Anti-CD20 antibodies or antigen binding fragments binding to CD20 and uses thereof

Abstract

The invention discloses an anti-CD20 antibody or an antigen binding fragment binding to CD20 and application of the anti-CD20 antibody or the antigen binding fragment binding to CD20. After the anti-CD20 antibody and the CD20 antigen binding fragment disclosed by the invention are specifically bound with a CD20 antigen on a cell, immune response is started to mediate B cell dissolution, so that the growth of tumor cells is effectively inhibited or the tumor cells are cleared, and the anti-CD20 antibody and the CD20 antigen binding fragment have a good patent medicine prospect.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 202211064962.4, application date September 1, 2022, and invention name “Anti-CD20 antibodies or antigen-binding fragments binding to CD20 and their applications”. Technical Field

[0002] The present invention belongs to the field of biomedicine, and specifically relates to an anti-CD20 antibody or an antigen binding fragment binding to CD20 and applications thereof. Background Art

[0003] CD20 is composed of 297 amino acids, with a molecular weight of 33-37kD, and is expressed on the surface of more than 95% of B cells. CD20 molecules exist in both normal B cells and malignant cells, especially in more than 90% of B cell non-Hodgkin's lymphoma (NHL), and are ideal target antigens for NHL. The CD20 molecule has four transmembrane regions, with the amino terminus and carboxyl terminus located on the inner side of the plasma membrane. Between the third and fourth transmembrane regions, there is a loop region composed of 43 amino acid residues, which constitutes the main epitope. The CD20 antigen molecule is relatively exposed and accessible. When CD20 approaches each other under the action of antibodies, the polymers formed by cross-linking or even hyper-cross-linking act as calcium ion channels, allowing extracellular calcium ions to flow into the cell; in addition, the tyrosine protein kinases of the Src family activate each other due to proximity. The initiation of signaling pathways and the activation of endogenous calcium stores all lead to an increase in intracellular calcium ion concentration, which in turn affects the operation of the cell cycle, regulates cell proliferation and differentiation, and even leads to the occurrence of cell apoptosis.

[0004] Anti-CD20 antibodies can kill B-cell derived tumors through antibody-dependent cytotoxicity, complement-dependent cytotoxicity, and inhibition of cell growth by binding to CD20 molecules. CD20-positive tumor diseases include B-cell lymphomas, such as diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, etc. CD20 provides an important target for antibody-mediated therapy, which can be used to control B cells involved in cancer and autoimmune diseases.

[0005] IDEC-C2B8, also known as Rituximab, trade name Approved for marketing by the FDA in 1997, it is a human-mouse chimeric antibody that contains the variable region of the mouse anti-CD20 monoclonal antibody 2B8 (Ibritumomab) and the constant region of the human IgG1 heavy chain and κ chain, and is used to treat B-cell lymphoma.

[0006] Clinical trial results show Combining it with the chemotherapy drug CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) for the treatment of low-grade or follicular NHL is more effective than using it alone. ) The cost of treating NHL is high, and some patients may also develop HAMA and HACA reactions. Therefore, it is of great practical significance to develop an effective anti-CD20 antibody independently developed in my country. Summary of the invention

[0007] In order to obtain an anti-CD20 antibody or CD20 antigen-binding fragment different from the prior art and to obtain a new drug independently developed, the present invention provides an anti-CD20 antibody or CD20 antigen-binding fragment, and the specific scheme thereof is as follows:

[0008] The anti-CD20 antibody or CD20 antigen-binding fragment can specifically bind to CD20 and comprises:

[0009] a1) three heavy chain complementary determining regions, HC-CDR1, HC-CDR2, and HC-CDR3, whose amino acid sequences are shown in the amino acid sequences encoded by SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; and

[0010] a2) three light chain complementary determining regions, LC-CDR1, LC-CDR2, and LC-CDR3, whose amino acid sequences are shown in the amino acid sequences encoded by SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively:

[0011] The anti-CD20 antibody is a polyclonal antibody or a monoclonal antibody;

[0012] The CD20 antigen binding fragment is:

[0013] b1) single chain Fv; or

[0014] b2) disulfide-linked Fv; or

[0015] b3) Fab fragment; or

[0016] b4)F(ab') 2 fragment; or

[0017] b5) Fab' fragment.

[0018] In some embodiments, it comprises a heavy chain variable region and a light chain variable region, whose amino acid sequences are shown in the amino acid sequences encoded by SEQ ID NO:3 and SEQ ID NO:4, respectively, or have at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequences encoded by SEQ ID NO:3 and SEQ ID NO:4, respectively; the heavy chain variable region comprises the three heavy chain complementary determining regions; the light chain variable region comprises the three light chain complementary determining regions.

[0019] In some embodiments, it further comprises one or more of a heavy chain constant region (including CH1, CH2 and CH3), a light chain constant region (CL), and an Fc region. Further, the light chain constant region is a kappa chain constant region. In some preferred embodiments, the anti-CD20 antibody is of IgG1 type. The anti-CD20 antibody or CD20 antigen-binding fragment is a chimeric antibody or a chimeric antigen-binding fragment. In certain embodiments, amino acid modifications may be introduced into the Fc region of the antibodies provided herein, and the amino acid modifications may be one or more, thereby producing Fc variants. The Fc variant may comprise a human Fc region sequence comprising amino acid modifications at one or more amino acid positions.

[0020] In some embodiments, the anti-CD20 antibody comprises a heavy chain and a light chain, whose amino acid sequences are shown in SEQ ID NO:1 and the amino acid sequences encoded by SEQ ID NO:2, respectively, or have at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequences encoded by SEQ ID NO:1 and SEQ ID NO:2, respectively; the heavy chain comprises the heavy chain variable region; the light chain comprises the light chain variable region.

[0021] In some embodiments, the anti-CD20 antibody or CD20 antigen-binding fragment has a fucose-containing glycoform area ratio of no more than 92.0% of the total glycoform area ratio.

[0022] In some embodiments, the anti-CD20 antibody or CD20 antigen-binding fragment contains a total defucosyl (high mannose) glycoform area ratio of not less than 8.0% of the total sugar area ratio; the anti-CD20 antibody or CD20 antigen-binding fragment contains a high mannose glycoform area ratio of not more than 8.0% of the total sugar area ratio.

