands) using targeted locus amplification (TLA) to confirm that the expressed TG-1101 antibody had the correct amino acid sequence and to check for the presence of low-level sequence variants. Testing for adventitious viruses, fungi, and microorganisms was performed and met the acceptance criteria established for WCB release, including the acceptance criteria for adventitious viruses, fungi, and microorganisms. The overall WCB test results confirmed the suitability of the MCB for establishment. Phenotypic characterization of the cell bank

Cell line phenotypic stability studies were performed on the MCB and two WCBs generated for the manufacture of TG-1101. One tube each of the MCB, WCB lot G140/R603/WCB001, and new WCB lot 127646-001 were thawed and subcultured for approximately 60 generations. VCD, cell viability, and titer samples were collected at each subculture. At intervals of approximately 15 cell generations, the cells were frozen and stored as a research cell bank (RCB). After approximately 60 generations, a vial of each RCB was thawed and expanded for 7 days. On day 7, shake flasks were inoculated, cultured under fed-batch conditions, and cultured for 12 days. VCD, cell viability, titer, specific productivity and quality attributes of the collected feed batch cultures were sampled daily to assess the stability of each cell bank at its corresponding generation. Comparability of cell growth, titer and product quality was used as a universal basis for determining the phenotypic stability of the cell banks. In addition, specific productivity results exceeding 70% of the results of low-generation control cultures were used as a clear basis for determining the phenotypic stability of the cell banks. The results of the three cell bank phenotypic stability studies are shown in Table 23 (MCB batch G071/MCB/070208), Table 24 (WCB batch G140/R603/WCB001) and Table 25 (WCB batch 127646). Table 23 : Phenotypic stability results for MCB batch G071/MCB/070208 ^a Fed-batch culture was started after thawing of MCB vials Table 24 : Phenotypic stability results of WCB batch G140/R603/WCB001 ^a Fed-batch culture was started after thawing of MCB vials Table 25 : Phenotypic stability results of WCB batch 127646 ^a MCB vials were thawed and fed-batch culture was started

The live cell density and cell viability results were tested with 15,000 L of thawed MCB and WCB used during the production of TG-1101 to evaluate and confirm the storage stability of the cell bank. (b)  Upstream process and process control

A flow chart of the upstream unit operations, including operational controls and in-process controls, is provided in Table 26. Among other controls, bioburden and endotoxins are measured in batch cultures during the inoculation and production bioreactor stages. Table 26 : Upstream Operations and In-Process Controls Cell culture medium and feed preparation

Cell culture growth medium (CDM4Mab) and feeds (BalanCD CHO Feed4, glucose feed, and glutamine feed) were prepared in water for injection (WFI). The medium and feeds were filtered (≦0.2 μm) into sterile containers and stored as needed before use. Cholesterol lipid concentrate was supplemented to CDM4Mab growth medium during preparation to support cell growth from the inoculum scale-up phase to the production bioreactor. In addition, cholesterol lipid concentrate was added to the production bioreactor as a fixed bolus feed addition on process days 0 and 4. The operation and in-process controls of the cell medium and feeds are described in Table 27 . Table 27 : Cell Culture Medium and Batch Preparation and Storage Control ^a Room temperature of SBL: 17-25°C ^b Operating temperature: 37.0°C (36.5 to 37.5°C) Inoculum expansion

The inoculum expansion steps involve thawing WCB vials and growing in shake flasks and/or cell bags, increasing size and volume to provide sufficient cell mass until the inoculum bioreactor stage. These steps are performed by growing in inoculum expansion growth medium (CDM4Mab). To start the process, thaw one tube of WCB G140/R603/WCB001 in pre-warmed water in a 37.0°C water bath. Transfer the thawed vial contents to pre-warmed medium and dilute to achieve a target inoculum density of 0.55 x ¹⁰⁶ viable cells/mL.

Cultures were initially grown in 125 mL shake flasks and grown for 1 day in a shaking incubator at 37.0°C/5.0% CO _2. Every 2 to 3 days, the cultures were expanded to larger sized shake flasks and/or multiple shake flasks. At each stage, the target inoculum density was 0.30 x 10 ⁶ viable cells/mL. The final inoculum preparation stage consisted of a 50 L bag of cells. After 2 to 3 days of growth, the viable cell density was checked and the cultures were further processed to the inoculum bioreactor stage. The operations and in-process controls for inoculum expansion are described in Table 28. Table 28 : Inoculum Expansion Controls ^a Rocker travel radius dependence parameter. Rocker travel radius: 22 mm inoculation bioreactor

The inoculation bioreactor phase further increases the volume and biomass of the cell culture prior to inoculating the production bioreactor. The media used in these phases was the inoculation expansion growth medium (CDM4Mab). The inoculation bioreactor phases were for 120 L, 600 L, and 3000 L stainless steel bioreactors. The bioreactors were equilibrated after the addition of media. The dissolved oxygen and pH probes were calibrated prior to use. Initial bioreactor set points included temperature, pH, dissolved oxygen, and agitation rate. Each bioreactor was inoculated with the cell culture from the previous phase, and the cultures were grown for 2 to 3 days. The inoculation bioreactor operations and in-process controls are summarized in Table 29. Table 29 : Inoculation Bioreactor Controls ^a Agitation depends on the volume size and bioreactor impeller. Target (NOR/AR) equals 20 (17-23) W/m ³ of power/volume at SBL. Production bioreactor

The production bioreactor stage is the final cell culture process stage, which further increases the volume and quality of the cell culture for TG-1101 antibody that expresses acceptable product quality. The basal medium used in this stage is the growth medium (CDM4Mab) and feed additions are made on designated days or schedules during the process. The production bioreactor is a 15,000L stainless steel bioreactor.

Equilibrate the production bioreactor after adding medium. Add inoculum to medium to initial volume target. Calibrate dissolved oxygen and pH probes before use. Initial bioreactor set points include temperature, pH, dissolved oxygen, and agitation rate. The bioreactor is inoculated with a cell culture from the N-1 inoculation bioreactor at a viable cell density target of 0.5 x ¹⁰⁶ viable cells/mL. During the production bioreactor process, pH is controlled as needed by base addition and _CO2 sparging. Dissolved oxygen is controlled as needed by oxygen and air sparging. Add antifoaming agent as needed to reduce foaming problems. Offline pH, pCO ₂ , pO ₂ , osmotic pressure and metabolites as well as viable cell density (VCD) and viability were monitored daily. BalanCD CHO Feed 4 was added at specified volumes (4.0% of initial bioreactor working volume) on days 3, 5, 7, and 9. Glucose solution was fed at calculated volumes (to 4.00 g/L) when daily bioreactor sample measurements were <3.00 g/L on a given process day. Glutamine solution was fed at fixed volumes (3.0% of initial bioreactor working volume) in batches on day 3 and at calculated volumes (to 4.00 mM) when daily bioreactor sample measurements were <3.00 mM on a given process day. Production bioreactors are harvested based on culture duration or cell viability criteria, whichever occurs first.

Unprocessed bulk samples were removed prior to operation of the clarification unit for foreign matter testing as described in Example 4. Production bioreactor operations and in-process controls are described in Table 30 below. Table 30 : Production Bioreactor Controls ^a Temporary variations are allowed. ^b Agitation depends on volume scale and bioreactor impeller. Target (NOR) corresponds to power/volume 50 (42-59) W/m ³ at SBL. AR corresponds to power/volume 42-70 W/m ^{3. c} Feed on days 3, 5, 7, 9. ^d Feed as needed on days 4-7 ^e Glucose/glutamine is not measured after addition of glucose/glutamine feed; values refer to target concentrations used in calculations to determine required glucose/glutamine feed amount. ^f Feed as needed on days 3 to 12. ^g Antifoam dosage is calculated based on the initial working volume of the production bioreactor. Target is a one-time addition. ^h Harvest occurs when the culture duration reaches its target/NOR or when the final cell viability drops below its NOR (whichever occurs first). Therefore, harvesting is possible when the final cell viability is below its NOR or the culture duration is below its NOR. (c) Downstream Processes and Process Control

A process flow diagram of the downstream steps including operational controls and in-process controls is provided in Table 31. As shown in Table 31 , in-process controls are incorporated into the process. Among other control measures, bioburden and endotoxins are measured during multiple stages of the downstream process. Table 31 : Downstream Operations and In-Process Controls ^a. Clarification of harvest at the beginning, middle and end of filling operations

Harvest and clarify cell culture supernatant from a 15,000 L bioreactor to remove cells and cell debris. Clarification was performed using continuous centrifugation followed by deep filtration.

The harvest clarification step is operated in a room with a controlled temperature range of 17 to 25°C; the harvest pool container is a jacketed drum that maintains the pool at 2 to 8°C. The centrifuge interval is set based on the percentage of packed cell volume (PCV) and adjusted to allow for 80% bowl fill. The centrifuge flow rate is actively controlled; the filter feed flow rate is the same as the centrifuge feed flow rate. Process parameters include centrate backpressure, deep filter operating pressure, inlet pressure, harvest weight, bioburden, and endotoxin.

The centrate was clarified using a three-stage filtration process by passing through 0.2 μm filters (Millistak A1HC POD deep filter, 1.2/0.5 μm filter, and 0.45/0.22 μm sterilizing grade filter) as needed. Prior to use, the filters were rinsed with WFI and then equilibrated. The centrate was pumped through the filters, which were monitored to ensure acceptable back pressure. The contents of the filters were evacuated with air and then the buffer was rinsed. The clarified harvest was stored at 2 to 8°C for ≤11 days. Protein A column chromatography (ProA)

Protein A column chromatography was performed in binding/elution mode using MabSuRe Select resin (Cytiva). This step captured and purified TG-1101, reduced process impurities such as cell culture components, HCPs, and residual DNA, and provided viral safety.

