Application of humanized DKK-1 monoclonal antibody in preparation of medicine for treating osteoporosis

Abstract

The invention discloses an application of a humanized DKK-1 monoclonal antibody in preparation of a medicine for treating osteoporosis. A light chain and a heavy chain of the humanized DKK-1 monoclonal antibody are shown as SEQ ID NO.1-2. The invention further discloses a preparation method of the humanized DKK-1 monoclonal antibody. Animal experiments prove that the humanized DKK-1 monoclonal antibody can remarkably improve the bone mineral density of lumbar vertebrae and tibia of an osteoporosis model mouse and can promote bone growth, and a new treatment scheme is provided for treating osteoporosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a humanized DKK-1 monoclonal antibody in the preparation of a drug for treating osteoporosis. Background Technology

[0002] Osteoporosis (OP) is a common, progressive, systemic skeletal disease characterized by decreased bone mineral density accompanied by microstructural degradation, leading to increased bone fragility and a heightened risk of fractures. Currently, bisphosphonates, calcitonin, estrogen, and raloxifene are commonly used medications for treating osteoporosis. However, the adverse effects of these drugs severely limit their clinical application. For example, hormone-related therapies such as estrogen can maintain bone density but increase the risk of cancer, blood clots, and heart disease; bisphosphonates can inhibit bone resorption and promote bone formation, but they can cause gastrointestinal problems such as esophagitis and gastric ulcers. Therefore, there is a need to develop more drugs with fewer adverse effects to prevent and treat OP.

[0003] Reported DKK-1 antibodies mainly include: DKN-01, BHQ-880, DKK1-A2, RH2-18, and Hetero-DS. Among them, DKN-01 is a humanized anti-DKK-1 IgG4 monoclonal antibody developed by Leap Therapeutics for the treatment of various tumors, such as cholangiocarcinoma, endometrial cancer, gastric cancer, esophageal cancer, liver cancer, ovarian cancer, and prostate cancer, and is currently in Phase II clinical trials. BHQ-880 can be used to treat multiple myeloma and osteosarcoma. DKK1-A2 recognizes the DKK1 P20 peptide and is used to treat hematologic malignancies and solid tumors. RH2-18 can treat postmenopausal osteoporosis. Hetero-DS can treat postmenopausal osteoporosis and fractures. In the applicant's prior authorized Chinese invention patent CN119318703B, the application of a humanized DKK-1 monoclonal antibody in the preparation of a drug for treating Alzheimer's disease is disclosed. Animal experiments have confirmed that the humanized DKK-1 monoclonal antibody can prolong the survival time of Alzheimer's mice, improve the cognitive level of Alzheimer's mice, improve the memory ability of Alzheimer's mice, and lower blood sugar, indicating that the humanized DKK-1 monoclonal antibody has the effect of treating Alzheimer's disease and can alleviate the symptoms of senile dementia.

[0004] As can be seen from the above, although there are reports of DKK-1 antibodies acting on osteoporosis, the existing DKK-1 antibodies have limited effect on improving osteoporosis. The inventors of this application, through further research, discovered that the above-mentioned humanized DKK-1 monoclonal antibody has a better effect on treating osteoporosis, and thus completed this invention. Summary of the Invention To address the aforementioned problems, this invention provides an application of a humanized DKK-1 monoclonal antibody in the preparation of a drug for treating osteoporosis, and specifically provides the following technical solution: This invention provides the use of a humanized DKK-1 monoclonal antibody in the preparation of a drug for treating osteoporosis, wherein the light and heavy chains of the humanized DKK-1 monoclonal antibody are as shown in SEQ ID NO.1-2.

[0005] In a preferred embodiment of the invention, the application is for the elderly.

[0006] In a preferred embodiment of the invention, the application includes increasing bone density, particularly increasing bone density of the lumbar vertebrae and tibia.

[0007] In a preferred embodiment of the present invention, the drug is an injectable preparation.

[0008] In a preferred embodiment of the present invention, the drug is an injectable preparation for subcutaneous injection.

[0009] In a preferred embodiment of the present invention, the drug further includes other pharmaceutical excipients or the drug is a lyophilized powder injection.

[0010] Beneficial effects: Animal experiments have demonstrated that the humanized DKK-1 monoclonal antibody of this invention can significantly improve bone density in the lumbar vertebrae and tibia of osteoporosis model mice and promote bone growth, providing a new treatment option for osteoporosis. Attached Figure Description

[0011] Figure 1 These are the statistical results of lumbar vertebral bone mineral density in laboratory mice. This indicates a statistically significant difference (p < 0.05). This indicates a statistically significant difference (p < 0.01). This indicates a statistically significant difference (p < 0.001).

[0012] Figure 2 These are the statistical results of tibial bone mineral density in laboratory mice. This indicates a statistically significant difference (p < 0.05). This indicates a statistically significant difference (p < 0.01). This indicates a statistically significant difference (p < 0.001).

