Application of CD40 antibody in preparation or screening of multiple myeloma bone disease products

Abstract

The invention discloses a therapeutic target CD40 related to multiple myeloma bone diseases and application of a monoclonal antibody of the therapeutic target CD40. On one hand, it is found that formation and osteogenic differentiation of primary cilia of BMSCs can be effectively recovered by adding a CD40 neutralizing antibody into an MM-BMSCs co-culture system; on the other hand, the CD40 neutralizing antibody is found to reduce bone destruction and tumor load in a Vk * MYC myeloma mouse model. CD40 is expected to become a potential target spot for treating multiple myeloma bone diseases and has a very wide application prospect in the field of medical research.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to the application of CD40 antibody in the preparation or screening of products for multiple myeloma bone disease. Background Art

[0002] Multiple myeloma bone disease (MBD) is a hallmark feature of multiple myeloma (MM), and approximately 80 - 90% of MM patients are clinically affected. These patients are at high risk of skeletal-related events, including pathologic fractures, spinal cord compression, and the need for surgical or radiotherapy intervention. Skeletal-related events greatly increase the disease burden of MM, both in terms of survival and quality of life, and in terms of public health costs. Its pathogenesis stems from the accumulation of tumor cells in the bone marrow and the subsequent bone destruction. The core pathological process of MBD is mainly manifested as the inhibition of osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and the enhanced osteoclastic activity of osteoclasts derived from macrophages, resulting in an imbalance in bone remodeling. In addition, MBD accelerates disease progression by forming a microenvironment (i.e., "myeloma niche") that is conducive to bone resorption and tumor proliferation. The complex pathogenesis of MBD makes it still incurable. Therefore, there is an urgent need to continue to search for effective new therapeutic targets. Although a large number of studies have explored the mechanism of impaired osteogenic differentiation in MBD, its core regulatory elements and molecular mechanisms are still unclear.

[0003] Bisphosphonates (BPs) are the gold standard for the prevention and treatment of MM-related bone disease. BPs are pyrophosphate analogs that bind to the exposed regions of hydroxyapatite crystals during bone remodeling. When osteoclasts resorb bone, the BPs embedded in the bone are released, causing rearrangement of the osteoclast cytoskeleton, which can lead to osteoclast apoptosis and prevent bone loss. All active MM patients can take bisphosphonates (i.e., zoledronic acid or pamidronic acid) regardless of the presence or absence of MM-related bone disease in imaging studies. Zoledronic acid is also suitable for the treatment of hypercalcemia caused by MM-related osteolytic disease and is superior to pamidronic acid in this case. For patients with smoldering multiple myeloma, monoclonal gammopathy of undetermined significance, or solitary plasmacytoma, bisphosphonates are only recommended in the presence of coexistent osteoporosis. Nevertheless, some patients still have poor responses, and long-term use may lead to a decrease in efficacy due to adaptive changes in osteoclasts; in addition, serious adverse reactions such as osteonecrosis of the jaw and acute renal failure induced by the use of bisphosphonates should be particularly emphasized, which seriously affects the efficacy of the MBD treatment regimen.

[0004] In-depth research on the potential pathophysiological mechanisms of multiple myeloma-related bone diseases has led to the clinical development of other targeted drugs, such as Denosumab. Denosumab is a fully human and highly specific monoclonal IgG2 antibody against RANKL, which reduces bone resorption by inhibiting the interaction between RANKL and RANK. Denosumab is used to treat newly diagnosed MM, as well as relapsed or refractory MM patients with evidence of multiple bone diseases, and is comparable to zoledronic acid in terms of delaying the time to the first skeletal-related event after MM diagnosis. In addition, it may prolong the progression-free survival of newly diagnosed MM and MBD patients eligible for autologous stem cell transplantation. In MM patients with renal dysfunction, Denosumab is superior to zoledronic acid and is used for patients with hypercalcemia related to myeloma, especially those who are ineffective in zoledronic acid treatment. So far, there is no difference in overall survival between zoledronic acid and Denosumab. However, compared with bisphosphonates, the treatment cost of Denosumab has increased significantly, which may limit its long-term application. In addition, significant inhibition of bone resorption may lead to severe hypocalcemia, especially in patients with renal insufficiency or vitamin D deficiency, and calcium and vitamin D need to be strictly monitored and supplemented.

