Anti-CD20 antibody preparations and use of anti-CD20 antibodies for the treatment of CD20-positive diseases

Abstract

Disclosed are anti-CD20 antibody formulations and uses of anti-CD20 antibodies for the treatment of CD20-positive diseases, including methods of using anti-CD20 antibodies to treat CD20-positive diseases, such as neuromyelitis optica spectrum disorder (NMOSD), non-Hodgkin's lymphoma (NHL), multiple sclerosis (MS), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), Wegener's granulomatosis (WG), microscopic polyangiitis (MPA), lupus nephritis, systemic lupus erythematosus, and chronic lymphocytic leukemia (CLL).

Description

[Technical Field]

[0001] This application is submitted with an electronic sequence listing. This sequence listing is provided as a file titled DSP1P202582J_SequenceListing, created on September 17, 2020, with a size of 8 kilobytes. The electronic information contained in this sequence listing is incorporated herein by reference in its entirety.

[0002] The present invention belongs to the field of biopharmaceuticals and relates to anti-CD20 antibody preparations and methods of using anti-CD20 antibodies to treat CD20-positive diseases such as neuromyelitis optica spectrum disorder (NMOSD), non-Hodgkin lymphoma (NHL), multiple sclerosis (MS), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), Wegener's granulomatosis (WG), microscopic polyangiitis (MPA), lupus nephritis, systemic lupus erythematosus, and chronic lymphocytic leukemia (CLL). [Background technology]

[0003] The CD20 molecule is a non-glycosylated phosphoprotein specifically labeled on the surface of human lymphocyte subgroups (B cells). It consists of 297 amino acids with a molecular weight of 33-37 kD and is expressed on the surface of over 95% of B cells. CD20 is present in both normal and malignant B cells, and is particularly expressed in over 90% of B-cell non-Hodgkin lymphomas. The CD20 molecule has four transmembrane domains, with the amino and carboxyl termini located inside the plasma membrane. Between the third and fourth transmembrane domains lies a loop region consisting of 43 amino acid residues that constitute the major epitope. The CD20 antigen molecule is relatively exposed and accessible. When CD20 molecules are brought close together by antibody action, the polymer formed by crosslinking or supercrosslinking functions as a calcium ion channel, allowing extracellular calcium ions to flow into the cell. Furthermore, Src family tyrosine protein kinases are in close proximity and therefore activate each other. The signaling pathway is initiated, endogenous calcium stores are mobilized, and both lead to an increase in intracellular calcium ion concentration, which then affects the execution of the cell cycle, regulates cell proliferation and differentiation, and ultimately leads to the onset of apoptosis.

[0004] While the exact role of CD20 in promoting B cell proliferation and differentiation is unclear, CD20 provides an important target for antibody-mediated therapies used to control B cells involved in cancer and autoimmune diseases.

[0005] Compared to conventional organic and inorganic drugs, anti-CD20 antibodies are larger and more complex. To maintain the biological activity of the antibody, formulations must preserve the overall conformational integrity of the protein's amino acid core sequence while simultaneously preventing the degradation of multiple functional groups of the antibody.

[0006] Neuromyelitis optica (NMO) is an immune-mediated inflammatory demyelinating disease of the central nervous system (CNS) that primarily affects the optic nerve and spinal cord, characterized by severe optic neuritis (ON) and transverse myelitis (LETM) that expands longitudinally. In-depth research has revealed that non-optical and non-spinal behaviors are also clinical features of NMO. The etiology of NMO is primarily associated with aquaporin 4 antibody (AQP4-IgG). Furthermore, there is a group of limited forms of demyelinating disorders that do not meet the clinical diagnostic criteria for NMO. These disorders have similar etiologies and clinical features to NMO, and some cases eventually develop into NMO. In 2007, these disorders were collectively named neuromyelitis optica spectrum disorder (NMOSD). In 2015, the International Panel for the Diagnosis of NMO (IPND) established a new international consensus on the diagnostic criteria for NMOSD, unifying the terms NMO and NMOSD under the single term NMOSD (Wingerchuk, et al., Neurology, 2015, 85(2): 177-189). [Overview of the project] [Problems that the invention aims to solve]

[0007] Based on this, the present invention provides an anti-CD20 antibody preparation and the use of an anti-CD20 antibody for the treatment of CD20-positive diseases. [Means for solving the problem]

[0008] In a first embodiment, the present invention provides a liquid formulation comprising an anti-CD20 antibody in a concentration of 10 mg / ml to 120 mg / ml, a buffer, a stabilizer, and a surfactant, and having a pH value in the range of 5 to 7.

[0009] In some embodiments, the solvent for the antibody preparation is water. In some embodiments, the solvent for the antibody preparation is sterile water for injection.

[0010] In some embodiments, the liquid formulation comprises 10 mg / ml to 120 mg / ml of anti-CD20 antibody, 10 mM to 30 mM of buffer, 58 mM to 292 mM of stabilizer, and 0.1 mg / ml to 0.5 mg / ml of surfactant, and the liquid formulation has a pH value in the range of 5.5 to 6.5.

[0011] In some embodiments, the liquid formulation comprises 15 mg / ml to 80 mg / ml of anti-CD20 antibody, 10 mM to 30 mM of buffer, 80 mM to 240 mM of stabilizer, and 0.1 mg / ml to 0.4 mg / ml of surfactant, and the liquid formulation has a pH value in the range of 5.5 to 6.2.

[0012] In some embodiments, the buffer is selected from succinate buffer, citrate buffer, phosphate buffer, histidine buffer, and acetate buffer.

[0013] In some embodiments, the stabilizer is selected from sucrose, trehalose, sorbitol, mannitol, and methionine, and the surfactant is selected from polysorbate-80 and polysorbate-20.

[0014] In some embodiments, the succinate buffer contains succinic acid and sodium succinate; the citrate buffer contains citric acid and sodium citrate; the histidine buffer contains L-histidine and L-histidine hydrochloride; and the acetate buffer contains acetic acid and sodium acetate.

[0015] In some embodiments, the concentration of the anti-CD20 antibody is about 15 mg / ml, 40 mg / ml, 60 mg / ml, 80 mg / ml (i.e., 8%), or a numerical value or range between any two of these values (including the endpoint values), the concentration of the buffer is about 10 mM, 15 mM, 26 mM, 30 mM, or a numerical value or range between any two of these values (including the endpoint values), the concentration of the stabilizer is about 80 mM, 140 mM, 190 mM, 240 mM, or a numerical value or range between any two of these values (including the endpoint values), the concentration of the surfactant is about 0.1 mg / ml, 0.3 mg / ml, 0.4 mg / ml (i.e., 0.04%), or a numerical value or range between any two of these values (including the endpoint values), and the pH value is about 5.5, 5.6, 5.7, 6.0, 6.2, or a numerical value or range between any two of these values (including the endpoint values).

[0016] In some embodiments, the anti-CD20 antibody is selected from monoclonal antibodies, CD20 binding fragments.

[0017] In some embodiments, the anti-CD20 antibody comprises a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 1 or a polypeptide having at least 80% sequence identity with SEQ ID NO: 1, and a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 2 or a polypeptide having at least 80% sequence identity with SEQ ID NO: 2.

[0018] In some embodiments, VL comprises a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a numerical value or range (including endpoints) between any two of these values of sequence identity with SEQ ID NO: 1. In some embodiments, VH comprises a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a numerical value or range (including endpoints) between any two of these values of sequence identity with SEQ ID NO: 2.

[0019] In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 1 and VH comprises the amino acid sequence of SEQ ID NO: 2.

[0020] In some embodiments, the anti-CD20 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 3 or a polypeptide having at least 80% sequence identity with SEQ ID NO: 3, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 or a polypeptide having at least 80% sequence identity with SEQ ID NO: 4.

[0021] In some embodiments, the light chain comprises a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a numerical value or range (including endpoints) between any two of these values of sequence identity with SEQ ID NO: 3. In some embodiments, the heavy chain comprises a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a numerical value or range (including endpoints) between any two of these values of sequence identity with SEQ ID NO: 4.

[0022] In some embodiments, the anti-CD20 antibody is A light chain containing the amino acid sequence of SEQ ID NO: 3, It contains a heavy chain containing the amino acid sequence of SEQ ID NO: 4.

[0023] In some embodiments, the anti-CD20 antibody is Each of the two light chains contains the amino acid sequence of Sequence ID No. 3, Each contains two heavy chains, each containing the amino acid sequence of SEQ ID NO: 4.

[0024] [Table A]

[0025] In some embodiments, the anti-CD20 antibody is BAT4406F. A light chain containing the amino acid sequence of SEQ ID NO: 3, It includes a heavy chain containing the amino acid sequence of SEQ ID NO: 4, It is characterized by a very low fucose content (less than 5%).

[0026] In some embodiments, the anti-CD20 antibody is BAT4406F. Each of the two light chains contains the amino acid sequence of Sequence ID No. 3, Each contains two heavy chains containing the amino acid sequence of Sequence ID No. 4, It is characterized by a very low fucose content (less than 5%).

[0027] BAT4406F is a glycosylation-optimized IgG1 subclass of a fully human monoclonal antibody described in international patent application PCT / CN2018 / 100008.

[0028] In some embodiments, the anti-CD20 mAb or its antigen-binding fragment (e.g., BAT4406F) is produced from a cell line capable of expressing an antibody with a low fucose content (e.g., 0-5%) and enhanced ADCC effect, in which the α-(1,6)-fucosyltransferase gene has been knocked out. In some embodiments, the cell line is the CHO-BAT-KF cell line described in international patent application PCT / CN2018 / 100008. The taxonomic name of the CHO host cell line is Chinese hamster ovary cell CHO-BAT-KF FUT8(- / -). The anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment can be expressed and purified as described in international patent application PCT / CN2018 / 100008. Purification can be carried out by conventional methods. For example, the cell suspension can be centrifuged to obtain the supernatant, which can then be removed by further centrifugation. The antibody can be purified using protein A affinity and an ion exchange column.

[0029] In some embodiments, glycosylation of an anti-CD20 mAb (e.g., BAT4406F) is characterized by one or more of the following: The fucose content of the antibody is very low (e.g., 0-5%). The galactose level of the antibody is low (e.g., 30% or less), The antibody mannose level is low (e.g., 5% or less), The antibody's high mannose level is low (e.g., 5% or less), The antibody G0 level is high (e.g., 60% or higher).

[0030] In some embodiments, the antibody has a low galactose level (e.g., 5% or less).

[0031] In some embodiments, the antibody has a high G0 level (e.g., 80% or higher).

[0032] In some embodiments, the glycosylation site of the antibody is an Asn residue on the heavy chain, such as Asn297 in SEQ ID NO: 4.

[0033] In some embodiments, at least about 50% of the anti-CD20 mAb (e.g., BAT4406F) may contain a G0 glycan at asparagine residue 297 (Asn297) of SEQ ID NO: 4, and / or up to about 10% of the anti-CD20 mAb may contain a Man5 glycan at amino acid residue 297 of SEQ ID NO: 4. In some embodiments, less than about 20% of the anti-CD20 mAb contains a fucosyl residue. In some embodiments, less than about 10% of the anti-CD20 mAb contains a fucosyl residue.

[0034] In some embodiments, less than 5% of the anti-CD20 mAb contains fucosyl residues. In some embodiments, less than 1% of the anti-CD20 mAb contains fucosyl residues.

[0035] In some embodiments, at least 50% of anti-CD20 mAbs (e.g., BAT4406F) contain G0 glycan at the N-glycosylation site of the constant region of the heavy chain of the anti-CD20 antibody. In some embodiments, at least 60% of anti-CD20 mAbs contain G0 glycan at the N-glycosylation site of the constant region of the heavy chain of the anti-CD20 antibody. In some embodiments, at least about 65% of anti-CD20 mAbs may contain G0 glycan at the N-glycosylation site of the constant region of the heavy chain of the anti-CD20 antibody. In some embodiments, up to 7% of anti-CD20 mAbs may contain Man5 glycan at the N-glycosylation site of the constant region of the heavy chain of the anti-CD20 antibody. In some embodiments, up to about 5% of anti-CD20 mAbs may contain Man5 glycan at the N-glycosylation site of the constant region of the heavy chain of the anti-CD20 antibody. In some embodiments, up to approximately 3% of anti-CD20 mAbs may contain Man5 glycan at the N-glycosylation site in the constant region of the heavy chain of the anti-CD20 antibody.

[0036] In some embodiments, the oligosaccharide in the Fc region of the anti-CD20 mAb (e.g., BAT4406F) in the formulation contains a fucosyl group content of about 10% or less. In some embodiments, the oligosaccharide in the Fc region of the anti-CD20 mAb contains a fucosyl group content of about 5% or less. In some embodiments, the oligosaccharide in the Fc region of the anti-CD20 mAb contains a fucosyl group content of about 1% or less.

[0037] In some embodiments, at least, or at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% of the anti-CD20 mAb in the formulation may contain a G0 glycan in the N-glycosylation site of the constant region of the heavy chain of the anti-CD20 mAb. In some embodiments, the maximum amount of anti-CD20 mAb in the formulation, or a numerical value or range (including endpoints) of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or any two of these values, may include any glycan other than G0 glycan in the N-glycosylation site of the constant region of the heavy chain of the anti-CD20 mAb.

[0038] In some embodiments, the maximum, or up to approximately 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or any two of these values ​​or a range (including endpoints) of the anti-CD20 mAb may contain G0-GN glycan in the N-glycosylation site of the steady region of the heavy chain. In some embodiments, the maximum or maximum percentage (including endpoints) of the anti-CD20 mAb, or approximately 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any two of these values, may contain Man5 glycan in the N-glycosylation site of the steady-state region of the heavy chain of the anti-CD20 mAb. In some embodiments, the maximum or approximately 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or any two of these values ​​or a range (including endpoints) of the anti-CD20 mAb may contain G1 glycans in the N-glycosylation sites of the steady-state region of the heavy chain. In some embodiments, the maximum or approximately 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or any two of these values ​​or a range (including endpoints) of the anti-CD20 mAb may contain G1' glycans in the N-glycosylation site of the steady-state region of the heavy chain of the anti-CD20 mAb.In some embodiments, the maximum or approximately 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or any two of these values ​​or a range (including endpoints) of the anti-CD20 mAb may contain G2 glycans in the N-glycosylation site of the steady-state region of the heavy chain.

[0039] In some embodiments, the fucose content of the anti-CD20 mAb (e.g., BAT4406F) in the formulation is very low. For example, the fucose content of the anti-CD20 mAb in the formulation is approximately, or up to 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any number or range between any two of these values. In some embodiments, the anti-CD20 mAb (e.g., BAT4406F) in the formulation has no fucose residues whatsoever; i.e., the formulation contains a non-fucosylated or defucosylated anti-CD20 mAb (e.g., non-fucosylated or defucosylated BAT4406F).

[0040] In some embodiments, the galactose level of the anti-CD20 mAb is low. For example, the galactose level of the antibody is approximately, or up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or a number or range between any two of these values ​​(including endpoints).

[0041] In some embodiments, the mannose content of anti-CD20 mAbs is very low. For example, the mannose content of anti-CD20 mAbs is approximately, or up to 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any number or range between any two of these values ​​(including endpoints).

[0042] In some embodiments, the high mannose content of anti-CD20 mAbs is very low. For example, the mannose content of anti-CD20 mAbs is approximately, or up to 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any number or range between any two of these values ​​(including endpoints).

