An anti-human cd20 antibody or antigen-binding fragment thereof, and preparation method and application thereof

Abstract

The application belongs to the technical field of biology and provides an anti-human CD20 antibody or an antigen binding fragment thereof, a light chain variable region of the anti-human CD20 antibody or the antigen binding fragment thereof has an amino acid sequence as shown in SEQ ID NO: 1-3 or at least one amino acid difference compared with the amino acid sequence, and a heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 4-6 or at least one amino acid difference compared with the amino acid sequence. The mouse anti-human CD20 antibody provided by the application is close to a commercial anti-human CD20 antibody in affinity, has good specificity and can specifically recognize a closed CD20 target antigen.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an anti-human CD20 antibody or its antigen-binding fragment, its preparation method, and its application. Background Technology

[0002] CD20, a crucial target for antibody drugs, is a transmembrane phosphoprotein composed of 279 amino acids. It is also a unique marker found on the surface of human B lymphocytes, with a relative molecular mass of 33,000-37,000. It has four transmembrane domains, with the primary antigenic epitope located between the third and fourth transmembrane regions on the inner side of the cytoplasmic membrane. CD20 is expressed on the surface of B cells at all stages of development and differentiation, except for plasma cells (B cells that secrete immunoglobulins). It directly regulates B cells by modulating transmembrane calcium ion flow. The B lymphocyte antigen CD20 is a glycosylated phosphoprotein expressed on the surface of B lymphocytes. It is a B cell differentiation antigen located only on pre-B cells and mature B cells, and is expressed in over 95% of B-cell lymphomas.

[0003] The differential expression of CD20 in normal physiological tissues and tumor tissues makes CD20 a well-identifiable target for B-cell diseases. Over the past 30 years, more than 80 antibody drugs targeting approximately 20 drug targets have been approved for marketing, with four of these targets having been extensively studied and widely developed into drugs. The antitumor activity of CD20-targeting antibodies mainly includes apoptosis induction, complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). Type I and type II CD20 antibodies act on different sites of CD20, and their main mechanisms of action also differ.

[0004] Chinese patent application 201910991541.8 discloses an anti-CD20 antibody formulation and its application. The antibody formulation comprises the following components at concentrations: 15-80 mg / mL anti-CD20 antibody, 10-30 mM buffer, 80-240 mM stabilizer, and 0.1-0.4 mg / mL surfactant. The pH value of the liquid formulation is 5.5-6.2. This antibody formulation exhibits good stability.

[0005] Chinese patent application 200610160713.X discloses a functional humanized anti-human CD20 antibody and its applications. This functional humanized anti-human CD20 antibody is specific to CD20. It exhibits low immunogenicity and has been experimentally demonstrated to possess the same specificity and affinity as its murine progenitor antibody. Drugs using the functional humanized anti-human CD20 antibody and its derivatives as active ingredients can be widely used in the treatment of diseases caused by high CD20 expression, such as rheumatoid arthritis and lupus erythematosus.

[0006] There is currently a need in this field for more anti-human CD20 antibodies. Summary of the Invention

[0007] In order to achieve the above-mentioned technical objectives, the present invention hereby proposes the following technical solution:

[0008] In a first aspect, the present invention provides an anti-human CD20 antibody or an antigen-binding fragment thereof, wherein the anti-human CD20 antibody or the antigen-binding fragment thereof comprises a light chain variable region and a heavy chain variable region;

[0009] The variable region of the light chain has:

[0010] (1) The amino acid sequences of LCDR1 as shown in SEQ ID NO:1, LCDR2 as shown in SEQ ID NO:2, and LCDR3 as shown in SEQ ID NO:3; or

[0011] (2) LCDR1 having at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:1, LCDR2 having at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:2, and LCDR3 having at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:3.

[0012] The heavy chain variable region has:

[0013] 1) HCDR1 with amino acid sequences as shown in SEQ ID NO:4, HCDR2 with amino acid sequences as shown in SEQ ID NO:5, and HCDR3 with amino acid sequences as shown in SEQ ID NO:6; or

[0014] 2) HCDR1, which has at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:4; HCDR2, which has at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:5; and HCDR3, which has at least one amino acid difference compared to the amino acid sequence shown in SEQ ID NO:6.