[0023] In some embodiments, the anti-CD20 antibody or CD20 antigen-binding fragment contains a β-galactose glycoform area ratio that does not exceed 44.6% of the total sugar area ratio; the anti-CD20 antibody or CD20 antigen-binding fragment contains a sialylated glycoform area ratio that does not exceed 4.8% of the total sugar area ratio.

[0024] In certain embodiments, the antibody can be further modified to add functional components, and suitable parts for antibody derivatization include, but are not limited to, the following examples, PEG, dextran, protein, lipid, therapeutic agent or toxin. Antibodies can be modified by phosphorylation, acetylation, glycosylation, pegylation, amidation, or connection with other proteins.

[0025] In some embodiments, the anti-CD20 antibody or CD20 antigen-binding fragment is a monospecific, bispecific or multispecific antibody or antigen-binding fragment. For example, the anti-CD20 antibody can be linked to another antibody or antibody fragment to produce a bispecific or multispecific antibody with a second or more binding specificity. The target of the other antibody or antibody fragment is one or more of CD19, CD3, CD39, CD40, CD70, Claudin18.2, CTLA4, GPRC5D, TNF-α, HER2, VEGF / VEGFR, PD1 / PDL1, CGRP / CGRPR, IL-15, IL-21, IL-6 / IL-6R, IL-23p19, EGFR, SARS-CoV-2, 4-1BB, LAG3, and CD47.

[0026] In a second aspect, the present invention also discloses a corresponding nucleic acid comprising a nucleotide sequence encoding the anti-CD20 antibody or CD20 antigen-binding fragment as described above.

[0027] In some embodiments, it is a nucleotide sequence encoding an anti-CD20 antibody or CD20 antigen-binding fragment as described above.

[0028] In some embodiments, it comprises nucleotide sequences encoding three heavy chain complementary determining regions, HC-CDR1, HC-CDR2, and HC-CDR3, as shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; and three light chain complementary determining regions, LC-CDR1, LC-CDR2, and LC-CDR3, as shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.

[0029] In some embodiments, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:3 and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:4.

[0030] In some embodiments, the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO:1 and the nucleotide sequence encoding the light chain is shown in SEQ ID NO:2.

[0031] In a third aspect, the present invention further discloses a composition comprising the anti-CD20 antibody or CD20 antigen-binding fragment as described above. Preferably, the composition is a pharmaceutical composition, further comprising a pharmaceutically acceptable carrier.

[0032] In a fourth aspect, the present invention discloses a biomaterial, which is:

[0033] c1) a vector, a host cell or a microorganism comprising a nucleic acid as described above; or

[0034] c2) the expression product, suspension or supernatant of c1).

[0035] In a fifth aspect, the present invention also provides a method for producing an antibody or an antigen-binding fragment, which comprises culturing a host cell or a microorganism comprising the nucleic acid as described above and recovering the antibody or the antigen-binding fragment from the culture.

[0036] In a sixth aspect, the present invention also discloses the use of the anti-CD20 antibody or CD20 antigen-binding fragment as described above, or the nucleic acid as described above, or the composition as described above, or the biomaterial as described above in the preparation of a drug for treating a tumor or autoimmune disease. Preferably, the tumor or autoimmune disease is a variety of blood tumors and solid tumors, such as diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, chronic lymphocytic leukemia, multiple sclerosis, rheumatoid arthritis and the like.

[0037] In some embodiments, the tumor or autoimmune disease includes but is not limited to 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).

[0038] In a seventh aspect, the present invention also discloses the use of the anti-CD20 antibody or CD20 antigen-binding fragment, or the nucleic acid as described above, or the composition as described above, or the biomaterial as described above in the preparation of a preparation for blocking CD20-expressing positive cells.

[0039] In an eighth aspect, the present invention also discloses the use of the anti-CD20 antibody or CD20 antigen binding fragment, or the nucleic acid as described above, or the composition as described above, or the biomaterial as described above and one or more other cancer therapeutic agents in the combined preparation of a drug for treating a tumor or autoimmune disease. Preferably, the tumor or autoimmune disease is a tissue that expresses positive CD20.

[0040] In some preferred embodiments, the other cancer or autoimmune disease therapeutic agents include but are not limited to chemotherapeutic agents, radiotherapeutic agents and biomacromolecule drugs. Further preferably, the biomacromolecule drug is a monoclonal antibody drug targeting tumor cell surface antigens, including tocilizumab, cetuximab or trastuzumab.

[0041] In a ninth aspect, the anti-CD20 antibody or CD20 antigen-binding fragment of the present invention is administered by bolus, infusion, injection, epithelial absorption or skin and mucosal absorption. Further, the injection includes but is not limited to intraperitoneal injection, intravenous injection, subcutaneous injection, intranasal injection or intramuscular injection.

[0042] After the anti-CD20 antibody or CD20 antigen binding fragment of the present invention specifically binds to the CD20 antigen on the cell, an immune response is initiated to mediate B cell lysis. The mechanism of B cell lysis may include: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), etc. The anti-CD20 antibody or CD20 antigen binding fragment of the present invention can effectively inhibit the growth of tumor cells or achieve the elimination of tumor cells, and has a good prospect for drug development.

[0043] definition:

[0044] In the present invention, the term "antibody" refers to an immunoglobulin that can specifically recognize and bind to an antigen, and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies or antibody fragments.

[0045] The term "variable region (Fv)" refers to the domain of the antibody heavy chain variable region and / or light chain variable region that recognizes and specifically binds to an antigen epitope.

[0046] CDR region or "complementarity determining region" refers to the region in the variable region of an antibody that is highly variable in sequence and forms a loop that is structurally determined and / or contains antigen contact amino acid residues. CDR is primarily responsible for the binding of the antibody to the antigen epitope and determines the specificity of the antibody. In a given heavy chain or light chain variable region amino acid sequence, the specific amino acid sequence of each CDR is determined using any one or a combination of many well-known numbering rules, including, for example, Kabat, Contact, AbM and Chothia. The CDR of the antibody of the present invention can be determined according to any rule or a combination of these techniques in the art.