[0043] In some embodiments, the G0 level of the anti-CD20 mAb is high. For example, the G0 level of anti-CD20 mAb is approximately, or at least, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or a number or range between any two of these values ​​(including the endpoints).

[0044] In some embodiments, the liquid formulation contains 20 mg / ml to 50 mg / ml of anti-CD20 antibody, wherein the concentration of anti-CD20 antibody is approximately 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, or a number or range between any two of these values ​​(including endpoints).

[0045] In some embodiments, the liquid formulation comprises 18 mM to 22 mM histidine buffer, wherein the concentration of the histidine buffer is approximately 18 mM, 19 mM, 20 mM, 22 mM, or a number or range between any two of these values ​​(including endpoints).

[0046] In some embodiments, the molar concentration ratio of L-histidine to L-histidine hydrochloride in the histidine buffer is 1 to 2:2.

[0047] In some embodiments, the liquid formulation comprises 158 mM to 225 mM trehalose, where the trehalose concentration is approximately 158 mM, 158.6 mM, 170 mM, 220 mM, 224.6 mM, 225 mM (224.6 mM is 7.68%), or a number or range between any two of these values ​​(including the endpoints).

[0048] In some embodiments, the liquid formulation contains polysorbate-80 in a concentration of 0.18 mg / ml to 0.22 mg / ml, where the concentration of polysorbate-80 is approximately 0.18 mg / ml, 0.19 mg / ml, 0.2 mg / ml, 0.22 mg / ml (i.e., 0.022%), or a numerical value or range between any two of these values ​​(including endpoint values).

[0049] In some embodiments, the pH value of the liquid formulation is in the range of 5.7 to 5.9, and the pH value is approximately 5.7, 5.8, 5.9, or a number or range between any two of these values ​​(including endpoint values).

[0050] In some embodiments, the liquid formulation contains approximately 20 mg / ml of anti-CD20 antibody, approximately 20 mM of histidine buffer, approximately 158.6 mM of trehalose (i.e., 54 mg / ml of trehalose), and approximately 0.2 mg / ml of polysorbate-80, with a pH of approximately 5.8, and at a pH of 5.8, the molar ratio of L-histidine to L-histidine hydrochloride in the histidine buffer is 2:3.

[0051] In some embodiments, the liquid formulation contains approximately 20 mg / ml of anti-CD20 antibody, approximately 20 mM of histidine buffer, approximately 224.6 mM of trehalose (i.e., 76.8 mg / ml of trehalose), and approximately 0.2 mg / ml of polysorbate-80, with a pH of approximately 5.8, and at a pH of 5.8, the molar ratio of L-histidine to L-histidine hydrochloride in the histidine buffer is 2:3.

[0052] In some embodiments, the liquid formulation contains approximately 50 mg / ml of anti-CD20 antibody, approximately 20 mM of histidine buffer, approximately 224.6 mM of trehalose (i.e., 76.8 mg / ml of trehalose), and approximately 0.2 mg / ml of polysorbate-80, with a pH of approximately 5.8, and at a pH of 5.8, the molar ratio of L-histidine to L-histidine hydrochloride in the histidine buffer is 2:3.

[0053] In some embodiments, the liquid formulation is a liquid formulation prepared according to Formulation 1, Formulation 2, Formulation 3, or Formulation 4.

[0054] In some embodiments, the liquid formulation can be used for injection, such as intravenous or subcutaneous injection.

[0055] In some embodiments, the liquid formulation prepared according to Formulation 1 is a formulation for intravenous injection.

[0056] In some embodiments, the liquid formulations prepared according to formulations 2, 3, or 4 are formulations for subcutaneous injection.

[0057] In some embodiments, the present invention further provides a formulation for intravenous injection comprising the liquid formulation and a diluent (e.g., water (such as sterile water for injection), an isotonic solution (such as a 0.9% NaCl solution for injection)). In some embodiments, the ratio of the liquid formulation to the diluent is 1:10 to 1:50, 1:20 to 1:40, or 1:25 to 1:35.

[0058] In a second embodiment, the present invention provides a method comprising administering an effective amount of the liquid formulation to a patient in need to treat a disease involving CD20-expressing cells in the patient.

[0059] In some embodiments, the patient is human.

[0060] In some embodiments, the drug is administered to the patient by injection, such as intravenous or subcutaneous injection.

[0061] In some embodiments, when administered to a patient by injection, the liquid formulation contains approximately 20 mg / ml of anti-CD20 antibody, approximately 20 mM of histidine buffer, approximately 158.6 mM of trehalose, and approximately 0.2 mg / ml of polysorbate-80, with a pH of approximately 5.8.

[0062] In some embodiments, when administered to a patient by injection, the liquid formulation contains approximately 20 mg / ml of anti-CD20 antibody, approximately 20 mM of histidine buffer, approximately 224.6 mM of trehalose, and approximately 0.2 mg / ml of polysorbate-80, with a pH of approximately 5.8.

[0063] In some embodiments, when administered to a patient by injection, the liquid formulation contains approximately 50 mg / ml of anti-CD20 antibody, approximately 20 mM of histidine buffer, approximately 224.6 mM of trehalose, and approximately 0.2 mg / ml of polysorbate-80, with a pH of approximately 5.8.

[0064] In some embodiments, the formulation is administered by intravenous injection, and the liquid formulation is diluted with a diluent (e.g., water (such as sterile water for injection), isotonic solution (such as a 0.9% NaCl solution for injection)) before intravenous injection to the patient. In some embodiments, the liquid formulation is diluted with the diluent at a ratio of 1:10 to 1:50, 1:20 to 1:40, or 1:25 to 1:35.

[0065] In some embodiments, the diseases involving CD20-expressing cells are selected from tumorigenic diseases and immunological diseases.

[0066] In some embodiments, neoplastic disorders include B-cell lymphomas, which include progenitor B-cell lymphocytic leukemia / lymphoma and mature B-cell tumors, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), low-grade, intermediate-grade and high-grade follicular lymphoma, cutaneous follicular central lymphoma, MALT type, nodular type and splenic type marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, plasmacytoma, plasmacytomyeloma, post-transplant lymphoproliferative disorders, Waldenström macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

[0067] In some embodiments, immune diseases include psoriasis, psoriatic arthritis, dermatitis, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjögren's syndrome, juvenile-onset diabetes mellitus, Reiter's disease, Behçet's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, neuromyelitis optica, immune-mediated thrombocytopenia, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, Omen's syndrome, chronic renal failure, acute infectious mononucleosis, and chronic obstructive pulmonary disease.

[0068] In some embodiments, diseases involving CD20-expressing cells are diseases or disorders that can be treated by B-cell depletion.

[0069] In some embodiments, the disease or disorder treatable by B cell depletion is selected from the group consisting of cancer, anaphylaxis, cardiovascular disease, inflammatory disease, autoimmune disease, metabolic disease, neurological disease, viral infection, and bacterial infection. For example, the disease may be cancer or anaphylaxis.

[0070] In some embodiments, the disease is pernicious anemia (Addison's disease), amyotrophic lateral sclerosis, ankylosing spondylitis, erythema multiforme, lupus nephritis, dermatomyositis, immune-mediated thrombocytopenia (e.g., acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura), Sydenham's chorea, rheumatic fever, polyglandular syndrome, Henoch-Schönlein purpura, post-streptococcal glomerulonephritis, erythema nodosum, Takayasu's arteritis, polyarteritis nodosa, Goodpasture syndrome, thromboangiitis obliterans, primary biliary cholangitis, Hashimoto's thyroiditis, thyroidopathy, chronic active hepatitis, polymyositis / dermatomyositis Myositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, tabes dorsalis, polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis and fibrous alveolitis, inflammatory skin diseases including psoriasis and dermatitis (e.g., atopic dermatitis), systemic scleroderma and sclerosis, diseases associated with inflammatory bowel disease (Crohn's disease, ulcerative colitis, etc.), respiratory distress syndrome (including adult respiratory distress syndrome (ARDS)), dermatitis, meningitis, encephalitis, colitis, glomerulonephritis, uveitis, allergic conditions such as eczema and asthma, and other conditions involving T cell infiltration or chronic inflammatory responses. Roaming arteriosclerosis, leukocyte adhesion disorders, rheumatoid arthritis, systemic lupus erythematosus (SLE), diabetes mellitus (e.g., type 1 diabetes or insulin-dependent diabetes mellitus), multiple sclerosis, Raynaud's syndrome, autoimmune thyroiditis, allergic encephalomyelitis, Sjögren's syndrome, juvenile diabetes mellitus, tuberculosis and sarcoidosis, polymyositis, granulomatous diseases and vasculitis, immune responses associated with cytokine and T lymphocyte-mediated acute and delayed hypersensitivity, diseases involving extravasation of leukocytes, central nervous system (CNS) inflammatory diseases, multiple organ dysfunction syndromes, lysis The following are selected from: hematologic anemia (including, but not limited to, cryoglobulinemia or Coombs-positive anemia), myasthenia gravis, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane diseases, antiphospholipid syndromes, allergic neuritis, Graves' disease, Lambert-Eaton myasthenic syndrome, bullous pemphigoid, pemphigus, polyglandular autoimmune syndrome, Reiter's disease, Stiff Person syndrome, Behçet's disease, giant cell arteritis, immune complex-type glomerulonephritis, IgA nephropathy, IgM polyneuropathy, immune thrombocytopenic purpura (ITP), or autoimmune thrombocytopenia.

[0071] In some embodiments, the disease is neuromyelitis optica spectrum disorder (NMOSD), non-Hodgkin lymphoma (NHL), multiple sclerosis (MS), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), Wegener's granulomatosis (WG), microscopic polyangiitis (MPA), lupus nephritis, systemic lupus erythematosus, or chronic lymphocytic leukemia (CLL).

[0072] In some embodiments, the disease is refractory or relapses after at least one prior treatment plan. In some embodiments, the disease is refractory or relapses after at least two prior treatment plans.

[0073] In a third embodiment, the present invention further provides the use of the liquid formulation for the preparation of a drug for treating a disease involving CD20-expressing cells in a patient, the disease involving CD20-expressing cells being selected from tumorigenic diseases and immunological diseases.

[0074] In some embodiments, the patient is human.

[0075] In some embodiments, neoplastic disorders include B-cell lymphomas, which include progenitor B-cell lymphocytic leukemia / lymphoma and mature B-cell tumors, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), low-grade, intermediate-grade and high-grade follicular lymphoma, cutaneous follicular central lymphoma, MALT type, nodular type and splenic type marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, plasmacytoma, plasmacytomyeloma, post-transplant lymphoproliferative disorders, Waldenström macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

[0076] In some embodiments, immune diseases include psoriasis, psoriatic arthritis, dermatitis, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjögren's syndrome, juvenile-onset diabetes mellitus, Reiter's disease, Behçet's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, neuromyelitis optica, immune-mediated thrombocytopenia, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, Omen's syndrome, chronic renal failure, acute infectious mononucleosis, and chronic obstructive pulmonary disease.

[0077] Since pH has been found to significantly affect the stability of a formulation during its development, the formulation of the present invention needs to maintain a specific pH range. The present invention maintains a pH value in the range of 5.5 to 6.2 by selecting an appropriate buffer system, such as succinate buffer, citrate buffer, phosphate buffer, histidine buffer, or acetate buffer. Trehalose, as a stabilizer, effectively protects the activity of the active ingredient in the formulation by reducing the aggregation and degradation of antibody proteins. Polysorbate-80, as a surfactant, plays an important role in preventing the aggregation of antibody proteins. The antibody formulation of the present invention has good stability.

[0078] In a fourth embodiment, the present invention further provides a method or use for treating a disease, the method or use being specific to human CD20, to a patient in need thereof. A light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 1 or a polypeptide having at least 80% sequence identity with SEQ ID NO: 1, The method involves administering a composition containing an effective amount of an anti-CD20 monoclonal antibody (mAb) or its antigen-binding fragment, which includes the amino acid sequence of SEQ ID NO: 2 or a heavy chain variable region (VH) containing a polypeptide having at least 80% sequence identity with SEQ ID NO: 2. However, the effective dose is approximately 10 mg to 3000 mg per dose.

[0079] In some embodiments, the composition is the formulation described herein.

[0080] In some embodiments, the patient has a disease characterized by CD20-positive cells. The disease may be neuromyelitis optica spectrum disorder (NMOSD), non-Hodgkin lymphoma (NHL), multiple sclerosis (MS), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), Wegener's granulomatosis (WG), microscopic polyangiitis (MPA), lupus nephritis, systemic lupus erythematosus, or chronic lymphocytic leukemia (CLL). The disease is refractory or likely to relapse after at least one previous standard treatment plan.

[0081] CD20-positive B cells are a specific type of immune cell. Studies suggest they play a crucial role in the pathogenesis of NMOSD and are considered a key factor leading to myelin and axonal damage. When blood-brain barrier permeability increases, AQP4-IgG enters the CNS and binds to the AQP4 antigen in the foot processes of astrocytes. This downregulates AQP4 expression, disrupting water homeostasis in the CNS and leading to the death of astrocytes, oligodendrocytes, and even neurons, causing demyelination and neuropathy of nerve cells. Studies suggest that since AQP4-IgG is produced only through B cell differentiation, the CD20 molecule is one of the ideal targets for the treatment of neuromyelitis optica. Studies have shown that B-cell depletion therapy using monoclonal antibodies against CD20 on the surface of B cells (such as rituximab) can reduce relapses in NMOSD and slow the progression of neurological dysfunction, demonstrating significant therapeutic effects. The main mechanisms by which these B cells are eliminated include antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cell-mediated cytotoxicity (CDC), and apoptosis.

[0082] In some embodiments, VL comprises a polypeptide having sequence identity with SEQ ID NO: 1 in a numerical or range (including endpoints) between at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any two of these values. In some embodiments, VH comprises a polypeptide having sequence identity with SEQ ID NO: 2 in a numerical or range (including endpoints) between at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any two of these values.

[0083] In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 1, and VH comprises the amino acid sequence of SEQ ID NO: 2.

[0084] In some embodiments, anti-CD20 mAb is, A light chain comprising the amino acid sequence of SEQ ID NO: 3 or a polypeptide having at least 80% sequence identity with SEQ ID NO: 3, The heavy chain comprises the amino acid sequence of SEQ ID NO: 4 or a polypeptide having at least 80% sequence identity with SEQ ID NO: 4.

[0085] In some embodiments, the light chain comprises a polypeptide having sequence identity with SEQ ID NO: 3 in a numerical or range (including endpoints) of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any two of these values. In some embodiments, the heavy chain comprises a polypeptide having sequence identity with SEQ ID NO: 4 in a numerical or range (including endpoints) of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any two of these values.

[0086] In some embodiments, anti-CD20 mAb is, A light chain containing the amino acid sequence of SEQ ID NO: 3, It contains a heavy chain containing the amino acid sequence of SEQ ID NO: 4.

[0087] In some embodiments, the anti-CD20 mAb is BAT4406F. A light chain containing the amino acid sequence of SEQ ID NO: 3, It includes a heavy chain containing the amino acid sequence of SEQ ID NO: 4, It is characterized by a very low fucose content (less than 5%).

[0088] BAT4406F is a glycosylation-optimized IgG1 subclass of a fully human monoclonal antibody described in international patent application PCT / CN2018 / 100008.

