Osteoclast fusion inhibitor and osteoclast fusion inhibitory composition

Abstract

To provide an osteoclast fusion inhibitor that is highly effective and has few side effects. [Solution] An osteoclast fusion inhibitor containing an anti-galectin-3 neutralizing antibody as an active ingredient.

Description

Technical Field

[0001] The present invention relates to an osteoclast fusion inhibitor and an osteoclast fusion inhibitory composition.

Background Art

[0002] Malignant bone tumors are tumors that occur in young children, adolescents, and young adults. Malignant bone tumors include osteosarcoma, Ewing sarcoma, chondrosarcoma, undifferentiated pleomorphic sarcoma, and the like. To date, surgical resection, chemotherapy, and radiotherapy are still the main treatment methods for malignant bone tumors.

[0003] For example, in Citation 1, it is described that a human antibody against human RANKL (denosumab, AMG162) inhibits the function of RANKL and is useful for treatment.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, depending on the location, size, metastasis, toxic side effects, and physical condition of the patient of the tumor, these treatment options are not always feasible. Regarding denosumab, osteonecrosis of the jaw and osteomyelitis of the jaw have been reported as serious side effects.

[0006] The present invention has been made in view of the above circumstances, and provides an osteoclast fusion inhibitor with high efficacy and few side effects.

Means for Solving the Problems

[0007] [[ID=***]] The present invention includes the following aspects. [1] An osteoclast fusion inhibitor containing an anti-galectin-3 neutralizing antibody as an active ingredient. [2] The osteoclast fusion inhibitor described in [1], wherein the anti-galectin-3 neutralizing antibody is an antibody against the β-galactoside carbohydrate recognition domain (CRD). [3] An osteoclast fusion inhibitor described in [1] that suppresses osteosarcoma. A composition that contains an osteoclast fusion inhibitor described in any one of [4][1] to [3] and a pharmaceutically acceptable additive. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide an osteoclast fusion inhibitor that is highly effective and has few side effects. [Brief explanation of the drawing]

[0009] [Figure 1] This graph shows the RNA expression of galectin-3 in malignant musculoskeletal tumors. Normalized expression values ​​were summarized using violin plots for each musculoskeletal tumor. [Figure 2] (A) Results of evaluating galectin-3 protein expression in malignant musculoskeletal tumors. To ensure objective evaluation, two examiners blinded the staining and used a staining score of 0-1 (negative) and 2-4 (positive) to determine the positive rate. (B) Graph showing the galectin-3 expression profile. (C) Graph showing the correlation between the score and grade or stage. Samples used in the study included clinical data such as stage, grade, and tumor size. *A difference of p<0.05 was considered statistically significant. [Figure 3]This image visualizes galectin-3 interactions in the bone tumor microenvironment (Saos-2, Hs-Os-1) of osteosarcoma. Dark arrows indicate the expression and interaction of each molecule, illustrating the feedback mechanism of galectin-3 interactions. The strength of the association is represented by the thickness of the arrow. Autocrine and paracrine galectin-3 secretion in the bone tumor microenvironment of osteosarcoma is shown. Light-colored arrows indicate interactions between galectin-3 and cellular components of the bone tumor microenvironment. [Figure 4] (A) Bright-field image of osteoclasts treated with monoclonal rat anti-CRD antibody against galectin-3. The control shows a bright-field image of osteoclasts treated with isotype IgG. Arrows indicate mature osteoclasts. (B) Graph showing the number of mature osteoclasts in osteoclasts treated with monoclonal rat anti-CRD antibody against galectin-3. [Figure 5] (A) Immunohistochemical results of osteosarcoma xenografts. The black bar represents 2.5 mm. (B) Scheme of the experimental treatment plan. (C) Graph showing the inhibitory effect of anti-galectin-3 (158-173) neutralizing antibodies on tumor volume. Targeting galectin-3 suppresses the growth of osteosarcoma in patient-derived orthotopic xenografts. (D) Graph showing weight changes during treatment to monitor toxic side effects. [Figure 6] This result demonstrates the inhibitory effect of anti-galectin-3 (158-173) neutralizing antibodies. Targeting galectin-3 suppressed osteosarcoma growth in the PDOX model. MRI scan data showed that anti-galectin-3 (158-173) neutralizing antibodies inhibited tumor progression. *A difference of p<0.05 was considered statistically significant. [Figure 7] (A) Representative CT images of the 3D structure of the fibula and tibia with and without anti-galectin-3 CRD antibody. (B) Non-decalcified sections were prepared using Villanueva-Goldner stained MMA resin-embedded specimens along the lines shown in Figure 7(A). The specimens revealed pathological changes in the bone mineralization components between control and treatment mice. [Modes for carrying out the invention]

