Anti-recombinant sCD40L murine monoclonal antibody as well as preparation method and application thereof

Abstract

The invention discloses an anti-recombinant sCD40L murine monoclonal antibody as well as a preparation method and application thereof, and belongs to the field of biological medicines. A recombinant sCD40L is used for immunizing a BALB / c mouse, four cell strains HYD-5048-01-04 capable of stably secreting monoclonal antibodies are obtained through a hybridoma technology, the subtypes of the monoclonal antibodies are IgG1 / kappa and IgG2b / kappa, the Kd value reaches nM level, and heavy and light chain variable region sequences (SEQ ID NO.1-16) are brand new sequences. Through ELISA (enzyme-linked immuno sorbent assay), Western blot and immunohistochemical verification, all the monoclonal antibodies can be specifically combined with the sCD40L. Wherein the F (ab ') 2 fragment (DT1) of 5048-02 can relieve the symptoms of thrombus and organ fibrosis; 5048-03 is used for targeting thrombotic diseases; 5048-02 / 04 has the potential of preventing and treating neurodegenerative diseases, autoimmune diseases and organ transplantation rejection. The monoclonal antibody provided by the invention has high specificity and strong affinity, and a brand new tool is provided for research and development of related drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to the development of genetically engineered recombinant proteins and therapeutic antibodies and the clinical application thereof. Specifically, the present application provides an anti-recombinant sCD40L protein mouse monoclonal antibody with important clinical application value, and a preparation method and use thereof. BACKGROUND

[0002] CD40 ligand (CD40L / CD154) as a key member of the tumor necrosis factor superfamily (TNFSF) is a type II transmembrane protein with important biological functions. This protein plays a core regulatory role in various physiological and pathological processes, including immune regulation, inflammatory response and thrombosis (Schönbeck et al., 2001, PMID: 11229815). In recent years, studies have shown that its soluble form (sCD40L) exhibits unique therapeutic value due to the retention of the complete receptor binding domain. Specifically, sCD40L binds to the platelet surface CD40 and GPⅡb / Ⅲa receptor, activates the PI3K-Akt signaling pathway (André et al., 2002, PMID: 11875495), and significantly enhances platelet aggregation and thrombus stability. Animal experiments have shown that exogenous sCD40L can shorten the secondary bleeding time after thrombus destruction by 40-60% (p<0.01), which provides a new idea for the development of drugs for treating traumatic massive hemorrhage and platelet dysfunction (such as Glanzmann thrombasthenia) (Lievens et al., 2010, PMID: 20705757). Excessive activation of the CD40L / CD40 signaling pathway can promote the release of inflammatory factors (TNF-α, IL-6) and induce endothelial cell activation (Henn et al., 1998). Clinical studies have shown that elevated sCD40L levels are significantly associated with atherosclerotic plaque instability (Heeschen et al., 2003, PMID: 12646667), systemic lupus erythematosus (Vakkalanka et al., 1999, PMID: 10323442) and other diseases.

[0003] Currently, the sCD40L preparation technology faces the following key problems: sCD40L produced by the traditional E. coli expression system (37℃, 1mMIPTG) mostly exists in the form of inclusion bodies, and its activity is severely lost after refolding (PMID: 12633569); while the mammalian cell expression system has low yield (<1mg / L) and is expensive, making it difficult to meet clinical needs. In terms of antibody development, the currently marketed anti-CD40L antibodies (such as Ruplizumab) have the following defects: (1) limited affinity (Kd>10nM); (2) easy to induce coagulation dysfunction (Kawai et al., 2000, PMID: 10655073); (3) lack of fragmentation modification technology, resulting in poor tissue penetration.

[0004] The necessity of technological innovation:

[0005] The following technical problems urgently need to be solved in this field: (1) establishing a recombinant expression process for sCD40L in E. coli in a soluble form (5.2 mg / L); (2) developing monoclonal antibodies with nM affinity and well-defined structural features (such as the Fab / F(ab')2 fragment); and (3) achieving bidirectional application of the same target: sCD40L can be used to promote hemostasis, while antibodies can block pathological processes. These breakthroughs will provide new solutions for the treatment of hemorrhagic diseases and immune-related diseases. Summary of the Invention

[0006] The purpose of this invention is to address the bidirectional regulatory needs of the CD40 / CD40L signaling pathway by creatively developing a murine monoclonal antibody against soluble recombinant sCD40L protein, along with its preparation method and applications. This invention establishes a complete technical system from genetic engineering and protein drug preparation to antibody drug development, and has significant clinical application value and industrialization prospects in the fields of hemostatic drugs, antithrombotic drugs, and immunomodulatory drugs.

[0007] The specific technical solution for achieving the objective of this invention is as follows:

[0008] A method for preparing a murine monoclonal antibody against recombinant sCD40L includes the following steps:

[0009] a) Immunogen preparation: Recombinant sCD40L was emulsified with Freund's adjuvant and immunized BALB / c mice by subcutaneous injection at multiple sites. The immunization dose was 30-50 μg / time, and a total of 4-6 immunizations were performed.

[0010] b) Hybridoma construction: Collect serum with a titer ≥1:10 4Spleen cells from immunized mice were fused with SP2 / 0 myeloma cells. After screening in HAT medium and subcloning using the limiting dilution method, four hybridoma cell lines that stably secrete anti-sCD40L antibodies were obtained and named HYD-5048-01 to HYD-5048-04. Among them, H: hypoxanthine, A: aminopterin, and T: thymine nucleotide.

[0011] c) Antibody production: 1~2×10⁶ hybridoma cells... 6 One cell / mouse was inoculated into the peritoneal cavity of paraffin oil-pre-excited BALB / c mice, and the ascites fluid was collected and purified by Protein G affinity to obtain monoclonal antibodies 5048-01 to 5048-04.