[0089] In some embodiments, the anti-CD20 mAb or its antigen-binding fragment (e.g., BAT4406F) is produced from a cell line capable of expressing an antibody with a low fucose content (e.g., 0-5%) and enhanced ADCC effect, in which the α-(1,6)-fucosyltransferase gene has been knocked out. In some embodiments, the cell line is the CHO-BAT-KF cell line described in international patent application PCT / CN2018 / 10008. The taxonomic name of the CHO host cell line is Chinese hamster ovary cell CHO-BAT-KF FUT8(- / -). The anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment can be expressed and purified as described in international patent application PCT / CN2018 / 100008. Purification can be carried out by conventional methods. For example, the cell suspension can be centrifuged to obtain the supernatant, which can then be removed by further centrifugation. The antibody can be purified using protein A affinity and an ion exchange column.

[0090] In some embodiments, glycosylation of an anti-CD20 mAb (e.g., BAT4406F) is characterized by one or more of the following: The fucose content of the antibody is very low (e.g., 0-5%). The galactose level of the antibody is low (e.g., 30% or less), The antibody mannose level is low (e.g., 5% or less), The antibody's high mannose level is low (e.g., 5% or less), The antibody G0 level is high (e.g., 60% or higher).

[0091] In some embodiments, the antibody has a low galactose level (e.g., 5% or less).

[0092] In some embodiments, the antibody has a high G0 level (e.g., 80% or higher).

[0093] In some embodiments, the glycosylation site of the antibody is an Asn residue on the heavy chain, such as Asn297 in SEQ ID NO: 4.

[0094] In some embodiments, at least about 50% of the anti-CD20 mAb (e.g., BAT4406F) may contain a G0 glycan at asparagine residue 297 (Asn297) of SEQ ID NO: 4, and / or up to about 10% of the anti-CD20 mAb may contain a Man5 glycan at amino acid residue 297 of SEQ ID NO: 4. In some embodiments, less than about 20% of the anti-CD20 mAb contains a fucosyl residue. In some embodiments, less than about 10% of the anti-CD20 mAb contains a fucosyl residue.

[0095] In some embodiments, less than 5% of the anti-CD20 mAb contains fucosyl residues. In some embodiments, less than 1% of the anti-CD20 mAb contains fucosyl residues.

[0096] In some embodiments, at least 50% of anti-CD20 mAbs (e.g., BAT4406F) contain G0 glycan at the N-glycosylation site of the constant region of the heavy chain of the anti-CD20 antibody. In some embodiments, at least 60% of anti-CD20 mAbs contain G0 glycan at the N-glycosylation site of the constant region of the heavy chain of the anti-CD20 antibody. In some embodiments, at least about 65% of anti-CD20 mAbs may contain G0 glycan at the N-glycosylation site of the constant region of the heavy chain of the anti-CD20 antibody. In some embodiments, up to 7% of anti-CD20 mAbs may contain Man5 glycan at the N-glycosylation site of the constant region of the heavy chain of the anti-CD20 antibody. In some embodiments, up to about 5% of anti-CD20 mAbs may contain Man5 glycan at the N-glycosylation site of the constant region of the heavy chain of the anti-CD20 antibody. In some embodiments, up to approximately 3% of anti-CD20 mAbs may contain Man5 glycan at the N-glycosylation site in the constant region of the heavy chain of the anti-CD20 antibody.

[0097] In some embodiments, the oligosaccharide in the Fc region of the anti-CD20 mAb (e.g., BAT4406F) in the composition contains a fucosyl group content of about 10% or less. In some embodiments, the oligosaccharide in the Fc region of the anti-CD20 mAb contains a fucosyl group content of about 5% or less. In some embodiments, the oligosaccharide in the Fc region of the anti-CD20 mAb contains a fucosyl group content of about 1% or less.

[0098] In some embodiments, at least, or at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% of the anti-CD20 mAb in the composition may contain G0 glycans in the N-glycosylation sites of the steady-state region of the heavy chain of the anti-CD20 mAb. In some embodiments, the maximum amount of anti-CD20 mAb in the composition, or a numerical value or range (including endpoints) of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or any two of these values, may include any glycan other than G0 glycan in the N-glycosylation site of the steady-state region of the heavy chain of anti-CD20 mAb.

[0099] In some embodiments, the maximum, or up to approximately 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or any two of these values ​​or a range (including endpoints) of the anti-CD20 mAb may contain G0-GN glycan in the N-glycosylation site of the steady region of the heavy chain. In some embodiments, the maximum or maximum percentage (including endpoints) of the anti-CD20 mAb, or approximately 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any two of these values, may contain Man5 glycan in the N-glycosylation site of the steady-state region of the heavy chain of the anti-CD20 mAb. In some embodiments, the maximum or approximately 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or any two of these values ​​or a range (including endpoints) of the anti-CD20 mAb may contain G1 glycans in the N-glycosylation sites of the steady-state region of the heavy chain. In some embodiments, the maximum or approximately 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or any two of these values ​​or a range (including endpoints) of the anti-CD20 mAb may contain G1' glycans in the N-glycosylation site of the steady-state region of the heavy chain of the anti-CD20 mAb.In some embodiments, the maximum or approximately 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or any two of these values ​​or a range (including endpoints) of the anti-CD20 mAb may contain G2 glycans in the N-glycosylation site of the steady-state region of the heavy chain.

[0100] In some embodiments, the fucose content of the anti-CD20 mAb (e.g., BAT4406F) in the composition is very low. For example, the fucose content of the anti-CD20 mAb in the composition is approximately, or up to, 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any number or range between any two of these values ​​(including endpoints). In some embodiments, the anti-CD20 mAb (e.g., BAT4406F) in the composition has no fucose residues, i.e., the composition contains a non-fucosylated or defucosylated anti-CD20 mAb (e.g., non-fucosylated or defucosylated BAT4406F).

[0101] In some embodiments, the galactose level of the anti-CD20 mAb is low. For example, the galactose level of the antibody is approximately, or up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or a number or range between any two of these values ​​(including endpoints).

[0102] In some embodiments, the mannose content of anti-CD20 mAbs is very low. For example, the mannose content of anti-CD20 mAbs is approximately, or up to 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any number or range between any two of these values ​​(including endpoints).

[0103] In some embodiments, the high mannose content of anti-CD20 mAbs is very low. For example, the mannose content of anti-CD20 mAbs is approximately, or up to 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any number or range between any two of these values ​​(including endpoints).

[0104] In some embodiments, the G0 level of the anti-CD20 mAb is high. For example, the G0 level of anti-CD20 mAb is approximately, or at least, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or a number or range between any two of these values ​​(including the endpoints).

[0105] In some embodiments, the disease treated by the methods or uses disclosed herein is a disease or disorder treatable by B cell depletion.

[0106] In some embodiments, the disease treated by the methods or uses disclosed herein is selected from the group consisting of cancer, anaphylaxis, cardiovascular disease, inflammatory disease, autoimmune disease, metabolic disease, neurological disease, viral infection, and bacterial infection. For example, the disease is cancer or anaphylaxis.

[0107] In some embodiments, the disease is pernicious anemia (Addison's disease), amyotrophic lateral sclerosis, ankylosing spondylitis, erythema multiforme, lupus nephritis, dermatomyositis, immune-mediated thrombocytopenia (e.g., acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura), Sydenham's chorea, rheumatic fever, polyglandular syndrome, Henoch-Schönlein purpura, post-streptococcal glomerulonephritis, erythema nodosum, Takayasu's arteritis, polyarteritis nodosa, Goodpasture syndrome, thromboangiitis obliterans, primary biliary cholangitis, Hashimoto's thyroiditis, thyroidopathy, chronic active hepatitis, polymyositis / dermatomyositis Myositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, tabes dorsalis, polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis and fibrous alveolitis, inflammatory skin diseases including psoriasis and dermatitis (e.g., atopic dermatitis), systemic scleroderma and sclerosis, diseases associated with inflammatory bowel disease (Crohn's disease, ulcerative colitis, etc.), respiratory distress syndrome (including adult respiratory distress syndrome (ARDS)), dermatitis, meningitis, encephalitis, colitis, glomerulonephritis, uveitis, allergic conditions such as eczema and asthma, and other conditions involving T cell infiltration or chronic inflammatory responses. Roaming arteriosclerosis, leukocyte adhesion disorders, rheumatoid arthritis, systemic lupus erythematosus (SLE), diabetes mellitus (e.g., type 1 diabetes or insulin-dependent diabetes mellitus), multiple sclerosis, Raynaud's syndrome, autoimmune thyroiditis, allergic encephalomyelitis, Sjögren's syndrome, juvenile diabetes mellitus, tuberculosis and sarcoidosis, polymyositis, granulomatous diseases and vasculitis, immune responses associated with cytokine and T lymphocyte-mediated acute and delayed hypersensitivity, diseases involving extravasation of leukocytes, central nervous system (CNS) inflammatory diseases, multiple organ dysfunction syndromes, lysis The following are selected from: hematologic anemia (including, but not limited to, cryoglobulinemia or Coombs-positive anemia), myasthenia gravis, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane diseases, antiphospholipid syndromes, allergic neuritis, Graves' disease, Lambert-Eaton myasthenic syndrome, bullous pemphigoid, pemphigus, polyglandular autoimmune syndrome, Reiter's disease, Stiff Person syndrome, Behçet's disease, giant cell arteritis, immune complex-type glomerulonephritis, IgA nephropathy, IgM polyneuropathy, immune thrombocytopenic purpura (ITP), or autoimmune thrombocytopenia.

[0108] In some embodiments, the disease is neuromyelitis optica spectrum disorder (NMOSD), non-Hodgkin lymphoma (NHL), multiple sclerosis (MS), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), Wegener's granulomatosis (WG), microscopic polyangiitis (MPA), lupus nephritis, systemic lupus erythematosus, or chronic lymphocytic leukemia (CLL).

[0109] In some embodiments, the disease is refractory or relapses after at least one prior treatment plan. In some embodiments, the disease is refractory or relapses after at least two prior treatment plans.

[0110] In some embodiments, a method is provided for treating neuromyelitis optica spectrum disorder in a patient in need thereof, comprising administering an effective amount of an anti-CD20 mAb described herein (e.g., BAT4406F) or its antigen-binding fragment, the effective amount being about 10 mg to about 3000 mg per dose.

[0111] In some embodiments, the use of an effective amount of an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment in the manufacture of a pharmacopoeia for treating the diseases described herein is provided, where the effective amount is about 0.5 mg / kg to about 30 mg / kg, or about 10 mg to about 3000 mg per dose.

[0112] In a fifth embodiment, a kit is provided comprising an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment for the treatment of the diseases described herein, and instructions for administering the anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment to a patient in need in the amount described herein.

[0113] Anti-CD20 mAbs (e.g., BAT4406F) or their antigen-binding fragments may be formulated as pharmaceutical compositions and administered parenterally to patients in various ways suitable for a selected route of administration, for example, intravenous (iv), intramuscular, topical, or subcutaneous routes. The amount of anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment may vary depending on the properties of the drug, the extent of drug internalization, transport, and release on the cell surface, the disease being treated, and the patient's condition, such as age, sex, and weight.

[0114] In some embodiments, the amount of anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment administered each time is 0.5 mg / kg to 20 mg / kg, or about those amounts. For example, the amount of anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment administered each time may be 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, or any number or range between any two of these values, or approximately those amounts. For example, the amount of antibody administered each time may be approximately 0.5 mg / kg to 10 mg / kg. For example, effective doses are approximately 0.5 mg / kg to 0.9 mg / kg, 0.7 mg / kg to 1.3 mg / kg, 1.5 mg / kg to 2.5 mg / kg, 3 mg / kg to 5 mg / kg, 5 mg / kg to 7 mg / kg, 7 mg / kg to 9 mg / kg, or 9 mg / kg to 10 mg / kg.

[0115] In some embodiments, an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment may be administered once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every three months, once every four months, once every five months, once every six months, or at a numerical value or range between any two of these values, or at approximately those frequencies. For example, the effective dose administered is approximately once a month, or once every two, three, four, five, or six months, ranging from approximately 0.5 mg / kg to approximately 10 mg / kg. The effective dose administered may be approximately 0.5 mg / kg to 0.9 mg / kg, 0.7 mg / kg to 1.3 mg / kg, 1.5 mg / kg to 2.5 mg / kg, 3 mg / kg to 5 mg / kg, 5 mg / kg to 7 mg / kg, 7 mg / kg to 9 mg / kg, or 9 mg / kg to 10 mg / kg, once a month, or once every two, three, four, five, or six months. The effective dose administered may also be approximately 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 6 mg / kg, 8 mg / kg, or 10 mg / kg, once every three weeks to six months.

[0116] In some embodiments, a method is provided comprising administering an effective dose of an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment to a patient in need to treat a disease, the effective dose being approximately 20 mg to approximately 2000 mg once every 3 weeks to 6 months.

[0117] In a sixth embodiment, the use of an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment in the manufacture of a pharmacopoeia for the treatment of the diseases described herein is provided, wherein the anti-CD20 mAb or its antigen-binding fragment is administered to the patient in amounts ranging from about 20 mg to about 2000 mg once every three weeks to six months.

[0118] In some embodiments, the effective dose is approximately 50 mg to approximately 1000 mg, administered monthly to every six months. In some embodiments, the effective dose is approximately 100 mg to approximately 500 mg, administered monthly, every two months, every three months, every four months, every five months, or every six months. In some embodiments, the effective dose is approximately 50 mg to approximately 300 mg, administered monthly, every two months, every three months, every four months, every five months, or every six months. In some embodiments, the effective dose is approximately 100 mg to approximately 300 mg, administered every two months or every three months. In some embodiments, the effective dose is approximately 100 mg, approximately 200 mg, or approximately 300 mg, administered every two months or every three months.

[0119] In some embodiments, the effective dose is approximately 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg, administered once a month to once every six months. In some embodiments, the effective dose is approximately 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg, administered once a month, once every two months, once every three months, once every four months, once every five months, or once every six months. In some embodiments, the effective dose is approximately 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, or 600 mg, administered once a month, once every two months, once every three months, once every four months, once every five months, or once every six months. In some embodiments, the effective dose is approximately 100 mg, 150 mg, 200 mg, 250 mg, or 300 mg, administered once a month, once every two months, once every three months, once every four months, once every five months, or once every six months. In some embodiments, the effective dose is approximately 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, or 600 mg, administered once every two months or once every three months. In some embodiments, the effective dose is approximately 100 mg, 150 mg, 200 mg, 250 mg, or 300 mg once every two months. In some embodiments, the effective dose is approximately 100 mg, 150 mg, 200 mg, 250 mg, or 300 mg once every three months.

[0120] In some embodiments, the total number of doses of an anti-CD20 mAb or its antigen-binding fragment may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or a number or range (including endpoints) between any two of these values, or about that number of doses. For example, an anti-CD20 mAb or its antigen-binding fragment may be administered about 4 times.

[0121] In some embodiments, the duration of administration may be 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or a number or range between any two of these values, or approximately those durations. For example, administration may be carried out over approximately 1 hour. In some embodiments, the duration of administration may depend on the circumstances of administration. In some embodiments, the duration of infusion may exceed 10 hours. In some embodiments, the duration of infusion may be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 14 hours, or a number or range between any two of these values ​​(including endpoints), or approximately those durations.

[0122] In some embodiments, an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment is administered by intravenous infusion.

[0123] In some embodiments, an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment is administered in combination with another therapeutic means. For example, an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment may be administered co-administered with another therapeutic means for treating the diseases described herein, such as immunosuppressants, glucocorticoids, corticosteroids, antimalarial agents, cytotoxic agents, integrin antagonists, cytokine agonists, or hormones.

