Pharmaceutical composition containing anti-RANKL-NGF bispecific antibody

Abstract

The present disclosure relates to a pharmaceutical composition comprising an anti-RANKL-NGF bispecific antibody. In particular, the present disclosure relates to pharmaceutical compositions comprising an anti-RANKL-NGF bispecific antibody and a buffer.

Description

A pharmaceutical composition containing an anti-RANKL-NGF bispecific antibody

[0001] This application claims priority to Chinese patent application (application number 202211438239.8, application date November 16, 2022). Technical Field

[0002] The present disclosure belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition containing an anti-RANKL-NGF bispecific antibody, and its use as a medicine. Background Art

[0003] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0004] Bone metastasis is a common symptom in the later stages of many cancers. The pathogenesis of bone metastasis is as follows: primary tumor cells shed into the bloodstream to form circulating tumor cells, which then metastasize to the bone to form transmissible tumor cells and become dormant in the bone microenvironment. This process may last for several years until the surviving tumor cells gradually adapt to the bone microenvironment, becoming activated and entering a proliferative state from a dormant state (Mantyh, PW, Curr Opin Support Palliat Care, 2014.8(2): p83-90). The proliferation of tumor cells affects the bone microenvironment and promotes the formation of osteoclasts. Osteoclasts accelerate bone resorption and release a variety of cytokines, further promoting the proliferation of tumor cells, thus forming a vicious cycle (Quayle, L., Current Cancer Drug Targets, 2015.15(6): p469-480). The continuous proliferation of tumor cells leads to the formation of multiple metastatic sites, causing patients to suffer from various problems such as bone damage and pain (Gartland, A., Journal of Bone Oncology, 2016.5(3): p100-103).

[0005] According to statistics, common cancers that metastasize to the bone include myeloma, renal cancer, melanoma, bladder cancer, thyroid cancer, lung cancer, breast cancer, and prostate cancer (Clezardin, P., Joint Bone Spine, 2017. 84(6): p677-684. Fidler, M.M. Fidler, Scandinavian Journal of Public Health, 2018. 46(1): p27-36).

[0006] In normal bone tissue, osteoblasts and osteoclasts are in a relatively balanced state to maintain normal bone growth and development, but the presence of cancer cells disrupts this balance. Cancer cells in the bone microenvironment release a series of cytokines to stimulate osteoblasts and osteocytes to secrete a large amount of Receptor Activator of Nuclear Factor-κB Ligand (RANKL). RANKL interacts with RANK receptors to promote the formation of osteoclasts, leading to increased bone resorption. Osteolysis caused by bone resorption releases other growth factors to promote the proliferation of cancer cells. This forms a cycle that is conducive to cancer cell metastasis (Body, JJ, Expert Rev Anticancer Ther, 2012.12(3): p307-322).

[0007] NGF (Nerve growth factor) is a nerve growth factor. The NGF signaling pathway mediates the growth and development of the nervous system and the conduction of pain signals. It is currently known that there are two NGF receptors on the cell surface, of which TrKA is a high-affinity receptor and p75NTR is a low-affinity receptor. The interaction between NGF and TrkA can simultaneously activate the Ras and PI3K pathways, promoting cell survival and nerve growth. The interaction between NGF and p75NTR can promote cell apoptosis. In addition, the PI3K pathway can also activate the phosphorylation of TRPV1, leading to the activation of ion channels to generate action potentials and transmit pain neuron signals (Kumar, V. and Mahal BA, Journal of Pain Research, 2012.5: p279-287).

[0008] Existing data show that monoclonal antibodies targeting NGF and RANKL have a certain therapeutic effect on bone damage and pain caused by bone metastasis (Body JJ. Expert Rev Anticancer Ther. 2012.12(3): p307-322. Sopata M., et al. 2015.156(9): p1703-1713).

[0009] Summary of the Invention

[0010] The present disclosure provides a pharmaceutical composition comprising an anti-RANKL-NGF bispecific antibody, which has therapeutic activity and the advantages of good stability.

[0011] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-RANKL-NGF bispecific antibody and a buffer, wherein the anti-RANKL-NGF bispecific antibody comprises a first antigen binding domain that specifically binds to RANKL and a second antigen binding domain that specifically binds to NGF, and the buffer is an acetate buffer, a histidine buffer, or a phosphate buffer.

[0012] In some embodiments, the buffer is acetic acid-sodium acetate buffer, histidine-histidine hydrochloride buffer, or citric acid-disodium hydrogen phosphate buffer.

[0013] In some embodiments, the buffer is acetic acid-sodium acetate buffer or histidine-histidine hydrochloride buffer.

[0014] In some specific embodiments, the buffer is acetic acid-sodium acetate buffer.

[0015] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the pH of the pharmaceutical composition is 4.2 to 7.8. In some embodiments, the pH of the pharmaceutical composition is 4.2 to 7.0. In some embodiments, the pH of the pharmaceutical composition is 4.2 to 5.4. In some embodiments, the pH of the pharmaceutical composition is 4.6 to 5.4. In some embodiments, the pH of the pharmaceutical composition is about 4.6. In some embodiments, the pH of the pharmaceutical composition is about 4.8. In some embodiments, the pH of the pharmaceutical composition is about 5.0. In some embodiments, the pH of the pharmaceutical composition is about 5.2. In some embodiments, the pH of the pharmaceutical composition is about 5.4. When a point value is mentioned in this disclosure, it should be understood that the point value includes an error range. This error range is due to factors such as laboratory environment, human operation, instrumentation, methodology, measurement error, etc. Taking pH as an example, when the measured value is about 5.0, it should be understood that it includes an error range. As an example, when the formulation is measured using an industrial pH meter, "about 5.0" means 5.0 ± 0.2 (ie, a pH of 4.8 to 5.2).

[0016] In some embodiments, the pH of the pharmaceutical composition is 4.6-6.6. In some embodiments, the pH of the pharmaceutical composition is 4.6-5.8. In some embodiments, the pH of the pharmaceutical composition is 5.0-5.8.

[0017] In some embodiments, the pH of the pharmaceutical composition is 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 or 7.8, or any range therebetween. In some embodiments, the pH of the pharmaceutical composition is 4.6. In some embodiments, the pH of the pharmaceutical composition is 4.8. In some embodiments, the pH of the pharmaceutical composition is 5.0. In some embodiments, the pH of the pharmaceutical composition is 5.2. In some embodiments, the pH of the pharmaceutical composition is 5.4.

[0018] Typically, the pH of the pharmaceutical composition obtained by replacing the buffer is almost the same as the buffer pH. At the same time, it is well known to those skilled in the art that during the process of pharmaceutical preparation, pH drift may sometimes occur, but the pH drift of the pharmaceutical preparation is generally small (e.g., within the range of ± 0.3). In some embodiments, the pH drift of the pharmaceutical preparation is within the range of ± 0.2. In some embodiments, the pH drift of the pharmaceutical preparation is within the range of ± 0.1.

[0019] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the concentration of the anti-RANKL-NGF bispecific antibody is 1 mg / mL to 150 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 1 mg / mL to 100 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 10 mg / mL to 80 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 20 mg / mL to 80 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 20 mg / mL to 77 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 20 mg / mL to 70 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 50 mg / mL to 77 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 56 mg / mL to 84 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 63 mg / mL to 77 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is about 77 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is about 70 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is about 50 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is about 20 mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 1mg / mL, 5mg / mL, 10mg / mL, 20mg / mL, 30mg / mL, 40mg / mL, 45mg / mL, 50mg / mL, 55mg / mL, 56mg / mL, 60mg / mL, 63mg / mL, 65mg / mL, 70mg / mL, 75mg / mL, 77mg / mL, 80mg / mL, 84mg / mL, 90mg / mL, 100mg / mL, 110mg / mL, 120mg / mL, 130mg / mL, 140mg / mL or 150mg / mL, or any range between these point values. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 77mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 70mg / mL. In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 50 mg / mL.In some embodiments, the concentration of the anti-RANKL-NGF bispecific antibody is 20 mg / mL.

[0020] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the pharmaceutical composition comprises a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from poloxamer (e.g., poloxamer 188), polysorbate (e.g., polysorbate 20, polysorbate 80), polyhydroxyalkylene, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, Myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, methyl cocoyl sodium, methyl oleyl taurate sodium, polyethylene glycol, polypropylene glycol, copolymer of ethylene and propylene glycol, etc. In some embodiments, the surfactant is polysorbate or poloxamer. In some embodiments, the surfactant is polysorbate 80, polysorbate 20 or poloxamer 188. In some embodiments, the surfactant is polysorbate 80 or polysorbate 20. In some embodiments, the surfactant is polysorbate 80.

[0021] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the surfactant concentration is 0.01 mg / mL to 1.0 mg / mL. In some embodiments, the surfactant concentration is 0.01 mg / mL to 0.8 mg / mL. In some embodiments, the surfactant concentration is 0.01 mg / mL to 0.6 mg / mL. In some embodiments, the surfactant concentration is 0.01 mg / mL to 0.4 mg / mL. In some embodiments, the surfactant concentration is 0.01 mg / mL to 0.2 mg / mL. In some embodiments, the surfactant concentration is 0.1 mg / mL to 0.4 mg / mL. In some embodiments, the surfactant concentration is 0.1 mg / mL to 0.2 mg / mL. In some embodiments, the surfactant concentration is 0.05 mg / mL to 0.15 mg / mL. In some embodiments, the surfactant concentration is 0.08 mg / mL to 0.12 mg / mL. In some embodiments, the surfactant concentration is 0.09 mg / mL to 0.11 mg / mL. In some embodiments, the surfactant concentration is about 0.4 mg / mL. In some embodiments, the surfactant concentration is about 0.2 mg / mL. In some embodiments, the surfactant concentration is about 0.1 mg / mL. In some embodiments, the surfactant concentration is 0.01 mg / mL, 0.05 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL or 1.0 mg / mL, or any range between these point values. In some embodiments, the surfactant concentration is 0.4 mg / mL. In some embodiments, the surfactant concentration is 0.2 mg / mL. In some embodiments, the surfactant concentration is 0.1 mg / mL.

[0022] In some embodiments, the surfactant is about 0.1 mg / mL of polysorbate 80. In some embodiments, the surfactant is about 0.1 mg / mL of polysorbate 80.

[0023] In some embodiments, the pharmaceutical composition as described in any one of the above, includes a stabilizer. In some embodiments, the stabilizer is a sugar (including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc.), an amino acid (including proline, arginine, glycine, cysteine, histidine, etc.) or a salt (sodium chloride, potassium chloride, calcium chloride, etc.). In some embodiments, the stabilizer is selected from one or more of the group consisting of proline, sucrose, trehalose, sorbitol, arginine, glycine and sodium chloride. In some embodiments, the stabilizer is proline, sucrose or sodium chloride. In some embodiments, the stabilizer is an amino acid. In some embodiments, the stabilizer is proline.

[0024] In some embodiments, the pharmaceutical composition of any of the above items, the stabilizer concentration is 1 mM to 300 mM. In some embodiments, the stabilizer concentration is 25 mM to 290 mM. In some embodiments, the stabilizer concentration is 25 mM to 250 mM. In some embodiments, the stabilizer concentration is 210 mM to 270 mM. In some embodiments, the stabilizer concentration is 216 mM to 264 mM. In some embodiments, the stabilizer concentration is 228 mM to 252 mM. In some embodiments, the stabilizer concentration is about 240 mM. In some embodiments, the stabilizer concentration is 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 216 mM, 220 mM, 228 mM, 230 mM, 235 mM, 240 mM, 245 mM, 250 mM, 252 mM, 260 mM, 264 mM, 270 mM, 280 mM, 290 mM or 300 mM, or any range therebetween. In some embodiments, the stabilizer concentration is 240 mM.