[0012] Further, the preparation of the recombinant sCD40L includes the following specific steps: a) obtaining the DNA sequence encoding amino acids 113-261 of human sCD40L through whole-genome synthesis, digesting it with BamHI and XhoI, and inserting it into the multiple cloning site of the pET28a vector to construct the recombinant expression plasmid pET28a-sCD40L; b) transforming the recombinant expression plasmid pET28a-sCD40L into Escherichia coli BL21(DE3)pLysS competent cells, and screening to obtain positive clones, namely the genetically engineered bacteria pET28a-sCD40L / BL21(DE3)pLysS; c) inoculating the genetically engineered bacteria into LB medium containing kanamycin, culturing at 37°C until the OD600nm value reaches 0.4-0.6, and adding kanamycin at a final concentration of 0.5-1 mM. IPTG, induce expression at 16-20℃ for 8-12 hours; d) collect bacterial cells by centrifugation, take the supernatant after sonication, and purify by nickel ion affinity chromatography to obtain recombinant sCD40L with at least 90% purity and natural activity, named 5048, with a molecular weight of 20 kDa and containing a 6×His tag.

[0013] Furthermore, the recombinant expression plasmid pET28a-sCD40L contains the recombinant sCD40L encoding gene, and the encoding gene is operatively linked to the following elements: a T7 promoter and a terminator; a kanamycin resistance selection marker; and a 6×His tag sequence.

[0014] Furthermore, the genetically engineered bacterium pET28a-sCD40L / BL21(DE3)pLysS is obtained by transforming the recombinant expression plasmid into Escherichia coli BL21(DE3)pLysS host cells.

[0015] A murine monoclonal antibody against recombinant sCD40L prepared by the above method, comprising monoclonal antibodies 5048-01 to 5048-04.

[0016] Furthermore, the biological characteristics of the monoclonal antibody are as follows:

[0017] 5048-01: IgG1 / κ type, Kd value 1.486 nM, targeting sCD40L (Q118).

[0018] 5048-02: IgG2b / κ type, Kd value 5.923 nM, targeting sCD40L (G116 / D117P120 / Q121 / R165).

[0019] 5048-03: IgG2b / κ type, Kd value 1.323 nM, targeting sCD40L (N119 / P120 / Q121 / R165).

[0020] 5048-04: IgG1 / κ type, Kd value 1.048 nM, targeting sCD40L (T134 / S136).

[0021] A derivative of the monoclonal antibody, the F(ab')2 fragment obtained by digesting the monoclonal antibody 5048-02 with pepsin, is named DT1.

[0022] A variable region sequence based on the monoclonal antibody, comprising the nucleotide and amino acid sequences of the heavy chain variable region VH and the light chain variable region VL of monoclonal antibodies 5048-01 to 5048-04, as shown in SEQ ID NO.1-16.

[0023] One pharmaceutical use of the monoclonal antibody is for the preparation of:

[0024] a) Inhibitors of the integrin αIIbβ3-CD40L signaling pathway and CD40-CD40L signaling pathway, used to prepare drugs for the treatment or prevention of the following diseases: a1) Thrombotic diseases, including acute pulmonary embolism, acute ischemic stroke, and atherosclerosis, as well as organ fibrosis corresponding to hypercoagulable states, wherein monoclonal antibody 5048-02 targets the 115-117KGD amino acid segment of sCD40L; F(ab')2 tablets prepared by pepsin digestion of monoclonal antibody 5048-02. a1) DT1, which is effective in the treatment of acute pulmonary embolism, acute ischemic stroke and liver and kidney fibrosis; a2) Autoimmune diseases and organ transplant rejection, including systemic lupus erythematosus, lupus nephritis and psoriasis, wherein the monoclonal antibody 5048-04 targets the T134 / S136 amino acid segment of sCD40L; a3) Neurodegenerative diseases, including Alzheimer's disease, wherein the fusion protein of the single-chain antibody scFv fragment of 5048-02 and the blood-brain barrier penetrating peptide Angiopep-2 is used.

[0025] b) Recombinant sCD40L detection reagent, including:

[0026] b1) Immunohistochemical detection reagents, using monoclonal antibody 5048-03, can be used to detect sCD40L expression in the hematoma area and surrounding tissues of cerebral hemorrhage pathological sections; b2) Monoclonal antibodies 5048-01 to 5048-04 are used for ELISA or Western blot to specifically detect sCD40L expression in biological samples.

[0027] A. Breakthrough in core technologies

[0028] A1. Recombinant sCD40L protein engineering design

[0029] a11) Design a truncated sCD40L (113-261aa) that retains the complete receptor-binding domain (KGD motif).

[0030] a12) Using the pET28a / BL21(DE3)pLysS expression system, some sCD40L was expressed in soluble form in Escherichia coli through codon optimization and low-temperature induction (20℃, 0.5mM IPTG);

[0031] a13) After purification, the nickel column has a purity of >95%, exhibits significant platelet activation and aggregation activity in vitro, and significantly shortens the time of secondary bleeding in the tail vein of mice.

[0032] A2. Preparation of high-affinity monoclonal antibodies

[0033] a21) Four monoclonal antibodies (5048-01 to 5048-04) were obtained using hybridoma technology. The affinity between the antibodies and antigens was detected using biomembrane interference (BLI) technology. The affinity Kd values ​​were 1.486 nM, 5.923 nM, 1.323 nM, and 1.048 nM, respectively.

[0034] a22) Antibody subtypes: IgG1 / κ type monoclonal antibodies (5048-01 and 5048-04) and IgG2b / κ type antibodies (5048-02, -03) were obtained through screening.

[0035] a23) The Fc fragment of the monoclonal antibody was removed by pepsin, and the enzyme-digested 5048-02-F(ab')2 fragment was developed and named DT1 to avoid the risk of thrombosis.

[0036] Methods for preparing B monoclonal antibodies

[0037] B1. Immunogen Preparation

[0038] b11) Ni-NTA purified sCD40L protein (purity >95%) was emulsified with Freund's adjuvant (1:1);

[0039] b12) BALB / c mice were subcutaneously immunized (50 μg / time, 2 weeks apart), and spleens were harvested when the serum titer reached 1:80000.

[0040] B2. Cell fusion and screening;

[0041] b21) SP2 / 0 cells were fused with spleen cells at a ratio of 1:5 (PEG1450 method).

