Application of monoclonal antibody 2F5 in autoimmune diseases and B cell regulation

Abstract

The invention discloses application of a monoclonal antibody 2F5 in autoimmune diseases and regulation and control of B cells, the monoclonal antibody 2F5 is obtained from SLE peripheral blood, and it is proved for the first time that the monoclonal antibody 2F5 can promote proliferation, activation and differentiation of the B cells through primary cell and cell line experimental models. The antibody provided by the invention is completely humanized and clear in targeting, can well simulate the blood microenvironment of the high-titer autoantibody in the peripheral blood of an autoimmune disease patient, is an ideal tool for defining the biological function of the autoantibody and researching the regulation and control of the autoantibody on the activation and differentiation of B cells, is beneficial to deepening the understanding of a mechanism causing immune disorder, and has a good application prospect. Therefore, the treatment intervention of immune pathological diseases caused by abnormal differentiation of B cells becomes possible, and an important foundation is laid for developing a new target spot of targeted pathogenic B cells for treating autoimmune diseases and tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibody preparation and immunological detection, and in particular to the application of monoclonal antibody 2F5 in autoimmune diseases and regulating B cells. Background Art

[0002] Autoimmune diseases (AIDs) are a class of diseases caused by the immune system's mistaken immune response to self-antigens. This pathological state causes the immune system to attack the body's own tissues, organs or cell components, ultimately causing organ damage or dysfunction. In recent years, the incidence of various autoimmune diseases has shown an upward trend, especially among young and middle-aged women, and has become one of the main causes of death. There are currently many known autoimmune diseases, nearly a hundred of which are more common, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), primary Sjogren's syndrome (pSS), and Graves disease (GD).

[0003] B cells are the main cells of humoral immunity, and they achieve immune effects by producing memory B cells that can respond quickly to subsequent antigen exposure and plasma cells that can continuously secrete antibodies. These B cell subsets can maintain humoral immunity and host protection for a long time and prevent repeated infections. Abnormal activation and differentiation of B cells play a key role in the pathological process of autoimmune diseases and are also one of the important factors for the increased overall tumor risk in patients with autoimmune diseases. B cells directly or indirectly participate in the autoimmune process by presenting antigens to T cells and producing proinflammatory cytokines or autoantibodies. Abnormal activation of B cells may be related to factors such as the destruction of immune tolerance and the imbalance of Th1 / Th2 cell balance, and is also affected by the imbalance of signal transduction molecules in B cells, such as Toll-like receptors (TLRs). CD11c present in healthy people, autoimmune diseases and (chronic) infected patients + Tbet + B cells have upregulated genes associated with antibody secreting cell (ASC) differentiation. Mouse experiments have shown that CD11c + Tbet + B cells are involved in preventing chronic viral infection. In healthy individuals, CD11c is highly expressed in + Tbet +B cells can prepare for ASC differentiation, and can secrete more IgM and IgG after culture in the presence of B cell antigen receptor ligation, TLR9 ligand, and interleukin-21, and can participate in protective immunity against infection or immunity as a separate germinal center (GC)-independent memory-like population. It can be said that autoantibodies are a double-edged sword, which can play a role in both protective immunity against infection and in promoting the occurrence of autoimmune diseases. However, although high titers of autoantibodies in the blood are a hallmark of many autoimmune diseases, their specific effects on B cell differentiation remain unclear.

[0004] The current method of using cytokines (such as interleukin-21) to regulate B cells has a small effective concentration window of cytokines and is prone to toxic side effects; while the method of using genome modification technology (such as knocking out the transcription factor Pax5) to regulate B cells has high technical requirements and has the risk of "off-target"; therefore, a simple and quick method to regulate it is urgently needed. Summary of the invention

[0005] The first objective of the present invention is to provide a method for preparing a drug for treating autoimmune diseases by using the monoclonal antibody 2F5 in order to address the deficiencies of the prior art. The monoclonal antibody 2F5 can specifically bind to the DEAD box helicase 5 (DDX5) protein, which is an autoantibody, i.e., an antibody against the body's own tissues, organs, cells and their components.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The use of monoclonal antibody 2F5 in the preparation of a drug for treating autoimmune diseases, wherein the autoimmune diseases are caused by abnormal differentiation of B cells, wherein the complementary determining regions CDR1, CDR2 and CDR3 of the light chain 2F5 are represented by LCDR1, LCDR2 and LCDR3, respectively; the LCDR1 sequence is QSVSSN, the LCDR2 sequence is GAS, and the LCDR3 sequence is QQYNNWPRT. The complementary determining regions CDR1, CDR2 and CDR3 of the heavy chain 2F5 are represented by HCDR1, HCDR2 and HCDR3, respectively; the HCDR1 sequence is GFTFSDYS, the HCDR2 sequence is ITSSSGYT, and the HCDR3 sequence is ARVRSSWGPIDS.