[0025] In some embodiments, the stabilizer is about 240 mM proline. In some embodiments, the stabilizer is 240 mM proline.

[0026] In some embodiments, the stabilizer is a sugar selected from the group consisting of glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol (also known as sorbitol), mannitol, milibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, maltitol, lactitol and iso-maltulose. In some embodiments, the stabilizer is selected from one or more of the group consisting of sucrose, trehalose, sorbitol, arginine, glycine and sodium chloride. In some embodiments, the stabilizer is a non-reducing disaccharide. In some embodiments, the stabilizer is trehalose or sucrose. In some embodiments, the stabilizer is sucrose.

[0027] In some embodiments, the stabilizer is 10 mg / mL to 100 mg / mL sucrose. In some embodiments, the stabilizer is 30 mg / mL to 80 mg / mL sucrose. In some embodiments, the stabilizer is 50 mg / mL to 80 mg / mL sucrose. In some embodiments, the stabilizer is 70 mg / mL to 80 mg / mL sucrose. In some embodiments, the stabilizer is 60 mg / mL to 90 mg / mL sucrose. In some embodiments, the stabilizer is 67.5 mg / mL to 82.5 mg / mL sucrose. In some embodiments, the stabilizer is about 75 mg / mL sucrose. In some embodiments, the stabilizer concentration includes, but is not limited to, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 65 mg / mL, 67.5 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 82.5 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, and any range therebetween. In some embodiments, the stabilizer is 75 mg / mL sucrose.

[0028] In some embodiments, the stabilizer is a salt. In some embodiments, the stabilizer is sodium chloride. In some embodiments, the stabilizer is 0.64% (w / v) to 0.96% (w / v) sodium chloride. In some embodiments, the stabilizer is 0.72% (w / v) to 0.88% (w / v) sodium chloride. In some embodiments, the stabilizer is about 0.8% (w / v) sodium chloride. In some embodiments, the stabilizer is 0.8% (w / v) sodium chloride.

[0029] In some embodiments, the pharmaceutical composition of any of the above, the concentration of the buffer is 5mM to 100mM. In some embodiments, the concentration of the buffer is 10mM to 50mM. In some embodiments, the concentration of the buffer is 10mM to 30mM. In some embodiments, the concentration of the buffer is 10mM to 20mM. In some embodiments, the concentration of the buffer is 16mM to 24mM. In some embodiments, the concentration of the buffer is 18mM to 22mM. In some embodiments, the concentration of the buffer is about 20mM. In some embodiments, the concentration of the buffer is about 10mM. In some embodiments, the concentration of the buffer is 5mM, 10mM, 15mM, 16mM, 18mM, 20mM, 22mM, 24mM, 25mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or 100mM, and any ranges therebetween. In some embodiments, the concentration of the buffer is 20mM. In some embodiments, the concentration of the buffer is 10mM.

[0030] In some embodiments, the buffer is about 10 mM acetic acid-sodium acetate buffer. In some embodiments, the buffer is about 20 mM acetic acid-sodium acetate buffer. In some embodiments, the buffer is 10 mM acetic acid-sodium acetate buffer. In some embodiments, the buffer is 20 mM acetic acid-sodium acetate buffer. In some embodiments, the buffer is 10 mM histidine-histidine hydrochloride buffer. In some embodiments, the buffer is 10 mM citric acid-disodium hydrogen phosphate buffer.

[0031] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-RANKL-NGF bispecific antibody comprises: at least one first antigen binding domain that specifically binds to RANKL and at least one second antigen binding domain that specifically binds to NGF.

[0032] In some embodiments, the pharmaceutical composition as described in any one of the above items, wherein the anti-RANKL-NGF bispecific antibody comprises: two first antigen-binding domains that specifically bind to RANKL and two second antigen-binding domains that specifically bind to NGF.

[0033] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-RANKL-NGF bispecific antibody has the structure as shown in Figure 1.

[0034] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein in the anti-RANKL-NGF bispecific antibody, the first antigen binding domain that specifically binds to RANKL comprises a heavy chain variable region and a light chain variable region, wherein:

[0035] The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0036] The light chain variable region comprises: LCDR1 comprising the amino acid sequence of SEQ ID NO: 4; LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0037] In some embodiments, a pharmaceutical composition as described in any of the above items, wherein in the anti-RANKL-NGF bispecific antibody, the first antigen binding domain that specifically binds to RANKL comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8.

[0038] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein in the anti-RANKL-NGF bispecific antibody, the second antigen binding domain that specifically binds to NGF comprises a heavy chain variable region and a light chain variable region, wherein:

[0039] The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 15; HCDR2 comprising the amino acid sequence of SEQ ID NO: 16; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 17; and

[0040] The light chain variable region comprises: LCDR1 comprising the amino acid sequence of SEQ ID NO: 18; LCDR2 comprising the amino acid sequence of SEQ ID NO: 19; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 20.

[0041] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein in the anti-RANKL-NGF bispecific antibody, the second antigen binding domain that specifically binds to NGF comprises a heavy chain variable region and a light chain variable region, wherein:

[0042] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21; and / or

[0043] The light chain variable region comprises the amino acid sequence of SEQ ID NO: 22.

[0044] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein in the anti-RANKL-NGF bispecific antibody, the second antigen binding domain that specifically binds to NGF comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 24, and the light chain comprises the amino acid sequence of SEQ ID NO: 25.

[0045] In some embodiments, the pharmaceutical composition as described in any one of the above items, wherein the anti-RANKL-NGF bispecific antibody,

[0046] (i) a first antigen-binding domain that specifically binds to RANKL comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0047] The light chain variable region comprises: LCDR1 comprising the amino acid sequence of SEQ ID NO: 4; LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6;

[0048] (ii) a second antigen-binding domain that specifically binds to NGF comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 15; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 16; and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 17; and

[0049] The light chain variable region comprises: LCDR1 comprising the amino acid sequence of SEQ ID NO: 18; LCDR2 comprising the amino acid sequence of SEQ ID NO: 19; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 20.

[0050] In some embodiments, the pharmaceutical composition as described in any one of the above items, wherein the anti-RANKL-NGF bispecific antibody,

[0051] (i) the first antigen-binding domain that specifically binds to RANKL comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8;

[0052] (ii) The second antigen-binding domain that specifically binds to NGF comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 22.

[0053] In some embodiments, the pharmaceutical composition as described in any one of the above items, wherein the anti-RANKL-NGF bispecific antibody,

[0054] (i) a first antigen-binding domain that specifically binds to RANKL comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0055] The light chain variable region comprises: LCDR1 comprising the amino acid sequence of SEQ ID NO: 4; LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6;

[0056] (ii) a second antigen-binding domain that specifically binds to NGF comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: a HCDR1 comprising the amino acid sequence of SEQ ID NO: 15; a HCDR2 comprising the amino acid sequence of SEQ ID NO: 16; and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 17; and

[0057] The light chain variable region comprises: LCDR1, which comprises the amino acid sequence of SEQ ID NO: 18; LCDR2, which comprises the amino acid sequence of SEQ ID NO: 19; and LCDR3, which comprises the amino acid sequence of SEQ ID NO: 20; and the anti-RANKL-NGF bispecific antibody has the structure shown in Figure 1.

[0058] In some embodiments, the pharmaceutical composition as described in any one of the above items, wherein the anti-RANKL-NGF bispecific antibody,

[0059] (i) the first antigen-binding domain that specifically binds to RANKL comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8;

[0060] (ii) The second antigen binding domain that specifically binds to NGF comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 22; and the anti-RANKL-NGF bispecific antibody has the structure shown in Figure 1.

[0061] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-RANKL-NGF bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 30, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 31.

[0062] In some embodiments, the anti-RANKL-NGF bispecific antibody comprises two first chains with identical sequences and two second chains with identical sequences, wherein: the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 30, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 31.

[0063] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0064] (a) 1 mg / mL to 150 mg / mL of the anti-RANKL-NGF bispecific antibody,

[0065] (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant,

[0066] (c) 1 mM to 300 mM of a stabilizer, and

[0067] (d) a 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 4.2 to 7.0.

[0068] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0069] (a) 1 mg / mL to 150 mg / mL of the anti-RANKL-NGF bispecific antibody,

[0070] (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant,

[0071] (c) 1 mM to 300 mM proline, and

[0072] (d) a 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 4.2 to 7.0.

[0073] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0074] (a) 10 mg / mL to 80 mg / mL of the anti-RANKL-NGF bispecific antibody,

[0075] (b) 0.01 mg / mL to 0.6 mg / mL of polysorbate 20 or polysorbate 80,

[0076] (c) 25 mM to 290 mM proline, and

[0077] (d) 10 mM to 50 mM acetate buffer, the pH of the pharmaceutical composition is 4.6 to 5.4.

[0078] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0079] (a) 10 mg / mL to 80 mg / mL of the anti-RANKL-NGF bispecific antibody,

[0080] (b) 0.01 mg / mL to 0.6 mg / mL of polysorbate 80,

[0081] (c) 25 mM to 250 mM proline, and

[0082] (d) 10 mM to 50 mM acetate buffer, the pH of the pharmaceutical composition is 4.6 to 5.4.

[0083] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0084] (a) 20 mg / mL to 80 mg / mL of the anti-RANKL-NGF bispecific antibody,

[0085] (b) 0.01 mg / mL to 0.4 mg / mL of polysorbate 80,

[0086] (c) 210 mM to 270 mM proline, and

[0087] (d) 10 mM to 30 mM acetic acid-sodium acetate buffer, the pH of the pharmaceutical composition is 4.6 to 5.4.

[0088] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0089] (a) 63 mg / mL to 77 mg / mL of an anti-RANKL-NGF bispecific antibody,

[0090] (b) 0.01 mg / mL to 0.2 mg / mL of polysorbate 80,

[0091] (c) 210 mM to 270 mM proline, and

[0092] (d) 10 mM to 30 mM acetic acid-sodium acetate buffer, the pH of the pharmaceutical composition is 4.8 to 5.2.

[0093] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0094] (a) 63 mg / mL to 77 mg / mL of an anti-RANKL-NGF bispecific antibody,

[0095] (b) 0.01 mg / mL to 0.2 mg / mL of polysorbate 80,

[0096] (c) 25 mM to 250 mM proline, and

[0097] (d) 10 mM to 20 mM acetic acid-sodium acetate buffer, the pH of the pharmaceutical composition is 4.8 to 5.2.

[0098] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0099] (a) 63 mg / mL to 77 mg / mL of an anti-RANKL-NGF bispecific antibody,

[0100] (b) 0.01 mg / mL to 0.2 mg / mL of polysorbate 80,

[0101] (c) 240 mM proline, and

[0102] (d) 10 mM to 20 mM acetic acid-sodium acetate buffer; the pH of the pharmaceutical composition is 4.8 to 5.2.

[0103] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0104] (a) 63 mg / mL to 77 mg / mL of an anti-RANKL-NGF bispecific antibody,

[0105] (b) 0.05 mg / mL to 0.15 mg / mL of polysorbate 80,

[0106] (c) 210 mM to 270 mM proline, and

[0107] (d) 16 mM to 24 mM acetic acid-sodium acetate buffer, the pH of the pharmaceutical composition is 4.8 to 5.2.

[0108] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0109] (a) 63 mg / mL to 77 mg / mL of an anti-RANKL-NGF bispecific antibody,

[0110] (b) 0.08 mg / mL to 0.12 mg / mL of polysorbate 80,

[0111] (c) 210 mM to 270 mM proline, and

[0112] (d) 16 mM to 24 mM acetic acid-sodium acetate buffer, the pH of the pharmaceutical composition is 4.8 to 5.2.