[0005] CD40 is an important member of the tumor necrosis factor receptor superfamily (TNFRSF5). As a type I transmembrane glycoprotein, it is mainly expressed on the surface of antigen-presenting cells such as B cells, dendritic cells, and macrophages. By binding to its ligand CD40L (CD154), it activates downstream signaling pathways such as NF-κB and MAPK, and plays a core regulatory role in adaptive immune responses such as B cell activation, antibody production, and germinal center formation. In addition, CD40 is also involved in tumor immune escape, inflammatory responses, and the occurrence of autoimmune diseases. It is both a therapeutic target for various B cell malignancies and an important co-target for immune checkpoint therapies (such as combined with PD-1 inhibitors). However, the regulatory mechanism and clinical treatment of CD40 for MBD have not been reported so far. Summary of the Invention

[0006] The present invention proposes the application of CD40 antibodies in the preparation or screening of products for multiple myeloma bone diseases in view of the problems existing in the traditional clinical treatment of multiple myeloma, specifically involving the application of the therapeutic target CD40 related to multiple myeloma bone diseases and its monoclonal antibodies.

[0007] In order to achieve the above object, the present invention is implemented by the following technical solutions: One object of the present invention is to provide a new use of a substance that blocks CD40-CD40L binding (i.e., a CD40 antibody), wherein the CD40 gene sequence is as shown in SEQ ID NO: 1, the CD40 protein sequence is as shown in SEQ ID NO: 2, the CD40L gene sequence is as shown in SEQ ID NO: 3, the CD40L protein sequence is as shown in SEQ ID NO: 4, the human CD40 antibody sequence is as shown in SEQ ID NO: 5 and SEQ ID NO: 6, and the human CD40 antibody gene sequence is as shown in SEQ ID NO: 7 and SEQ ID NO: 8.

[0008] Specifically, the present invention provides the application of a CD40 antibody in any one or more of the following products A1-A4: A1: Preparing or screening products for treating multiple myeloma and / or bone diseases.

[0009] A2: Preparing or screening products for blocking the interaction between multiple myeloma cells and BMSCs.

[0010] A3: Preparing or screening products for relieving myeloma bone destruction.

[0011] A4: Preparing or screening products for reducing the tumor burden of multiple myeloma.

[0012] Another object of the present invention is to provide the application of a CD40 antibody as a substance for blocking the interaction between multiple myeloma cells and BMSCs through CD40-CD40L. The application of the product includes any one or more of the above A1-A4.

[0013] In any of the above applications, the CD40 has at least one of the following characteristics B1 and B2: B1: Expressing on the surface of multiple myeloma cells and binding to CD40L on the surface of BMSCs.

[0014] B2: Regulating the formation of primary cilia and osteogenic differentiation of BMSCs in the myeloma bone marrow microenvironment.

[0015] That is, the present invention proposes that the CD40 regulates the formation of primary cilia of BMSCs by binding to the ligand CD40L on the surface of BMSCs on the surface of myeloma cells, or the CD40 regulates osteogenesis by binding to the ligand CD40L on the surface of BMSCs on the surface of myeloma cells.

[0016] In any of the above products, it contains any substance known to those skilled in the art that can block CD40-CD40L communication, such as CD40 neutralizing antibodies, polypeptides, small molecule compounds, etc.

[0017] The present invention provides the use of a CD40 neutralizing antibody in screening, treating or adjuvant treating myeloma products, and the CD40 neutralizing antibody can block the binding of CD40 to its ligand CD40L.

[0018] In any of the above-mentioned uses or products, the myeloma is multiple myeloma.

[0019] The present invention provides the use of CD40 as a target in developing or designing products for treating or adjuvant treating multiple myeloma.

[0020] The present invention provides the use of a substance that regulates the binding of CD40 on the surface of myeloma cells to CD40L on the surface of BMSCs and affects the formation of primary cilia of BMSCs.

[0021] The present invention further provides the use of a substance that regulates the binding of CD40 on the surface of myeloma cells to CD40L on the surface of BMSCs and affects the osteogenic differentiation of BMSCs.

[0022] The myeloma cells are multiple myeloma cells, specifically human multiple myeloma cells derived from cell lines or multiple myeloma CD138 from patients + bone marrow cells.

[0023] The present invention first detected the effect of a CD40 neutralizing antibody on the formation of primary cilia of BMSCs in a co-culture system. The results showed that direct contact co-culture of myeloma cells ARH-77 and BMSCs cells for 24 hours inhibited the formation of primary cilia and osteogenic differentiation of BMSCs; after adding a CD40 neutralizing antibody with a final concentration of 5 μg / mL to the co-culture system, the ability to form primary cilia and osteogenic differentiation of BMSCs could be restored.