[0010] Embodiments of the present invention will be described in detail below. ≪Osteoclast Fusion Inhibitor≫ In one embodiment, the present invention provides an osteoclast fusion inhibitor containing an anti-galectin-3 neutralizing antibody as an active ingredient.

[0011] Galectin-3 is a 31 kDa protein belonging to the lectin family. Galectin-3 is present on the cell surface, in the cytoplasm, and in the nucleus, and is secreted into the extracellular environment via non-classical secretory pathways, including vesicular release and exosome secretion. Furthermore, galectin-3 is a crucial molecule involved in Wnt signaling, the Ras / Raf / MAPK pathway, and the PI3K / AKT pathway, respectively, through binding to β-catenin, K-Ras, and AKT, and thus significantly impacts the tumor microenvironment. Galectin-3 secreted from tumors has been shown to induce osteolytic bone remodeling in bone tumors.

[0012] Osteosarcoma is a bone-derived tumor and is known as a representative primary malignant bone tumor. Osteosarcoma cells independently produce osteoclast-forming factors. As shown in the examples, galectin-3 expression is high in osteosarcoma patients, and cytoplasmic galectin-3 enhances malignancy. Osteosarcoma cells secrete galectin-3, inducing osteolytic effects. In addition to tumor cells, galectin-3-positive osteoclast precursor cells accumulate near mature osteoclasts in the osteosarcoma microenvironment. Therefore, galectin-3 promotes the progression of osteosarcoma and osteoclast formation.

[0013] An anti-galectin-3 neutralizing antibody is an antibody that binds immunospecifically to galectin and inhibits its function. In this invention, we found that an anti-galectin-3 neutralizing antibody suppresses osteoclast fusion. In the present embodiment, the antibody includes not only a complete antibody molecule but also a fragment of an antibody molecule that functions as a neutralizing antibody. Examples of antibodies include polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, multispecific antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab’, scFv, nanobodies, minibodies, and the like.

[0014] Galectin-3 binds to various sugar receptors such as EGFR, VEGFR-2, TGFβR, p-glycoprotein, Notch1, CD44, and integrin via its β-galactoside carbohydrate recognition domain (CRD), and stimulates cell migration, proliferation, angiogenesis, and the like. Therefore, the anti-galectin-3 neutralizing antibody is preferably an antibody against the β-galactoside carbohydrate recognition domain (CRD).

[0015] Diseases associated with abnormal regulation of osteoclasts include osteosarcoma, synovial sarcoma, fibrosarcoma, chondrosarcoma, multiple myeloma, giant cell tumor of bone, and the like, and osteosarcoma is preferably mentioned. That is, in one embodiment, the present invention provides an osteosarcoma inhibitor containing an anti-galectin-3 neutralizing antibody as an active ingredient.

[0016] The amount of the anti-galectin-3 neutralizing antibody contained in the inhibitor of the present embodiment can be appropriately determined in consideration of various factors such as the sex, body weight, age, and symptoms of the subject. In the case of oral administration, for example, an active ingredient of 1 μg to 10 g in terms of solid matter per day, for example, 10 μg to 2 g in terms of solid matter per day may be administered. In the case of an injection, for example, an active ingredient of 0.1 μg to 1 g in terms of solid matter per day, for example, 1 μg to 200 mg in terms of solid matter per day may be administered.