[0113] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0114] (a) about 70 mg / mL of an anti-RANKL-NGF bispecific antibody,

[0115] (b) about 0.1 mg / mL of polysorbate 80,

[0116] (c) about 240 mM proline, and

[0117] (d) about 20 mM acetic acid-sodium acetate buffer, the pH of the pharmaceutical composition is 4.8 to 5.2.

[0118] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0119] (a) 70 mg / mL of anti-RANKL-NGF bispecific antibody,

[0120] (b) 0.1 mg / mL of polysorbate 80,

[0121] (c) 240 mM proline, and

[0122] (d) 20 mM acetic acid-sodium acetate buffer, the pH of the pharmaceutical composition is 4.8 to 5.2.

[0123] In some embodiments, the pharmaceutical composition as described above is a liquid formulation. In some embodiments, the solvent of the liquid formulation is water.

[0124] The present disclosure also provides a lyophilized preparation, characterized in that the lyophilized preparation can form any of the pharmaceutical compositions described above after reconstitution.

[0125] The present disclosure also provides a lyophilized preparation, which is a lyophilized preparation of the pharmaceutical composition as described in any one of the above items.

[0126] The present disclosure also provides a method for preparing a lyophilized formulation, comprising freeze-drying the pharmaceutical composition as described in any one of the above items. In some embodiments, the freeze-drying as described in any one of the above items sequentially comprises the steps of pre-freezing, primary drying, and secondary drying.

[0127] The present disclosure also provides a lyophilized preparation, which is obtained by freeze-drying the pharmaceutical composition as described above.

[0128] The present disclosure also provides a reconstituted solution, characterized in that the reconstituted solution is prepared by reconstituted the lyophilized preparation as described in any one of the above items.

[0129] The present disclosure also provides a reconstituted solution, which is a reconstituted preparation of any of the above lyophilized preparations.

[0130] In some embodiments, the components and contents of the reconstituted solution as described in any of the above items are the same as those of the aforementioned pharmaceutical composition.

[0131] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0132] (a) 1 mg / mL to 150 mg / mL of the anti-RANKL-NGF bispecific antibody, (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant, (c) 1 mM to 300 mM of a stabilizer, and (d) 5 mM to 100 mM of a buffer, wherein the pH of the pharmaceutical composition is 4.2 to 7.0.

[0133] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0134] (a) 1 mg / mL to 150 mg / mL of the anti-RANKL-NGF bispecific antibody as described in any of the above items, (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant, (c) 1 mM to 300 mM proline, and (d) 5 mM to 100 mM buffer, the pH of the pharmaceutical composition being 4.2 to 7.0.

[0135] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0136] (a) 10 mg / mL to 80 mg / mL of the anti-RANKL-NGF bispecific antibody, (b) 0.01 mg / mL to 0.6 mg / mL of polysorbate 20 or polysorbate 80, (c) 25 mM to 290 mM proline, and (d) 10 mM to 50 mM acetate buffer, the pH of the pharmaceutical composition being 4.6 to 5.4.

[0137] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0138] (a) 10 mg / mL to 80 mg / mL of the anti-RANKL-NGF bispecific antibody, (b) 0.01 mg / mL to 0.6 mg / mL of polysorbate 80, (c) 25 mM to 250 mM proline, and (d) 10 mM to 50 mM acetate buffer, the pH of the pharmaceutical composition being 4.6 to 5.4.

[0139] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0140] (a) 20 mg / mL to 80 mg / mL of the anti-RANKL-NGF bispecific antibody, (b) 0.01 mg / mL to 0.4 mg / mL of polysorbate 80, (c) 210 mM to 270 mM proline, and (d) 10 mM to 30 mM acetate buffer, wherein the pH of the pharmaceutical composition is 4.6 to 5.4.

[0141] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0142] (a) 63 mg / mL to 77 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) 0.01 mg / mL to 0.2 mg / mL of polysorbate 80, (c) 210 mM to 270 mM proline, and (d) 10 mM to 30 mM acetic acid-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.8 to 5.2.

[0143] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0144] (a) 63 mg / mL to 77 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) 0.01 mg / mL to 0.2 mg / mL of polysorbate 80, (c) 25 mM to 250 mM proline, and (d) 10 mM to 20 mM acetic acid-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.8 to 5.2.

[0145] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0146] (a) 63 mg / mL to 77 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) 0.01 mg / mL to 0.2 mg / mL of polysorbate 80, (c) 240 mM proline, and (d) 10 mM to 20 mM acetic acid-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.8 to 5.2.

[0147] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0148] (a) 63 mg / mL to 77 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) 0.05 mg / mL to 0.15 mg / mL of polysorbate 80, (c) 210 mM to 270 mM proline, and (d) 16 mM to 24 mM acetic acid-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.8 to 5.2.

[0149] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0150] (a) 63 mg / mL to 77 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) 0.08 mg / mL to 0.12 mg / mL of polysorbate 80, (c) 210 mM to 270 mM proline, and (d) 16 mM to 24 mM acetic acid-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.8 to 5.2.

[0151] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0152] (a) about 70 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) about 0.1 mg / mL of polysorbate 80, (c) about 240 mM proline, and (d) about 20 mM acetic acid-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.8 to 5.2.

[0153] In some embodiments, the reconstitution solution as described in any of the above items comprises the following components:

[0154] (a) 70 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) 0.1 mg / mL of polysorbate 80, (c) 240 mM proline, and (d) 20 mM acetic acid-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.8 to 5.2.

[0155] In some embodiments, the pharmaceutical composition or reconstituted solution as described in any one of the above is a subcutaneous injection preparation, an intravenous injection preparation, an intraperitoneal injection preparation or an intramuscular injection preparation. In some embodiments, the pharmaceutical composition or reconstituted solution as described in any one of the above is a subcutaneous injection preparation.

[0156] In some embodiments, the pharmaceutical composition or reconstituted solution as described in any one of the above items is suitable for subcutaneous injection, intravenous injection, intraperitoneal injection or intramuscular injection; preferably, it is suitable for subcutaneous injection.

[0157] In some embodiments, the pharmaceutical composition, reconstituted solution or lyophilized preparation as described in any one of the above items is used to prepare a drug for subcutaneous injection, intravenous injection, intraperitoneal injection or intramuscular injection; preferably, it is used to prepare a drug for subcutaneous injection.

[0158] The present disclosure also provides a drug kit comprising at least one container, each container independently containing the pharmaceutical composition as described in any one of the above items, the lyophilized formulation as described in any one of the above items, or the reconstituted solution as described in any one of the above items.

[0159] In some embodiments, the present disclosure also provides a method for diagnosing, treating, or alleviating a condition in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition, lyophilized formulation, reconstituted solution, or kit as described above.

[0160] In some embodiments, the present disclosure also provides use of the pharmaceutical composition as described in any of the above, the lyophilized preparation as described in any of the above, the reconstituted solution as described in any of the above, or the medicine kit as described in any of the above in the preparation of a drug for treating or preventing a disease.

[0161] In some embodiments, the present disclosure also provides a method for treating or preventing a disease, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition as described in any one of the above, the lyophilized formulation as described in any one of the above, the reconstituted solution as described in any one of the above, or the drug kit as described in any one of the above.

[0162] In some embodiments, the present disclosure further provides the pharmaceutical composition as described in any of the above, the lyophilized formulation as described in any of the above, the reconstituted solution as described in any of the above, or the drug kit as described in any of the above, for use in treating or preventing a disease.

[0163] In one aspect, the present disclosure also provides use of the pharmaceutical composition as described in any of the preceding items, the lyophilized formulation as described in any of the preceding items, the reconstituted solution as described in any of the preceding items, or the kit as described in any of the preceding items in the preparation of a medicament for preventing or treating a disease or condition.

[0164] In some embodiments, the disease described in any of the above items is pain, joint stiffness, or bone loss.

[0165] In some embodiments, the pain is selected from the group consisting of: osteoarticular pain, rheumatoid arthritis pain, gout, bone cancer pain, fracture pain, postoperative pain, cancer pain, painful bladder syndrome, musculoskeletal pain, prostatitis (e.g., chronic prostatitis), pelvic pain (e.g., chronic pelvic pain), interstitial cystitis, low back pain, dysmenorrhea, pain associated with bone diseases, trigeminal neuralgia, postherpetic neuralgia, herpes zoster infection, sciatica, migraine, diabetic neuropathy, and peripheral nerve-related pain.

[0166] In some embodiments, the bone loss is associated with at least one condition selected from the group consisting of osteoporosis, Paget's disease, osteomyelitis, hypercalcemia, osteopenia, osteoporosis, osteonecrosis, bone injury, bone resorption, bone dysplasia, inflammation, autoimmune disease, inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, periodontal bone resorption, osteolytic metastasis, and cancer.

[0167] In some embodiments, the cancer is selected from breast cancer, prostate cancer, thyroid cancer, kidney cancer, lung cancer, esophageal cancer, rectal cancer, bladder cancer, cervical cancer, ovarian cancer, liver cancer, gastrointestinal cancer, melanoma, multiple myeloma, osteosarcoma, lymphoma, non-small cell lung cancer, bone tumor and Hodgkin's disease.

[0168] In some embodiments, the aforementioned disease is a disease associated with NGF or RANKL.

[0169] In some embodiments, the aforementioned disease is a disease expressing NGF or RANKL.

[0170] In one aspect, the present disclosure provides a method for treating or preventing a disease associated with NGF or RANKL, the method comprising administering to a subject a preventively effective amount or a therapeutically effective amount of the pharmaceutical composition as described in any of the preceding items, the lyophilized preparation as described in any of the preceding items, the reconstituted solution as described in any of the preceding items, or the drug kit as described in any of the preceding items.

[0171] In some embodiments, the disease associated with NGF or RANKL is pain, joint stiffness, or bone loss.

[0172] In one aspect, the pharmaceutical composition of any of the preceding claims, the lyophilized formulation of any of the preceding claims, the reconstituted solution of any of the preceding claims, or the kit of any of the preceding claims of the present disclosure can be used as a medicament. In some embodiments, it is used as a medicament for treating pain, joint stiffness, or bone loss. In some embodiments, it is used as a medicament for treating osteoarthritis, rheumatoid arthritis pain, gout, bone cancer pain, fracture pain, postoperative pain, cancer pain, painful bladder syndrome, musculoskeletal pain, prostatitis (e.g., chronic prostatitis), pelvic pain (e.g., chronic pelvic pain), interstitial cystitis, low back pain, dysmenorrhea, pain associated with bone diseases, trigeminal neuralgia, postherpetic neuralgia, herpes zoster infection, sciatica, migraine, diabetic neuropathy, and pain associated with peripheral nerves. In some embodiments, it is used as a drug for the treatment of osteoporosis, Paget's disease, osteomyelitis, hypercalcemia, osteopenia, osteoporosis, osteonecrosis, bone injury, bone resorption, bone dysplasia, inflammation, autoimmune diseases, enteritis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, periodontal bone resorption, osteolytic metastasis and bone loss caused by cancer.

[0173] In one aspect, the present disclosure provides a pharmaceutical composition as described in any of the preceding items, a lyophilized formulation as described in any of the preceding items, a reconstituted solution as described in any of the preceding items, or a kit as described in any of the preceding items for the preparation of a medicament for preventing or treating a disease associated with NGF or RANKL. In some embodiments, the disease associated with NGF or RANKL is pain, joint stiffness, or bone loss.

[0174] In one aspect, the pharmaceutical composition as described in any of the preceding items, the lyophilized formulation as described in any of the preceding items, the reconstituted solution as described in any of the preceding items, or the drug kit as described in any of the preceding items provided herein can be used as a drug for preventing or treating a disease associated with NGF or RANKL. In some embodiments, the disease associated with NGF or RANKL is pain, joint stiffness, or bone loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0175] Figure 1: Schematic diagram of DVD-IgG structure.