[0007] Preferably, the monoclonal antibody 2F5 promotes B cell proliferation, activation and differentiation.

[0008] The second object of the present invention is to provide the use of the monoclonal antibody 2F5 in the preparation of a preparation for promoting B cell proliferation, activation or differentiation.

[0009] Preferably, the B cells include RAMOS, OCI-LY-19 and primary B cells.

[0010] The third object of the present invention is to provide a method for specifically inducing B cell proliferation, activation or differentiation for non-diagnostic and non-therapeutic purposes, the method comprising the following steps: The purified monoclonal antibody 2F5 was diluted to prepare a 2F5 antibody diluent, and then the B cells were treated.

[0011] Preferably, the B cells include RAMOS, OCI-LY-19 and primary B cells.

[0012] The fourth object of the present invention is to provide a composition for treating autoimmune diseases, characterized in that the composition comprises one or more of the following ingredients: (a) Monoclonal antibody 2F5; (b) nucleic acid encoding monoclonal antibody 2F5; (c) An expression vector encoding the monoclonal antibody 2F5 nucleic acid.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses the monoclonal autoantibody 2F5 obtained from SLE peripheral blood, and confirms for the first time through primary cell and cell line experimental models that it can promote B cell proliferation, activation and differentiation, expanding its application in the field of immune regulation. The antibody is fully humanized and has a clear target. It can perfectly simulate the blood microenvironment of high-titer autoantibodies in the peripheral blood of patients with autoimmune diseases. It is an ideal tool for clarifying the biological functions of autoantibodies and studying the regulation of B cell activation and differentiation by autoantibodies.

[0014] The method of the monoclonal antibody 2F5 targeted stimulation of B cells in the present invention induces the differentiation of specific B cell subsets, reduces the impact on non-target cells, has strong specificity, high controllability, better safety and economy, and antibody-mediated B cell activation can occur rapidly, which provides the possibility for rapid induction of immune response. The present invention realizes the precise regulation of B cell differentiation, makes it possible to treat immunopathological diseases caused by abnormal differentiation of B cells, and lays an important foundation for the development of new targets for targeting pathogenic B cells to treat autoimmune diseases and tumors. At the same time, a small amount of correct antibodies can also significantly improve the responsiveness of low-affinity B cells, thereby generating high-affinity antibodies. Therefore, the precise regulation of B cell response by the present invention is of great significance for vaccine design, improving vaccine effectiveness, and blocking the spread of infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1The results of 2F5 antibody promoting the proliferation of PBMC and B cell lines, where A: 2F5 antibody promotes PBMC cell proliferation, and the optimal time point for proliferation is 48h; B: 2F5 supernatant and 2F5 antibody can promote OCI-LY-19 cell proliferation, and the optimal time point for proliferation is 48h; C: 2F5 supernatant and 2F5 antibody can promote RAMOS cell proliferation, and the optimal time point for proliferation is 24h. ****P <0.0001, ***P <0.001, **P<0.01, *P <0.05, ns P >0.05.

[0016] Figure 2 The results of the promotion of B cell line activation by 2F5 antibody, wherein A: surface fluorescence intensity of OCI-LY-19 cells treated with 2F5 antibody; B: surface fluorescence intensity of RAMOS cells treated with 2F5 antibody. The scale bar is 50 μm.

[0017] Figure 3 The results of the promotion of 2F5 antibody supernatant on the activation of two B cell lines, where A: indirect immunofluorescence detection of the surface fluorescence intensity of OCI-LY-19 cells treated with 2F5 supernatant; B: indirect immunofluorescence detection of the surface fluorescence intensity of RAMOS cells treated with 2F5 supernatant. The scale bar is 50 μm.

[0018] Figure 4 The results show that 2F5 antibody promotes the activation of primary B cells. The scale bar indicates 50 μm.

