Anti-DNMT3A antibody and application thereof in osteoarthritis
By screening and combining the anti-DNMT3A monoclonal antibody scFv-A7 with phage display technology and a nuclear-targeted delivery system, the shortcomings of existing methods in the treatment of osteoarthritis have been overcome. This approach achieves highly efficient targeting of DNMT3A, significantly improves inflammation and function, and slows disease progression.
Patent Information
- Application Number
- CN202511784580.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-01
- Publication Date
- 2026-02-24
AI Technical Summary
Current treatments for osteoarthritis primarily relieve symptoms but cannot reverse disease progression. Biologics targeting inflammatory factors exhibit individual differences and side effects. Existing DNMT3A inhibitors have poor selectivity and off-target effects, and there is a lack of effective new therapies.
A high-affinity anti-DNMT3A monoclonal antibody, scFv-A7, was obtained through phage display technology and combined with a nuclear-targeted delivery system to directly target the epigenetic pathway of DNMT3A, forming nanoparticles that enter the cell nucleus and inhibit its activity.
It achieves highly specific targeting of DNMT3A, significantly reduces inflammatory factor levels, improves joint function, and delays cartilage degeneration, providing a novel disease modification therapy strategy for osteoarthritis.
Smart Images
Figure CN121554589A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and more specifically, to an anti-DNMT3A antibody and its application in osteoarthritis. Background Technology
[0002] Osteoarthritis (OA) is a common degenerative joint disease characterized by cartilage destruction, synovitis, and osteophyte formation, severely impacting patients' quality of life and joint function. Globally, approximately 250 million people suffer from OA, and its incidence continues to rise with an aging population. Current treatments for OA primarily include nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injections of corticosteroids or hyaluronic acid, and physical therapy; however, these methods only relieve symptoms and cannot reverse disease progression. In recent years, biologics targeting inflammatory factors (such as IL-1β and TNF-α) have shown some effectiveness, but issues such as significant individual variability and side effects exist. Therefore, developing novel therapies that can fundamentally slow the progression of OA is an urgent need.
[0003] DNA methyltransferase 3A (DNMT3A) is a key enzyme in epigenetic regulation, participating in DNA methylation and playing a crucial role in cell differentiation and apoptosis. Recent studies have shown that DNMT3A is highly expressed in degenerated human cartilage tissue and is positively correlated with the expression of apoptosis-related proteins (such as Caspase-3), suggesting that DNMT3A may exacerbate the pathological process of osteoarthritis (OA) by promoting chondrocyte apoptosis. Inhibition of DNMT3A expression has been shown to delay cartilage degeneration, but existing DNMT3A inhibitors (such as small molecule compounds) have limitations such as poor selectivity and off-target effects. Monoclonal antibodies are widely used in the treatment of tumors and autoimmune diseases due to their high specificity and low toxicity; however, studies on antibodies targeting DNMT3A in the treatment of OA have not yet been reported.
[0004] This invention addresses the aforementioned technological gap by using phage display technology to screen for a high-affinity anti-DNMT3A monoclonal antibody, scFv-A7, and innovatively combining it with a nuclear-targeted delivery system to validate its therapeutic potential in an OA model. This antibody directly targets the epigenetic pathway of DNMT3A, providing a novel strategy for OA treatment that breaks through existing symptom-relief modalities. Summary of the Invention
[0005] To address the aforementioned problems, the present invention first provides an anti-DNMT3A antibody, wherein the heavy chain variable region (VH) comprises a complementarity-determining region (CDR) sequence selected from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the light chain variable region (VL) comprises a CDR sequence selected from SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
[0006] In some embodiments, the amino acid sequence is shown in SEQ ID NO:7.
[0007] The present invention also provides a nucleic acid molecule that encodes the aforementioned antibody.
[0008] The present invention also provides an expression vector comprising the above-described nucleic acid molecules.
[0009] The present invention also provides a host cell comprising the above-described expression vector, wherein the host cell is a HEK293F cell.
[0010] The present invention also provides a pharmaceutical composition comprising the antibody described above and a pharmaceutically acceptable carrier.
[0011] The present invention also provides the use of the above-described antibody in the preparation of a medicament for treating osteoarthritis.
[0012] The present invention also provides a method for treating osteoarthritis, comprising administering a therapeutically effective amount of the above-mentioned antibody to an individual in need.
[0013] In some embodiments, the antibody forms nanoparticles with protamine to enhance nuclear delivery within cells.
[0014] Finally, the present invention provides a kit comprising the aforementioned antibody and instructions for use for intra-articular injection.