[0176] Figures 2A to 2D: Figure 2A shows the statistical results of pain behavior in mice on day 14 of combined treatment; Figure 2B shows the statistical results of pain behavior in mice on day 21 of combined treatment; Figure 2C shows the bone injury score results in mice on day 14 of combined treatment; Figure 2D shows the bone injury score results in mice on day 21 of combined treatment. **** vs vehicle indicates P < 0.0001; *** indicates P < 0.001; ** indicates P < 0.01; * indicates P < 0.05. Blank control; sham control; vehicle control.

[0177] Figures 3A and 3B: Figure 3A shows the statistical results of pain behavior in mice on day 14 after treatment with bispecific antibody 1; Figure 3B shows the statistical results of pain behavior in mice on day 21 after treatment with bispecific antibody 1. **** vs. vehicle: P < 0.0001; *** vs. vehicle: P < 0.001; ** vs. vehicle: P < 0.01; * vs. vehicle: P < 0.05. Vehicle control.

[0178] Figures 4A to 4D: Figure 4A shows the statistical results of pain behavior in mice on day 15 after treatment with bispecific antibody 1; Figure 4B shows the statistical results of pain behavior in mice on day 21 after treatment with bispecific antibody 1; Figure 4C shows the bone injury score results in mice on day 15 after treatment with bispecific antibody 1; Figure 4D shows the bone injury score results in mice on day 21 after treatment with bispecific antibody 1. **** vs vehicle indicates P < 0.0001; *** indicates P < 0.001; ** indicates P < 0.01; * indicates P < 0.05. Sham group; vehicle control. DETAILED DESCRIPTION

[0179] the term

[0180] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present disclosure belongs.

[0181] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0182] Unless the context clearly requires otherwise, in the patent specification and claims, the words "comprising," "having," "including," and the like should be construed in the sense of "including but not limited to," rather than in an exclusive or exhaustive sense.

[0183] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and where it does not.

[0184] It will be understood by those skilled in the art that when reference is made to a numerical range, a cutoff value, or a specific value, "about" can be expressed as being within one or more than one standard deviation. Alternatively, "about" can be expressed as being within a range of up to 20% (i.e., ± 20%). Since many numerical values ​​used herein are determined experimentally, it will be understood by those skilled in the art that such determinations can differ between different experiments and typically differ between different experiments. Due to this inherent difference, it is believed that the values ​​used herein should not be overly limited. Therefore, the term "about" is used to encompass variations of ± 20% or less, ± 10% or less, ± 5% or less, ± 1% or less, ± 0.5% or less, or ± 0.1% or less from a specified value.

[0185] Although the present disclosure provides content ranges or content values, persons of ordinary skill in the art understand that the content ranges or content values ​​encompass an acceptable error range for the specific values ​​determined.

[0186] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0187] "Anti-RANKL antibody" refers to an antibody that can bind to RANKL or an epitope thereof and inhibit the biological activity of RANKL and / or inhibit the downstream pathway of RANKL.

[0188] The term "NGF", nerve growth factor, means nerve growth factor and variants thereof that retain at least a portion of the biological activity of NGF. When used herein, NGF includes wild-type sequence NGF or naturally occurring variants thereof of all mammalian species, including humans, mice, monkeys, dogs, cats, horses or cows.

[0189] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.

[0190] "Antibody" is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. "Native antibody" refers to naturally occurring immunoglobulin molecules. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains bound by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy domain, a heavy chain variable region, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also known as a variable light domain, or a light chain variable domain, followed by a constant light domain (light chain constant region, CL). The term "bispecific antibody" refers to an antibody (including an antibody or an antigen-binding fragment thereof, such as a single-chain antibody) that can specifically bind to two different antigens or at least two different antigenic epitopes of the same antigen.

[0191] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain involved in antigen binding. Herein, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved framework regions (FRs) and three complementarity determining regions (CDRs). The term "complementarity determining region" or "CDR" refers to the region within the variable region that primarily contributes to antigen binding; "framework" or "FR" refers to the variable domain residues other than the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen-binding specificity.

[0192] The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering convention, "ABM" numbering convention, "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc. The correspondence between various numbering systems is well known to those skilled in the art. The numbering convention of the present disclosure is shown in Table 1 below.

[0193] Table 1. Relationships between CDR numbering systems

[0194] Unless otherwise indicated, the variable region and CDR sequences in the disclosed embodiments are all subject to the "Kabat" numbering convention. Although in specific embodiments, the Kabat numbering convention is used to define amino acid residues, corresponding technical solutions provided by other numbering systems will be considered equivalent technical solutions.

[0195] A "pharmaceutical composition" refers to a composition containing one or more bispecific antibodies described herein, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and enabling its biological activity. As used herein, the terms "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0196] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0197] "Pharmaceutically acceptable carriers" or "pharmaceutically acceptable excipients" include any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods.

[0198] "Buffer" refers to a buffer that tolerates changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0199] "Histidine buffer" is a buffer containing histidine. Examples of histidine buffers include histidine-histidine hydrochloride, histidine-histidine acetate, histidine-histidine phosphate, histidine-histidine sulfate, and the like, with histidine-histidine hydrochloride buffer being preferred. Histidine-histidine hydrochloride buffer can be prepared from histidine and hydrochloric acid, or from histidine and histidine hydrochloride.

[0200] "Citrate buffer" is a buffer comprising citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate.

[0201] A "succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. A preferred succinate buffer is succinic acid-sodium succinate. For example, the succinic acid-sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0202] "Phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include citric acid-disodium hydrogen phosphate, disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like. A preferred phosphate buffer is citric acid-disodium hydrogen phosphate.

[0203] An "acetate buffer" is a buffer comprising acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine-histidine acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like. A preferred acetate buffer is acetic acid-sodium acetate.

[0204] "Poloxamer" is a block copolymer of ethylene oxide and propylene oxide that is water-soluble and used as a surfactant in pharmaceutical formulations. Examples of poloxamers include poloxamer 188.

[0205] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.

[0206] The pharmaceutical compositions disclosed herein can achieve a stable effect: the antibody therein substantially retains its physical stability and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, there are various analytical techniques for measuring protein stability after storage at a selected temperature for a selected period of time.

[0207] A stable formulation is one in which no significant change is observed after storage at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. Additionally, stable liquid formulations include those that exhibit the desired characteristics after storage at 25°C for a period of time, including 2 weeks, 4 weeks, 1 month, 3 months, or 6 months. Furthermore, stable liquid formulations also include those that exhibit the desired characteristics after storage at 40°C for a period of time, including 2 weeks, 4 weeks, 1 month, 3 months, or 6 months. Typical examples of stability include: typically no more than about 10%, preferably no more than about 5%, of the antibody aggregates or degrades as measured by SEC-HPLC. Visually, the formulation is a pale yellow, nearly colorless, clear liquid, or a colorless, clear liquid, or clear to slightly opalescent. The concentration, pH, and osmolality of the formulation vary by no more than ±10%, preferably no more than ±5%. The formulation typically exhibits no more than about 10%, preferably no more than about 5%, of aggregation.

[0208] An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured by visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

[0209] An antibody "retains its chemical stability" in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0210] An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared.

[0211] "Administer," "give," and "treat," as they apply to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer," "give," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "give," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as it applies to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0212] "Treatment" means administering an internal or external therapeutic agent, such as a pharmaceutical composition comprising any of the present disclosures, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, a therapeutic agent is administered to a patient or population treated in an amount effective to alleviate one or more symptoms of a disease, to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0213] The pharmaceutical composition of the present disclosure can be applied by any suitable means, including parenteral, intrapulmonary and intranasal, and if local treatment is needed, then intralesional application. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any appropriate route, for example, by injection, such as intravenous or subcutaneous injection. A variety of dosing time schemes are contemplated herein, including but not limited to, single or multiple administrations at multiple time points, push administration and pulse infusion. In some embodiments, the pharmaceutical composition of the present disclosure is applied by subcutaneous injection.

[0214] The pharmaceutical composition of the present disclosure will be prepared, administered and used in a manner consistent with good medical practice. Factors considered in this context include the specific illness treated, the specific mammal treated, the clinical condition of the individual patient, the cause of the illness, the delivery site of the reagent, the method of administration, the administration time arrangement and other factors known to medical practitioners. Optionally, the pharmaceutical composition can also be prepared together with one or more other reagents for preventing or treating the illness. The effective amount of such other reagents depends on the amount of the antigen binding molecules present in the pharmaceutical composition, the type of illness or treatment and other factors. Can be used with the same dosage and administration route as described herein, or with about 1 to 99% of the dosage described herein, or with any dosage, and be determined to be suitable any approach use by experience / clinical practice.

[0215] There is no limitation on the diseases related to NGF or RANKL in the present disclosure, as long as it is a disease related to NGF or RANKL. For example, the therapeutic response induced by the antibodies disclosed herein can be achieved by binding to human NGF or RANKL and then inhibiting the binding of NGF or RANKL to its receptor, or killing cells that overexpress NGF or RANKL; or inhibiting the growth of cells that overexpress NGF or RANKL.

[0216] The details of one or more embodiments of the present disclosure are set forth in the above description. Although any methods and materials similar or identical to those described herein can be used to implement or test the present disclosure, preferred methods and materials are described below. Other features, objects, and advantages of the present disclosure will be apparent from the description and claims. In the description and claims, unless the context clearly indicates otherwise, the singular includes the case of plural referents. Unless otherwise defined, all technical and scientific terms used herein have the common meaning understood by those of ordinary skill in the art to which the present disclosure belongs. All patents and publications cited in the description are incorporated by reference. The following examples are presented to more fully illustrate the preferred embodiments of the present disclosure. These examples should not be construed in any way to limit the scope of the present disclosure, which is defined by the claims.

[0217] Example - Preparation and detection of anti-RANKL-NGF bispecific antibodies

[0218] PCT / CN2022 / 092333 (application date: 2022.05.12; priority patent application number: CN202110515444.9) is incorporated into this disclosure in its entirety by reference.

[0219] Example 1: Preparation of anti-RANKL antibodies

[0220] A yeast library was constructed by designing primers. The library was enriched and screened using RANKL protein (Sino biological, 11682-HNCH) to obtain candidate clones. The amino acid sequences of the light and heavy chain variable regions of the antibodies in the clones were determined by sequencing. The sequences of the exemplary anti-RANKL antibodies obtained are as follows:

[0221] Table 2. CDR region sequences of anti-RANKL antibodies

[0222] Table 3. RANKL antibody variable region sequences

[0223] The above variable regions are fused with the human light chain constant region and heavy chain constant region to form complete antibody light and heavy chains. The constant region sequences of exemplary antibodies are as follows:

[0224] Human IgG4 heavy chain constant region:

[0225] Human kappa light chain constant region:

[0226] The sequences of exemplary anti-RANKL antibodies are as follows:

[0227] D75H heavy chain:

[0228] D75H light chain:

[0229] The sequence of the control anti-RANKL antibody is as follows:

[0230] Denosumab heavy chain sequence:

[0231] Denosumab light chain sequence:

[0232] Example 2: Preparation of anti-RANKL / NGF bispecific antibody

[0233] The second antigen-binding domain that binds to NGF of the bispecific antibodies of the present disclosure can be derived from the antigen-binding portion of any suitable antibody. Particularly suitable antibodies are described in, for example, International Application WO2004058184A2 (incorporated herein by reference in its entirety).