[0019] Figure 5 Figure 2 shows the results of 2F5 antibody promoting the differentiation of PBMC and RAMOS cells. A: The proportion of CD19+CD27+CD38+B cells in PBMCs stimulated by 2F5 purified antibody increased, *P < 0.05, ns P > 0.05; B: Both 2F5 purified antibody and IgG antibody can promote the expression of CD138 on the cell surface, that is, promote B cell differentiation, but compared with IgG, the effect of 2F5 in promoting B cell differentiation is more significant.

[0020] Figure 6 The morphological effects of 2F5 antibody on two B cell lines, A: Wright-Giemsa staining to observe the morphological changes of OCI-LY-19 cells treated with 2F5 antibody; B: Wright-Giemsa staining to observe the morphological changes of RAMOS cells treated with 2F5 antibody. The scale bar is 50 μm.

[0021] Figure 7 The effect of 2F5 antibody on the differentiation of primary B cells was detected by ELISA. **P < 0.01, *P < 0.05, nsP > 0.05. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with embodiments and drawings.

[0023] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0024] 1. Experimental Materials 1.1 Experimental cells (1) RAMOS (human B lymphoblastoma cells): grow in suspension, highly express CD38, can secrete IgM, and express membrane-type and secretory immunoglobulins. Purchased from Guangzhou Geneo Cell Center.

[0025] (2) OCI-LY-19 (human diffuse large B-cell lymphoma cells): grows in suspension and highly expresses CD38. As a type of diffuse large B-cell lymphoma cell line, it is currently often used as an experimental cell in scientific research to explore the mechanism of diffuse large B-cell lymphoma. Purchased from Guangzhou Geneo Cell Center.

[0026] (3) PBMCs (Peripheral Blood Mononuclear Cells) were obtained from healthy volunteers, including 4 males and 6 females aged between 18 and 26 years old. 30 mL of peripheral cubital venous blood was drawn from the volunteers in the early morning on an empty stomach and anticoagulated with ethylenediaminetetraacetic acid (EDTA). All subjects followed the principle of informed consent and signed the informed consent form.

[0027] 1.2 Experimental reagents and equipment RPMI-1640 (Thermofisher, 11875093), human peripheral blood mononuclear cell extraction kit (Solaibo, P8680), human primary B cell extraction kit (Thermofisher, 11351D), human IgM ELISA kit (ABclonal, RK00097), CCK-8 kit (Dojindo, CK04), CD80 rabbit monoclonal antibody (ABclonal, A25138), FITC-labeled goat anti-rabbit IgG secondary antibody (ABclonal, AS011), PE-CD19 antibody (Lianke Biotechnology, F11019A02), APC-CD27 antibody (Abcam, ab239293), FITC-CD38 antibody (Lianke Biotechnology, F1103801) 2. Experimental Methods 2.1 Preparation of 2F5 antibody A fully human monoclonal antibody library was established using human B cell cloning technology. The purified DDX5 protein was used as an antigen to coat the ELISA reaction plate at 4°C overnight. The reaction plate was then blocked with 4% BSA / PBST for 1 hour at room temperature. After blocking, the hybridoma cell culture supernatant of the antibody library was added to the reaction plate and reacted at room temperature for 1 hour. Washed 3 times with PBST for 5 minutes each. Goat anti-human horseradish peroxidase-labeled secondary antibody was added for 1 hour at room temperature. Washed 3 times with PBST-PBS for 5 minutes each. The hybridoma cell line with positive reaction was screened by color development, namely 2F5. Subsequently, the culture was expanded and the serum-free cell culture supernatant was collected. After purification with protein A affinity column, desalting and concentration, the purified monoclonal antibody 2F5 was obtained.

[0028] The amino acid sequence of the monoclonal antibody 2F5 is shown in Table 1 and Table 2 below: Table 1 CDR (complementarity determining region) amino acid sequence of monoclonal antibody 2F5 Table 2 Amino acid sequence of the variable region of monoclonal antibody 2F5 V L VH 2F5 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWPRT (SEQ ID NO.6) QVQLVESGGDLVKPGGSLRLSCAASGFTFSDYSMNWVRQTPGKGLEWLSYITSSSGYTNYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVRSSWGPIDS (SEQ ID NO.7) 2.2 2F5, IgG antibody configuration (1) Take a 15 mL centrifuge tube, add the prepared 1640 medium, take a tube of purified 2F5 antibody (1 mg / mL, 480 μL), add 120 / 240 μL of 2F5 antibody to the centrifuge tube to make 80 / 160 μM antibody diluent, mix evenly. Filter with a 0.22 nm bacterial filter membrane and store in a 4°C refrigerator.