[0047] The light chain variable region (VL) and the heavy chain variable region (VH) both include three complementary determining regions (CDR1, CDR2, CDR3) and four framework regions (FR1, FR2, FR3, FR4). The three CDRs of the light chain variable region (VL) are LC-CDR1, LC-CDR2, and LC-CDR3; the three CDRs of the heavy chain variable region (VH) are HC-CDR1, HC-CDR2, and HC-CDR3.

[0048] The anti-CD20 antibodies or antigen-binding fragments thereof of the present invention comprise substitutions, insertions or deletions. The anti-CD20 antibodies of the present invention comprise modifications to the light chain variable region, the heavy chain variable region, the light chain or the heavy chain, and the amino acid sequence thereof after modification is different from the amino acid sequence from which the antibody is derived. For example, the amino acid sequence derived from the same specified protein may be similar to the starting sequence, for example, having a certain percentage identity, for example, it may have a percentage identity of 90%, 92%, 96%, 98% with the starting sequence.

[0049] In the present invention, "identity" refers to the percentage of bases (or amino acids) in the two sequences being compared when the sequences are aligned between two peptides or between two nucleic acid molecules. Software programs known in the art can be used to determine the alignment and homology percentage or sequence identity, such as BLASTN and BLASTP.

[0050] The "antibodies and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, multispecific, recombinant, heterologous, chimeric, humanized, de-immunized antibodies, or Fab fragments, Fab' fragments, F(ab') 2 Fragments, single-chain antibodies, nanobodies, and epitope-binding fragments of any of the foregoing.

[0051] The term "antigen specificity" refers to a specific antigen or its epitope selectively recognized by an antigen binding molecule. The terms "treatment" or "treatment" or "relief" or "improvement" are used interchangeably herein and refer to methods for obtaining beneficial or desired results (including but not limited to therapeutic benefits and / or preventive benefits). As used herein, therapeutic benefits generally refer to eradication or mitigation of the severity of the underlying condition being treated. In addition, by eradicating, mitigating severity or reducing the incidence of one or more physiological symptoms associated with the underlying condition, so that improvements are observed in animals (although the animal may still be afflicted with the underlying condition) to achieve therapeutic benefits. For preventive benefits, the risk of morbidity of animals at risk of developing a specific disease can be reduced. As used herein, the term "therapeutic effect" generally includes therapeutic benefits and / or preventive benefits as described above. Preventive effects include delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing down, stopping or reversing the progress of a disease or condition, or any combination thereof.

[0052] The term "cell proliferation" generally refers to the phenomenon that the number of cells changes due to division. For example, cell proliferation can lead to an increase in the number of cells. The term also includes cell growth through which the cell morphology has changed (e.g., increased in size), which is consistent with a proliferation signal. As used herein, the term "proliferation inhibition" or "inhibition of cell proliferation" generally refers to a decrease in the growth rate and / or proliferation rate of cancer cells. For example, this can include the death of cancer cells (e.g., by apoptosis). In some embodiments, the term may also refer to inhibiting the growth and / or proliferation of solid tumors and / or inducing a reduction in the size or elimination of tumors.

[0053] The term "subject" or "individual" or "animal" or "patient" used in this application refers to a human or non-human animal, including mammals or primates, for whom diagnosis, prognosis, alleviation, prevention and / or treatment of a disease or condition is desired. Mammalian subjects include humans, livestock animals, farm animals, and zoo or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, bears, etc.

[0054] As used herein, the term "in vivo" generally refers to events that occur within an animal's body.

[0055] As used herein, the term "in vitro" generally refers to an event that occurs outside an animal. For example, an in vitro cell function test or any animal in vitro assay. In vitro assays include cell-based assays in which dead or live cells are used. In vitro assays also include cell-free assays in which intact cells are not used.

[0056] As used herein, the term "administering" refers to delivering a therapeutically effective amount of a pharmaceutical composition comprising a recombinant protein or fusion protein of the present invention to a subject. Administration may be systemic or topical. Administration may be performed by an administration device, such as a syringe. Modes of administration include, but are not limited to, embedding, nasal inhalation, spraying, injection, and the like. Routes of administration include inhalation, intranasal, oral, intravenous, subcutaneous, or intramuscular administration, and the like.

[0057] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is the agarose gel electrophoresis of total RNA of M3-4 cells. 1: M3-4 total RNA; M: DL2000.

[0059] Figure 2 This is an agarose gel electrophoresis diagram of the antibody light and heavy chain Fd gene. Left: 1: light chain Fd fragment; M: DL2000; Right: 1: heavy chain Fd fragment; M: DL2000.

[0060] Figure 3 This is the restriction enzyme electrophoresis diagram of antibody light and heavy chain Fd fragment cloning vector. M: DL2000; 1: heavy chain Fd fragment cloning vector; 2: light chain Fd fragment cloning vector.

[0061] Figure 4 This is the agarose gel electrophoresis of the M3-4 light and heavy chain variable region genes. VH: heavy chain variable region gene; Vκ: light chain variable region gene; M: DL2000.

[0062] Figure 5 This is the electrophoresis diagram of the pCMV163 vector PuvII / BstEII digestion. M: high molecular weight DNA standard; pCMV163: pCMV163 vector.

[0063] Figure 6 This is the electrophoresis diagram of pCMV163 / M3-4 / VH vector PuvII / BstEII digestion. 1: High molecular weight DNA standard; 2: Positive plasmid PuvII / BstEII digestion; 3: DL2000 standard.

[0064] Figure 7 This is the electrophoresis diagram of Ecor V / Xho I digestion of pCMV163 / M3-4 / VH / VL vector. 1: High molecular weight DNA standard; 2: Positive plasmid Ecor V / Xho I digestion identification; 3: DL2000 standard.

[0065] Figure 8 This is a 9% non-reducing SDS-PAGE electrophoretogram. 1: 2: The peak sample of the supernatant of transfected cells; 3: The peak sample of the washed transfected cells.

[0066] Fig. 9 This is a 12% reducing SDS-PAGE electrophoretogram. 1: 2: The peak sample of the supernatant of transfected cells; 3: The peak sample of the washed transfected cells.

