Engineered compositions for bone-targeted therapy - Patents.com

Abstract

The present disclosure provides methods for treating bone diseases, such as bone cancer or bone metastasis of cancer, by administering an engineered bone-targeting composition, such as an antibody or one or more polypeptides, such as an antibody or polypeptide engineered to include a bone-homing peptide. Further provided herein are bone-targeting compositions, such as an antibody or one or more polypeptides, such as an antibody or polypeptide engineered to include a bone-homing peptide. TIFF2024526920000016.tif48128

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 223,875, filed July 20, 2021, the entire contents of which are incorporated herein by reference.

[0002] Inclusion of sequence listing This application contains a sequence listing XML, which has been submitted electronically and is incorporated herein by reference in its entirety. The XML sequence listing, created on July 18, 2022, is named RICEP0085WO.xml and is 30,327 bytes in size.

[0003] 1. Field The present disclosure relates generally to the field of molecular biology, and more specifically to methods for site-specific delivery of antibodies or polypeptides. [Background technology]

[0004] 2. Related Technologies Antibody-based therapeutics entered clinical practice more than 30 years ago and have been used to treat malignant tumors. 1,2 , infectious diseases 3,4 , and transplant rejection 5 These biologic therapeutics have become the mainstream treatment option for patients with cancer. Compared with traditional chemotherapy, these biologic therapeutics preferentially target cells that present tumor-associated antigens, resulting in improved treatment outcomes and reduced side effects. 6,7,8,9 Despite their high affinity for tumor antigens, poor tumor tissue penetration and uneven distribution of therapeutic antibodies in brain and bone significantly limits their effectiveness in treating disease in these tissues. The inability to deliver an effective antibody dose throughout the tumor in these tissues leads not only to treatment failure but also to the development of acquired drug resistance. 10 Exposure to sub-therapeutic antibody levels has been shown to promote the ability of tumor cells to evade antibody-mediated killing. 11,12Furthermore, attempts to ensure effective concentrations of antibodies in the tumor niche usually result in high concentrations in other tissues, resulting in adverse systemic side effects that can limit or eliminate the use of the therapeutic. Thus, strategies to improve tumor penetration and distribution of antibodies in specific tissues after systemic delivery are needed to optimize the clinical potential of these agents.

[0005] Despite a 5-year survival rate of over 90%, 20-40% of breast cancer survivors eventually experience distant metastases, even years after initial treatment. 13 Bone is the most frequent tissue of breast cancer metastasis. 14,15 Dosing into the bone microenvironment has proven difficult due to the relatively low density of angiogenesis and the presence of physical barriers to penetration. Antibody-based therapies face special distribution challenges due to the large molecular size of these agents. Thus, therapeutic antibodies that show excellent efficacy in treating primary breast tumors only produce suboptimal responses in patients with bone metastases. For example, the trastuzumab (Herceptin) antibody, which successfully targets human epidermal growth factor receptor 2 (HER2) in primary breast tumors, is also being evaluated as a treatment option for patients with metastatic breast cancer. Although some breast cancer patients benefit from these therapies, the majority of breast cancer patients with bone metastases experience further tumor progression within one year, and only a few patients achieve long-term remission. 16 Thus, the effectiveness of therapeutic antibodies appears to be particularly limited in cases of bone metastases. Summary of the Invention

[0006] overview In certain embodiments, the present disclosure provides methods for bone-targeting polypeptides or proteins (e.g., antibodies) engineered to include at least one bone-homing peptide that selectively binds to bone hydroxyapatite (HA). In one embodiment, the present disclosure provides methods for bone-targeting antibodies engineered to include at least one bone-homing peptide that selectively binds to bone hydroxyapatite (HA). In another embodiment, the present disclosure provides one or more bone-targeting polypeptides engineered to include at least one bone-homing peptide that selectively binds to bone HA.

[0007] In some embodiments, at least one bone-homing peptide is inserted into a permissive internal site of the antibody, hi certain embodiments, at least one bone-homing peptide is inserted into the light chain (LC), heavy chain (CH1), and / or C-terminus (CT) of the antibody.

[0008] In some embodiments, at least one bone-homing peptide is inserted at the C-terminus or N-terminus of one or more polypeptides.

[0009] In certain embodiments, the antibody or polypeptide comprises two, three, or four bone-homing peptides. In certain embodiments, the bone-homing peptides are L-Asp3, L-Asp4, L-Asp5, L-Asp6, L-Asp7, L-Asp8, L-Asp9, or L-Asp 10 In some embodiments, the bone homing peptide comprises at least 3 consecutive aspartic acids, such as at least 4, 5, 6, 7, 8, 9, or 10 aspartic acids. In a specific embodiment, the bone homing peptide is L-Asp6.

[0010] In some aspects, the antibody is a monoclonal antibody, a bispecific antibody, a Fab', a F(ab')2, a F(ab')3, a monovalent scFv, a bivalent scFv, a single domain antibody, or a nanobody. In certain aspects, the antibody is an immune checkpoint inhibitor. In some embodiments, the antibody is an anti-HER2 antibody, an anti-CD99 antibody, an anti-IGF-IR antibody, an anti-PD-L1, an anti-PD-1, an anti-CTLA4 antibody, an anti-Siglec-2 antibody, an anti-Siglec-3 antibody, an anti-Siglec-5 antibody, an anti-Siglec-6 antibody, an anti-Siglec-7 antibody, an anti-Siglec-8 antibody, an anti-Siglec9 antibody, an anti-Siglec-10 antibody, an anti-Siglec-11 antibody, an anti-Siglec-15 antibody, an anti-RANKL antibody, an anti-EGFR antibody, an anti-VEGFR antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD52 antibody, an anti-Trop-2 antibody, an anti-CD30 antibody, an anti-CD152 antibody, an anti-IL-6R antibody, an anti-GD2 antibody, or an anti-TGFβ antibody. In some embodiments, the antibody is an anti-CD99 antibody.

[0011] In some embodiments, the one or more polypeptides comprise an adrenergic agonist, an anti-apoptotic factor, an apoptosis inhibitor, a cytokine receptor, a cytokine, a cytotoxin, an erythropoietic agent, a glutamic acid decarboxylase, a glycoprotein, a growth factor, a growth factor receptor, a hormone, a hormone receptor, an interferon, an interleukin, an interleukin receptor, a kinase, a kinase inhibitor, a nerve growth factor, a netrin, a neuroactive peptide, a neuroactive peptide receptor, a neurogenic factor, a neurogenic factor receptor, a neuropilin, a neurotrophic factor, a neurotrophin, a neurotrophin receptor, an N-methyl-D-aspartate antagonist, a plexin, a protease, a protease inhibitor, a protein decarboxylase, a protein kinase, a protein kinase inhibitor, a proteolytic protein, a proteolytic protein inhibitor, a semaphorin, a semaphorin receptor, a serotonin transporter protein, a serotonin uptake inhibitor, a serotonin receptor, a serpin, a serpin receptor, or a tumor suppressor. In certain embodiments, the polypeptide comprises a cytokine. In a specific embodiment, the cytokine is IL-6. In a particular embodiment, the IL-6 comprises a bone homing peptide at the C-terminus.

[0012] In further aspects, the antibody or polypeptide is conjugated to a drug. In some aspects, the drug is monomethyl auristatin E (MMAE), ozogamicin, tiuxetan, vedotin, pasudotox tdfx, vedotin, mafodotin, calicheamicin, maytansine, or deruxtecan. In certain aspects, the drug is an antimitotic drug, such as monomethyl auristatin E (MMAE).

[0013] In some embodiments, the antibody is an anti-HER2 antibody. For example, the antibody is trastuzumab (Herceptin), pertuzumab (Perjeta), or atezolizumab. In certain embodiments, the antibody is trastuzumab, for example, trastuzumab is conjugated to MMAE. In specific embodiments, the bone-homing peptide is inserted into residues A153, A165, and / or G449 of trastuzumab.

[0014] In certain aspects, the antibody has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to Tras-LC (SEQ ID NOs:3-4), Tras-CH1 (SEQ ID NOs:5-6), Tras-CT (SEQ ID NOs:7-8), Tras-LC / CH1 (SEQ ID NOs:9-10), Tras-LC / CT (SEQ ID NOs:11-12), Tras-CH1 / CT (SEQ ID NOs:13-14), or Tras-LC / CH1 / CT (SEQ ID NOs:15-16). In certain aspects, the antibody comprises Tras-LC (SEQ ID NO:3-4), Tras-CH1 (SEQ ID NO:5-6), Tras-CT (SEQ ID NO:7-8), Tras-LC / CH1 (SEQ ID NO:9-10), Tras-LC / CT (SEQ ID NO:11-12), Tras-CH1 / CT (SEQ ID NO:13-14), or Tras-LC / CH1 / CT (SEQ ID NO:15-16).

[0015] In some embodiments, the bone-targeted antibody or polypeptide has increased binding affinity for HA compared to an antibody or polypeptide that does not include a bone-homing peptide, hi certain embodiments, the bone-targeted antibody has a 2- to 3-fold higher binding affinity for HA compared to an antibody or polypeptide that does not include a bone-homing peptide.

[0016] Further embodiments provide methods of treating or preventing a bone disease (e.g., a bone tumor) in a subject, comprising administering to the subject an effective amount of a bone-targeted antibody or polypeptide of any of the embodiments and aspects thereof (e.g., a bone-targeted antibody or polypeptide engineered to contain at least one bone-homing peptide that selectively binds to bone hydroxyapatite (HA)).

[0017] In some embodiments, the subject has bone cancer or bone metastases. In certain embodiments, the bone cancer is Ewing's sarcoma, osteosarcoma, or chondrosarcoma. In certain embodiments, the bone metastases are from breast cancer, myeloma, renal cancer, lung cancer, prostate cancer, thyroid cancer, or bladder cancer. In specific embodiments, the breast cancer is triple negative breast cancer, HER2 negative breast cancer, or HER2 positive breast cancer.

[0018] In certain aspects, the bone disease is osteoporosis, osteomalacia, periodontitis, rheumatoid arthritis, metabolic bone disease, parathyroid disorders, steroid-induced osteoporosis, chemotherapy-induced bone loss, premenopausal bone loss, fragility and recurrent fractures, renal osteodystrophy, bone infection, or Paget's disease. The methods and compositions provided herein can be used to reduce cortical and / or trabecular bone loss, reduce cortical and / or trabecular bone mineral content loss, improve bone biomechanical resistance, increase bone formation, and / or reduce bone resorption.

[0019] In certain embodiments, the bone-targeting antibody or polypeptide results in an increased concentration of the therapeutic antibody or polypeptide in the bone tumor niche, inhibiting cancer development in bone and / or limiting secondary metastasis to other organs. In some embodiments, the bone-targeting antibody or polypeptide results in a reduction in micrometastasis-induced osteolytic lesions.

[0020] In further aspects, the method further comprises administering an additional anti-cancer therapy. In some aspects, the additional anti-cancer therapy comprises surgery, chemotherapy, radiation therapy, hormonal therapy, immunotherapy, or cytokine therapy. For example, the additional anti-cancer therapy comprises immunotherapy or chemotherapy.

[0021] Another embodiment provides the use of a bone-targeted antibody or polypeptide of any of this embodiment and its aspects (e.g., a bone-targeted antibody engineered to contain at least one bone-homing peptide that selectively binds to bone hydroxyapatite (HA)) for the treatment or prevention of a bone disease (e.g., a bone tumor) in a subject with cancer.

[0022] In some embodiments, the subject has bone cancer or bone metastases. In certain embodiments, the bone cancer is Ewing's sarcoma, osteosarcoma, or chondrosarcoma. In certain embodiments, the bone metastases are from breast cancer, myeloma, renal cancer, lung cancer, prostate cancer, thyroid cancer, or bladder cancer. In specific embodiments, the breast cancer is triple negative breast cancer, HER2 negative breast cancer, or HER2 positive breast cancer.

[0023] In certain aspects, the bone disease is osteoporosis, osteomalacia, periodontitis, rheumatoid arthritis, metabolic bone disease, parathyroid disorders, steroid-induced osteoporosis, chemotherapy-induced bone loss, premenopausal bone loss, fragility and recurrent fractures, renal osteodystrophy, bone infection, or Paget's disease. The methods and compositions provided herein can be used to reduce cortical and / or trabecular bone loss, reduce cortical and / or trabecular bone mineral content loss, improve bone biomechanical resistance, increase bone formation, and / or reduce bone resorption.

[0024] In certain embodiments, the bone-targeting antibody or polypeptide results in an increased concentration of the therapeutic antibody or polypeptide in the bone tumor niche, inhibiting cancer development in bone and / or limiting secondary metastasis to other organs. In some embodiments, the bone-targeting antibody results in a reduction in micrometastasis-induced osteolytic lesions.

[0025] In further aspects, the use further comprises an additional anti-cancer therapy. In some aspects, the additional anti-cancer therapy comprises surgery, chemotherapy, radiation therapy, hormonal therapy, immunotherapy, or cytokine therapy. For example, the additional anti-cancer therapy comprises immunotherapy or chemotherapy.

[0026] Another embodiment provides a method for engineering a bone-targeted antibody or polypeptide of any of this embodiment and its aspects (e.g., a bone-targeted antibody or polypeptide engineered to contain at least one bone-homing peptide that selectively binds to bone hydroxyapatite (HA)), comprising inserting at least one bone-homing peptide into a permissive internal site of the antibody or at the C-terminus or N-terminus of the polypeptide.

[0027] In some embodiments, at least one bone-homing peptide is inserted into a permissive internal site of the antibody, hi certain embodiments, at least one bone-homing peptide is inserted into the light chain (LC), heavy chain (CH1), and / or C-terminus (CT) of the antibody.

[0028] In certain embodiments, the antibody or polypeptide comprises two, three, or four bone-homing peptides. In certain embodiments, the bone-homing peptides are L-Asp3, L-Asp4, L-Asp5, L-Asp6, L-Asp7, L-Asp8, L-Asp9, or L-Asp 10 In some embodiments, the bone homing peptide comprises at least 3 consecutive aspartic acids, such as at least 4, 5, 6, 7, 8, 9, or 10 aspartic acids. In a specific embodiment, the bone homing peptide is L-Asp6.

[0029] In some aspects, the antibody is a monoclonal antibody, a bispecific antibody, a Fab', a F(ab')2, a F(ab')3, a monovalent scFv, a bivalent scFv, a single domain antibody, or a nanobody. In certain aspects, the antibody is an immune checkpoint inhibitor. In some embodiments, the antibodies are anti-HER2 antibodies, anti-CD99 antibodies, anti-IGF-IR antibodies, anti-PD-L1, anti-PD-1, anti-CTLA4 antibodies, anti-Siglec-2 antibodies, anti-Siglec-3 antibodies, anti-Siglec-5 antibodies, anti-Siglec-6 antibodies, anti-Siglec-7 antibodies, anti-Siglec-8 antibodies, anti-Siglec9 antibodies, Anti-Siglec-10 antibody, anti-Siglec-11 antibody, anti-Siglec-15 antibody, anti-RANKL antibody, anti-EGFR antibody, anti-VEGFR antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD52 antibody, anti-Trop-2 antibody, anti-CD30 antibody, anti-CD152 antibody, anti-IL-6R antibody, anti-GD2 antibody, or anti-TGFβ antibody.