[0029] (2) Take a 15 mL centrifuge tube, add the prepared 1640 medium, take a tube of IgG antibody (10 mg / mL, 45 μL), add 15 / 30 μL of IgG antibody to the centrifuge tube to make 80 / 160 μM antibody diluent, mix evenly. Filter with a 0.22 nm bacterial filter membrane and store in a 4°C refrigerator.

[0030] 2.3 Configuration Grouping (1) 2F5 group (80 μM): 120 μL of 2F5 stock solution was added to 9.88 mL of 1640 medium to dilute to 80 μM 2F5 antibody diluent.

[0031] (2) 2F5 group (160 μM): 240 μL of 2F5 stock solution was added to 9.76 mL of 1640 culture medium to dilute to 160 μM 2F5 antibody diluent.

[0032] (3) IgG group (80 μM): 15 μL of IgG stock solution was added to 12.485 mL of 1640 culture medium to dilute to 80 μM IgG antibody diluent.

[0033] (4) IgG group (80 μM): 30 μL of IgG stock solution was added to 12.47 mL of 1640 medium and diluted to 160 μM IgG antibody diluent.

[0034] (5) NC group (normal culture in 1640 medium).

[0035] (6) Contral group: only secondary antibody was added without primary antibody.

[0036] 2.4 Indirect immunofluorescence (1) Coating: Coat a 48-well plate with poly-D-lysine (200 μg / mL) and incubate at 37°C for more than 1 h.

[0037] (2) Inoculate the cells and place them in a 37°C cell culture incubator for 24 hours. After the cells sink to the bottom, carefully remove the supernatant. Add different treatment solutions to each well, gently shake the cells to mix, and then place the cells in the incubator for another 24 hours (primary B cells are cultured for 48 hours).

[0038] (3) Carefully drain the supernatant and heat in a metal bath at 100°C for 5-10 min (bake dry). Wash once with PBS.

[0039] (4) Fixation: Add 200 μL of 4% paraformaldehyde to each well for fixation and let stand at room temperature for 10 min.

[0040] (5) Washing: Add 600 μL PBST to each well and wash twice for 5 min each time. Add 600 μL PBS and wash once for 5 min at room temperature.

[0041] (6) Blocking: Add 200 μL of blocking solution (10% FBS, 2% BSA in PBS) to each well for blocking and place on a shaker for 1 hour to allow the well to shake evenly.

[0042] (7) Add primary antibody: Add primary antibody (Rb CD80 mAb) diluted 1:100 with PBS, put into a sealed bag, and refrigerator at 4°C overnight.

[0043] (8) Washing: Add 600 μL PBST to each well and wash twice for 5 min each, then wash once with PBS for 5 min at room temperature.

[0044] (9) Add secondary antibody: Add fluorescent secondary antibody (goat anti-rabbit IgG-FITC) diluted 1:100 with PBS (be careful to avoid light) and place on a shaker for 2-3 hours to allow the antibody to fully contact the cells.

[0045] (10) Washing: Add 600 μL PBST to each well and wash twice for 5 min each, then wash once with PBS for 5 min at room temperature.

[0046] (11) Nuclear staining: Add 100 μL Hoechest staining solution (1:10,000) to each well, protect from light, and incubate at room temperature for 10 min.

[0047] (12) Washing: Add 600 μL PBST to each well and wash twice for 5 min each, then wash once with PBS for 5 min at room temperature.

[0048] (13) Sealing: Aspirate the liquid in the wells and add 50 μL of sealing medium (50% glycerol in PBS) to each well.

[0049] (14) Observation under fluorescence microscope 2.5 Cell Counting Kit-8 (CCK-8) (1) Add 200 μL of PBS to each well around the outermost circle of the 96-well plate to prevent the liquid in the target well from evaporating.

[0050] (2) Cell counting: Concentrate the cell density to 1×10 3 Pieces / μL.

[0051] (3) Add 100 μL of different treatment solutions to each well, and pipette 10 μL (treated to the required concentration into 10 μL) of the original cell solution into each well.

[0052] (4) Place the 96-well plate in a 37°C incubator for 12 / 24 / 48 h.