[0067] Fig.10 The transfected cells were washed and then analyzed by western blot. 2, 3, 4: Wash the off-peak samples on transfected cells.

[0068] Fig.11 This is the FACS analysis of TGLA on Daudi cells. The dotted line represents the GAHIgG-FITC control group, and the solid line represents the antibody + GAHIgG-FITC group.

[0069] Fig.12This is the FACS analysis of TGLA on Raji cells, where the dotted line represents the GAHIgG-FITC control group, and the solid line represents the antibody+GAHIgG-FITC group.

[0070] Fig.13 is the TGLA affinity constant.

[0071] Fig.14 yes Affinity constant.

[0072] Fig.15 It is TGLA and Analysis of Daudi cell proliferation activity.

[0073] Fig.16 It is TGLA and Analysis of proliferation activity of Raji cells.

[0074] Fig.17 It is a nude mouse subcutaneous human B lymphocyte transplant tumor membrane type. Among them, A1: Daudi cell inoculation density 1.0×10 7 ; A2: Raji cell inoculation density 1.0×10 7 .

[0075] Fig.18 This is the FACS analysis of CD20+ cells in Daudi tumor tissue. Among them, B1a: GAHIgG-FITC control; B1b: CD20 antibody group.

[0076] Fig.19 CD20 in Raji tumor tissue + Cell FACS analysis. Among them, B2a: GAHIgG-FITC control; B2b: CD20 antibody group.

[0077] Fig. 20 This is the survival curve of nude mice bearing Raji cells treated with antibodies.

[0078] Fig.21 This is an observation of the efficacy of the antibody on Raji tumor growth.

[0079] Fig. 22 The effect of antibody treatment on the body weight of Raji tumor-bearing mice. DETAILED DESCRIPTION

[0080] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described below with reference to specific diagrams. However, the invention is not limited to the following implementation cases.

[0081] The reagents involved in the following examples, Trizol was purchased from Sigma; AMV reverse transcription kit and T-easy vector were purchased from Promega; Escherichia coli (E.coli) XL1-Blue was purchased from Invitrogen; restriction endonucleases were all products of Biolabs unless otherwise specified; rTaq DNA polymerase, T4 DNA ligase, dNTP, DNA molecular weight standard DL2000, and related buffers were purchased from Takara; DNA gel recovery kit was from OMEGA Biotechnology; premixTaq was purchased from NEB; PvuⅡ and BstEⅡ, EcoR V and Xho I enzymes were all purchased from NEB; plasmid extraction kit and DNA recovery kit were Qiagen products. Primers were synthesized by Beijing Aoke Biotechnology Co., Ltd. The nucleotide sequences involved in the following examples are shown in Table 1.

[0082] Table 1. Nucleotide sequences

[0083]

[0084]

[0085]

[0086] Example 1: Extraction of total RNA

[0087] Take 2×10 6 M3-4 hybridoma cells [mouse anti-human CD20 hybridoma cell line (M3-4, IgG1 subtype, type k), from the Molecular Immunology Department of the Institute of Basic Medicine, Academy of Military Medical Sciences], washed three times with 10mM PBS, and the supernatant was discarded. Add 0.5ml of Trizol, mix well, shake vigorously for 20 seconds, and stand at room temperature for 5 minutes. Add 0.2ml of chloroform, shake for 30 seconds, stand at room temperature for 15 minutes, and centrifuge at 12000rpm, 4℃ for 15 minutes. Take the upper liquid part of the centrifuge tube and mix it with 0.5ml of isopropanol, stand at room temperature for 10 minutes, and centrifuge at 12000rpm, 4℃ for 15 minutes. Discard the supernatant, wash the precipitate with 75% cold ethanol, and centrifuge at 12000rpm, 4℃ for 10 minutes. Discard the supernatant, add 20μL of RNAase-free water after the ethanol evaporates, detect OD260, OD280 values ​​and OD260 / 280 ratio, and store total RNA at -80℃.

[0088] Example 2: Extraction of antibody light and heavy chain Fd gene fragments

[0089] cDNA reverse transcription system: Use AMV reverse transcription kit for reverse transcription, total RNA 1μg, AMV5×Buffer 4μL, Oligo(dT)0.5μL, 2.5mM dNTP 2μL, RNasin 0.5μL, water 12μL, mix well and add AMV 1μL; react at 42℃ for 1 hour and 95℃ for 5 minutes.

[0090] Light chain FLC PCR: Take 3μL cDNA, 12.5μL premixTaq, 1μL light chain upstream primer (MuLC1, MuLC2, MuLC3, MuLC4, MuLC5, MuLC6, MuLC7 equal amounts), 1μL light chain downstream primer (MuCK), 2.5μL water. After mixing, perform PCR: 95℃ for 5 minutes, 94℃ for 30 seconds, 60℃ for 30 seconds, 72℃ for 1 minute, a total of 25 cycles; 72℃ for 10 minutes, 1 cycle, 1.5% agarose electrophoresis for identification.

[0091] Heavy chain Fd PCR: 3 μL cDNA, 12.5 μL premixTaq, 1 μL heavy chain upstream primer (MuHC1, MuHC2, MuHC3, MuHC4, MuHC5, MuHC6, MuHC7, MuHC8 equal amounts), 1 μL heavy chain downstream primer (MuIgG1), 2.5 μL water. After mixing, perform PCR: 95°C for 5 minutes, 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute, a total of 25 cycles; 72°C for 10 minutes, 1 cycle, 1.5% agarose electrophoresis for identification.

[0092] The total RNA of hybridoma cell lines was identified by 1.5% agarose gel electrophoresis, and clear 28s, 18s and 5s3 bands appeared ( Figure 1 ).

[0093] The extracted total RNA was used as a template to obtain cDNA by reverse transcription; the target fragment was obtained by PCR amplification using universal primers for mouse antibody light and heavy chains. Figure 2 ) showed that the light chain Fd fragment size was approximately 670 bp, and the heavy chain Fd fragment size was approximately 690 bp.