[0042] b22) After screening in HAT medium, the samples were screened by ELISA (coating concentration 1 μg / mL).

[0043] B3. Large-scale antibody production

[0044] The ascites method yields 15-20 mg / mL, and the recovery rate of Protein G after purification is >85%.

[0045] The technological innovation of this invention is reflected in the following aspects:

[0046] 1) Antibody Development Innovation:

[0047] 1-1) A method for preparing high-affinity (nM-level Kd) monoclonal antibodies based on this recombinant protein was developed;

[0048] 1-2) Four stable hybridoma cell lines secreting antibodies were obtained, and the variable regions of the antibody heavy chain and light chain were sequenced.

[0049] 1-3) Establish antibody fragmentation (Fab / F(ab')2) enzyme digestion process to improve the efficacy and safety of drug treatment.

[0050] 2) Innovation in clinical application:

[0051] For the first time, bidirectional drug therapy targeting the same protein / antibody has been achieved:

[0052] 2-1) Use of sCD40L in the preparation of medicaments for the treatment of diseases selected from: platelet dysfunction, platelet reservoir deficiency or severe multiple injuries, and post-burn hemorrhage, wherein the sCD40L protein enhances hemostatic efficacy by promoting platelet aggregation.

[0053] 2-2) Use of an anti-CD40L monoclonal antibody in the preparation of a medicament for treating diseases selected from thrombotic diseases, neurodegenerative diseases, autoimmune diseases, organ transplant rejection or fibrosis, wherein the monoclonal antibody is capable of specifically inhibiting the integrin αIIbβ3-CD40L and CD40L-CD40 signaling pathways. Attached Figure Description

[0054] Figure 1 Schematic diagram of the construction and sequence verification of the recombinant plasmid pET28a-sCD40L prokaryotic expression system;

[0055] Figure 2 Schematic diagram of the expression and purification of recombinant sCD40L protein;

[0056] Figure 3 Schematic diagram showing how sCD40L promotes platelet aggregation and shortens the time to secondary bleeding after thrombus destruction in mice;

[0057] Figure 4 Schematic diagram of purity identification for monoclonal antibodies 5048-01 to 5048-04;

[0058] Figure 5 Schematic diagram of affinity assay between monoclonal antibodies 5048-01 to 5048-04 and sCD40L;

[0059] Figure 6. Computational structural model and molecular docking schematic diagram of anti-sCD40L monoclonal antibody;

[0060] Figure 7 Schematic diagram of ELISA binding characteristics analysis of monoclonal antibodies 5048-01 to 5048-04 with sCD40L;

[0061] Figure 8 Schematic diagram of Western blot specificity detection of monoclonal antibodies 5048-01 to 5048-04;

[0062] Figure 9 Schematic diagram of immunohistochemical localization analysis of monoclonal antibody 5048-03 in pathological tissue of cerebral hemorrhage;

[0063] Figure 10 Schematic diagram of purity identification of F(ab')2 fragment (DT1) after pepsin digestion of monoclonal antibody 5048-02;

[0064] Figure 11 A schematic diagram illustrating how DT1 significantly inhibits the formation of collagen I (COL Ⅰ)-induced extracorporeal platelet aggregates;

[0065] Figure 12 Schematic diagram showing that DT1 significantly inhibits collagen I / adrenaline-induced acute pulmonary embolism;

[0066] Figure 13 Schematic diagram of the therapeutic effect of DT1 on infarct volume in acute stroke;

[0067] Figure 14A schematic diagram showing how DT1 significantly reduced abnormal collagen deposition in the livers of mice with carbon tetrachloride (CCl4)-induced liver fibrosis.

[0068] Figure 15 . Schematic diagram showing that DT1 significantly reduces abnormal collagen deposition in renal fibrosis induced by unilateral ureteral ligation (UUO) in mice. Detailed Implementation

[0069] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, reagents, experimental methods, etc., for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0070] Example 1: Construction of pET-28a-sCD40L expression vector and expression and purification of recombinant sCD40L protein

[0071] 1. Gene Construction

[0072] 1-1) Based on the human CD40L gene sequence (NCBI accession number NC000023.11), the sCD40L113-261aa fragment was synthesized from the whole genome.

[0073] 1-2) The fragment was cloned into the pET28a vector (BamHI / XhoI site) and transformed into BL21(DE3)pLysS competent cells.

[0074] 2. Protein expression optimization

[0075] 2-1) Select positive clones and inoculate them into LB medium containing kanamycin (50 μg / mL), and culture at 37°C with shaking until OD600 = 0.4-0.6;

[0076] 2-2) Optimization of induction conditions: 20℃, 0.5mM IPTG induction for 12 hours (control: 37℃ induction for 4 hours)

[0077] 2-3) After ultrasonic lysis, the protein was centrifuged at 12000g for 30 minutes at 4℃. The soluble protein content in the supernatant was 12.6±3.8% (SDS-PAGE analysis).

[0078] 3. Purification process

[0079] 3-1) Nickel column affinity chromatography: binding buffer (50mM PBS, 150mM NaCl, pH 7.4)

[0080] 3-2) Gradient elution (50-1500 mM imidazole), collect the 500-1000 mM elution peak.

[0081] 3-3) Repeat the experiment and select the parts with higher purity to combine, then concentrate by ultrafiltration (the final product concentration and purity are greater than 95%).

[0082] Vector construction: The gene fragment encoding sCD40L (113-261aa, 450bp) was cloned into the pET28a vector using BamHI / XhoI double restriction sites to construct the recombinant plasmid pET28a-sCD40L; the vector contains a T7 promoter, an N-terminal His tag, and a kanamycin resistance gene ( Figure 1 A).

[0083] Sequence validation: Bidirectional Sanger sequencing confirmed that the inserted fragment was 100% identical to the designed sequence. Figure 1 B).