[0053] (5) Add 10 μL CCK-8 reagent to each well. (Keep away from light)

[0054] (6) After incubation at 37°C for 4 hours, the cells were detected using an ELISA reader at 450 nm. (Detection within 5 minutes)

[0055] 2.6 Wright-Giemsa staining (1) Cytological smear ①Cells were cultured in a 6-well plate for 5 days, then aspirated into a 1.5 mL EP tube using a 1 mL pipette tip and centrifuged. The supernatant was discarded, 1 mL PBS was added for washing, and centrifuged.

[0056] ② Aspirate the supernatant, leaving about 20 μL of liquid in each tube. Aspirate with a 200 μL pipette tip, spread evenly in a circular motion on the center of the absorbent slide, and then place in an oven to dry.

[0057] ③ Add 4% paraformaldehyde to the slide for fixation (just cover the cells), discard after 15 min. Wash with PBS for 2 min (just cover the cells), discard. Bake in a 37℃ oven to dry.

[0058] (2) Dyeing ① Add Wright-Giemsa A solution (about 0.5 mL-0.8 mL) to the smear and allow the stain to cover the entire specimen. Stain for 1 min.

[0059] ② Add Giemsa B solution on top of solution A (the amount added is 2-3 times that of solution A), use an ear bulb to blow a breeze to create ripples on the liquid surface to fully mix the two liquids, and dye for 3-10 minutes.

[0060] ③ Wash with water (do not pour out the dye solution first when rinsing, it should be rinsed away with running water to prevent sediment from settling on the specimen).

[0061] ④ Drying and microscopic examination.

[0062] 2.7 Enzyme Linked Immunosorbent Assay (ELISA) (1) Sample preparation: Culture cells in a 6-well plate for 24 h. Then take out the cell culture dish and place it under a microscope to observe the cell status. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add different groups of treatment solutions to each well and culture again in a 37°C cell culture incubator for 5-7 days. Collect different groups of cells in a centrifuge tube, centrifuge at 1000 rpm for 5 min, and collect the supernatant.

[0063] (2) Dilute the sample and standard in a certain ratio using the sample / standard diluent (R1). Then remove the microwell strip from the plate frame.

[0064] (3) Add 350 μL of 1× washing buffer to each well, let it stand for 40 s, then discard the liquid in the well. Repeat this step three times.

[0065] (4) Add 100 μL of sample / standard diluent (R1) to the blank well.

[0066] (5) Add 100 μL of standard / sample of different concentrations to other wells, seal the wells with the provided sealing film, and incubate at 37°C for 2 h.

[0067] (6) Prepare biotinylated antibody (1×) 15 min before use (7) Discard the liquid in the wells and repeat the washing steps in step 3.

[0068] (8) Add biotinylated antibody working solution (100 μL / well) to each well, cover with a new sealing film, and incubate at 37°C for 1 h.

[0069] (9) Prepare streptavidin-HRP (1×) 15 min before use.

[0070] (10) Add streptavidin-HRP working solution (100 μL / well) to each well, cover with a new sealing film, and incubate at 37°C for 30 min.

[0071] (11) Preheat the microplate reader.

[0072] (12) Discard the liquid in the wells and repeat the washing steps in step 3.

[0073] (13) Add TMB substrate (100 μL / well) to the wells and incubate at 37°C in the dark for 15-20 min.

[0074] (14) Add stop solution (50 μL / well) and immediately place in the microplate reader for detection.

[0075] 2.8 Collection of human peripheral blood and isolation of PBMCs (1) Take 5 mL of venous blood from a healthy person and place it in a purple-capped EDTA anticoagulant tube. Dilute the whole blood with an equal volume of whole blood and tissue diluent or PBS.

[0076] (2) Take a 15 mL sterile centrifuge tube, first add 3.6 mL of reagent A, then add 2.4 mL of reagent D, so that the two form a gradient interface (the volume ratio of reagent A: reagent D is 3:2, and the total amount of reagent is equal to the volume of the diluted blood sample). Note that the stratification of the two reagents must be clear.

[0077] (3) Use a Pasteur pipette to spread the diluted blood evenly on top of the separation liquid, making sure the interface between the two liquid surfaces is clear. (Because of the density difference between the two, a clear stratified interface will be formed.)

[0078] (4) Centrifuge at room temperature in a horizontal rotor at 500-800 × g for 20-30 min.

[0079] (5) After centrifugation, obvious stratification will appear: the first layer is the plasma layer; the second layer is the white mononuclear cell layer; the third layer is the transparent reagent D liquid layer; the fourth layer is the translucent reagent A liquid layer; and the fifth layer is the red blood cell layer.