[0017] The administration form is not particularly limited and is appropriately selected as needed. Generally, oral preparations such as tablets, capsules, granules, fine granules, powders, liquids, syrups, suspensions, emulsions, and elixirs, or injections, drip infusions, suppositories, inhalants, and transmucosal absorbents can be administered. In addition, the injection can be administered intravenously alone or mixed with ordinary replenishing solutions such as glucose and amino acids, and furthermore, if necessary, it can be administered intraarterially, intramuscularly, intradermally, subcutaneously or intraperitoneally. Suppositories are administered rectally. Topical skin preparations are applied, affixed or sprayed onto the affected area.

[0018] The administration route of the inhibitor of this embodiment may be administered, for example, by injection, drip infusion, oral, transdermal, nasal, topical, vaginal, rectal, airway, inhalation, or spray administration.

[0019] In addition, the number of administrations may be three times a day, twice a day, once a day, once every two days, once every three days, once a week, once every two weeks, once a month, etc. Also, the administration period may be one day, two days, three days, one week, two weeks, one month, six months, one year, or more.

[0020] [Composition for inhibiting osteoclast fusion] In one embodiment, the present invention provides a composition for inhibiting osteoclast fusion, which contains the above therapeutic or prophylactic agent and a pharmaceutically acceptable additive.

[0021] The composition of this embodiment can be administered parenterally in the form of, for example, sprays, injections, drip infusions, suppositories, inhalants, transmucosal absorbents, etc., or orally in the form of tablets, capsules, granules, fine granules, powders, liquids, syrups, suspensions, emulsions, and elixirs.

[0022] The composition of this embodiment may use, without particular limitation, pharmaceutically acceptable additives commonly used in the formulation of pharmaceutical compositions. More specifically, examples include excipients such as starch and crystalline cellulose; binders such as gelatin, corn starch, tragacanth gum, and gum arabic; leavening agents such as alginic acid; solvents for injection such as water, ethanol, and glycerin; adhesives such as rubber-based adhesives and silicone-based adhesives; lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavoring agents such as peppermint and red ginger oil; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; and preservatives. Additives can be used individually or in combination of two or more types. The additives may be used individually or in combination of two or more as carriers for therapeutic or prophylactic drugs.

[0023] [Other embodiments] In one embodiment, the present invention provides an anti-galectin-3 neutralizing antibody for inhibiting osteoclast fusion.

[0024] In one embodiment, the present invention provides a method for inhibiting osteoclast fusion, comprising administering an effective amount of an anti-galectin-3 neutralizing antibody to a patient in need of treatment or prevention.

[0025] In one embodiment, the present invention provides the use of an anti-galectin-3 neutralizing antibody for producing an osteoclast fusion inhibitor. [Examples]

[0026] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples. [Galectin-3 is widely prevalent in musculoskeletal tumors.] To investigate the expression distribution of galectin-3 in musculoskeletal tumors, we first analyzed the RNA expression profile of galectin-3 in musculoskeletal patients. Galectin-3 levels were analyzed for all 1366 patients, by histological group. Compared to non-cancerous bone as a control sample, galectin-3 was significantly expressed in bone metastases of breast cancer (p=0.0007), giant cell tumor of bone (p=0.0016), rhabdomyosarcoma (p=0.0012), fibrosarcoma (p=0.0421), and angiosarcoma (p<0.0001) (see Figure 1). Higher expression was also observed in specific patient cohorts with osteosarcoma, undifferentiated pleomorphic sarcoma, chondrosarcoma, chordoma, synovial sarcoma, liposarcoma, leiomyosarcoma, and angiopericytoma. These results indicate that galectin-3 is widely prevalent in musculoskeletal tumors.

[0027] [Osteosarcoma may be a disease for which galectin-3 targeted therapy is applicable.] Next, we analyzed the protein expression profile of galectin-3 in musculoskeletal patients using an immunohistochemistry score (0-4). For objective evaluation, two examiners blinded the staining and examined the positivity rate using a staining score of 0-1 (negative) and 2-4 (positive) (see Figure 2(A)). For all 584 clinical samples, the galectin-3 positivity rates compared to non-cancerous bone or synovial tissue control samples were 69.3% for osteosarcoma (n=127), 74.5% for synovial sarcoma (n=98), 75.0% for fibrosarcoma (n=96), 100% for chondrosarcoma (n=11), 66.7% for multiple myeloma (n=40), 100% for giant cell tumor of bone (n=40), 76.0% for bone metastases from breast cancer (n=17), and 13.0% for bone metastases from prostate cancer (n=77) (see Figure 2(B)). Samples used in the study included clinical data such as stage, grade, and tumor size. For osteosarcoma, a positive correlation was observed between the score and grade or stage (see Figure 2(C)), but not with tumor size. Further statistical analysis was performed using Pearson's correlation test. The correlation coefficient between score and grade was 0.522 (p-value = 0.000000292). The correlation coefficient between score and stage was 0.557 (p-value = 0.00000003). This data suggests that galectin-3 is involved in the progression of osteosarcoma. These results indicate that osteosarcoma may be a target for galectin-3 targeted therapy.