[0013] Figure 3The results of calcein fluorescence labeling in mouse tibial tissue are shown. Two green fluorescent bands are visible in the image, corresponding to the newly formed bone deposition sites marked on day 13 and day 3 before tissue collection, respectively. The width of the fluorescent bands (as indicated by the arrows) reflects the bone formation rate during that time period; a wider band indicates better bone growth.

[0014] Figure 4 These are statistical results of mineral deposition rate in laboratory mice. This indicates a statistically significant difference (p < 0.05). Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0016] Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are all commercially available.

[0017] Example 1: Preparation of DKK-1 monoclonal antibody The preparation and bioactivity verification of the humanized DKK-1 monoclonal antibody are described in prior art patent 202410979827.5 (application number) and are incorporated herein by reference. Specifically, the light and heavy chain amino acid sequences of the DKK-1 monoclonal antibody used in this embodiment are as follows: Light chain sequence (SEQ ID NO.1): DIQMTQSPSSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKLLISYTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYSKLPLTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain sequence (SEQ ID NO.2): EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSSISTGGTTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVSEIYWPMDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Example 2: Verification of the efficacy of a humanized DKK-1 monoclonal antibody in the treatment of osteoporosis Thirty-two 12-month-old female C57BL / 6 mice were randomly divided into four groups (n=8 per group): sham operation group, osteoporosis model + IgG group, osteoporosis model + DKK-1 monoclonal antibody treatment group, and positive control osteoporosis model + DKK-1-01 treatment group. In this study, the DKK-1 treatment group received weekly subcutaneous injections of DKK-1 monoclonal antibody (25 mg / kg), diluted in 100 μL PBS; the DKK-1-01 treatment group received weekly subcutaneous injections of DKK-1-01 monoclonal antibody (25 mg / kg, commercially available as Sirexatambaab, whose light and heavy chain amino acid sequences are shown in SEQ ID No. 3 and SEQ ID No. 4), diluted in 100 μL PBS; and the control group and the model + IgG group received weekly injections of an equal volume of PBS or isotype control IgG. The experiment used 12-week-old female C57BL / 6 mice. An osteoporosis model was established through ovariectomy (OVX) surgery (bilateral ovariectomy (OVX) was used to construct the osteoporosis model. After anesthesia, the skin and muscle layers were incised below the costal margin on the back, and both ovaries were ligated and removed. The control group underwent sham surgery: only the ovaries were exposed without removal). A postmenopausal osteoporosis model was established, and a sham surgery group was set up as a control. After modeling, the OVX group was divided into an IgG control group (OVX-control), a DKK-1 monoclonal antibody (OVX-Ab-DKK-1) (monoclonal antibody dose of 25 mg / kg, once a week, subcutaneous injection for three months), and a DKK-1-01 monoclonal antibody. Three months later, samples were collected, and the isolated bone mineral density (BMD) of the mouse lumbar vertebrae and tibia was analyzed using dual-energy X-ray absorptiometry (DXA). The results are as follows. Figure 1 and 2 As shown, the results indicate that, compared with the untreated OVX model group, DKK-1 monoclonal antibody treatment significantly improved bone health and effectively increased bone mineral density. Compared with the positive control DKK-1-01 monoclonal antibody, DKK-1 monoclonal antibody treatment also significantly improved bone health and effectively increased bone mineral density.

[0018] Light chain sequence (SEQ ID NO.3): EIVLTQSPATLSLSPGERATLSCHASDSISNSLHWYQQKPGQAPRLLIYYARQSIQGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSESWPLHFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain sequence (SEQ ID NO.4): EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQAPGKGLEWVATISGGGFGTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPGYNNYYFDIWGQ GTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG To further evaluate bone formation kinetics, all mice were administered calcein (10 mg / kg, intraperitoneally) twice, on days 13 and 3 before the experimental endpoint (sampling). Fluorescent labeling analysis of bone tissue sections yielded the following results: Figure 3 The experimental results showed that, compared with the model control group, the DKK-1 monoclonal antibody treatment group had a significantly wider calcein marker band spacing, indicating faster bone regeneration. Based on this, the calculated mineral deposition rate (MAR) was significantly higher. Figure 4 This demonstrates that the monoclonal antibody can effectively promote bone growth.

[0019] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of a humanized DKK-1 monoclonal antibody in the preparation of a medicament for treating osteoporosis, wherein the light and heavy chains of the humanized DKK-1 monoclonal antibody are as shown in SEQ ID NO.1-2.

2. The application according to claim 1, wherein the application is for the elderly.

3. The application according to claim 1, wherein the application includes increasing bone density.

4. The application according to claim 1, wherein the application includes increasing bone mineral density of the lumbar spine and tibia.

5. The application according to claim 1, wherein the drug is an injectable preparation.

6. The application according to claim 5, wherein the drug is an injectable preparation for subcutaneous injection.

7. In the application according to claim 5, the drug further includes other pharmaceutical excipients or the drug is a lyophilized powder for injection.

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