[0124] In a seventh aspect, a pharmaceutical composition comprising one or more antibodies described herein is provided, for example, such a composition containing 0.1% or more of the antibody. The percentage of the antibody may vary and may be between about 2% and about 90% of the weight of the dosage form of interest. Depending on the amount of antibody in such a therapeutically useful composition, an effective dose can be administered.

[0125] Examples of pharmaceutical compositions suitable for injection or infusion include sterile aqueous solutions or dispersions in pharmaceutically acceptable liquid carriers or vehicles, or sterile powders containing active ingredients optionally encapsulated in liposomes, suitable for immediate preparation of sterile injectable or injectable solutions or dispersions. Other forms of pharmaceutical compositions include topical formulations such as gels, ointments, creams, lotions, or transdermal patches. Pharmaceutical compositions involve the use of techniques familiar to those skilled in the art. Suitable pharmaceutically acceptable carriers other than those described herein are known in the art. See, for example, Remington, The Science and Practice of Pharmacy, 20th Edition, 2000, Lippincott Williams & Wilkins, (Editors: Gennaro, AR, et al.).

[0126] In an eighth embodiment, a method for producing a pharmaceutical composition is provided, comprising mixing the antibody described herein with a pharmaceutically acceptable carrier. In some embodiments, a method for producing a formulation described herein is provided, comprising mixing the antibody described herein with an excipient such as a buffer, stabilizer, or surfactant described herein. Methods for mixing an antibody with a pharmaceutically acceptable carrier are generally known in the art, for example, by uniformly mixing the antibody with a liquid or finely divided solid carrier, or both, in the required ratio, and then, if necessary, shaping the resulting mixture into a desired form. In some embodiments, the method comprises mixing an aqueous antibody solution with an aqueous solution containing an excipient such as a buffer, stabilizer, or surfactant described herein.

[0127] In some embodiments, the antibody is formulated as an injectable substance, for example, at a concentration of 2 to 50 mg / mL in an aqueous solution containing 4 to 10 mg / mL of sodium chloride and / or 5 to 12 mg / mL of sodium acetate, or at a concentration of 2 to 50 mg / mL in an aqueous solution containing 5 to 10 mg / mL of sodium chloride, 1 to 5 mg / mL of disodium hydrogen phosphate heptahydrate, and 0.1 to 0.5 mg / mL of sodium dihydrogen phosphate monohydrate.

[0128] Other examples of antibody preparations include injectable preparations having an antibody concentration of 2 to 100 mg / mL in an aqueous solution containing 0.5-1.0% sodium chloride, 0.05-0.10% sodium dihydrogen phosphate dihydrate, 1.0-2.0% disodium hydrogen phosphate dihydrate, 0.01-0.05% sodium citrate, 0.10-0.20% citric acid monohydrate, 1.0-2.0% mannitol, 0.1-0.2% polysorbate 80, and water for injection (USP standard). Sodium hydroxide may be added to adjust the pH as needed.

[0129] In some embodiments, the antibody is formulated as described herein. [Brief explanation of the drawing]

[0130] [Figure 1] This shows the number of particles (particles / mL) in samples containing different surfactants. [Figure 2] This shows the turbidity of samples containing different surfactants after shaking. [Figure 3] The image shows particles of samples containing different surfactants after five cycles of freezing and thawing. [Modes for carrying out the invention]

[0131] definition As used herein, unless otherwise specified, the following definitions apply:

[0132] Where used herein, unless otherwise specified, the singular forms "1" and "one" include references to plural forms. Therefore, for example, a reference to "compound" includes multiple compounds.

[0133] As used herein, "about" can be understood by those skilled in the art and may vary to some extent depending on the context in which it is used. In some embodiments, "about" means up to ±10%, ±5%, or ±1% of a particular term. "About ×" includes "×".

[0134] An "amino acid" refers to an α-aminocarboxylic acid, which may be encoded directly or as a precursor by nucleic acids. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The encoding of the same amino acid by different codons is called "genetic code degeneracy." As used in this application, the term "amino acid" refers to naturally occurring α-aminocarboxylic acids, and includes alanine (3-letter code ala, or 1-letter code A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0135] The term "antibody" is used in its broadest sense, encompassing monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity.

[0136] An "antibody fragment" generally refers to a portion of a full-length antibody that includes its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, as well as diabodies, linear antibodies, single-chain antibodies, and multispecific antibodies formed by antibody fragments.

[0137] A monoclonal antibody (mAb) is an antibody prepared by a single immune cell, and all immune cells are clones from a single parent cell. Monoclonal antibodies can have monovalent affinity because they bind to the same epitope (the antigenic portion recognized by the antibody). In contrast, polyclonal antibodies bind to multiple epitopes and are generally secreted by several different plasma cells. Monoclonal antibodies can also be modified into bispecific monoclonal antibodies by increasing the therapeutic target of a single monoclonal antibody to two epitopes. Monoclonal antibodies can be prepared by hybridoma, recombination, transgenes, or other techniques known to those skilled in the art.

[0138] In this invention, the amount of buffer refers to the total amount of buffer pairs in the buffer system that constitutes the buffer. In some embodiments, molar concentration is used as the unit of amount of buffer, and its value refers to the molar concentration of buffer pairs in the buffer system. For example, when a histidine buffer composed of L-histidine and L-histidine hydrochloride is used, the target concentration of the histidine buffer (e.g., 10 mM) is the sum of the concentrations of L-histidine and L-histidine hydrochloride (e.g., L-histidine is 5 mM, L-histidine hydrochloride is 5 mM, or L-histidine is 6 mM, L-histidine hydrochloride is 4 mM, or L-histidine is 3.46 mM, L-histidine hydrochloride is 6.54 mM, etc.).

[0139] The formulations of the present invention may be prepared using histidine hydrochloride, also known as histidine monohydrate, which may be an excipient or a hydrate thereof, such as anhydrous histidine hydrochloride or histidine hydrochloride monohydrate. "5 mM histidine hydrochloride" may be 5 mmol of histidine hydrochloride or histidine hydrochloride hydrate dissolved in a solvent to obtain 1 L of solution, and 958 mg of histidine hydrochloride may contain 958 mg of histidine hydrochloride or an equivalent amount of hydrate.

[0140] The following are some examples of the amount of anhydrous trehalose corresponding to the amount of trehalose hydrate. 1) 158.6 mM trehalose means that 158.6 mM anhydrous trehalose (158.6 mM anhydrous trehalose is equivalent to approximately 5.4% anhydrous trehalose) or 158.6 mM trehalose dihydrate (158.6 mM trehalose dihydrate is equivalent to approximately 6% trehalose dihydrate) may be added. 2) 224.6 mM trehalose means that 224.6 mM anhydrous trehalose (224.6 mM anhydrous trehalose is equivalent to approximately 7.68% anhydrous trehalose) or 224.6 mM trehalose dihydrate (224.6 mM trehalose dihydrate is equivalent to approximately 8.5% trehalose dihydrate) may be added.

[0141] In this invention, the concentration "%" represents a mass-volume concentration in units of g / ml. For example, a 0.9% sodium chloride solution means that 0.9 g of sodium chloride is dissolved in a solvent to obtain 100 ml of solution, which means that the solution contains 0.9 g / 100 ml of sodium chloride.

[0142] Where used herein, the term “contains” is intended to mean that a composition or method includes the elements described, but does not exclude others. Where used to define a composition or method, “substantially composed of” means excluding other elements that have any substantial significance to the combination. For example, a composition substantially composed of the elements defined herein does not exclude other elements that do not substantially affect the basic and novel characteristics of the claimed invention. “Composed of” means excluding trace amounts or more of other components and steps of the substantial method described. Embodiments limited by each of these transitional terms are within the scope of this disclosure.

[0143] "Patient" or "subject" refers to a mammal, including humans and non-human mammals. In some embodiments, the patient is human. In some embodiments, the patient is a non-human mammal such as a wild animal, livestock, or farm animal. In other embodiments, the patient is a primate such as a dog, cat, mouse, rat, rabbit, guinea pig, or crab-eating macaque.

[0144] "To treat" or "to cure" a patient's disease means (1) preventing the onset of the disease in a patient who is predisposed to the disease or who has not yet shown symptoms of the disease, (2) suppressing the disease or its symptoms, or preventing its onset, or (3) improving or regressing the disease or its symptoms.

[0145] "Effective dose" is intended to mean the amount of an active compound or drug that elicits a biological or medical response in a tissue, system, animal, individual, or human, including the treatment of a disease, as required by researchers, veterinarians, physicians, or other clinicians.

[0146] "Administering" a composition can be achieved by injection, infusion, parenteral, intravenous, transmucosal, sublingual, intramuscular, intradermal, intranasal, intraperitoneal, intra-arterial, subcutaneous absorption, or any other method in combination with other known techniques. "Administering" may also mean formulating a composition that includes instructions for administration using one or more of the above techniques. In some embodiments, administration is systemic.

[0147] Where used herein, the phrase “needs it” means that a patient has been identified as needing a particular method or treatment. In some embodiments, identification may be by any diagnostic means. For example, neuromyelitis optica spectrum disorder can be diagnosed according to Appendix 1. A patient may need any of the methods and treatments described herein.

[0148] In the following example, AUC (0~inf) AUC represents the area under the concentration-time curve from time zero to time infinity.(0~t) This represents the area under the concentration-time curve from time zero to the last measurable time point, C max represents the peak blood concentration, T max t represents the time to reach peak blood concentration, 1 / 2 This represents the time it takes for the blood concentration to decrease by half, CL t Vd represents plasma clearance, Vd represents apparent volume of distribution, and MRT represents mean residence time.

[0149] All publications are incorporated herein by reference in their entirety for all purposes.

[0150] Disclosed in one embodiment is a liquid formulation containing an anti-CD20 antibody, comprising 10 mg / ml to 120 mg / ml of anti-CD20 antibody, a buffer, a stabilizer, and a surfactant as components, wherein the liquid formulation has a pH value in the range of 5 to 7.

[0151] In some embodiments, the liquid formulation comprises 10 mg / ml to 120 mg / ml of anti-CD20 antibody, 10 mM to 30 mM of buffer, 58 mM to 292 mM of stabilizer, and 0.1 mg / ml to 0.5 mg / ml of surfactant, and the liquid formulation has a pH value in the range of 5.5 to 6.5.

[0152] In some embodiments, the liquid formulation comprises 15 mg / ml to 80 mg / ml of anti-CD20 antibody, 10 mM to 30 mM of buffer, 80 mM to 240 mM of stabilizer, and 0.1 mg / ml to 0.4 mg / ml of surfactant, and the liquid formulation has a pH value in the range of 5.5 to 6.2.

[0153] In some embodiments, the buffer is selected from succinate buffer, citrate buffer, phosphate buffer, histidine buffer, and acetate buffer.

[0154] In some embodiments, the stabilizer is selected from sucrose, trehalose, sorbitol, mannitol, and methionine, and the surfactant is selected from polysorbate-80 and polysorbate-20.

[0155] In some embodiments, the concentration of the anti-CD20 antibody is approximately 15 mg / ml, 40 mg / ml, 60 mg / ml, 80 mg / ml (i.e., 8%), or a numerical value or range between any two of these values ​​(including endpoint values); the concentration of the buffer is approximately 10 mM, 15 mM, 26 mM, 30 mM, or a numerical value or range between any two of these values ​​(including endpoint values); the concentration of the stabilizer is approximately 80 mM, 140 mM, 190 mM, 240 mM, or a numerical value or range between any two of these values ​​(including endpoint values); the concentration of the surfactant is approximately 0.1 mg / ml, 0.3 mg / ml, 0.4 mg / ml (i.e., 0.04%), or a numerical value or range between any two of these values ​​(including endpoint values); and the pH value is approximately 5.5, 5.6, 5.7, 6.0, 6.2, or a numerical value or range between any two of these values ​​(including endpoint values).

[0156] In some embodiments, the anti-CD20 antibody is A light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 1 or a polypeptide having at least 80% sequence identity with SEQ ID NO: 1, It comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 2 or a polypeptide having at least 80% sequence identity with SEQ ID NO: 2.

[0157] In some embodiments, the anti-CD20 antibody is BAT4406F.

[0158] Disclosed in a further embodiment is a method comprising administering an effective amount of the liquid formulation to a patient in need to treat a disease involving CD20-expressing cells in the patient.

[0159] In some embodiments, the patient is human.

[0160] In some embodiments, the drug is administered to the patient by injection, such as intravenous or subcutaneous injection.

[0161] In some embodiments, when administered to a patient by injection, the liquid formulation contains approximately 20 mg / ml of anti-CD20 antibody, approximately 20 mM of histidine buffer, approximately 158.6 mM of trehalose, and approximately 0.2 mg / ml of polysorbate-80, with a pH of approximately 5.8.

[0162] In some embodiments, when administered to a patient by injection, the liquid formulation contains approximately 20 mg / ml of anti-CD20 antibody, approximately 20 mM of histidine buffer, approximately 224.6 mM of trehalose, and approximately 0.2 mg / ml of polysorbate-80, with a pH of approximately 5.8.

[0163] In some embodiments, when administered to a patient by injection, the liquid formulation contains approximately 50 mg / ml of anti-CD20 antibody, approximately 20 mM of histidine buffer, approximately 224.6 mM of trehalose, and approximately 0.2 mg / ml of polysorbate-80, with a pH of approximately 5.8.

[0164] In some embodiments, the diseases involving CD20-expressing cells are selected from tumorigenic diseases and immunological diseases.

[0165] In some embodiments, neoplastic disorders include B-cell lymphomas, which include progenitor B-cell lymphocytic leukemia / lymphoma and mature B-cell tumors, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), low-grade, intermediate-grade and high-grade follicular lymphoma, cutaneous follicular central lymphoma, MALT type, nodular type and splenic type marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, plasmacytoma, plasmacytomyeloma, post-transplant lymphoproliferative disorders, Waldenström macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

[0166] In some embodiments, neoplastic diseases include immune diseases, which include psoriasis, psoriatic arthritis, dermatitis, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjögren's syndrome, juvenile-onset diabetes mellitus, Reiter's disease, Behçet's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, neuromyelitis optica, immune-mediated thrombocytopenia, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, Omen's syndrome, chronic renal failure, acute infectious mononucleosis, and chronic obstructive pulmonary disease.

[0167] In some embodiments, diseases involving CD20-expressing cells are diseases or disorders that can be treated by B-cell depletion.

[0168] In some embodiments, the disease or disorder treatable by B cell depletion is selected from the group consisting of cancer, anaphylaxis, cardiovascular disease, inflammatory disease, autoimmune disease, metabolic disease, neurological disease, viral infection, and bacterial infection. For example, the disease may be cancer or anaphylaxis.

