An antagonistic anti-cd40 antibody and methods of making and uses thereof

Abstract

The application belongs to the field of biological products, and particularly relates to an antagonistic anti-CD40 antibody and a preparation method and application thereof. The antagonistic anti-CD40 antibody is H-CD40-ab-DYS-1~3 nanobody or polyclonal antibody containing H-CD40-ab-DYS-1~3 nanobody. The antagonistic anti-CD40 antibody of the application has an effect equivalent to that of a commercially available anti-CD40L monoclonal antibody hu5c8 on the inhibition rate of B cell proliferation, and is even more superior, which provides a broad prospect for application of the antagonistic anti-CD40 antibody to autoimmune diseases and inhibition of immune rejection of organ transplantation.

Description

Technical Field

[0001] This invention belongs to the field of biological products, specifically relating to an antagonistic anti-CD40 antibody, its preparation method, and its uses. Background Technology

[0002] CD40 is a 48 kDa type I membrane glycoprotein belonging to the Tumor Necrosis Factor Receptor Superfamily (TNFRSF), widely expressed on the surface of immune cells such as B cells, dendritic cells, and macrophages, as well as non-immune cells such as endothelial cells. CD40 ligand (CD40L, CD154) is a 39 kDa type II membrane glycoprotein of the TNF family, expressed on B cells, activated T cells, and natural killer cells. CD40 and CD40L, as a pair of co-stimulatory molecules, play important roles in the regulation of cellular and humoral immunity. Studies have demonstrated that the CD40-CD40L co-stimulatory pathway has a significant bidirectional regulatory role in immune regulation. Agonistant anti-CD40 antibodies can target this pathway to promote B cell activation and proliferation, regulate T cell responses, promote the production of inflammatory cytokines, and enhance the antigen-presenting capacity of dendritic cells, making them suitable for tumor immunotherapy. Antagonistic CD40 antibodies can target this pathway to inhibit B cell activation and reduce the production of inflammatory cytokines. They can be applied to the treatment of autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome, as well as in the field of xenotransplantation immune rejection, reducing immune rejection after xenotransplantation and prolonging graft survival time.

[0003] Current domestic research on CD40 antibodies largely focuses on agonist anti-CD40 antibodies, while research on antagonist anti-CD40 antibodies is relatively limited. Existing antagonist anti-CD40 antibodies have only demonstrated their binding to the CD40 protein through ELISA experiments; however, the lack of further in vitro validation of their biological activity prevents their practical application in clinical disease treatment. Therefore, it is necessary to provide antagonist anti-CD40 antibodies with clearly defined biological activity to enrich the therapeutic options for autoimmune diseases and xenotransplantation, enabling patients to benefit from them. Summary of the Invention

[0004] To achieve the above objectives, the present invention provides an antagonistic anti-CD40 antibody, which is an H-CD40-ab-DYS-1 nanobody with an amino acid sequence as shown in SEQ ID NO. 3, an H-CD40-ab-DYS-2 nanobody with an amino acid sequence as shown in SEQ ID NO. 4, or an H-CD40-ab-DYS-3 nanobody with an amino acid sequence as shown in SEQ ID NO. 5.

[0005] The present invention also provides an antagonistic anti-CD40 antibody, which is a polyclonal antibody containing any number of nanobodies with amino acid sequences such as SEQ ID NO. 3~5.

[0006] Furthermore, the polyclonal antibody is an antibody obtained by purifying the serum of alpacas immunized with CD40 protein.

[0007] The amino acid sequence of the CD40 protein is shown in SEQ ID NO. 1.

[0008] The present invention also provides a gene fragment encoding the aforementioned antagonistic anti-CD40 antibody, which is a nucleic acid encoding H-CD40-ab-DYS-1 nanobody with a nucleotide sequence as shown in SEQ ID NO. 7, H-CD40-ab-DYS-2 nanobody with a nucleotide sequence as shown in SEQ ID NO. 8, or H-CD40-ab-DYS-3 nanobody with a nucleotide sequence as shown in SEQ ID NO. 9.

[0009] The present invention also provides a recombinant plasmid, which is a plasmid connected with a gene fragment whose nucleotide sequence is shown in any one of SEQ ID NO. 7 to 9; the plasmid includes the TY3H-D-S7 plasmid.

[0010] The present invention also provides a recombinant cell, which is an animal cell containing the aforementioned recombinant plasmid; said animal cell includes Expi293F cells.

[0011] The present invention also provides a method for preparing the aforementioned antagonistic anti-CD40 antibody, comprising the following steps:

[0012] Gene fragments with nucleotide sequences as shown in any one of SEQ ID NO.7~9 are ligated into plasmids, introduced into animal cells for protein expression, and then collected and purified.

[0013] The plasmid includes TY3H-D-S7 plasmid; the animal cells include Expi293F cells; the purification was performed using Protein At Beads affinity chromatography medium.

[0014] The present invention also provides a method for preparing the aforementioned antagonistic anti-CD40 antibody, comprising the following steps:

[0015] CD40 protein is emulsified and injected into alpacas. Alpaca serum is then collected, purified, and the final product is obtained.

[0016] The emulsification was performed sequentially with Freund's complete adjuvant and Freund's incomplete adjuvant.

[0017] The injection dose is 1 mg of CD40 protein once every 2 weeks, for a total of 5 times;

[0018] The purification was performed using CD40 protein-coupled chromatography medium.