[0015] Compared with the prior art, the present invention has at least the following beneficial effects: (1) High affinity and specificity: The scFv-A7 antibody provided in this invention was obtained through phage panning optimization. Its EC50 value for binding to DNMT3A protein is 5.2 nM, classifying it as a high-affinity antibody. It can specifically target DNMT3A, avoiding off-target effects. For example... Figure 4 As shown, ELISA detection revealed significant antibody binding activity.
[0016] (2) Effective nuclear delivery and targeting: The antibody's C-terminus is embedded with the SV40 nuclear localization signal (NLS). After forming nanoparticles with protamine, it can enter the cell via endocytosis and be actively transported to the nucleus under the mediation of importin-α / β. The abundance of scFv in the nucleus reaches 9-12 ng / μg nucleoprotein (high-dose group), directly inhibiting DNMT3A activity and expression (enzyme activity decreased to 0.6±0.2, and the grayscale ratio of expression level was 0.7±0.2).
[0017] (3) Significant therapeutic improvement: In a mouse model of osteoarthritis, antibody treatment reduced serum levels of inflammatory factors IL-1β and IL-6 (e.g., Figure 5 (as shown), and improves joint function.
[0018] (4) Innovative treatment strategy: The anti-DNMT3A antibody was applied to the treatment of OA for the first time. It delayed cartilage degeneration through epigenetic regulation, made up for the poor selectivity of small molecule inhibitors, and provided a new approach for the disease modification treatment of OA. Attached Figure Description
[0019] Figure 1 The image shows the results of ELISA detection of serum antibody titers in mice after immunization.
[0020] Figure 2 Results of ELISA screening for phage clone affinity.
[0021] Figure 3 This is an SDS-PAGE plot on A7.
[0022] Figure 4 This is a graph showing the binding activity of the A7 antibody detected by ELISA.
[0023] Figure 5 This is a graph showing the detection of serum inflammatory factors after treatment. Figure 5 In the middle, A represents the detection result of IL1β; Figure 5 B represents the detection result of IL6. Detailed Implementation
[0024] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.
[0025] Example 1: Preparation of Monoclonal Antibodies (1) Expression of recombinant protein DNMT3A According to NCBI records, the catalytic domain (628-912) of DNMT3A [Homo sapiens] was selected for expression. Six histidine residues (His6) and a SUMO tag were added before the target protein to construct a pET28b vector, which was then transformed into E. coli BL21 competent cells for expression. IPTG was added for induction when the OD600 reached approximately 0.8 at 37°C, and the cells were incubated overnight at 16°C. His6-SUMO-DNMT3A was purified using a Ni column, followed by removal of the His6-SUMO tag by ULP1 restriction enzyme digestion. Finally, further purification was achieved through ion exchange and molecular sieve filtration to obtain the target recombinant protein.
[0026] (2) Immunized animals 50 μg of DNMT3A was mixed with an equal volume of complete Freund's adjuvant (CFA) and emulsified at 4°C to prepare a fully emulsified solution, which was then administered subcutaneously at multiple sites in 5 Balb / c mice. Two weeks after the primary immunization, 50 μg of DNMT3A was mixed with an equal volume of incomplete Freund's adjuvant (IFA) and emulsified at 4°C for a second subcutaneous immunization. Two weeks later, a third immunization was administered after emulsifying the protein and IFA again. Seven days after the third immunization, 100 ng of DNMT3A protein was coated onto the emulsion, and a small amount of serum was serially diluted and incubated for 2 hours. Antibody titers were detected by ELISA using anti-mouse IgG-HRP. The results are as follows. Figure 1 . Figure 1 The results showed that the serum after the third immunization was still positive after being diluted 364,500 times, indicating that the immunogenicity of the DNMT3A recombinant protein had met the requirements for library construction.
[0027] (3) Construction of the immune repositories Two weeks after the third immunization, mice were sacrificed, and their spleens were isolated. RNA was extracted and reverse transcribed to obtain cDNA. Using the cDNA as a template, a first round of multi-primer PCR was performed to amplify VH and VL cells separately. A second round of extension PCR was performed to construct scFv fragments from VH and VL, with a product of approximately 750 bp. The recovered product was ligated into the pComb vector, and all cells were electroporated into XL1-Blue competent cells. After transformation, the cells were added to culture medium for one hour of recovery and then plated. 10 μl of the stock solution was serially diluted and plated. The library size was determined based on the number of clones, and the positive rate was determined by PCR. The next day, a portion of the bacteria was scraped from the plate and inoculated into 2XYT medium at 37°C. When the OD600 reached 0.8, helper phages were added, and the cells were incubated statically at 37°C for 30 min, followed by overnight incubation at 220 rpm. The supernatant was collected by centrifugation, concentrated with PEG8000 / NaCl, and resuspended in PBS after high-speed centrifugation to obtain the phage library.