[0030] In some embodiments, the one or more polypeptides comprise an adrenergic agonist, an anti-apoptotic factor, an apoptosis inhibitor, a cytokine receptor, a cytokine, a cytotoxin, an erythropoietic agent, a glutamic acid decarboxylase, a glycoprotein, a growth factor, a growth factor receptor, a hormone, a hormone receptor, an interferon, an interleukin, an interleukin receptor, a kinase, a kinase inhibitor, a nerve growth factor, a netrin, a neuroactive peptide, a neuroactive peptide receptor, a neurogenic factor, a neurogenic factor receptor, a neuropilin, a neurotrophic factor, a neurotrophin, a neurotrophin receptor, an N-methyl-D-aspartate antagonist, a plexin, a protease, a protease inhibitor, a protein decarboxylase, a protein kinase, a protein kinase inhibitor, a proteolytic protein, a proteolytic protein inhibitor, a semaphorin, a semaphorin receptor, a serotonin transporter protein, a serotonin uptake inhibitor, a serotonin receptor, a serpin, a serpin receptor, or a tumor suppressor. In certain embodiments, the polypeptide comprises a cytokine. In a specific embodiment, the cytokine is IL-6. In a particular embodiment, the IL-6 comprises a bone homing peptide at the C-terminus.

[0031] In further aspects, the antibody or polypeptide is conjugated to a drug. In some aspects, the drug is monomethyl auristatin E (MMAE), ozogamicin, tiuxetan, vedotin, pasudotox tdfx, vedotin, mafodotin, calicheamicin, maytansine, or deruxtecan. In certain aspects, the drug is an antimitotic drug, such as monomethyl auristatin E (MMAE).

[0032] In some embodiments, the antibody is an anti-HER2 antibody. For example, the antibody is trastuzumab (Herceptin), pertuzumab (Perjeta), or atezolizumab. In certain embodiments, the antibody is trastuzumab, for example, trastuzumab is conjugated to MMAE. In specific embodiments, the bone-homing peptide is inserted into residues A153, A165, and / or G449 of trastuzumab.

[0033] In certain aspects, the antibody has an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to Tras-LC (SEQ ID NOs:3-4), Tras-CH1 (SEQ ID NOs:5-6), Tras-CT (SEQ ID NOs:7-8), Tras-LC / CH1 (SEQ ID NOs:9-10), Tras-LC / CT (SEQ ID NOs:11-12), Tras-CH1 / CT (SEQ ID NOs:13-14), or Tras-LC / CH1 / CT (SEQ ID NOs:15-16). In certain aspects, the antibody comprises Tras-LC (SEQ ID NO:3-4), Tras-CH1 (SEQ ID NO:5-6), Tras-CT (SEQ ID NO:7-8), Tras-LC / CH1 (SEQ ID NO:9-10), Tras-LC / CT (SEQ ID NO:11-12), Tras-CH1 / CT (SEQ ID NO:13-14), or Tras-LC / CH1 / CT (SEQ ID NO:15-16).

[0034] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. For example, a compound synthesized by one method can be used to prepare a final compound by a different method.