HETP performance of packed columns was evaluated using sodium acetate/benzyl alcohol buffer. During manufacturing, all column operations were performed at 13 to 25 °C. Prior to loading, the columns were sanitized with 0.5 M sodium hydroxide and rinsed with WFI. The columns were equilibrated with Equilibration Buffer (25 mM Tris, 25 mM NaCl, 5 mM EDTA, pH 7.1). The clarified harvest was mixed briefly and then loaded onto the column using a maximum of 36 g of TG-1101/L resin and the column was washed with Wash 1 Buffer (equilibration buffer), followed by a second wash with high-salt Wash 2 Buffer (25 mM Tris, 1.2 M NaCl, 5 mM EDTA, pH 7.1), followed by an additional wash with Wash 3 Buffer (equilibration buffer). The elution peak was collected based on A280 and bound TG-1101 was eluted with elution buffer (25 mM sodium citrate, pH 3.6) at 200 to 220 cm/h, not exceeding 2.4 times the column volume. The precipitate was collected in a bucket containing neutralization buffer (2.0 M Tris, pH 7.5) and filtered through a 0.2 μm filter and then transferred to a different bucket. The Protein A column was sanitized with 0.5 M sodium hydroxide. A maximum of three cycles could be run per batch; if the Protein A process required multiple cycles, the column was re-equilibrated with equilibration buffer for the next cycle. After sanitization, the Protein A column was neutralized with equilibration buffer and stored at 13 to 25 °C in 200 mM sodium acetate, 2% benzyl alcohol, pH 5.0. Dilute the combined (if more than one cycle) neutralized precipitates to a concentration of ≤10 g/L with 5 mM sodium phosphate (pH 7.2) and store at 13 to 25°C for ≤24 hours or at 2 to 8°C for ≤11 days. Solvent Detergent Virus Inactivation (SDVI)

The Protein A capture chromatography step is followed by a solvent detergent viral inactivation (SDVI) step to inactivate potential viral agents. In the current validated manufacturing method, the Protein A lysate pool is diluted and treated with 3.5% (v/v) TnBP, 12% (w/v) polysorbate 80, and maintained at 24.0 to 26.0°C for at least 120 minutes while mixing. The SDVI pool is filtered with a 0.2 μm filter before being transferred to a different bucket, where it is diluted to 50 mOsm/kg with 5 mM sodium phosphate (pH 7.2) and the pH is adjusted to 7.2 as needed. After pH adjustment, the pool is maintained at 13 to 25°C for ≤ 30 hours before entering the CEX column. Cation Exchange Chromatography (CEX)

Cation exchange column chromatography was performed using SP Sepharose Fast Flow (Cytiva) in bind/elute mode. This step further purified TG-1101 and removed residual process impurities (HCP, DNA, residual polysorbate 80, and TnBP).

The HETP performance of the packed columns was evaluated using a buffer containing sodium acetate/benzyl alcohol. During manufacturing, all column operations were performed at 13 to 25 °C. In the current validated manufacturing method, the chromatography columns were sanitized with 0.5 M sodium hydroxide and rinsed with WFI before loading. The columns were equilibrated using equilibration buffer (20 mM sodium phosphate, pH 7.2). The virus inactivation/dilution solution was loaded onto the column at a maximum load of 65 g/L resin. The column was washed with Wash 1 Buffer (equilibration buffer) and then washed a second time with Wash 2 Buffer (equilibration buffer in reverse). The bound TG-1101 was eluted using 20 mM sodium phosphate, 150 mM NaCl, pH 7.2, and the elution peak was collected based on A280 monitoring. The eluted solution was filtered (0.2 μm) and stored at 13 to 25°C for ≤ 72 hours or at 2 to 8°C for ≤ 11 days. After elution, the column was back-extracted with 2 M NaCl and then sanitized with 0.5 M sodium hydroxide. One cycle was allowed for each batch. Upon completion, the column was sanitized (0.5 M sodium hydroxide) and stored in storage buffer (200 mM sodium acetate, 2% benzyl alcohol, pH 5.0). Anion Exchange Membrane Chromatography (AEX)

Anion exchange membrane chromatography was performed in flow-through mode using Mustang Q (Pall Corporation) membrane absorber (MA) filters. This step further purifies TG-1101; the product flows through the membrane, while the remaining impurities (DNA, HCP, and viruses) are retained on the membrane. The membrane is disposable (i.e., each individual membrane cannot be reused) and multiple membrane capsules can be used per batch at the appropriate loading level.

To perform this step, in the current validated manufacturing method, the effluent from the cation exchange column chromatography step is diluted with 20 mM sodium phosphate, pH 8.0, and then the pH is adjusted to 8.0. The concentration is determined and the number of cycles is calculated based on the protein concentration to allow a load of 200 to 700 g TG-1101/L membrane. The membrane is sanitized with 0.5 M sodium hydroxide, rinsed with 2 M NaCl, then rinsed with WFI, and then equilibrated with Equilibration Buffer (20 mM sodium phosphate, 75 mM NaCl, pH 8.0) in preparation for loading. After loading, the membrane is rinsed with 20 mM sodium phosphate, 75 mM NaCl, pH 8.0 to maximize recovery. The product containing effluent collected from all cycles was filtered (0.5/0.2 μm) and diluted to ≤6 g/L with 75 mM sodium citrate, 312 mM NaCl, pH 6.0, and the pH was adjusted to 6.8. The conditioned AEX pool was stored at 13 to 25°C for ≤72 hours or at 2 to 8°C for ≤11 days. Virus Filtration (VF)

Virus filtration was performed using a Viresolve pre-filter in series with a Viresolve Pro virus filter (Millipore Sigma) at an operating temperature target range of 13 to 25°C. Sufficient filters were used to meet the loading limit. This step is designed to remove potential viruses, including small viruses such as parvoviruses.

For filtration, filters were set up in series and flushed with WFI, integrity tested, and then sterilized with 0.5 M sodium hydroxide. This was followed by flushing with equilibration buffer (25 mM sodium citrate, 154 mM NaCl, pH 6.5). The filtered Mustang Q membrane effluent was processed through a virus reduction filter.

Protein concentration is measured and used to confirm membrane loading is ≤600 g/m ² . After loading, the membrane is rinsed with equilibration buffer and tested for post-use integrity. Virus filtrate is stored at 13 to 25°C for ≤72 hours or at 2 to 8°C for ≤11 days. Ultrafiltration / filtration (UFDF) control

The UFDF step was used to concentrate the virus filtrate and buffer exchange into the filtration buffer at an operating temperature target range of 13 to 25°C. The method used tangential flow filters with a molecular weight cutoff of 30 kDa. The setup included enough filters to meet a loading limit of ≤ 250 g/ ^m2 . To perform this operation, the membrane was sanitized with 0.5M NaOH, flushed with WFI, and flushed/equilibrated with filtration buffer before the start of unit operation.

The unit operation consists of an initial ultrafiltration (UF1) step, in which TG-1101 is first concentrated to a target value of 39 mg/mL. This is followed by filtration (DF) into filtration buffer (25 mM sodium citrate 154 mM NaCl, pH 6.5) at 8 filtration volumes (upper limit ≤ 9.2 filtration volumes). The ultrafiltration system is flushed with filtration buffer to maximize recovery; the flush is transferred to a preparation vessel and combined with the UF/DF cell. The membrane is rinsed with WFI, cleaned with 0.5 M NaOH, 250 ppm sodium hypochlorite, rinsed with WFI, and stored in 0.1 M sodium hydroxide. The diluted UFDF pool is stored at 15 to 25°C for ≤ 18 hours.

The formulation step involved the addition of concentrated polysorbate 80 in the formulation buffer to obtain the final drug substance formulation at the target operating temperature range of 17 to 25°C. This step was performed by adding a stabilizing buffer (25 mM sodium citrate, 154 mM NaCl, 10 g/L polysorbate 80, pH 6.5). After addition of stabilization buffer, the pool was diluted to a target of 23.5 to 26.5 mg/mL with 25 mM sodium citrate, 154 mM sodium chloride, 700 mg/L polysorbate 80, pH 6.5, resulting in a ready-to-fill drug substance in a formulation buffer of 25 mM sodium citrate, 154 mM NaCl, 0.07% polysorbate 80, pH 6.5.

The formulated bulk drug substance was transferred and 0.2 μm filtered into 6L Celsius® FFT bags in sealed single-use systems to a target fill volume of 5.50 L. After filling, the formulated bulk drug substance was frozen at ≤-60°C for >17 hours and stored at ≤-35°C. 8.16 Example 16 - Development and maintenance of no disease activity (NEDA) with Utuximab

This example illustrates the time to no disease activity (NEDA) with utuximab and the proportion of participants maintaining NEDA in a pooled post hoc analysis of ULTIMATE I and II. ULTIMATE I (NCT03277261) and ULTIMATE II (NCT03277248) were identical Phase 3, randomized, multicenter, double-blind, active-controlled studies evaluating the efficacy and safety of utuximab versus teriflunomide in participants with relapsing multiple sclerosis (RMS). Utuximab is a novel monoclonal antibody that targets a unique epitope on CD20. Utuximab was glycoengineered to enhance antibody-dependent cytotoxicity and was administered as an infusion over 1 hour after the first infusion. In ULTIMATE I and II, utuximab significantly improved the annualized relapse rate and the number of gadolinium-enhancing (Gd+) T1 lesions and new/enlarging T2 lesions, and a higher proportion of participants achieved 3-parameter NEDA (NEDA-3) rates with utuximab compared with teriflunomide. In a pooled post hoc analysis, NEDA rates were 44.6% and 12.4% for the utuximab and teriflunomide groups at weeks 0 to 96 and 82.1% and 22.5%, respectively, at weeks 24 to 96 (rebaselined; P < .0001 for both). During Weeks 24 to 96 (rebaseline), 17.9% of participants treated with utuximab had EDA, with relapse being the most common component (11.4% with utuximab and 22.9% with teriflunomide). In contrast, 77.5% of participants treated with teriflunomide had EDA, with the most common component being new/enlarging T2 lesions (71.6% with teriflunomide and 3.1% with utuximab). (a)  Methods

The Phase 3 ULTIMATE I (N=549) and II (N=545) studies evaluated RMS participants with either utuximab 450 mg intravenously every 24 weeks (following a 150 mg infusion on day 1 and a 450 mg infusion on day 15) or teriflunomide 14 mg orally once daily for 96 weeks. Clinical assessments were performed every 12 weeks, and magnetic resonance imaging (MRI) was performed at weeks 24, 48, and 96. Pooled post hoc analyses assessed the proportion of participants who achieved and maintained NEDA at weeks 0 to 24, 24 to 96, 24 to 48, and 48 to 96. NEDA (e.g., NEDA-3) was defined as no confirmed relapse, no Gd+ T1 lesions, no new/enlarging T2 lesions, and no confirmed disability progression at 12 weeks. The results are depicted in Figures 16A to 16C . (b) Results