[0094] Example 3: Connection and sequencing of antibody light and heavy chain variable region genes and T vectors

[0095] The antibody light and heavy chain variable region genes were connected to the T vector: The antibody light and heavy chain Fd gene fragments were recovered by 1.5% agarose electrophoresis and connected to the T-easy vector. Connection system: 1μL vector, 3μL enzyme digestion to recover the antibody gene fragment, 5μL connection buffer, 0.5μL T4 DNA ligase, sterile water to 10μL, 16℃ connection overnight. Take 10μL of the connection product, transform E.coli XL1-Blue, inoculate ampicillin resistant agar plate, culture at 37℃, pick the clone and use the EcoR I restriction site at both ends of the T-easy vector cloning site for restriction digestion identification, 1.5% agarose electrophoresis results ( Figure 3 ) showed that the heavy chain and light chain Fd genes were successfully cloned into the T-easy vector. The clones identified by restriction digestion were sent to Beijing Nosai Genome Research Center Co., Ltd. for sequencing analysis.

[0096] The obtained antibody light and heavy chain variable region nucleic acid sequences were compared and analyzed in the KABAT database to determine that the coding sequence was mouse immunoglobulin light and heavy chain genes. The antibody variable region base sequences obtained by sequencing were translated using www.expasy.ch to obtain their amino acid sequences.

[0097] The monoclonal antibody with correct sequencing was named M3-4, among which the T-easy vector for cloning the heavy chain variable region gene was named T-easy / M3-4-VH, and the T-easy vector for cloning the light chain variable region gene was named T-easy / M3-4-Vκ.

[0098] Example 4: Obtaining the gene sequences of the light and heavy chain variable regions of M3-4

[0099] The M3-4 / VH and M3-4 / Vκ gene fragments were amplified by PCR using the correctly sequenced T-easy / M3-4VH and T-easy / M3-4-Vκ clones as templates and the light chain upstream and downstream primers (LC1, LC2) and heavy chain upstream and downstream primers (HC1, HC2) as primers. Agarose electrophoresis results ( Figure 4 ) showed that the VH and Vκ target gene fragments with corresponding restriction sites introduced upstream and downstream were about 350 bp and 320 bp, respectively, and were named M3-4 / VH and M3-4 / Vκ.

[0100] Example 5: pCMV163 vector extraction, enzyme digestion identification and recovery

[0101] After the pCMV16 vector (from the Institute of Virology, Chinese Center for Disease Control) was extracted with a plasmid extraction kit, it was digested with PuvII and BstEII to identify and recover the 10Kb vector fragment. The enzyme electrophoresis pattern is shown in Figure 5 shown.

[0102] Example 6: Construction of pCMV163 / M3-4 / VH / VL plasmid vector

[0103] The M3-4 / VH gene fragment (about 350 bp) was connected to pCMV163 at a ratio of 10:1. The ligation product was transformed and cloned. The plasmid was extracted and identified by PuvII / BstEII restriction enzyme digestion. The agarose electrophoresis results ( Figure 6 ) showed that the positive clone (i.e., pCMV163 connected with M3-4 / VH, named pCMV163 / M3-4 / VH) could digest the target fragment of about 350 bp (see Figure 6 (where the arrow points).

[0104] The M3-4 / VL gene fragment (about 320 bp) was connected to pCMV163 / M3-4 / VH at a ratio of 10:1. The ligation product was transformed and cloned. The plasmid was extracted and identified by EcoR V / Xho I digestion. The agarose electrophoresis results were ( Figure 7 ) showed that the positive clones could be digested into a target fragment of 320 bp (see Figure 7 (where the arrow points).

[0105] Through the above experiments, the variable region gene fragments of antibody light and heavy chains were successfully obtained, and the expression plasmid pCMV163 / M3-4 / VH / VL, which can be used for mammalian cell transfection, was constructed and named HGE.

[0106] Example 7: TGLA Characterization

[0107] Exponentially growing adherent 293T cells were used for transient transfection, and the expression plasmid HGE and Lipofectamine TM 2000 293T cells were transfected according to the instructions of the transfection kit, and the serum-free culture system was replaced. The cell supernatant was collected after 60-72 hours. After affinity purification of the supernatant, the purified and collected samples were compared with the purified samples by non-reducing 9% SDS-PAGE. Comparative analysis revealed that a band of about 150 KDa was visible in the elution peak samples of the supernatant of transfected cells after affinity purification ( Figure 8 ), in the reducing 12% SDS-PAGE results Both antibody light and heavy chain bands of 50KDa and 25KDa appeared ( Fig. 9 ). The immunoblotting method (WB) analysis of the purified samples also confirmed that the 150KDa protein molecule purified from the supernatant of transfected cells could be specifically recognized by anti-human IgG antibodies, and the position of the recognition molecule band was similar to that of same( Fig.10The above results preliminarily confirmed that after the expression plasmid HGE was transfected into 293T cells, a 150KDa antibody could be purified from the supernatant of the transfected cells. The 293T cells transfected with HGE were named TGLA cells, and the antibodies or antigen-binding fragments separated and purified from the cells were named TGLA.

[0108] Example 8: TGLA cell-specific binding assay

[0109] (1) TGLA on CD20 + Specific recognition analysis of target cells

[0110] Select for expression of CD20 + The B lymphocyte cell lines Daudi cells and Raji cells (both from the Molecular Immunology Department of the Institute of Basic Medicine, Academy of Military Medical Sciences) were used as target cells for TGLA detection. The specific binding activity of TGLA was analyzed by indirect immunofluorescence labeling and flow cytometry (FACS). The results are shown in Figure 2. Fig.11 , Fig.12 And as shown in Table 2.

[0111] Table 2. Effect of TGLA on CD20 + Specific recognition analysis of target cells

[0112]

[0113] FACS results showed that TGLA had a specific recognition rate of more than 97% for CD20-positive (CD20+) Daudi cells and Raji cells, and a specific recognition rate of more than 97% for CD20-negative (CD20 - The positive rate of binding to Jurkat cells (Molecular Immunology Department, Institute of Basic Medicine, Academy of Military Medical Sciences) was <1.0%, and The reaction specificity was consistent.

[0114] (2) Relative affinity of TGLA for CD20 molecules

[0115] After the antibody binds to the cell surface CD20 molecule and reaches equilibrium, the concentration of unbound free antibody in the reaction solution is detected by ELISA, and the corresponding bound antibody concentration is calculated. The ratio of bound antibody concentration to free antibody concentration is plotted to calculate the relative affinity constant of the antibody binding to the cell membrane CD20 molecule. The result is as follows: Fig.13 , 14 shown.