[0035] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0036] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0037] [Figure 1] 1A-1B: (FIG. 1A) Protein sequences of hydroxyapatite binding proteins (SEQ ID NOs: 25-26). (FIG. 1B) Therapeutic antibodies can be delivered specifically to bone by introducing a bone-homing peptide sequence that binds to the bone hydroxyapatite matrix. [Diagram 2]Figures 2A-2H: Preparation and characterization of bone-targeting antibodies. (Figure 2A) Bone-homing peptides were inserted into three locations: light chain (LC), heavy chain (CH1), and c-terminus (CT). (Figure 2B) SDS-PAGE analysis of bone-targeting antibodies in the presence (left) and absence (right) of reducing reagent. (Figure 2CC) Mass spectrometry analysis of bone-targeting antibodies. (Figure 2D) Binding kinetics of Tras, Tras-CH1, Tras-LC, Tras-CT, Tras-LC / CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT to hydroxyapatite (HA). (Figure 2E) Differential bone-targeting ability of Tras and bone-targeting conjugates. Non-demineralized bone sections from C57 / BL6 mice were incubated overnight with 50 μg / mL of Tras or bone-targeting conjugates, followed by staining with fluorescein isothiocyanate (FITC)-labeled anti-human IgG and 4 μg / mL of xylenol orange (XO, known to label bone). Scale bar, 200 μm. (FIG. 2F) Flow cytometry profiles of Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT binding to SK-BR-3 (HER2+++) and MDA-MB-468 (HER2-) cells. (FIG. 2G) In vitro cytotoxicity of Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT to SK-BR-3 and MDA-MB-468 cells. (FIG. 2H) Ex vivo fluorescence images of the lower limbs of athymic nude mice bearing MDA-MB-361 tumors 72 or 120 hours after retro-orbital injection of Cy7.5-labeled Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT antibodies. Tumor cells were inoculated into the right tibia of nude mice via para-tibial injection. [Diagram 3]Figures 3A-3S: Bone-targeted antibodies inhibit breast cancer bone metastasis. (Figure 3A) MDA-MB-361 cells were injected paratibially into the right hind leg of nude mice followed by treatment with PBS, Tras (1 mg / kg in sterile PBS, retro-orbital sinus, twice weekly), Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT (same as Tras). Tumor burden was monitored by weekly bioluminescence imaging. (Figures 3B-3C) Fold change in mean luminescence intensity of MDA-MB-361 tumors in mice treated as described in Figure 3A. p values ​​are based on two-way ANOVA test. (Figure 3D) Kaplan-Meier plot of time to euthanasia for mice treated as described in Figure 3A. For each individual mouse, the end point was considered to be when the BLI signal in the whole body reached 107 photons-sec-1. (Figure 3E) Body weight change of tumor-bearing mice over time. (Fig. 3F) MicroCT scans in supine position of PBS, Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT treated groups. (Fig. 3G) Quantitative analysis of bone volume (BV). (Fig. 3H) Quantitative analysis of bone surface / bone volume ratio (BS / BV). (Fig. 3I) Quantitative analysis of bone volume / tissue volume ratio (BV / TV). (Fig. 3J) Quantitative analysis of trabecular thickness (Tb.Th). (Fig. 3K) Quantitative analysis of trabecular bone mineral density (BMD). (Fig. 3L) Representative longitudinal, midsagittal hematoxylin and eosin (H&E) stained sections of tibia / femur from each group. T: tumor; B: bone; BM: bone marrow. (Fig. 3M) Representative images of HER2 and TRAP staining of bone sections from each group. (Fig. 3N) Number of osteoclasts per image calculated at the tumor-bone interface in each group (pink cells in Fig. 3K were considered as osteoclast-positive cells). (Fig. 3O) MDA-MB-361 cells were injected paratibially into the right hind limb of nude mice, followed by treatment with Tras (10 mg / kg retro-orbital sinus in sterile PBS, every 2 weeks for 2 months) and Tras-CH1 / CT (same as Tras). Tumor burden was monitored by weekly bioluminescence imaging. (Fig. 3P) Fold change in mean luminescence intensity of MDA-MB-361 tumors in mice treated as described in Fig. 3O.(Fig. 3Q) Fold change in individual luminescence intensity of MDA-MB-361 tumors in mice treated as described in (O). (Fig. 3R) Kaplan-Meier plot of time to euthanasia for mice treated as described in Fig. 3O. Reaching a BLI signal of 108 photons s-1 in the whole body was considered as the endpoint for each individual mouse. (Fig. 3S) Body weight change of tumor-bearing mice in Fig. 3O over time. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, and nsP>0.05. [Figure 4] Figure 4A-4C: (Figure 4A) Bone lesions more easily give rise to secondary metastasis to multiple organs. (Figure 4B) Secondary metastases observed in various organs of mice treated with Tras or Tras-CH1 / CT. (Figure 4C) Heatmap of ex vivo BLI intensity and status of metastatic involvement in tissues from mice treated with PBS, Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT. Each column represents an individual animal and each row represents a tissue type. Presence of metastasis was defined as the presence of a BLI signal above 18 counts / pixel under an exposure time of 120 seconds. Multisite metastasis was defined as metastatic involvement of at least three tissues. p-values ​​were determined by Fisher's exact test for frequency of metastatic involvement, whereas Mann-Whitney test for metastatic burden. [Diagram 5] ESI-MS spectrum of Tras. [Figure 6] ESI-MS spectrum of Tras-LC. [Figure 7] ESI-MS spectrum of Tras-CH1. [Figure 8] ESI-MS spectrum of Tras-CT. [Figure 9] ESI-MS spectrum of Tras-LC / CH1. [Figure 10] ESI-MS spectrum of Tras-LC / CT. [Figure 11] ESI-MS spectrum of Tras-CH1 / CT. [Figure 12]ESI-MS spectrum of Tras-LC / CH1 / CT. [Figure 13] Differential cancellous bone targeting ability of Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT. Non-demineralized bone sections from C57 / BL6 mice were incubated overnight with 50 μg / mL of Tras, Tras-CT, Tras-CH1 / CT, or Tras-LC / CH1 / CT, followed by staining with fluorescein isothiocyanate (FITC)-conjugated anti-human IgG and 4 μg / mL of xylenol orange (XO, known to label bone). Scale bar, 200 μm. [Figure 14] Tras binding to SK-BR-3 cells. SK-BR-3 cells were incubated with increasing concentrations of Tras and fluorescence was measured by flow cytometer. KD was determined as follows: 1 / F=1 / Fmax+(KD / Fmax)(1 / [Ab]). [Figure 15] Tras-LC binding to SK-BR-3 cells. SK-BR-3 cells were incubated with increasing concentrations of Tras-LC and fluorescence was measured by flow cytometer. KD was determined as follows: 1 / F=1 / Fmax+(KD / Fmax)(1 / [Ab]). [Figure 16] Tras-CT binding to SK-BR-3 cells. SK-BR-3 cells were incubated with increasing concentrations of Tras-CT and fluorescence was measured by flow cytometer. KD was determined as follows: 1 / F=1 / Fmax+(KD / Fmax)(1 / [Ab]). [Figure 17] Tras-CH1 binding to SK-BR-3 cells. SK-BR-3 cells were incubated with increasing concentrations of Tras-CH1 and fluorescence was measured by flow cytometer. KD was determined as follows: 1 / F=1 / Fmax+(KD / Fmax)(1 / [Ab]). [Figure 18]Tras-LC / CT binding to SK-BR-3 cells. SK-BR-3 cells were incubated with increasing concentrations of Tras-LC / CT and fluorescence was measured by flow cytometer. KD was determined as follows: 1 / F=1 / Fmax+(KD / Fmax)(1 / [Ab]). [Figure 19] Tras-CH1 / CT binding to SK-BR-3 cells. SK-BR-3 cells were incubated with increasing concentrations of Tras-CH1 / CT and fluorescence was measured by flow cytometer. KD was determined as follows: 1 / F=1 / Fmax+(KD / Fmax)(1 / [Ab]). [Figure 20] Tras-CH1 / CT binding to SK-BR-3 cells. SK-BR-3 cells were incubated with increasing concentrations of Tras-CH1 / CT and fluorescence was measured by flow cytometer. KD was determined as follows: 1 / F=1 / Fmax+(KD / Fmax)(1 / [Ab]). [Figure 21] Tras-LC / CH1 binding to SK-BR-3 cells. SK-BR-3 cells were incubated with increasing concentrations of Tras-LC / CH1 and fluorescence was measured by flow cytometer. KD was determined as follows: 1 / F=1 / Fmax+(KD / Fmax)(1 / [Ab]). [Figure 22] Tras-LC / CH1 / CT binding to SK-BR-3 cells. SK-BR-3 cells were incubated with increasing concentrations of Tras and fluorescence was measured by flow cytometer. KD was determined as follows: 1 / F=1 / Fmax+(KD / Fmax)(1 / [Ab]). [Diagram 23] Cell surface binding of Tras-CH1, Tras-LC, Tras-LC / CT, and Tras-LC / CH1 to SK-BR-3 and MDA-MB-468 cells. Flow cytometry profiles of Tras-CH1, Tras-LC, Tras-LC / CT, and Tras-LC / CH1 binding to SK-BR-3 (HER2+++) and MDA-MB-468 (HER2-) cells. [Figure 24]Ex vivo fluorescence images of major organs: heart, liver, spleen, lung, kidney, and brain of C57 / BL6 mice 48 hours after retro-orbital injection of Cy7.5-labeled Tras, Tras-CT, and Tras-LC / CH1 / CT. [Diagram 25] 25A-25C: In vivo and ex vivo fluorescence image analysis of the biodistribution of Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT. (Fig. 25A) 72 or 120 hours after retro-orbital injection of Cy7.5-labeled Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT. Bones and major organs (heart, liver, spleen, lung, kidney, brain) were collected and analyzed. (Fig. 25B) Quantitative analysis of the distribution of Tras and bone-targeting antibodies in various tissues (heart, liver, spleen, lung, kidney, brain, and bone). (Fig. 25C) Histological analysis of heart with H&E staining after treatment with PBS, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT. *P<0.05, and nsP>0.05. [Figure 26] Figures 26A-26C: Fold change in BLI signal intensity during treatment. MDA-MB-361 cells were injected paratibially into the right hind leg of nude mice followed by treatment with PBS, Tras (1 mg / kg retro-orbital sinus in sterile PBS, twice weekly), Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT (same as Tras). Tumor burden was monitored by weekly bioluminescence imaging. (Figure 26A) Fold change in mean luminescence intensity of MDA-MB-361 tumors in mice treated as described in Figure 3A. Two-way ANOVA comparing Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT groups. (Figure 26B) Two-way ANOVA comparing fold change in mean luminescence intensity between Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT groups. (Figure 26C) Two-way ANOVA comparing the fold changes of individual emission intensities among Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT. [Figure 27]Figures 27A-27D: Quantification of BLI signaling. (Figure 27A) BLI from each treatment group quantified by radiance detected in the region of interest. (Figure 27B) Two-way ANOVA comparing BLI among Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT groups. (Figure 27C) Two-way ANOVA comparing BLI among Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT groups. (Figure 27D) Individual luminescence intensity of the various treatment groups as described in Figure 27C. **P<0.01. [Figure 28] 3D rendering of bone based on MicroCT. Images of cortical bone, cortical compartment show cortical bone destruction in Control (Con), Tras, Tras-CT, Tras-CH1 / CT, and Tras-CH1 / CL / CT treatment groups. Images of trabecular bone, cortical compartment show trabecular bone destruction in Control, Tras, Tras-CT, Tras-CH1 / CT, and Tras-CH1 / CL / CT treatment groups. [Figure 29] Representative microCT slices from each treatment group proximal to the growth plate (lower panels), 1.25 mm distal to the growth plate (middle panel, considered to be cancellous bone), and 3.25 mm distal to the growth plate (middle plate, considered to be cortical bone). [Diagram 30] Representative images of HER2 staining of bone sections from each group. [Diagram 31] TRAP staining of bone sections from control and Tras groups. [Diagram 32] TRAP staining of bone sections from Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT. [Diagram 33] Figures 33A-33B: Effect of bone-targeting antibodies on the MDA-MB-361 model: Analysis of serum TRACP 5b and calcium levels. (Figure 33A) Serum TRAcP 5b levels in mice treated with PBS, Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT. (Figure 33B) Serum calcium levels in mice from each treatment group. **P<0.01, *P<0.05, and nsP>0.05. [Diagram 34]Figures 34A-34B: Effect of bone-targeted antibodies on multi-organ metastasis in MDA-MB-361 cell line. (Figure 34A) Secondary metastases observed in various organs of mice treated with PBS, Tras-CT, or Tras-LC / CH1 / CT. (Figure 34B) Heatmap of ex vivo BLI intensity and status of metastatic involvement in tissues from mice treated with PBS, Tras, Tras-CT, and Tras-LC / CH1 / CT. Each column represents an individual animal and each row represents a tissue type. Presence of metastasis was defined as the presence of a BLI signal above 18 counts / pixel under an exposure time of 120 seconds. Multisite metastasis was defined as metastatic involvement of at least three tissues. p-values ​​were determined by Fisher's exact test for frequency of metastatic involvement, whereas Mann-Whitney test for metastatic burden. [Diagram 35] Trastuzumab-monomethylauristatin E ADC, an antibody-drug conjugate modified with a bone-homing peptide, demonstrated better antitumor activity. [Diagram 36] Figures 36A-36E: (Figure 36A) MCF-7 cells were injected paratibially into the right hind leg of nude mice followed by treatment with Tras (1 mg / kg in sterile PBS, retro-orbital sinus, twice weekly for 2 months) and Tras-CH1 / CT (same as Tras). Tumor burden was monitored by weekly bioluminescence imaging. (Figure 36B) Fold change in mean luminescence intensity of MCF-7 tumors in mice treated as described in Figure 36A. p-values ​​are based on two-way ANOVA test. (Figure 36C) Fold change in individual luminescence intensity of MCF-7 tumors in mice treated as described in Figure 36A. (Figure 36D) Kaplan-Meier plot of time to euthanasia for mice treated as described in Figure 36A. For each individual mouse, a BLI signal in the whole body reaching 5x107 photons s-1 was considered as the endpoint. (Figure 36E) Body weight change of tumor-bearing mice in Figure 36A over time. ****P<0.0001, *P<0.05, and nsP>0.05. [Figure 37]Figures 37A-37K: (Figure 37A) Preparation of bone-targeting antibody-drug conjugates. Tras antibody was first modified with bone-homing peptides at the heavy chain (CH1) and c-terminus (CT), followed by modification with MMAE using pClick antibody conjugation technology. (Figure 37B) SDS-PAGE analysis of Tras-MMAE and Tras-CH1 / CT-MMAE under non-reducing (left) and reducing (right) conditions. (Figure 37C, D) In ​​vitro cytotoxicity of MMAE, Tras-MMAE, and Tras-CH1 / CT-MMAE against SK-BR-3 and MDA-MB-468 cancer cells. (Figure 37E) Differential bone-targeting ability of Tras-MMAE and Tras-CH1 / CT-MMAE. Non-decalcified bone sections from C57 / BL6 mice were incubated overnight with 50 μg / mL Tras-MMAE and Tras-CH1 / CT-MMAE, followed by staining with fluorescein isothiocyanate (FITC)-labeled anti-human IgG and 4 μg / mL xylenol orange (XO, known to label bone). (Figure 37F) MDA-MB-361 cells were injected paratibially into the right hind limb of nude mice, followed by treatment with Tras-MMAE (retro-orbital sinus at 0.5 mg / kg in sterile PBS, weekly for 2 months) and Tras-CH1 / CT-MMAE (same as Tras). Tumor burden was monitored by weekly bioluminescence imaging. (Figure 37G) Fold change in mean luminescence intensity of MDA-MB-361 tumors in mice treated as described in Figure 37F. (Fig. 37H) Fold change in individual luminescence intensity of MDA-MB-361 tumors in mice treated as described in Fig. 37F. (Fig. 37I) Body weight change of tumor-bearing mice in Fig. 37F over time. (Fig. 37J) Micro-CT scans of bones from mice treated with Tras-MMAE and Tras-CH1 / CT-MMAE after tumor implantation. (Fig. 37K) Heatmap of ex vivo BLI intensity and status of metastatic involvement in tissues from mice treated with Tras-MMAE and Tras-CH1 / CT-MMAE. Each column represents an individual animal and each row represents a tissue type. Presence of metastasis was defined as the presence of a BLI signal greater than 18 counts / pixel under an exposure time of 120 seconds. Multisite metastasis was defined as metastatic involvement of at least three tissues.p values ​​were determined by Fisher's exact test for frequency of metastatic involvement and by Mann-Whitney test for metastatic burden. ****P<0.0001 and nsP>0.05. [Figure 38] Figures 38A-38B: Safety evaluation of Tras and Tras-CH1 / CT in (Figure 38A) RAW264.7 cells and (Figure 38B) MC3T3-E1 cells. [Figure 39] 39A-39G: Analysis of tumor burden at bone sites. (Fig. 39A) MicroCT scans in supine position of PBS, Tras, Tras-CT, Tras-CH1 / CT, Tras-LC / CH1 / CT, and Sham treated groups. (Fig. 39B) Quantitative analysis of bone volume (BV). (Fig. 39C) Quantitative analysis of bone volume / tissue volume ratio (BV / TV). (Fig. 39D) Quantitative analysis of trabecular bone mineral density (BMD). (Fig. 39E) Quantitative analysis of trabecular bone thickness (Tb.Th). (Fig. 39F) Quantitative analysis of bone surface / bone volume ratio (BS / BV). ***P<0.001, **P<0.01, *P<0.05, and nsP>0.05. (Fig. 39G) Representative longitudinal and midsagittal H&E stained sections of tibia / femur from each group. T: tumor; B: bone; BM: bone marrow. [Diagram 40] Figures 40A-40B: In vitro stability of Tras-CH1 / CT. (Figure 40A) ESI-MS spectrum of Tras-CH1 / CT after 3 months of incubation in PBS at 4°C. (Figure 40B) SDS-PAGE analysis of Tras and Tras-CH1 / CT after 3 months of incubation in PBS at 4°C. [Diagram 41] Pharmacokinetic profile of Tras and Tras-CH1 / CT. Tumor-bearing athymic nude mice (3 months after surgery) were retro-orbitally injected with 1 mg / kg Tras and Tras-CH1 / CT in PBS. Antibody concentrations in serum were determined by ELISA kit (data presented as mean ± SD of three independent replicates). [Diagram 42] High-dose bone-targeted antibodies inhibit breast cancer bone metastasis. Tumor burden was monitored by weekly BLI. [Diagram 43]Figures 43A-43B: Quantification of BLI signaling from high doses in MDA-MB-361 treatment groups. (Figure 43A) BLI from each treatment group quantified by radiance detected in the region of interest. Two-way ANOVA comparing BLI between Tras and Tras-CH1 / CT groups. (Figure 43B) Individual luminescence intensity of the various treatment groups as described in Figure 3O. ****P<0.0001. [Diagram 44] Figures 44A-44B: Quantification of BLI signaling from treatment groups in MCF-7 model. (Figure 44A) BLI from each treatment group quantified by radiance detected in the region of interest. Two-way ANOVA comparing BLI between Tras and Tras-CH1 / CT groups. (Figure 44B) Individual luminescence intensity of various treatment groups as described in Figures 4A-C. ****P<0.0001. [Diagram 45] Figures 45A-45C: Therapeutic effect of Tras-CH1 / CT on mice bearing primary tumors. (Figure 45A) Tumor tissues surgically removed from nude mice 21 days after inoculation. (Figure 45B) Tumor volumes of mice treated with PBS, Tras, or Tras-CH1 / CT at various times after inoculation. (Figure 45C) Tumor volumes of mice treated with Tras or Tras-CH1 / CT at various times after inoculation. nsP>0.05. [Figure 46] Figures 46A-46E: Evaluation of immune responses in mice after treatment with Tras-CH1 / CT. (Figure 46A) B cells, monocytes, neutrophils, macrophages, CD4+T cells, and CD8+T cell suspensions obtained from blood were stained with various surface markers and analyzed by flow cytometry. (Figure 46B) IFNγ levels in CD4+T, CD8+T, and B cells. (Figures 46C, 46D, 46E) Analysis of IL2, IL4, and IFNγ in plasma samples from mice. nsP>0.05. [Figure 47] Anti-trastuzumab antibody levels developed in mice injected with Tras and Tras-CH1 / CT.Tras and Tras-CH1 / CT groups: Mice were treated with Tras and Tras-CH1 / CT (5 mg / kg) twice a week for one month. [Figure 48]ESI-MS spectrum of Tras-MMAE. [Figure 49] ESI-MS spectrum of Tras-CH1 / CT-MMAE. [Figure 50] Cell surface binding of Tras-MMAE and Tras-CH1 / CT-MMAE to SKBR-3 and MDA-MB-468 cells. Cells were incubated with 30 nM Tras-ALN in medium at 37° C. for 30 min and stained with Hoechst nuclear stain (blue fluorescence). [Figure 51] Figures 51A-51B: Fold change in BLI signal intensity during Tras-CH1 / CT-MMAE treatment. (Figure 51A) Fold change in mean luminescence intensity of MDA-MB-361 tumors in mice treated as described in Figure 37F. Two-way ANOVA comparing Tras-MMAE and Tras-CH1 / CT-MMAE groups. (Figure 51B) Two-way ANOVA comparing fold change in individual luminescence intensity between Tras-MMAE and Tras-CH1 / CT-MMAE. ****P<0.0001. [Figure 52] Figures 52A-52D: Quantification of BLI signaling from Tras-CH1 / CT-MMAE treatment groups. (Figure 52A) BLI from each treatment group quantified by radiance detected in the region of interest. Two-way ANOVA comparing BLI between PBS, Tras-MMAE, and Tras-CH1 / CTMMAE groups. (Figure 52B) Individual emission intensities of PBS, Tras-MMAE, and Tras-CH1 / CT-MMAE treatment groups. (Figure 52C) Two-way ANOVA comparing BLI between Tras-MMAE and Tras-CH1 / CT-MMAE groups. (Figure 52D) Individual emission intensities of Tras-MMAE and Tras-CH1 / CT-MMAE treatment groups. ****P<0.0001. [Diagram 53] Representative microCT slices of cortical and trabecular bone from each treatment group. [Figure 54]Figures 54A-54B: Effect of Tras-CH1 / CT-MMAE on MDA-MB-361 model: Analysis of BV / TV and Tb.Th. (Figure 54A) Quantitative analysis of bone volume / tissue volume ratio (BV / TV). (Figure 54B) Quantitative analysis of cancellous bone thickness (Tb.Th). **P<0.01 and nsP>0.05. [Figure 55] Figures 55A-55B: Comparison of the Tras-CH1 / CT-MMAE treatment group with other treatment groups. (Figure 55A) Representative longitudinal, midsagittal hematoxylin and eosin (H&E) stained sections of tibia / femur from each group. (Figure 55B) Representative images of HER2 staining of bone sections from each group. [Figure 56] Secondary metastases observed in various organs of mice treated with Tras-MMAE or Tras-CH1 / CT-MMAE. [Figure 57] Figures 57A-57D: Preparation and characterization of bone-targeting antibodies. (Figure 57A) Bone-homing peptides were inserted into three locations: light chain (LC), heavy chain (CH1), and c-terminus (CT). (Figure 57B) ESI-MS analysis of antibodies. (Figure 57C) Binding of αCD99 antibodies to Ewing's sarcoma and osteosarcoma cell lines. (Figure 57D) Cell apoptosis induced by αCD99 antibodies in ES cells detected by flow cytometry. [Figure 58] Figures 58A-58D: (Figure 58A) Binding kinetics of αCD99 antibody to hydroxyapatite (HA). (Figure 58B) Differential bone targeting ability of wild-type αCD99 and bone-targeted αCD99 mutants. Scale bar, 200 μm. (Figure 58C) Ex vivo fluorescence images of the lower limbs of athymic nude mice bearing SK-ES-1 tumors 7 or 9 days after retro-orbital injection of NIR-labeled αCD99 and αCD99-CH1 / CT. (Figures 58D-58E) In vitro cytotoxicity of αCD99 against HUVEC cells and monocytes. [Figure 59]59A-59D: 12E7 antibody inhibits Ewing's sarcoma tumors in a xenograft model. (Fig. 59A) SK-ES-1 cells were injected intra-tibially into the right hind leg of nude mice followed by treatment with PBS and 12E7 antibody (retro-orbital sinus at 1 mg / kg in sterile PBS, twice weekly). Tumor burden was monitored by weekly bioluminescence imaging. (Fig. 59B) Signal progression of Fluc activity in mice treated as described in Fig. 59A. p-values ​​are based on two-way ANOVA test. (Fig. 59C) Body weight of mice treated as described in Fig. 59A. (Fig. 59D) SK-ES-1 cells were injected intramuscularly adjacent to the tibia of nude mice followed by treatment with PBS and 12E7 antibody (retro-orbital sinus at 1 mg / kg in sterile PBS, twice weekly). (Fig. 59E) Signal progression of Fluc activity in mice treated as described in Fig. 59D. p-values ​​are based on two-way ANOVA test. (FIG. 59F) Body weight of mice treated as described in FIG. 59D. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, ns>0.05. [Figure 60] Pulmonary and skeletal metastases observed in various organs of mice after establishment of an intratibial introduced bone lesion in the right leg. [Figure 61] Figures 61A-61B: (Figure 61A) Expression and purification of IL6-6D. (Figure 61B) Binding kinetics of IL6-6D to hydroxyapatite. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Description of exemplary embodiments Therapeutic antibodies have made great strides in realizing their clinical potential and have become useful in the treatment of various pathologies. Despite the rapid evolution of therapeutic antibodies, their clinical effectiveness in the treatment of bone tumors has been hindered by the inadequate pharmacokinetics of these large macromolecules and poor accessibility to bone tissue. Thus, in certain embodiments, the present disclosure provides methods for engineering therapeutic antibodies or polypeptides to include bone-homing peptide sequences that improve their concentration in the bone metastasis niche, resulting in a significant reduction in breast cancer bone metastasis survival and progression. To improve the bone tumor targeting ability of the engineered antibodies, various numbers of bone-homing peptides can be introduced into permissive internal sites of the antibody. Compared to unmodified antibodies, engineered bone-targeting antibodies were shown to have similar pharmacokinetics and in vitro cytotoxic activity, but showed improved bone tumor distribution in vivo. These results demonstrate that the addition of bone-specific targeting to antibody therapy results in robust delivery of therapeutic antibodies to the bone tumor niche. This provides a powerful strategy to overcome the inadequate treatment of bone cancer and the development of acquired resistance to treatment.

[0039] Thus, in certain embodiments, the present disclosure provides an innovative bone-targeting technology that allows for specific delivery of therapeutic antibodies or polypeptides to bone via engineering of the antibody or polypeptide to contain a bone-homing peptide. This type of specific delivery of therapeutic antibodies or polypeptides to bone has the potential to improve both the breadth and efficacy of antibody therapy for bone-related diseases.

[0040] I. Definition As used herein, "essentially free" with respect to a particular component is used herein to mean that none of the particular components are intentionally formulated into the composition and / or are present only as contaminants or only in trace amounts.Thus, the total amount of the particular component resulting from any unintentional contaminants of the composition is well below 0.05%, preferably below 0.01%.Most preferred is a composition in which the amount of the particular component cannot be detected by standard analytical methods.

[0041] As used herein, "a" or "an" may mean one or more. As used herein in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one.

[0042] Use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer to alternatives only or the alternatives are mutually exclusive, however, the present disclosure supports a definition that refers only to alternatives and "and / or." As used herein, "another" can mean at least a second, or more.

[0043] The term "about" generally means within the standard deviation of the stated value, as determined using standard analytical techniques to measure the stated value. The term may also be used to refer to plus or minus 5% of the stated value.