[0080] (6) Carefully transfer the second layer of white flocculent mononuclear cells to another sterile 15 mL centrifuge tube, add 10 mL of cell washing buffer or PBS, invert to mix, and centrifuge at 250 × g for 10 min.

[0081] (7) Discard the supernatant, resuspend the cells in 5 mL of cell washing buffer or PBS, and centrifuge at 250 × g for 10 min.

[0082] (8) Repeat step 7.

[0083] (9) Discard the supernatant and resuspend the cells for later use.

[0084] 2.9 Isolation of primary B lymphocytes (1) Use an appropriate volume of ImunoSep TM Resuspend PBMCs in Buffer and adjust the cell concentration to 1×10 8 / mL to prepare a single cell suspension.

[0085] (2) Place the required number of cells in a 15 mL centrifuge tube, but do not exceed 2 × 10 8 indivual.

[0086] (3) Add 20 μL of sorting reagent A to every 100 μL of cell suspension. Mix well by vortexing or pipetting 5 times with a 1 mL pipette tip, and incubate at room temperature for 10 min.

[0087] (4) Add ImunoSep to cells TM Buffer was added to 4 mL, cells were washed, and then centrifuged at 300 × g for 5 min at room temperature.

[0088] (5) Discard the supernatant and use ImunoSep TM Resuspend cells to their original volume in Buffer.

[0089] (6) Add 20 μL of sorting reagent B to every 100 μL of cell suspension, mix well using a vortexer or pipette five times with a 1 mL pipette tip, and incubate at room temperature for 5 min.

[0090] (7) Add ImunoSep TM Buffer to 2.5 mL. Mix well by pipetting 3 times with a 1 mL pipette tip. Be careful not to use a vortexer to mix well.

[0091] (8) Insert the centrifuge tube containing the cell suspension into the magnetic pole, allowing the bottom of the centrifuge tube to pass through the hole at the bottom of the magnetic pole until it touches the work surface. Let it stand at room temperature for 5 minutes.

[0092] (9) Keep the centrifuge tube in the magnet, lift the magnet and the centrifuge tube together, and quickly pour the supernatant into the 50 mL centrifuge tube. (The time should not exceed 2 seconds)

[0093] 2.10 Flow cytometry (1) The cells were cultured in a 6-well plate for 24 h, then centrifuged and the medium was replaced with different treatment solutions, and then placed back in the cell culture incubator for 5-7 days (the number of cells filled with cells in a 6-well plate is about 2×10 6 The cells of different groups were collected in centrifuge tubes, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded.

[0094] (2) Wash the cells with 1 mL washing buffer (PBS containing 0.1% BSA) and mix well using a 1 mL pipette. Then place the cells in a centrifuge at 1000 rpm for 5 min and discard the supernatant.

[0095] (3) Add Fc receptor blocker (5 μL / 10 6 Use a 1 mL pipette tip to mix the blocking agent and sample cells evenly and let stand at room temperature for 10 min.

[0096] (4) After blocking, no washing is required. Add fluorescently labeled primary antibodies (PE-CD19, FITC-CD38, APC-CD27) to each centrifuge tube according to the antibody instructions and mix well using a vortex.

[0097] (5) Place the sample in a 4°C refrigerator and incubate for 35 min.

[0098] (6) After incubation, add washing buffer (2 mL to each 15 mL centrifuge tube), centrifuge at 1000 rpm for 5 min, and discard the supernatant. Repeat the washing process 3 times.

[0099] (7) Resuspend the cells in 500 μL of washing buffer. Detect on a flow cytometer and analyze the results.

[0100] 3. Results 3.1 2F5 promotes the proliferation of PBMC and B cell lines The CCK8 method was used to detect the effect of 2F5 antibody on the proliferation of PBMC cells and different B lymphocyte cell lines. PBMC cells, OCI-LY-19, and RAMOS were treated with 2F5 supernatant or purified 2F5 antibody, respectively. The results showed that compared with the NC control group, both 2F5 antibody and IgG antibody could promote the proliferation of PBMC cells, but the ability of 2F5 antibody group to promote PBMC cell proliferation was stronger than that of IgG control group, and the optimal time point for the proliferation of 2F5 antibody was 48h ( Figure 1A). Further investigation of the effect of 2F5 antibody on the proliferation of different B lymphocyte cell lines revealed that 2F5 supernatant promoted the proliferation of OCI-LY-19 cells, and this promoting effect was independent of the components of HT culture medium. 2F5 antibody could also promote the proliferation of OCI-LY-19 cells, and the optimal time point for the proliferation of 2F5 antibody was 48h ( Figure 1 B). In addition, both 2F5 supernatant and 2F5 antibody can promote the proliferation of RAMOS cells, and the optimal time point for the proliferation of 2F5 antibody is 24h ( Figure 1 C).