[0169] In some embodiments, the disease is pernicious anemia (Addison's disease), amyotrophic lateral sclerosis, ankylosing spondylitis, erythema multiforme, lupus nephritis, dermatomyositis, immune-mediated thrombocytopenia (e.g., acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura), Sydenham's chorea, rheumatic fever, polyglandular syndrome, Henoch-Schönlein purpura, post-streptococcal glomerulonephritis, erythema nodosum, Takayasu's arteritis, polyarteritis nodosa, Goodpasture syndrome, thromboangiitis obliterans, primary biliary cholangitis, Hashimoto's thyroiditis, thyroidopathy, chronic active hepatitis, polymyositis / dermatomyositis Myositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, tabes dorsalis, polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis and fibrous alveolitis, inflammatory skin diseases including psoriasis and dermatitis (e.g., atopic dermatitis), systemic scleroderma and sclerosis, diseases associated with inflammatory bowel disease (Crohn's disease, ulcerative colitis, etc.), respiratory distress syndrome (including adult respiratory distress syndrome (ARDS)), dermatitis, meningitis, encephalitis, colitis, glomerulonephritis, uveitis, allergic conditions such as eczema and asthma, and other conditions involving T cell infiltration or chronic inflammatory responses. Roaming arteriosclerosis, leukocyte adhesion disorders, rheumatoid arthritis, systemic lupus erythematosus (SLE), diabetes mellitus (e.g., type 1 diabetes or insulin-dependent diabetes mellitus), multiple sclerosis, Raynaud's syndrome, autoimmune thyroiditis, allergic encephalomyelitis, Sjögren's syndrome, juvenile diabetes mellitus, tuberculosis and sarcoidosis, polymyositis, granulomatous diseases and vasculitis, immune responses associated with cytokine and T lymphocyte-mediated acute and delayed hypersensitivity, diseases involving extravasation of leukocytes, central nervous system (CNS) inflammatory diseases, multiple organ dysfunction syndromes, lysis The following are selected from: hematologic anemia (including, but not limited to, cryoglobulinemia or Coombs-positive anemia), myasthenia gravis, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane diseases, antiphospholipid syndromes, allergic neuritis, Graves' disease, Lambert-Eaton myasthenic syndrome, bullous pemphigoid, pemphigus, polyglandular autoimmune syndrome, Reiter's disease, Stiff Person syndrome, Behçet's disease, giant cell arteritis, immune complex-type glomerulonephritis, IgA nephropathy, IgM polyneuropathy, immune thrombocytopenic purpura (ITP), or autoimmune thrombocytopenia.

[0170] In some embodiments, the disease is neuromyelitis optica spectrum disorder (NMOSD), non-Hodgkin lymphoma (NHL), multiple sclerosis (MS), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), Wegener's granulomatosis (WG), microscopic polyangiitis (MPA), lupus nephritis, systemic lupus erythematosus, or chronic lymphocytic leukemia (CLL).

[0171] In some embodiments, the disease is refractory or relapses after at least one prior treatment plan. In some embodiments, the disease is refractory or relapses after at least two prior treatment plans.

[0172] Disclosed in a further embodiment is the use of the liquid formulation for the formulation of a drug for treating a disease involving CD20-expressing cells in a patient, the disease involving CD20-expressing cells being selected from tumorigenic diseases and immunological diseases. In some embodiments, the patient is human.

[0173] In some embodiments, neoplastic disorders include B-cell lymphomas, which include progenitor B-cell lymphocytic leukemia / lymphoma and mature B-cell tumors, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), low-grade, intermediate-grade and high-grade follicular lymphoma, cutaneous follicular central lymphoma, MALT type, nodular type and splenic type marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, plasmacytoma, plasmacytomyeloma, post-transplant lymphoproliferative disorders, Waldenström macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

[0174] In some embodiments, neoplastic diseases include immune diseases, which include psoriasis, psoriatic arthritis, dermatitis, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud's syndrome, Sjögren's syndrome, juvenile-onset diabetes mellitus, Reiter's disease, Behçet's disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathy, neuromyelitis optica, immune-mediated thrombocytopenia, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, Omen's syndrome, chronic renal failure, acute infectious mononucleosis, and chronic obstructive pulmonary disease.

[0175] Disclosed in further embodiments are methods or uses for treating diseases. In some embodiments, the method or use provides to a patient in need of it a substance specific to human CD20, A light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 1 or a polypeptide having at least 80% sequence identity with SEQ ID NO: 1, The method involves administering a composition containing an effective amount of an anti-CD20 monoclonal antibody (mAb) or its antigen-binding fragment, which includes the amino acid sequence of SEQ ID NO: 2 or a heavy chain variable region (VH) containing a polypeptide having at least 80% sequence identity with SEQ ID NO: 2. However, the effective dose is approximately 10 mg to 3000 mg per dose.

[0176] In some embodiments, the anti-CD20 monoclonal antibody (mAb) is BAT4406F.

[0177] In some embodiments, the disease treated by the methods or uses disclosed herein is a disease or disorder treatable by B cell depletion.

[0178] In some embodiments, the disease treated by the methods or uses disclosed herein is selected from the group consisting of cancer, anaphylaxis, cardiovascular disease, inflammatory disease, autoimmune disease, metabolic disease, neurological disease, viral infection, and bacterial infection. For example, the disease is cancer or anaphylaxis.

[0179] In some embodiments, the disease is pernicious anemia (Addison's disease), amyotrophic lateral sclerosis, ankylosing spondylitis, erythema multiforme, lupus nephritis, dermatomyositis, immune-mediated thrombocytopenia (e.g., acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura), Sydenham's chorea, rheumatic fever, polyglandular syndrome, Henoch-Schönlein purpura, post-streptococcal glomerulonephritis, erythema nodosum, Takayasu's arteritis, polyarteritis nodosa, Goodpasture syndrome, thromboangiitis obliterans, primary biliary cholangitis, Hashimoto's thyroiditis, thyroidopathy, chronic active hepatitis, polymyositis / dermatomyositis Myositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, tabes dorsalis, polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis and fibrous alveolitis, inflammatory skin diseases including psoriasis and dermatitis (e.g., atopic dermatitis), systemic scleroderma and sclerosis, diseases associated with inflammatory bowel disease (Crohn's disease, ulcerative colitis, etc.), respiratory distress syndrome (including adult respiratory distress syndrome (ARDS)), dermatitis, meningitis, encephalitis, colitis, glomerulonephritis, uveitis, allergic conditions such as eczema and asthma, and other conditions involving T cell infiltration or chronic inflammatory responses. Roaming arteriosclerosis, leukocyte adhesion disorders, rheumatoid arthritis, systemic lupus erythematosus (SLE), diabetes mellitus (e.g., type 1 diabetes or insulin-dependent diabetes mellitus), multiple sclerosis, Raynaud's syndrome, autoimmune thyroiditis, allergic encephalomyelitis, Sjögren's syndrome, juvenile diabetes mellitus, tuberculosis and sarcoidosis, polymyositis, granulomatous diseases and vasculitis, immune responses associated with cytokine and T lymphocyte-mediated acute and delayed hypersensitivity, diseases involving extravasation of leukocytes, central nervous system (CNS) inflammatory diseases, multiple organ dysfunction syndromes, lysis The following are selected from: hematologic anemia (including, but not limited to, cryoglobulinemia or Coombs-positive anemia), myasthenia gravis, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane diseases, antiphospholipid syndromes, allergic neuritis, Graves' disease, Lambert-Eaton myasthenic syndrome, bullous pemphigoid, pemphigus, polyglandular autoimmune syndrome, Reiter's disease, Stiff Person syndrome, Behçet's disease, giant cell arteritis, immune complex-type glomerulonephritis, IgA nephropathy, IgM polyneuropathy, immune thrombocytopenic purpura (ITP), or autoimmune thrombocytopenia.

[0180] In some embodiments, the disease is neuromyelitis optica spectrum disorder (NMOSD), non-Hodgkin lymphoma (NHL), multiple sclerosis (MS), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), Wegener's granulomatosis (WG), microscopic polyangiitis (MPA), lupus nephritis, systemic lupus erythematosus, or chronic lymphocytic leukemia (CLL).

[0181] In some embodiments, the disease is neuromyelitis optica spectrum disorder (NMOSD), including neuromyelitis optica (NMO).

[0182] In some embodiments, NMOSD is characterized by optic neuritis (ON) and transverse myelitis extending longitudinally (LETM). In some embodiments, neuromyelitis optica spectrum disorder (NMOSD) is diagnosed according to the NMOSD diagnostic criteria established by the International Panel for NMO Diagnosis (IPND), e.g., as described in Appendix 1. In some embodiments, NMOSD is AQP4-IgG positive. In some embodiments, NMOSD is AQP4-IgG negative or unknown.

[0183] In some embodiments, NMOSD is characterized by optic neuritis. In some embodiments, NMOSD is characterized by acute myelitis. In some embodiments, NMOSD is characterized by area last syndrome (APS). In some embodiments, NMOSD is characterized by acute brainstem syndrome. In some embodiments, NMOSD is characterized by symptomatic narcolepsy. In some embodiments, NMOSD is characterized by acute diencephalic clinical syndrome. In some embodiments, NMOSD is characterized by symptomatic brain syndrome.

[0184] In some embodiments, a method is provided for treating neuromyelitis optica spectrum disorder in patients requiring it, comprising administering an effective amount of an anti-CD20 mAb described herein (e.g., BAT4406F) or its antigen-binding fragment, the effective amount being about 20 mg to about 2000 mg per treatment cycle or multiple treatment cycles. In some embodiments, a method is provided for treating neuromyelitis optica spectrum disorder in patients requiring it, comprising administering an effective amount of an anti-CD20 mAb described herein (e.g., BAT4406F) or its antigen-binding fragment, the effective amount being about 300 mg to about 2000 mg per treatment cycle or multiple treatment cycles. Each treatment cycle may be administered by a single dose (e.g., a single injection) or by multiple doses (e.g., two, three, four, or five injections) over a treatment cycle period such as one week, two weeks, three weeks, four weeks, or one month.

[0185] In some embodiments, the disease is neuromyelitis optica spectrum disorder (NMOSD), including neuromyelitis optica (NMO).

[0186] In some embodiments, NMOSD is characterized by optic neuritis (ON) and transverse myelitis extending longitudinally (LETM). In some embodiments, neuromyelitis optica spectrum disorder (NMOSD) is diagnosed according to the NMOSD diagnostic criteria established by the International Panel for NMO Diagnosis (IPND), e.g., as described in Appendix 1. In some embodiments, NMOSD is AQP4-IgG positive. In some embodiments, NMOSD is AQP4-IgG negative or unknown.

[0187] In some embodiments, NMOSD is characterized by optic neuritis. In some embodiments, NMOSD is characterized by acute myelitis. In some embodiments, NMOSD is characterized by area last syndrome (APS). In some embodiments, NMOSD is characterized by acute brainstem syndrome. In some embodiments, NMOSD is characterized by symptomatic narcolepsy. In some embodiments, NMOSD is characterized by acute diencephalic clinical syndrome. In some embodiments, NMOSD is characterized by symptomatic brain syndrome.

[0188] In some embodiments, a method is provided for treating neuromyelitis optica spectrum disorder in patients requiring it, comprising administering an effective amount of an anti-CD20 mAb described herein (e.g., BAT4406F) or its antigen-binding fragment, the effective amount being about 20 mg to 2000 mg per treatment cycle or multiple treatment cycles. In some embodiments, a method is provided for treating neuromyelitis optica spectrum disorder in patients requiring it, comprising administering an effective amount of an anti-CD20 mAb described herein (e.g., BAT4406F) or its antigen-binding fragment, the effective amount being about 300 mg to 2000 mg per treatment cycle or multiple treatment cycles. Each treatment cycle may be administered by a single dose (e.g., a single injection) or by multiple doses (e.g., two, three, four, or five injections) over a treatment cycle period such as one week, two weeks, three weeks, four weeks, or one month. In some embodiments, the disease is refractory or relapses after at least one previous treatment plan. In some embodiments, the disease is refractory or relapses after at least two prior treatment plans. In some embodiments, the prior treatment plans include standard treatment plans used to treat acute attacks, such as corticosteroids (e.g., methylprednisolone) and plasmapheresis. In some embodiments, the prior treatment plans include standard treatment plans used to prevent attacks, such as eculizumab (humanized mAb) and other immunosuppressants (e.g., azathioprine, rituximab, mycophenolate mofetil).

[0189] Anti-CD20 mAbs (e.g., BAT4406F) or their antigen-binding fragments may be formulated as pharmaceutical compositions and administered parenterally to patients in various ways suitable for a selected route of administration, for example, intravenous (iv), intramuscular, topical, or subcutaneous routes. The amount of anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment may vary depending on the properties of the drug, the extent of drug internalization, transport, and release on the cell surface, the disease being treated, and the patient's condition, such as age, sex, and weight.

[0190] In some embodiments, the amount of anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment administered each time is 5 mg / kg to 30 mg / kg, or about those amounts. For example, the amount of anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment administered each time is 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, or a number or range between any two of these values ​​(including the endpoints), or about those amounts.

[0191] In some embodiments, methods are provided for treating NMOSD, comprising administering an effective dose of an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment to patients in need, the effective dose ranging from approximately 20 mg to 2000 mg per treatment cycle or multiple treatment cycles. In some embodiments, methods are provided for treating NMOSD, comprising administering an effective dose of an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment to patients in need, the effective dose ranging from approximately 300 mg to 2000 mg per treatment cycle or multiple treatment cycles. The dosage plan and dosage form are determined by a benefit-risk assessment of anti-CD20 mAb (e.g., BAT4406F) in a patient population with a specific disease and general clinical guidelines. In some embodiments, the effective dose is approximately 20 mg to approximately 2000 mg of anti-CD20 mAb (e.g., BAT4406F) per treatment cycle, with one treatment cycle administered monthly to once every 12 months. In some embodiments, the effective dose is approximately 20 mg to approximately 300 mg of anti-CD20 mAb (e.g., BAT4406F) per treatment cycle, with one treatment cycle administered monthly to once every 12 months. In some embodiments, the effective dose is approximately 300 mg to approximately 2000 mg of anti-CD20 mAb (e.g., BAT4406F) per treatment cycle, with one treatment cycle administered monthly to once every 12 months. In some embodiments, the effective dose is approximately 300 mg to approximately 2000 mg per treatment cycle, with one treatment cycle administered once monthly, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, or once every twelve months. In some embodiments, the effective dose is approximately 500 mg to approximately 2000 mg per treatment cycle, with one treatment cycle administered once monthly, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, or once every twelve months.In some embodiments, the effective dose is approximately 300 mg, approximately 500 mg, approximately 1000 mg, approximately 1500 mg, or approximately 2000 mg per treatment cycle, or any range between any two of these values ​​(including endpoints), and one treatment cycle is administered every two months, every three months, every six months, or every twelve months.

[0192] In some embodiments, the effective dose is approximately 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 260 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg per treatment cycle, with one treatment cycle administered once a month to once every 12 months. In some embodiments, the effective dose is approximately 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 260 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg per treatment cycle, with each treatment cycle administered once monthly, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, or once every twelve months. In some embodiments, the effective dose is approximately 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 260 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, or 600 mg per treatment cycle, with each treatment cycle administered once monthly, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, or once every twelve months. In some embodiments, the effective dose is approximately 300 mg per treatment cycle, with each treatment cycle administered once monthly, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, or once every twelve months.In some embodiments, the effective dose is approximately 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 260 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, or 600 mg per treatment cycle, with one treatment cycle administered every two months or every three months. In some embodiments, the effective dose is approximately 300 mg per treatment cycle, with one treatment cycle administered every two months. In some embodiments, the effective dose is approximately 300 mg per treatment cycle or a range between any two of these values ​​(including endpoints), with one treatment cycle administered every three months. In some embodiments, the effective dose is approximately 500 mg, 1000 mg, 1500 mg, or 2000 mg per treatment cycle, or a range between any two of these values ​​(including endpoints), with one treatment cycle administered every six months or every twelve months.