[0116] The ELISA method was used to determine the concentration of unbound antibody [F] at different dilutions of TGLA. [Ab]-[F] was the concentration of bound antibody [B]. [B] / [F] was used for software statistical analysis of [B]. The correlation coefficient R of TGLA was 2=0.9774, relative affinity constant K of TGLA a =2.25×10 8 M;

[0117] The same method detects Relative affinity constant K a =2.03×10 8 M.

[0118] The comparison of relative affinity confirmed that TGLA and The affinity for CD20 molecules is similar.

[0119] Example 9: Identification of TGLA Biological Activity

[0120] (1) Growth inhibition of Daudi cells and Raji cells by TGLA

[0121] The MTT method was used to determine the growth inhibitory effect of TGLA on Daudi cells and Raji cells. TGLA had no significant growth inhibitory effect on Daudi cells and Raji cells. When the concentration of TGLA increased to 50 μg / ml, the proliferation inhibition rate of Daudi cells was 29.92±5.63, and the proliferation inhibition rate of Raji cells was 31.55±7.22 (P>0.05); similarly At the same dose, the inhibition rate of Daudi and Raji cell proliferation was less than 30% (P>0.05), and compared with CD20-negative Jurkat cells, it did not show a significant proliferation inhibition effect.

[0122] In vitro cell proliferation test results ( Fig.15 , 16 ) shows: TGLA and There was no significant inhibitory effect on the proliferation of target cells.

[0123] (2) Tumor-bearing animal experiments

[0124] ①Establishment and identification of transplanted tumor model

[0125] Different numbers of Daudi and Raji cells were inoculated subcutaneously in nude mice. Tumors of varying degrees enlarged subcutaneously on days 7 to 16 after inoculation. Fig.17 ), the tumor formation rate was 100%, and the time of tumor formation was related to the inoculation cell density. The inoculation density of Daudi and Raji cells was 1.2×10 7 Tumor formation was observed 7±2 days after inoculation, and the inoculation density was reduced to 1.0×10 7 Tumor formation was observed on day 10±2, and the seeding density was reduced to 0.7×10 7 Tumor formation can be seen on the 15th to 16th ± 2th day.

[0126] ②Immunological detection of transplanted tumor

[0127] Subcutaneous transplanted tumor tissues from Daudi and Raji tumor-bearing animals were respectively taken to prepare cell suspensions, and the cell components in the transplanted tumor tissues were analyzed by FACS using immunofluorescence staining.

[0128] The results of FACS analysis confirmed that more than 95% of the cells in the transplanted tumor tissue showed a positive reaction rate to anti-CD20 monoclonal antibody, thus proving that the transplanted tumor tissue was human B lymphoma ( Fig.18 , 19 ), the tumor-bearing animal model established is a human B lymphoma model.

[0129] ③ Antibody therapy

[0130] Based on the tumor-bearing animal model established above, on the 5th day after Raji cell inoculation, the animals randomly divided into groups were treated with TGLA (500 μg / animal), The survival of the tumor-bearing animals in the saline treatment group was observed. All the tumor-bearing animals in the saline treatment group died on the 24th day, while the TGLA treatment group and All tumor-bearing animals in the treatment group died on the 30th day ( Fig. 20 Statistical analysis of the surviving animals showed that TGLA and Treatment can prolong the survival time of tumor-bearing animals, and the survival rate increased by 1.7 times compared with the saline treatment group (P<0.05). There was no significant difference between the TGLA group and the TGLA group.

[0131] At the same time, the TGLA treatment group (500 μg / mouse) The tumor growth volume of the treatment group (500 μg / mouse) and the saline treatment group, Fig.21 The results showed that the tumor volume of the saline-treated group increased significantly over time, while that of the TGLA-treated group and The tumor proliferation rate in the treatment group was significantly lower than that in the saline treatment group. On the 12th day after treatment, the tumor proliferation rate in the saline group reached 401.0%, while that in the TGLA group was 191.0%. The tumor proliferation rate in the treatment group was 205.0%. On the 16th day after treatment, the tumor proliferation rate in the saline group reached 554.0%, and the tumor proliferation rate in the TGLA treatment group was 161.0%. The tumor proliferation rate in the treatment group was 232.0%. Both treatments could effectively inhibit tumor growth, and TGLA showed a more obvious tumor inhibition effect (P<0.05).

[0132] In addition, the TGLA treatment group, The body weight changes of mice in the treatment group and the saline treatment group showed that the body weight of all three groups decreased with time. Fig. 22 The results showed that TGLA and The effect of treatment on the body weight of mice was not as significant as that of the saline-treated group.

[0133] (3) Abnormal toxicity of antibodies

[0134] Seven days after mice were treated with 0.5 mg TGLA and guinea pigs were treated with 5 mg TGLA, the survival of mice and guinea pigs was observed to be normal, and there was no significant decrease in body weight compared with before treatment (Table 3).

[0135] Table 3. Changes in body weight of mice and guinea pigs before and after injection of TGLA

[0136]

[0137] The results showed that there was no significant change in body weight of mice and guinea pigs after TGLA treatment for 7 days, indicating that the antibody had no obvious toxicity.

[0138] The above test results confirmed that the plasmid constructed with the TGLA light and heavy chain variable region gene fragments expressed TGLA with the physical and chemical properties of a complete IgG antibody molecule and had specific recognition activity for CD20 molecules. The results of in vitro and in vivo biological tests confirmed that TGLA had a specific recognition activity for CD20. + The antibody has killing and tumor-suppressing activity on lymphoma cells. Its antibody physicochemical properties, specific recognition activity, relative affinity and tumor-suppressing effect are related to Highly similar.