[0024] The present invention further explored the effect of a CD40 neutralizing antibody on alleviating bone destruction in vivo. The results showed that a Vk*MYC MM mouse model was constructed, and a CD40 neutralizing antibody was injected via the tail vein, 5 μg CD40 neutralizing antibody / 100 μl 1×PBS / 20 g mouse, injected for 3 weeks, twice a week, and the CD40 neutralizing antibody could effectively alleviate bone destruction.

[0025] The present invention further detected the in vitro and in vivo cytotoxic effects of a CD40 neutralizing antibody on myeloma cells and BMSCs. To clarify the correlation between tumor burden and improvement of bone injury, the in vitro and in vivo cytotoxic effects of a CD40 neutralizing antibody on myeloma cells were detected. In vitro experiments showed that antibody doses below 10 µg / mL failed to induce the death of myeloma cells, while significant cytotoxicity was exhibited when the dose exceeded 10 µg / mL. The results of the in vivo Vk*MYC mouse model showed that the proportion of plasma cells in the bone marrow in the CD40 neutralizing antibody treatment group (CD138 + B220- The proportions of M protein in both bone marrow aspirates and peripheral blood serum decreased. However, the CD40 neutralizing antibody did not show cytotoxicity to human- and mouse-derived BMSCs.

[0026] The present invention further examined the effect of the CD40 neutralizing antibody on osteoclasts in Vk*MYC mice. The results of TRAP staining showed that the number of osteoclasts in Vk*MYC mice treated with the CD40 neutralizing antibody was significantly reduced compared to the IgG control group. The mouse CD40 antibody sequences are shown in SEQ ID NO: 9 and SEQ ID NO: 10, and the mouse CD40 antibody gene sequences are shown in SEQ ID NO: 11 and SEQ ID NO: 12.

[0027] Compared with the prior art, the advantages and positive effects of the present invention are as follows: On the one hand, the experimental results of the present invention show that blocking the CD40-CD40L pathway using the CD40 neutralizing antibody can provide a promising therapeutic strategy for alleviating the disease progression and bone destruction of multiple myeloma patients; on the other hand, it shows that in the bone marrow microenvironment of multiple myeloma, MM cells inhibit the primary cilia formation and osteogenic differentiation of BMSCs. These research findings bring hope for accelerating the acquisition of reliable preclinical results and are crucial for proposing innovative treatment methods for MBD and MM. Primary cilia may regulate bone growth and development through a basic mechanism, which will promote its combination with different treatment methods and further drive the progress in the fields of bone tissue engineering and bone disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 are the immunofluorescence and alizarin red osteogenic staining results of BMSCs after treatment with the CD40 neutralizing antibody in the co-culture system. Among them, Figure 1 A shows the expression and localization of primary cilia acetyl-α tubulin and OFD1 detected by immunofluorescence of BMSCs in the non-co-culture group; Figure 1 B shows the expression and localization of primary cilia acetyl-α tubulin and OFD1 detected by immunofluorescence of BMSCs in the co-culture group with ARH-77 after treatment with the IgG antibody; Figure 1 C shows the expression and localization of primary cilia acetyl-α tubulin and OFD1 detected by immunofluorescence of BMSCs in the co-culture system after treatment with the CD40 neutralizing antibody; Figure 1 D is a statistical chart of the number of BMSCs cells with primary cilia; Figure 1 E is a statistical chart of the length of primary cilia of BMSCs; Figure 1 F is the number of BMSCs cells in which OFD1 is localized to the centriolar satellite; Figure 1G shows the alizarin red staining results of BMSCs in the non-coculture group after being induced with osteogenic induction medium for 14 days; Figure 1 H shows the alizarin red staining results of BMSCs in the group co-cultured with ARH-77 after treatment with IgG antibody and being induced with osteogenic induction medium for 14 days; Figure 1 I shows the alizarin red staining results of BMSCs in the co-culture system after treatment with CD40 neutralizing antibody and being induced with osteogenic induction medium for 14 days.

[0029] Figure 2 It shows the effect of CD40 neutralizing antibody on bone destruction in the Vk*MYC myeloma mouse model. Among them, Figure 2 The left figure in A shows the effect of CD40 neutralizing antibody on bone injury in Mock group mice analyzed by micro-CT, and the right figure shows the results of H&E staining and Masson staining; Figure 2 The left figure in B shows the effect of CD40 neutralizing antibody on bone injury in Vk*MYC group mice analyzed by micro-CT, and the right figure shows the results of H&E staining and Masson staining; Figure 2 C-G show the quantitative analysis results of bone injury. Among them, Figure 2 C is the statistical chart of bone volume fraction, BV / TV: percentages of bone volume density; Figure 2 D is the statistical chart of trabecular thickness, Tb.Th: trabecula thickness; Figure 2 E is the statistical chart of trabecular number, Tb.N: trabecula numbers; Figure 2 F is the statistical chart of trabecular porosity, Tb.Sp: trabecula separation; Figure 2 G is the statistical chart of bone mineral density, BMD: bone mineral density.