[0234] The CDR and variable region sequences of the NGF binding domain of an exemplary bispecific antibody are shown below:

[0235] Table 4. CDRs of the NGF binding domain

[0236] NGF binding domain heavy chain variable region:

[0237] NGF binding domain light chain variable region:

[0238] The variable region of the NGF binding domain was fused to the heavy chain constant region of SEQ ID NO: 9 or SEQ ID NO: 23 and the light chain constant region of SEQ ID NO: 10, respectively, to obtain an anti-NGF antibody. The sequence is as follows:

[0239] Anti-NGF antibody (N-mAb) heavy chain:

[0240] Anti-NGF antibody (N-mAb) light chain:

[0241] Sequence of control anti-NGF antibody:

[0242] Tanezumab heavy chain:

[0243] Tanezumab light chain:

[0244] For bone metastasis indications, the current clinical dosages of monoclonal antibodies targeting NGF and RANKL vary significantly. Therefore, when preparing bispecific antibodies targeting NGF and RANKL, it is necessary to balance the activities of the NGF and RANKL arms of the bispecific antibody to determine the appropriate dosage of the bispecific antibody, avoiding side effects caused by excessively high dosages or ineffective bispecific antibody function due to excessively low dosages.

[0245] The bispecific antibodies prepared by the present disclosure have a DVD-IgG structure (as shown in Figure 1). The light chain variable domains (VL) of the anti-RANKL antibody and the anti-NGF antibody are directly or connected in series via a short linker by recombinant DNA technology, followed by the light chain constant domain. Similarly, the heavy chain comprises two heavy chain variable domains (VH) connected in series, followed by the constant domain CH1 and Fc region. The linker includes but is not limited to the following peptide linkers: ASTKGP (SEQ ID NO: 28), TVAAP (SEQ ID NO: 29).

[0246] The sequences of exemplary bispecific antibodies are as follows:

[0247] First chain of bispecific antibody 1

[0248] Second chain of bispecific antibody 1

[0249] Note: The single underline in the sequence is the variable region of RANKL antibody, the double underline is the variable region of NGF antibody, the bold is the linker, and the rest of the sequence is the constant region.

[0250] Test Case

[0251] Test Example 1. Anti-RANKL Antibody Affinity Test

[0252] A certain amount of the test antibody was affinity-captured using a biosensor chip Protein A (GE, 29127556). A series of antigen concentration gradients, including human RANKL protein (Sino Biological, 11682-HNCH) and human NGF protein (Sino Biological, 11050-HNAC), were then passed over the chip surface. Binding and dissociation curves were generated using Biacore real-time monitoring. After each dissociation cycle, the biochip was washed and regenerated with pH 1.5 glycine-HCl regeneration solution (GE, BR-1003-54). The experimental data were fitted using BIAevaluation version 4.1 software using a 1:1 model to determine affinity values. The results are shown in Table 5 below.

[0253] Table 5. Affinity KD values ​​of anti-RANKL antibodies

[0254] The results showed that the affinity of the modified antibody D75H was more than 6 times higher than that of the Denosumab antibody.

[0255] Test Example 2: Detection of the ability of anti-RANKL antibodies to block the binding of RANKL to RANK

[0256] The activity of the antibodies blocking ligand and receptor binding was tested by ELISA. Human RANK protein was diluted to 2 μg / mL in PBS (Source Bio, B320) at pH 7.4 and added to a 96-well ELISA plate (Corning, 3590) at a volume of 100 μL / well. The plate was incubated overnight at 4°C. After discarding the liquid, 200 μL of 1% Casein blocking solution (Thermo, 37528) was added to each well for blocking and incubation at 37°C for 2 hours. After blocking, the blocking solution was discarded and the plate was washed three times with PBST buffer (pH 7.4 PBS containing 0.1% tween-20) before use. A fixed concentration of biotin-labeled human RANKL protein (Sino Biological, 11682-HNCH) was mixed with serially diluted antibodies and pre-incubated at 37°C for 30 minutes before being added to the blocked ELISA plate and incubated at 37°C for 1.5 hours. After incubation, wash the plate three times with PBST, add 100 μL of streptavidin-HRP (Invitrogen, 434323, 1:4000 dilution) to each well, and incubate at 37°C for 1 hour. Remove the supernatant, wash the plate three times with PBST, and add 100 μL of TMB colorimetric substrate (KPL, 5120-0077) to each well. Incubate at room temperature for 10 to 15 minutes. Add 50 μL of 1M H2SO4 to each well to terminate the reaction. Read the absorbance at 450 nm using a microplate reader. Use software to fit the curve of inhibition of ligand and receptor binding and calculate the IC 50 The results are shown in Table 6 below.

[0257] Table 6. Ability of anti-RANKL antibodies to block the binding of RANKL to RANK

[0258] The results showed that the anti-RANKL antibody D75H could block the binding of RANKL to RANK, and its blocking activity was better than that of Denosumab antibody.

[0259] Test Example 3: ELISA to detect the binding activity of bispecific antibodies to antigens from different species

[0260] The binding activity of the bispecific antibody disclosed herein to human RANKL (Sino biological, 11682-HNCH), monkey RANKL (Sino Biological, 90301-C01H), mouse RANKL (R&D Systems, 462-TR / CF), human NGF (the sequence of monkey NGF is identical to that of human NGF) (Sino biological, 11050-HNAC), and mouse NGF (Sino Biological, 50385-MNAC) was detected by ELISA as follows:

[0261] The antigen was diluted to 1 μg / mL in PBS buffer (Source Bio, B320) at pH 7.4 and added to a 96-well microtiter plate (Corning, 9018) at a volume of 100 μL / well. The plate was incubated at 4°C overnight. After discarding the liquid, 300 μL / well of blocking solution (5% skim milk (BD, 232100) diluted in PBS) was added and incubated at 37°C for 2 hours. After blocking, the blocking solution was discarded and the plate was washed three times with PBST buffer (PBS, pH 7.4 containing 0.1% tween-20). Then, 100 μL of bispecific antibody diluted in sample diluent (PBS, pH 7.4 containing 1% BSA) at different concentrations was added to each well and incubated in a 37°C incubator for 1 hour. After incubation, wash the plate three times with PBST, add 100 μL of HRP-labeled anti-human Fc secondary antibody (Abcam, ab97225) diluted in sample diluent to each well, and incubate at 37°C for 1 hour. After washing the plate three times with PBST, add 100 μL of TMB colorimetric substrate (KPL, 5120-0077) to each well and incubate at room temperature for 10 to 15 minutes. Add 50 μL of 1M H2SO4 to each well to terminate the reaction. Read the absorbance at 450 nm using a microplate reader to calculate the EC value of the bispecific antibody binding to the antigen. 50 The results are shown in Table 7 below.

[0262] Table 7. EC values ​​of bispecific antibodies binding to antigens from different species 50

[0263] The results showed that the binding activity of bispecific antibody 1 to human RANKL and monkey RANKL was superior to that of the control Denosumab antibody. However, neither bispecific antibody 1 nor Denosumab cross-binds to mouse RANKL. The binding activity of bispecific antibody 1 to human NGF and mouse NGF was significantly weaker than that of Tanezumab, indicating that the bispecific antibody 1 disclosed herein successfully reduced the binding activity of the Tanezumab end (the second antigen binding domain that specifically binds to NGF).

[0264] Test Example 4: Bispecific Antibody Affinity Test

[0265] A certain amount of the test antibody was affinity-captured using a biosensor chip Protein A (GE, 29127556). A series of antigen concentration gradients, including human RANKL protein (Sino Biological, 11682-HNCH) and human NGF protein (Sino Biological, 11050-HNAC), were then passed over the chip surface. The reaction signals were monitored in real time using Biacore to generate binding and dissociation curves. After each dissociation cycle, the biochip was washed and regenerated with pH 1.5 glycine-HCl regeneration solution (GE, BR-1003-54). The experimental data were fitted using BIAevaluation version 4.1 software using a 1:1 model to determine affinity values. The results are shown in Table 8 below.

[0266] Table 8. Affinity of bispecific antibodies to different antigens

[0267] The results showed that the affinity of bispecific antibody 1 for human RANKL was 3.8 times that of Denosumab, and the affinity of bispecific antibody 1 for human NGF was significantly lower than that of Tanezumab.

[0268] Test Example 5: Ligand and receptor blocking experiment

[0269] The blocking activity of antibodies against ligands and receptors was tested by ELISA. The receptor protein (RANK or TrkA) was diluted to 2 μg / mL in PBS (Source Bio, B320) at pH 7.4 and added to a 96-well ELISA plate (Corning, 3590) at a volume of 100 μL / well. The plate was incubated overnight at 4°C. After discarding the liquid, 200 μL of 1% Casein blocking solution (Thermo, 37528) was added to each well for blocking and incubation at 37°C for 2 hours. After blocking, the blocking solution was discarded and the plate was washed three times with PBST buffer (pH 7.4 PBS containing 0.1% tween-20) before use. A fixed concentration of biotin-labeled ligand protein (RANKL or NGF) was mixed with a gradient dilution of the antibody or fusion protein, pre-incubated at 37°C for 30 minutes, and then added to the blocked ELISA plate and incubated at 37°C for 1.5 hours. After incubation, wash the plate three times with PBST, add 100 μL streptavidin-HRP (Invitrogen, 434323, 1:4000 dilution) to each well, and incubate at 37°C for 1 hour. Remove the supernatant, wash the plate three times with PBST, and add 100 μL TMB colorimetric substrate (KPL, 5120-0077) to each well. Incubate at room temperature for 10 to 15 minutes. Add 50 μL 1 M H2SO4 to each well to terminate the reaction. Read the absorbance at 450 nm using a microplate reader. Use software to fit the curve of inhibition of ligand and receptor binding, and calculate the IC 50 value.

[0270] The ligand and receptor protein information used in the experiment are as follows: human RANKL protein (Sino Biological, 11682-HNCH), human RANK protein (Sino Biological, 16078-H02H), human TrkA protein (Sino Biological, 11073-H03H), and human NGF protein (Sino biological, 11050-HNAC).

[0271] The experimental results are shown in Table 9 below.

[0272] Table 9. Blocking activity of bispecific antibodies

[0273] The results showed that bispecific antibody 1 had better blocking activity against RANKL than Denosumab, and significantly weaker blocking activity against NGF than Tanezumab. This suggests that by reducing the binding activity of bispecific antibody 1 to the NGF end, its blocking activity against NGF was also significantly reduced.

[0274] Test Example 6: Osteoclast differentiation experiment

[0275] The main function of RANKL is to promote the differentiation and maturation of osteoclasts. Therefore, the inhibitory activity of antibodies against RANKL can be tested through osteoclast differentiation experiments. The experimental method is as follows:

[0276] Raw264.7 cells (ECACC, 91062702) were plated in 96-well cell culture plates (Corning, 3599) and cultured overnight in a 37°C incubator. The next day, a fixed concentration of human RANKL protein (Sino Biological, 11682-HNCH) and a serial dilution of the bispecific antibody to be tested were added to the 96-well cell plate and cultured at 37°C for 4 days. The 96-well plate was removed, the supernatant was removed, and 100 μL of cell lysis buffer (Biyuntian, P0013J) was added to each well. After pipetting and mixing, the cell lysate was transferred to a centrifuge tube, placed on ice for 10 minutes, and centrifuged to obtain the supernatant. Referring to the method in the tartrate-resistant acid phosphatase detection kit (Biyuntian, P0332), 40 μL of the sample to be tested was added with 40 μL of the colorimetric substrate and 5 μL of tartaric acid, mixed evenly, and placed in a 37°C incubator for 10 min. 160 μL of stop solution was added, and the OD 405 nm value was detected by a microplate reader. The data were fitted with software to obtain the IC 50 The experimental results are shown in Table 10 below.

[0277] Table 10. Activity of bispecific antibodies in inhibiting osteoclast differentiation

[0278] The results showed that the activity of bispecific antibody 1 in inhibiting osteoclast differentiation was more than 3.3 times that of Denosumab.