[0084] Protein expression and purification: After IPTG induction (0.5 mM, 37℃ for 4 hours or 20℃ for 12 hours), the target band was detected at 20 kDa by SDS-PAGE. Figure 2 AB); Ni-NTA purification showed that 500-1000 mM imidazole was the optimal elution condition, and Western blot confirmed protein specificity. Figure 2 CE).

[0085] Example 2: Application of sCD40L in platelet aggregation and thrombosis models

[0086] In vitro platelet aggregation assay: sCD40L (1-10 μg / mL) combined with type I collagen (1-5 μg / mL) significantly enhanced platelet aggregation area. Figure 3 AB).

[0087] In vivo hemostasis model: Local application of sCD40L (10 μg / cm²) shortened the time of secondary bleeding in mice. Figure 3 C).

[0088] Example 3: Preparation and characterization of anti-sCD40L monoclonal antibody

[0089] The antibody preparation steps are as follows:

[0090] 1. Immunization regimen

[0091] 1-1) Immunogen: sCD40L protein obtained in Example 1 (purity > 95%);

[0092] 1-2) Subcutaneous immunization of female BALB / c mice at 6-8 weeks of age (50 μg / dose, alternating Freund's complete / incomplete adjuvant);

[0093] 1-3) After four immunizations, the serum titer reached 1:80000 (indirect ELISA method).

[0094] 2. Cell fusion

[0095] 2-1) Fusing the highest titer mouse spleen cells with SP2 / 0 cells (ratio 5:1) under the action of 50% PEG1450;

[0096] 2-2) After culturing in HAT selective medium, hybridoma clones can be seen after 10 days.

[0097] 3. Antibody screening

[0098] 3-1) Initial screening: ELISA (1 μg / mL sCD40L coating, 1:1000 secondary antibody);

[0099] 3-2) Secondary screening: After three subclonings, five stable antibody-secreting hybridomas (HYD-5048-01 to 5048-05) were obtained.

[0100] 4. Monoclonal antibody production

[0101] 4-1) Resuscitation of hybridoma cells

[0102] ① Take one cell from liquid nitrogen and quickly place it in a 37°C water bath while stirring continuously;

[0103] ② After the cells are completely thawed, wipe the cryovial with 75% alcohol and place it in a clean bench. Aspirate the cells and transfer them into 10 ml of DMEM medium. Centrifuge at 1200 rpm for 5 minutes.

[0104] ③ Discard the supernatant, loosen the precipitate, mix it with 5ml of complete culture medium, transfer it into a T-25 square bottle, and incubate it in an incubator (37℃, 5% CO2).

[0105] 4-2) Inoculation of hybridoma cells

[0106] ①Pre-inject paraffin oil into mice via intraperitoneal injection (0.5 ml / mouse; paraffin oil can only be used to prepare ascites at least 7 days after injection);

[0107] ② Collect the cells when they have basically covered the bottom of the flask and are in good condition. Prepare a suspension by pipetting the hybridoma cells with a bent pipette, count them, and centrifuge at 1200 rpm for 5 minutes.

[0108] ③ Discard the supernatant, resuspend the cells in a small amount of DMEM, and inject them intraperitoneally into paraffin-embedded mice (1-2 × 10⁶ cells per mouse). 6 cell).

[0109] 4-3) Collection and preservation of ascites

[0110] ① After mice are inoculated with hybridoma cells, their condition should be observed daily;

[0111] ② Before the mice were dying, they were euthanized by dislocating their cervical vertebrae. The skin was cut open with surgical scissors, the skin was torn open to expose the peritoneum, and a small incision was made in the peritoneum. The ascites was then suctioned out with a dropper.

[0112] ③ Centrifuge the ascites fluid at 3000 rpm for 30 minutes, collect the supernatant, aliquot and freeze at -70℃, and take a small amount of ELISA to detect the ascites fluid titer.

[0113] 4-4) Antibody purification

[0114] ① Take 1 ml of ascites fluid and centrifuge at 12000 rpm for 4 minutes. Take the supernatant and dilute it with 4 ml of PBS.

[0115] ②Take out the proteinG column (column volume 1ml), equilibrate with 10ml PBS, wait for the PBS to drain completely, load the ascites fluid through the column, and collect the flow-through.

[0116] ③ Equilibrate with 10ml PBS, drain, add 1ml of 0.1M pH 2.5 glycine solution, drain again, add 2.5ml of 0.1M pH 2.5 glycine solution, collect the eluent, and neutralize with 250ul of 1M Tris solution (pH 8.8);

[0117] ④ Add 10ml of PBS to equilibrate, drain to dryness, then add 2ml of 20% ethanol and store the column at 4 degrees Celsius.

[0118] ⑤ Dialyze the antibody eluent with PBS overnight, and then detect the titer using ELISA.

[0119] The methods for cloning and sequencing the variable region gene of monoclonal antibodies are as follows:

[0120] 1) mRNA extraction and preparation and cDNA synthesis: Hybridoma cell lines secreting antibodies were cultured, and hybridoma cells in good growth condition were collected, with a cell count of approximately 1 × 10⁻⁶. 6-7 / mL. Total mRNA was extracted and reverse transcribed into cDNA;

[0121] 2) Cloning of antibody light and heavy chain variable region genes: Using extracted cDNA as a template, the designed degenerate primers P1 / P2 were used to amplify the antibody light / heavy chain variable region genes; the PCR reaction system and amplification conditions for the light and heavy chain variable region genes were consistent. PCR system: 10×Ex buffer (with added Mg2+) 2+2.5 μL of dNTPs (2.5 μmol / L each), 1 μL each of forward and reverse primers (20 μmol / L), 0.3 μL of ExTaq polymerase, 1 μL of cDNA, and 17.2 μL of distilled water, for a total volume of 25 μL. PCR reaction conditions: 94℃ for 5 min; 94℃ for 30 s, 52℃ for 30 s, 72℃ for 30 s, for a total of 30 cycles; 72℃ for 10 min.

[0122] 3) T vector construction and sequencing: The light chain variable region gene and heavy chain variable region gene fragments obtained by PCR were recovered by gel extraction and ligated with the pMD19-T vector. The ligation product was transformed into E. coli DH5α competent cells, and 4-6 clones were selected for bacterial PCR and plasmid extraction and enzyme digestion identification.