Among participants treated with utuximab, 53.4% achieved NEDA during Weeks 0 to 24, and 45.2% achieved NEDA during Weeks 0 to 24 and maintained NEDA during Weeks 24 to 96. In addition, 36.9% of participants had EDA during Weeks 0-24 but subsequently achieved NEDA during Weeks 24 to 96, bringing the total proportion of participants with NEDA during Weeks 24 to 96 to 82.1% (re-baseline; Figure 16A ). After approximately 6 months of treatment, 93.6% of participants achieved NEDA during Weeks 24 to 48, and 83.5% of participants achieved NEDA during Weeks 24 to 48 and maintained NEDA during Weeks 48 to 96. An additional 4.8% of participants had EDA during weeks 24 to 48 but achieved NEDA during weeks 48 to 96, for a total of 88.2% of participants with NEDA during weeks 48 to 96 ( Figure 16B ). The main cause of disease activity during weeks 0 to 24 was new/enlarging T2 lesions (occurring in 42.4% of participants). However, this MRI activity decreased during weeks 24 to 48 and continued to decrease during weeks 48 to 96 ( Figure 16C ). (c) Conclusions

Utuximab treatment in the Phase 3 ULTIMATE study resulted in a high percentage of participants achieving and maintaining NEDA. More than half of participants (53.4%) achieved NEDA by Week 24, increasing to more than 82% at Week 96. The majority of participants maintained NEDA once achieved. 8.17 Example 17 - Characterization of Cytopenias with Utuximab in the Phase 3 ULTMATE I and II Studies in Participants with Relapsing Multiple Sclerosis

This example describes the use of utuximab to characterize cytopenias in the ULTIMATE I and II studies. With continued B cell depletion, pharmacodynamic (PD) studies of utuximab have reported a transient decrease in the percentage of total T cells and NK cells and a corresponding increase in bone marrow cells after an initial dose of utuximab on day 2 (Fox EJ, et al., ACTRIMS (February 24-26, 2022), West Palm Beach, FL, Poster P105; Lovett-Racke AE, et al. J Neuroimmunol. 2021, 359:577676). Pivotal trials of other anti-CD20 agents have not evaluated day 2 lymphocyte counts (Hauser SL, et al. N Engl J Med. 2017, 376(3):221-234; Hauser SL, et al. N Engl J Med. 2020, 383(6):546-557). Additional evaluation of the ULTIMATE I and II data was performed to understand the incidence and kinetics of cytopenias with utuximab and potential associations with infections. In the phase 3 ULTIMATE I and II studies in RMS participants, the adverse event profile of utuximab was not overall imbalanced compared with teriflunomide and was consistent with the anti-CD20 class. (a)  Methods

ULTIMATE I and II enrolled a total of 1,094 adults from 10 countries diagnosed with RMS with active disease (remitting or secondary progressive) (Steinman L, et al. N Engl J Med. 2022, 387(8):704-714). The ULTIMATE I (N=549) and II (N=545) studies evaluated utuximab 450 mg intravenously every 24 weeks or teriflunomide 14 mg orally once daily for 96 weeks (following a 150 mg infusion on day 1 and a 450 mg infusion on day 15) in participants with RMS. Laboratory assessments were performed on Day 1 (predose), Day 2 (one day after the first infusion), Day 8, and Day 15 (predose); every 4 weeks from Weeks 4 to 24; every 12 weeks until Week 96; and at Weeks 100 and 104. Hematopenias were assessed as laboratory abnormalities (graded by cell count) and treatment-emergent adverse events (TEAEs) based on medical judgment as clinically significant (graded by the investigator for severity) according to the Common Terminology Criteria for Adverse Events, version 4.0. The safety population (pooled from both studies) included all participants who received at least one dose of study drug. The results are depicted in Figures 17A to 17C . (b) Results

In the pooled analysis, 25.7%/12.1% of utuximab participants reported cytopenias as TEAEs (any grade/grade ≥3). The most commonly reported cytopenias (all grades/grade ≥3) were lymphopenia (18.7%/9.5%), neutropenia (3.3%/2.0%), leukopenia (3.3%/0.2%), and anemia (2.9%/0.0%). The median time to resolution of TEAEs ≥ Grade 3 with utoximab (quartile 1, quartile 3) was 6.0 days (6.0, 6.0) for lymphopenia and 13.0 days (7.0, 21.0) for neutropenia until the next assessment. Excluding lymphopenia, the incidence of TEAEs ≥ Grade 3 was similar between participants treated with utoximab (14.1%) and those treated with teriflunomide (13.5%). Among participants treated with utoximab, cytopenia was more frequently reported as a laboratory abnormality than a TEAE at all study visits, and the majority of these laboratory abnormalities were Grade 1 or 2 events (Figure 17A). Lymphopenias, leukocytes, hemoglobin, and neutropenias were reported in 90.8%, 38.1%, 23.6%, and 15.1% of participants receiving utuximab, respectively. Excluding day 2, the proportion of participants with lymphopenia decreased from 90.8% to 32.2% (Figure 17B). On day 2, 91% of participants had low lymphocyte counts (39.2% ≥ grade 3); this resulted in a dramatic decrease of approximately 12-fold to only 7.8% of participants with low lymphocyte counts (<1% ≥ grade 3) on day 8, suggesting a temporary post-infusion outcome (Figure 17C). On Day 2, <1% of participants had low neutrophil counts; only 1 participant had a Grade 2 event, and 16.6% had low hemoglobin; all were Grade 1 or 2 events. After Day 2, a low incidence of lymphocyte counts below the lower limit of normal (<LLN) was observed for the remainder of the study. Of the 102 participants with a TEAE of lymphopenia, 81 (79.4%) reported an event once on Day 2 and resolved by Day 8 without recurrence. Of the 52 participants with a TEAE of ≥ Grade 3 lymphopenia or decreased lymphocyte count, only 2 participants experienced a TEAE outside of the Day 2 time point. Excluding lymphocytopenia, the incidence of grade ≥3 TEAEs was 14.1% for utuximab and 13.5% for teriflunomide.

Severe infections are usually not associated with a decrease in lymphocyte or neutrophil counts < LLN. Approximately 10.7% of severe infections occurred when lymphocyte counts were < LLN within 1 month before or after the infection, and 3.6% of severe infections occurred when neutrophils were < LLN within 1 month before or after the infection. (c)  Conclusions

The unique design of the ULTIMATE I and II studies included laboratory values assessed on Day 2, which showed that the majority of participants treated with utuximab experienced a transient decrease in lymphocytes that returned to normal by Day 8. Excluding lymphopenia, the incidence of Grade ≥ 3 TEAEs with utuximab was comparable to that with teriflunomide. 8.18 Example 18 - Phase 3 Results of Utuximab Versus Teriflunomide in Patients with Relapsing Forms of Multiple Sclerosis (RMS) - Annualized Relapse Rate (ARR)

ULTIMATE I and ULTIMATE II are two identical, independent, phase 3 randomized, multicenter, double-blind, active-controlled clinical trials conducted in parallel, recruiting 1,094 patients with relapsed forms of multiple sclerosis (RMS) in 10 countries to evaluate the safety and efficacy of the glycoengineered anti-CD20 antibody utuximab compared with teriflunomide in these patients.

Clinical assessments were performed every 12 weeks, and CNS magnetic resonance imaging (MRI) was performed at weeks 12, 24, 48, and 96. The clinical application of MRI in the diagnosis of MS is known (Rovira, A. et al ., Nat Rev Neurol 11 : 471-482 (2015)) and MRI lesion assessment results are used as a surrogate marker for MS relapse (Sormani et al., Ann. Neurol. 65 (3): 268-275 (2009); Sormani et al., Lancet Neurol. 12 : 669-676 (2013)). See also Kraunzer, UW and Gauthier, SA, Ther Adv Neurol Disord. 10 (6) 247-261 (2017).

The primary endpoint of the Phase 3 ULTIMATE I and ULTIMATE II studies was the annualized relapse rate (ARR) after 96 weeks of treatment. The ARR is the ratio of the sum of the subjects' RMS relapse counts divided by the sum of the subjects' treatment duration (in years).

Key secondary endpoints for ULTIMATE I and ULTIMATE II included the total number of gadolinium-enhancing T1 lesions on MRI through Week 96; the total number of new and/or enlarging T2 hyperintense lesions through Week 96; and the proportion of subjects with no disease activity (NEDA) from Week 24 to Week 96.

Key secondary outcomes in the prespecified meta-analysis included time to confirmed disability progression (CDP) of at least 12 weeks. Tertiary analyses included time to confirmed disability progression (CDP) of at least 24 weeks, time to confirmed disability improvement (CDI) of at least 12 weeks; and time to confirmed disability improvement (CDI) of at least 24 weeks.

These results are presented in this example and the following examples and associated figures.

As mentioned above, the primary endpoint of the Phase 3 ULTIMATE I and ULTIMATE II clinical studies was the annualized relapse rate (ARR) after 96 weeks of treatment. Both ULTIMATE I and ULTIMATE II studies achieved their primary endpoints with treatment with Utuximab, demonstrating a significant reduction in the annualized relapse rate (ARR) over the 96-week period (p < 0.005 in each trial). Utuximab treatment resulted in an ARR of 0.10 in both ULTIMATE I and ULTIMATE II.

More specifically, referring to FIG. 18A , in ULTIMATE I, treatment with utuximab (N = 271) resulted in an ARR of 0.076 compared to 0.188 with teriflunomide (N = 274), representing a relative reduction of 59.4% (p < 0.0001).