[0019] The amino acid sequence of the CD40 protein is shown in SEQ ID NO. 1.

[0020] The present invention also provides the use of the aforementioned antagonistic anti-CD40 antibody in the preparation of a drug for inhibiting B cell proliferation.

[0021] Finally, this invention provides a drug for inhibiting B cell proliferation, wherein the drug has the aforementioned antagonistic anti-CD40 antibody as its active ingredient.

[0022] This invention relates to an antagonistic anti-CD40 antibody, which is a polyclonal antibody obtained by eukaryotic expression of human CD40 CDs sequence after substitution and optimization, resulting in a CD40 with intact antigenic immunogenic structure and function. This CD40 is then used to immunize alpacas, and the antibody is purified from the alpaca serum. This alpaca-derived anti-CD40 polyclonal antibody is comparable in its effectiveness in inhibiting B cell proliferation to the commercially available anti-CD40L monoclonal antibody hu5c8. This invention, based on anti-CD40 polyclonal antibodies, utilizes CD40 antigen to immunize alpaca PBMCs and constructs a phage library via nested PCR. Four specific nanobodies were discovered from this library. These nanobodies exhibit excellent inhibitory effects on B cell proliferation. H-CD40-ab-DYS-1~3 show comparable inhibition of human and monkey B cell proliferation to the commercially available anti-CD40L monoclonal antibody hu5c8. H-CD40-ab-DYS-1 and H-CD40-ab-DYS-3 are even slightly more effective than the commercially available anti-CD40L monoclonal antibody hu5c8 at certain concentrations. The application of anti-CD40 polyclonal antibodies and the three anti-CD40 nanobodies in autoimmune diseases and the suppression of organ transplant immune rejection shows broad application prospects.

[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0025] Figure 1Figure 1. Partial sequencing results of CD40 recombinant plasmid and comparison results with optimized CD40 sequence.

[0026] Figure 2 SDS-PAGE electrophoresis results and WB results

[0027] Figure 3 Antibody concentration-OD 450 nm value concentration curve;

[0028] Figure 4 Serum antibody concentration results graph;

[0029] Figure 5 Image of SDS-PAGE electrophoresis results of size-specific bands of alpaca heavy chains;

[0030] Figure 6 Figure showing the inhibition of B cell proliferation rate by polyclonal antibodies;

[0031] Figure 7 RNA electrophoresis image;

[0032] Figure 8 First round of nested PCR electrophoresis image;

[0033] Figure 9 PCR electrophoresis image of VHH fragment;

[0034] Figure 10 Antibody library electrophoresis image;

[0035] Figure 11 Figure of amino acid sequencing results of antibody library;

[0036] Figure 12 Image of positive clone antibody ELISA results at OD 450 nm;

[0037] Figure 13 Spatial structure diagram of anti-CD40 nanobody;

[0038] Figure 14 TY3H-D-S7 plasmid map;

[0039] Figure 15 Image of SDS-PAGE electrophoresis results of anti-CD40 nanobody;

[0040] Figure 16 Figure showing the inhibition of B cell proliferation rate by anti-CD40 nanobody. Detailed Implementation

[0041] Example 1: Preparation and efficacy study of antagonistic anti-CD40 nanobodies

[0042] 1. Optimized synthesis of CD40 CDs sequences

[0043] The human CD40 CDs sequence >KAI4005855.1 CD40 molecule [Homo sapiens] in NCBI was analyzed to determine its transmembrane region, signal peptide, and functional region. Then, a design was developed to replace the signal peptide and transmembrane region with a mouse-derived signal peptide and a flexible linker, respectively, optimizing the nucleotide sequence encoding the functional region to eliminate AvrII, Bstz17I, HindIII, EcoRI, NruI, NheI, XhoI, and PacI restriction sites. The optimized CD40 CDs sequence is as follows:

[0044] H-CD40-CDs amino acid sequence (SEQ ID NO. 1):

[0045] MLRGPPGGLLLAVLCLGTAVRCTEAEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIAT GVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRGSGGGSGGGSVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQGSGSGHHHHHHHHHHH

[0046] H-CD40-CDs nucleotide sequence (SEQ ID NO. 2):

[0047] ATGCTACGAGGTCCAGGTCCAGGTCTACTACTGGCTGTGCTGTGCCTGGGCACCGCTGTTAGATGTACAGAGGCCGAGCCTCCTACCGCCTGCAGAGAGAAACAGTACCTGATCAACTCCCAGTGCTGCTCCCTGTGTCAGCCTGGCCAGAAGCTGGTGTCCGACTGCACCGAGTTCACCGAGACCGAGTGCCTCCCATGTGGCGAGTCCGAGTTCCTGGACACTTGGAACCGGGAAACCCACTGCCACCAGCACAAGTACTGCGACCCTAACCTGGGCCTGAGAGTGCAACAGAAGGGCACATCCGAGACAGATACCATCTGCACATGTGAGGAAGGCTGGCACTGTACCTCTGAGGCCTGCGAGTCTTGCGTGCTGCACAGATCTTGTTCTCCCGGCTTTGGCGTGAAGCAGATCGCCACCGGAGTGTCTGATACCATCTGCGAGCCCTGCCCCGTCGGCTTCTTCTCCAACGTGTCCAGCGCCTTCGAGAAGTGCCATCCTTGGACCTCCTGCGAAACCAAGGACCTGGTCGTGCAGCAGGCTGGCACCAACAAGACCGACGTGGTGTGCGGACCTCAGGACAGACTGCGGGGCTCTGGCGGTGGCAGCGGAGGCGGCTCTGTGGCCAAGAAGCCTACCAATAAGGCCCCTCACCCTAAGCAGGAACCACAAGAGATCAACTTCCCTGACGATCTGCCTGGCAGCAACACCGCCGCTCCTGTGCAAGAAACCCTGCACGGCTGCCAGCCCGTGACCCAGGAGGACGGCAAAGAATCCCGGATCTCCGTGCAGGAGCGCCAGGGCAGTGGATCTGGCCATCACCATCATCACCACCACCACCACCAC