[0030] Figure 3 It shows the detection of the cytotoxic effect of CD40 neutralizing antibody on myeloma cells. Among them, Figure 3 A shows the CCK8 assay results after treating U266 myeloma cells with different concentrations of CD40 neutralizing antibody for 24 hours and 48 hours; Figure 3 B shows the CCK8 assay results after treating LP-1 myeloma cells with different concentrations of CD40 neutralizing antibody for 24 hours and 48 hours; Figure 3 C shows the CCK8 assay results after treating MM.1S myeloma cells with different concentrations of CD40 neutralizing antibody for 24 hours and 48 hours; Figure 3 D shows the CCK8 assay results after treating ARH-77 myeloma cells with different concentrations of CD40 neutralizing antibody for 24 hours and 48 hours.

[0031] Figure 4 To detect the effect of CD40 neutralizing antibody on Vk*MYC murine myeloma cells. Among them, Figure 4 A shows the M protein band in the peripheral blood serum of Mock group mice after treatment with IgG or CD40 neutralizing antibody detected by Serum Protein Electrophoresis (SPEP); Figure 4 B shows the M protein band in the peripheral blood serum of Vk*MYC group mice after treatment with IgG or CD40 neutralizing antibody detected by Serum Protein Electrophoresis; Figure 4 C is the statistical analysis chart of the percentage of M protein in the peripheral blood serum of Vk*MYC group mice after treatment with IgG or CD40 neutralizing antibody; Figure 4 D is the statistical analysis chart of the proportion of plasma cells in the bone marrow of Vk*MYC group mice after treatment with IgG or CD40 neutralizing antibody.

[0032] Figure 5 To detect the cytotoxic effect of CD40 neutralizing antibody on BMSCs. Among them, Figure 5 A shows the CCK8 assay results after treating human-derived BMSCs cells with different concentrations of CD40 neutralizing antibody for 24 hours and 48 hours; Figure 5 B shows the CCK8 assay results after treating mouse-derived BMSCs cells with different concentrations of CD40 neutralizing antibody for 24 hours and 48 hours.

[0033] Figure 6 To detect the effect of CD40 neutralizing antibody on bone resorption in Vk*MYC mice. Among them, Figure 6 A shows the tartrate resistant acid phosphatase (TRAP) staining results of the femur tissues of Vk*MYC group mice after treatment with IgG or CD40 neutralizing antibody. Among them, TRAP: tartrate resistant acid phosphatase; Figure 6 B shows the quantitative statistical results of osteoclast staining of the femur tissues of Vk*MYC group mice after treatment with IgG or CD40 neutralizing antibody. Among them, the number of osteoclasts per unit tissue area, N.Oc / T.A: the number of osteoclasts per unit tissue area. Detailed implementation manners

[0034] In order to more clearly understand the above objects, features and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be noted that, without conflict, the embodiments of the present application and the features in the embodiments can be combined with each other.

[0035] In the following description, numerous specific details are set forth to provide a thorough understanding of the present invention. However, the present invention may be practiced in other ways than those specifically described herein. Therefore, the present invention is not limited to the limitations of the specific embodiments disclosed in the following specification.

[0036] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0037] Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available. The percentage of substances not specifically stated in the following examples is the mass fraction.

[0038] Cells: Multiple myeloma cell lines include: MM.1S (Catalog number: 1101HUM-PUMC000680), U266 (Catalog number: 1101HUM-PUMC000684) purchased from the National Experimental Cell Resource Sharing Platform (Beijing); ARH-77 (Catalog number CRL-1621) purchased from ATCC (American Type Culture Collection); and the LP-1 cell line kindly provided by Dr. Robert Z. Orlowski of the Lymphoma and Myeloma Department of UT MD Anderson Cancer Center. The cells were cultured in RPMI-1640 medium (gbico) containing 15% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, and 2 mM L-glutamine. Human BMSCs were isolated and purified from human bone marrow blood by density gradient centrifugation and differential adherence (the samples were from the Proton Center Laboratory, with the patients informed and consenting). When the confluence of BMSCs reached 80-90%, they were passaged into 2 culture dishes, and the third-generation cells were used for subsequent experiments after growing to the required density. The culture was carried out in DMEM medium (gbico) containing 15% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and 2 mM L-glutamine. Mouse BMSCs were derived from mouse bone marrow blood and cultured in αMEM medium (gbico) containing 15% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mM L-glutamine, 10 nM dexamethasone, and 55 μM 2-ME.