See Figure 18B , in ULTIMATE II, treatment with utuximab (N = 272) resulted in an ARR of 0.091, compared to 0.178 with teriflunomide (N = 272), representing a relative reduction of 49.1% (p=0.0022). 8.19 Example 19 - MRI Results: In the Phase 3 ULTIMATE I and II studies in patients with RMS , the total number of gadolinium (Gd) -enhancing T1 lesions was reduced with utuximab compared to teriflunomide

See Figure 19A (ULTIMATE I) and Figure 19B (ULTIMATE II). At 96 weeks, treatment with utuximab resulted in a 96.7% (N=265) and 96.5% (N=267) reduction in the total number of gadolinium-enhancing (Gd+) lesions on MRI, compared to treatment with teriflunomide (N=270; N=267) in ULTIMATE I and ULTIMATE II, respectively (p<0.0001). 8.20 Example 20 - MRI Results : In the Phase 3 ULTIMATE I and II studies in patients with RMS , treatment with utuximab resulted in a 96.7% reduction in the number of new or enlarging T2 lesions , compared to treatment with teriflunomide (N=270; N=267).

See Figure 20A (ULTIMATE I) and Figure 20B (ULTIMATE II). At 96 weeks, treatment with utuximab resulted in a 92.4% (N=265) and 90.0% (N=272) reduction in the number of new or enlarged T2 lesions on MRI in ULTIMATE I and ULTIMATE II, respectively, compared with treatment with teriflunomide (N=270 and N=267, respectively (p<0.0001). 8.21 Example 21 - Utuximab was associated with an improvement in the free disease activity (NEDA) status relative to teriflunomide

See Figure 21A . In the ULTIMATE I study, 44.6% of patients treated with utuximab (N=271) achieved no disease activity (NEDA) status, compared to 15.0% of patients treated with teriflunomide (N=274), representing a 197% improvement over treatment with teriflunomide (p < 0.0001).

See Figure 21B . In the ULTIMATE II study, 43% of patients treated with utuximab (N=272) achieved no disease activity (NEDA) status, compared to 11.4% of patients treated with teriflunomide (N=272), representing a 277% improvement over treatment with teriflunomide (p<0.0001). 8.22 Example 22 - In the Phase 3 ULTIMATE I and II studies in patients with RMS , T1 hypointense lesions ( “black holes” ) were reduced with utuximab compared to teriflunomide.

Background : In this example, the effect of utuximab on T1 hypointense lesions based on MRI was evaluated in the ULTIMATE I and ULTIMATE II studies. T1 "black holes" are common hypointense lesions on T1-weighted images in patients with multiple sclerosis and represent a chronic stage of the disease associated with white matter destruction, axonal loss, and irreversible clinical outcomes. See Kocsis, K. et al ., Frontiers in Neurology 12 : 1-8 (March 2021).

Methods : In the phase 3 ULTIMATE I (N=549) and ULTIMATE II (N=545) studies, T1 hypointense lesion volume was a tertiary assessment in individual studies. Pooled analyses of T1 hypointense lesions were performed post hoc. T1 hypointense lesion volume was assessed using mixed models with repeated measures of change from baseline in cube-root-transformed volume at scheduled visits up to 96 weeks.

Results : In the pooled analysis of ULTIMATE I and II, at week 96, the mean change from baseline in the volume of T1 hypointense lesions was 0.0101 mL in patients treated with utuximab and 0.0491 mL in patients treated with teriflunomide, a difference of -0.0390 (95% confidence interval, -0.0585 to -0.0195; P<0.0001 for all postbaseline time points). The mean volume (mL) of T1 hypointense lesions at baseline, week 24, week 48, and week 96 were 3.280, 3.266, 3.244, and 3.132 for utuximab, and 3.343, 3.360, 3.330, and 3.475 for teriflunomide, respectively. The mean number of new T1 hypointense lesions at week 96 was significantly reduced compared with teriflunomide (1.5±3.55 vs 5.4±10.67; P<0.0001).

The percentage change (%) reduction in T1 lesion volume and T1 lesion number between utuximab and teriflunomide was 9.9% and 72.2%, respectively.

Conclusions: In a pooled post hoc analysis of the Phase 3 ULTIMATE 1 and ULTIMATE II trials, a significant reduction in both the volume and number of new T1 low-intensity lesions was observed with utuximab compared with teriflunomide at week 96. 8.23 Example 23 - Pharmacodynamics (PD) and Pharmacokinetics (PK) of B- cell Depletion with Utuximab in Patients with RMS in ULTIMATE I and ULTIMATE II

In this example, the pharmacodynamics (PD) and pharmacokinetics (PK) of B cell depletion with utuximab treatment in patients with RMS were evaluated in the ULTIMATE I and ULTIMATE II Phase 3 clinical studies.

Methods : In patients with RMS, ULTIMATE I (N=549) and ULTIMATE II (N=545) evaluated utuximab 450 mg given as a 1-hour intravenous infusion every 24 weeks or teriflunomide 14 mg orally once daily for 96 weeks (following 150 mg on day 1 and 450 mg on day 15). The last dose of utuximab was given at week 72, and retreatment was initiated after a mean of (mean) 51 to 52 weeks for patients enrolled in the open-label expansion (OLE) study. PD and PK analyses were performed in the modified intention-to-treat population at prespecified intervals.

Results : In a pooled post hoc analysis of ULTIMATE I and ULTIMATE II (N=543), participants who received utuximab had a median CD19+ B cell count (cells/μL) of 201.0 at baseline. Beginning at Week 1, Day 2, participants had a significant decrease in median CD19+ B cell count relative to baseline (-193.0 [96.0% decrease]) and remained consistent at Week 104 (32 weeks after the last infusion) (-183.0 [91.0% decrease]). Prior to the first OLE infusion, median B cell counts had increased to 22.4% of baseline. Pharmacokinetics and Absorption

The pharmacokinetics (PK) of utuximab after repeated IV infusions were adequately described by a two-compartment model with first-order clearance. In patients with RMS, utuximab exposure increased in a dose-proportional manner (i.e., linear pharmacokinetics) over the dose range of 150 to 600 mg.

Utuximab was administered by intravenous infusion resulting in a geometric mean steady-state AUC of 3000 mcg/mL (CV = 28%) and a mean maximum concentration of 139 mcg/mL (CV = 15%) per day.

The population PK estimate for the central distribution volume was 3.18 L.

The mean terminal half-life of utuximab was estimated to be 22 days.

Conclusions : In the Phase 3 ULTIMATE I and ULTIMATE II trials, peripheral B cell counts declined rapidly after the first infusion of utuximab and remained low during treatment, which may be consistent with the mechanism of action of utuximab. 8.24 Example 24 - Neutralizing Antibodies (NAbs) and Anti-Drug Antibodies (ADAs) in the Phase 3 ULTIMATE I and II Studies of Utuximab in Patients with RMS

In this example, the incidence of neutralizing antibodies (NAbs) and anti-drug antibodies (ADA) to utuximab was evaluated in the ULTIMATE I and II studies.

Methods : In patients with RMS, the ULTIMATE I (N=549) and ULTIMATE II (N=545) studies evaluated utuximab 450 mg intravenously every 24 weeks (following 150 mg infusion on day 1 and 450 mg infusion on day 15) or teriflunomide 14 mg orally once daily for 96 weeks. NAb and ADA analyses were performed in patients who received at least 1 dose of utuximab (safety population) and were evaluable for both NAb and ADA; ULTIMATE I and II data were pooled (N=534).

Results : The proportion of patients receiving utuximab who tested positive for NAbs and ADAs was 2.4% and 17.8% at baseline and 6.4% and 86.5% post-baseline, respectively. Patients who tested positive at baseline did not necessarily test positive at post-baseline time points. Treatment-emergent (TE)-NAbs were present in 4.3%, 3.4%, 1.1%, and 1.1% of patients at Weeks 24, 48, 72, and 96, respectively. The proportion of patients with TE-ADAs was 48.9%, 69.1%, 48.1%, 28.1%, and 16.5% at Weeks 3, 24, 48, 72, and 96, respectively. The annualized relapse rate (ARR) was 0.03 in NAb-positive patients (post-baseline) (n=34), 0.11 in NAb-negative patients (post-baseline) (n=500), 0.10 in TE-ADA-positive patients (n=434), and 0.12 in TE-ADA-negative patients (n=100); the number of patients in the NAb-positive group was smaller. The incidence of infusion-related reactions (IRR) of all grades/≥3 was 48.4%/3.0% in TE-ADA-positive patients and 42.0%/2.0% in TE-ADA-negative patients. IRR AEs (all grades) that occurred more than 2% more frequently in TE-ADA-positive patients compared with TE-ADA-negative patients included fever (10.1% vs 7.0%), chills (8.3% vs 6.0%), nausea (3.7% vs 1.0%), and lymphopenia (3.2% vs 1.0%). Post-baseline NAb and TE-ADA status did not appear to be associated with specific baseline demographics or disease characteristics.

Conclusions : In the Phase 3 Utuximab studies, a low incidence of TE-NAbs was observed, and most patients developed TE-ADA. After 24 weeks, the proportion of patients with TE-NAbs and TE-ADA decreased. 8.25 Example 25 - Prespecified Pooled Disability Outcomes - Confirmed Disability Progression (CDP) and Confirmed Disability Improvement (CDI) in the Phase 3 ULTIMATE I and II Studies of Utuximab in Patients with RMS

CDP : As used herein, CDP refers to the proportion (%) of patients who achieve CDP results from baseline to week 96 for at least 12 weeks (CDP 12) or for 24 weeks (CDP 24) while receiving treatment with utuximab or teriflunomide. In addition, CDP refers to the time from baseline to week 96 when the CDP results are achieved for at least 12 weeks (CDP 12) or for 24 weeks (CDP 24) while receiving treatment with utuximab or teriflunomide. See Ontaneda, D. et al., Lancet 389 : 1357-1366 (2017).

Very low rates of disability progression were observed in all treatment groups (i.e., utuximab and teriflunomide). Only 5.2% of RMS patients treated with utuximab achieved confirmed disability progression (CDP) at 12 weeks, compared to 5.9% of RMS patients treated with teriflunomide. See Figure 22A . Only 3.3% of RMS patients treated with utuximab demonstrated a time to CDP at 24 weeks, compared to 4.8% of RMS patients treated with teriflunomide, see Figure 22B . Both results were not statistically different.

CDI : As used herein, CDI refers to the proportion (%) of patients who achieve a CDI result lasting at least 12 weeks (CDI 12) or lasting at least 24 weeks (CDI 24) from baseline to week 96 while receiving treatment with utuximab or teriflunomide. In addition, CDI refers to the time to achieve a CDI result lasting at least 12 weeks (CDI 12) or lasting 24 weeks (CDI 24) from baseline to week 96 while receiving treatment with utuximab or teriflunomide. See Cree, B. et al., Mult. Scler. 27 (14):2219-2231 (2021).