[0015] The amino acid differences mentioned above are achieved through the addition, substitution, deletion, replacement, or modification of amino acids.

[0016] In some embodiments, the light chain variable region of the anti-human CD20 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:7, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:7.

[0017] In some embodiments, the heavy chain variable region of the anti-human CD20 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:8, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:8.

[0018] In some embodiments, the light chain of the anti-human CD20 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:9, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:9.

[0019] In some embodiments, the heavy chain of the anti-human CD20 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:10, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:10.

[0020] SEQ ID NO:1: SASSFITYMH.

[0021] SEQ ID NO:2: DTSKLAS.

[0022] SEQ ID NO:3: QQWNSNPPT.

[0023] SEQ ID NO:4: SYNIH.

[0024] SEQ ID NO:5: AIYPGNGDTSHNQKFKG.

[0025] SEQ ID NO:6:SRRYDGDWSFDV.

[0026] SEQ ID NO:7:

[0027] QIVLTQSPAIMSASPGEKVTMTCSASSFITYMHWFQQKSGTSPKRWIYDTSKLASGVP ARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNSNPPTFGGGTKLEIKRA.

[0028] SEQ ID NO:8:

[0029] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNIHWVKQTPGQGLEWIGAIYPGNG DTSHNQKFKGKATLTADRSSSTAYMQFSSLTSEDSAVYYCARSRRYDGDWSFDVWGAGT TVTVSS.

[0030] SEQ ID NO:9:

[0031] QIVLTQSPAIMSASPGEKVTMTCSASSFITYMHWFQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNSNPPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0032] The nucleotide sequence encoding SEQ ID NO:9 is shown in SEQ ID NO:11:

[0033] CAGATCGTGCTGACACAGAGCCCTGCCATCATGAGCGCTAGCCCTGGCGAGAAGGTGA

[0034] CCATGACCTGCAGCGCTAGCAGCTTCATCACCTACATGCACTGGTTTCAGCAGAAGAG

[0035] CGGCACAAGCCCTAAGAGATGGATCTACGACACAAGCAAGCTGGCTAGCGGCGTGCCT

[0036] GCTAGATTCAGCGGCAGCGGCAGCGGCACAAGCTACAGCCTGACCATCAGCAGCATGG

[0037] AGGCCGAGGACGCCGCCACCTACTACTGTCAGCAGTGGAACAGCAACCCTCCTACCTT

[0038] CGGCGGAGGCACCAAGCTGGAGATCAAGAGAGCCGACGCCGCCCCTACCGTGAGCAT

[0039] CTTCCCTCCTAGCAGCGAGCAGCTGACAAGCGGCGGCGCTAGCGTGGTGTGCTTCCTG

[0040] AACAACTTCTACCCTAAGGACATCAACGTGAAGTGGAAGATCGACGGCAGCGAGAGA

[0041] CAGAACGGCGTGCTGAACAGCTGGACCGACCAAGACAGCAAGGACAGCACCTACAGC

[0042] ATGAGCAGCACCCTGACCCTGACCAAGGACGAGTACGAGAGACACAACAGCTACACC

[0043] TGCGAGGCCACCCACAAGACAAGCACAAGCCCTATCGTGAAGAGCTTCAACAGAAACGAGTGC。

[0044] SEQ ID NO:10:

[0045] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNIHWVKQTPGQGLEWIGAIYPGNGDTSHNQKFKGKATLTADRSSSTAYMQFSSLTSEDSAVYYCARSRRYDGDWSFDVWGAGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

[0046] The nucleotide sequence encoding SEQ ID NO: 10 is shown in SEQ ID NO: 12:

[0047]

[0048] Secondly, the present invention provides an isolated polynucleotide encoding any of the aforementioned CD20 antibodies or their antigen-binding fragments.

[0049] Thirdly, the present invention provides a recombinant expression vector comprising the aforementioned isolated polynucleotides.

[0050] In some embodiments, the recombinant expression vector includes a eukaryotic expression vector and a prokaryotic expression vector.

[0051] Fourthly, the present invention provides a host cell comprising the aforementioned recombinant expression vector.