[0279] Test Example 7: TF-1 cell proliferation assay

[0280] NGF can promote the proliferation of TF-1 cells in vitro, so the inhibitory activity of the antibody against NGF was evaluated by TF-1 cell proliferation assay. The experimental method is as follows:

[0281] TF-1 cells (ATCC, CRL-2003) were resuspended in 1640 medium without GM-CSF (Gibco, 22400-105) and plated in 96-well cell culture plates (Corning, 3903) and cultured overnight in a 37°C incubator. The next day, a fixed concentration of human NGF protein (Sino biological, 11050-HNAC) and gradient dilutions of the test antibody were added to the 96-well cell plate and cultured in a 37°C incubator for 72 hours. The cell culture plate was removed, 50 μL of Cell-titer Glo (Promega, G755B) solution was added to each well, gently shaken to mix for 10 minutes, allowed to stand at room temperature for 10 minutes, and the bioluminescent signal was detected with PE Victor 3. The data were fitted using software to obtain IC 50 The experimental results are shown in Table 11 below.

[0282] Table 11. Activity of bispecific antibodies in inhibiting TF-1 cell proliferation

[0283] The results showed that bispecific antibody 1 can still inhibit the proliferation of TF-1 cells, among which Tanezumab's inhibitory activity is about 4.2 times that of bispecific antibody 1, indicating that bispecific antibody 1 significantly reduced the activity of the NGF end.

[0284] Therefore, under the premise that in vitro activity is correlated with dosage, we anticipate that the dosage of bispecific antibody 1 for the RANKL end can be reduced to approximately 40 mg while still maintaining the in vivo efficacy of the RANKL end; while the dosage of the NGF end can be increased to approximately 80 mg while still maintaining a good safety profile. Furthermore, the current clinical dosing cycle of the NGF-targeting antibody Tanezumab is twice that of the anti-RANKL antibody Denosumab, so the exemplary bispecific antibody 1 disclosed herein can achieve a balanced dosage for both targets.

[0285] Test Example 8: In vivo efficacy in bone metastasis animal models

[0286] A bone metastasis animal model was used to evaluate the analgesic and bone protective effects of the antibody.

[0287] The model was established using SPF-grade male C57BL / 6 mice purchased from Changzhou Cavens Laboratory Animal Co., Ltd. They were housed in a laboratory environment with a 12 / 12 hour light / dark cycle, a temperature of 23 ± 1°C, and a humidity of 40-50%. The animals were fed a standard sterilized mouse chow diet with free access to food and water. Modeling was initiated when mice reached approximately 25 g in weight.

[0288] After the mice were anesthetized with 1% sodium barbital injected intraperitoneally, the skin was prepared and the mice were placed on a heating pad. Using ophthalmic scissors, a 1 cm incision was made on the skin on the outside of the left hind knee parallel to the femur to expose the muscle. The skin and muscle layer were bluntly separated. Using ophthalmic scissors, the connective tissue line was used as a guide to cut between the rectus femoris and vastus medialis muscles. The rectus femoris and patella were moved to the medial side of the knee joint with curved forceps to expose the femoral condyle without cutting the patellar ligament. A 0.45 mm diameter needle was used to make a hole from the top to the middle of the femur at the intermalleolar fossa, and the injection channel was opened by entering the intramedullary space 1 to 1.5 cm. A Hamilton microsyringe (50 μL, Model 1705 RN SYR, 26 gauge needle, model ga26 / 51mm / pst3, catalog number 7768-02) was inserted into the bone marrow cavity through the incision and 10 μL (5×10 4 )LLC1 cells (ATCC, CRL-1642). After the injection is completed, the syringe needle is pulled out, the leg is straightened and the patella and ligament are repositioned with curved forceps, the muscle is restored to its original shape, antibiotic powder is sprinkled, and the skin is sutured with automatic wound clips (Roboz, Reflex 7 Clip 7mm). After surgery, no more than three animals should be kept in a cage, and the wound clips should be removed after seven days. The sham group (Sham) was operated in the same way as above, and the same volume of PBS was injected into the bone marrow cavity; the blank group was kept normally without any treatment. Drug administration began 7 days after modeling, and the drug was administered once every 5 days, for a total of 3 times. The pain behavior of the mice and the leg bone damage were scored on the 14th day (or 15th day) and the 21st day respectively.

[0289] Pain behavior statistics: Animals were placed on a floor surrounded by plexiglass and a wire grid below. They were first acclimated for 30 min (until the animals' exploration and major grooming activities in the box ceased). Then their movements were observed and the avoidance behavior of the affected limb pain within 5 min was evaluated. The duration was measured with a stopwatch.

[0290] Pain avoidance behavior is defined as:

[0291] (1) Complete protection (lift the affected limb when walking and do not put weight on the ground);

[0292] (2) The five fingers of the affected limb curl up and pierce the mesh (the limbs of normal mice should be stretched out with five fingers flat on the mesh, and supported by the forefoot. The affected limb of mice with tumor bone metastasis will curl up with five fingers. If the hind heel can be used for support at this time, the pain is considered mild and is not counted in the measurement time. If the entire sole of the foot does not touch the mesh or pierce the mesh, it is counted in the measurement time);

[0293] (3) licking the affected limb;

[0294] (4) sporadic single-leg hopping;

[0295] (5) Unilateral hind limb stance (with both forelimbs raised).

[0296] Bone injury scoring method:

[0297] The left femur was carefully removed in the prone position, and bone destruction was detected using an MX-20 digital cabinet X-ray system (Faxitron / Bioptics). Bone damage was scored by a fixed professional.

[0298] The scoring criteria are as follows:

[0299] 0 - relatively normal, 1 - mild - localized damage, 2 - moderate - localized damage, 3 - moderate - multiple damage, 4 - severe - diffuse damage. Scores are added together for the distal and proximal femurs, with the most severe bone destruction being assigned a score of 4 + 4 = 8.

[0300] 1. Testing the effects of RANLK antagonists and NGF antagonists on analgesia and bone protection

[0301] Since Denosumab has no cross-binding activity against mouse RANKL, we used the mouse RANKL antibody AMR2 in combination with Tanezumab to evaluate whether the in vivo efficacy of the two antibodies combined is superior to that of a single antibody.

[0302] The variable region sequences of the AMR2 antibody were derived from WO2013176469A1. The variable region heavy and light chain sequences were fused with human IgG4 and human lambda constant regions, respectively, to construct the full-length antibody AMR2. The relevant sequences of AMR2 are as follows:

[0303] Table 12. CDRs of AMR2 Antibody

[0304] AMR2 heavy chain variable region:

[0305] AMR2 light chain variable region:

[0306] AMR2 heavy chain

[0307] AMR2 light chain

[0308] The groups and dosages are shown in Table 13 below:

[0309] Table 13. Grouping and dosage Note: Q5d*3 means administration once every 5 days, for a total of 3 times; ip means intraperitoneal injection.

[0310] The experimental results are shown in Figures 2A to 2D below.

[0311] Statistical analysis of pain behavior in mice showed that on day 14, all three treatment groups exhibited significant analgesic effects compared to the negative control group, with the combined treatment group exhibiting a stronger analgesic effect than the monoclonal antibody group. On day 21, only the combined treatment group demonstrated a significant analgesic effect. While both monoclonal antibody groups also showed some analgesic effect, no statistical difference was observed compared to the negative control group (vs vehicle: *P<0.05, **P<0.01, ***P<0.0001).

[0312] Bone damage scoring results showed that on day 14, the combined drug group exhibited a certain bone protective effect. On day 21, the combined drug group showed a significant bone protective effect. The above experimental results show that the combined drug group showed efficacy in both analgesia and bone protection. Therefore, the inventors subsequently further verified the efficacy of the bispecific antibody molecules disclosed herein in vivo.

[0313] 2. Evaluation of the in vivo efficacy of bispecific antibody NGF

[0314] Since the in vitro activity of the NGF end of bispecific antibody 1 is about 4 times lower than that of the NGF monoclonal antibody Tanezumab, this efficacy experiment was conducted to determine whether the NGF end of bispecific antibody 1 still has efficacy in mice. The experimental groups are shown in the following table:

[0315] Table 14. Dosage and grouping Note: Q5d means administration once every 5 days; ip means intraperitoneal injection.

[0316] The experimental results are shown in FIG3A and FIG3B .

[0317] The results showed that on the 14th and 21st days after administration, bispecific antibody 1 showed significant analgesic effect compared with the negative group, indicating that the NGF end of bispecific antibody 1 still has significant pharmacological efficacy.

[0318] 3. In vivo efficacy evaluation of bispecific antibodies

[0319] A bone metastasis animal model was established using human RANKL transgenic mice (purchased from Biocytogen) to evaluate the in vivo efficacy of the bispecific antibody. The experimental groups are shown in Table 15 below:

[0320] Table 15. Dosage and grouping Note: Q5d means administration every 5 days; ip means intraperitoneal injection.

[0321] The experimental results are shown in Figures 4A to 4D.

[0322] The results showed that bispecific antibody 1 demonstrated significant analgesic and bone protective effects. On days 15 and 21, both the high-dose group of bispecific antibody 1 demonstrated significant analgesic effects, and both the high- and low-dose groups demonstrated significant protective effects against bone damage.

[0323] Test Example 9: Rat PK Test

[0324] In vivo pharmacokinetic testing was performed in SD rats. Male SD rats (Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were divided into groups of four and administered intravenously with bispecific antibody 1 at a dose of 4 mg / kg. 0.2 mL of whole blood (without anticoagulation) was collected before dosing and 5 minutes, 8 hours, 24 hours, 48 ​​hours, 84 hours, 9 days, 10 days, 14 days, 21 days, and 28 days after dosing. After blood collection, the blood was incubated at 4°C for 30 minutes, centrifuged at 1000 g for 15 minutes, and the supernatant serum was collected and placed in an EP tube and stored at -80°C.

[0325] The blood drug concentration in serum was detected by ELISA method, and the pharmacokinetic parameters of the antibody to be tested were calculated using Winnolin software. The test results are shown below:

[0326] Table 16. PK results of bispecific antibody 1

[0327] The results showed that bispecific antibody 1 had good PK performance in rats, and the half-lives at both ends of RANKL and NGF were relatively close, at 15.09 days and 14.3 days, respectively.

[0328] Preparation Example - Anti-RANKL-NGF Bispecific Antibody Preparation

[0329] Exemplary Antibody Pharmaceutical Composition (Formulation) Preparation Process

[0330] Step 1: The RANKL-NGF bispecific antibody and the stabilizer are formulated into a preparation stock solution containing the antibody, which is sterilized and filtered through a 0.22 μm filter element, and the filtrate is collected.

[0331] Step 2: Adjust the filling volume (target filling volume is 1.0 mL / bottle), select vials for filling, and take samples at the beginning, middle, and end of filling to detect filling volume differences.

[0332] Step 3: Turn on the capping machine, add aluminum caps, and start capping.

[0333] Step 4: Visual inspection to confirm that the product has no defects such as inaccurate filling quantity and poor appearance. Print carton labels, fold cartons, pack cartons, and apply carton labels.

[0334] Appearance:

[0335] Using the visual method, wipe the sample bottle clean and observe the sample color, clarity and visible foreign matter on a clarity tester with a white background and a black background under a light intensity of 1000-1500lx.

[0336] Appearance inspection instrument: Jingtuo Instrument YB-2A clarity tester.

[0337] Melting temperature (Tm) and aggregation temperature (Tagg):

[0338] Tm is the temperature at which 50% of the protein components are denatured during heating; Tagg is the temperature at which protein aggregation occurs during heating. Load the sample into a Uni tube and run the assay at a thermal ramp of 25-95°C.