A higher proportion of participants achieved CDI lasting at least 12 weeks in the utuximab group (12.0%) compared with the teriflunomide group (6.0%). Using a hierarchical log-rank test, the difference between treatment groups in the time to achieve CDI lasting at least 12 weeks was statistically significant in favor of utuximab (116% increase; p=0.0003]).

A higher proportion of participants in the utuximab group achieved CDI lasting at least 24 weeks (9.6%) compared to the teriflunomide group (5.1%). Using a stratified log-rank test, the difference between treatment groups in time to achieve CDI lasting at least 24 weeks was statistically significant in favor of utuximab (103% increase; p=0.0026). See Figure 23A (12-week CDI analysis) and Figure 23B (24-week CDI analysis). 8.26 Example 26 - Utuximab is associated with significant improvements in the Multiple Sclerosis Functional Composite (MSFC) score compared to teriflunomide : Results from the Phase 3 ULTIMATE I and ULTIMATE II studies

Disability improvement was observed with utuximab in both the ULTIMATE I and ULTIMATE II studies, with significantly more patients achieving confirmed disability improvement (CDI) at 12 or 24 weeks compared with teriflunomide. See Example 25.

In this example, the effect of utuximab on the Multiple Sclerosis Functional Composite (MSFC) score and its components was investigated compared with teriflunomide. The components of the MSFC include the 9-hole peg test (9-HPT), which assesses arm and hand function; the timed 25-foot walk (T25FW), which assesses leg function; and the timed auditory serial addition test (PASAT), which assesses cognitive function. Fischer, JS et al ., Mult. Scler . 5:244-250 (1999).

Design / Methods : In the ULTIMATE I and ULTIMATE II studies, 1094 patients with RMS from 10 countries were studied and received either utuximab 450 mg IV infusion every 24 weeks (following a 150 mg infusion on day 1) or teriflunomide 14 mg orally once daily for 96 weeks. MSFC (tertiary outcome measure in ULTIMATE 1 and II) was performed at baseline and every 12 weeks thereafter. MSFC and components (9-HPT, PASAT, T25FW; post hoc analysis) were prespecified outcomes and analyzed using a modified intention-to-treat (ITT) population. Changes from baseline in MSFC and components were analyzed using mixed models with repeated measures of all postbaseline time points. The model included treatment, region, baseline EDSS strata, visit, visit-treatment interaction, and baseline value as covariates and used an unstructured covariate matrix, restricted maximum likelihood estimation, and the Satterthwaite degree method.

Results : Ultuximab treatment was associated with significant improvements from baseline in the global Multiple Sclerosis Functional Composite (MSFC) score in RMS patients compared with patients treated with teriflunomide in ULTIMATE I and ULTIMATE II, respectively (p=0.0484 and p=0.0171).

In ULTIMATE I, patients treated with utuximab (N=271) had a 76.4% improvement in MSFC scores compared to patients treated with teriflunomide (N=274). See Figure 24A . In ULTIMATE II, patients treated with utuximab (N=272) had an 89.6% improvement in MSFC scores compared to patients treated with teriflunomide (N=272). See Figure 24B ).

In both ULTIMATE I and ULTIMATE II, utuximab significantly improved mean timed 25-foot walk (T25FW) scores from baseline to 96 weeks compared with teriflunomide. See Figure 25A (ULTIMATE I) and Figure 25B (ULTIMATE II). T25FW was statistically significant in ULTIMATE II but not in ULTIMATE I (p=0.0446 and p=0.2861, respectively).

The change in mean Paced Auditory Serial Addition Test (PASAT) scores from baseline to 96 weeks was similar between utuximab and teriflunomide in ULTIMATE I and ULTIMATE II, as shown in Figure 26A (ULTIMATE I) and Figure 26B (ULTIMATE II). The PASAT scores were not statistically significant in ULTIMATE I or II, respectively (p=0.7444 and p=0.7915).

In both ULTIMATE I and ULTIMATE II, the mean 9-hole plunger test (9-HPT) scores increased significantly from baseline to Week 96 for utuximab compared to teriflunomide. 9-HPT was statistically significant in both ULTIMATE I and II (p=0.0084 and p=0.0106, respectively). See Figure 27A (ULTIMATE I) and Figure 27B (ULTIMATE II).

Conclusions : In the Phase 3 ULTIMATE I and ULTIMATE II studies, treatment with utuximab was associated with significant improvements in the Multiple Sclerosis Functional Composite (MSFC) score compared with teriflunomide. The improvement in MSFC score was driven by improvements in disability as measured by the 9-hole peg test (9-HPT) and the timed 25-foot walk (T25FW). 8.27 Example 27 - Percent Brain Volume Change (PBVC) Based on MRI

Referring to FIG. 28A , in ULTIMATE I, a post hoc analysis of the difference in brain volume between Week 24 and Week 96 demonstrated no difference in brain volume reduction between the utuximab and teriflunomide groups as measured by mean PBVC.

See Figure 28B , in ULTIMATE II, the decrease in brain volume as measured by mean PBVC was less in the utuximab group than in the teriflunomide group. 8.28 Example 28 - Infusion-Related Reactions (IRRs) with Utuximab in the Phase 3 ULTIMATE I and ULTIMATE II Studies

In a post hoc analysis of the Phase 3 ULTIMATE I and ULTIMATE II studies, the pooled IRR rates from the two trials were analyzed and characterized according to schedule and severity.

Methods : ULTIMATE I (N=549) and II (N=545) evaluated utuximab 450 mg intravenously as a 1-hour infusion every 24 weeks (following a 150-mg infusion on day 1 and a 450-mg infusion on day 15) or teriflunomide 14 mg orally once daily for 96 weeks. The teriflunomide group received a placebo infusion; the utuximab group received an oral placebo. Participants received premedication with antihistamines and corticosteroids 30 to 60 minutes before utuximab/placebo infusions. Pooled IRR data from both trials were analyzed.

Results : In the ULTIMATE study, the total number of infusions was 2644 (utuximab) and 2637 (placebo). The IRR rates were 47.7% (utuximab) and 12.2% (placebo). 43.3% of patients treated with utuximab experienced an IRR at the first infusion. 68.8% (n=181) had only 1 IRR; of these, 90.6% experienced an IRR at the first dose. Among patients who experienced >1 IRR (n=82), 87.8% experienced their first IRR during the first dose; thus, among patients with a first-dose IRR, 69.5% did not have a recurrence of an IRR. Fever was the most common IRR (9.5% of patients treated with utuximab). One patient had a grade 4 IRR (anaphylaxis) to utuximab on day 15 (drug interruption/withdrawal) after two grade 1 IRRs (both reported as influenza-like syndrome) on the first infusion; all IRRs resolved. Overall, 96.6% of total utuximab infusions were completed without interruption.

Conclusions : In the ULTIMATE I & II Phase 3 trials, IRRs (most commonly pyrexia) were the major adverse events of utuximab. Most IRRs occurred with the first dose and had minimal impact on infusion completion. 8.29 Example 29 - Utuximab reduced thalamic volume loss and new lesion formation in participants in the ULTIMATE I & II Phase 3 studies

Thalamic integrity is a measure of deep gray matter that is affected by damage to nerve tracts that project into and out of the thalamus. The ULTIMATE I and II studies evaluated the effects of utuximab on thalamic volume and the formation of new lesions during and after the first year.

Methods: The placebo-controlled ULTIMATE I (N=549) and II (N=545) studies evaluated utuximab 450 mg intravenously every 24 weeks (following 150 mg on day 1 and 450 mg on day 15) versus teriflunomide 14 mg orally once daily for 96 weeks in patients with RMS. Paired Jacobian integration was used to measure percent change in thalamic volume from baseline to week 96 and at annual intervals. Post hoc analyses were performed for changes in T1 hypointense lesion volume, new T1 hypointense lesion counts, and new/enlarged T2 lesion counts at 2-year and annual intervals.

Results : Utuximab treatment significantly reduced thalamic volume loss by 22% over 2 years compared with teriflunomide. The least squares (LS) mean percentage change from baseline to week 96 was -1.34 (95% CI: -1.56, -1.12) for utuximab vs -1.71 (95% CI: -1.93, -1.50) for teriflunomide ( P = .0013). For T1 hypointense lesion volume (mL), the LS mean percentage change from baseline to week 96 was 6.32 (95% CI: -0.82, 13.46) for utuximab vs 24.87 (95% CI: 17.77, 31.96) for teriflunomide ( P < .0001). The reduction in the volume of T1 hypointense lesions was more pronounced in the second year of treatment than in the first year, and the same pattern was observed for the count of T1 hypointense lesions and new/enlarged T2 lesions.

Conclusions : Compared with teriflunomide, utuximab reduced thalamic volume loss and the volume of T1 hypointense lesions (a marker of brain tissue loss). After the first year of treatment, the formation of new T1 and T2 lesions was almost completely suppressed.

Other aspects, advantages, and modifications are within the scope of the appended claims.

without

6. Simple diagram

Figure 1 illustrates the structures and abbreviations of various N-glycans.

FIG2 illustrates the glycosylation profile of the anti -CD20 antibody protein samples provided herein.

FIG3 illustrates the complete MS spectrum of the anti -CD20 antibody protein sample provided herein.

FIG. 4 illustrates dose-response curves of antibody-dependent cytotoxicity (ADCC) activity using Raji cells and KILR CD16a cells.

FIG5 illustrates dose-response curves of ADCC activity using Raji cells and primary NK cells.

FIG6 illustrates the dose-response curve of antibody-dependent cellular phagocytosis (ADCP) activity.

FIG. 7 illustrates the dose-response curve of complement-dependent cytotoxicity (CDC) activity.

Figure 8 illustrates the CD20 binding dose response curve.

Figure 9 illustrates the CD20 binding dose response curve using FACS.

Figure 10 illustrates the C1q binding dose response curve.

Figures 11A to C illustrate human whole blood B cell depletion from three donors. B cell depletion was calculated based on data using CD19 as a B cell marker.

Figure 12 illustrates the goodness-of-fit (GOF) diagnosis of the final TG-1101 model.