[0052] Fifthly, the present invention provides a method for preparing any of the aforementioned anti-human CD20 antibodies or their antigen-binding fragments, the preparation method comprising at least one of the following steps:

[0053] (I) The aforementioned isolated polynucleotide or recombinant expression vector is introduced into host cells, and after culturing the cells, the anti-human CD20 antibody or its antigen-binding fragment is purified and collected;

[0054] (II) Culturing the aforementioned host cells, and purifying and collecting anti-human CD20 antibodies or their antigen-binding fragments after culturing the cells.

[0055] Sixthly, the present invention provides a method for detecting CD20 protein for non-diagnostic purposes, characterized in that the detection method comprises:

[0056] I) Incubate the sample to be tested with any of the aforementioned anti-human CD20 antibodies or their antigen-binding fragments, or with a fluorescently labeled antibody containing any of the aforementioned anti-human CD20 antibodies or their antigen-binding fragments;

[0057] Ⅱ) Detect whether there are antibody-antigen conjugates in the sample, or detect the presence of fluorescence by flow cytometry.

[0058] The mouse anti-human CD20 antibody provided by this invention has a similar affinity to commercially available anti-human CD20 antibodies and good specificity, and can specifically recognize and block CD20 target antigens. Attached Figure Description

[0059] Figure 1 The SDS gel electrophoresis images of the commercially available mouse anti-human CD20 antibody Biolengend [2H7] and the ZXClone-HCD20-07 antibody of the present invention are shown.

[0060] Figure 2 Flow cytometry results for commercial Biolegend mouse anti-human CD20[2H7]-FITC.

[0061] Figure 3 The flow cytometry result of mouse anti-human CD20 [ZXCloneHCD20-07]-FITC is shown in the present invention.

[0062] Figure 4 Flow cytometry results for commercial Biolegend mouse anti-human CD20[2H7]-PE.

[0063] Figure 5 The flow cytometry result of the mouse anti-human CD20 [ZXCloneHCD20-07]-PE of this invention is shown.

[0064] Figure 6 and Figure 7 A graph showing the potency of commercially available Biolegend mouse anti-human CD20[2H7].

[0065] Figure 8 and Figure 9 This is a graph showing the potency of the mouse anti-human CD20 [ZXCloneHCD20-07] of the present invention.

[0066] Figure 10 This is the specific detection result for the control group.

[0067] Figure 11 This is a specificity detection diagram of mouse anti-human CD20 [ZXCloneHCD20-07] of the present invention. Detailed Implementation

[0068] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0069] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0070] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or those recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques are also described in many publications, such as *Molecular Cloning: A Laboratory Manual (Fourth Edition)* (Cold Spring Harbor Laboratory Science Press), Ausubel, FM et al., *Current Protocols in Molecular Biology*, Greene Publishing Assoc., and Wiley-Interscience.

[0071] As used herein, the term "antibody" refers to any form of antibody possessing the desired biological activity. Therefore, it is used in the broadest sense, specifically including, but not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camel-derived single-domain antibodies. The term "antibody" also refers to a glycoprotein comprising a heavy chain (H) and a light chain (L) linked together by disulfide bonds (SS). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (VH). The light chain constant region consists of one domain: CL. Light chains are divided into two categories: kappa-type light chains and lambda-type light chains (for example, in this invention, the light chain constant region Cκ / λ indicates that the light chain constant region is a kappa-type light chain or a lambda-type light chain). The VH and VL regions can be further subdivided into hypervariable regions (also called complementarity-determining regions (CDRs)), interspersed with more conserved framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Antibodies include monospecific antibodies, bispecific antibodies, and multispecific antibodies, as long as they exhibit the desired biological activity or function.

[0072] As used herein, the term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of an antibody that typically include at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules, such as scFv; nanobodies formed from antibody fragments; and multispecific antibodies. When the antigen-binding activity is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or higher of the antigen-binding affinity of the parent antibody. It is also anticipated that the antigen-binding fragment of an antibody may include conserved or non-conserved amino acid substitutions (referred to as "conserved variants" or "functionally conserved variants" of the antibody) that do not significantly alter its biological activity. The term "binding compound" refers to both the antibody and its binding fragment.