[0044] The phrase "effective amount" or "therapeutically effective" refers to a dosage of a drug or agent sufficient to produce a desired result, which may be a subjective or objective improvement in the recipient of the dosage, increased lung growth, increased lung repair, reduced tissue edema, increased DNA repair, reduced apoptosis, reduced tumor size, reduced rate of cancer cell growth, reduced metastasis, or any combination of the above.

[0045] As used herein, the term "antibody" refers to immunoglobulins, derivatives thereof that maintain specific binding ability, and proteins having binding domains that are homologous or largely homologous to immunoglobulin binding domains. These proteins may be derived from natural sources or may be partially or fully synthetically produced. Antibodies may be monoclonal or polyclonal. Antibodies may be members of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. Antibodies may be bispecific antibodies. In exemplary embodiments, antibodies used with the methods and compositions described herein are derivatives of the IgG class. The term antibody also refers to antigen-binding antibody fragments. Examples of such antibody fragments include, but are not limited to, Fab, Faby, F(aby)2, scFv, Fv, dsFv diabody, and Fd fragments. Antibody fragments may be produced by any means. For example, antibody fragments may be produced enzymatically or chemically by fragmentation of an intact antibody, recombinantly produced from a gene encoding a partial antibody sequence, or fully or partially synthetically produced. The antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may comprise multiple chains linked together, for example, by disulfide bonds. The fragment may also optionally be a multimolecular complex. A functional antibody fragment will typically comprise at least about 10 amino acids, and more typically at least about 200 amino acids.

[0046] "Subject" and "patient" refer to either humans or non-humans, such as primates, mammals, and vertebrates. In certain embodiments, the subject is a human.

[0047] As used herein, the terms "treat", "treatment", "treating" or "amelioration" when used in reference to a disease, disorder or medical condition refer to the therapeutic treatment of a condition, the purpose of which is to reverse, alleviate, ameliorate, inhibit, slow or stop the progression or severity of a symptom or condition. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of a condition is reduced or stopped. That is, "treatment" includes not only the improvement of a symptom or marker, but also the halting or at least slowing of the progression or worsening of a symptom that would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, a reduction in the extent of a defect, a stabilized (i.e., not worsening) state of a tumor or malignancy, a delay or slowing of tumor growth and / or metastasis, and an increase in life span compared to that expected in the absence of treatment.

[0048] II. Bone targeting antibodies Bone is primarily composed of hydroxyapatite (HA) crystals, an insoluble salt of calcium and phosphorus. The limited distribution of HA in hard tissues such as bone makes it an attractive target for selective bone targeting. Nature has evolved a variety of HA-binding proteins, including sialoproteins and osteopontin, that provide sites for cell anchoring and for regulating the bone mineralization process. Interestingly, sequence analysis has revealed that these proteins possess repeat sequences of acidic amino acids that represent potential bone-binding sites (Figure 1A). 17 Short bone-homing peptides consisting of aspartic acid (Asp) have been tested for the specific delivery of small molecules, microRNAs, and nanoparticles to the bone niche. 18~22 These short peptides have been shown to favor binding to HA surfaces with higher levels of crystallinity, which are characterized by the presence of bone-resorbing surfaces and are known as the osteolytic bone metastasis niche. 23The formation of osteolytic bone lesions is driven by paracrine crosstalk between cancer cells, osteoblasts, and osteoclasts. 22~26 Specifically, cancer cells secrete molecules such as parathyroid hormone-related protein (PTHrP) and interleukin 8, which directly or indirectly stimulate osteoclast formation by acting to regulate the expression of osteoblast genes such as receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG). The resulting increase in bone resorption leads to the release of growth factors (e.g., IGF1) that reciprocally stimulate tumor growth. Thus, selective delivery of therapeutic agents to the bone metastatic niche has the potential to interrupt this vicious osteolytic cycle. Thus, methods are provided herein for engineering antibodies capable of improved targeting of bone tumors. Following insertion of bone-homing peptide sequences into antitumor antibodies, the utility of these engineered antibodies was demonstrated for the treatment of breast cancer metastases in bone (Figure 1B).

[0049] In certain embodiments, methods and compositions are provided herein relating to bone-targeting antibodies, such as antibodies engineered to include one or more bone-homing peptides. In some aspects, the bone-homing peptide is a peptide that binds to bone hydroxyapatite (HA) matrix, such as L-Asp6.

[0050] The bone homing peptide may be inserted into the antibody at a "permissive internal site", referred to herein as a site where the insertion of the peptide is minimally disruptive to the structure and function of native IgG, while still allowing retention of the high affinity of the peptide for bone matrix. To screen for permissive internal sites, flow cytometry may be used to ensure that it does not disrupt antibody function. The binding affinity of the bone-targeting antibody may be evaluated using an HA binding assay. The bone homing peptide may be inserted into the light chain (LC), heavy chain (CH1), and / or C-terminus (CT) of the antibody. For example, the antibody may be trastuzumab. In specific aspects, the antibody may comprise a protein sequence of SEQ ID NO:3-16, or a protein sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3-16. Exemplary amino acid sequences of unmodified and modified trastuzumab are provided below, with the bone-homing peptide underlined. TIFF2024526920000002.tif62159TIFF2024526920000003.tif238160TIFF2024526920000004.tif243159TIFF2024526920000005.tif255158

[0051] The antibody may be conjugated to an imaging or diagnostic agent.

[0052] As used herein, a "therapeutic agent" refers to any agent that can be administered to a subject for the purpose of obtaining a therapeutic benefit for a disease or health-related condition. For example, an antibody conjugated to a therapeutic agent can be administered to a subject for the purpose of reducing the size of a tumor, reducing or inhibiting the local invasiveness of a tumor, or reducing the risk of developing metastases.

[0053] As used herein, a "diagnostic agent" or "imaging agent" (interchangeably referred to) refers to any agent that can be administered to a subject for the purpose of diagnosing a disease or health-related condition in the subject. Diagnosis can include determining whether a disease is present, whether a disease is progressing, or any change in the disease state.

[0054] The therapeutic or diagnostic agent can be a small molecule, peptide, protein, polypeptide, antibody, antibody fragment, DNA, or RNA.

[0055] A. Formulation and Administration The present disclosure provides pharmaceutical compositions comprising bone-targeting antibodies. Such compositions comprise a prophylactically or therapeutically effective amount of an antibody or fragment thereof, or a peptide immunogen, and a pharmaceutically acceptable carrier. In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, more specifically in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which a therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, a particular carrier is water. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0056] If desired, the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral formulations may contain standard carriers of pharmaceutical grade, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical agents are described in "Remington's Pharmaceutical Sciences". Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation should be compatible with the mode of administration, which may be oral, intravenous, intraarterial, buccal, intranasal, aerosol, bronchial inhalation, or delivered by mechanical ventilation.

[0057] When antibodies such as those disclosed are produced in vivo in subjects at risk of poxvirus infection, active vaccines are also envisioned. Such vaccines can be formulated for parenteral administration, for example, for injection via intradermal, intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Intradermal and intramuscular administration are contemplated. Alternatively, the vaccine can be administered directly by topical routes to the mucous membrane, for example, by nasal drops, inhalation, or nebulizer. Pharmaceutically acceptable salts include those formed with acid salts and inorganic acids, such as, for example, hydrochloric or phosphoric acid, or organic acids, such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0058] Passive transfer of antibodies, known as artificially acquired passive immunity, will generally involve the use of intravenous or intramuscular injections. The form of the antibodies can be human or animal plasma or serum, as pooled human immune globulin for intravenous (IVIG) or intramuscular (IG) use, as high titer human IVIG or IG from donors who have been immunized or are recovering from disease, and as monoclonal antibodies (MAbs). Such immunity generally lasts only for a short period of time, and there are also potential risks of hypersensitivity reactions and serum sickness, especially from gamma globulins of non-human origin. Passive immunity, however, provides immediate protection. The antibodies will be formulated in a carrier suitable for injection, i.e., a sterile and injectable carrier.

[0059] In general, the components of the composition of the present disclosure are supplied separately or mixed together in unit dosage form, for example as a dry lyophilized powder or water-free concentrate in an airtight container such as an ampoule or sachet indicating the amount of active agent.If the composition is to be administered by injection, it can be dispensed using an infusion bottle containing sterile water or saline of pharmaceutical grade.If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0060] The compositions of the present disclosure may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0061] B. Hyperproliferative disorders Hyperproliferative disease may be associated with any disease that causes cells to start reproducing uncontrollably, but the prototypical example is cancer. One of the key elements of cancer is that the normal apoptosis cycle of cells is interrupted, and therefore drugs that interrupt cell growth are important as therapeutic agents for treating these diseases. In the present disclosure, bone-targeting antibodies can be used to treat various types of cancer, such as bone cancer and cancer that metastasizes to bone.

[0062] Cancer cells that can be treated with the compounds of the present disclosure include, but are not limited to, cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testis, tongue, cervix, or uterus. In addition, the cancer may be of the following histological types, but is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; ciliary body carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; cavernous adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyps; adenocarcinoma, familial multiple adenomatous polyposis; solid tumors; Carcinoid tumors, malignant;Bronchioloalveolar carcinoma;Papillary adenocarcinoma;Chromophobe cell carcinoma;Eosinophilic carcinoma;Osinophilic adenocarcinoma;Basophilic carcinoma;Clear cell adenocarcinoma;Granular cell carcinoma;Follicle adenocarcinoma;Papillary and follicular adenocarcinoma;Nonencapsulated sclerosing carcinoma;Adrenal cortical carcinoma;Endometrial carcinoma;Cutaneous adnexal carcinoma;Apocrine gland carcinoma;Sebaceous gland carcinoma;Earwax gland carcinoma;Mucoepidermoid carcinoma;Cystic line carcinoma;Papillary cystadenocarcinoma;Papillary serous cystadenocarcinoma;Mucinous cystic adenocarcinoma;Mucinous adenocarcinoma;Signet ring cell carcinoma;Invasive ductal carcinoma;Medullary carcinoma;Lobular carcinoma ;inflammatory carcinoma;Paget's disease, breast;Acinic cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Thymoma, malignant;Ovarian stromal tumor, malignant;Theca cell tumor, malignant;Granulosa cell tumor, malignant;Male germinoma, malignant;Sertoli cell carcinoma;Leydig cell tumor, malignant;Adipocytic tumor, malignant;Paragaglioma, malignant;Extramammary paraganglioma, malignant;Pheochromocytoma;Hemangiosarcoma;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Malig melanoma in giant pigmented nevus noma;epithelioid cell melanoma;blue nevus, malignant;sarcoma;fibrosarcoma;fibrous histiocytoma, malignant;myxosarcoma;liposarcoma;leiomyosarcoma;rhabdomyosarcoma;embryonal rhabdomyosarcoma;alveolar rhabdomyosarcoma;stromal sarcoma;mixed tumor, malignant;mixed Müllerian tumor;nephroblastoma;hepatoblastoma;carcinosarcoma;mesenchymoma, malignant;Brenner tumor, malignant;phyllodes tumor, malignant;synovial sarcoma;mesothelioma, malignant;dysgerminoma;embryonal carcinoma;teratoma, malignant;ovarian goiter, malignant;choriocarcinoma;mesonephroma, malignant;angiosarcoma;hemangioendothelioma, malignant;Kaposi's sarcoma;Hemangiopericytoma, malignant;Lymphangiosarcoma;Osteosarcoma;Parocytic osteosarcoma;Chondrosarcoma;Chondrosarcoma;Chondroblastoma, malignant;Mesenchymal chondrosarcoma;Giant cell tumor of bone;Ewing's sarcoma;Odontogenic tumor, malignant;Ameloblastic sarcoma;Ameloblastoma, malignant;Ameloblastic fibrosarcoma;Pinealoma, malignant;Chordoma;Glioma, malignant;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrillary astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Neuroectodermal;Cerebellar sarcoma;Ganglioblastoma;Neuroblastoma;Retinoblastoma;Olfactory neurogenic tumor;Meningioma, malignant;Neurofibrosarcoma;Neuro Sheath tumor, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's; lateral granuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specific non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. In certain aspects, the tumor may include osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing's sarcoma, glioblastoma, neuroblastoma, or leukemia. ;

[0063] C. Treatment Methods In particular, compositions are disclosed herein that can be used to treat cancer in a subject (e.g., a human subject). The compositions are preferably administered to a mammal (e.g., rodent, human, non-human primate, dog, cow, sheep, horse, cat, etc.) in an effective amount, i.e., an amount capable of producing the desired result in the treated subject (e.g., causing apoptosis of cancerous cells or killing bacterial cells). The toxicity and therapeutic efficacy of the compositions utilized in the methods of the present disclosure can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary fields, the dosage for any one animal depends on many factors, including the subject's size, body surface area, weight, age, the particular composition administered, the time and route of administration, general health, clinical symptoms of infection or cancer, and other drugs administered concomitantly. The compositions described herein are typically administered in a dosage that induces the death of cancerous cells (e.g., induces apoptosis of cancer cells) as assayed by identifying a reduction in the growth or proliferation of cancer cells.

[0064] The therapeutic methods (including prophylactic treatments) of the present disclosure generally comprise administering to a subject, including a mammal, particularly a human, in need thereof a therapeutically effective amount of a composition as described herein. Such treatments would be suitably administered to subjects, particularly humans, who suffer from, have, are susceptible to, or are at risk for a disease, disorder, or symptoms thereof. The determination of these "at risk" subjects may be made by any objective or subjective determination (e.g., genetic testing, enzyme or protein markers, Markers (as defined herein), family history, etc.) by diagnostic testing or the opinion of the subject or a health care provider.

[0065] In one embodiment, the present disclosure provides a method for monitoring the progress of a treatment. The method includes determining the level of changes in hematological parameters and / or cancer stem cell (CSC) analysis using cell surface proteins as diagnostic markers (e.g., including but not limited to, CD34, CD38, CD90, and CD117) or diagnostic measurements (e.g., screening, assays) in a subject suffering from or susceptible to a disorder associated with cancer (e.g., leukemia) or symptoms thereof, where the subject has been administered a therapeutic amount of a composition described herein. The level of the marker determined by the method can be compared to known levels of the marker in either healthy normal controls or other affected patients to establish the disease status of the subject. In a preferred embodiment, a second level of the marker in the subject is determined at a time point subsequent to the determination of the first level, and the two levels are compared to monitor the progress of the disease or the effectiveness of the treatment. In certain preferred embodiments, a pre-treatment level of the marker in the subject is determined before initiating treatment according to the methods described herein, and then this pre-treatment level of the marker can be compared to the level of the marker in the subject after initiating treatment to determine the effectiveness of the treatment.

[0066] D. Additional Therapies In certain embodiments, the compositions and methods of the present invention comprise a bone-targeting antibody in combination with a second or additional therapy. Such therapy can be applied to the treatment of any disease involving bone tumors. For example, the disease can be bone cancer or bone metastasis.

[0067] In certain embodiments, the compositions and methods of the present invention include a bone-targeting antibody in combination with at least one additional therapy. The additional therapy can be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the above. The additional therapy can be in the form of adjuvant therapy or neoadjuvant therapy.