[0123] Antibody screening: Four monoclonal antibodies (5048-01 to 5048-04) were obtained, and their purity was analyzed by 12% reducing polyacrylamide gel electrophoresis (SDS-PAGE). Results showed that each antibody exhibited typical heavy chain (approximately 50 kDa) and light chain (approximately 25 kDa) bands under reducing conditions; no significant contaminating protein bands were observed, and the purity of all antibodies was >95% (quantitative analysis via gel scanning). Figure 4 ).

[0124] Affinity assay: Real-time binding sensing of monoclonal antibody-sCD40L interaction based on biomembrane interference (BLI) technology. All antibodies showed concentration-dependent signal growth (RU value); the initial binding slope reflected the difference in binding rate, and the signal decay rate reflected the stability of the antibody-antigen complex. Figure 5 A). Affinity parameters: Kon represents the efficiency of antibody-antigen molecule binding to form a complex per unit time (per second), reflecting the speed of molecular recognition; Kd is the equilibrium dissociation constant; KD (dissociation constant) is a key thermodynamic parameter characterizing the binding strength of biomolecules (such as antibody-antigen), and its unit is nanomolar (nM, 10⁻⁻¹). 9 The smaller the value, the stronger the affinity between the two. BLI analysis showed that the KD value reached the nanomolar level (nM), with the 5048-04 monoclonal antibody having the best KD value (M). Figure 5 B).

[0125] Structural prediction: Predicted single-chain antibody structures were generated using AlphaFold2 (v2.3.0). All variable regions scored greater than 85 on the predicted local distance difference test (pLDDT). Heavy and light chains are represented in red and blue, respectively. Figure 6Among them, 5048-01 targets sCD40L (Q118); 5048-02 targets sCD40L (G116 / D117P120 / Q121 / R165).

[0126] 5048-03 targets sCD40L (N119 / P120 / Q121 / R165); 5048-04 targets sCD40L (T134 / S136).

[0127] Epitope recognition: ELISA confirmed that all antibodies specifically bound to sCD40L (113-190aa) Figure 7 Western blot analysis confirmed that all antibodies could recognize sCD40L in activated platelets (…). Figure 8 Monoclonal antibody 5048-03 can be used for immunohistochemical staining, showing specific brown staining in the perihemorrhagic area of ​​pathological tissues of cerebral hemorrhage. Figure 9 ).

[0128] Example 4: Preparation and functional verification of F(ab')2 fragment (DT1)

[0129] Fragment preparation: DT1 was obtained by digestion of 5048-02 with pepsin. SDS-PAGE confirmed that the purity under non-reducing conditions was ≥95% (110 kDa). Figure 10 ).

[0130] In vitro inhibition of platelet aggregation: DT1 dose-dependently inhibits collagen-induced platelet aggregate formation ( Figure 11 (a) shows representative images of platelet aggregates in vitro after treatment with COL Ⅰ and COL Ⅰ + DT1. COL Ⅰ, 5 μg / mL; DT1, 10 or 25 μg / mL. Scale bar: 50 μm. (b) shows the quantitative analysis of platelet aggregate numbers. Platelets were derived from mixed blood from 3 mice. Five to six different fields of view were observed and quantitatively analyzed in each group, with three different biological replicates. Data are expressed as mean ± standard deviation (n = 3). Statistical significance was determined by one-way ANOVA and Tukey's post-hoc test; no significant difference was found in ns.

[0131] The allergic effect of DT1 in disease models:

[0132] Pulmonary embolism: DT1 reduces the area of ​​pulmonary embolism caused by CD41⁺ platelets ( Figure 12(a) shows representative immunohistochemical staining images of platelet (CD41) in lung tissue sections from each group of mice. The scale bar is 100 μm. (b) shows the quantitative analysis of the CD41-positive area in lung tissue sections from each group of mice. Each point represents one individual (n = 3 / group). For each individual, 5-6 different fields of view were observed and quantitatively analyzed. Data are expressed as mean ± standard deviation. Statistical significance was determined by one-way ANOVA and Tukey's post-hoc test; no significant difference was found in ns.

[0133] Stroke: DT1 (25 μg / animal) reduces infarct volume ( Figure 13 a, b) and improved neurological function scores ( Figure 13 c); and does not pose a risk of bleeding ( Figure 13 d). Among them, (ab) the infarct volume was detected by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The results showed that the model group presented large white infarct lesions (n ​​= 5), while the DT1 group (25 μg / animal, n = 5) significantly reduced the infarct area. (c) Quantification of neurological function severity scores (n = 8; significant difference compared with the control group was obtained by Student's t test). (d) Results of tail bleeding detection during hemorrhage time in different groups. Ns indicates no significant difference.

[0134] Liver fibrosis: Male C57BL / 6 mice were randomly divided into two groups (prevention group and treatment group). Mice were intraperitoneally injected with 1.0 ml / kg body weight of CCl4 (dissolved in corn oil at a 1:4 ratio) on Mondays and Thursdays around 2 PM. The prevention group received the injection for four weeks, and the treatment group for six weeks. From week one, each mouse in the prevention group received an intraperitoneal injection daily of either control IgG F(ab')2 fragment or DT1 (25 μg / mouse) for four weeks. From week four, each mouse in the treatment group received an intraperitoneal injection daily of either control IgG F(ab')2 fragment or DT1 (25 μg / mouse) for three weeks. Mice were sacrificed 48 hours after the last injection for pathological analysis. Results showed that DT1 significantly reduced liver collagen deposition in both the prevention and treatment groups. Figure 14The images show: (a) Representative images of Masson staining liver sections from mice in the prevention groups (Normal, Veh, and DT1 groups) (n = 4 / group). Scale bar: 100 μm. (b) Representative images of Masson staining liver sections from mice in the treatment groups (Normal, Veh, and DT1 groups) (n = 4 / group). Scale bar: 100 μm. (c, d) Quantitative analysis of the blue collagen-positive area in liver sections from mice in the prevention group (c) and treatment group (d). Each point represents one individual. For each individual, 5-6 different fields of view were observed and quantitatively analyzed. Data are expressed as mean ± standard deviation. Statistical significance was determined by one-way ANOVA and Tukey's post-hoc test; no significant difference was found in ns.