[0073] As used herein, the terms “hypervariant region,” “hypervariant region,” “complementarity-determining region,” “HVR,” or “CDR” refer to regions in the variable domain region of an antibody that are highly variable in sequence and / or form structurally defined loops (“hypervariant loops”). Typically, a natural tetrachain antibody contains six HVRs or CDRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Based on Chothia’s definition, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L26–L32 (L1), L50–L52 (L2), L91–L96 (L3), H26–H32 (H1), H52–H56 (H2), and H96–H101 (H3) (Chothia et al., J. Mol. Biol. 196:901–917 (1987)). Based on Kabat's definition rules, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L24-L34 (L1), L50-L56 (L2), L89-L97 (L3), H31-H35 (H1), H50-H65 (H2), and H95-H102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, the fifth edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Based on the IMGT definition rules, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) are located at amino acid residues L27-L32 (L1), L50-L51 (L2), L89-L97 (L3), H26-H33 (H1), H51-H56 (H2), and H93-H102 (H3) (Honjo, T. and Alt, FW (1995) Immunoglobulin genes. Academic Press pp. 3-443). It is well known to those skilled in the art that antibody CDRs can be defined using various methods, such as the Kabat definition rule based on sequence variability, the Chothia definition rule based on the location of structural loop regions, and reference tools for antibody humanization design based on CDR transplantation (see J MolBiol. 273:927-48, 1997).Those skilled in the art will understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined as described in any of the known schemes described herein. While the scope of this disclosure is based on the sequences defined by the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of this invention.

[0074] Example 1: Preparation and Detection of Anti-Human CD20 Antibody

[0075] 1.1 Preparation of anti-human CD20 antibody

[0076] The inventors prepared an anti-human CD20 antibody, numbered ZXCloneHCD20-07.

[0077] The light chain amino acid sequence of CD20[ZXCloneHCD20-07] is shown in SEQ ID NO:9, and the heavy chain amino acid sequence is shown in SEQ ID NO:10. A nucleotide sequence for expressing CD20[ZXCloneHCD20-07] was designed using conventional methods. The nucleotide sequence encoding SEQ ID NO:9 is shown in SEQ ID NO:11, and the nucleotide sequence encoding SEQ ID NO:10 is shown in SEQ ID NO:12. After cloning the above nucleotide sequences into a stable CHO cell line, antibody expression was performed. The constructed vector was then introduced into CHO cells, and after cell culture, the antibody was purified using protein A affinity chromatography to obtain CD20[ZXCloneHCD20-07].

[0078] The specific steps for antibody preparation are as follows:

[0079] (1) CHO cells were cultured in CHO CD medium at a concentration of 2 × 10⁶. 5 Inoculate cells / mL with 4 mM glutamine. Build up cell density to 4 × 10⁶ cells / mL. 6 Cells were collected at a density of 1 × 10⁶ cells / mL with a viability greater than 95%. Cells were collected by centrifugation, resuspended in electroporation buffer, and adjusted to a density of 1 × 10⁶ cells / mL. 7 cells / mL.

[0080] (2) Take a sterile EP tube, add 0.8 mL of cell suspension and 5 μg of plasmid, mix gently, incubate for 15 min, and then incubate on ice for 5 min. Set the electroporator parameters as follows: voltage 280V, pulse count 3, pulse duration 5 ms, pulse interval 0.784 s, and electroporation cuvette inner diameter 4 mm. Add 0.5 mL of the cell and plasmid mixture to each electroporation cuvette, place it in the electroporator, and start the electroporation program. After completion, quickly aspirate the cells from the electroporation cuvette and transfer them to a 125 mL Erlenmeyer flask containing 20 mL of CHO CD medium. Repeat the above steps to electroporate the second electroporation cuvette. Gently mix the cells in the shake flask, then tighten the cap and incubate in a shaker at 37°C.

[0081] (3) After 48 hours, transfer the cells to centrifuge tubes and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and resuspend the cells in 20 mL of CHO CD medium containing 2, 5, and 20 μM MSX. Calculate cell density and viability. The cell density should be around 1.2 × 10⁻⁶ cells / mL. 6 Approximately [number] cells / mL.

[0082] (4) Transfer the cells to new shake flasks. Starting from day seven, observe cell density and viability every two days. Cell density should be increased from an initial 1.2 × 10⁻⁶. 6 The cell density decreases from 10 to 15 days, reaching its lowest point between days 5 and 7. The cell density then begins to increase. When the cell density reaches 1 × 10⁻⁶, the density rises. 6 After reaching a cell density of 3 × 10⁶ cells / mL, the cells were passaged to a cell density of 3 × 10⁶ cells / mL using CD OPM medium containing 50 μM MSX. 5 cells / mL, continue culturing.