[0028] (4) Antibody panning 100 μl of DNMT3A recombinant protein (1 μg / ml) was used to coat the plate overnight at 4°C. The plate was then blocked with 3% MPBS (PBS + 3% skim milk powder) at room temperature for 1 h. After incubation with a phage library diluted in MPBS for 1 h, the plate was washed 5 times with PBST (PBS + 0.05% Tween-20) for a total of 30 min. Finally, the plate was digested with 1 mg / ml Trypsin for 20 min and then infecting XL1-Blue. The plate was then incubated at 37°C for 30 min, followed by incubation at 220 rpm for 30 min before plating. The next day, the "over-infection, overnight expression, and precipitation" steps were repeated for the next round of screening. After three rounds of screening, 30 single clones were selected from the library. Phage supernatant treated with blocking buffer was added to the DNMT3A recombinant protein-coated ELISA plate to allow specific binding. The plates were then incubated with anti-M13-HRP antibody for affinity identification and screening. The ELISA results were analyzed, and strains with strong positive reactions were selected for sequencing to obtain their sequences. The results are as follows: Figure 2 . Figure 2 The results showed that strain 7, which had the strongest positive reaction, had the highest OD450 value, indicating that strain 7 had the strongest affinity for DNMT3A. Therefore, it was selected for sequencing analysis and expression purification.
[0029] Example 2: Purification and Expression of scFv Based on the ELISA results, strain 7 was selected for sequencing and expression purification. Strain 7 was inoculated into 2 mL of LB liquid medium containing appropriate antibiotics and cultured at 37°C with shaking at 220 rpm for 12–16 hours. Plasmid DNA was then extracted using a plasmid miniprep kit, and the amino acid sequence was obtained through sequencing (see Table 1).
[0030] Table 1. Amino acid sequence of scFv
[0031] Primers were designed to construct VL-linker-VH on the pFuse expression vector. To ensure the subsequent transport of scFv into the nucleus for function, SV40 NLS (PKKKRKV) was inserted at its C-terminus. The target plasmid was obtained using a plasmid large-scale extraction kit, achieving a viability of over 95% and a density of 2 x 10⁻⁶ on HEK293F. 6 Transfection was performed at the appropriate time. Taking 30ml of 293F cells as an example, solutions A and B were prepared separately. Solution A consisted of 1ml opti-MEM + 30μg plasmid, and solution B consisted of 1ml opti-MEM + 60μl PEI. After mixing solutions A and B and allowing them to stand for 30min, the mixture was added to HEK293F cells to induce protein expression. When the cell viability decreased to 70%, the cell supernatant was collected and purified using a Ni column to obtain the target protein, named A7. The SDS-PAGE of scFv is shown below. Figure 3As shown.
[0032] Example 3: Affinity activity of A7 100 ng of recombinant protein DNMT3A was coated onto an ELISA plate and incubated overnight at 4°C. After blocking with 2% BSA for 1 h, the plate was washed three times with PBST, incubated with serially diluted A7 at room temperature for 2 h, washed three times with PBST, incubated with anti-his-HRP antibody for 1 h, washed five times with PBST, and the reaction was terminated with 1M sulfuric acid after TMB color development. The OD450 was measured using an ELISA reader to obtain the binding EC50. The results are as follows: Figure 4 . Figure 4 Affinity assay results showed that the purified A7 antibody could specifically bind to DNMT3A protein with an EC50 value of 5.2 nM, classifying it as a high-affinity antibody.
[0033] Example 4: Application of A7 in a mouse model of osteoarthritis To enable scFv to successfully target DNMT3A, A7 and protamine were mixed in a 1:1 ratio and incubated at room temperature for 20 min, followed by electrostatic self-assembly into nanoparticles. On one hand, protamine, through its surface charge, can electrostatically attract and induce endocytosis of the negatively charged cell membrane, while a large amount of buffered H+ causes vesicle rupture, releasing the nanoparticles into the cytoplasm. On the other hand, the released free scFv, guided by NLS, can be recognized by importin-α / β to form a transport complex, actively transporting into the nucleus along the nuclear pore complex.