[0135] Renal fibrosis: A unilateral ureteroscopic ligation (UUO) renal fibrosis model was established using 8-week-old male C57BL / 6 mice. Mice were euthanized on day 14 post-surgery to assess the intervention effect of DT1. In the prevention regimen, mice were intraperitoneally injected daily with either control IgG F(ab')2 fragment (control group) or DT1 (25 μg / mouse) from day 1 to day 7 post-surgery, starting from day 7 after UUO model establishment (fibrosis formation stage). In the treatment regimen, intervention was initiated on day 7 after UUO model establishment (fibrosis formation stage), with mice receiving intraperitoneal injections of either DT1 or control IgG F(ab')2 fragment from day 7 to day 14 post-surgery. Detection indicators included collagen deposition in the renal tubulointerstitium and glomerular regions (via Masson trichrome staining) and myofibroblast activation; α-smooth muscle actin (α-SMA) expression levels were detected by immunohistochemistry and Western blot. Results showed that both the DT1 prevention and treatment groups significantly reduced collagen deposition (…). Figure 15 The images show: (a) Representative images of Masson staining on kidney sections from mice in the prevention groups (Normal, Veh, and DT1 groups) (n = 4 / group). Scale bar: 100 μm. (b) Representative images of Masson staining on kidney sections from mice in the treatment groups (Normal, Veh, and DT1 groups) (n = 4 / group). Scale bar: 100 μm. (c, d) Quantitative analysis of the blue collagen-positive area in kidney sections from mice in the prevention group (c) and treatment group (d). Each point represents one individual. For each individual, 5-6 different fields of view were observed and quantitatively analyzed. Data are expressed as mean ± standard deviation. Statistical significance was determined by one-way ANOVA and Tukey's post-hoc test; no significant differences were found.

[0136] This invention provides a total of 16 sequences, including 8 nucleic acid sequences (encoding the variable region of monoclonal antibodies) and 8 corresponding amino acid sequences (monoclonal antibody variable region proteins).

[0137] Nucleic acid sequence:

[0138] SEQ ID NO: 1: A 390 bp DNA sequence derived from a mouse (Mus musculus). This sequence encodes the heavy chain variable region (VH) nucleotide sequence of the anti-sCD40L monoclonal antibody 5048-01.

[0139] SEQ ID NO: 2: A 395 bp DNA sequence derived from a mouse (Mus musculus). This sequence encodes the light chain variable region (VL) nucleotide sequence of the anti-sCD40L monoclonal antibody 5048-01.

[0140] SEQ ID NO: 3: A 342 bp DNA sequence derived from a mouse (Mus musculus). This sequence encodes the heavy chain variable region (VH) nucleotide sequence of the anti-sCD40L monoclonal antibody 5048-02.

[0141] SEQ ID NO: 4: A 336 bp DNA sequence derived from a mouse (Mus musculus). This sequence encodes the light chain variable region (VL) nucleotide sequence of the anti-sCD40L monoclonal antibody 5048-02.

[0142] SEQ ID NO: 5: A 399 bp DNA sequence derived from a mouse (Mus musculus). This sequence encodes the heavy chain variable region (VH) nucleotide sequence of the anti-sCD40L monoclonal antibody 5048-03.

[0143] SEQ ID NO: 6: A 396 bp DNA sequence derived from a mouse (Mus musculus). This sequence encodes the light chain variable region (VL) nucleotide sequence of the anti-sCD40L monoclonal antibody 5048-03.

[0144] SEQ ID NO: 7: A 399 bp DNA sequence derived from a mouse (Mus musculus). This sequence encodes the heavy chain variable region (VH) nucleotide sequence of the anti-sCD40L monoclonal antibody 5048-04.

[0145] SEQ ID NO: 8: A 378 bp DNA sequence derived from a mouse (Mus musculus). This sequence encodes the light chain variable region (VL) nucleotide sequence of the anti-sCD40L monoclonal antibody 5048-04.

[0146] amino acid sequence:

[0147] SEQ ID NO: 9: A protein sequence of 112 amino acids derived from mouse (Mus musculus). This sequence is the heavy chain variable region (VH) amino acid sequence of anti-sCD40L monoclonal antibody 5048-01, encoded by SEQ ID NO: 1.

[0148] SEQ ID NO: 10: A protein sequence of 112 amino acids derived from mouse (Mus musculus). This sequence is the light chain variable region (VL) amino acid sequence of anti-sCD40L monoclonal antibody 5048-01, encoded by SEQ ID NO: 2.

[0149] SEQ ID NO: 11: A protein sequence of 114 amino acids derived from mouse (Mus musculus). This sequence is the heavy chain variable region (VH) amino acid sequence of anti-sCD40L monoclonal antibody 5048-02, encoded by SEQ ID NO: 3.

[0150] SEQ ID NO: 12: A protein sequence of 112 amino acids derived from mouse (Mus musculus). This sequence is the light chain variable region (VL) amino acid sequence of anti-sCD40L monoclonal antibody 5048-02, encoded by SEQ ID NO: 4.

[0151] SEQ ID NO: 13: A protein sequence of 114 amino acids derived from mouse (Mus musculus). This sequence is the heavy chain variable region (VH) amino acid sequence of anti-sCD40L monoclonal antibody 5048-03, encoded by SEQ ID NO: 5.

[0152] SEQ ID NO: 14: A protein sequence of 112 amino acids, derived from mouse (Mus musculus). This sequence is the light chain variable region (VL) amino acid sequence of anti-sCD40L monoclonal antibody 5048-03, encoded by SEQ ID NO: 6.

[0153] SEQ ID NO: 15: A protein sequence of 114 amino acids derived from mouse (Mus musculus). This sequence is the heavy chain variable region (VH) amino acid sequence of anti-sCD40L monoclonal antibody 5048-04, encoded by SEQ ID NO: 7.