[0083] (5) When the cell density increases to 4×10 6 After reaching a cell / mL level, cells were expanded or passaged using CD OPM medium containing 50 μM MSX, with a minimum seeding density of 3 × 10⁻⁶ cells / mL. 5 When the number of cells / mL reaches a certain level, preparations for cryopreservation or large-scale culture can begin.

[0084] (6) Antibody collection method: Antibody purification was performed using protein A affinity chromatography.

[0085] 1.2 Antibody Detection

[0086] Take 10 μg of commercially available CD20 antibody (Biolengend, catalog number: 302350). Mix 10 μg of the prepared CD20 [ZXCloneHCD20-07] with the loading buffer (antibody to loading buffer volume ratio 4:1), incubate at 100℃ for 30 min, and centrifuge at 10000 rpm for 1 min at room temperature to obtain the sample to be tested. Take out the pre-cast gel and install it in the electrophoresis tank, inject the electrophoresis buffer, remove the comb, and load the sample. 10 μL of protein marker and 10 μg of sample to be tested are loaded. Electrophoresis settings: voltage 80V, electrophoresis time 90 min, stop when the bromophenol blue indicator reaches the bottom. Remove the gel and stain with Coomassie Brilliant Blue for 30 min, then destain in destaining solution for 1 h. Repeat 5 times and take pictures.

[0087] The results showed that the anti-human CD20 antibody prepared in this embodiment had intact heavy and light chains, clear bands, and a purity of over 90%. (See attached figures.) Figure 1 .

[0088] Example 2 Anti-human CD20 antibody labeling

[0089] FITC (fluorescein isothiocyanate) and PE were used to label CD20 [ZXCloneHCD20-07].

[0090] 2.1 FITC Marking

[0091] Take 0.2 mg of CD20 [ZXCloneHCD20-07] into an ultrafiltration tube, wash the antibody three times with 1 mM PBS. Centrifuge the ultrafiltration tube inverted at 4000 g for 2 min, collect the concentrated antibody, and dilute it to 10 mg / mL with 1 mM PBS.

[0092] (2) Take the small molecule dye FITC (add dye at a ratio of n (molar mass) antibody: n (molar mass) FITC = 1:15) and add it to the monoclonal antibody reaction solution and mix well.

[0093] (3) React at room temperature of 25℃ for 45 minutes in the dark on a shaker.

[0094] (4) Transfer the reaction solution to an ultrafiltration centrifuge tube, wash the antibody 5 times with 1mM PBS. Centrifuge the ultrafiltration tube inverted at 4000g for 2min to collect the concentrated antibody, and dilute the antibody with 1mM PBS to a final concentration of 0.5mg / mL.

[0095] The labeled antibody is designated as CD20[ZXCloneHCD20-07]-FITC.

[0096] 2.2 PE Marking

[0097] CD20[ZXCloneHCD20-07] was labeled according to the method described in the patent "Method and Kit for Labeling Fluorescent Proteins and / or Coupled Proteins (CN202010972671.X)". The labeled antibody was denoted as CD20[ZXCloneHCD20-07]-PE.

[0098] 2.3 Detection of the effect of fluorescently labeled antibodies

[0099] 1) Add 100 μL of cell quality control material (Beckman Coulter, catalog number: 6607077) to a 1.5 mL centrifuge tube;

[0100] 2) Add 1 μL of the fluorescently labeled antibodies CD20[2H7]-FITC and CD20[2H7]-PE to the sample respectively; add 5 μL of the control fluorescent antibodies CD20[2H7]-FITC (Biolegend, catalog number: 302303) and CD20-[2H7]-PE (Biolegend, catalog number: 980214).

[0101] 3) All four samples were reacted in the dark for 15 minutes.

[0102] 4) Add 1 mL of hemolysin and continue the reaction for 15 minutes. The hemolysin used is the one from Zhengxi flow cytometer. Hemolysin: Zhejiang Medical Device Registration No. 20200133.

[0103] 5) Centrifuge at 4000 rpm for 2 min, discard the supernatant, and resuspend the sample to be tested in 500 μL PBS.