[0048] 2. Eukaryotic recombinant expression of CD40 CDs sequence

[0049] The optimized CD40 CDs sequence, i.e., the H-CD40-CDs nucleotide sequence, was digested and recombined into the expression vector plasmid pCDNA3.1 (purchased from Sangon Biotech Co., Ltd.) and sequenced. The results of the forward and reverse sequencing splicing of the plasmid are shown below. Figure 1 The sequence at positions 450-580 indicates that the recombinant plasmid sequencing results are of good quality. Figure 1 The comparison between the sequence at positions 450-520 and the optimized CD40 sequence indicates that the recombinant plasmid has been successfully constructed. Figure 1 ).

[0050] The successfully constructed recombinant plasmid was transfected into Chinese hamster ovary cells (CHO) for recombinant expression. The histidine (His)-tagged target protein was purified using nickel affinity chromatography. After elution, the absorbance of the target protein at 280 nm was measured, and its concentration was calculated to be approximately 2.1 mg / mL using the protein extinction coefficient. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (WB) results confirmed the successful extraction of CD40 protein. Figure 2 ).

[0051] 3. Alpaca immunization and antibody concentration detection

[0052] (1) Emulsify 1 mg of CD40 protein sequentially with Freund's complete / incomplete adjuvant in equal volumes, and then immunize healthy alpacas without immunization records with the emulsified CD40 protein antigen (inject the emulsified antigen containing 0.5 mg of CD40 protein into the left and right cervical lymph nodes, once every 2 weeks, for a total of 5 immunizations).

[0053] (2) The change in antibody titer after immunization of alpacas was detected using enzyme-linked immunosorbent assay (ELISA). The specific method is as follows:

[0054] ① Plot the antibody concentration-OD 450 nm value concentration curve.

[0055] Take 100 μL of 2x10 3Coat an ELISA plate with ng / mL CD40 protein and incubate overnight at 4°C. Add 4% skim milk solution and incubate at 37°C for 60 min. After washing, add CD40 monoclonal antibody 2C10R4 (10.06 mg / mL) diluted in the following proportions (1:2kJ, 1:4kJ, 1:8kJ, 1:16kJ, 1:32kJ, 1:64kJ, 1:128kJ, 1:256kJ), and incubate at 37°C for 30 min. Add 100 μL of Goat anti-LLama IgG horseradish peroxidase (HRP) antibody (1:10000), and incubate at 37°C for 30 min. After washing, add 50 μL of 2 M sulfuric acid solution to terminate the reaction. Quickly read the value at OD450 nm using an ELISA reader. Plot an antibody concentration-OD450 nm value concentration curve based on the experimental results. (See attached image). Figure 3 .

[0056] ② Calculate the concentration of anti-CD40 antibodies in the serum of alpacas after immunization.

[0057] Take 100 μL of 2x10 3 Coat an ELISA plate with ng / mL CD40 protein and incubate overnight at 4°C. Add 4% skim milk powder solution and incubate at 37°C for 60 min. After washing, add pre-immunization alpaca serum and serum from days 7 after each immunization (D0, D7, D21, D35, D49, D63) (dilution ratios of 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000), and incubate at 37°C for 30 min. Add 100 μL of Goat anti-LLama IgG (HRP) antibody (1:10000), and incubate at 37°C for 30 min. After washing, add 50 μL of 2 M sulfuric acid solution to terminate the reaction. Quickly read the value at OD450 nm using an ELISA reader. Calculate the serum antibody concentration using an antibody concentration-OD450 nm value concentration curve. Results are shown in [Figure missing]. Figure 4 .

[0058] from Figure 4 The experimental results show that as the number of immunizations increases, the serum antibody titer of alpaca-derived anti-CD40 gradually increases, and the serum antibody concentration can reach 53 mg / mL after 5 immunizations (the dilution ratio is calculated to be 1:128000).

[0059] 4. Polyclonal antibody purification

[0060] After five immunizations, 100 mL of blood was collected from alpacas, and serum was separated. The serum was purified by gravity using CD40 protein-conjugated chromatography medium to obtain alpaca-derived anti-CD40 polyclonal antibodies. The SDS-PAGE electrophoresis results of the CD40 polyclonal antibody are shown below. Figure 5The alpaca heavy chain showed a size-specific band, and the antibody concentration was determined to be 2.2 mg / mL by ultraviolet spectrophotometer.