[0068] Methods and compositions that include combination therapy improve the therapeutic or protective effect and / or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. The therapeutic and prophylactic methods and compositions can be provided in combined amounts effective to achieve a desired effect, such as killing cancer cells and / or inhibiting cell hyperproliferation. This process can include contacting cells with both the antibody or antibody fragment and the second therapy. The tissue, tumor, or cells may be contacted with one or more compositions or pharmacological formulations that include one or more of the agents (i.e., antibody or antibody fragment, or anti-cancer agent), or by contacting the tissue, tumor, and / or cells with two or more separate compositions or formulations, where one composition provides both 1) the antibody or antibody fragment, 2) the anti-cancer agent, or 3) the antibody or antibody fragment, and the anti-cancer agent. It is also contemplated that such combination therapy can be used simultaneously with chemotherapy, radiation therapy, surgery, or immunotherapy.

[0069] The terms "contacting" and "exposing," as applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to or placed in direct juxtaposition with a target cell. To achieve cell killing, for example, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent the cell from dividing.

[0070] The inhibitory antibody may be administered before, during, after, or in various combinations relative to the anti-cancer therapy. Administration may be at intervals ranging from simultaneous to minutes to days to weeks. In embodiments in which the antibody or antibody fragment is provided to the patient separately from the anti-cancer drug, it will generally be ensured that no significant period of time passes between the respective delivery times so that the two compounds are still able to exert a combined effect beneficial to the patient. In such cases, it is contemplated that the antibody therapy and the anti-cancer therapy may be provided to the patient within about 12 to 24 or 72 hours of each other, more specifically within about 6 to 12 hours of each other. In some circumstances, it may be desirable to significantly extend the treatment period, with days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) passing between the respective administrations.

[0071] In certain embodiments, the course of treatment will last from 1 to 90 days or more (such ranges including intervening days). It is contemplated that one agent may be administered on any day from day 1 to day 90 (such ranges including intervening days) or any combination thereof, and another agent may be administered on any day from day 1 to day 90 (such ranges including intervening days) or any combination thereof. Within a day (24 hour period), the patient may receive one or more doses of the agents. It is further contemplated that after the course of treatment, there will be a period during which no anti-cancer treatment is administered. This period may last from 1 to 7 days, and / or 1 to 5 weeks, and / or 1 to 12 months or more (such ranges including intervening days), depending on the patient's prognosis, strength, health, etc. It is anticipated that the treatment cycle will be repeated as necessary.

[0072] In some embodiments, the additional therapy is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional therapy is administration of a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of treatment, such as an anti-nausea agent). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventative agent. The additional therapy can be one or more of chemotherapeutic agents known in the art.

[0073] Various combinations can be used. In the following example, the bone-targeting antibody is "A" and the anti-cancer therapy is "B": TIFF2024526920000006.tif18128

[0074] Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for administering such compounds, taking into account the toxicity of the drug, if any. Thus, in some embodiments, there is a step of monitoring for toxicity resulting from the combination therapy.

[0075] 1.Chemotherapy A wide variety of chemotherapeutic agents may be used in accordance with this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within the cell, for example, whether and at what stage they affect the cell cycle. Alternatively, agents may be characterized based on their ability to directly crosslink DNA, to intercalate into DNA, or to induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0076] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic relatives). analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifomide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, fenestrone nitrogen mustards and uracil mustards such as phosphatidylcholine, prednimustine, trofosfamide; nitrosurea such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma Il and calicheamicin omega Il); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin. , daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, and peplomycin. , potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folate analogs such as denopterin, pteropterin, and trimetrexate; fludarabine, 6-mercapto Purine analogues such as purine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as mitotane and trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diazicon; elformithine; elliptinium acetate; epothilone; etoglucide;Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Phenamet; Pirarubicin; Rosoxantrone; Podophyllinic acid acid;2-ethylhydrazine;procarbazine;PSK polysaccharide complex;razoxane;rhizoxin;schizofiran;spirogermanium;tenuazonic acid;triaziquone;2,2',2"-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine);urethane;vindesine;dacarbazine;mannomustine;mitobronitol;mitolactol;pipobroman;gacytosine;arabinosides ("Ara-C");cyclophosphamide;taxoids, such as paclitaxel and docetaxel. Gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS200 0; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and pharma- ceutically acceptable salts, acids, or derivatives of any of the above;

[0077] 2. Radiation therapy Other agents that cause DNA damage and are widely used include what are commonly known as gamma radiation, X-rays, and / or direct delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents such as microwaves, proton beam irradiation (U.S. Patents. 5,760,395 and 4,870,287), and UV irradiation are also contemplated. All of these agents most likely affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. Dose ranges for X-rays range from daily doses of 50-200 roentgens to single doses of 2000-6000 roentgens over prolonged periods (3-4 weeks). Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by the neoplastic cells.

[0078] 3. Immunotherapy Those skilled in the art will understand that additional immunotherapy can be used in combination or simultaneously with the method of the embodiment. In the context of cancer treatment, immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector can be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody alone can function as an effector of treatment or can actually recruit other cells to affect cell killing. The antibody can also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and function as a targeting agent. Alternatively, the effector can be a lymphocyte carrying a surface molecule that directly or indirectly interacts with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0079] Antibody-drug conjugates have emerged as a breakthrough approach to the development of cancer therapeutics. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently linked to a cell-killing drug. This approach combines the high specificity of MAbs for antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells and enrich the levels of the antigen (Carter et al., 2008; Teicher 2014; Leal et al., 2014). Targeted delivery of the drug also minimizes exposure in normal tissues, resulting in reduced toxicity and improved therapeutic index. The FDA approval of two ADC drugs, ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013, validated this approach. There are currently more than 30 ADC drug candidates for cancer treatment in various stages of clinical trials (Leal et al., 2014). As antibody engineering and linker-payload optimization become more mature, the discovery and development of new ADCs increasingly relies on the identification and validation of new targets suitable for this approach, and the generation of targeting MAbs (Teicher 2009). Two criteria for ADC targets are up-regulation / high levels of expression in tumor cells, and robust internalization.

[0080] In one aspect of immunotherapy, tumor cells must carry some marker that is easy to target, i.e., not present on most other cells. Many tumor markers exist, any of which may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erbB, and p155. An alternative aspect of immunotherapy is to combine anti-cancer effects with immune stimulatory effects. There are also immune stimulatory molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0081] Examples of immunotherapies currently under investigation or in use include immune adjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy such as interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Antibody therapies include, but are not limited to, monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Villaseca, 1998; U.S. Patent Nos. 5,830,880 and 5,846,945), and monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used in conjunction with the antibody therapies described herein.

[0082] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints are molecules of the immune system that either raise a signal (e.g., a costimulatory molecule) or lower a signal. Inhibitory checkpoint molecules that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0083] The immune checkpoint inhibitor may be a drug, such as a small molecule, a recombinant form of a ligand or receptor, or may be an antibody, particularly a human antibody (e.g., International Patent Publication No. 2015 / 016718, Pardoll, Nat Rev Cancer, 12(4):252-64, 2012, both of which are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof may be used, particularly chimeric, humanized, or human forms of antibodies. As will be appreciated by those skilled in the art, alternative and / or equivalent names may be used for the specific antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of the present invention. For example, lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0084] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain aspects, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In certain aspects, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In certain aspects, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Pat. Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, such as those described in U.S. Patent Application Nos. 2014 / 0294898, 2014 / 022021, and 2011 / 0008369, all of which are incorporated by reference herein.

[0085] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin that includes an extracellular portion or a PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, Lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0086] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to CD28, a T cell costimulatory protein, and both molecules bind to CD80 and CD86 (also called B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. Activation of T cells via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0087] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0088] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, anti-CTLA-4 antibodies disclosed in US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab, formerly ticilimumab), U.S. Patent No. 6,207,156, Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17):10067-10071; Camacho et al. (2004) J Clin Oncology 22(145):Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304 may be used in the methods disclosed herein. The teachings of each of the foregoing publications are incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. 2001 / 014424, 2000 / 037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0089] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WOO1 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0090] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors such as those described in U.S. Pat. Nos. 5,844,905, 5,885,796, and International Patent Applications Nos. 1995 / 001994 and 1998 / 042752, all of which are incorporated herein by reference, and immunoadhesins such as those described in U.S. Pat. No. 8,329,867, which is incorporated herein by reference.

[0091] 4.Surgery Approximately 60% of people with cancer will undergo some type of surgery, including preventive, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection, which physically removes, cuts out, and / or destroys all or part of the cancerous tissue, and may be used simultaneously with other therapies, such as the treatment of the present embodiments, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery).

[0092] Removal of part or all of the cancerous cells, tissues, or tumors may result in the formation of a cavity in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also be of varying dosages.

[0093] 5. Other drugs It is contemplated that other agents may be used in combination with certain aspects of the present embodiment to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing cell-cell signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the present embodiment to improve the anti-hyperproliferative efficacy of the treatment. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents, such as the antibody c225, that increase the sensitivity of hyperproliferative cells to apoptosis, may be used in combination with certain aspects of the present embodiment to improve the therapeutic efficacy.

[0094] III. Kit In various aspects of the embodiments, kits are contemplated that contain the therapeutic agent and / or other therapeutic and delivery agents. In some embodiments, the present embodiments contemplate kits for preparing and / or administering the antibody compositions of the embodiments. The kits may include one or more sealed vials containing any of the pharmaceutical compositions of the present embodiments. The kits may include, for example, engineered antibodies and reagents for preparing, formulating, and / or administering the components of the present embodiments or performing one or more steps of the methods of the present invention. In some embodiments, the kits may also include suitable containers, such as Eppendorf tubes, assay plates, syringes, bottles, or tubes, that will not react with the components of the kit. The containers may be made of sterilizable materials, such as plastic or glass.

[0095] The kit may further include instructions outlining the procedural steps of the methods described herein, following substantially the same procedures as those described herein or known to those of skill in the art. The instruction information may be present in a computer readable medium containing machine readable instructions that, when executed using a computer, display an actual or virtual procedure for the delivery of a pharma- ceutical effective amount of a therapeutic agent. EXAMPLES

[0096] IV. Working Examples The following examples are included to demonstrate preferred embodiments of the present disclosure. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present disclosure, and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should understand in light of the present disclosure that many changes can be made to the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the present disclosure.

[0097] Example 1 - Engineering a bone-targeting antibody Modification of trastuzumab with bone-homing peptides To harness the power of bone-homing peptides for selective delivery of antibodies to bone cancer sites, we first engineered a library of antibodies bearing the bone-homing peptide L-Asp6 at various sites within the immunoglobulin molecule. The design principle was to incorporate the bone-homing sequence at a site that would be minimally disruptive to the structure and function of native IgG and still allow retention of the high affinity of the peptide for bone matrix. Based on the crystal structure of an IgG1 monoclonal antibody, the L-Asp6 peptide was inserted into permissive internal sites in the light chain (LC, A153), heavy chain (CH1, A165), and C-terminus (CT, G449) of trastuzumab, yielding Tras-LC, Tras-CH1, and Tras-CT, respectively (Figure 2A). These internal sites have been shown to be stable to peptide insertion by screening of antibody peptide configuration libraries. 27 To modulate the bone tumor targeting ability of the engineered antibodies, the number of bone-homing peptide sequences per immunoglobulin molecule was also varied, generating trastuzumab species with two L-Asp6 peptide sequences (Tras-LC / CT, Tras-CH1 / CT, and Tras-LC / CH1) and a trastuzumab species with three L-Asp6 peptide sequences (Tras-LC / CH1 / CT). The seven resulting constructs were expressed in ExpiCHO-S cells by transient transfection, followed by purification of the immunoglobulins using protein G chromatography and analysis of the expressed proteins by SDS-PAGE. All of these antibody variants were expressed with good yields (50–100 mg / L). SDS-PAGE and ESI-MS analysis confirmed the successful insertion of the bone-homing peptides (Figures 2B, 2C, 5–12). Among the antibody variants, the Tras-LC / CH1 species, which contains the L-Asp6 peptide sequence in both the light and heavy chains, showed significant aggregation, and therefore this antibody variant was not studied further.

[0098] In vitro evaluation of bone-targeting antibodies With the bone-targeting antibody variants in hand, we first used an HA binding assay to examine their binding to mineralized bone. Briefly, the bone-targeting antibody species were incubated with HA for varying lengths of time, and the unbound antibody remaining in solution was measured using a UV-Vis spectrophotometer. As shown in Figure 2D, unmodified Tras showed only slight affinity for HA, while the L-Asp6 peptide-modified antibody bound to HA in a time-dependent manner. Antibodies with multiple L-Asp6 peptides, namely Tras-CH1 / CT and Tras-LC / CH1 / CT, showed the highest HA binding capacity, with more than 80% of the antibody bound after 9 hours of incubation (Table 2). For the three antibody species containing a single L-Asp6 peptide, the C-terminal construct (Tras-CT) showed the highest HA binding capacity. Therefore, fluorescein isothiocyanate (FITC)-labeled Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT were used to stain non-demineralized bone sections from C57BL / 6 mice. Sections treated with unmodified Tras did not show any fluorescence after overnight incubation (Figure 2E). In contrast, FITC signals were observed in all sections stained with the three L-Asp6 peptide-containing variants. These FITC antibody signals correlated well with the xylenol orange (XO) signals from bone (Figures 2E, 13).

[0099] To demonstrate that the insertion of the L-Asp6 sequence has negligible effect on the binding and specificity of the Tras antibody, FITC-labeled Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT antibodies were tested for binding to HER2-positive and HER2-negative cell lines. By flow cytometry, none of these antibodies bound to HER2-negative MDA-MB-468 cells, whereas each of the bone-targeting antibodies bound to HER2-expressing SK-BR-3 cells with a K similar to that of unmodified Tras. d(7.09 nM) (Figure 2F, 14–23 and Table 3). The Tras-CT, Tras-LC, Tras-CH1, Tras-CH1 / CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT species had slightly higher K than Tras, presumably due to increased electrostatic repulsion mediated by the inserted negatively charged residues. d The bone-targeted antibodies had EC values ​​of 14.60 nM, 19.39 nM, 12.99 nM, 19.47 nM, 19.47 nM, and 25.20 nM, respectively (Figures 14-22). Next, the in vitro cytotoxicity of the bone-targeted antibodies was evaluated against HER2-positive and HER2-negative cell lines. Consistent with the flow cytometry data, the Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT antibodies kill SK-BR-3 cells with efficiencies similar to that of unmodified Tras (EC values ​​of 5.97 ± 5.64 nM, 13.07 ± 12.09 nM, and 21.10 ± 20.25 nM, respectively). 50 Values ​​(Figure 2G)). In contrast, none of the antibodies show cytotoxicity against HER2-negative MDA-MB-468 cells under the same experimental conditions (Figure 2G). These results indicate that the introduction of a bone-homing sequence into the antibody significantly improved its bone affinity while maintaining its antitumor activity. To examine the potential toxicity of Tras-CH1 / CT to bone cells, mouse pre-osteoclast cell line RAW264.7 and osteoblast cell line MC3T3-E1 were incubated with various concentrations of Tras or Tras-CH1 / CT for 4 days and cell survival was evaluated. As shown in Figure 38, neither Tras nor Tras-CH1 / CT showed significant toxicity. These results indicate that despite the improved binding to bone, bone-targeting antibodies are unlikely to cause significant toxicity to bone stromal cells.