[0104] 6) Flow cytometry (Beckman DxFLEX flow cytometer) is used to detect the proportion of positive cells in the sample, the clustering of the chromatogram, and other indicators, thereby determining whether CD20[2H7]-FITC and CD20[2H7]-PE can be labeled with the fluorescent dyes FITC and PE, and whether the labeled fluorescent antibody is correctly used to detect the target antigen.

[0105] The results showed that FITC successfully labeled mouse anti-human CD20 [ZXCloneHCD20-07], and the ratio of mouse anti-human CD20 [ZXCloneHCD20-07] to commercial CD20 [2H7]-FITC was similar ( Figures 2-5 ).

[0106] Example 3: Determination of Anti-human CD20 Antibody Titer

[0107] First, commercially available CD20[2H7] antibody (Bioegend, catalog number: 302350) and antibody CD20[ZXCloneHCD20-07] at known concentrations were diluted to 1 mg / mL. The antibody was then further diluted with PBS at ratios of 1:100, 1:1000, 1:3000, 1:6000, 1:12000, 1:24000, 1:48000, 1:60000, and 1:96000. CD20[ZXCloneHCD20-07] served as the experimental group, and the commercially available CD20 antibody served as the control group.

[0108] The experimental group consisted of two subgroups, and the control group consisted of one subgroup. Each subgroup received 100 μL of healthy human blood or a manufacturer's reference sample. 1 mL of hemolysin was added to each of the three experimental groups. Hemolysin (Zhejiang Medical Device Registration No. 20200133), continue the reaction for 15 min. Centrifuge at 4000 rpm for 2 min, and discard the supernatant.

[0109] The experimental group was given 100 μL of CD20 [ZXCloneHCD20-07] diluted according to the specified ratio, while the control group was given 100 μL of commercially available anti-human CD20 [2H7] antibody diluted according to the specified ratio.

[0110] Vortex to mix and incubate at room temperature in the dark for 15 min. Resuspend the sample in 900 μL of PBS, centrifuge at 4000 rpm for 2 min, and discard the supernatant. Add 100 μL of PBS and vortex to mix. Add 1 μL of commercially available goat anti-mouse IgG-PE flow cytometry antibody (Biolgend, catalog number: 405307) to each group, vortex to mix, and incubate at room temperature in the dark for 15 min. Resuspend the sample in 900 μL of PBS, centrifuge at 4000 rpm for 2 min, and discard the supernatant. Add another 1000 μL of PBS, resuspend the sample, centrifuge at 4000 rpm for 2 min, and discard the supernatant. Resuspend the sample to be tested in 500 μL of PBS and analyze using a flow cytometer.

[0111] The results showed that the commercially available CD20[2H7] antibody had an affinity (kd) of 195.4 (1 mg / mL), while the self-made mouse anti-human CD20[ZXCloneHCD20-07] antibody had an affinity (kd) of 189.8 (1 mg / mL). (See [link to relevant documentation]). Figures 6-9 ).

[0112] The results showed that CD20[ZXCloneHCD20-07] and commercial CD20[2H7] had similar affinities.

[0113] Example 4: Specificity assay of anti-human CD20 antibody

[0114] The antibody selected in Example 1 is:

[0115] Commercial CD20 antibody (Biolegend, catalog number: 302350)

[0116] CD20 [ZXCloneHCD20-07] prepared in Example 1;

[0117] Mouse isotype control antibody (Biolegend, catalog number: 400100)

[0118] Commercial fluorescent antibody CD20[2H7]-PE (Manufacturer: Biolegend, Catalog No.: 980214)

[0119] Antibody-specific detection methods:

[0120] Lymphocytes were obtained by collecting 100 μL of human peripheral blood sample (Huzhou Zhengxi Medical Laboratory, sample number: 02-056). 1 mL of hemolysin was added, and the reaction was continued for 15 minutes. The hemolysin used was the one from Zhengxi flow cytometry. Hemolysin: Zhejiang Medical Device Registration No. 20200133. Centrifuge at 4000 rpm for 2 min, discard the supernatant, and resuspend the sample in 100 μL of PBS.