[0039] Antibodies: Monoclonal antibody against acetylated α-tubulin (Lys40) (Invitrogen, catalog number 32-2700); Polyclonal antibody against OFD1 (NOVUS, catalog number NBP1-89355); Alexa Fluor™ 488-conjugated goat anti-mouse IgG (H+L) secondary antibody (Invitrogen, catalog number A11001), Alexa Fluor™ 555-conjugated donkey anti-rabbit IgG (H+L) secondary antibody (Invitrogen, catalog number A31572); Human CD40 neutralizing antibody (bioxcell, BE0189); Mouse CD40 neutralizing antibody (bioxcell, BE0016-2).

[0040] Experimental animals: 6-8-week-old sex-matched C57BL / 6J wild-type mice, purchased from Beijing Spearf Bio-Technology Co., Ltd.

[0041] Data analysis: All data were performed in three independent replicate experiments, and the results were expressed as mean ± standard error of the mean (mean ± SEM). Student's t-test was used for analysis of differences between groups. A P value < 0.05 was considered statistically significant.

[0042] Example 1 Effect of CD40 neutralizing antibody on primary cilia formation and osteogenic differentiation of BMSCs in an in vitro co-culture system.

[0043] First, BMSCs cells were seeded in advance to allow them to adhere to the wall. Then, the myeloma cell line ARH-77 was added to the adherent BMSCs cells at a ratio of 5:1, and co-cultured for 24 hours. Meanwhile, human CD40 neutralizing antibody or human IgG at a working concentration of 5 μg / mL was added. Myeloma cells were removed by CD138 magnetic beads, and BMSCs cells were collected for immunofluorescence staining to detect the expression and localization of primary cilia (acetylated α-tubulin) and OFD1, and alizarin red staining was performed after 14 days of induction with osteogenic induction medium to detect osteogenic differentiation.

[0044] The specific steps are as follows: 1. Construction of an in vitro co-culture model of myeloma cells and bone marrow mesenchymal stem cells First, 2×10 6 BMSCs from personal sources were seeded in a 10-cm culture dish and allowed to adhere statically for 12 hours. Subsequently, 1×10 7ARH-77 myeloma cells were resuspended in 10 mL of complete medium and seeded into a culture dish pre-coated with BMSCs for co-culture for 24 hours. After collecting the co-cultured cells, they were washed with 1×PBS and the myeloma cells were removed using human CD138 immunomagnetic beads according to the operating procedure to sort and purify BMSCs. In the experiment, the temporarily mixed BMSCs-ARH-77 group was used as a control, and human CD138 immunomagnetic beads were also used to remove myeloma cells to sort and purify BMSCs. The finally obtained co-culture group and control group BMSCs with 100% purity were respectively used for subsequent experimental analysis.

[0045] 2. Removal of myeloma cells using human CD138 immunomagnetic beads 1) Resuspend the above-collected cell pellet with 80 μL of separation buffer (BSA (100 mg / mL): PBS = 1:20, filtered and sterilized with 0.22 µm filter), add 20 μL of human CD138 magnetic beads, mix well by inverting up and down, and incubate at 2 - 8 °C for 15 minutes.

[0046] 2) During the incubation period, prepare a magnetic stand, install the magnet, select a suitable magnetic column, install the magnetic column on the magnet, place a 1.5 mL centrifuge tube under the magnetic column to collect the dripping liquid, and pre-wash the magnetic column with 0.5% BSA solution, and it can be observed that the washing solution drips down.

[0047] 3) Wash the incubated cells with 1 mL of separation buffer, centrifuge at 300 g for 10 minutes, discard the supernatant, and resuspend with 500 μL of separation buffer.

[0048] 4) Transfer the resuspended cells to the magnetic column. The first dripping cells are CD138 - positive cells. Collect the cells into a 1.5 mL centrifuge tube and count (repeat the column washing three times to obtain CD138 - negative cells, that is, BMSCs).

[0049] 5) CD138 + positive cells are adsorbed on the magnetic column. Remove the magnetic column from the magnet, place it in a 1.5 mL centrifuge tube, add 1 mL of separation buffer to the magnetic column, and immediately push the piston to wash down the CD138 + positive cells and count them, that is, myeloma cells, and perform sample preservation.