[0100] In vivo distribution of bone-targeting antibodies The effect of bone-homing peptides on Tras antibody distribution was further investigated in a mouse xenograft model. Paratibial injection was used to administer 2 × 10 5HER2-expressing MDA-MB-361 breast cancer cells were first introduced into the right leg of nude mice, followed by administration of sulfo-Cy7.5-labeled Tras, Tras-CT, Tras-CH1 / CT, or Tras-LC / CH1 / CT via retro-orbital injection. At 72 and 120 hours after antibody injection, the main organs were harvested and the antibody distribution was imaged. The intensity of the interosseous fluorescence signal was stronger in Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT injected animals than in Tras injected mice (Figures 2H, 24, and 25). As shown in Figure 25, the ex vivo signals from various tissues quantified suggest that significantly higher signals of bone-targeted antibodies could be observed in skeletal tissues but not in other tissues (Figure 25B). Furthermore, we performed histological analysis of the collected cardiac tissues and observed no obvious pathological changes in cardiac tissues upon different treatments, indicating good biocompatibility of the bone-targeting antibody (Figure 25C). Moreover, a higher amount of bone-targeting antibody was present in the tumor-bearing right leg bone compared with the left healthy bone, probably due to L-Asp6 peptide-mediated targeting to the bone resorption niche. Overall, these results showed that the introduction of the L-Asp6 sequence can significantly increase the concentration of therapeutic antibody at the bone tumor site. This effect has the potential to improve antitumor activity at the tumor site while simultaneously reducing systemic toxicity.

[0101] In vivo therapeutic activity of bone-targeted antibodies against bone micrometastases To determine whether bone-targeting antibodies can serve as novel therapeutic entities for the treatment of breast cancer metastasis to bone, in vivo antitumor experiments were performed in nude mice bearing MDA-MB-361 tumors. 2 × 10 5MDA-MB-361 breast cancer cells were inoculated into the right leg of nude mice via paratibial injection. One week after injection, wild-type Tras and bone-targeted Tras antibodies were administered by retro-orbital injection. As shown in FIG. 3A, mice receiving 1 mg / kg unmodified Tras did not respond well to this treatment. Despite an initial inhibitory effect during the first 2 weeks of treatment, unmodified Tras was unable to control long-term tumor growth, prolonging the median mouse survival time of subjects by only 9.7 days (FIG. 3B and 3C). In contrast, when tumor-bearing mice were treated with bone-targeted antibodies, tumor growth was significantly inhibited. The Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT treatment groups showed a significant delay in tumor growth of 27.5, 35.8, and 18.9 days, respectively (FIG. 3B and 3C). Figures 3A, 3B, 3C, 26, 27, and Table 4 show the bioluminescence (BLI) signals of each treatment group from day 1 to day 80. In the PBS-treated control group, there was a progressive increase in the BLI signal over time. The BLI signals from day 1 to day 80 demonstrated that the bone-targeted Tras antibody treatment group experienced a significant delay in tumor growth compared to the Tras treatment group (Figures 3B and 3C). Mice treated with Tras-CH1 / CT showed a minimal increase in tumor size (Tras-CH1 / CT vs. Tras: 9.3±4.2 vs. 1562.7±801.6, p<0.0001). Furthermore, Tras-CH1 / CT-treated mice experienced a survival rate of 62.5%, a significant improvement over that seen in Tras-treated mice (Figure 3D). Thus, it appears that treatment with Tras-CH1 / CT resulted in a more effective inhibition of the progression of micrometastases than that seen in Tras-treated mice. Treatment with the bone-targeting antibody was well tolerated, and no obvious signs of toxicity were observed in any of the treatment groups, e.g., no differences in body weight were observed across the various treatment groups (Figure 3E).

[0102] At the end of the experiment (day 81), tibiae (from the tumor-bearing leg) were harvested and scanned by micro-computed tomography (mico-CT). Micro-CT analysis revealed extensive osteolytic bone destruction in both PBS- and Tras-treated groups, whereas bone loss was significantly reduced in the bone-targeting antibody-treated group (Figures 3F, 28, and 29). Compared with PBS- and Tras-treated mice, Tras-CH1 / CT-treated mice showed larger bone volume (BV, Figure 3G), larger bone volume / tissue volume ratio (BV / TV, Figure 3H), larger bone mineral density (BMD, Figure 3I), and thicker cancellous bone (Tb.Th, Figure 3J), but smaller bone surface / bone volume ratio (BS / BV, Figure 3K and Table 5). All these parameters indicate a significant delay of micrometastasis-induced osteolysis by the bone-homing peptide-modified antibody. Histological analysis further revealed infiltration of tumor cells into the bone matrix and adjacent tissues in the Tras-treated group (Figure 3L). Histology also confirmed the reduced intratibial tumor burden in these mice indicated by the BLI results. Bone sections from Tras-CH1 / CT-treated mice revealed reduced tumor growth and relatively normal bone morphology in these mice, consistent with significant inhibition of tumor invasion and bone destruction (Figure 3L). Bone samples from the various treatment groups were also analyzed for bone-resorbing TRAP (tartrate-resistant acid phosphatase)-positive multinucleated osteoclasts (shown as pink cells) and HER-expressing cancer cells (Figures 3M, 3N, 30-32). Compared to Tras treatment, Tras-CH1 / CT treatment significantly reduced the number of both osteoclasts and HER2-positive cells in the bone niche, again consistent with the ability of bone-targeted antibodies to inhibit the progression of micrometastases (Figure 3N). Because tumor-induced hypercalcemia and TRACP 5b protein are indicators of osteolytic bone destruction, the effect of bone-targeting antibody therapy was evaluated. The results suggested that the Tras-CH1 / CT treatment group had a better therapeutic effect (Figure 33).

[0103] Consistent with the microCT and histological analysis, Tras-CH1 / CT treatment showed the lowest level of bone destruction as assessed by serum hypercalcemia and TRACP 5b protein levels (Figure 33). To evaluate the stability of bone-targeting antibodies in vitro, Tras and Tras-CH1 / CT (1 mg / mL, 100 μL) were incubated in PBS at 4°C for 3 months. SDS-PAGE and ESI-MS analysis revealed that no significant degradation or aggregation of bone-targeting antibodies was observed (Figure 40). Meanwhile, serum levels of both Tras and Tras-CH1 / CT showed similar pharmacokinetics in vivo (Figure 41), suggesting that the addition of bone-homing peptide did not alter the stability of the antibodies. Next, the benefit of bone-targeting antibodies for bone metastasis was evaluated at higher doses. Nude mice bearing MDA-MB-361 bone metastases were treated with 10 mg / kg Tras or Tras-CH1 / CT every 2 weeks. Notably, Tras-CH1 / CT treatment resulted in statistically significant growth inhibition and prolonged overall survival compared to treatment with 10 mg / kg Tras (Figures 3O-R, 42, 43). Furthermore, no weight loss was observed with the high dose of the bone-targeting antibody (Figure 3S).

[0104] The improved therapeutic efficacy of bone-targeting antibodies was also evaluated in a secondary bone metastasis model using MCF-7 breast cancer cells. Consistent with the MDA-MB-361 model, a significant reduction in metastatic burden and increased mouse survival time was observed in the Tras-CH1 / CT treatment group compared to the Tras treatment group (Figures 36A-D, 44). Tras-CH1 / CT treatment did not alter the additional body weight change in the animals (Figure 36E).

[0105] To investigate the efficacy of Tras-CH1 / CT in treating primary tumors, MDA-MB-361 cells (1 × 10 6) into the mammary fat pad, followed by treatment with PBS, Tras (1 mg / kg), or Tras-CH1 / CT (1 mg / kg). As shown in Figures 45A-C, both Tras and Tras-CH1 / CT treatments significantly reduced tumor growth in the mammary fat pad, but there was no statistical difference in tumor size between the two treatments (Figure 45C). Taken together, these data suggest that the introduction of a bone-homing peptide into an antibody can significantly inhibit breast cancer bone metastasis without compromising its antitumor activity in the primary tumor.

[0106] Immunogenicity evaluation of bone-targeting antibodies To understand whether bone-targeted antibody treatment induces a specific immune response, immunocompetent C57BL / 6J mice were treated with PBS, Tras, or Tras-CH1 / CT twice a week for 2 weeks. As shown in Figure S37A, immune profiling determined by flow cytometry suggests that mice treated with either Tras or Tras-CH1 / CT had similar immune composition, except for CD4+ T cells. Furthermore, IFNγ staining of CD4+ T cells had no significant difference in Tras-treated and Tras-CH1 / CT-treated mice, indicating that Tras-CH1 / CT does not significantly alter the functional activity of CD4+ T cells (Figure 46B). Furthermore, there was no significant difference in serum levels of INFγ, IL-2, and IL-4 levels between treatment with wild-type or bone-targeted antibodies (Figure 46C-E). The results showed that the addition of L-Asp6 to the antibody did not induce any obvious extra immune response. Furthermore, the in vivo immune response to the bone-targeting antibody can also be tested by an immunogenicity test based on an anti-trastuzumab antibody ELISA-based assay as previously reported. Similar anti-trastuzumab antibody levels were observed in animals treated with Tras (5 mg / kg) or Tras-CH1 / CT (5 mg / kg), suggesting that the addition of the bone-targeting peptide does not alter the immunogenicity of the antibody (Figure 47).

[0107] Bone-targeted antibodies inhibit secondary metastases from bone lesions In more than two-thirds of patients, breast cancer metastases are not restricted to the skeleton but occur subsequently in other organs 28、29、30、31 Recent genomic analyses suggest that these metastases, which are a major cause of morbidity and mortality, do not originate from the primary tumor but are seeded from other metastatic sites. Utilizing a recently developed approach to selectively deliver cancer cells to hindlimb bones, frequent "metastasis-to-metastasis" seeding from established bone lesions to multiple other organs has been observed. 32~34 Therefore, we evaluated whether bone-targeting antibodies could inhibit these secondary metastases originating from bone lesions. Using the paratibial injection method, highly bone-specific tibial-bearing tumors were at an early stage of development. As the bone lesions progressed, metastases marked by bioluminescent signals began to appear in many other organs, including other bones, lungs, heart, liver, spleen, kidneys, and brain. Using this model, we were able to obtain 2 × 10 5 Luciferase-labeled MDA-MB-361 cells were introduced into the right hind limb of nude mice via paratibial injection, followed by treatment with unmodified Tras antibody and bone-targeted Tras antibody. Mice were euthanized 81 days after the start of treatment, and organs were harvested for ex vivo bioluminescence imaging. Seven organs, including the right hind limb bone and six soft tissue organs, were isolated for evaluation of metastasis. As shown in Figures 4A, 4B, and 34, treatment with Tras-CH1 / CT significantly reduced the frequency of secondary metastasis to the contralateral hind limb (left hind limb bone), heart, and liver. The ex vivo BLI intensity of secondary metastasis from primary bone lesions was reduced in the experimental groups treated with bone-targeted antibodies, especially in the Tras-CH1 / CT treatment group. Compared with treatment with unmodified Tras, metastatic signals from the lung and liver were significantly reduced after Tras-CH1 / CT treatment (Figure 4B). Taken together, these data demonstrate the improved therapeutic efficacy of bone-targeted antibodies against both primary bone metastases and secondary metastases from bone to other distant organs.

[0108] Modification of antibody-drug conjugates with bone-homing peptides shows improved therapeutic efficacy in vivo Antibody-drug conjugates (ADCs), which combine the tumor specificity of antibodies with the high toxicity of chemotherapy drugs, have emerged as an important class of anticancer drugs for breast cancer patients, especially those with advanced breast cancer. Following the initial FDA approval of trastuzumab emtansine (T-DM1) for HER2-positive breast cancer, trastuzumab deruxtecan was recently approved for the treatment of adults with unresectable or metastatic HER2-positive breast cancer. To test whether bone-targeted ADCs can further improve their efficacy in treating bone metastases, pClick conjugation technology was first used to site-specifically conjugate monomethyl auristatin E (MMAE) to both wild-type antibody Tras and bone-targeted antibody Tras-CH1 / CT (Figure 37E). Successful conjugation was demonstrated by SDS-PAGE and ESI-MS (Figures 37B, 48, 49). To confirm that toxin conjugation did not alter antigen targeting ability and specificity, in vitro binding assays were performed using HER2-positive and HER2-negative cells (Figure 50). Tras-CH1 / CT-MMAE showed high binding affinity to HER2-positive SK-BR-3 cells but not to HER2-negative MDA-MB-468 cells. Next, the in vitro cytotoxicity of these ADCs was evaluated in SK-BR-3 and MDA-MB-468 breast cancer cell lines (Figure 37C-D). Both Tras-MMAE and Tras-CH1 / CT-MMAE showed high potency only in SK-BR-3 (EC50: 0.18±0.82nM and 0.49±0.30nM, respectively), and no significant toxicity was observed in MDA-MB-468 cells. To test the bone targeting ability of Tras-CH1 / CT-MMAE in vitro, either Tras-CH1 / CT-MMAE or Tras-MMAE was incubated with non-demineralized bone sections. As expected, only the signal of Tras-CH1 / CT-MMAE correlated well with the XO signal, confirming its bone targeting ability (Figure 37E).

[0109] Next, the therapeutic efficacy of bone-targeted ADC was examined in a xenograft model of bone metastasis. Weekly administration of Tras-CH1 / CT-MMAE resulted in significant inhibition of metastatic growth in bone compared to unmodified Tras-MMAE (Figures 37F-H, 51, 52). Furthermore, bone-targeted ADC showed no obvious toxicity, as indicated by the continuous increase in body weight across the various groups during treatment (Figure 37I). Micro-CT analysis revealed extensive osteolytic bone destruction in both PBS- and Tras-MMAE-treated groups, but not in the Tras-CH1 / CT-MMAE-treated group (Figures 37J, 53). Compared with mice in the PBS- and Tras-MMAE-treated groups, Tras-CH1 / CT-MMAE-treated mice exhibited a higher bone volume / tissue volume ratio (BV / TV, Figure S46A) and thicker cancellous bone (Tb.Th, Figure 54B). Histology also confirms the reduction of intratibial tumor burden in Tras-CH1 / CT-MMAE mice (Figure 55). Consistently, bone-targeted ADC also significantly reduced the frequency and size of secondary metastases originating from bone lesions (Figures 37K, 56).

[0110] Thus, it was demonstrated that the addition of bone specificity to antibody therapy results in increased antibody concentration in the bone metastasis niche compared to other tissues. This approach results in targeted therapy for bone metastasis and for secondary multiorgan metastatic seeding from bone lesions. Using a xenograft model of bone metastasis, it was found that unmodified trastuzumab has poor bone tissue penetration and distribution, thus reducing the antibody's contact with its target and limiting its efficacy against cancer cells in the bone microenvironment. Compared to unmodified antibodies, trastuzumab modified with a bone homing peptide sequence (L-Asp6) showed improved targeting to the site of bone metastasis. This resulted in improved activity against breast cancer metastasis to bone and metastatic seeding from bone lesions to other organs. Most importantly, it was demonstrated that modified antibodies with moderate bone binding capacity had optimal efficacy in vivo. In contrast, antibodies with higher bone binding capacity have suboptimal activity against bone metastasis. This may be due to slow release of the latter entities from the bone matrix or increased electrostatic repulsion due to the increased number of negatively charged peptides. The addition of bone specificity to antibody therapy has enabled the specific delivery of these agents to bone. This has resulted in not only improved therapeutic efficacy but also reduced adverse side effects associated with systemic distribution of drugs. Thus, a new strategy is provided herein to transition antibody-based therapy from antigen-specific to both antigen- and tissue-specific, thus offering a promising new path to advance antibody therapy towards clinical translation.