[0121] Experimental group: Add 3 μL of CD20[ZXCloneHCD20-07] to one of the above samples, react in the dark for 15 minutes, then resuspend the sample in 500 μL of PBS, centrifuge at 4000 rpm for 2 min, discard the supernatant, and then resuspend the sample in 100 μL of PBS. Add 5 μL of CD3-APC and 5 μL of BiolegendCD20[2H7]-PE to the sample blocked with pure anti-CD20[ZXCloneHCD20-07].

[0122] Control group: Add 3 μL of pure mouse isotype control antibody to one of the above samples, react in the dark for 15 minutes, then add 500 μL of PBS to resuspend the sample, centrifuge at 4000 rpm for 2 min, discard the supernatant, and add another 100 μL of PBS to resuspend the sample. Add 5 μL of CD3-APC and 5 μL of Biolegend CD20[2H7]-PE to the resuspended sample.

[0123] After reacting all samples in the dark for 15 minutes, resuspend the samples in 500 μL of PBS, centrifuge at 4000 rpm for 2 min, discard the supernatant, and then resuspend the samples in 500 μL of PBS.

[0124] Flow cytometry (Agilent NovoCyte flow cytometer) was used to detect indicators such as the proportion of positive cells in the sample and the clustering of cell patterns, in order to determine whether CD20 [ZXCloneHCD20-07] can specifically recognize the CD20 target antigen.

[0125] The results showed that the experimental group had CD3-APC signal but no signal from the CD20[2H7]-PE fluorescent antibody. In the control group, the isotype control antibody could not block the antibody, but there was a fluorescent signal from CD20[2H7]-PE. This indicates that the two CD20 antibodies prepared from CHO, clone number CD20[ZXCloneHCD20-07], can specifically recognize and block the CD20 target antigen (…). Figures 10-11 ).

[0126] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An anti-human CD20 antibody or its antigen-binding fragment, characterized in that, The anti-human CD20 antibody or its antigen-binding fragment includes a light chain variable region and a heavy chain variable region; The variable region of the light chain has: The amino acid sequences of LCDR1 (SEQ ID NO:1), LCDR2 (SEQ ID NO:2), and LCDR3 (SEQ ID NO:3) are shown in SEQ ID NO:

1. The heavy chain variable region has: The amino acid sequences are HCDR1 as shown in SEQ ID NO:4, HCDR2 as shown in SEQ ID NO:5, and HCDR3 as shown in SEQ ID NO:

6.

2. The anti-human CD20 antibody or its antigen-binding fragment according to claim 1, characterized in that, The light chain variable region of the anti-human CD20 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:

7.

3. The anti-human CD20 antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The heavy chain variable region of the anti-human CD20 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:

8.

4. The anti-human CD20 antibody or its antigen-binding fragment according to claim 3, characterized in that, The light chain of the anti-human CD20 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:

9.

5. The anti-human CD20 antibody or its antigen-binding fragment according to claim 4, characterized in that, The heavy chain of the anti-human CD20 antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO:

10.

6. An isolated polynucleotide, characterized in that, It encodes the anti-human CD20 antibody or its antigen-binding fragment as described in any one of claims 1-5.

7. A recombinant expression vector, characterized in that, It contains the isolated polynucleotide as described in claim 6.

8. A host cell, characterized in that, It comprises the recombinant expression vector as described in claim 7.

9. A method for preparing an anti-human CD20 antibody or its antigen-binding fragment according to any one of claims 1-5, characterized in that, The preparation method includes at least one of the following steps: (I) The isolated polynucleotide of claim 6 or the recombinant expression vector of claim 7 is introduced into a host cell, and the cell is cultured and then purified and collected for anti-human CD20 antibody or its antigen-binding fragment. (II) Cultivate the host cells as described in claim 8, and purify and collect the anti-human CD20 antibody or its antigen-binding fragment after culturing the cells.

10. A method for detecting CD20 protein for non-disease diagnostic purposes, characterized in that, The detection method includes: I) Incubate the sample to be tested with any of the anti-human CD20 antibodies or their antigen-binding fragments as described in claims 1-5, or with a fluorescently labeled antibody containing any of the anti-human CD20 antibodies or their antigen-binding fragments as described in claims 1-5; Ⅱ) Detect whether there are antibody-antigen conjugates in the sample, or detect the presence of fluorescence by flow cytometry.

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