[0050] 3. Immunofluorescence staining The BMSCs cells obtained by the above magnetic bead sorting were fixed with 4% paraformaldehyde fixative at room temperature for 20 minutes, permeabilized with 0.5% (V / V) TritonX-100 for 15 minutes, and then blocked with 5% bovine serum albumin (BSA) at room temperature for 30 minutes. The primary antibodies (acetylated α-tubulin (Lys40) monoclonal antibody and OFD1 polyclonal antibody) were incubated overnight at 4°C. Subsequently, the corresponding fluorescent secondary antibodies (488-labeled goat anti-mouse IgG (H+L) secondary antibody and 555-labeled donkey anti-rabbit IgG (H+L) secondary antibody) were incubated in the dark at room temperature for 60 minutes, and the nuclei were counterstained with DAPI. Image acquisition was performed using an Olympus FV1000 IX81-SIM confocal microscope (Tokyo, Japan), and subsequent analysis was carried out using ImageJ software.

[0051] 4. Preparation of Osteogenic Induction Medium for Human BMSCs The cells were standardized at the initial stage of the experiment to ensure that the BMSCs cells reached a consistent confluence. When the cell confluence reached 80%, the medium was changed to osteogenic induction medium, and the cells were induced for 14 days (mature osteoblast stage), during which the medium was changed every 3 days.

[0052] The osteogenic induction medium for human BMSCs (hBMSCs) was composed of DMEM complete medium supplemented with L-ascorbic acid (50 μg / mL), dexamethasone (0.1 μmol / L), and β-glycerophosphate (10 mmol / L). Alizarin red S staining was used to evaluate the osteogenic differentiation efficiency.

[0053] 5. Alizarin Red S Staining Steps of alizarin red S staining: 1) Discard the original medium and wash twice with PBS; 2) Fix with fresh 70% ethanol at 4°C for 60 minutes or fresh 95% ethanol for 10 minutes; 3) Then wash 2-3 times with distilled water and add 1 mL / well of alizarin red solution with a concentration of 40 mM (pH: 4.2), stain at room temperature for 5-10 minutes, and observe the staining degree with the naked eye; 4) Wash 3-5 times with distilled water, gently pipette to wash away the remaining staining solution. Observe the staining under an inverted microscope and obtain the well plate scan image.

[0054] The results showed that: BMSCs had primary cilia and the OFD1 protein was localized at the basal body (as shown in Figure 1 Figure A); When BMSCs were co-cultured with ARH-77 myeloma cells and treated with IgG antibody, the formation (quantity and length) of BMSCs primary cilia was inhibited, and the OFD1 protein aggregated at the centriolar satellites (as shown in Figure 1as shown in Figure B); in the co-culture system, compared with the IgG control group, the CD40 neutralizing antibody can restore the formation (quantity and length) of primary cilia of BMSCs in the co-culture group, localize the OFD1 protein at the basal body instead of aggregating at the centriolar satellites (as Figure 1 shown in Figure C); under co-culture conditions, the quantity of primary cilia of BMSCs in the IgG group decreases, and the CD40 neutralizing antibody group can restore the quantity of primary cilia of BMSCs to a certain extent (as Figure 1 shown in Figure D); under co-culture conditions, the length of primary cilia of BMSCs in the IgG group shortens, and the CD40 neutralizing antibody group can restore the length of primary cilia of BMSCs to a certain extent (as Figure 1 shown in Figure E); under co-culture conditions, the OFD1 localized at the centriolar satellites of BMSCs in the IgG group increases, while that in the CD40 neutralizing antibody group decreases (as Figure 1 shown in Figure F); the alizarin red staining results demonstrate the osteogenic differentiation ability of human BMSCs (as Figure 1 shown in Figure G); the osteogenic differentiation ability of BMSCs treated with IgG in the co-culture group decreases (as Figure 1 shown in Figure H); for BMSCs treated with the CD40 neutralizing antibody in the co-culture group, compared with the IgG group, its osteogenic differentiation ability is improved (as Figure 1 shown in Figure I).

[0055] Example 2 Effect of CD40 neutralizing antibody on bone destruction in the Vk*MYC mouse model

[0056] In the constructed Vk*MYC model, CD40 neutralizing antibody was injected via the tail vein. The experiment was divided into four groups: control + IgG group, control + CD40 neutralizing antibody group, Vk*MYC + IgG group, and Vk*MYC + CD40 neutralizing antibody group, with 6 mice in each group. Bone destruction was analyzed by micro-CT, H&E staining, and Masson staining.