[0193] In some embodiments, the effective dose is approximately 20 mg to approximately 2000 mg of anti-CD20 mAb (e.g., BAT4406F) per treatment cycle, administered once every four months. In some embodiments, the effective dose is approximately 20 mg to approximately 300 mg of anti-CD20 mAb (e.g., BAT4406F) per treatment cycle, administered once every four months. In some embodiments, the effective dose is approximately 300 mg to approximately 2000 mg of anti-CD20 mAb (e.g., BAT4406F) per treatment cycle, administered once every four months. In some embodiments, the effective dose is approximately 500 mg to approximately 1000 mg per treatment cycle, with one treatment cycle administered once every four months. In some embodiments, the effective dose is approximately 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg per treatment cycle, or any range between any two of these values ​​(including endpoints), with one treatment cycle administered once every four months.

[0194] In some embodiments, the effective dose is approximately 20 mg to approximately 2000 mg of anti-CD20 mAb (e.g., BAT4406F) per treatment cycle, with one treatment cycle administered every 5 months. In some embodiments, the effective dose is approximately 20 mg to approximately 300 mg of anti-CD20 mAb (e.g., BAT4406F) per treatment cycle, with one treatment cycle administered every 5 months. In some embodiments, the effective dose is approximately 300 mg to approximately 2000 mg of anti-CD20 mAb (e.g., BAT4406F) per treatment cycle, with one treatment cycle administered every 5 months. In some embodiments, the effective dose is approximately 500 mg to approximately 1000 mg per treatment cycle, with one treatment cycle administered every 5 months. In some embodiments, the effective dose is approximately 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg per treatment cycle, or any range between any two of these values ​​(including endpoints), with one treatment cycle administered once every five months.

[0195] In some embodiments, the effective dose is approximately 20 mg to approximately 2000 mg of anti-CD20 mAb (e.g., BAT4406F) per treatment cycle, with one treatment cycle administered every six months. In some embodiments, the effective dose is approximately 20 mg to approximately 300 mg of anti-CD20 mAb (e.g., BAT4406F) per treatment cycle, with one treatment cycle administered every six months. In some embodiments, the effective dose is approximately 300 mg to approximately 2000 mg of anti-CD20 mAb (e.g., BAT4406F) per treatment cycle, with one treatment cycle administered every six months. In some embodiments, the effective dose is approximately 500 mg to approximately 1000 mg per treatment cycle, with one treatment cycle administered every six months. In some embodiments, the effective dose is approximately 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg per treatment cycle, or any range between any two of these values ​​(including endpoints), with one treatment cycle administered once every six months.

[0196] In some embodiments, the effective dose is approximately 20 mg to approximately 2000 mg of anti-CD20 mAb (e.g., BAT4406F) per treatment cycle, with one treatment cycle administered once every 12 months. In some embodiments, the effective dose is approximately 20 mg to approximately 300 mg of anti-CD20 mAb (e.g., BAT4406F) per treatment cycle, with one treatment cycle administered once every 12 months. In some embodiments, the effective dose is approximately 300 mg to approximately 2000 mg of anti-CD20 mAb (e.g., BAT4406F) per treatment cycle, with one treatment cycle administered once every 12 months. In some embodiments, the effective dose is approximately 500 mg to approximately 1000 mg per treatment cycle, with one treatment cycle administered once every 12 months. In some embodiments, the effective dose is approximately 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg per treatment cycle, or any range between any two of these values ​​(including endpoints), with one treatment cycle administered once every 12 months.

[0197] In some embodiments, the treatment cycle is administered once. In some embodiments, the treatment cycle is administered multiple times, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, or in a range between any two of these values. For example, the treatment cycle is administered only once or four times.

[0198] In some embodiments, the amount of the treatment cycle is from about 20 mg to about 2000 mg, the treatment cycle is administered once, and is not repeated until required due to recurrence of the disease. In some embodiments, the amount of the treatment cycle is from about 20 mg to about 300 mg, the treatment cycle is administered once, and is not repeated until required due to recurrence of the disease. In some embodiments, the amount of the treatment cycle is from about 300 mg to about 2000 mg, the treatment cycle is administered once, and is not repeated until required due to recurrence of the disease.

[0199] In some embodiments, the amount of the treatment cycle is about 300 mg, about 500 mg, about 1000 mg, about 1500 mg, about 2000 mg, about 2500 mg or about 3000 mg, or any range between any two of these values, including the endpoints. In some embodiments, the amount of the treatment cycle is about 500 mg. In some embodiments, the amount of the treatment cycle is about 1000 mg. In some embodiments, the amount of the treatment cycle is about 1500 mg. In some embodiments, the amount of the treatment cycle is about 2000 mg. In some embodiments, there is no recurrence of the disease for at least about 3 months after administration. In some embodiments, there is no recurrence of the disease for at least about 6 months after administration. In some embodiments, there is no recurrence of the disease for at least about 9 months after administration. In some embodiments, there is no recurrence of the disease for at least about 12 months after administration. In some embodiments, there is no recurrence of the disease after administration. Recurrence of the disease includes recurrence of symptoms of the disease, diagnosis of the disease based on conventional diagnostic criteria of the disease, or detection of the occurrence or potential occurrence of the disease by detection of one or more biomarker levels (e.g., total T cells, total B cells, naive B cells, NK cells, CD3 + CD4 + CD8 + CD19 + CD20 + CD27 + and / or CD40 + levels, etc.).

[0200] In some embodiments, the treatment cycle dose is administered by a single injection (e.g., intravenous or subcutaneous injection). In some embodiments, the treatment cycle dose is approximately 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg per treatment cycle, or any range between any two of these values ​​(including endpoints), administered by a single injection, and the treatment cycle is repeated every 6 months, every 12 months, or as needed for disease relapse.

[0201] In some embodiments, the treatment cycle dose is administered by two injections (e.g., intravenous or subcutaneous) at intervals of approximately one, two, three, or four weeks. In some embodiments, the treatment cycle dose is administered by four injections (e.g., intravenous or subcutaneous), each administered weekly for four consecutive weeks. In some embodiments, the treatment cycle dose is administered by five injections (e.g., intravenous or subcutaneous), each administered weekly for five consecutive weeks.

[0202] In some embodiments, the treatment cycle dose is approximately 300 mg, administered by a first injection of approximately 150 mg and a second injection of approximately 150 mg two weeks after the first injection. In some embodiments, the treatment cycle dose is approximately 300 mg, administered by three injections of approximately 100 mg each, administered weekly for three consecutive weeks. In some embodiments, the treatment cycle is repeated every six months, every twelve months, or as needed for disease relapse.

[0203] In some embodiments, the treatment cycle dose is approximately 500 mg and is administered by two injections. In some embodiments, the treatment cycle dose is administered by a first injection of approximately 250 mg and a second injection of approximately 250 mg two weeks after the first injection. In some embodiments, the treatment cycle is repeated every six months, every twelve months, or as needed for disease relapse.

[0204] In some embodiments, the treatment cycle dose is approximately 1000 mg and is administered by two injections. In some embodiments, the treatment cycle dose is administered by a first injection of approximately 500 mg and a second injection of approximately 500 mg two weeks after the first injection. In some embodiments, the treatment cycle is repeated every six months, every twelve months, or as needed for disease relapse.

[0205] In some embodiments, the treatment cycle dose is approximately 2000 mg and is administered by two injections. In some embodiments, the treatment cycle dose is administered by a first injection of approximately 1000 mg and a second injection of approximately 1000 mg two weeks after the first injection. In some embodiments, the treatment cycle is repeated every 6 months, every 12 months, or as needed for disease relapse.

[0206] In some embodiments, the treatment cycle dose is approximately 400 mg, administered by four injections (e.g., approximately 100 mg per injection). In some embodiments, each injection is administered weekly for four consecutive weeks. In some embodiments, the treatment cycle is repeated every six months, every twelve months, or as needed for disease relapses.

[0207] In some embodiments, the treatment cycle dose is approximately 500 mg and is administered by four injections (e.g., approximately 125 mg per injection). In some embodiments, each injection is administered weekly for four consecutive weeks. In some embodiments, the treatment cycle dose is approximately 500 mg and is administered by five injections (e.g., approximately 100 mg per injection). In some embodiments, each injection is administered weekly for five consecutive weeks. In some embodiments, the treatment cycle is repeated every six months, every twelve months, or as needed for disease relapse.

[0208] In some embodiments, the treatment cycle dose is approximately 1000 mg and is administered by four injections (e.g., approximately 250 mg per injection). In some embodiments, each injection is administered weekly for four consecutive weeks. In some embodiments, the treatment cycle dose is approximately 1000 mg and is administered by five injections (e.g., approximately 200 mg per injection). In some embodiments, each injection is administered weekly for five consecutive weeks. In some embodiments, the treatment cycle is repeated every six months, every twelve months, or as needed for disease relapse.

[0209] In some embodiments, the treatment cycle dose is approximately 2000 mg and is administered by four injections (e.g., approximately 500 mg per injection). In some embodiments, each injection is administered weekly for four consecutive weeks. In some embodiments, the treatment cycle dose is approximately 2000 mg and is administered by five injections (e.g., approximately 400 mg per injection). In some embodiments, each injection is administered weekly for five consecutive weeks. In some embodiments, the treatment cycle is repeated every six months, every twelve months, or as needed for disease relapse.

[0210] In some embodiments, an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment is administered by injection, such as subcutaneous (sc) injection, intraperitoneal (ip) injection, parenteral injection, intra-arterial injection, intravenous (iv) injection, or intravenous (iv) infusion. In some embodiments, an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment is administered by intravenous infusion. In some embodiments, an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment is administered by subcutaneous injection.

[0211] In some embodiments, an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment is administered by intravenous (iv) infusion. In some embodiments, the duration of intravenous infusion depends on the circumstances of administration. In some embodiments, the duration of intravenous infusion is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 14 hours, or a number or range (including endpoints) between any two of these values, or approximately those durations. For example, the administration may take place over approximately 1 hour.

[0212] In some embodiments, an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment is administered in combination with another therapeutic means. For example, an anti-CD20 mAb (e.g., BAT4406F) or its antigen-binding fragment may be administered co-administered with another therapeutic means for treating the diseases described herein, such as immunosuppressants, glucocorticoids, corticosteroids, antimalarial agents, cytotoxic agents, integrin antagonists, cytokine agonists, or hormones.

[0213] The technical solutions of the present invention are further illustrated by the following specific embodiments, but these embodiments do not limit the scope of protection of the present invention. Several other substantial modifications and adjustments made in accordance with the spirit of the present invention are still within the scope of protection of the present invention.

[0214] Unless otherwise specified, the solvent for the liquid formulations in the following examples is water, such as water for injection, or ordinary physiological saline for injection. [Examples]

[0215] Example 1: In the following examples, the anti-CD20 antibody is BAT4406F, which contains the heavy chain shown in SEQ ID NO: 4 and the light chain shown in SEQ ID NO: 3. EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQG TTVTVSSASTKGPSVFPLAPGSSKSTSGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 4), EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC (Sequence ID 3).

[0216] pH range screening Two buffer solutions with a pH range of 5.0 to 6.5 were selected: 20 mM histidine buffer (His) and 20 mM acetate buffer (NaAc). Purified BAT4406F (the heavy chain of the antibody was as shown in SEQ ID NO: 4, and the light chain of the antibody was as shown in SEQ ID NO: 3) was ultrafiltered, and the solvent was changed to prepare six formulations (buffer solutions and pH values ​​were His 5.5, His 6.0, His 6.5, NaAc 5.0, NaAc 5.5, and NaAc 6.0, with an antibody concentration of 18 mg / mL). The specific components are shown in Table 1.

[0217] Samples of the formulation were subjected to a high-temperature test at 40°C and analyzed by SEC-HPLC and IEC-HPLC on day 0 (0d), week 1 (1W), week 2 (2W), and week 3 (3W). The results are shown in Table 2.

[0218] [Table 1]

[0219] [Table 2]

[0220] As shown in the SEC-HPLC monomer purity data in Table 2, His 6.0 and His 5.5 showed less degradation in SEC monomer purity and performed better than His 6.5, NaAc 5.5, NaAc 5.0, and NaAc 6.0. The monomers in His buffer had good stability when the pH was in the range of 5.5 to 6.0. From the IEC-HPLC main peak content data, it was found that His 6.0, His 5.5, and NaAc 6.0 performed better than NaAc 5.5, NaAc 5.0, and His 6.5, indicating that the IEC main peak content in the formulations had good stability in buffers with a pH in the range of 5.5 to 6.0.

[0221] According to the results of SEC-HPLC and IEC-HPLC, BAT4406F exhibited good stability at high temperatures (40°C) when the pH of the antibody sample was in the range of 5.5 to 6.0.

[0222] Screening of buffer systems The antibody exhibited good stability in the pH range of 5.5 to 6.0. Therefore, buffers with a buffering capacity in the pH range of 5.5 to 6.0 were selected for screening. Five different buffers were prepared: succinate-sodium succinate buffer (Sua), citrate-sodium citrate buffer (CB), phosphate buffer (PB), histidine-histidine hydrochloride buffer (His), and acetate-sodium acetate buffer (NaAc) (see Table 3). Purified BAT4406F was ultrafiltered and replaced with the five buffers mentioned above to adjust the antibody concentration to 20 mg / mL. Samples of the resulting formulations were subjected to a high-temperature test at 40°C and analyzed by SEC-HPLC and IEC-HPLC at day 0, week 1, week 2, and week 3, respectively. The results are shown in Table 4.

[0223] [Table 3]

[0224] [Table 4]

[0225] As shown in Table 4, His buffer was optimal for maintaining the purity of SEC-HPLC monomers under high-temperature conditions. IEC-HPLC main peak content data shows that His buffer and NaAc buffer perform better than other buffers.

[0226] The results from SEC-HPLC and IEC-HPLC showed that antibody samples in His buffer exhibited better stability at higher temperatures (40°C) during SEC-HPLC and IEC-HPLC compared to other buffers.

[0227] pH level optimization (1) Optimization of pH value 1 The antibody sample exhibited good stability in histidine buffer with a pH range of approximately 5.5 to 6.0. pH optimization was performed. Purified BAT4406F was ultrafiltered and the solvent was changed to prepare five formulations with different pH values: histidine buffer (pH 5.6), histidine buffer (pH 5.8), histidine buffer (pH 6.0), histidine buffer (pH 6.2), and histidine buffer (pH 6.4). The antibody concentration was adjusted to 20 mg / mL for all formulations. The samples were subjected to a high-temperature test at 40°C, and samples were taken on day 0, week 1, week 2, and week 3, and analyzed by SEC-HPLC and IEC-HPLC, respectively. The results are shown in Table 5.

[0228] [Table 5]

[0229] As shown in Table 5, 1) the formulations had similar SEC-HPLC monomer purity at high temperatures, and 2) the IEC-HPLC main peak of the samples decreased slowly when the pH was in the range of 5.6 to 6.2, indicating good stability.

[0230] According to the results of SEC-HPLC and IEC-HPLC, the antibody sample exhibited good stability when the buffer pH was in the range of 5.6 to 6.2.