[0139] Example 10: Construction of recombinant human-mouse chimeric anti-CD20 monoclonal antibody engineering cells

[0140] The HGE recombinant plasmid containing the light and heavy chain coding sequences of the recombinant human-mouse chimeric anti-CD20 monoclonal antibody was used to transfect CHO dhfr- cells under liposome-mediated transfection. The clones with positive antibody secretion were screened with hygromycin (250 μg / ml) and the cells were transfected with methotrexate (MTX) at different doses (10 -7 M, 3×10 -7 M, 6×10 -7 M and 10 -6M) was screened under pressure, and finally a cell line with high antibody expression, TGLA / II4, was obtained. TGLA / II4 was acclimated in serum-free suspension culture, and monocloned using the limiting dilution method. Five high-yield cell lines, TGLA / SC1, TGLA / SC4, TGLA / SC6, TGLA / SC8, and TGLA / SC11, were screened based on antibody expression. TGLA / SC1 was selected as the production cell line based on the shortest doubling time during culture, and a comprehensive identification and analysis was performed on it, including FISH in situ hybridization monoclonality, gene copy number, light and heavy chain peptide fingerprint analysis, antibody molecular weight determination, antibody isoelectric point determination, antibody light and heavy chain N-terminal sequence analysis, etc.

[0141] Example 11: Large-scale expression, purification and quality analysis of recombinant human-mouse chimeric anti-CD20 monoclonal antibody

[0142] A large-scale mammalian cell culture and protein purification system for recombinant human-mouse chimeric anti-CD20 monoclonal antibody was established, and large-scale expression of recombinant human-mouse chimeric anti-CD20 monoclonal antibody protein was achieved by using a large-scale mammalian cell culture and purification platform.

[0143] The glycoform components of this product and Rituximab were qualitatively analyzed by LC-MS. The proportion of N-sugar glycoforms is shown in Table 4.

[0144] Table 4. Summary of sugar ratios of this product and Rituximab

[0145]

[0146] Note: *: average value of 6 batches of this product; #: average value of 3 batches of Rituximab; % refers to area percentage.

[0147] The glycosylation level of the Fc segment of a monoclonal antibody can affect antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), and can change the pharmacokinetic properties of the antibody. Some glycoform structures can also affect the immunogenicity of the antibody. Reducing the proportion of fucose in the core sugar structure can improve the ADCC effect; reducing the proportion of high mannose glycoforms can enhance C1q affinity and CDC activity, but at the same time reduce ADCC activity; terminal galactose can improve the CDC effect; the higher the sialic acid content, the lower the affinity of the antibody to the Fc receptor, which leads to reduced ADCC activity and weakened binding to antigens on the cell surface, and also affects the half-life of the drug in vivo.

[0148] Example 12: CDC Biological Activity

[0149] Rabbit complement mediated:

[0150] Raji cells with high expression of CD20 and rabbit complement were used to analyze the CDC activity.

[0151] The results showed that the biological activity of this product was 98±5%. The biological activity was 310±35%.

[0152] The CDC biological activity of this product is lower than

[0153] Human complement mediates:

[0154] Raji cell line expressing CD20 on cell membrane and human complement were used to analyze the CDC activity.

[0155] The results showed that the CDC biological activity of this product was 89.8±4.0%. The CDC biological activity of this product is 1226.2±129.0%.

[0156] Example 13: ADCC biological activity (NK92 cell method)

[0157] The LDH release method was used to analyze the effects of this product and Raji cells expressing CD20 on the cell membrane as target cells and NK-92MI-CD16a cells expressing FcγRⅢa (CD16a) on the membrane (from Huabo Biotechnology) as effector cells. ADCC biological activity.

[0158] The results showed that the ADCC activity of this product was 102.9±5.9%. The ADCC activity was 26.5±4.7%.

[0159] The ADCC activity of this product is higher than

[0160] This product has a different amino acid sequence from Rituximab and has different action epitopes. Its CDC is weaker than that of Rituximab, but its ADCC activity is 3 to 4 times that of Rituximab. It may be based on this mechanism that this product has a stronger and more thorough killing effect on CD20-positive cells. At the same time, it showed a larger steady-state distribution volume and lower serum drug exposure level in human pharmacokinetic studies, which led to the observation of an ORR that was non-inferior to Rituximab and a more advantageous CR in clinical efficacy studies.

[0161] Example 14: Affinity detection with FcγRIIIa / CD16a (F176)

[0162] FcγRIIIa, also known as CD16a, is a transmembrane protein that is expressed in activated monocytes and macrophages, NK cells, and T cells. At the same time, FcγRIIIa is an Fc receptor with high affinity for IgG, mainly binding to the Fc segment of IgG1, IgG3, and IgG4. The affinity value of FcγRIIIa to IgG can be measured by the BLI method.

[0163] In this study, BLI was used to analyze the Dissociation equilibrium constant K for binding to FcγRIIIa / CD16a (F176) [from ACROBiosystems Inc.] D Conduct testing.

[0164] The results showed that this product and Dissociation equilibrium constant K for binding to FcγRIIIa / CD16a (F176) D They are (2.9±0.2)×10 -7 M, (6.0±0.4)×10 -7 M. The affinity of this product to FcγRIIIa / CD16a (F176) is higher than

[0165] Example 15: Clinical Study Results

[0166] Clinical trial results showed that at the end of 6 cycles of treatment for patients with newly diagnosed CD20-positive diffuse large B-cell lymphoma, the CR rate of this product was higher than trend.

[0167] In a multicenter, randomized, double-blind, active-drug parallel-controlled, 3-year clinical study (HS006-III), a total of 483 patients with newly diagnosed diffuse large B-cell non-Hodgkin's lymphoma received 375 mg / m 2 This product or Combined with standard CHOP (cyclophosphamide 750 mg / m 2 , day 1; doxorubicin 50 mg / m 2 , day 1; vincristine 1.4 mg / m 2 , up to 2 mg on day 1; and prednisone 100 mg / day on days 1-5), 3 weeks as a cycle, 6 cycles in total.