FIG13 illustrates the pcVPC of the final PK model for TG-1101 by study. Blue dots are observed concentrations corrected for predictions; blue lines are the 50th (solid), 5th (dashed), and 95th (dashed) percentiles of observed concentrations; and black lines are the simulated 50th (solid), 5th (dashed), and 95th (dashed) percentiles. Gray bands are the 95% PI of the corresponding black lines based on 500 simulations. Yellow dashes indicate bin intervals. Numbers 201, 301, 302, and 101-304-703 represent study numbers TG1101-RMS201, TG1101-RMS301, and TG1101-RMS302, respectively, and studies targeting individuals with hematological malignancies in the previous analysis set. Abbreviations: pcVPC - prediction-corrected visual prediction test; PI - prediction interval; pred-corr - prediction-corrected; popPK - population pharmacodynamics.

FIG14 illustrates a forest plot of the covariate effects of TG-1101 drug exposure. The first and second vertical dashed lines correspond to ratios of 0.8 and 1.25, respectively. The vertical solid line corresponds to a ratio of 1 and represents a typical subject. The dots and whiskers represent estimates and 90% confidence intervals, respectively. The blue-grey horizontal bars show the range of exposure due to inter-subject variability. The typical subject is defined as a male subject from North America/Western Europe, weighing 73 kg, ADA negative, and having been treated for <416 days. Abbreviations: ADA-antidrug antibodies; AUCss-area under the serum TG-1101 concentration-time curve at steady state; BSV-intersubject or interindividual variability; CI-confidence interval; CMAXSS-maximum TG-1101 concentration at steady state; CMINSS-minimum TG-1101 concentration at steady state; N/A-not applicable.

Figure 15 depicts a map of the expression vector HK463-25, which contains the immunoglobulin heavy and light chain cDNA sequences derived from the anti-CD20 antibody TG-1101 described herein.

Figures 16A to 16C depict the development and maintenance of free disease activity (NEDA) with utuximab. Figure 16A is a bar graph showing NEDA-3 ratios by treatment with EPOCH (Weeks 0-24 vs. Weeks 24-96). ^a The denominator is based on participants in the Week 24-96 analysis. Pooled post hoc analysis. Modified intention-to-treat population. Figure 16B is a bar graph showing NEDA-3 ratios by treatment with EPOCH (Weeks 24-48 vs. Weeks 48-96). ^a The denominator is based on participants in the Week 48-96 analysis. Pooled post hoc analysis. Modified intention-to-treat population. FIG. 16C is a bar graph showing components driving EDA in participants treated with utuximab. ^a Participants may have >1 EDA component. Pooled post hoc analyses. Modified intention-to-treat population. CDP, confirmed disability progression; EDA, evidence of disease activity; Gd+, gadoid enhancement; n/e, new/enhanced; NEDA, no disease activity; NEDA-3, 3-parameter NEDA; EPOCH, combination of etoposide phosphate, prednisone, vincristine sulfate (Oncovin), cyclophosphamide, and doxorubicin hydrochloride (hydroxydaunorubicin).

Figures 17A to 17C depict the characterization of cytopenias with utuximab in the ULTMATE I and II Phase 3 studies in participants with relapsing multiple sclerosis. Figure 17A is a bar graph showing hematology laboratory abnormalities in participants treated with utuximab (all study visits). ^a (493/543). Percentages are based on the number of participants with no missing baseline and at least 1 post-baseline assessment. Pooled post hoc analysis. Safety population. Figure 17B is a bar graph showing hematology laboratory abnormalities in participants treated with utuximab (excluding Day 2). ^a (175/543). Percentages are based on the number of participants with no missing baseline and at least 1 post-baseline assessment. Pooled post hoc analysis. Safety population. Figure 17C is a bar graph showing the proportion of participants treated with utuximab who had low lymphocyte counts by visit. ^a (476/543). Pooled post hoc analysis. Safety population. Percentages are based on the number of participants in the population/treatment group. BL, baseline.

Figures 18A-18B are bar graphs showing the relative reduction in annualized relapse outcomes (ARR) in RMS patients treated with utuximab versus teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. The ARR results for ULTIMATE I ( Figure 18A ) and ULTIMATE II ( Figure 18B ) are shown.

Figures 19A-19B are bar graphs showing the relative reduction in the total number of gadolinium (Gd)+T1 lesions on MRI in patients with RMS treated with utuximab versus teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Results from ULTIMATE I ( Figure 19A ) and ULTIMATE II ( Figure 19B ) are shown.

Figures 20A-20B are bar graphs showing a relative reduction in the number of new or enlarged T2 lesions on MRI in RMS patients treated with utuximab versus teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Results from ULTIMATE I ( Figure 20A ) and ULTIMATE II ( Figure 20B ) are shown.

Figures 21A-21B are bar graphs showing the improvement in the proportion (%) of patients with no disease activity (NEDA) status in RMS patients administered utuximab relative to teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. NEDA results are shown for ULTIMATE I ( Figure 21A ) and ULTIMATE II ( Figure 21B ).

Figures 22A-22B are graphs comparing the percentage of RMS patients with confirmed disability progression (CDP) in a pre-specified pooled analysis for patients administered utuximab versus teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Figure 22A shows the 12-week CDP, while Figure 22B shows the 24-week CDP.

Figures 23A-23B are graphs comparing the percentage of RMS patients who achieved confirmed disability improvement (CDI) in a pre-specified pooled analysis for patients administered utuximab versus teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Figure 23A shows the 12-week CDI, while Figure 23B shows the 24-week CDI.

Figures 24A-24B are bar graphs showing that in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples, mean Multiple Sclerosis Functional Composite (MSFC) scores in RMS patients treated with utuximab versus teriflunomide significantly improved from baseline to Week 96. Results from ULTIMATE I ( Figure 24A ) and ULTIMATE II ( Figure 24B ) are shown.

Figures 25A-25B are bar graphs showing that in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples, mean timed 25-foot walk (T25FW) scores of RMS patients treated with utuximab versus teriflunomide significantly improved from baseline to 96 weeks. Results from ULTIMATE I ( Figure 25A ) and ULTIMATE II ( Figure 25B ) are shown.

Figures 26A-26B are bar graphs showing the change in mean Timed Auditory Serial Addition Test (PASAT) scores from baseline to 96 weeks in RMS patients administered utuximab versus teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Results are shown for ULTIMATE I ( Figure 26A ) and ULTIMATE II ( Figure 26B ). The change in mean PASAT scores was similar between treatment groups.

Figures 27A-27B are bar graphs showing that the mean 9-hole plunger test (9-HPT) scores of RMS patients treated with utuximab versus teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples increased significantly from baseline to Week 96. Results from ULTIMATE I ( Figure 27A ) and ULTIMATE II ( Figure 27B ) are shown.

Figures 28A-28B are graphs showing percent brain volume change (PBVC) based on MRI from Week 24 to Week 96 (post hoc analysis) in RMS patients administered utuximab and teriflunomide in the Phase 3 clinical studies ULTIMATE I and ULTIMATE II described in the Examples. Results are shown for ULTIMATE I ( Figure 28A ) and ULTIMATE II ( Figure 28B ).

without

TW202413404A_112120372_SEQL.xml

Claims (73)