[0154] SEQ ID NO: 16: A protein sequence of 106 amino acids derived from mouse (Mus musculus). This sequence is the light chain variable region (VL) amino acid sequence of anti-sCD40L monoclonal antibody 5048-04, encoded by SEQ ID NO: 8.

[0155] SEQ ID NO.1:

[0156] atggaatggaggatctttctcttcatcctgtcaggatctgcaggtgtccactcccaggttcagctgcagcagtctggacctgagctggtgaagcctggggcttcagtgaagatgtcctgcaaggcttctggatacacattcactgactatgttataagctgggtgaagcagagaactggacagggccttgagtgg attggagagctttatcctggaagtagtacttactacaatgagaagttcaagggcaaggccacactgactgcagacatatcctccaacacagcctacctgcagttcagcagcctgacatctgaggactctgcggtctatttctgtgcacgttctttggactactggggtctaggaacctcagtcaccgtctcctca

[0157] SEQ ID NO.2:

[0158] atgatgagtcctgcccagttcctgtttctgttagtgctctggattcaggaaaccaacggtgatgttgtgatgacccagacgccactcactttgtcggttaccattggacaaccagcctctatttcttgtaagtcaagtcagagcctcttatatagtaatggaaaaacctatttgaattggttattacagaggccaggccagtctccaaagcgcctactctatctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacactgaaaatcagcggagtggaggctgaggatttgggagtttattactgcgtgcaaggtacacattatccattcacgttcggctcggggacaaagttggaaataaa

[0159] SEQ ID NO.3:

[0160] caggttcagctgcagcagtctggaactgagttgatgaagcctggggcctcagtgaagatatcctgcaagacttctggctacacattcaatgactactggatagagtggataaaacagaggcctggacatggccttgagtggattggagagattttacctggaagtggtagtactaactacaatgagaaattcaagggcaaggccacattcactgcagatgcgtcctcctccacagtctacatacaactcaccaccctgacatctgaggactctgccgtctattactgtgcaagagataaatatcctaactggggccaaggcaccactctcacagtctccaca

[0161] SEQ ID NO.4:

[0162] gattttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctccatctcttgcaggtcaagtcagagcctcttatatagtgatggagagacgtatttgaattggttgttacagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggtacacattttccattcacgttcggctcggggacaaagttggagataaaa

[0163] SEQ ID NO.5:

[0164] atggaatggacctgggtctttctcttcctcctgtcagtaactgcaggtgtccactcccaggttcagctgcagcagtctggagctgaactgatggagccgggggcctcagtgaagatatcctgcaaggctactggctacacattcaatagctactggatagaatggataaaacagaggcctggacatggccttgagtggattggagagattttacctggaagtgatagtactaactacaatgagaagttcgaggacagggccacattcactgcagatacctcctccaacacagcctacatgcaactcagcagcctgacatctgaggactctgccgtctattactgtgcaagagataagtaccctcactggggccaaggcaccactctcacagtctcctca

[0165] SEQ ID NO.6:

[0166] atgatgagtcctgcccagttcctgtttctgttagtgctctggattcgggaaaccaacggtgattttgtggtgacccagactccactcactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttatatagtgatggaaagacatatttgagttggttgttacagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggtacacattttccattcacgttcggctcggggacaaagttggaaataaaa

[0167] SEQ ID NO.7:

[0168] atgaacttcgggctcagattgattttccttgtccttactttaaaaggtgtccagtgtgacgtgaagctggtggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtagctctaccatgtcttgggttcgccagactccggaaaagaggctggagtgggtcgcaaccattagtagtggtggtcattacacctactttccagacagtgtgaagggccgattcaccatctccagagacaatgccaagaacaccctgtacctacaaatgcgcagtctggagtctgaagacacagcaatgtattactgtgcaagggggatccccgatgtctggggcgcagggaccacggtcaccgtctcctca

[0169] SEQ ID NO.8:

[0170] atggagtcacagactcaggtctttgtatacatgttgctgtggttgtctggtgttgatggagacattgtaatgacccagtctcaaaaatccctgtccacatcagttggagacagggtcaacgtcacctgcaaggccagtcagaatgtgggtactgatgtagcctggtatcaacagaaaacagggcaatctcctaaaccactgatttactcggcatcctcccgattcagtggagtccctgatcgcttcgcgggcagtggatctgggacagatttcactctcaccatcagcgatgtgcagtctgaagacttggcagattatttctgtcaccaatataagacttatctcacgttcggtgctgggaccaagctggagttgaaa

[0171] SEQ ID NO.9:

[0172] QVQLQQSGPELVKPGASVKMSCKASGYTFTDYVISWVKQRTGQGLEWIGELYPGSSTYYNEKFKGKATLTADISSNTAYLQFSSLTSEDSAVYFCARSLDYWGLGTSVTVSS

[0173] SEQ ID NO.10:

[0174] DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLLYLVSKLDSGVPDRFTGSGSGTDFTLKISGVEAEDLGVYYCVQGTHYPFTFGSGTKLEIK

[0175] SEQ ID NO.11:

[0176] QVQLQQSGTELMKPGASVKISCKTSGYTFNDYWIEWIKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTAD ASSSTVYIQLTTLTSEDSAVYYCARDKYPNWGQGTTLTVST

[0177] SEQ ID NO.12:

[0178] DFVMTQTPLTLSVTIGQPASISCRSSQSLLYSDGETYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSG TDFTLKISRVEAEDLGVYYCWQGTHFPFTFGSGTKLEIK

[0179] SEQ ID NO.13:

[0180] QVQLQQSGAELMEPGASVKISCKATGYTFNSYWIEWIKQRPGHGLEWIGEILPGSDSTNYNEKFEDRATFTADTSSNTAYMQLSSLTSEDSAVYYCARDKYPHWGQGTTLTVSS

[0181] SEQ ID NO.14:

[0182] DFVVTQTPLTLSVTIGQPASISCKSSQSLLYSDGKTYLSWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPFTFGSGTKLEIK

[0183] SEQ ID NO.15:

[0184] DVKLVESGGGLVKPGGSLKLSCAASGFTFSSSTMSWVRQTPEKRLEWVATISSGGHYTYFPDSVKGRFTISRDNAKNTLYLQMRSLESEDTAMYYCARGIPDVWGAGTTVTVSS

[0185] SEQ ID NO.16:

[0186] DIVMTQSQKSLSTSVGDRVNVTCKASQNVGTDVAWYQQKTGQSPKPLIYSASSRFSGVPDRFAGSGSGTDFTLTISDVQSEDLADYFCHQYKTYLTFGAGTKLELK

Claims

1. A method for preparing a murine monoclonal antibody against recombinant sCD40L, characterized in that, Includes the following steps: a) Immunogen preparation: Recombinant sCD40L was emulsified with Freund's adjuvant and immunized BALB / c mice by subcutaneous injection at multiple sites. The immunization dose was 30-50 μg / time, and a total of 4-6 immunizations were performed. b) Hybridoma construction: Collect serum with a titer ≥1:10 4 Spleen cells from immunized mice were fused with SP2 / 0 myeloma cells. After screening in HAT medium and subcloning using the limiting dilution method, four hybridoma cell lines that stably secrete anti-sCD40L antibodies were obtained and named HYD-5048-01 to HYD-5048-04. Among them, H: hypoxanthine, A: aminopterin, and T: thymine nucleotide. c) Antibody production: 1~2×10⁶ hybridoma cells... 6 One cell / mouse was inoculated into the peritoneal cavity of paraffin oil-pre-excited BALB / c mice, and the ascites fluid was collected and purified by Protein G affinity to obtain monoclonal antibodies 5048-01 to 5048-04.

2. The preparation method according to claim 1, characterized in that, The recombinant sCD40L is prepared by the following specific steps: a) The DNA sequence encoding amino acids 113-261 of human sCD40L was obtained by whole-genome synthesis, and after double digestion with BamHI and XhoI, it was inserted into the multiple cloning site of the pET28a vector to construct the recombinant expression plasmid pET28a-sCD40L. b) Transform the recombinant expression plasmid pET28a-sCD40L into Escherichia coli BL21(DE3)pLysS competent cells and screen to obtain positive clones, namely the genetically engineered bacteria pET28a-sCD40L / BL21(DE3)pLysS. c) Inoculate the genetically engineered bacteria into LB medium containing kanamycin and culture at 37°C until the OD600nm value is 0.4-0.

6. Then add IPTG at a final concentration of 0.5-1 mM and induce expression at 16-20°C for 8-12 hours. d) Collect bacterial cells by centrifugation, and after sonication, take the supernatant and purify it by nickel ion affinity chromatography to obtain recombinant sCD40L with at least 90% purity and natural activity, named 5048, with a molecular weight of 20 kDa and containing a 6×His tag.

3. The preparation method according to claim 2, characterized in that, The recombinant expression plasmid pET28a-sCD40L contains the recombinant sCD40L encoding gene, and the encoding gene is operatively linked to the following elements: a T7 promoter and a terminator; a kanamycin resistance selection marker; and a 6×His tag sequence.

4. A murine monoclonal antibody against recombinant sCD40L prepared by the method of claim 1, comprising monoclonal antibodies 5048-01 to 5048-04.

5. The monoclonal antibody according to claim 4, characterized in that, Its biological characteristics are: 5048-01: IgG1 / κ type, Kd value 1.486 nM, targeting sCD40L (Q118). 5048-02: IgG2b / κ type, Kd value 5.923 nM, targeting sCD40L (G116 / D117P120 / Q121 / R165). 5048-03: IgG2b / κ type, Kd value 1.323 nM, targeting sCD40L (N119 / P120 / Q121 / R165). 5048-04: IgG1 / κ type, Kd value 1.048 nM, targeting sCD40L (T134 / S136).

6. A derivative based on the monoclonal antibody of claim 4, characterized in that, The F(ab')2 fragment obtained by digesting the monoclonal antibody 5048-02 described in claim 4 with pepsin is named DT1.

7. A variable region sequence based on the monoclonal antibody of claim 4, characterized in that, The nucleotide and amino acid sequences of the heavy chain variable region VH and light chain variable region VL of monoclonal antibodies 5048-01 to 5048-04 are shown in SEQ ID NO. 1-16.

8. A pharmaceutical use of the monoclonal antibody according to claim 4, characterized in that, Used for preparation: a) Inhibitors of the integrin αIIbβ3-CD40L signaling pathway and CD40-CD40L signaling pathway, used to prepare drugs for the treatment or prevention of the following diseases: a1) Thrombotic diseases, including acute pulmonary embolism, acute ischemic stroke, and atherosclerosis, as well as organ fibrosis corresponding to hypercoagulable states. Monoclonal antibody 5048-02 targets the 115-117 KGD amino acid segment of sCD40L. The F(ab')2 fragment DT1 prepared by pepsin digestion of monoclonal antibody 5048-02 is effective in the treatment of acute pulmonary embolism, acute ischemic stroke, and liver and kidney fibrosis. a2) Autoimmune diseases and organ transplant rejection, including systemic lupus erythematosus, lupus nephritis and psoriasis, wherein the monoclonal antibody 5048-04 targets the T134 / S136 amino acid region of sCD40L. a3) Neurodegenerative diseases, including Alzheimer's disease, in which a fusion protein of the single-chain antibody scFv fragment of 5048-02 and the blood-brain barrier penetrating peptide Angiopep-2 is used; b) Recombinant sCD40L protein detection reagent, including: b1) Immunohistochemical detection reagents, using monoclonal antibody 5048-03, can be used to detect sCD40L expression in the hematoma area and surrounding tissues of cerebral hemorrhage pathological sections; b2) Monoclonal antibodies 5048-01 to 5048-04 are used for the specific detection of sCD40L expression in biological samples by ELISA or Western blot.

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