[0057] The specific steps are as follows: 1. Vk*MYC multiple myeloma mouse model The Vk*MYC cell line (Vk12653) was kindly provided by Dr. Peter Leif Bergsagel of the Mayo Clinic in the United States in 2021. The establishment of this transgenic and transplanted mouse model strictly followed the operation protocol published by the team of Dr. Bergsagel PL. To exclude host lymphocyte contamination, the cell line was Rag2 - / - Il2rg - / -Passage culture in immunodeficient mice. Four to five weeks after injecting Vk*MYC myeloma cells, a large number of malignant plasma cells infiltrated the spleens of the mice. The splenocytes with a malignant plasma cell proportion > 50% were collected and cryopreserved for later use. 5×10 5 donor Vk*MYC mouse splenocytes were injected into C57BL / 6J mice via the tail vein. One week after inoculating Vk*MYC cells, CD40 neutralizing antibody was injected via the tail vein, 5 μg CD40 neutralizing antibody / 100 μl 1×PBS / 20 g mouse, for 3 weeks, twice a week, with IgG being equal in amount to the CD40 neutralizing antibody. Starting one week after inoculating Vk*MYC cells, blood was collected from the tail vein every week and serum protein electrophoresis (SPEP) analysis was performed using the Sebia HYDRASYS 2 electrophoresis system. The myeloma burden (CD138 + B220 - cell proportion) was evaluated by flushing the femoral marrow cavity with 5 mL syringe of PBS, and osteolytic lesions were detected using the Skyscan 1276 micro-CT system.

[0058] 2. Histological analysis of mouse bone After fixing the femoral tissues of the above mice with 4% paraformaldehyde (PFA), complete decalcification treatment was carried out and paraffin embedding was performed. The embedded tissues were cut into 3-μm thick sections and stained as follows: hematoxylin-eosin (H&E) staining and Masson staining. The stained sections were observed and analyzed using a ZEISS fully automatic digital slide scanner or iViewer software.

[0059] The results showed that: compared with the IgG group, the CD40 neutralizing antibody had basically no effect on bone destruction in the Mock group mice (as shown in Figure 2 A), but could effectively relieve bone destruction in Vk*MYC myeloma mice (as shown in Figure 2 B); in Vk*MYC mice, compared with the IgG group, the use of the CD40 neutralizing antibody increased the bone volume fraction (as shown in Figure 2 C), trabecular bone thickness (as shown in Figure 2 D), trabecular bone number (as shown in Figure 2 E), bone mineral density (as shown in Figure 2 G), and decreased the trabecular bone separation (as shown in Figure 2 F).

[0060] Example 3 Cytotoxic effect of CD40 neutralizing antibody on myeloma cell lines.

[0061] In four myeloma cell lines, namely U266, LP-1, MM.1S, and ARH-77, different concentrations of CD40 neutralizing antibody were added and incubated for 24 hours and 48 hours, and the cell viability was detected using CCK8.

[0062] The specific steps are as follows: Add 100 μL of cell suspension to each well of a 96-well plate and culture for 24 hours. Add human CD40 neutralizing antibody at different working concentrations (0, 1, 5, 10, 25, 50 μg / mL) and incubate for 24 hours and 48 hours respectively. Then add 10 μL of CCK-8 reagent (APExBIO) to each well, mix gently, and react for 1 hour in the dark. Detect the OD value at 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader to reflect cell viability. Calculate the cell survival rate and plot the antibody concentration-effect curve.

[0063] The results showed that: in U266 (as shown in Figure 3 A), LP-1 (as shown in Figure 3 B), MM.1S (as shown in Figure 3 C), and ARH-77 (as shown in Figure 3 D), CD40 neutralizing antibody doses below 10 μg / mL failed to induce myeloma cell death, while doses above 10 μg / mL showed significant cytotoxicity.

[0064] Example 4 Effect of CD40 neutralizing antibody on the tumor burden of Vk*MYC mice.

[0065] A Vk*MYC mouse model was constructed and treated with IgG and CD40 neutralizing antibody respectively. One week after inoculation with Vk*MYC cells, CD40 neutralizing antibody was injected via the tail vein, 5 μg CD40 neutralizing antibody / 100 μl 1×PBS / 20 g mouse, injected for 3 weeks, twice a week, and IgG and CD40 neutralizing antibody were of equal amount. The M protein band in peripheral blood serum was detected by serum protein electrophoresis, and the proportion of M protein and the proportion of plasma cells in bone marrow blood were quantitatively analyzed.

[0066] The specific steps are the same as those in Example 2.