[0061] 5. Evaluation of the effectiveness of polyclonal antibodies

[0062] Peripheral blood mononuclear cells (PBMCs) were collected from rhesus monkeys and isolated. The PBMCs were labeled using the eBioscience™ Carboxyfluorescein Diacetate Succinimide Ester (CFSE) Cell Proliferation Kit, and the concentration was adjusted to 2 × 10⁻⁶. 6 / mL; successfully resuscitated mouse embryonic fibroblasts (3T3) cells transfected with CD40L (provided by the Organ Transplantation Institute of Tongji Hospital) and adjusted to a concentration of 2×10⁻⁶. 5 / mL; take 100 μL of each and add it to a 24-well plate (PBMC: 2×10⁻⁶). 5 / well, 3T3 cells: 2×10 4 ( / well), add pre-cooled culture medium to make up to 1 mL of culture system, the culture medium system is shown in Table 1.

[0063] Table 1 Culture medium system

[0064]

[0065] The wells of the plates were divided into experimental and positive control groups. The experimental and positive control groups were incubated for 8 days with purified polyclonal antibody and anti-CD40L monoclonal antibody hu5c8 (provided by the Organ Transplantation Institute of Tongji Hospital), respectively (the drug concentrations for each group were 0 μg / mL, 0.78 μg / mL, 3.125 μg / mL, 12.5 μg / mL, and 50 μg / mL, respectively). B cells were labeled with CD19 fluorescent antibody (CD19 PoLycLonaL Antibody), and the B cell proliferation rate was detected by BD FACSCeLesta flow cytometry. The rank-sum test was used to compare the differences between the experimental and positive control groups.

[0066] result( Figure 6 The results showed that there was no statistically significant difference in B cell proliferation rate between alpaca-derived anti-CD40 polyclonal antibody and anti-CD40L monoclonal antibody hu5c8 at different drug concentrations (rank-sum test).

[0067] 6. Phage Library Construction

[0068] 6.1 Extraction of total RNA

[0069] Peripheral blood cells (PBMCs) were isolated from alpacas after immunization with CD40 protein emulsion antigen. Total RNA was extracted using the TrizoL method, and the results were analyzed by 1% agarose gel electrophoresis. Figure 7 This indicates that the total RNA integrity is good.

[0070] 6.2 Reverse transcription of cDNA

[0071] Prepare the following reaction mixture in a Microtube: 1 μL OLigo dT Primer (50 μM), 4 μL dNTP Mixture (2.5 mM each), 5 μg total RNA, and RNase-free ddH2O to a final volume of 10 μL. Incubate at 65°C for 5 min, then rapidly cool on ice. Prepare the following reverse transcription reaction mixture in a Microtube tube: 10 μL of the above denaturing reaction mixture, 4 μL 5×PrimeScript II Buffer, 0.5 μL RNase Inhibitor (20 U), 1 μL PrimeScript II RTase (200 U), and RNase-free ddH2O to a final volume of 20 μL to obtain cDNA.

[0072] 6.3 Nested PCR for obtaining nanobodies (VHH)

[0073] Biosynthesized nested PCR primers 1 [FP (SEQ ID NO. 11): 5'-GAGCTGGGTGGTCCTGGCTGCTC-3', RP (SEQ ID NO. 12): 5'-GGTACGTGCTGTTGAACTGTTCC-3'] and 2 [FP (SEQ ID NO. 13): 5'-GCTACCGTGGCCCAGGCGGCCSAGGTGCAGSTSGTGGAGTCTG-3', RP (SEQ ID NO. 14): 5'-ATGGTGCTGGCCGGCCTGGCCTGAGGACACGGTGCCCAGGTG-3'] were used for the first round of PCR amplification of VHH using primer 1. The 600bp target fragment was recovered by 2% gel electrophoresis. Figure 8 The recovered target fragment was amplified by PCR in the second round using primer 2. The recovered VHH fragment (approximately 360 bp) was detected by 2% gel electrophoresis. Figure 9 ).

[0074] 6.4 Phage Library Construction and Size Calculation

[0075] (1) Pcomb3XSS phage was used as the vector (purchased from Changsha Aibiwei Biotechnology Co., Ltd.). After digestion of the vector and VHH fragment, the fragment was recovered and ligated with T4 DNA ligase to obtain the vector overnight (vector:VHH molar ratio = 1:3). Approximately 2 μg of the ligation product was electroporated into TG1 competent cells, and the cells were incubated in 2YT medium at 37℃ for 60 min. 100 μL of the medium was then serially diluted to 10⁻⁶.-4 and 10 -5 Then, 20 μL was spread on LB medium and incubated overnight to determine the library capacity. The result was 3.18 × 10⁻⁶. 9 pfu.

[0076] (2) Moisten the plate with 10 mL of 2YT medium, scrape and collect the bacterial growth into a closed centrifuge tube, wash it once with 3 mL of LB medium, add sterile glycerol to make a final concentration of 30%, and measure the glycerol bacterial OD. 600 When the value is 1, record the number of units. Collect the bacterial culture and freeze it at -80°C to obtain the original bacterial library.

[0077] (3) Take 10 μL of phage library and serially dilute it to 10 μL using 2 YT medium. -10 Take 10 μL and add it to 200 μL of TG1 bacterial culture (OD). 600 >1) Mix gently. Incubate at 37°C for 30 min, then spread onto Amp resistance plates and incubate overnight at 37°C. Calculate the titer based on the number of single colonies on the Amp resistance plates. Tit determination result: 1.21 × 10⁻⁶ 13 pfu / mL.

[0078] (4) Spread the remaining bacterial culture evenly on Amp-resistant plates and incubate overnight. Randomly select 24 single colonies from the plates for PCR amplification. 2% agarose gel electrophoresis results ( Figure 10 ) 24 target bands, each around 360bp in size, were observed, with an insertion rate of 100% and a qualified antibody library positivity rate.