[0339] Tm and Taag determination instruments: Uncle, instrument manufacturer is Unchained.

[0340] SEC size exclusion chromatography:

[0341] An analytical method for separating solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0342] SEC% (SEC monomer content percentage) = A monomer / A total × 100% (A monomer is the peak area of ​​the main peak monomer in the sample, A total is the sum of all peak areas). ΔSEC% = SEC% of the formulation after the stability experiment - SEC% of the formulation before the stability experiment.

[0343] SEC measurement instrument: Agilent HPLC 1260.

[0344] Column: Tosoh, TSKgel G3000SWXL (7.8 mm×30 cm, 5 μm).

[0345] NR-CE capillary gel electrophoresis:

[0346] A method of electrophoresis in which gel is transferred to a capillary tube as a supporting medium and samples are separated according to their molecular weight at a certain voltage.

[0347] NR-CE% (non-reduced CE purity percentage) = Amainpeak / Atotal × 100% (Amainpeak is the peak area of ​​the main peak in the sample, and Atotal is the sum of all peak areas.) ΔNR-CE% = NR-CE% of the preparation after the stability experiment - NR-CE% of the preparation before the stability experiment.

[0348] CE measurement instrument: Beckman capillary electrophoresis instrument, model PA800plus.

[0349] icIEF imaging capillary isoelectric focusing electrophoresis:

[0350] Using the capillary as the separation channel, a DC voltage is applied to both ends of the capillary. The amphoteric electrolyte solution in the capillary forms a pH gradient within a certain range. Each component migrates to its respective isoelectric point according to the difference in charge and focuses into a very narrow segment, thereby achieving component separation.

[0351] icIEF% (main peak content percentage) = A main peak area / A total area × 100% (A total area is the sum of the acidic peak, main peak and basic peak areas). ΔicIEF% = icIEF% of the preparation after the stability test - icIEF% of the preparation before the stability test.

[0352] Instrument for icIEF measurement: Protein Simple, model Maurice.

[0353] The RANKL-NGF bispecific antibody used in the following preparation examples is the bispecific antibody 1 described above, sometimes referred to simply as "antibody."

[0354] Preparation Example 1. pH Screening of RANKL-NGF Bispecific Antibody Preparation

[0355] A 10mM citric acid-sodium hydrogen phosphate buffer system was used, and 10 different pH values ​​(4.2, 4.6, 5.0, 5.4, 5.8, 6.2, 6.6, 7.0, 7.4, and 7.8) were designed to prepare 10 formulations of the RANKL-NGF bispecific antibody at a concentration of 20mg / mL. The melting temperature (Tm) of the samples was measured to investigate the conformational stability of the antibody at different pH conditions. The aggregation temperature (Tg) of the samples was also measured to investigate the colloidal stability of the antibody at different pH conditions. The thermal stability of the antibody at different pH conditions was also investigated by measuring the appearance, SEC, and NRCE of the samples at 40°C.

[0356] 1) 10 mM citric acid-disodium hydrogen phosphate, pH 4.2;

[0357] 2) 10 mM citric acid-disodium hydrogen phosphate, pH 4.6;

[0358] 3) 10 mM citric acid-disodium hydrogen phosphate, pH 5.0;

[0359] 4) 10 mM citric acid-disodium hydrogen phosphate, pH 5.4;

[0360] 5) 10 mM citric acid-disodium hydrogen phosphate, pH 5.8;

[0361] 6) 10 mM citric acid-disodium hydrogen phosphate, pH 6.2;

[0362] 7) 10 mM citric acid-disodium hydrogen phosphate, pH 6.6;

[0363] 8) 10 mM citric acid-disodium hydrogen phosphate, pH 7.0;

[0364] 9) 10 mM citric acid-disodium hydrogen phosphate, pH 7.4;

[0365] 10) 10 mM citric acid-disodium hydrogen phosphate, pH 7.8.

[0366] Table 17. RANKL-NGF bispecific antibody formulation pH screening results 1

[0367] Table 18. RANKL-NGF bispecific antibody formulation pH screening results 2

[0368] Table 19. RANKL-NGF bispecific antibody formulation pH screening results 3

[0369] The results showed that the RANKL-NGF bispecific antibody had high Tm and Tagg values ​​at pH 4.6-5.4, indicating good conformational and colloidal stability within this pH range. After one week of high-temperature (40°C) testing, a small amount of visible protein particles appeared in the pH 4.2-5.4 range, while larger amounts appeared at other pH levels. Regarding purity, with the exception of the significant aggregation observed after 7 days of incubation at 40°C in the pH 4.2, 7.4, and 7.8 systems, and a small amount of aggregation observed at pH 7.0, no significant aggregation was observed in the remaining pH systems, and no significant differences were observed between groups. NRCE results showed that the protein fragmentation content in the pH 4.6-5.8 system was relatively low at T0, and the fragmentation ratio of pH 5.0-5.8 was less than 4% after 7 days of incubation at 40°C. Based on the molecular characteristics, appearance and SEC results of the above samples, it was found that the antibody performed best in the pH 4.6-5.4 system, so the intermediate pH 5.0 was selected as the preferred pH condition for subsequent studies.

[0370] Preparation Example 2. Screening of buffer systems for RANKL-NGF bispecific antibody preparations

[0371] Three buffer systems, 10 mM acetic acid-sodium acetate at pH 4.8 and 5.2, and 10 mM histidine-histidine hydrochloride at pH 5.2, were selected. Using 75 mg / mL sucrose as a stabilizer, and a RANKL-NGF bispecific antibody concentration of 20 mg / mL, three formulations were prepared. The stability of the three formulations was evaluated by testing sample appearance, SEC-HPLC, NRCE, and iCIEF purity. The stability of the three formulations was evaluated after exposure to high temperature (40°C) for 2 weeks, 25°C for 2 weeks and 4 weeks, 2-8°C for 2 weeks and 4 weeks, and repeated freeze-thaw cycles (-35°C and room temperature) for 3 and 5 times, to identify the optimal buffer system.

[0372] 1) 10 mM acetic acid-sodium acetate, pH 4.8, 75 mg / mL sucrose, 20 mg / mL antibody;

[0373] 2) 10 mM acetic acid-sodium acetate, pH 5.2, 75 mg / mL sucrose, 20 mg / mL antibody;

[0374] 3) 10 mM histidine-histidine hydrochloride, pH 5.2, 75 mg / mL sucrose, 20 mg / mL antibody.

[0375] Table 20. RANKL-NGF bispecific antibody preparation buffer system screening results 1

[0376] Table 21. RANKL-NGF bispecific antibody preparation buffer system screening results 2

[0377] Table 22. RANKL-NGF bispecific antibody preparation buffer system screening results 3

[0378] Table 23. RANKL-NGF bispecific antibody preparation buffer system screening results 4

[0379] Results showed that after two weeks of incubation at 25°C and 40°C, the protein in the 10mM acetic acid-sodium acetate system (pH 5.2) exhibited opalescence, while the protein in the 10mM acetic acid-sodium acetate system (pH 4.8) exhibited slight opalescence. A small amount of protein particles appeared in both formulations. Furthermore, the protein in the 10mM histidine-histidine hydrochloride system (pH 5.2) exhibited significant protein particles after two weeks of incubation at 40°C. This visual observation suggests that the protein exhibited greater stability in the 10mM acetic acid-sodium acetate buffer system. Regarding purity, the proportions of aggregates, monomers, and fragments in each formulation under different stability conditions did not significantly change compared to T0, and there were no differences between the groups, indicating good purity and stability as determined by SEC-HPLC. Under high temperature conditions, the 10mM histidine-histidine hydrochloride system exhibited a more pronounced increase in NRCE fragmentation compared to the acetate buffer system. iCIEF analysis revealed no significant differences among the formulations. Therefore, the 10mM acetic acid-sodium acetate buffer system was selected for subsequent formulation development.

[0380] Preparation Example 3. RANKL-NGF bispecific antibody preparation concentration and stabilizer screening

[0381] To ensure a better pH buffering effect of the buffer system, the buffer system concentration was increased to 20mM. 20mM acetic acid-sodium acetate, pH 5.0, was selected as the buffer system, 0.2mg / mL polysorbate 80 was selected as the surfactant, 75mg / mL sucrose or 240mM proline or 0.8% (w / v) sodium chloride was selected as the stabilizer, and the antibody concentrations were 20mg / mL, 50mg / mL, and 70mg / mL, respectively. A total of five groups of formulation samples were prepared, and the sample stability was investigated under shaking (300rpm / 25℃), repeated freeze-thaw cycles (-35℃ / room temperature), high temperature (40℃), 25℃, and light conditions.

[0382] 1) 20 mM acetic acid-sodium acetate, pH 5.0, 75 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 50 mg / mL antibody;

[0383] 2) 20 mM acetic acid-sodium acetate, pH 5.0, 240 mM proline, 0.2 mg / mL polysorbate 80, 50 mg / mL antibody;

[0384] 3) 20 mM acetic acid-sodium acetate, pH 5.0, 0.8% (w / v) sodium chloride, 0.2 mg / mL polysorbate 80, 50 mg / mL antibody;

[0385] 4) 20 mM acetic acid-sodium acetate, pH 5.0, 75 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 20 mg / mL antibody;

[0386] 5) 20 mM acetic acid-sodium acetate, pH 5.0, 75 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 70 mg / mL antibody.

[0387] Table 24. RANKL-NGF bispecific antibody formulation concentration and stabilizer screening - Results 1

[0388] Table 25. RANKL-NGF bispecific antibody formulation concentration and stabilizer screening - Results 2

[0389] Table 26. RANKL-NGF bispecific antibody formulation concentration and stabilizer screening - Results 3

[0390] Table 27. RANKL-NGF bispecific antibody formulation concentration and stabilizer screening - Results 4

[0391] Results showed that compared with the other formulations, formulation 3 had a slightly heavier opalescence and a slightly inferior appearance. Regarding purity, the SEC-HPLC aggregate and NRCE fragment content remained unchanged across all formulations under conditions of shaking, illumination, freeze-thaw cycles, and 25°C. At 40°C, the SEC-HPLC aggregate content of formulation 3 increased significantly, indicating poor stability. The NRCE fragment content of formulation 2 increased less, suggesting that proline, as a stabilizer, significantly improved antibody stability. Higher antibody concentrations were associated with higher NRCE purity (with relatively small differences between the 50 mg / mL and 70 mg / mL formulations). The iCIEF acidic, neutral, and basic peaks remained unchanged across all formulations under conditions of shaking, freeze-thaw cycles, and 25°C. However, the acidic peak increased after 4 weeks at 40°C. Formulation 2 had the highest main peak content, with relatively small differences between the 50 mg / mL and 70 mg / mL formulations. Based on these results, proline was selected as the stabilizer, with an antibody concentration of 70 mg / mL and a pH of 5.0.

[0392] Preparation Example 4. Screening of surfactant concentrations for RANKL-NGF bispecific antibody preparations

[0393] A 20mM acetic acid-sodium acetate, pH 5.0 buffer system was selected, 240mM proline was used as a stabilizer, and the concentration of RANKL-NGF bispecific antibody was 70mg / mL. Three groups of formulation samples with polysorbate 80 concentrations of 0.1mg / mL, 0.2mg / mL, and 0.4mg / mL were prepared. The stability of the samples under shaking (300rpm / 25℃), repeated freeze-thaw cycles (-35℃ / room temperature), and high temperature (40℃) conditions were investigated.