[0111] Example 2 - Materials and Methods material Unless otherwise stated, chemicals and solvents used were of analytical grade and were used as received from commercial sources. LB agar was ordered from Fisher. Oligonucleotide primers were purchased from Eurofins Genomics (Table 1). Plasmid DNA preparation was performed with the GenCatch™ Plus Plasmid DNA Miniprep Kit and the GenCatch™ Advanced Gel Extraction Kit. PNGase F was purchased from New England Biolabs to remove glycans prior to ESI-MS analysis. NuPAGE 4-12% Bis-Tris Gel was purchased from Invitrogen. SDS-PAGE Sample Loading Buffer [6x] was purchased from BIOSCIENCES. PM2500 ExcelBand 3-color Regular Range Protein Marker was purchased from SMOBIO. ExpiCHO Expression Medium, ExpiFectamine CHO Transfection Kit, and OptiPRO SFM Complexation Medium were purchased from Thermofisher. Hoechst 33342 (catalog number: H1399) was purchased from Life Technologies™. 3,3-Dioctadecyloxacarbocyanine perchlorate (DiIC18, catalog number: M1197) was purchased from Marker Gene Technologies, Inc.

[0112] cell line MDA-MB-361, BT474, SK-BR-3, and MDA-MB-468 cell lines were cultured according to ATCC instructions. Firefly luciferase and RFP-labeled MDA-MB-361 cell lines were generated as previously described. 40 .

[0113] SDS-PAGE analysis Intact and reduced antibody samples were analyzed using Invitrogen NuPAGE 4-12% Bis-Tris gels. 20 μL of 0.2 mg / mL antibody sample was mixed with 4 μL of sample loading dye before loading onto the gel. Gels were run in MES buffer under 160 V for 35 min and stained with Coomassie Blue buffer. Gel images were captured by Amersham Imager 600 and analyzed by ImageQuanTL software.

[0114] ESI-MS analysis Antibodies were analyzed using a single quadrupole mass spectrometer (Agilent: G7129A) coupled to a 1260 infinity II Quaternary Pump (Agilent: G7111B). (Column: Pursuit 5 Diphenyl 150 x 2.0 mm). The elution conditions for the antibodies were as follows: mobile phase A = 0.1% formic acid in water; mobile phase B = 0.1% formic acid in acetonitrile; gradient 0-0.1 min, 10-15% B; 0.1-8 min, 15-50% B; 8-8.1 min, 50-10% B; flow rate = 0.5 mL / min. Absorbance was measured at 280 nm. Automated data processing was performed with MassHunter BioConfirm software (Agilent) to analyze the as-reduced and reduced MS spectra.

[0115] HA binding assay Briefly, 1 mg of Tras or bone-targeting antibody was diluted in 0.5 mL of PBS (pH 7.4) in an Eppendorf tube. HA (20 eq., 20 mg) was suspended in 0.5 mL of PBS. The antibody and HA were then mixed by vortexing, and the resulting suspension was shaken at 220 rpm at 37°C. A sample without HA was used as a control. After 0.25, 0.5, 1, 2, 3, 6, and 8 hours, the suspension was centrifuged (3000 rpm, 3 min) and the absorbance of the supernatant at 280 nm was measured by Nanodrop. The percentage of binding to HA was calculated as follows: where OD stands for optical density. [(OD HAを含まない -OD HAを含む) / (OD HAを含まない )] × 100%

[0116] In vitro cytotoxicity of Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT 2 x 10 in 96-well plate 3 cells / well of BT474 and MDA-MB-468 cells. After 24 h of incubation, the cells were treated with various concentrations of Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT, and then incubated for 4 days. Then, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg / mL) was added to each well and incubated for another 4 h. The medium was aspirated and 150 μL of DMSO was added to each well. To quantify live cells, the absorbance at 565 nm was measured by a microplate reader (Infinite M Plex by Tecan).

[0117] Flow cytometry Cancer cells (3 × 10 5 ) were incubated with 30 μg / mL of Tras and bone-targeting antibodies for 30 min at 4° C. After washing away unbound antibodies, bound antibodies were detected with fluorescein (FITC) AffiniPure Goat Anti-Human IgG(H+L) (code: 109-095-003, Jackson Immunology) for 30 min at 4° C. Fluorescence intensity was determined using BD FACSVerse (BD Biosciences).

[0118] K d Determining Values The functional affinity of bone-targeting antibodies to HER2 was determined as reported 41 In short, the sum is 2 × 10 5SK-BR-3 or MDA-MB-468 cells were incubated with graded concentrations of Tras, Tras-LC, Tras-CT, Tras-CH1, Tras-LC / CT, Tras-CH1 / CT, Tras-LC / CH1, and Tras-LC / CH1 / CT for 4 hours on ice. Bound antibodies were then detected by fluorescein (FITC) AffiniPure Goat Anti-Human IgG(H+L) (Jackson Immunology). Cells were analyzed for fluorescence intensity after propidium iodide (Molecular Probes, Eugene, OR) staining. The linear portion of the saturation curve was used to calculate the dissociation constant, KD, using the Lineweaver-Burk method, plotting the reciprocal of the median fluorescence as a function of the reciprocal of the antibody concentration. KD was determined as follows: 1 / F=1 / Fmax+(K d / Fmax)(1 / [Ab]), where F corresponds to the background-subtracted median fluorescence, and Fmax was calculated from the plot.

[0119] Binding to bone frozen sections Non-decalcified long bone sections from C57BL / 6 mice were incubated with 50 μg / mL of Tras, Tras-CT, Tras-CH1 / CT, or Tras-LC / CH1 / CT conjugated overnight at 4° C., followed by staining with fluorescein isothiocyanate (FITC)-labeled anti-human IgG for 60 min at room temperature. After three washes with PBS, specimens were incubated with xylenol orange (XO) (stock: 2 mg / ml, 1:500 dilution, dilution buffer: PBS at pH 6.5) for 30 min at 37° C. After three washes with PBS, specimens were stained with Hoechst33342 (stock 10 mg / ml, 1:2000 dilution) for 10 min. Slides were then washed with PBS, air-dried, and sealed with PROLONG™ gold antifade mounting medium (from ThermoFisher).

[0120] In vivo evaluation of Tras, Tras-CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT To establish bone metastases, firefly luciferase- and RFP-labeled MDA-MB-361 cells (2 × 10 5 1000 ng / mL) were inoculated into the tibia of 3-4 week old female athymic nude mice using paratibial injection method. Seven days after surgery, mice were ranked / randomly divided to obtain similar tumor burden in each group. PBS and antibody (1.0 mg / kg) were injected via retro-orbital injection twice weekly. Animals were imaged weekly using IVIS Lumina II (Advanced Molecular Vision) according to recommended procedures and manufacturer settings. All of the mice were euthanized on day 81 after blood collection and all organs (tumor-bearing tibia, heart, liver, spleen, lungs, brain, and kidneys) were collected for further studies.

[0121] Analysis of metastasis from ex vivo metastases At the end point, live animals were given D-luciferin and immediately dissected. Tissues were examined by ex vivo BLI imaging. The entire process of organ collection procedure and ex vivo imaging process should be completed within 15 minutes for each mouse.

[0122] Bone histology and immunohistochemistry On day 81, mice were euthanized and tibiae were harvested, fixed, and then decalcified in 12% EDTA for 10 days. Tibiae were embedded in paraffin and sectioned. Tumor burden was assessed on hematoxylin and eosin (H&E) sections. Osteoclasts within the tumor and on the bone-tumor interface were counted after staining with Acid Phosphatase, Leukocyte (TRAP) Kit (Sigma). Immunohistochemistry analysis was performed on decalcified paraffin-embedded tissue sections using HRP / DAB ABC IHC KIT (abcam) according to the manufacturer's protocol.

[0123] Radiographic analysis Tibiae were dissected, fixed and scanned by micro-computed tomography (micro-CT, Skyscan1272, Aartselaar, Belgium) with a resolution of 16.16 μm / pixel. Raw images were reconstructed with NReconn and analyzed with CTan (SkyScan, Aartselaar, Belgium) using regions of interest (ROI). Analyzed bone parameters included trabecular bone thickness (Tb.Th), bone volume fraction (BV / TV), bone mineral density (BMD), and bone surface / bone volume ratio (BS / BV).

[0124] Biodistribution Female athymic nude mice were inoculated with MDA-MB-361 cells (2 × 10 5 Mice were injected with 100 μg / ml of 10 ...

[0125] For wild-type mice, Cy7.5-labeled Tras, Tras-CT, and Tras-LC / CH1 / CT were administered to C57BL / 6 mice via retro-orbital injection. After 48 hours, organs were dissected and imaged using IVIS.

[0126] Quantification of TRAP and calcium levels in serum On day 81, blood was collected by cardiac puncture and centrifuged at 3,000 rpm for 15 minutes to obtain serum. The concentration of osteoclast-derived TRACP 5b was measured by using Mouse ACP5 / TRAP ELISA Kit (Cat. No. IT5180, GBiosciences). Serum calcium level was determined colorimetrically using a calcium detection kit (Cat. No. DICA-500, Bioassays).

[0127] statistical methods Data are presented as the mean plus or minus SEM and statistically analyzed using GraphPad Prism software version 6 (GraphPad software, San Diego, CA). For all data collected over time, a two-way analysis of variance followed by Sidak's multiple comparisons was used. For Micro-CT data, a one-way analysis of variance followed by Tukey's multiple comparisons was used. For multi-organ metastasis data, an unpaired Student's t-test was used. P<0.05 was considered to represent statistical significance.

[0128] Plasmid construction pCDNA-Tras-LC: The 6D gene was inserted into the LC strand (border residues A153, Q155) of the Tras sequence by PCR using primers RG03, RG04, RG07, and RG08 and pCDNA-Tras as a template. The gene fragments were annealed by using the Gibson assembly method.

[0129] pCDNA-Tras-CH1: The 6D gene was inserted into the CH1 strand (border residues A165, G169) of the Tras sequence by PCR using primers RG05, RG06, RG07, and RG008 and pCDNA-Tras as a template. The gene fragments were annealed by using the Gibson assembly method.

[0130] pCDNA-Tras-CT: The 6D gene was inserted into the C-terminus (border residue G449) of the Tras sequence by PCR using primers RG01, RG02, RG07, and RG008 and pCDNA-Tras as a template. The gene fragments were annealed by using the Gibson assembly method.

[0131] pCDNA-Tras-LCCH1 / LCCT / CH1CT / LCCH1CT-6D: By using the above primers and the constructed plasmid as a template, the 6D gene was inserted into the Tras sequence. The gene fragments were annealed by using the Gibson assembly method.

[0132] (Table 1) DNA oligomers TIFF2024526920000007.tif90160

[0133] Expression and purification of antibody variants Tras and 6D insertion mutants were expressed in ExpiCHO-S cells following Thermofisher's ExpiCHO expression protocol. Cells were grown on an orbital shaker platform (125 rpm) in a 37°C incubator with >80% relative humidity and 8% CO2 until the cultures reached 4×10 6 ~6×10 6 They were grown and subcultured until a density of viable cells / mL was reached. Prior to transfection, ExpiFectamine CHO / plasmid DNA complexes were prepared and incubated at room temperature for 5 min and then slowly added to the cell culture. 18-22 h after transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were added to the cell culture. After 12 days of expression, secreted antibodies were harvested by centrifugation at 9000 rpm for 30 min and purified with Protein G resin according to the manufacturer's instructions. Prior to injection, each antibody sample was buffer exchanged into PBS via a PD-10 desalting column and the concentration was measured using a NanoDrop Lite (Thermofisher). All antibody samples were characterized by ESI-MS and SDS-PAGE analysis.

[0134] Table 2. Adsorption of 6D insertion mutants that bind to HA TIFF2024526920000008.tif49128 antibody (1 mg / ml) was incubated with 20 mg / ml hydroxyapatite for 9 hours at 37° C. The percentage of hydroxyapatite-bound fraction is shown (mean ± SEM).

[0135] Table 3. Potency and cell surface reactivity of Tras, Tras-CT, Tras-LC, Tras-CH1, Tras-LC / CH1, Tras-LC / CT, Tras-CH1 / CT, and Tras-LC / CH1 / CT against breast cancer epithelial cell lines. TIFF2024526920000009.tif32159Abbreviations: MFI, median fluorescence intensity. Binding was determined as the mean fold increase in median fluorescence over PBS control.

[0136] Table 4. Comparison of various treatment groups across multiple assays. TIFF2024526920000010.tif22282Abbreviations: BLI, bioluminescence imaging; TRAP, tartrate-resistant acid phosphatase. a Whole-body BLI signal intensity over the course of the experiment. b Fold increase in BLI signal intensity after treatment (BLI on day 80 / BLI on day 1). C Osteoclast number measurements from TRAP stained tibia sections at the end of the experiment. d Serum TRACP 5b concentrations at the end of the experiment. e Serum calcium concentrations at the end of the experiment. f Weight progression over the course of the experiment. g 10 7 A mouse BLI intensity of > 100 was considered as reaching the endpoint. a、b were statistically analyzed by using two-way repeated measures analysis of variance followed by Sidak's multiple comparison test. c、d、e、f were analyzed by using one-way analysis of variance followed by Tukey's multiple comparison test. g were analyzed by using the log-rank test. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, ns indicates P>0.05.

[0137] Table 5. Comparison of microCT parameters from different treatment groups. TIFF2024526920000011.tif85159Abbreviations: BV: bone volume; BV / TV: bone volume / tissue volume ratio; BS / BV: bone surface / bone volume ratio; BMD: bone mineral density; Tb.Th: corpus trabecularis thickness. Data presented as mean ± sem. MicroCT data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. ****P<0.0001, ***P<0.001, **P<0.001, *P<0.05, ns indicates P>0.05.

[0138] Example 3 - Engineering an αCD99 antibody for Ewing's sarcoma with a bone homing peptide Using peptides, we engineered an αCD99 antibody for bone targeting. ES tumors have much higher CD99 expression levels than normal cells. To achieve specific targeting of ES tumors, which highly express CD99, but not normal human cells, we first expressed a full-length antibody with a relatively low CD99 affinity (clone: ​​12E7, K D =10 nM, FIG. 57). The antibody clone has been recently used in clinical trials and has demonstrated an excellent safety profile. It was demonstrated that this purified αCD99 antibody can specifically bind to CD99-positive RD-ES cells, SK-ES-1 cells, but not to CD99-negative U2-OS osteosarcoma cells (FIG. 57C). Furthermore, 12E7 antibody treatment induced apoptosis in more than 60% and 70% of RD-ES cells and SK-ES-1 cells, respectively (FIG. 57D). The in vivo therapeutic efficacy of this αCD99 antibody was also verified by the inventors in a xenograft model (FIG. 59).

[0139] To prepare an antibody that targets the bone resorption niche, the bone-homing peptide L-Asp6 peptide was introduced into permissive internal sites in the light chain (LC, A153), heavy chain (CH1, A165), and C-terminus (CT, G449) of the αCD99 antibody to obtain a bone-targeted αCD99 antibody (Figures 57A and B). These internal sites were shown to support additional peptide insertions by screening an antibody peptide configuration library. αCD99 with one L-Asp6 peptide was prepared (αCD99-CT, Figure 57B).