[0079] 6.5 Amino acid sequencing of positive clones

[0080] Twenty-four positive bacterial cultures were sent for sequencing, yielding 23 sequences. These sequences were translated into amino acid sequences and compared. The results are shown below. Figure 11 The results showed that most of the 23 sequences were identical and there were no repetitive sequences, indicating good library diversity.

[0081] 7. Screening of phage library-specific antibodies

[0082] 7.1 Liquid phase-affinity screening

[0083] (1) Blocking the phage library: 1×10 12 The phage library was added to the blocking solution, placed in a 1.5 mL tube, and incubated at 37°C. The tube was then rotated at 20 rpm for 1 hour.

[0084] (2) Blocking magnetic beads: Take two 1.5 mL tubes. Add 80 μL of avidin-coated magnetic beads to the "negative pan" and 60 μL of avidin-coated magnetic beads to the "positive pan". Use these as "negative pan" and "positive pan" respectively. Remove the protective solution, wash twice with 0.5‰ PBST, and add 1 mL of blocking solution and mix well. Place the tubes on a magnetic bead separator for 1 min to adsorb the magnetic beads, remove the supernatant, add 1 mL of blocking solution, invert and mix well, place the tubes on a rotary mixer, and incubate at 37°C for 20 rpm for 1 h.

[0085] (3) Preparation of positive selection antigen: Aspirate the supernatant from the "positive selection" tube, wash once with 0.5‰ PBST, add 1 mL of biotinylated antigen, and place on a rotary mixer to incubate at 37°C at 20 rpm for 1 h. (The amount of antigen used is 1-2 times the capacity of the magnetic beads.)

[0086] (4) Negative panning: Remove the supernatant from the negative panning tube, wash with 0.5‰ PBST, add the blocked phage library to the negative panning tube, mix thoroughly by inverting, place on a rotary mixer, and incubate at 37℃ for 20 rpm for 1 h. Place on a magnetic bead separator for 1 min to adsorb magnetic beads, transfer the supernatant to a new EP tube, which is the blocked and negative panned phage library.

[0087] (5) Antigen-antibody binding: Discard the liquid in the "positive panning" tube, wash with 0.5‰ PBST, add the blocked and negative panning phage library, mix thoroughly by inverting, place on a rotary mixer, and incubate at 37°C for 1 hour at 20 rpm.

[0088] (6) Washing: Place the "positive selection" tube on the magnetic bead separator for 1 min to adsorb the magnetic beads, remove the supernatant, add PBST, mix well and let stand for 10 s, then place the tube on the magnetic bead separator for 1 min to adsorb the magnetic beads, remove the supernatant. Repeat this step 8-10 times.

[0089] (7) Elution and collection of phages: Place the "positive panning" tube on the magnetic bead separator for 1 min to adsorb the magnetic beads, aspirate the supernatant, add 600 μL of elution buffer (glycine hydrochloride, pH 2.2), invert to mix thoroughly, place on a rotary mixer, and incubate at 37°C for 20 rpm for 10 min. Then place the "positive panning" EP tube on the magnetic bead separator for 1 min to adsorb the magnetic beads, aspirate the supernatant and add it to a new 1.5 mL EP tube, and immediately add 200 μL of neutralization buffer (1 mM Tris hydrochloride, pH 8.0). After filtering the solution through a 0.22 μm sterile filter membrane, place the sample on ice.

[0090] 7.2 Amplification of R1 elution products

[0091] (1) Pick a single TG1 clone from the plate, add it to 20 mL of 2YT, and incubate at 37℃ and 200 rpm until the logarithmic phase OD600 = 0.6-0.8.

[0092] (2) Add the eluted antibody library and mix well. Incubate at 37°C for 60 min.

[0093] (3) Add 4uL / Amp and incubate at 37℃ and 180rpm for 60min.

[0094] (4) Add M13K07 (helper cells TG1>=20:1) to the culture, mix well, and let stand at 37℃ for 30 min.

[0095] (5) Add 30 mL of 2YT and 6 μL of Amp, and incubate at 37°C and 180 rpm.

[0096] (6) Centrifuge at 5000 rpm for 10 min at room temperature, remove the supernatant, collect the bacterial pellet, add 50 mL of 2YTAK (Amp: 100 ug / mL, Kana: 50 ug / mL) to resuspend the pellet, and incubate overnight at 30°C and 220 rpm in a 200 mL Erlenmeyer flask.

[0097] 7.3 Recovery of R1 amplified library precipitation

[0098] (1) Collect all bacterial culture and centrifuge at 12,000 rpm at 4℃; collect the supernatant and add 1 / 4 volume of PEG-4000NaCL solution and mix thoroughly.

[0099] (2) Incubate at 4℃ in an ice-water bath for more than 3 hours, then centrifuge at 12000 rpm at 4℃ for 15 minutes, and discard the supernatant. At this time, the R1 amplification library will precipitate at the bottom of the centrifuge tube. Resuspend in 1 mL of PBS and centrifuge at 12000 rpm for 5 minutes. Take the supernatant as the concentrated library, add 500 uL of 50% volume, and store at -80℃ for a long time.

[0100] 7.4 R2 liquid phase screening

[0101] Take the R1 screening amplification library and follow the R1 screening protocol; increase the library cleaning intensity for R2 screening and detect the titer.