A composition comprising a population of anti-CD20 antibody proteins, wherein the anti-CD20 antibodies in the population are represented by one or more nucleic acid sequences encoding a heavy chain comprising an amino acid sequence of SEQ ID NO: 1 and a light chain comprising an amino acid sequence of SEQ ID NO: 2, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising about 20 to 40% fucosylated glycans and optionally about 10 to 20% galactosylated glycans. The composition of claim 1, wherein: the N-glycan profile comprises 23% to 36% fucosylated polysaccharides, preferably about 30% fucosylated polysaccharides; and/or the N-glycan profile comprises 16% to 18% galactosylated polysaccharides, preferably about 17% galactosylated polysaccharides. The composition of claim 1 or 2, wherein: the relative abundance of the fucosylated polysaccharide is the percentage of the fucosylated polysaccharide in all the polysaccharides in the N-glycan spectrum; and/or the relative abundance of the galactosylated polysaccharide is the percentage of the galactosylated polysaccharide in all the polysaccharides in the N-glycan spectrum. A composition as in any one of claims 1 to 3, wherein the N-glycan profile comprises 12% to 30% bisecting N-glycan, preferably about 18% bisecting N-glycan. The composition of claim 4, wherein the bisected N-glycans comprise one or more of G0B, G0FB, G1FB, G2FBS1 and G2FBS2. A composition as claimed in any one of claims 1 to 5, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising less than 5% sialylated glycans, optionally wherein the N-glycan profile comprises less than 4%, 3%, 2.5%, 2%, 1% or 0.5% sialylated glycans, optionally wherein the N-glycan profile does not comprise detectable amounts of sialylated glycans. A composition as claimed in any one of claims 1 to 6, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising 0.1% to 1.5% Man5 N-glycans, optionally wherein the N-glycan profile comprises 0.4% to 0.7% Man5 N-glycans, optionally wherein the N-glycan profile comprises approximately 0.6% Man5 N-glycans, optionally wherein the Man5 N-glycans are the only high mannose species in the N-glycan profile. A composition as claimed in any one of claims 1 to 7, wherein the population of anti-CD20 antibody proteins comprises 0.20 to 0.40 mol isoaspartic acid/mol protein, optionally 0.25 to 0.35 mol isoaspartic acid/mol protein. The composition of any one of claims 1 to 8, wherein the glutamine at heavy chain position 1 is pyroglutamine and the glutamine at light chain position 1 is pyroglutamine. A composition as in any one of claims 1 to 9, wherein the population of anti-CD20 antibody proteins has an N-glycan profile comprising N-glycans having a relative abundance ratio of G1 to G0 of 0.1 to 0.15 and/or N-glycans having a relative abundance ratio of G1F to G1 of 0.5 to 0.9. A composition as claimed in any one of claims 1 to 10, wherein the population of anti-CD20 antibody proteins further comprises at least two N-glycans within the following relative abundance ranges: (a) 0.3% to 2% G0-GN; (b) 0.1% to 2% G0F-GN; (c) 30% to 60% G0; (d) 0.1% to 1% G1-GN; (e) 5% to 20% G0B; (f) 5% to 30% G0F; (g) 0.1% to 1.5% Man5; (h) 1% to 15% G0FB; (i) 1% to 13% G1; (j) 0.5% to 10% G1'; (k) 0.5% to 6% G1B; (l) 0.5% to 12% G1F; (m) 0.1% to 3% G1F'; (n) 0.1% to 3% G1FB; (o) 0.1% to 2% G2; and (p) 0.1% to 2% G2F, whereby the anti-CD20 antibody protein further comprises at least two N-glycans in the following relative abundance ranges: (a) 0.8% to 1.1% G0-GN; (b) 0.5% to 1.1% G0F-GN; (c) 42.5% to 48.8% G0; (d) 0.3% to 0.6% G1-GN; (e) 9.5% to 14.1% G0B; (f) 12.8% to 19.7% G0F; (g) 0.4% to 0.7% Man5; (h) 5.1% to 7.0% G0FB; (i) 5.7% to 6.4% G1; (j) 2.7% to 3.3% G1'; (k) 1.4% to 2.0% G1B; (l) 2.6% to 4.2% G1F; (m) 1.1% to 1.6% G1F'; (n) 1.1% to 1.8% G1FB; (o) 0.5% to 0.7% G2; and (p) 0.3% to 0.5% G2F, whereby the anti-CD20 antibody protein further comprises at least two N-glycans within the following relative abundance ranges: (a) 0.9% G0-GN; (b) 0.8% G0F-GN; (c) 46.1% G0; (d) 0.5% G1-GN; (e) 10.9% G0B; (f) 17.0% G0F; (g) 0.6% Man5; (h) 6.0% G0FB; (i) 6.1% G1; (j) 2.9% G1'; (k) 1.6% G1B; (l) 3.2% G1F; (m) 1.3% G1F'; (n) 1.3 G1FB; (o) 0.5% G2; and (p) 0.3% G2F. A composition as claimed in claim 11, wherein the group of anti-CD20 antibody proteins further comprises at least three, four or five N-glycans within the following relative abundance ranges: (a) 0.3% to 2% G0-GN; (b) 0.1% to 2% G0F-GN; (c) 30% to 60% G0; (d) 0.1% to 1% G1-GN; (e) 5% to 20% G0B; (f) 5% to 30% G0F; (g) 0.1% to 1.5% Man5; (h) 1% to 15% G0FB; (i) 1% to 13% G1; (j) 0.5% to 10% G1'; (k) 0.5% to 6% G1B; (l) 0.5% to 12% G1F; (m) 0.1% to 3% G1F'; (n) 0.1% to 3% G1FB; (o) 0.1% to 2% G2; and (p) 0.1% to 2% G2F. A composition as claimed in any one of claims 1 to 12, wherein the N-glycan profile of the population of anti-CD20 antibody proteins is determined using a method comprising: (a) incubating the population of anti-CD20 antibody proteins with an enzyme, wherein the enzyme catalyzes the release of N-glycans from the anti-CD20 antibodies; (b) using one or more methods selected from chromatography, mass spectrometry, capillary electrophoresis and combinations thereof to measure the relative abundance of the released N-glycans, optionally wherein the method further comprises the following steps after step (a) and before step (b): (c) purifying the N-glycans; and (d) labeling the N-glycans with a fluorescent compound, optionally wherein the enzyme is PNGase F and/or the fluorescent compound is 2-aminobenzamide (2-AB). A composition as claimed in any one of claims 1 to 13, wherein less than 10% of the anti-CD20 antibody proteins in the population are non-glycosylated, optionally wherein less than 5% of the anti-CD20 antibody proteins in the population are non-glycosylated, optionally wherein less than 1% of the anti-CD20 antibody proteins in the population are non-glycosylated. A composition as claimed in any one of claims 1 to 14, wherein the group of anti-CD20 antibody proteins comprises two or more of the following secondary structures as determined by circular dichroism at 205 nm to 260 nm: (a) 8.0% to 10.0% α-helix; (b) 32.0% to 36.0% antiparallel β-sheets; (c) 5.0% to 6.0% parallel β-sheets; (d) 16.0% to 18.0% β-turns; and (e) 35.0% to 36.0% random coils, whereby the group of anti-CD20 antibody proteins comprises the following secondary structures as determined by circular dichroism at 205 nm to 260 nm: (a) 8.0% to 10.0% α-helix; (b) 32.0% to 36.0% antiparallel β-sheets; (c) 5.0% to 6.0% parallel β-sheets; (d) 16.0% to 18.0% β-turns; and (e) 35.0% to 36.0% random coils, whereby the anti-CD20 antibody protein comprises two or more of the following secondary structures as determined by circular dichroism at 205 nm to 260 nm: (a) approximately 9.0% α-helices; (b) approximately 33.0% antiparallel β-sheets; (c) approximately 5.6% parallel β-sheets; (d) approximately 17.5% β-turns; and (e) approximately 35.2% random coils. The composition of any one of claims 1 to 15, wherein the population of anti-CD20 antibody proteins further comprises one or more of the following post-translational modifications in a specified abundance: Optionally, one or more of the post-translational modifications are measured by peptide mapping using liquid chromatography-mass spectrometry (LC-MS). The composition of any one of claims 1 to 16, wherein the population has a total protein content of 25.5-25.8 mg/mL as measured by absorbance at 280 nm. The composition of any one of claims 1 to 17, wherein the group of anti-CD20 antibody proteins induces greater cytotoxicity in a cell-based antibody-dependent cytotoxicity (ADCC) assay compared to obinutuzumab, ofatumumab, rituximab, veltuzumab, ibritumomab tiuxetan and/or ocrelizumab. The composition of any one of claims 1 to 18, wherein the population: has a relative potency of 90% to 163% in a cell-based ADCC assay compared to a commercial reference standard; has a relative potency of 78% to 116% or 73% to 128% in a cell-based complement-dependent cytotoxicity (CDC) assay compared to a commercial reference standard; has a relative potency of 92% to 118% or 82% to 138% in a cell-based CD20 binding activity bioassay compared to a commercial reference standard; has a _KD value of 30 to 70 nM in an FcγRIIIa-158V binding assay as measured by surface plasmon resonance; has a KD value of 500 to 1000 nM in an FcγRIIIa 158F binding assay as measured by surface plasmon resonance; nM _KD value; significantly higher binding affinity to FcγRIIIa 158V or FcγRIIIa 158F than rituximab; 88% to 113% or 86% to 116% relative potency in a C1q binding assay as measured by ELISA compared to a commercial reference standard; and/or 106% to 126% relative potency in a CD16 activity assay compared to a commercial reference standard. A composition as in any of claims 1 to 19, wherein the population has: 99.2% to 99.9% monomers as detected by size exclusion chromatography (SEC); 0.1% to 0.8% dimers as detected by SEC; no detectable aggregates as detected by SEC; and/or no detectable fragments as detected by SEC. A composition as in any of claims 1 to 20, wherein the population: has 93.6 to 95.9% IgG after purification by non-reducing capillary gel electrophoresis (CGE); has 0.1 to 0.3% high molecular weight species (HMWS) after purification by non-reducing CGE; has 0.7 to 1.2% free light chains (LC) after purification by non-reducing CGE; and/or has 97.7 to 98.0% heavy chain plus light chain species (HC+LC) after purification by reducing CGE. A composition as in any of claims 1 to 21, wherein the population: has 20% to 25% acidic isoforms as detected by imaged capillary isoelectric focusing (iCIEF); has 50% to 60% major isoforms as detected by iCIEF; has 20% to 30% basic isoforms as detected by iCIEF; and/or has an average molar ratio of free thiols to anti-CD20 antibodies of about 2.0 to 2.2. A composition as claimed in any one of claims 1 to 22, wherein the amino acid sequence of the anti-CD20 antibodies in the group comprises a deletion of N-terminal residues, optionally a deletion of up to 5 N-terminal residues, optionally a deletion of up to 10 N-terminal residues; and/or the heavy chain terminal lysine residue of the anti-CD20 antibodies in the group is truncated. A pharmaceutical formulation comprising the composition of any one of claims 1 to 23, wherein the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 10 mg/mL to 50 mg/mL, optionally wherein the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 25 mg/mL. The pharmaceutical formulation of claim 24, further comprising one or more of the following: sodium chloride, trisodium citrate dehydrate, polysorbate 80, and hydrochloric acid. The pharmaceutical formulation of claim 25, comprising about 9.0 mg/mL of sodium chloride, about 7.4 mg/mL of trisodium citrate dehydrate, about 0.7 mg/mL of polysorbate 80, and/or about 0.4 mg/mL of hydrochloric acid. The pharmaceutical formulation of any one of claims 24 to 26, wherein the anti-CD20 antibody is present in a single dosage form. A pharmaceutical formulation comprising: (i) a composition as claimed in any one of claims 1 to 23, wherein the composition comprises a single dosage form of the group of anti-CD20 antibody proteins, wherein the anti-CD20 antibody is present in the pharmaceutical formulation at a concentration of about 25 mg/mL, (ii) about 9.0 mg/mL of sodium chloride, (iii) about 7.4 mg/mL of dehydrated trisodium citrate, (iv) about 0.7 mg/mL of polysorbate 80, and (v) about 0.4 mg/mL of hydrochloric acid. A single batch preparation of the population of anti-CD20 antibody proteins as described in any one of claims 1 to 23 or the pharmaceutical formulation of any one of claims 24 to 28, wherein the single batch comprises at least 100 g, at least 120 g, or at least 150 g of the anti-CD20 antibody protein. A population of anti-CD20 antibody proteins as described in any one of claims 1 to 23 or a pharmaceutical formulation as described in any one of claims 24 to 28, which is produced in a 15,000 L or 20,000 L bioreactor. A method for treating multiple sclerosis (MS) in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a composition as described in any one of claims 1 to 23 or a pharmaceutical formulation