[0028] [Galectin-3 may have a significant impact on the microenvironment of bone tumors.] We visualized and analyzed the bone tumor microenvironment of osteosarcoma to investigate the putative role of galectin-3 in the bone tumor microenvironment of osteosarcoma, including osteodegrading / forming cells such as mature osteoclasts, osteoclast precursors, osteoblasts, and osteocytes; immune cells such as CD4 T cells, CD8 T cells, macrophages, and dendritic cells; and bone marrow stromal cells, bone marrow mesenchymal stem cells, and endothelial cells. All cells were analyzed simultaneously to construct a bone microenvironment model. The data showed that among the above cellular components, mature osteoclasts, osteoclast precursors, and bone marrow stromal cells interacted with osteosarcoma via tumor-derived galectin-3 (see Figure 3). Immune cells such as CD4 T cells, CD8 T cells, macrophages, and dendritic cells were not involved in galectin-3 interactions. Furthermore, endothelial cells, bone marrow mesenchymal stem cells, osteoblasts, and osteocytes were not involved in interactions. Next, to establish the importance of molecular interactions, highly related interactions between ligands and receptors were investigated. Osteosarcoma-derived galectin-3 was found to bind to fibronectin (FN1), integrin subunit beta-1 (ITGB1), myosin 2A (MYH9), CD44, integrin subunit beta-2 (ITGB2), and lysosome-associated membrane protein 1 (LAMP1) (see Figure 3). These molecules may alter the state of mature osteoclasts, osteoclast progenitor cells, bone marrow stromal cells, and osteosarcoma. Subsequently, to quantify the molecular interactions of galectin-3 in the osteosarcoma bone microenvironment, TPM (transcripts per million kilobases), representing the strength of molecular binding, was calculated. As a result, it was revealed that LGALS3-ITGB1 (258750.52 TPM), LGALS-ITGB2 (215914.52 TPM), LGALS3-MYH9 (167738.75 TPM), and LGALS3-ITGAV (148589.23 TPM) are important interactions between osteosarcoma and mature osteoclasts. Regarding osteoclast precursors, clear molecular binding was confirmed between LGALS3-ITGB2 (211469.62 TPM) and LGALS3-LAMP1 (102571.46 TPM). On the other hand, in bone marrow stromal cells, LGALS3-FN1 (2039817.92 TPM), LGALS3-ITGB1 (489899.96 TPM), LGALS3-MYH9 (305725.77 TPM), and LGALS3-CD44 (215277.43 TPM) showed strong galectin-3 intermolecular relationships. This result suggests that galectin-3-mediated interactions may cause significant molecular changes in the bone microenvironment of osteosarcoma.

[0029] [Targeting the galectin-3 CRD domain suppresses the growth of osteosarcoma.] Monoclonal rat anti-CRD antibodies against galectin-3 were prepared using the following method. Animals were immunized with a KLH-linked peptide (HFNPRFNENNRRVIVC). The CRD domain of galectin-3, amino acids 158-175 (HFNPRFNENNRRVIVCNT), is a conserved sequence across species, particularly between humans and mice. After establishing hybridoma clones, the supernatant was tested for neutralization effects using Raw264.7, a mouse osteoclast precursor. Cells were cultured in α-MEM medium supplemented with 10% fetal bovine serum, and differentiation was induced with 100 μg / ml RANKL (Biolegend, San Diego, CA). After confirming the neutralization effect, the supernatant of the hybridoma clones was purified with Protein G Sepharose. As shown in Figure 4(A), the generated monoclonal rat anti-CRD antibody against galectin-3 suppressed osteoclast fusion. As shown in Figure 4(B), the generated monoclonal rat anti-CRD antibody against galectin-3 suppressed osteoclast fusion dose-dependently.