[0102] 7.5 Monoclonal Elisa

[0103] (1) Randomly pick 96 single clones from the plate for screening and titer determination into a 96-well deep plate, add 300uL of 2YT medium at 180rpm, incubate at 37℃ to the logarithmic phase, add M13K07 helper phage for infection, change the medium, and incubate overnight at 30℃.

[0104] (2) Dilute the target protein to a concentration of 0.1 ug / mL with PBS, then add 100 uL / well to a 96-well SA-ELISA plate and incubate at 37°C for 1 h.

[0105] (3) Discard the coating solution in the ELISA plate, pat dry on sterile absorbent paper, wash 3 times with PBS for 60s each time, then add 300uL of blocking solution and incubate at 37℃ for 1h. Discard the blocking solution, pat dry on sterile absorbent paper, wash 2 times with PBS for 60s each time, and add the centrifuged monoclonal supernatant and 1% skim milk powder 1:1 to the corresponding ELISA wells, 100uL / well, incubate at 37℃ for 1h. Discard the supernatant, wash 5 times with 0.1% PBST for 60s each time, add M13 secondary antibody (1% skim milk powder diluted 1:5000), 100uL / well, and incubate at 37℃ for 1h. Discard the secondary antibody, wash 5 times with 0.1% PBST, and once with PBS, 60 s each time. Add 100 μL TMB to each well, incubate in the dark for 8 min, and stop the reaction by adding 50 μL 2M dilute sulfuric acid. Quickly read the OD450 value using a microplate reader. Determine the positive wells based on the P / N value (>2.1). See the results below. Figure 12 .

[0106] Eighty-three positive phage clones obtained from the ELISA experiment were sequenced and analyzed, ultimately yielding 20 specific nanobody sequences. Preliminary experiments indicated that four of these specific nanobodies exhibited good inhibitory effects on B cell proliferation. Therefore, these four specific nanobodies, H-CD40-ab-DYS-1~4, were further evaluated for their bioefficacy.

[0107] The spatial structure of the specific nanobody H-CD40-ab-DYS-1~4 is shown in [reference needed]. Figure 13 The amino acid sequence is as follows:

[0108] H-CD40-ab-DYS-1 (SEQ ID NO.3):

[0109] QVQLVESGGGLAQPGGSLRLSCAASGFTFSDYAMSWVRQGPGKGLEWVSSISEPGGTINYADSVKGRFTISRDNAKNTLYLRMNSLKPEDTAVYYCAKDPYYRSSYYDLEGSQQGQGTQVTVSS

[0110] H-CD40-ab-DYS-2 (SEQ ID NO.4):

[0111] QVQLVESGGGLVQPGGSLRLSCAVSGFTFSGYAMSWVRQAPGKGLEWVSSISERGGTKDYADSVKGRFTISRDNAKNTLHLQMNSLKPEDTAVYYCVKDPYYKATYYTISEGPGGGMQLVRGQGTQVTVSS

[0112] H-CD40-ab-DYS-3 (SEQ ID NO.5):

[0113] EVQLVESGGGLVKPGSSLRLSCVASGFTFTDSAMSWVRQGPGKGLEWISSISEPGGLKNYADSVKGRFTISRDIAQNTLYLQMNSLRPEDTAVYYCAKDPYYRASYYNLEGSQRGQGTQVIVSS

[0114] H-CD40-ab-DYS-4 (SEQ ID NO.6):

[0115] EVQVVESGGGLVQPGSSLRLSCATSGFTFADSAMSWVRQGPGKGLEWVSSISEPGGIKNYADSVKGRFTIARDNARDTLYLHMNTLIPEDTAVYYCAKDPYYRASYYDLEGSQRGQGTQVTVSS

[0116] 8. Evaluation of the effectiveness of specific nanobodies

[0117] 8.1 Gene Synthesis and Plasmid Extraction

[0118] The gene encoding the H-CD40-ab-DYS-1~4 nanoantibody was synthesized at Changzhou Hongmeng Biotechnology Co., Ltd., and the above gene was recombinated and ligated into the TY3H-D-S7 plasmid (plasmid map shown). Figure 14 The bacterial culture transfected with the recombinant plasmid was inoculated into 80 mL of LB medium at a ratio of 1:400 and cultured overnight at 37°C and 180 rpm; the plasmid was extracted using the Plasmid Purification MaxiPrep kit.

[0119] The nucleotide sequence of the gene encoding the H-CD40-ab-DYS-1 nanobody (SEQ ID NO. 7):

[0120] CAGGTTCAGCTGGTGGAGAGCGGAGGAGGACTGGCTCAACCAGGAGGAAGCCTGAGACTGTCCTGCGCTGCTTCCGGATTCACATTCAGCGACTACGCCATGAGCTGGGTGAGACAGGGCCCTGGAAAGGGACTGGAGTGGGTTTCCTCCATCAGCGAGCCCGGAGGAACCATCAATTACGCCGATTCCGTGAAGGGCAGATTCACAATCTCCAGGGACAACGCCAAGAACACCCTGTACCTGAGAATGAACTCCCTGAAGCCTGAGGACACCGCCGTGTACTACTGCGCTAAGGACCCTTACTACAGGTCCAGCTACTACGATCTGGAGGGCAGCCAGCAGGGCCAAGGAACCCAGGTGACAGTGTCCAGC;