[0101] 3.2 2F5 promotes B cell lineage activation The indirect immunofluorescence method was used to evaluate the effect of 2F5 purified antibody on the activation state of different B lymphocyte cell lines. OCI-LY-19 cells and RAMOS cells were treated with 2F5 purified antibody. The results showed that compared with the blank control group, both cells treated with 2F5 purified antibody and IgG antibody showed significant green fluorescence. In particular, cells treated with 2F5 purified antibody showed more specific green fluorescence signals, which were mainly distributed on the cell membrane. In contrast, the green fluorescence signal of the IgG control group was evenly distributed in the cells, and the overall fluorescence intensity of the IgG control group was lower than that of the 2F5 antibody group ( Figure 2 ). This suggests that both 2F5 purified antibody (80 μM) and IgG antibody (80 μM) can promote the expression of CD80 on the cell surface and promote the activation of B cell lines, but compared with the IgG control group, 2F5 purified antibody showed a more significant effect in promoting B cell activation. In addition, 2F5 supernatant can also promote the expression of CD80 on the membrane surface of two types of B cells, that is, promote the activation of B cell lines, and this promoting effect is unrelated to the components of HT culture medium ( Figure 3 ).

[0102] 3.3 2F5 promotes primary B cell activation The indirect immunofluorescence method was used to detect the effect of purified 2F5 antibody on the activation of primary B cells. Primary B cells were treated with purified 2F5 antibody (160 μM). The results showed that compared with the blank control group, the membrane surface of primary B cells cultured with 2F5 antibody, IgG antibody, and 1640 medium all had green fluorescence signals, but the fluorescence intensity was significantly different. Compared with the IgG control group and the NC control group, the membrane surface of B cells treated with 2F5 antibody showed more significant and specific fluorescence signals. These fluorescence signals were distributed on the cell membrane surface in the form of stars and high brightness. This phenomenon is consistent with the typical biological characteristics of the cell membrane surface activation marker protein CD80 ( Figure 4 ). This indicates that the expression of activation marker CD80 on the membrane surface of primary B cells treated with 2F5 antibody increased significantly, and cell activation was more obvious.

[0103] 3.4 2F5 promotes differentiation of PBMC, B cell lines and primary B cells Flow cytometry was used to detect the effect of 2F5 on the differentiation of PBMC cells and RAMOS cells. PBMC cells and RAMOS cells were treated with 2F5 purified antibodies. The results showed that in PBMC cells, compared with the NC control group, the CD19 + CD27 + CD38 + The proportion of B cells increased significantly, and the CD19 + CD27 + CD38 + There was no significant difference in the proportion of B cells ( Figure 5 A). This indicates that after treatment with 2F5 purified antibody, B cell differentiation was more obvious, and the proportion of plasma cells was larger. In RAMOS cells, compared with the NC control group, both the 2F5 antibody group and the IgG control group showed a significant increase in the expression of CD138, a B cell surface differentiation marker, but the expression level of the IgG control group was slightly lower than that of the 2F5 antibody group ( Figure 5 B). This suggests that both 2F5 purified antibody and IgG antibody have the ability to promote the expression of CD138 on the surface of B cells and promote B cell differentiation, but compared with IgG, 2F5 is more effective in promoting B cell differentiation; in addition, the results of Giemsa staining show that ( Figure 6 ), compared with the NC control group and the IgG control group, OCI-LY-19 cells and RAMOS cells treated with 2F5 purified antibody showed significant morphological changes: the cell volume became larger, the nucleus was darkly stained, occupying most or the entire cell and biased to one side. These characteristics are typical manifestations of the B cell differentiation process. Therefore, the significant changes in the morphology of the two B cell lines also suggest that the 2F5 antibody (80μM) may have induced the differentiation of the two B cell lines. In order to further explore the effect of 2F5 antibody on the differentiation of primary B cells, we quantitatively detected the secretion level of IgM in the culture supernatant by ELISA, and the results showed that ( Figure 7 ), compared with the NC control group, the IgM content in the cell culture supernatant of the 2F5 antibody group and the IgG control group increased, but the IgM antibody content in the cell culture supernatant of the 2F5 antibody group was significantly higher than that of the IgG group. These results confirm the important role of 2F5 in the differentiation of PBMC, B cell lines and primary B cells.