[0231] 2) Optimization of pH value 2 From pH optimization 1, it was found that antibody samples exhibited good stability when the pH was in the range of 5.6 to 6.2, and that IEC-HPLC characteristics were poor when the pH exceeded 6.2. However, since the stability of the sample could not be determined when the pH was below 5.6, a pH screening test was performed again. Because the pH of His buffer is greatly affected by temperature, citrate-PB(CP) was used as the buffer this time. After ultrafiltration and solvent exchange, six samples of BAT4406F with different pH values ​​were obtained: pH 5.0, pH 5.3, pH 5.6, pH 5.9, pH 6.2, and pH 6.5. The antibody samples were subjected to a high-temperature test at 40°C, and samples were taken on day 0, week 1, week 2, and week 3, and analyzed by SEC-HPLC and IEC-HPLC, respectively. The results are shown in Table 6.

[0232] [Table 6]

[0233] As shown in Table 6, 1) the monomer purity of the samples decreased after standing at high temperature for 4 weeks, and monomer purity was poor when the pH was less than 5.3, while 2) when the pH was in the range of 5.6 to 6.5, the IEC-HPLC main peak of the samples decreased slowly and showed good stability.

[0234] pH is an important factor for liquid formulations. While the antibody exhibited different stabilities in buffers with varying pH values, fluctuations within a specific pH range did not significantly affect antibody quality. Based on the above studies involving both preliminary and advanced pH screening, a preliminary determination was made that the antibody exhibited good stability within the pH range of 5.5 to 6.2.

[0235] Screening for stabilizers BAT4406F solutions (50 mg / ml) containing seven types of stabilizers—sodium chloride (0.9%), sucrose (9%), trehalose (9%), mannitol (4%), sorbitol (4%), arginine (1%), and methionine (1%)—were each prepared, and the specific formulations are shown in Table 7. In Table 7, Tre (trehalose) refers to trehalose dihydrate.

[0236] [Table 7]

[0237] (1) High temperature test We compared samples containing a stabilizer with samples without a stabilizer at high temperature (40°C) to investigate changes in sample quality. Samples were analyzed by SEC-HPLC at weeks 1, 2, 3, and 4. The results are shown in Table 8.

[0238] [Table 8]

[0239] As shown in Table 8, Tre, Sor, and Suc exhibited the best SEC-HPLC monomer purity.

[0240] (2) Test under light irradiation conditions The quality of samples was investigated by comparing samples containing stabilizers under light irradiation. Samples were analyzed in the first and second weeks using SEC-HPLC and IEC-HPLC detection. The results are shown in Table 9.

[0241] [Table 9]

[0242] As shown in Table 9, 1) the SEC-HPLC monomer purity of sample Met showed the least decrease, followed by sample Tre, Suc, and others, and 2) the IEC-HPLC main peak content of Met and Arg was good, followed by Tre, Suc, Sor, etc.

[0243] Results under high temperature and light irradiation conditions showed that trehalose performed well in both SEC-HPLC and IEC-HPLC.

[0244] Screening of surfactants Compatibility test with normal physiological saline Since antibody preparations need to be formulated with physiological saline for clinical purposes when administered by intravenous injection, a compatibility test was performed. Antibody sample solutions containing different concentrations and types of surfactants were prepared, and the details of the formulations are shown in Table 10. The concentration of BAT4406F in the sample solution was 30 mg / mL. Sample solutions containing different concentrations of surfactants in the same volume were added to 0.9% sodium chloride solution at a compatibility ratio (1 mL sample: 29 mL of normal physiological saline), lightly mixed thoroughly, and their properties were observed. After standing for 2 hours, insoluble particle analysis was performed on the antibody solution. The results are shown in Figure 1.

[0245] [Table 10]

[0246] As shown in Figure 1, the blank without surfactant had the highest particle count, while the blank containing surfactant (polysorbate-80 (Tween 80) or polysorbate-20 (Tween 20)) generally had a lower particle count. As the surfactant content increased, the particle count decreased slightly, but increased again once the surfactant concentration reached a certain level. The particle count in the formulation was low when the surfactant was polysorbate-80 at a concentration of 0.02%.

[0247] Shaking test Vibration cannot be avoided during the transportation or transfer of samples, which may promote protein aggregation. By adding a surfactant to the formulation, protein aggregation can be effectively prevented. Sample solutions of BAT4406F (20 mg / ml) containing different concentrations and different types of surfactants were prepared, and the details of the formulation are shown in Table 11. The samples were placed on a shaker and the antibody solutions were subjected to a shaking test at 200 rpm at room temperature. To examine the change in the quality of the antibody solutions, light irradiation tests and turbidity (OD340 value) analyses were performed on the solutions at 0 h, 2 h, 4 h, 8 h, 24 h, 48 h, and 72 h. The results are shown in Figure 2 (Note: The OD value was the value obtained by detecting the protein solution at UV340 nm). In Table 11, Tre refers to trehalose dihydrate.

[0248]

Table 11

[0249] As shown in Figure 2, the blank sample and the sample with only 6% Tre started to become turbid after 4 hours of shaking, while the samples containing Tween remained clear even after 72 hours of shaking. This indicates that polysorbate-80 or polysorbate-20 has a certain protective effect on the samples under shaking, can prevent protein aggregation and turbidity, and plays a solubilizing role. For both polysorbate-80 and polysorbate-20, the samples with 0.02% and 0.03% had the lowest turbidity, and the samples with 0.01% and 0.04% had relatively higher turbidity, indicating that it is not good if the content of polysorbate is too low or too high. 0.02% of polysorbate is preferred.

[0250] Repeated Freezing and Thawing Test 1 For repeated freeze-thaw tests, samples of BAT4406F (20 mg / ml) with different polysorbate (e.g., Tween) content and type were prepared (details of the formulations are shown in Table 12, where Tre refers to trehalose dihydrate). After freezing at -80°C for 24 hours, the sample solutions were allowed to stand at 25°C for 24 hours, and five cycles of freezing and thawing were performed. Insoluble particle analysis was performed on the frozen and thawed samples, and the results are shown in Figure 3.

[0251] [Table 12]

[0252] As shown in Figure 3, after five cycles of freezing and thawing, the number of particles in the sample containing Tween was significantly lower than that in the sample without Tween, indicating that Tween effectively prevents protein aggregation during repeated freezing and thawing cycles.

[0253] Example 2: A liquid formulation containing BAT4406F was provided, consisting of approximately 15 mg / ml of BAT4406F, approximately 10 mM of succinate buffer, approximately 80 mM of sucrose, and approximately 0.1 mg / ml of polysorbate-20. The pH of the liquid formulation was approximately 5.5.

[0254] Example 3: A liquid formulation containing BAT4406F was provided, consisting of approximately 40 mg / ml of anti-CD20 antibody, approximately 15 mM of citrate buffer, approximately 140 mM of sorbitol, and approximately 0.3 mg / ml of polysorbate-80. The pH value of the liquid formulation was approximately 5.6.

[0255] Example 4: A liquid formulation containing BAT4406F was provided, consisting of approximately 60 mg / ml of anti-CD20 antibody, approximately 26 mM of acetate buffer, approximately 190 mM of sorbitol, and approximately 0.35 mg / ml of polysorbate-20. The pH value of the liquid formulation was approximately 5.7.

[0256] Example 5: A liquid formulation containing BAT4406F was provided, consisting of approximately 70 mg / ml of anti-CD20 antibody, approximately 26 mM of phosphate buffer, approximately 240 mM of methionine, and approximately 0.38 mg / ml of polysorbate-20. The pH value of the liquid formulation was approximately 6.0.

[0257] Example 6: A liquid formulation containing BAT4406F was provided, consisting of approximately 80 mg / ml of anti-CD20 antibody, approximately 30 mM of phosphate buffer, approximately 158 mM of mannitol, and approximately 0.4 mg / ml of polysorbate-20. The pH value of the liquid formulation was approximately 6.2.

[0258] Example 7: A liquid formulation containing BAT4406F was provided, consisting of approximately 20 mg / ml of anti-CD20 antibody, approximately 20 mM of histidine buffer, approximately 158.6 mM of trehalose, and approximately 0.2 mg / ml of polysorbate-80. The pH of the liquid formulation was approximately 5.8. At a pH of 5.8, the molar ratio of L-histidine to L-histidine hydrochloride in the histidine buffer was 2:3.

[0259] Example 8: A liquid formulation containing BAT4406F was provided, consisting of approximately 20 mg / ml of anti-CD20 antibody, approximately 20 mM of histidine buffer, approximately 224.6 mM of trehalose, and approximately 0.2 mg / ml of polysorbate-80. The pH of the liquid formulation was approximately 5.8. At a pH of 5.8, the molar ratio of L-histidine to L-histidine hydrochloride in the histidine buffer was 2:3.

[0260] Example 9: A liquid preparation containing BAT4406F, which is composed of components including an anti-CD20 antibody at about 50 mg / ml, a histidine buffer at about 20 mM, trehalose at about 224.6 mM, and polysorbate-80 at about 0.2 mg / ml, was provided, and the pH value of the liquid preparation was about 5.8. When the pH value was 5.8, the molar ratio of L-histidine to L-histidine hydrochloride in the histidine buffer was 2:3.

[0261] Example 10: A liquid preparation containing BAT4406F, which is composed of components including an anti-CD20 antibody at about 30 mg / ml, a histidine buffer at about 18 mM, trehalose at about 170 mM, and polysorbate-80 at about 0.18 mg / ml, was provided, and the pH value of the liquid preparation was about 5.7.

[0262] Example 11: A liquid preparation containing BAT4406F, which is composed of components including an anti-CD20 antibody at about 40 mg / ml, a histidine buffer at about 19 mM, trehalose at about 225 mM, and polysorbate-80 at about 0.19 mg / ml, was provided, and the pH value of the liquid preparation was about 5.9.

[0263] Example 12: A liquid preparation containing BAT4406F, which is composed of components including an anti-CD20 antibody at about 46 mg / ml, a histidine buffer at about 22 mM, trehalose at about 220 mM, and polysorbate-80 at about 0.22 mg / ml, was provided, and the pH value of the liquid preparation was about 6.0.

[0264] Comparison of stability For stability analysis, the preparations disclosed in Chinese Patent CN101820912B (Formulation A, the antibody is ofatumumab) (antibody concentration 20 mg / mL), a comparative preparation (using the same excipients as the preparation of CN101820912B, but the antibody was BAT4406F, Formulation B), Formulation 1 (see Example 7), Formulation 2 (Example 8), Formulation 3 (Example 9), and Formulation 4 were used. The formulations are shown in Table 13.

[0265] [Table 13]

[0266] 1) Dissolution temperature, coagulation temperature, particle size [Table 14]

[0267] As shown in Table 14, 1) when the anti-CD20 antibody in the original formulation A was replaced with BAT4406F, the Tm1, Tag, and DLS (particle size and uniformity) of the two formulations were basically the same; 2) compared to the original formulation A, both the Tm1 and Tag of formulation 1 were higher, the PDI of particle size was less than 0.25, and the uniformity of the formulation was good, indicating that formulation 1 had good stability; and 3) when the trehalose was adjusted from 158.6 mM to 224.6 mM, and other parameters remained unchanged, the Tm1, Tag, and DLS of formulations 2 and 3 were basically the same as those of formulation 1, and their stability was relatively good.

[0268] 2) 40℃ high temperature test Samples of the formulation were subjected to a high-temperature test at 40°C and analyzed by SEC-HPLC on day 0, week 1, week 2, week 3, and week 4. The results are shown in Table 15.

[0269] As shown in the SEC-HPLC monomer purity data in Table 15, formulations 1, 2, and 3 were more stable than formulation B, with formulations 1 and 2 being the most stable.

[0270] [Table 15]

[0271] 3) Light irradiation condition test After exposing the formulation to light irradiation, monomer purity was analyzed by SEC-HPLC on days 0, 5, and 10. The results are shown in Table 16.

[0272] As shown in Table 16, the monomer purity of formulations 1, 2, and 3 measured by SEC-HPLC showed better stability, with formulations 1 and 2 exhibiting particularly better stability compared to formulation B.

[0273] [Table 16]

[0274] suitability analysis The liquid formulation of the present invention exhibits good compatibility with drug vials and met packaging requirements overall in both accelerated and long-term stability tests.

[0275] Example 13: Clinical trial of BAT4406F This study is an open-label, dose-escalation Phase I clinical trial evaluating the safety, tolerability, pharmacokinetics, and preliminary efficacy of BAT4406F in the treatment of patients with neuromyelitis optica spectrum disorder (NMOSD).

[0276] Subject registration / screening The inclusion criteria are as follows: 1) Meets the NMOSD diagnostic criteria established by the International Panel for the Diagnosis of NMO (IPND) in 2015 (see Appendix 1). 2) Ages 18 to 65. 3) At least two relapses within two years prior to screening, or at least one relapse within one year prior to screening. 4) Immunosuppressants such as azathioprine, tacrolimus, mycophenolate mofetil, cyclophosphamide, and methotrexate should be discontinued within 28 days prior to baseline. 5) The amount of corticosteroids administered should be equivalent to 30 mg or less of prednisone and must be discontinued within one month of administration of the study drug. 6) EDSS score is 6 or less.

[0277] The exclusion criteria are as follows: 1) History of treatment with monoclonal antibodies within 6 months prior to administration. 2) Currently undergoing treatment with anti-CD20 mAb. 3) The patient receives a live vaccine within four weeks prior to screening. 4) Prior to baseline, the patient had participated in another clinical trial within one month or within five half-lives of the drug (whichever is longer). 5) History of allergy to monoclonal antibodies, or history of severe allergic reactions to specific foods or medications. 6) Abnormal liver function, kidney function, and bone marrow reserve. a. ALT is 2.5 × ULN or higher, and / or AST is 2.5 × ULN or higher, and / or TBIL is 1.5 × ULN or higher. b. Serum creatinine is ≥1.5 × ULN, and creatinine clearance is ≤60 mL / min (calculated using the Cockcroft formula). c. Blood neutrophil count is 1.5 × 10 9 Less than / L and / or platelet count of 75 × 10 9 Patients with a / L level of less than / L 7) The patient has a history of an infectious disease that the principal investigator has determined to be unsuitable for the trial. 8) Patients who showed acute respiratory failure, pulmonary interstitial infiltration, and edema on chest CT. 9) Patients with a clear history of heart disease (such as angina pectoris, atrial flutter, atrial fibrillation, conduction block, other arrhythmias, or heart failure). In the case of patients with first-degree atrioventricular block, the principal investigator will make a determination by referring to the patient's medical history. 10) Patients diagnosed with malignant tumors.

[0278] Test drug, usage, and dosage: BAT4406F is administered intravenously in doses of 20 mg, 50 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg. A 6-month observation period follows each dose.

[0279] Before initiating BAT4406F administration, prophylactic administration of antihistamines (such as diphenhydramine and promethazine hydrochloride) and glucocorticoids (such as methylprednisolone and dexamethasone) is performed.

[0280] The initial intravenous infusion rate is approximately 12 ml / hour. If no infusion response occurs, the rate is increased by 12 ml / hour every 30 minutes until the maximum infusion rate of 96 ml / hour is reached.

[0281] Safety, tolerability, clinical pharmacokinetics (C max , T max , t 1 / 2 CL t Vd, MRT, AUC (0~t) AUC (0~inf) Pharmacokinetics, immunogenicity, and preliminary efficacy (e.g., assessed by annual relapse rate (ARR), time to first relapse, diastolic impairment status scale (EDSS), MRI, etc.) are evaluated. MRI may be evaluated according to the international consensus diagnostic criteria for neuromyelitis optica spectrum disorder (Neurology, 2015;85:177-189). After treatment, patients are expected to achieve complete response, partial response, or stability. In some embodiments, patients experience a decrease in ARR and EDSS scores (e.g., a decrease of at least 0.5).