[0121] Nucleotide sequence of the gene encoding H-CD40-ab-DYS-2 nanobody (SEQ ID NO. 8):

[0122] CAGGTTCAGCTGGTGGAGAGCGGAGGAGGACTGGTTCAGCCAGGAGGAAGCCTGAGGCTGAGCTGTGCTGTGAGCGGATTCACCTTTAGCGGCTACGCCATGAGCTGGGTGAGGCAAGCTCCTGGAAAGGGCCTGGAGTGGGTGTCTAGCATCTCCGAGAGGGGCGGAACCAAGGACTACGCTGATTCCGTGAAGGGCAGGTTCACAATCTCCAGAGACAATGCCAAGAATACCCTGCACCTGCAGATGAACTCCCTGAAGCCCGAGGACACCGCCGTTTACTACTGCGTGAAGGACCCCTACTACAAGGCCACCTACTACACAATCTCCGAGGGCCCCGGCGGAGGAATGCAGCTGGTGAGGGGACAAGGCACCCAGGTTACCGTGAGCTCC;

[0123] Nucleotide sequence of the gene encoding H-CD40-ab-DYS-3 nanobody (SEQ ID NO. 9):

[0124] GAGGTTCAGCTGGTGGAGTCCGGAGGAGGACTGGTTAAGCCCGGAAGCAGCCTGAGGCTGTCCTGTGTTGCCTCCGGATTCACCTTCACAGACAGCGCCATGAGCTGGGTGAGGCAGGGACCTGGAAAGGGACTGGAGTGGATCTCCAGCATCTCCGAGCCCGGAGGACTGAAGAATTACGCCGATTCCGTGAAGGGCAGGTTCACAATCAGCAGAGACATCGCCCAGAACACCCTGTACCTGCAGATGAATAGCCTGAGACCTGAGGATACAGCCGTGTACTACTGCGCCAAGGACCCCTACTACAGAGCCAGCTACTACAATCTGGAGGGCAGCCAGAGGGGCCAGGGAACCCAGGTCATTGTGTCCTCC;

[0125] Nucleotide sequence of the gene encoding H-CD40-ab-DYS-4 nanobody (SEQ ID NO. 10):

[0126] GAGGTTCAGGTGGTGGAGAGCGGAGGAGGACTGGTTCAGCCTGGAAGCAGCCTGAGACTGTCCTGTGCCACCAGCGGATTCACTTTCGCCGATAGCGCCATGTCCTGGGTGAGGCAAGGCCCTGGAAAGGGACTGGAGTGGGTGTCTAGCATCAGCGAGCCTGGCGGAATCAAGAACTACGCCGATAGCGTGAAGGGCAGGTTTACAATCGCCAGAGACAATGCCAGGGACACCCTGTACCTGCACATGAATACCCTGATCCCTGAGGATACCGCCGTGTACTACTGCGCCAAGGACCCTTACTACAGAGCCAGCTACTACGACCTGGAGGGCAGCCAAAGGGGCCAAGGAACACAGGTGACAGTGTCCAGC;

[0127] 8.2 Cell culture and expression

[0128] 8.2.1 Resuscitation and subculture of Expi293F cells

[0129] (1) Preheat the medium at 37 °C for half an hour in advance.

[0130] (2) Take the frozen Expi293F cells out of the liquid nitrogen tank and immediately put them into a 42°C water bath and shake them gently to thaw them quickly.

[0131] (3) Wipe the outer wall with a 75% alcohol cotton ball, place it in the biosafety cabinet, transfer the cells to a 1.5 mL centrifuge tube, and centrifuge at 800×g for 3 min.

[0132] (4) After centrifugation, remove the supernatant containing DMSO from the cells, take 500 μL of fresh culture medium to resuspend the cells, and then transfer them to a culture flask pre-filled with 30 mL of fresh culture medium for culture at 125 rpm, 37°C, and 5% CO2.

[0133] (5) Culture cells for 2-3 days until the density reaches 3.0 × 10⁻⁶. 6 The cells were passaged when the volume was around 1 mL.

[0134] (6) The cell passage density is generally 0.5 × 10⁻⁶. 6 / mL, cells do not need to be centrifuged during passage; simply add them directly to fresh culture medium for dilution.

[0135] 8.2.2 Cell transfection

[0136] Plasmid transfection was performed using PEI max transfection reagent: 100 mL (concentration 1 mg / mL, stored at 4℃, filtered through a 0.22 μm filter membrane before use).

[0137] (1) One day before transfection, administer 1-1.5×10 6 Cells were seeded at 37°C, 125 rpm, and 5% CO2.

[0138] (2) Perform cell counting before transfection, with the cell count reaching 2.5-3 × 10⁻⁶. 6 Transfection is possible when the viability is ≥96% per mL.

[0139] (3) Add 0.3 mL of PEI max to 5 mL of serum-free culture medium, mix by inverting the container, and let stand at room temperature for 5 min.

[0140] (4) Add 100 μg of plasmid to 5 mL of serum-free culture medium and mix by inverting. Let stand at room temperature for 5 min.

[0141] (5) Add the PEI max mixture to the plasmid mixture, let it stand at room temperature for 10 min, and then add it to 100 mL of cells to complete the transfection;

[0142] (6) Add 5 mL of feed solution 20 h after transfection;

[0143] (7) After 5 days of expression, centrifuge at 6000 rpm for 6 min and collect the supernatant for purification.