Claims

1. Use of monoclonal antibody 2F5 in the preparation of a drug for treating autoimmune diseases, characterized in that: The autoimmune disease is caused by abnormal differentiation of B cells; in the monoclonal antibody 2F5: The amino acid sequence of the complementarity determining region CDR1 of the light chain 2F5 is: QSVSSN; The amino acid sequence of the complementarity determining region CDR2 of the light chain 2F5 is: GAS; The amino acid sequence of the complementarity determining region CDR3 of the light chain 2F5 is: QQYNNWPRT; The amino acid sequence of the complementarity determining region CDR1 of the heavy chain 2F5 is: GFTFSDYS; The amino acid sequence of the complementarity determining region CDR2 of the heavy chain 2F5 is: ITSSSGYT; The amino acid sequence of the complementarity determining region CDR1 of the heavy chain 2F5 is: ARVRSSWGPIDS.

2. The use according to claim 1, characterized in that: The monoclonal antibody 2F5 promotes B cell proliferation, activation and differentiation.

3. Use of monoclonal antibody 2F5 in the preparation of a preparation for promoting B cell proliferation, activation or differentiation, characterized in that: In the monoclonal antibody 2F5: The amino acid sequence of the complementarity determining region CDR1 of the light chain 2F5 is: QSVSSN; The amino acid sequence of the complementarity determining region CDR2 of the light chain 2F5 is: GAS; The amino acid sequence of the complementarity determining region CDR3 of the light chain 2F5 is: QQYNNWPRT; The amino acid sequence of the complementarity determining region CDR1 of the heavy chain 2F5 is: GFTFSDYS; The amino acid sequence of the complementarity determining region CDR2 of the heavy chain 2F5 is: ITSSSGYT; The amino acid sequence of the complementarity determining region CDR1 of the heavy chain 2F5 is: ARVRSSWGPIDS.

4. The use according to claim 3, characterized in that: The B cells include RAMOS, OCI-LY-19 and primary B cells.

5. A method for specifically inducing B cell proliferation, activation or differentiation for non-diagnostic and non-therapeutic purposes, characterized in that: The method comprises the following steps: The purified monoclonal antibody 2F5 is diluted to prepare a 2F5 antibody diluent, and then the B cells are treated; wherein the monoclonal antibody 2F5 comprises: The amino acid sequence of the complementarity determining region CDR1 of the light chain 2F5 is: QSVSSN; The amino acid sequence of the complementarity determining region CDR2 of the light chain 2F5 is: GAS; The amino acid sequence of the complementarity determining region CDR3 of the light chain 2F5 is: QQYNNWPRT; The amino acid sequence of the complementarity determining region CDR1 of the heavy chain 2F5 is: GFTFSDYS; The amino acid sequence of the complementarity determining region CDR2 of the heavy chain 2F5 is: ITSSSGYT; The amino acid sequence of the complementarity determining region CDR1 of the heavy chain 2F5 is: ARVRSSWGPIDS.

6. The method according to claim 5, characterized in that The B cells include RAMOS, OCI-LY-19 and primary B cells.

7. A composition for treating autoimmune diseases, characterized in that: The composition includes one or more of the following ingredients: (a) Monoclonal antibody 2F5; (b) nucleic acid encoding monoclonal antibody 2F5; (c) an expression vector encoding the monoclonal antibody 2F5 nucleic acid; In the monoclonal antibody 2F5: The amino acid sequence of the complementarity determining region CDR1 of the light chain 2F5 is: QSVSSN; The amino acid sequence of the complementarity determining region CDR2 of the light chain 2F5 is: GAS; The amino acid sequence of the complementarity determining region CDR3 of the light chain 2F5 is: QQYNNWPRT; The amino acid sequence of the complementarity determining region CDR1 of the heavy chain 2F5 is: GFTFSDYS; The amino acid sequence of the complementarity determining region CDR2 of the heavy chain 2F5 is: ITSSSGYT; The amino acid sequence of the complementarity determining region CDR1 of the heavy chain 2F5 is: ARVRSSWGPIDS.

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