as described in any one of claims 24 to 28. The method of claim 31, wherein the MS comprises a relapsing form of MS (RMS), wherein the RMS is selected from a clinically isolated syndrome (CIS); a relapsing-remitting MS (RRMS); or an active secondary progressive MS (SPMS). The method of claim 31 or 32, wherein the composition or the pharmaceutical formulation is administered as follows: i) a first infusion at a dose of about 150 mg of the anti-CD20 antibody protein, ii) a second infusion two weeks later at a dose of about 450 mg of the anti-CD20 antibody protein, and iii) subsequent infusions every 24 weeks or six months at a dose of about 450 mg of the anti-CD20 antibody protein. The method of any of claims 31 to 33, wherein administration of the composition or pharmaceutical formulation to the subject results in one or more of the following pharmacokinetic parameters: (a) an AUC between 2,160 μg/mL and 3,840 μg/mL; (b) a Cmax between 118,011 ng/mL and 159,989 ng/mL; (c) a Cmin between 40 ng/mL and 375 ng/mL; and (d) a Cavg between 6,437 ng/mL and 11,443 ng/mL, whereby administration of the composition or pharmaceutical formulation to the subject results in one or more of the following pharmacokinetic parameters: (a) an AUC of approximately 3,000 μg/mL; (b) a Cmax of approximately 139,000 ng/mL; (c) Cmin of approximately 139 ng/mL; and (d) Cavg of approximately 8,940 ng/mL. The method of any one of claims 31 to 34, wherein the method comprises a treatment period of at least 96 weeks. The method of any one of claims 31 to 35, wherein the individual has been pre-administered with a corticosteroid 30 to 60 minutes prior to administration of the composition or the pharmaceutical formulation, wherein the corticosteroid is methylprednisone or dexamethasone, wherein the methylprednisone is administered in a dose of about 100 mg and/or the dexamethasone is administered in a dose of about 10 to 20 mg. The method of any one of claims 31 to 36, wherein the subject has been pre-administered with an antihistamine 30 to 60 minutes prior to administration of the composition or pharmaceutical formulation, optionally wherein the antihistamine is diphenhydramine HCl, optionally wherein the diphenhydramine HCl is administered in an amount of about 25 to 50 mg. The method of any one of claims 31 to 37, wherein the subject has been pre-administered with an antipyretic 30 to 60 minutes prior to administration of the composition or the pharmaceutical formulation, wherein the antipyretic is acetaminophen or an antipyretic bioequivalent thereto. The method of any one of claims 31 to 38, wherein the individual has an Expanded Disability Status Scale (EDSS) score of 0 to 5.5 before treatment. The method of any one of claims 31 to 39, wherein the individual is diagnosed with RMS according to the McDonald Criteria (2010). A method for treating multiple sclerosis (MS) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition as described in any one of claims 1 to 23 or a pharmaceutical formulation as described in any one of claims 24 to 28, wherein administration of the composition or the pharmaceutical formulation results in no evidence of disease activity (NEDA) in the subject 24 to 96 weeks after administration. The method of claim 41, wherein administering the composition or the pharmaceutical formulation causes NEDA in the subject 24 weeks after administration. A method for treating multiple sclerosis (MS) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition as described in any one of claims 1 to 23 or a pharmaceutical formulation as described in any one of claims 24 to 28, wherein administration of the composition or the pharmaceutical formulation results in a temporary decrease in lymphocyte count in the subject. The method of claim 43, wherein the lymphocyte count is normal before day 8 of administration. A method as in any one of claims 41 to 44, wherein the MS is a recurrent form of MS (RMS). A method for reducing the annualized relapse rate (ARR) of an individual with relapsing forms of multiple sclerosis (MS), comprising administering to the individual an effective amount of a composition as described in any one of claims 1 to 23 or a pharmaceutical formulation as described in any one of claims 24 to 28, the method comprising: Administering the composition or pharmaceutical formulation for intravenous infusion in a multiple infusion dosage regimen, the dosage regimen comprising: a) a first infusion on day 1, comprising 150 mg of anti-CD20 antibody protein; b) a second infusion about 2 weeks after the first infusion, comprising 450 mg of anti-CD20 antibody protein; c) a first subsequent infusion about 24 weeks or about six months after the first infusion, comprising 450 mg of anti-CD20 antibody protein; and d) One or more subsequent infusions containing 450 mg of anti-CD20 antibody protein approximately 24 weeks or approximately six months after the previous infusion. The method of claim 46, wherein the effective amount of the composition or the pharmaceutical formulation is sufficient to achieve an ARR of 0.091 or an ARR of 0.076. The method of claim 46 or 47, wherein the second infusion, the first subsequent infusion, and the one or more subsequent infusions of the anti-CD20 antibody protein last for about one hour. A method for treating relapsing forms of multiple sclerosis (MS) in an individual in need thereof, comprising administering to the individual an effective amount of a composition as described in any one of claims 1 to 23 or a pharmaceutical formulation as described in any one of claims 24 to 28, the method comprising: Administering the composition or pharmaceutical formulation as an intravenous infusion in a multiple infusion dosage regimen, the dosage regimen comprising: e)     The first infusion on day 1 comprises 150 mg of anti-CD20 antibody protein; f)     The second infusion approximately 2 weeks after the first infusion comprises 450 mg of anti-CD20 antibody protein; g)    The first subsequent infusion approximately 24 weeks or approximately six months after the first infusion comprises 450 mg of anti-CD20 antibody protein; and h)   One or more subsequent infusions about 24 weeks or about six months after the previous infusion, comprising 450 mg of anti-CD20 antibody protein, wherein the second infusion, the first subsequent infusion, and the one or more subsequent infusions of anti-CD20 antibody protein are about one hour in duration. The method of any one of claims 41 to 49, further comprising pre-administering a corticosteroid and an antihistamine to the individual 30 to 60 minutes prior to administering the composition or the pharmaceutical formulation. The method of claim 50, wherein the corticosteroid is methylprednisolone or dexamethasone, as appropriate, wherein the methylprednisolone is administered in a dose of about 100 mg and/or the dexamethasone is administered in a dose of about 10 to 20 mg. The method of any one of claims 46 to 51, wherein the composition or pharmaceutical formulation for intravenous infusion is prepared in 250 mL of 0.9% sodium chloride injection. The method of any one of claims 46 to 52, wherein the first subsequent infusion is about 24 weeks after the first infusion. The method of any one of claims 46 to 53, wherein the one or more subsequent infusions are about 24 weeks from the previous infusion. The method of any one of claims 46 to 54, wherein the first subsequent infusion is about 6 months from the first infusion. The method of any of claims 46 to 55, wherein the one or more subsequent infusions are about 6 months from the previous infusion. The method of any of claims 46 to 56, wherein the duration of the first infusion of the anti-CD20 antibody protein is about four hours, optionally wherein the first infusion of the anti-CD20 antibody protein is infused at the following rates: 10 mL per hour for the first 30 minutes; 20 mL per hour for the next 30 minutes; 35 mL per hour for the next hour; and 100 mL per hour for the remaining two hours. The method of any one of claims 48 to 57, wherein the second infusion, the first subsequent infusion, and the one or more subsequent infusions of the anti-CD20 antibody protein are infused at the following rate: 100 mL per hour for the first 30 minutes and 400 mL per hour for the remaining 30 minutes. The method of any one of claims 46 to 58, wherein the multiple infusion dosing regimen of the anti-CD20 antibody protein reduces or delays the progression of MS symptoms. The method of any of claims 46 to 59, wherein the subject administered the multiple infusion dosing regimen of the anti-CD20 antibody protein achieves a reduction in the total number of gadolinium-enhancing T1 lesions as measured by MRI scans compared to subjects receiving 14 mg teriflunomide administered orally daily during the same treatment period. The method of any of claims 46 to 60, wherein the subject administered the multiple infusion dosing regimen of the anti-CD20 antibody protein achieves a reduction in the total number of new and enlarging T2 hyperintense lesions as measured by MRI scans compared to subjects receiving 14 mg of teriflunomide administered orally daily during the same treatment period. The method of any of claims 46 to 61, wherein the subject administered the multiple infusion dosing regimen of the anti-CD20 antibody protein achieves an increase in the free disease activity (NEDA) status compared to a subject receiving daily oral administration of 14 mg teriflunomide during the same treatment period. The method of any of claims 46 to 62, wherein the subject administered the multiple infusion dosing regimen of the anti-CD20 antibody protein achieves an increase in Confirmed Disability Improvement (CDI) compared to subjects receiving 14 mg teriflunomide orally daily during the same treatment period. The method of any of claims 46 to 63, wherein the subject administered the multiple infusion dosing regimen of the anti-CD20 antibody protein achieves an increase in the Multiple Sclerosis Functional Composite (MSFC) score compared to subjects receiving 14 mg teriflunomide orally daily during the same treatment period. The method of any of claims 46 to 64, wherein the subject administered the multiple infusion dosing regimen of the anti-CD20 antibody protein achieves an improvement in the Timed 25-Foot Walk (T25FW) score compared to subjects receiving 14 mg teriflunomide orally daily during the same treatment period. The method of any of claims 46 to 65, wherein the subject administered the multiple infusion dosing regimen of the anti-CD20 antibody protein achieves an improvement in the 9-Hole Peg test (9-HPT) score compared to subjects receiving 14 mg teriflunomide orally daily during the same treatment period. The method of any of claims 46 to 66, wherein the subject administered the multiple infusion dosing regimen of the anti-CD20 antibody protein achieves a significant reduction in the volume and number of new T1 hypointense lesions as measured by MRI scans compared to subjects receiving 14 mg of teriflunomide orally daily during the same treatment period. The method of any one of claims 46 to 67, wherein the multiple infusion dosing regimen of the anti-CD20 antibody protein results in a geometric mean steady-state AUC of 3000 mcg/mL (CV = 28%) per day and a mean maximum concentration of 139 mcg/mL (CV = 15%). The method of any of claims 45 to 68, wherein RMS comprises clinically isolated syndrome ("CIS"); relapsing remitting MS ("RRMS"); or active secondary progressive MS ("SPMS"). The method of any one of claims 45 to 69, wherein the individual is diagnosed with RMS according to the McDonald Criteria (2010). The method of any one of claims 31 to 70, wherein the individual is a human. A method for inactivating viruses or adventitious agents in rat myeloma cells expressing an anti-CD20 antibody protein in a composition as claimed in any one of claims 1 to 23, wherein the method maintains suitability for antibody production in a 15,000 L or 20,000 L bioreactor. A method for reducing the immunogenicity of an anti-CD20 antibody protein in a composition as claimed in any one of claims 1 to 23, wherein the method maintains the suitability of producing the antibody in a 15,000 L or 20,000 L bioreactor.