[0144] 8.3 Protein purification and identification

[0145] (1) After centrifuging the cells, discard the precipitate and take the supernatant. The culture medium supernatant is directly purified with Protein At Beads. The packing material is placed in a gravity column tube.

[0146] (2) The column packing volume is determined according to the culture volume. Pack 1.5 ml of Protein At Beads into the column for every 100 ml of supernatant.

[0147] (3) Equilibrate with 10 column volumes of equilibration buffer (1xPBS, pH=7.4). Do not control the equilibration rate. The equilibration buffer washes the packing material by gravity.

[0148] (4) After equilibration, load the sample at a flow rate of approximately 1 ml / min;

[0149] (5) After the sample loading is completed, rinse again with 20 column volumes of equilibration solution. The same equilibration process is not controlled in speed, and the packing is rinsed by gravity.

[0150] (6) Collect the eluent in separate tubes during elution. The eluent is 0.1M glycine at pH 3.0. Collect one column volume in each tube, and collect a total of 5 tubes.

[0151] (7) SDS-PAGE electrophoresis was used to detect purity and concentration. Figure 15 Endotoxins are controlled throughout the protein purification process;

[0152] (8) The sample was dialyzed overnight into 0.01M PBS (pH=7.4) solution to obtain specific nanobodies H-CD40-ab-DYS-1~4.

[0153] 8.4 Validity Evaluation

[0154] The efficacy of the specific nanobody H-CD40-ab-DYS-1~4 was evaluated using human B cells and monkey B cells, following the methods described in the aforementioned "Evaluation of Polyclonal Antibody Efficacy" section. Results of the in vitro B cell proliferation inhibition assay are shown below. Figure 16As shown in the figure, the four anti-CD40 nanobodies can bind to the CD40 protein on human B cells and monkey B cells, and exhibit good inhibitory effects on B cell proliferation. Their inhibitory effect on B cell proliferation is similar to that of the positive control anti-CD40L monoclonal antibody hu5c8. In particular, H-CD40-ab-DYS-4 is slightly superior to antibody hu5c8 in inhibiting the proliferation of both human and monkey B cells. This indicates that the above four anti-CD40 nanobodies, especially H-CD40-ab-DYS-4, have broad application prospects in autoimmune diseases and in suppressing immune rejection in organ transplantation.

[0155] In summary, this invention, using CD40 as an antigen to immunize alpacas, yielded polyclonal antibodies isolated and purified from alpaca serum that, compared to the commercially available anti-CD40L monoclonal antibody hu5c8, showed comparable efficacy in inhibiting B cell proliferation. Based on the anti-CD40 polyclonal antibody, four specific nanobodies, H-CD40-ab-DYS-1~4, were also discovered, exhibiting excellent inhibitory effects on B cell proliferation. Among them, H-CD40-ab-DYS-1~3 were comparable to the commercially available anti-CD40L monoclonal antibody hu5c8 in inhibiting the proliferation of human and monkey B cells, with H-CD40-ab-DYS-1 and H-CD40-ab-DYS-3 showing slightly better efficacy than the commercially available anti-CD40L monoclonal antibody hu5c8 at certain concentrations. The application of anti-CD40 polyclonal antibodies and the three anti-CD40 nanobodies in autoimmune diseases and the inhibition of organ transplant immune rejection shows broad application prospects.

Claims

1. An antagonistic anti-CD40 antibody, characterized in that: It is the H-CD40-ab-DYS-1 nanobody with the amino acid sequence shown in SEQ ID NO.3, the H-CD40-ab-DYS-2 nanobody with the amino acid sequence shown in SEQ ID NO.4, or the H-CD40-ab-DYS-3 nanobody with the amino acid sequence shown in SEQ ID NO.

5.

2. The gene fragment encoding the antagonistic anti-CD40 antibody of claim 1, characterized in that: It is a nucleic acid encoding H-CD40-ab-DYS-1 nanobody with a nucleotide sequence as shown in SEQ ID NO. 7, a nucleic acid encoding H-CD40-ab-DYS-2 nanobody with a nucleotide sequence as shown in SEQ ID NO. 8, or a nucleic acid encoding H-CD40-ab-DYS-3 nanobody with a nucleotide sequence as shown in SEQ ID NO.

9.

3. A recombinant plasmid, characterized in that: It is a plasmid that links gene fragments with nucleotide sequences as shown in any one of SEQ ID NO. 7 to 9.

4. A recombinant cell, characterized in that: It is an animal cell containing the recombinant plasmid of claim 3; the animal cell is selected from Expi293F cells.

5. A method for preparing the antagonistic anti-CD40 antibody of claim 1, characterized in that: Includes the following steps: Gene fragments with nucleotide sequences as shown in any one of SEQ ID NO.7~9 are ligated into plasmids, then introduced into eukaryotic cells for protein expression, and collected and purified to obtain the final product. The eukaryotic cells were selected from Expi293F cells; the purification was performed using Protein At Beads affinity chromatography medium.

6. The use of the antagonistic anti-CD40 antibody of claim 1 in the preparation of a medicament for treating autoimmune diseases or inhibiting immune rejection in organ transplantation, characterized in that: The autoimmune diseases mentioned are systemic lupus erythematosus, rheumatoid arthritis, or Sjögren's syndrome.

7. A drug for inhibiting B cell proliferation, characterized in that: The drug uses the antagonistic anti-CD40 antibody as the active ingredient as described in claim 1.

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