[0030] Next, prior to in vivo studies using PDOX (Patient-Derived-Orthotopic Xenograft), the expression status of the established xenografts was evaluated. As described above, immunohistochemistry was performed using a customized galectin-3 monoclonal rat anti-CRD antibody.

[0031] We examined the expression status of galectin-3 in four osteosarcoma patients. Galectin-3 was strongly detected in three samples, and weakly positive in one sample (see Figure 5(A)). Notably, galectin-3 expression was detected mainly in the cell membrane and / or cytoplasm of osteosarcoma cells, suggesting its influence on the bone microenvironment. Therefore, from these samples, we selected a xenograft (#2) originating from a 14-year-old patient with conventional osteoblastic high-grade osteosarcoma of the focal pelvis who had not received chemotherapy, radiotherapy, or other treatments at the time of biopsy sampling. The xenograft was used in the following PDOX in vivo study.

[0032] Regarding the treatment plan, the PDOX model was randomly divided into two groups: one was a control group, meaning no treatment was given, and isotype IgG was administered intraperitoneally; the other was a treatment group, where anti-galectin-3 antibodies were administered intraperitoneally. Tumor length, width, and mouse body weight were measured twice a week (see Figure 5(B)). As a result, in the treatment group, the increase in tumor volume was suppressed in a time-dependent manner, particularly on post-transplant day 14, and a statistically significant difference was observed between the control group and the treatment group (p=0.0495) (see Figure 5(C)). There was no significant difference between the two groups in weight changes due to side effects of the neutralizing antibodies, such as loss of appetite, indicating that the antibodies produced were not harmful, and at least partially, no acute side effects were observed (see Figure 5(D)). To confirm the acute toxicity of the anti-galectin-3 neutralizing antibodies, various organs such as the heart, lungs, liver, kidneys, stomach, small / large intestine, muscles, spleen, pancreas, bladder, hip joint, and mandible were pathologically examined. However, no abnormal lesions were found, including osteonecrosis of the jaw, a secondary toxic condition in which osteoclasts die due to osteoclast apoptosis-inducing substances such as zoledronate. Furthermore, visualization of osteoclasts by TRAP staining showed no difference in the number of normal osteoclasts around the epiphyseal cartilage. These findings indicated that galectin-3 targeted therapy is well tolerated without side effects. MRI scans were performed to measure accurate volume changes, regardless of the presence or absence of anti-galectin-3 antibodies. The image data showed a reduction in tumor volume with anti-galectin-3 antibody treatment (a reduction of 16.3% to 22.6% compared to control mice), and surprisingly, a clear therapeutic response was observed in one of the three mice (see Figure 6). Furthermore, CT scans were performed to analyze the skeletal structure. The tibia and fibula of control mice were severely lysed, but bone remained intact in mice treated with anti-galectin-3 antibody (see Figure 7(A)). Consistently, skeletal microstructure, including bone mass, cortical thickness, and bone density, showed statistically significant differences between control and treated mice. This finding indicates that anti-galectin-3 antibodies have an inhibitory effect on tumor-induced bone destruction (see Figure 7(B)). These results suggest that neutralizing galectin-3 in the tumor bone microenvironment reduces osteosarcoma progression. [Industrial applicability]

[0033] According to the present invention, it is possible to provide an osteoclast fusion inhibitor that is highly effective and has few side effects.

Claims

1. An osteoclast fusion inhibitor containing an anti-galectin-3 neutralizing antibody as an active ingredient.

2. The osteoclast fusion inhibitor according to claim 1, wherein the anti-galectin-3 neutralizing antibody is an antibody against the β-galactoside carbohydrate recognition domain (CRD).

3. An osteoclast fusion inhibitor according to claim 1, which suppresses osteosarcoma.

4. An osteoclast fusion inhibitory composition comprising an osteoclast fusion inhibitor according to any one of claims 1 to 3 and a pharmaceutically acceptable additive.

Images