[0034] Eight-week-old C57BL / 6J mice underwent DMM (medial meniscus instability) surgery to induce osteoarthritis (OA). Postoperatively, OA pathological features (such as cartilage degeneration and synovitis) were confirmed, and intra-articular injection of drugs was initiated one week later. To expand the nuclear pore and increase the effective dose of intranuclear scFv, importazole (an importin-α / β inhibitor, 40 mg / kg) was administered intraperitoneally 30 minutes after each injection as adjuvant therapy.
[0035] The specific groupings (n=10) are as follows: Control group: Healthy mice + 10 μL PBS, administered on days 0, 3, 6, and 9; Model group: OA model + 10μL PBS, administered on days 0, 3, 6, and 9; Low-dose treatment group: OA model + 10 μL A7 (0.5 mg / kg), administered on days 0, 3, 6, and 9; High-dose treatment group: OA model + 10 μL A7 (2 mg / kg), administered on days 0, 3, 6, and 9; After group intervention, the treatment effect was evaluated. First, 24 hours after the last administration, blood was collected from the orbital region of mice to detect the serum levels of inflammatory factors IL6 and IL1β. The results are as follows: Figure 5 As shown, the levels of inflammatory factors IL1β and IL6 in serum decreased after treatment compared with the model group.
[0036] One week later, the pain-function changes before and after treatment were monitored using the HSS score, and the results are shown in Table 2.
[0037] Table 2 HSS Scores
[0038] Compared with the control group, the model group mice showed significant changes in pain and function, and pain and function recovered after treatment, especially after high-dose treatment.
[0039] Finally, at the time of mouse sacrifice, articular cartilage was collected, ground with liquid nitrogen, lysed with hypotonic solution, centrifuged, and the precipitate was resuspended to obtain nuclear extract. After BCA quantification, the same amount of protein was used for detection. On the one hand, the quantitative concentration of intranuclear scFv can be monitored by ELISA. A standard curve was prepared by serially diluting purified scFv, and the abundance of scFv in the nuclear extract was detected by anti-his-HRP. The results are shown in Table 3.
[0040] Table 3. Abundance of scFv in the nucleus
[0041] scFv was detected in the nucleus after drug administration, with the high concentration group showing an abundance of approximately 10 ng / μg, indicating that the nanoparticles can carry scFv into the nucleus. On the other hand, the same amount of protein was loaded, and the kit was used to detect DNMT3A activity levels. Western blotting analysis of anti-DNMT3A expression changes was performed, and the results are shown in Table 4.
[0042] Table 4. Analysis of DNMT3A activity and expression levels
[0043] Compared with the model group, the enzyme activity and expression level of DNMT3A decreased significantly after drug administration.
[0044] The above results demonstrate that nanoparticles carrying scFv-A7 into the nucleus can effectively reduce the inflammatory response of IL6 and IL1β by targeting DNMT3A to inhibit its activity and expression, thereby alleviating pain and functional symptoms in a mouse OA model and providing a novel candidate strategy for OA treatment.
[0045] In summary, this invention, through phage screening, prepared and screened scFv-A7 antibodies against DNMT3A, determined the amino acid sequence of the antibody's variable region, and evaluated the therapeutic effect of A7 in a mouse osteoarthritis (OA) model. DNA methyltransferase 3α (DNMT3A) is highly expressed in human degenerated cartilage tissue, along with apoptosis-related proteins. Strategies to inhibit DNMT3A expression and effectively delay OA progression warrant further investigation. The screened anti-DNMT3A monoclonal antibody shows promise as a treatment option for osteoarthritis.
[0046] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. An anti-DNMT3A antibody, characterized in that, Its heavy chain variable region (VH) contains complementarity determination region (CDR) sequences selected from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and its light chain variable region (VL) contains CDR sequences selected from SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:
6.
2. The antibody according to claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO:
7.
3. A nucleic acid molecule, characterized in that, It encodes the antibody as described in claim 1 or 2.
4. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 3.
5. A host cell, characterized in that, The expression vector of claim 4 is included, wherein the host cell is HEK293F cell.
6. A pharmaceutical composition, characterized in that, It comprises the antibody as described in claim 1 or 2 and a pharmaceutically acceptable carrier.
7. Use of the antibody according to claim 1 or 2 in the preparation of a medicament for treating osteoarthritis.
8. A method for treating osteoarthritis, characterized in that, This includes administering a therapeutically effective amount of the antibody as described in claim 1 or 2 to an individual in need.
9. The method according to claim 8, characterized in that, The antibody forms nanoparticles with protamine to enhance delivery into the cell nucleus.
10. A reagent kit, characterized in that, It includes the antibody as described in claim 1 or 2 and instructions for use for intra-articular injection.