[0067] The results showed that: in Mock control group mice treated with IgG or CD40 neutralizing antibody, the SPEP results showed no M protein band (as shown in Figure 4 A); in Vk*MYC group mice, the SPEP results showed the appearance of M protein band, but the treatment with CD40 neutralizing antibody reduced the M protein band (as shown in Figure 4 B); in Vk*MYC mice, compared with the IgG group, CD40 neutralizing antibody could reduce the proportion of M protein in peripheral blood serum of mice (as shown in Figure 4as shown in C), reducing the proportion of plasma cells in the bone marrow (such as Figure 4 as shown in D).

[0068] Example 5 Cytotoxic effect of CD40 neutralizing antibody on BMSCs.

[0069] Add different concentrations of CD40 neutralizing antibody to human and mouse BMSCs, and incubate for 24 hours and 48 hours. Use CCK8 to detect cell viability.

[0070] The specific steps are the same as those in Example 3.

[0071] The results showed that human CD40 neutralizing antibody did not show cytotoxicity to human BMSCs (such as Figure 5 shown in A); mouse CD40 neutralizing antibody also did not show cytotoxicity to mouse-derived BMSCs (such as Figure 5 shown in B).

[0072] Example 6 Effect of CD40 neutralizing antibody on osteoclasts in Vk*MYC mice.

[0073] In the established Vk*MYC model, inject CD40 neutralizing antibody via the tail vein. Divide the experiment into two groups: Vk*MYC + IgG group and Vk*MYC + CD40 neutralizing antibody group, with 6 mice in each group. Analyze the osteoclast situation in the femoral tissue by TRAP staining.

[0074] The specific steps are as follows: 1. Vk*MYC multiple myeloma mouse model The specific steps are the same as those in Example 2.

[0075] 2. Histological analysis of mouse bone Take the femoral tissue of the above mice, fix it with 4% paraformaldehyde (PFA), perform complete decalcification and paraffin embedding. Cut the embedded tissue into 3μm thick sections, perform tartrate resistant acid phosphatase (TRAP) staining, and observe and analyze the stained sections using a ZEISS fully automatic digital slide scanner or iViewer software.

[0076] 3. TRAP staining The paraffin sections were routinely dewaxed to water. The sections were washed with pure water for 5 minutes. After using a histochemical pen to draw a circle on the section tissue, they were placed in a wet box containing pure water and incubated with pure water at 37 °C for 2 hours. After pouring off the pure water on the tissue, the sections were put back into the wet box, and freshly prepared and filtered TRAP working solution was added dropwise to completely cover the section tissue, and then placed in an oven at 37 °C for 30 minutes for light-avoiding staining. The staining solution was poured off, and the glass slides were gently washed with water. Mayer hematoxylin staining solution was added dropwise or used for immersion staining for 2 minutes, and then washed with water. The sections were dehydrated with absolute ethanol three times, 5 minutes each time, and cleared with xylene twice, 5 minutes each time, and then a neutral balsam mounting agent was added dropwise for mounting. Osteoclasts were wine red, and cell nuclei were blue.

[0077] The results showed that: The results of TRAP staining showed that compared with the IgG control group, the number of osteoclasts stained wine red in Vk*MYC mice treated with CD40 neutralizing antibody decreased (as Figure 6 shown in A); The quantitative analysis results showed that the number of osteoclasts per unit tissue area decreased significantly (as Figure 6 shown in B).

[0078] As described above, it is only a preferred embodiment of the present invention, and it is not a limitation to the present invention in other forms. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes and apply them to other fields. However, as long as it does not depart from the technical solution content of the present invention, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. Use of a CD40 antibody in any one or more of the following products A1 - A4: A1: Preparing or screening a product for treating multiple myeloma and / or bone diseases; A2: Preparing or screening a product for blocking the interaction between multiple myeloma cells and BMSCs; A3: Preparing or screening a product for alleviating myeloma bone destruction; A4: Preparing or screening a product for reducing the tumor burden of multiple myeloma; The CD40 gene sequence is as shown in SEQ ID NO: 1, and the CD40 antibody sequences are as shown in SEQ ID NO: 5 and SEQ ID NO:

6.

2. The application according to claim 1, wherein The mode of action of the CD40 antibody is to block the interaction between multiple myeloma cells and BMSCs.

3. The application according to claim 1, characterized in that The mode of action of the CD40 antibody is to inhibit the formation of primary cilia or osteogenic differentiation of BMSCs.

4. The application according to claim 1 or 2, characterized in that The multiple myeloma refers to human multiple myeloma cells derived from a cell line or CD138 + plasma cells derived from the bone marrow of a multiple myeloma patient.

Images