[0394] 1) 20 mM acetic acid-sodium acetate, pH 5.0, 240 mM proline, 0.1 mg / mL polysorbate 80, 70 mg / mL antibody;

[0395] 2) 20 mM acetic acid-sodium acetate, pH 5.0, 240 mM proline, 0.2 mg / mL polysorbate 80, 70 mg / mL antibody;

[0396] 3) 20 mM acetic acid-sodium acetate, pH 5.0, 240 mM proline, 0.4 mg / mL polysorbate 80, 70 mg / mL antibody.

[0397] Table 28. Surfactant concentration screening of RANKL-NGF bispecific antibody formulations - Results 1

[0398] Table 29. Surfactant concentration screening of RANKL-NGF bispecific antibody formulations - Results 2

[0399] Table 30. Surfactant concentration screening of RANKL-NGF bispecific antibody formulations - Results 3

[0400] Table 31. Surfactant concentration screening of RANKL-NGF bispecific antibody formulations - Results 4

[0401] Result shows: along with polysorbate 80 concentration increases, polymer, fragment and acid-base peak content all show increasing trend, and when polysorbate 80 concentration is 0.4mg / mL, antibody stability is relatively poor.Therefore, polysorbate 80 concentration is preferably 0.1mg / mL.

[0402] Preparation Example 5. Stability Study of the Preferred Formulation of RANKL-NGF Bispecific Antibody

[0403] A 20 mM acetic acid-sodium acetate, pH 5.0 buffer system was selected, 240 mM proline was used as a stabilizer, the RANKL-NGF bispecific antibody concentration was 70 mg / mL, and a polysorbate 80 concentration of 0.1 mg / mL was prepared. The stability of the formulation under accelerated conditions (25±2°C / 60%RH±5RH) and long-term conditions (2-8°C) was investigated.

[0404] Table 32. Accelerated stability results of RANKL-NGF bispecific antibody formulations Note: NT means not tested.

[0405] Table 33. Long-term stability results of RANKL-NGF bispecific antibody formulations Note: NT means not tested.

[0406] The results showed that after storage for 6 months under accelerated conditions (25±2°C / 60%RH±5RH), the main peak of iCIEF decreased by about 12.7%, and there was no significant change in other test items; while after storage for 12 months under long-term conditions (2-8°C), all test items showed no significant change compared with time 0, indicating that the RANKL-NGF bispecific antibody preparation has good long-term stability.

[0407] Preparation Example 6. Optional formulation formula

[0408] The present disclosure provides a RANKL-NGF bispecific antibody pharmaceutical formulation comprising 50-77 mg / mL RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate, pH 4.6-5.4, including but not limited to:

[0409] (1) 70 mg / mL RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate pH 4.6;

[0410] (2) 70 mg / mL RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate pH 5.0;

[0411] (3) 70 mg / mL RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate pH 5.4;

[0412] (4) 50 mg / ml RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate pH 4.6;

[0413] (5) 50 mg / ml RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate pH 5.0;

[0414] (6) 50 mg / ml RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate pH 5.4;

[0415] (7) 77 mg / ml RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate pH 4.6;

[0416] (8) 77 mg / ml RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate pH 5.0;

[0417] (9) 77 mg / ml RANKL-NGF bispecific antibody, 240 mM proline, 0.1 mg / mL polysorbate 80, 20 mM acetic acid-sodium acetate pH 5.4.

[0418] The experimental results show that the RANKL-NGF bispecific antibody preparations of the above-mentioned formulations have good stability and can be used in the preparation of RANKL-NGF bispecific antibody drugs.

Claims

1. A pharmaceutical composition comprising an anti-RANKL-NGF bispecific antibody and a buffer, wherein: The anti-RANKL-NGF bispecific antibody comprises a first antigen-binding domain that specifically binds to RANKL and a second antigen-binding domain that specifically binds to NGF; The buffer is an acetate buffer, a histidine buffer or a phosphate buffer; Preferably, the buffer is acetic acid-sodium acetate buffer, histidine-histidine hydrochloride buffer or citric acid-disodium hydrogen phosphate buffer; More preferably, the buffer is acetic acid-sodium acetate buffer or histidine-histidine hydrochloride buffer; Most preferably, the buffer is acetic acid-sodium acetate buffer.

2. The pharmaceutical composition according to claim 1, wherein the pH of the pharmaceutical composition is 4.2 to 7.0; Preferably, the pH of the pharmaceutical composition is 4.6 to 5.4; More preferably, the pH of the pharmaceutical composition is 4.8 to 5.

2.

3. The pharmaceutical composition according to claim 1 or 2, wherein the concentration of the anti-RANKL-NGF bispecific antibody is 1 mg / mL to 150 mg / mL; Preferably, the concentration of the anti-RANKL-NGF bispecific antibody is 10 mg / mL to 80 mg / mL; More preferably, the concentration of the anti-RANKL-NGF bispecific antibody is 63 mg / mL to 77 mg / mL.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition comprises a surfactant; Preferably, the surfactant is polysorbate or poloxamer; More preferably, the surfactant is polysorbate 20 or polysorbate 80; Most preferably, the surfactant is polysorbate 80.

5. The pharmaceutical composition according to claim 4, wherein the concentration of the surfactant is 0.01 mg / mL to 1.0 mg / mL; Preferably, the concentration of the surfactant is 0.01 mg / mL to 0.6 mg / mL; More preferably, the concentration of the surfactant is 0.01 mg / mL to 0.2 mg / mL; Most preferably, the concentration of the surfactant is 0.05 mg / mL to 0.15 mg / mL.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition comprises a stabilizer; Preferably, the stabilizer is one or more selected from the group consisting of proline, sucrose, trehalose, sorbitol, arginine, glycine and sodium chloride; More preferably, the stabilizer is proline, sucrose or sodium chloride; Most preferably, the stabilizer is proline.

7. The pharmaceutical composition according to claim 6, wherein the concentration of the stabilizer is 1 mM to 300 mM; Preferably, the concentration of the stabilizer is 25 mM to 290 mM; More preferably, the concentration of the stabilizer is 210 mM to 270 mM.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the concentration of the buffer is 5 mM to 100 mM; Preferably, the concentration of the buffer is 10 mM to 50 mM; More preferably, the concentration of the buffer is 10 mM to 30 mM; Most preferably, the concentration of the buffer is 16 mM to 24 mM.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein: The anti-RANKL-NGF bispecific antibody comprises: at least one first antigen binding domain that specifically binds RANKL, and at least one second antigen binding domain that specifically binds NGF; Preferably, The anti-RANKL-NGF bispecific antibody comprises: two first antigen-binding domains that specifically bind to RANKL, and two second antigen-binding domains that specifically bind to NGF; More preferably, the anti-RANKL-NGF bispecific antibody has the structure as shown in FIG1 .

10. The pharmaceutical composition according to claim 9, wherein in the anti-RANKL-NGF bispecific antibody, the first antigen-binding domain that specifically binds to RANKL comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 1; HCDR2 comprising the amino acid sequence of SEQ ID NO: 2; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 3; and The light chain variable region comprises: LCDR1 comprising the amino acid sequence of SEQ ID NO: 4; LCDR2 comprising the amino acid sequence of SEQ ID NO: 5; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6; Preferably, In the anti-RANKL-NGF bispecific antibody, the first antigen binding domain that specifically binds to RANKL comprises a heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7; and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO:

8.

11. The pharmaceutical composition according to claim 9 or 10, wherein in the anti-RANKL-NGF bispecific antibody, the second antigen-binding domain that specifically binds to NGF comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of SEQ ID NO: 15; HCDR2 comprising the amino acid sequence of SEQ ID NO: 16; and HCDR3 comprising the amino acid sequence of SEQ ID NO: 17; and The light chain variable region comprises: LCDR1 comprising the amino acid sequence of SEQ ID NO: 18; LCDR2 comprising the amino acid sequence of SEQ ID NO: 19; and LCDR3 comprising the amino acid sequence of SEQ ID NO: 20; Preferably, In the anti-RANKL-NGF bispecific antibody, the second antigen binding domain that specifically binds to NGF comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 21; and / or the light chain variable region comprising the amino acid sequence of SEQ ID NO: 22; More preferably, In the anti-RANKL-NGF bispecific antibody, the second antigen-binding domain that specifically binds to NGF comprises a heavy chain and a light chain, the heavy chain comprises the amino acid sequence of SEQ ID NO: 24, and the light chain comprises the amino acid sequence of SEQ ID NO:

25.

12. The pharmaceutical composition according to any one of claims 1 to 11, comprising the following components: (a) 1 mg / mL to 150 mg / mL of the anti-RANKL-NGF bispecific antibody, (b) 0.01 mg / mL to 1.0 mg / mL of a surfactant, (c) 1 mM to 300 mM of a stabilizer, and (d) 5 mM to 100 mM buffer, wherein the pH of the pharmaceutical composition is 4.2 to 7.0; Preferably, the pharmaceutical composition comprises the following components: (a) 10 mg / mL to 80 mg / mL of the anti-RANKL-NGF bispecific antibody, (b) 0.01 mg / mL to 0.6 mg / mL of polysorbate 20 or polysorbate 80, (c) 25 mM to 290 mM proline, and (d) 10 mM to 50 mM acetate buffer, wherein the pH of the pharmaceutical composition is 4.6 to 5.4; More preferably, the pharmaceutical composition comprises the following components: (a) 63 mg / mL to 77 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) 0.01 mg / mL to 0.2 mg / mL of polysorbate 80, (c) 210 mM to 270 mM proline, and (d) 10 mM to 30 mM acetic acid-sodium acetate buffer, the pH of the pharmaceutical composition is 4.8 to 5.2; Most preferably, the pharmaceutical composition comprises the following components: (a) 63 mg / mL to 77 mg / mL of an anti-RANKL-NGF bispecific antibody, (b) 0.05 mg / mL to 0.15 mg / mL of polysorbate 80, (c) 210 mM to 270 mM proline, and (d) 16 mM to 24 mM acetic acid-sodium acetate buffer, the pH of the pharmaceutical composition is 4.8 to 5.

2.

13. A lyophilized preparation, which can form the pharmaceutical composition according to any one of claims 1 to 12 after being reconstituted.

14. A method for preparing a lyophilized preparation, comprising the step of freeze-drying the pharmaceutical composition according to any one of claims 1 to 12.

15. A freeze-dried preparation obtained by the method according to claim 14.

16. The pharmaceutical composition according to any one of claims 1 to 12, which is a subcutaneous injection preparation, an intravenous injection preparation, an intraperitoneal injection preparation or an intramuscular injection preparation; preferably a subcutaneous injection preparation.

17. A method for treating or preventing a disease, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 12, or the lyophilized preparation according to claim 13 or 15; Preferably, the disease is pain, joint stiffness or bone loss; More preferably, the pain is selected from the group consisting of: bone and joint pain, rheumatoid arthritis pain, gout, bone cancer pain, fracture pain, postoperative pain, cancer pain, painful bladder syndrome, musculoskeletal pain, prostatitis, pelvic pain, interstitial cystitis, low back pain, dysmenorrhea, pain associated with bone diseases, trigeminal neuralgia, postherpetic neuralgia, herpes zoster infection, sciatica, migraine, diabetic neuropathy and peripheral nerve-related pain; in, The bone loss is associated with at least one condition selected from the group consisting of osteoporosis, Paget's disease, osteomyelitis, hypercalcemia, osteopenia, osteoporosis, osteonecrosis, bone injury, bone resorption, bone dysplasia, inflammation, autoimmune disease, inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, periodontal bone resorption, osteolytic metastasis, and cancer.

18. The method of claim 17, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, thyroid cancer, kidney cancer, lung cancer, esophageal cancer, rectal cancer, bladder cancer, cervical cancer, ovarian cancer, liver cancer, gastrointestinal cancer, melanoma, multiple myeloma, osteosarcoma, lymphoma, non-small cell lung cancer, bone tumor, and Hodgkin's disease.

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