[0282] clinical results Two subjects enrolled in the 20 mg dose group experienced an infusion reaction on the day of administration, but it resolved / resolved spontaneously without treatment. The first subject completed the DLT observation period. This indicates that the current 20 mg dose is well tolerated.

[0283] Tests were conducted on days 4, 8, and 15 after administration of 20 mg of BAT4406F, and CD19 was observed in both subjects. + The proportion of B cells had decreased from over 80% to over 90%.

[0284] Example 14: Non-clinical study of BAT4406F 1. Pharmacodynamic research Pharmacodynamic studies of B-cell depletion in cynomolgus monkeys showed that the onset time of BAT4406F was not significantly associated with the dose within the tested dose range (2, 5, 15 mg / kg). On day 2 post-administration, CD20 was observed in all three dose groups. + The lymphocyte subgroup was less than 0.5%, and this ratio was maintained until day 21. On day 28 after administration, CD20 + The proportions of lymphocyte subgroups recovered to 3.87%, 2.43%, and 0.3% in the low, intermediate, and high-dose groups, respectively. BAT4406F demonstrated superior B-cell depletion ability compared to the commercially available drug rituximab at the same dose of 5 mg / kg.

[0285] In vitro pharmacodynamic studies using healthy human whole blood showed that both 10 ng / mL and 50 ng / mL of BAT4406F exhibited potent B-cell depletion ability after incubation with whole blood for 4 hours. The mean B-cell depletion percentages at the above concentrations were 37.16% and 61.99%, respectively, and efficacy was dose-related. Compared to commercially available anti-CD20 monoclonal antibodies, B-cell depletion ability weakened in the order of BAT4406F, ofatumumab, ocrelizumab, and rituximab.

[0286] For further details, please refer to Table 17.

[0287] [Table 17]

[0288] 2. Pharmacological studies The target of BAT4406F is the CD20 antigen expressed by B cells. After binding to the CD20 antigen, BAT4406F can initiate an immune response that mediates B cell lysis. The depletion mechanism involves antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cell-mediated cytotoxicity (CDC), and CD20 + This includes direct induction of apoptosis in B cells.

[0289] ADCC activity was evaluated using PBMC cytotoxicity and reporter gene assays. Different anti-CD20 monoclonal antibodies—BAT4406F, BAT4406 (a fucose-modified protein expressed by wild-type cell CHO-BAT), ofatumumab, ocrelizumab, and rituximab—were able to induce ADCC effects. The ADCC effect of BAT4406F, obtained by knocking out fucose, was enhanced approximately 10-fold, significantly stronger than the other four anti-CD20 monoclonal antibodies, indicating that the ADCC effect is one of the main mechanisms of action of BAT4406F.

[0290] For details, see Table 18.

[0291] [Table 18]

[0292] 3.Toxicology research Long-term toxicity studies were conducted in cynomolgus monkeys with five weekly doses of BAT4406F at doses of 10 mg / kg, 30 mg / kg, and 100 mg / kg over four consecutive weeks, with a recovery period of 20 weeks. No significant drug-related abnormalities were observed in body weight, body temperature, electrocardiogram, blood pressure, coagulation function, clinical biochemistry, serum complement C3 and C4, immunoglobulins IgG, IgM and IgA, ophthalmological examination, bone marrow smear, organ weight, organ-to-body weight ratio, or organ-to-brain weight ratio in any of the dose groups. Lymphocyte counts were measured in female animals in each dose group on the day after the third dose (D16) (×10 9 A decrease in ( / L), an increase in mono(%) in male animals in the 30 mg / kg group, an increase in mono(%) in male animals in the 10 mg / kg group on the day after the last dose (D30), a decrease in MCHC in male and female animals, an increase in retic(%) in female animals in the 30 mg / kg group, and a decrease in retic(×10) in female animals in the 10 mg / kg and 30 mg / kg groups. 12 Peripheral blood CD20 showed an increase in / L. + and CD40 + B lymphocytes are depleted or decreased, CD3 +The proportion of T lymphocytes increased. This is a manifestation of the pharmacological effect or extended pharmacological effect of BAT4406F. Antidrug antibodies were detected in individual animals in each dose group.

[0293] Some male animals (2 / 5) in the BAT4406F 30 mg / kg group showed moderate glomerular mesangial hyperplasia and mild tubular dilation. Both electron microscopy and immunohistochemical staining results showed deposition of immune complexes in the renal glomeruli of the animals' kidneys. Some animals (1 / 10, 1 / 10, and 2 / 10, respectively) in the BAT4406F 10 mg / kg, 30 mg / kg, and 100 mg / kg groups showed visible epidermal necrosis / scarring / thickening, acute inflammation of the epidermis and dermis, and subacute or chronic active inflammation of the dermis and subcutaneous tissue of the fore or hind leg skin, with vasculitis and / or perivascular inflammation, which are thought to be secondary infections caused by skin trauma or pharmacological effects. At the end of the 20-week recovery period (D169), some animals (2 / 10) in the BAT4406F 100 mg / kg group had recovered from all other lesions except for drug-related lesions in the spleen.

[0294] The NOAEL (No Observed Adverse Effect Level) for this study was considered to be 10 mg / kg.

[0295] 4. Pharmacokinetic studies Exposure to BAT4406F in cynomolgus monkeys increased with increasing dose, and after administering BAT4406F to cynomolgus monkeys via weekly intravenous infusion at doses of 2 mg / kg, 5 mg / kg, 15 mg / kg, or 10-100 mg / kg for four consecutive weeks, the major pharmacokinetic parameters (C) increased in each dose group. max No significant sex differences were observed in AUC and MRT. Antibodies were produced in the animals two weeks after BAT4406F administration, resulting in a significant decrease in drug concentrations at the end of the concentration-time curve. Therefore, elimination, half-life, clearance, and other parameters are for reference only. See Tables 19 and 20 for further details.

[0296] [Table 19]

[0297] [Table 20]

[0298] Appendix 1: 2015 International Consensus on Diagnostic Criteria for Neuromyelitis Optica Spectrum Disorder (IPND, 2015) Diagnostic criteria for NMOSD using AQP4-IgG (1) At least one main clinical feature (2) Positive testing for AQP4-IgG using the best available detection method (cell-based assays are strongly recommended). (3) Exclusion of alternative diagnoses Diagnostic criteria for NMOSD with no or unknown AQP4-IgG status. (1) At least two principal clinical features resulting from one or more clinical seizures that meet all of the following requirements: 1) At least one principal clinical feature must be optic neuritis, acute myelitis with LETM, or field last syndrome; 2) Spatial polymorphism (two or more principal clinical features); 3) Meeting additional MRI requirements. (2) Negative test for AQP4-IgG using the best available detection method, or undetectable. (3) Exclusion of alternative diagnoses Main clinical characteristics (1) Optic neuritis (2) Acute myelitis (3) Last field syndrome: episodes of hiccups, nausea, and vomiting of unknown cause (4) Acute brainstem syndrome (5) Brain MRI in symptomatic narcolepsy, diencephalic syndrome, and NMOSD - typical diencephalic lesions (6) Brain syndromes with NMOSD - typical brain lesions Additional MRI requirements for NMOSD with no or unknown AQP4-IgG status (1) Acute optic neuritis: A brain MRI showing one of the following is required: 1) Normal brain MRI or only nonspecific white matter lesions. 2) Optic nerve with T2 hyperintensity lesions or T1 enhancement lesions that exceed half the length of the optic nerve or involve the optic chiasm. (2) Acute myelitis: In patients with a history of myelitis, an associated intramedullary lesion spanning three adjacent vertebral segments or three or more adjacent vertebral segments of focal spinal atrophy is required. (3) Field-Last Syndrome: A related dorsal medulla oblongata / field-last lesion is required. (4) Acute brainstem syndrome: requires associated peripituitary brainstem lesions. Appendix 2: Extended Disability Scale Score (EDSS) 0. Standard neurological examination (all FS grade 0) 1.0 No faults, minimal indications in one FS (one FS grade 1) 1.5 No failures, minimal indications in one or more FS (one or more FS grade 1) 2.0 Minimal failures in one FS (one FS grade 2, others 0 or 1) 2.5 Minimal failures in two FS (two FS grade 2, others 0 or 1) 3.0 Fully ambulating but with moderate impairment in one FS (one FS grade 3, others 0 or 1), or fully ambulating but with mild impairment in three or four FS (three / four FS grade 2, others 0 or 1). 3.5 Fully amblyable, but with moderate impairment in one FS (one FS grade 3), mild impairment in one or two FS (one / two FS grade 2), and 0 or 1 in the others, or fully amblyable with two FS grade 3 (others 0 or 1), or fully amblyable with five FS grade 2 (others 0 or 1) 4.0 Able to walk more than 500 meters without assistance or rest, and despite a relatively severe disability consisting of one FS grade 4 (others 0 or 1) or a combination of lower grades exceeding the limits of the previous stage, the individual is able to get up and move around for approximately 12 hours a day. 4.5 Able to walk more than 300 meters without assistance or rest, and despite a relatively severe disability consisting of one FS grade 4 (others 0 or 1) or a combination of lower grades exceeding the limit of the previous stage, the individual is able to get up and move around for approximately 12 hours a day. 5.0 Walking 200 meters or more without assistance or rest (Normal FS equivalent includes at least one FS Grade 5, or a combination of lower grades exceeding the content of a normal stage 4.5) 5.5 Walking more than 100 meters without assistance or rest. 6.0 Regardless of whether breaks are taken, assistance (cane or crutch) is required for walking at least 100 meters. 6.5 Whether or not rest is taken, both types of assistance (cane or crutches) are consistently required for walking at least 20 meters. 7.0 Unable to walk more than 5 meters even with assistance. Basically confined to a wheelchair. Moves around independently by turning the wheels on their own. Uses a wheelchair for approximately 12 hours a day. 7.5 Unable to walk more than a certain number of steps. Restricted to a wheelchair and may require assistance with movement and steering. 8.0 Primarily confined to bed or chair, or able to move around in a wheelchair, but spends most of the day out of bed. Maintains many self-care abilities. Generally able to use both arms effectively. 8.5 Basically confined to bed all day. Some degree of effective use of arms. Retains some self-care abilities. 9.0 A helpless, bedridden patient. Still able to communicate and eat. 9.5 Completely helpless, bedridden patient. Unable to communicate effectively or eat / drink. 10. Deaths due to multiple sclerosis

Claims

1. The solution contains 15 mg / ml to 80 mg / ml of anti-CD20 antibody, 10 mM to 30 mM of buffer, 80 mM to 240 mM of stabilizer, and 0.1 mg / ml to 0.4 mg / ml of surfactant, with a pH in the range of 5.5 to 6.

2. The buffer is selected from succinate buffer, citrate buffer, phosphate buffer, histidine buffer, and acetate buffer. The aforementioned stabilizer is selected from sucrose, trehalose, sorbitol, mannitol, and methionine. The surfactant is selected from polysorbate-80 and polysorbate-20, and the anti-CD20 antibody is a liquid formulation. A liquid formulation comprising a light chain containing the amino acid sequence of SEQ ID NO: 3 and a heavy chain containing the amino acid sequence of SEQ ID NO:

4.

2. The liquid formulation according to claim 1, wherein at least about 60% of the anti-CD20 antibody has a G0 glycan in the N-glycosylation site of its constant region.

3. The liquid formulation according to claim 1 or 2, wherein the fucose content of the anti-CD20 antibody is approximately 5% or less.

4. The liquid formulation according to any one of claims 1 to 3, wherein the anti-CD20 antibody is produced from a CHO cell line in which the α-(1,6)-fucosyltransferase gene has been knocked out.

5. A liquid formulation according to any one of claims 1 to 4, comprising 20 mg / ml to 50 mg / ml of anti-CD20 antibody.

6. A liquid formulation according to any one of claims 1 to 5, comprising 18 mM to 22 mM histidine buffer.

7. A liquid formulation according to any one of claims 1 to 6, comprising 158 mM to 225 mM trehalose.

8. A liquid formulation according to any one of claims 1 to 7, comprising 0.18 mg / ml to 0.22 mg / ml of polysorbate-80.

9. A liquid formulation according to any one of claims 1 to 8, wherein the pH is in the range of 5.7 to 5.

9.

10. A liquid formulation according to claim 1, comprising 20 mg / ml of anti-CD20 antibody, 20 mM histidine buffer, 158.6 mM trehalose, and 0.2 mg / ml of polysorbate-80, with a pH of 5.

8.

11. A liquid formulation according to claim 1, comprising 20 mg / ml of anti-CD20 antibody, 20 mM histidine buffer, 224.6 mM trehalose, and 0.2 mg / ml of polysorbate-80, with a pH of 5.

8.

12. A liquid formulation according to claim 1, comprising 50 mg / ml of anti-CD20 antibody, 20 mM histidine buffer, 224.6 mM trehalose, and 0.2 mg / ml of polysorbate-80, with a pH of 5.

8.

13. Use of a liquid formulation according to any one of claims 1 to 12 in the preparation of a pharmaceutical product for the treatment of CD20-positive diseases.

14. The use according to claim 13, wherein the preparation is administered by intravenous injection or subcutaneous injection.

15. The use according to claim 13 or 14, wherein the CD20-positive disease is selected from neoplastic diseases and immunological diseases.

16. The use according to claim 15, wherein the tumorigenic disease includes B-cell lymphoma, the B-cell lymphoma includes Hodgkin lymphoma and non-Hodgkin lymphoma, and the immune disease includes multiple sclerosis, immune thrombocytopenia, neuromyelitis optica, myasthenia gravis, rheumatoid arthritis, psoriasis, and psoriatic arthritis.

17. The use according to claim 13, wherein the CD20-positive disease is neuromyelitis optica spectrum disorder (NMOSD), non-Hodgkin lymphoma (NHL), multiple sclerosis (MS), immune thrombocytopenia (ITP), rheumatoid arthritis (RA), Wegener's granulomatosis (WG), microscopic polyangiitis (MPA), lupus nephritis, systemic lupus erythematosus, or chronic lymphocytic leukemia (CLL).

18. The use according to any one of claims 13 to 17, wherein the CD20-positive disease is refractory or relapses after at least one prior treatment regimen.

19. The use according to any one of claims 13 to 18, wherein the effective amount of the anti-CD20 antibody is about 10 mg to about 3000 mg per dose.

20. The use according to claim 19, wherein the effective amount is approximately 300 mg to approximately 3000 mg per dose.

21. The use according to claim 19 or 20, wherein the effective dose is approximately 100 mg to approximately 1000 mg once a month, once every two months, once every three months, once every four months, once every five months, or once every six months.

22. The use according to claim 19 or 20, wherein the effective dose is approximately 100 mg to approximately 500 mg once every two months or once every three months.

23. The use according to claim 19 or 20, wherein the effective dose is approximately 300 mg to approximately 2000 mg per treatment cycle, and one treatment cycle is administered once a month, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, or once every twelve months.

24. The use according to claim 19 or 20, wherein the effective dose is approximately 500 mg to approximately 2000 mg and is administered as needed for recurrence of the disease.

25. A pharmaceutical composition comprising a liquid formulation according to any one of claims 1 to 12, containing the anti-CD20 antibody in an amount of 10 mg to about 3000 mg per dose.

26. A method for producing the pharmaceutical composition according to claim 25, comprising mixing the anti-CD20 antibody with a buffer, a stabilizer, and a surfactant.

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