[0168] For all patients (375 mg / m 2 This product was combined with CHOP to treat 327 patients, 375 mg / m 2 The efficacy of the two groups was analyzed in combination with CHOP (156 patients). The two groups were balanced in terms of pre-treatment demographics and disease status. The results showed that the objective response rate of this product after 6 cycles of treatment was non-inferior to (375mg / m 2 This product combined with CHOP is 82.87%, 375mg / m 2 The rate of complete remission in the combined CHOP group was 81.41%, and the 95% confidence interval of the difference between the two groups was -5.88 to 8.81%); the complete remission rate in the protocol-compliant group was higher than (375mg / m 2 This product combined with CHOP is 85.3%, 375mg / m 2 The 1-year progression-free survival rate was 77.34% in the combined CHOP group and 77.34% in the control group (P=0.0481). There was a statistically significant difference (hazard ratio HR = 0.60, P = 0.0425). In the germinal center B cell-like patient subgroup, the complete remission rate of this product after 6 cycles of treatment was higher than that of (375mg / m 2 This product combined with CHOP is 77.45%, 375mg / m 2 The 1-year duration of remission, progression-free survival and There was a statistically significant difference (duration of remission: hazard ratio HR = 0.60, P = 0.0425; progression-free survival: hazard ratio HR = 0.43, P = 0.0052). In the subgroup of patients without bone marrow involvement, the complete remission rate of this product after 6 cycles of treatment was higher in both the remission evaluable set and the protocol-compliant set. (Response evaluable set: 375 mg / m 2 This product combined with CHOP is 77.45%, 375mg / m 2 Combined with CHOP: 68.21%, P = 0.0333; per protocol set: 375 mg / m 2 This product combined with CHOP is 85.82%, 375mg / m 2 Combined with CHOP, the rate was 76.8%, P = 0.0268).

[0169] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. An anti-CD20 antibody or CD20 antigen-binding fragment, It is characterized in that Capable of specifically binding to CD20 and comprising: a1) three heavy chain complementary determining regions, HC-CDR1, HC-CDR2, and HC-CDR3, whose amino acid sequences are shown in the amino acid sequences encoded by SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively; and a2) three light chain complementary determining regions, LC-CDR1, LC-CDR2, and LC-CDR3, whose amino acid sequences are shown in the amino acid sequences encoded by SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively; and The anti-CD20 antibody or CD20 antigen-binding fragment contains a total defucosylation glycoform area ratio of not less than 8.0% of the total sugar area.

2. The anti-CD20 antibody or CD20 antigen-binding fragment according to claim 1, It is characterized in that in, The total defucosylated glycoforms include high mannose glycoforms.

3. The anti-CD20 antibody or CD20 antigen-binding fragment according to claim 1 or 2, It is characterized in that The anti-CD20 antibody or CD20 antigen-binding fragment contains a high mannose glycoform area ratio that does not exceed 8.0% of the total sugar area.

4. The anti-CD20 antibody or CD20 antigen-binding fragment according to any one of claims 1 to 3, It is characterized in that The total defucosyl glycoform area ratio is 13.57±2.78% of the total sugar area; preferably, the anti-CD20 antibody or CD20 antigen-binding fragment contains a high mannose glycoform area of ​​5.76±1.84% of the total sugar area.

5. The anti-CD20 antibody or CD20 antigen-binding fragment according to any one of claims 1 to 4, It is characterized in that The anti-CD20 antibody or CD20 antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, and the amino acid sequences thereof are shown in the amino acid sequences encoded by SEQ ID NO:3 and SEQ ID NO:4, respectively, or have at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequences encoded by SEQ ID NO:3 and SEQ ID NO:4, respectively.

6. The anti-CD20 antibody or CD20 antigen-binding fragment according to any one of claims 1 to 5, It is characterized in that The anti-CD20 antibody comprises a heavy chain and a light chain, and the amino acid sequences thereof are shown in the amino acid sequences encoded by SEQ ID NO:1 and SEQ ID NO:2, respectively, or have at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequences encoded by SEQ ID NO:1 and SEQ ID NO:2, respectively.

7. The anti-CD20 antibody or CD20 antigen-binding fragment according to any one of claims 1 to 6, It is characterized in that The CD20 antigen binding fragment is: b1) single chain Fv; or b2) disulfide-linked Fv; or b3) Fab fragment; or b4)F(ab') 2 fragment; or b5) Fab' fragment.

8. The anti-CD20 antibody or CD20 antigen-binding fragment according to any one of claims 1 to 7, It is characterized in that The ratio of the fucose glycoform area of ​​the anti-CD20 antibody or CD20 antigen-binding fragment does not exceed 92.0% of the total sugar area; preferably, the ratio of the fucose glycoform area of ​​the anti-CD20 antibody or CD20 antigen-binding fragment is 83.44±3.12% of the total sugar area.

9. The anti-CD20 antibody or CD20 antigen-binding fragment according to any one of claims 1 to 7, It is characterized in that The anti-CD20 antibody or CD20 antigen-binding fragment contains a β-galactose glycoform area ratio of no more than 44.6% of the total sugar area; the anti-CD20 antibody or CD20 antigen-binding fragment contains a sialylated glycoform area ratio of no more than 4.8% of the total sugar area; preferably, The anti-CD20 antibody or CD20 antigen binding fragment contains β-galactose glycoform area ratio of 37.00±2.54% of the total sugar area; the anti-CD20 antibody or CD20 antigen binding fragment contains sialylated glycoform area ratio of 3.32±0.48% of the total sugar area.

10. A composition comprising the anti-CD20 antibody or CD20 antigen-binding fragment according to any one of claims 1 to 9.

11. Use of the anti-CD20 antibody or CD20 antigen-binding fragment according to any one of claims 1 to 9, or the composition according to claim 10 in the preparation of a medicament for treating a tumor or an autoimmune disease; preferably, the tumor is a blood tumor or a solid tumor; more preferably, the tumor or autoimmune disease is selected from the group consisting of diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, chronic lymphocytic leukemia, multiple sclerosis, rheumatoid arthritis, neuromyelitis optica spectrum disorder, non-Hodgkin's lymphoma, immune thrombocytopenia, Wegener's granulomatosis, microscopic polyangiitis, lupus nephritis and systemic lupus erythematosus; most preferably, the tissue cells of the tumor or autoimmune disease express CD20.

12. The use according to claim 11, comprising further using it in combination with one or more therapeutic agents for cancer or autoimmune diseases; preferably, the one or more therapeutic agents for cancer or autoimmune diseases are selected from chemotherapeutic agents, radiotherapeutic agents and biomacromolecule drugs.

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