[0140] Antibodies with various numbers of L-Asp6 peptides were prepared and their bone targeting activity was evaluated. L-Asp6 peptides were cloned into two (CH1 and CT) and three permissive internal sites (LC, CH1, and CT) to obtain αCD99 antibody variants with two and three L-Asp6 peptides, respectively. The successful preparation of αCD99-CH1 / CT was confirmed by ESI-MS (Figure 57B).

[0141] The difference in bone affinity was evaluated among the bone-targeting antibodies. αCD99, αCD99-CT, αCD99-CH1 / CT, and αCD99-LC / CH1 / CT antibodies were incubated with hydroxyapatite / natural bone. The introduction of L-Asp6 sequence into the antibody can significantly improve its bone-targeting ability. As shown in Figure 58A, wild-type αCD99 showed little binding affinity with HA, while 80%-95% of the antibodies containing the L-Asp6 peptide sequence bound to HA within 4 hours. In general, bone-targeting antibodies with more bone-homing peptides show better HA binding affinity (Figure 58A). Furthermore, FITC-labeled αCD99, αCD99-CT, and αCD99-CH1 / CT mutants were further used to stain non-demineralized bone sections from C57BL / 6 mice, as shown in Figure 58B. FITC signal was observed in sections stained with αCD99-CT, the αCD99-CH1 / CT antibody variants, but not with wild-type αCD99 (FIG. 58B).

[0142] No cytotoxicity of 12E7 antibody was observed in human macrophages and endothelial cells (Figure 58D and E). To investigate the feasibility of using bone-specific antibodies to treat ES, the effect of αCD99 antibody engineered with L-Asp6 sequence on orthotopic models was examined using established cell lines and patient-derived xenograft models (Figure 59A-C). A xenograft nude mouse model was established to demonstrate that L-Asp6 sequence modification can improve the bone-binding affinity of the antibody. 5 × 10 5 SK-ES-1 (CD99+++) ES cells were inoculated into the right leg of nude mice via intratibial injection (Figure 58C). Wild-type αCD99 modified with NIR dye (1 mg / kg in sterile PBS, retro-orbital) and αCD99-CH1 / CT (same regimen as Tras) were introduced using retro-orbital injection. Seven and nine days after administration, the main organs were removed and analyzed using a Caliper IVIS Lumina II in vivo imager. The fluorescence intensity in bone tissue was higher for the antibody with the L-Asp6 peptide sequence than for the wild-type αCD99 (Figure 58C).

[0143] To demonstrate the ability of bone-targeted αCD99 to inhibit ES development in bone and soft tissues in vivo, a xenograft model was established to study the in vivo therapeutic efficacy of αCD99 against ES tumors in bone. 5 SK-ES-1 cells were inoculated into nude mice via intratibial injection. As shown in Figures 59A and 59B, whole-body bioluminescence imaging (BLI) signals suggested that treatment with bone-targeted αCD99 resulted in more significant inhibition of ES tumor progression compared to that seen in wild-type αCD99-treated mice (Figures 59A-C).

[0144] To evaluate the efficacy of bone-targeted αCD99 for the treatment of non-bone ES tumors, 5 × 10 5SK-ES-1 cells were injected adjacent to the tibia. The bone-targeted αCD99 antibody showed superior and similar efficacy for treating ES located in soft tissue compared to wild-type αCD99 (Figure 59A-C).

[0145] Example 4 - Bone-targeted IL-6 Furthermore, bone-targeted interleukin-6 (IL6) was generated by inserting six aspartic acids into the C-terminus of IL-6 (Figure 61A). The resulting bone-targeted IL6 (IL6-6D) showed improved binding affinity to hydroxyapatite structures (Figure 61B).

[0146] All of the methods disclosed and claimed herein can be made and performed without undue experimentation in light of this disclosure. Although the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that modifications may be applied to the methods and steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0147] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2024526920000012.tif201160TIFF2024526920000013.tif253160TIFF2024526920000014.tif238160TIFF2024526920000015.tif244160

Claims

1. An antibody targeted to bone, engineered to include at least one bone-homing peptide that selectively binds to bone hydroxyapatite (HA).

2. The antibody according to claim 1, wherein the at least one bone-homing peptide is inserted into an acceptable internal site of the antibody.

3. The antibody according to claim 1, wherein the at least one bone-homing peptide is inserted into the light chain (LC), heavy chain (CH1), and / or C-terminus (CT) of the antibody.

4. The antibody according to claim 1, comprising two, three, or four bone-homing peptides.

5. The bone homing peptide is L-Asp ３ , L-Asp ４ , L-Asp ５ , L-Asp ６ , L-Asp ７ , L-Asp ８ , L-Asp ９ , or L-Asp １０ , the antibody according to claim 1.

6. The bone-homing peptide is L-Asp ６ The antibody of claim 1,

7. The antibody according to claim 1, which is a monoclonal antibody, bispecific antibody, Fab’, F(ab’)2, F(ab’)3, monovalent scFv, divalent scFv, single domain antibody, or nanobody.

8. The antibody according to claim 1, which is an immune checkpoint inhibitor.

9. The antibody according to claim 1, which is an anti-HER2 antibody, anti-CD99 antibody, anti-IGF-IR antibody, anti-PD-L1, anti-PD-1, anti-CTLA4 antibody, anti-Siglec-2 antibody, anti-Siglec-3 antibody, anti-Siglec-5 antibody, anti-Siglec-6 antibody, anti-Siglec-7 antibody, anti-Siglec-8 antibody, anti-Siglec9 antibody, anti-Siglec-10 antibody, anti-Siglec-11 antibody, anti-Siglec-15 antibody, anti-RANKL antibody, anti-EGFR antibody, anti-VEGFR antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD52 antibody, anti-Trop-2 antibody, anti-CD30 antibody, anti-CD152 antibody, anti-IL-6R antibody, anti-GD2 antibody, or anti-TGFβ antibody.

10. The antibody according to claim 1, which is an anti-CD99 antibody.

11. The antibody according to claim 1, which is conjugated to a drug.

12. The antibody according to claim 11, wherein the drug is monomethyl auristatin E (MMAE), ozogamicin, thiouxianthene, vedotin, pasdotox tdfx, vedotin, mafodotin, calicheamicin, maytansine, or deruxtecan.

13. The antibody according to claim 11, wherein the drug is an anti-mitotic agent.

14. The antibody according to claim 13, wherein the anti-mitotic agent is monomethyl auristatin E (MMAE).

15. The antibody according to claim 1, which is an anti-HER2 antibody.

16. The antibody of claim 1, which is trastuzumab, pertuzumab, or atezolizumab.

17. The antibody of claim 1, which is trastuzumab.

18. 18. The antibody of claim 17, wherein the trastuzumab is conjugated to MMAE.

19. 18. The antibody of claim 17, wherein the bone-homing peptide is inserted at residues A153, A165, and / or G449 of trastuzumab.

20. 2. The antibody of claim 1, having an amino acid sequence that has at least 90% sequence identity to Tras-LC (SEQ ID NOs: 3-4), Tras-CH1 (SEQ ID NOs: 5-6), Tras-CT (SEQ ID NOs: 7-8), Tras-LC / CH1 (SEQ ID NOs: 9-10), Tras-LC / CT (SEQ ID NOs: 11-12), Tras-CH1 / CT (SEQ ID NOs: 13-14), or Tras-LC / CH1 / CT (SEQ ID NOs: 15-16).

21. 2. The antibody of claim 1, having an amino acid sequence that has at least 95% sequence identity to Tras-LC (SEQ ID NOs: 3-4), Tras-CH1 (SEQ ID NOs: 5-6), Tras-CT (SEQ ID NOs: 7-8), Tras-LC / CH1 (SEQ ID NOs: 9-10), Tras-LC / CT (SEQ ID NOs: 11-12), Tras-CH1 / CT (SEQ ID NOs: 13-14), or Tras-LC / CH1 / CT (SEQ ID NOs: 15-16).

22. 2. The antibody of claim 1, comprising Tras-LC (SEQ ID NOs: 3-4), Tras-CH1 (SEQ ID NOs: 5-6), Tras-CT (SEQ ID NOs: 7-8), Tras-LC / CH1 (SEQ ID NOs: 9-10), Tras-LC / CT (SEQ ID NOs: 11-12), Tras-CH1 / CT (SEQ ID NOs: 13-14), or Tras-LC / CH1 / CT (SEQ ID NOs: 15-16).

23. The antibody of claim 1 , wherein the bone-targeting antibody has increased binding affinity to HA compared to an antibody that does not contain a bone-homing peptide.

24. The antibody according to claim 1, wherein the bone-targeted antibody has a binding affinity that is 2 to 3 times higher for HA as compared to an antibody that does not contain a bone-homing peptide.

25. A pharmaceutical composition for treating or preventing a bone disease in a subject, comprising an effective amount of the bone-targeted antibody according to any one of claims 1 to 23.

26. The pharmaceutical composition according to claim 25, wherein the subject has bone cancer or bone metastasis.

27. The pharmaceutical composition according to claim 26, wherein the bone cancer is Ewing's sarcoma, osteosarcoma, or chondrosarcoma.

28. The pharmaceutical composition according to claim 26, wherein the bone metastasis is from breast cancer, myeloma, renal cancer, lung cancer, prostate cancer, thyroid cancer, or bladder cancer.

29. The pharmaceutical composition according to claim 28, wherein the breast cancer is triple-negative breast cancer.

30. The pharmaceutical composition according to claim 28, wherein the breast cancer is HER2-negative breast cancer.

31. The pharmaceutical composition according to claim 28, wherein the breast cancer is HER2-positive breast cancer.

32. The pharmaceutical composition according to claim 25, wherein the bone disease is osteoporosis, osteomalacia, periodontitis, rheumatoid arthritis, metabolic bone disease, parathyroid disorder, steroid-induced osteoporosis, chemotherapy-induced bone loss, premenopausal bone loss, fragility and recurrent fractures, renal osteodystrophy, bone infection, or Paget's disease.

33. The pharmaceutical composition according to claim 25, wherein the bone-targeted antibody results in an increase in the concentration of the therapeutic antibody in the bone tumor niche, inhibits cancer development in the bone, and / or limits secondary metastasis to other organs.

34. The pharmaceutical composition according to claim 25, wherein the bone-targeted antibody results in a decrease in micro-metastasis-induced osteolytic lesions.

35. The pharmaceutical composition according to claim 25, further administered with an additional anti-cancer therapy.

36. The pharmaceutical composition according to claim 35, wherein the additional anti-cancer therapy includes surgery, chemotherapy, radiation therapy, hormone therapy, immunotherapy, or cytokine therapy.

37. The pharmaceutical composition according to claim 35, wherein the additional anti-cancer therapy includes immunotherapy or chemotherapy.

38. Use of the bone-targeted antibody according to any one of claims 1 to 23 in the manufacture of a pharmaceutical composition for treating or preventing bone tumors in a subject having cancer.

39. The use according to claim 38, wherein the subject has bone cancer or bone metastasis.

40. The use according to claim 39, wherein the bone cancer is Ewing's sarcoma, osteosarcoma, or chondrosarcoma.

41. The use according to claim 39, wherein the bone metastasis is from breast cancer, myeloma, renal cancer, lung cancer, prostate cancer, thyroid cancer, or bladder cancer.

42. A method for engineering a bone-targeted antibody according to any one of claims 1 to 23, the method comprising inserting at least one bone-homing peptide into an acceptable internal site of the antibody.

43. The method according to claim 42, wherein the antibody comprises two, three, or four bone-homing peptides.

44. The bone-homing peptide is L-Asp ６ 43. The method of claim 42, wherein:

45. A bone-targeted composition comprising one or more polypeptides engineered to comprise at least one bone-homing peptide that selectively binds to bone hydroxyapatite (HA).

46. The antibody according to claim 45, wherein the at least one bone-homing peptide is inserted into an acceptable internal site of the polypeptide.

47. The antibody according to claim 45, wherein the at least one bone-homing peptide is inserted at the C-terminus or N-terminus of the polypeptide.

48. The antibody according to claim 45, wherein the polypeptide comprises two, three, or four bone-homing peptides.

49. The bone homing peptide is L-Asp ３ 、L-Asp ４ 、L-Asp ５ 、L-Asp ６ 、L-Asp ７ 、L-Asp ８ 、L-Asp ９ 、or L-Asp １０ The antibody according to claim 45, wherein the antibody is

50. The bone homing peptide is L-Asp ６ The antibody according to claim 45, wherein the antibody is as described above. ６

51. The antibody according to claim 45, wherein the one or more polypeptides comprise an adrenergic agonist, an anti-apoptotic factor, an apoptosis inhibitor, a cytokine receptor, a cytokine, a cytotoxin, an erythropoietin, glutamic acid decarboxylase, a glycoprotein, a growth factor, a growth factor receptor, a hormone, a hormone receptor, an interferon, an interleukin, an interleukin receptor, a kinase, a kinase inhibitor, a nerve growth factor, a netrin, a bioactive peptide, a bioactive peptide receptor, a neurogenic factor, a neurogenic factor receptor, a neuropilin, a neurotrophic factor, a neurotrophin, a neurotrophin receptor, an N-methyl-D-aspartate antagonist, a plexin, a protease, a protease inhibitor, a protein decarboxylase, a protein kinase, a protein kinase inhibitor, a proteolytic protein, a proteolytic protein inhibitor, a semaphorin, a semaphorin receptor, a serotonin transporter protein, a serotonin uptake inhibitor, a serotonin receptor, a serpin, a serpin receptor, or a tumor suppressor.

52. The antibody according to claim 45, wherein the one or more polypeptides comprise a cytokine.

53. The antibody according to claim 52, wherein the cytokine is IL-6.

54. The antibody according to claim 45, wherein the one or more polypeptides are conjugated to a drug.

55. The antibody according to claim 54, wherein the drug is monomethyl auristatin E (MMAE), ozogamicin, thiouixanthene, vedotin, pasdotox td fx, vedotin, mafodotin, calicheamicin, maytansine, or deruxtecan.

56. The antibody according to claim 54, wherein the drug is an anti-mitotic agent.

57. The antibody according to claim 56, wherein the anti-mitotic agent is monomethyl auristatin E (MMAE).

58. The antibody according to claim 45, wherein the bone-targeting composition has an increased binding affinity for HA as compared to a polypeptide that does not include a bone-homing peptide.

59. The antibody according to claim 45, wherein the bone-targeting composition has a binding affinity for HA that is 2 to 3 times higher as compared to a polypeptide that does not include a bone-homing peptide.

60. A pharmaceutical composition for treating or preventing a bone disease in a subject, the pharmaceutical composition comprising an effective amount of the bone-targeted composition according to any one of claims 45 to 59.

61. The pharmaceutical composition according to claim 60, wherein the subject has bone cancer or bone metastasis.

62. The pharmaceutical composition according to claim 61, wherein the bone cancer is Ewing's sarcoma, osteosarcoma, or chondrosarcoma.

63. The pharmaceutical composition according to claim 61, wherein the bone metastasis is from breast cancer, multiple myeloma, kidney cancer, lung cancer, prostate cancer, thyroid cancer, or bladder cancer.

64. The pharmaceutical composition according to claim 63, wherein the breast cancer is triple-negative breast cancer.

65. The pharmaceutical composition according to claim 63, wherein the breast cancer is HER2-negative breast cancer.

66. The pharmaceutical composition according to claim 63, wherein the breast cancer is HER2-positive breast cancer.

67. The pharmaceutical composition according to claim 60, wherein the bone disease is osteoporosis, osteomalacia, periodontitis, rheumatoid arthritis, metabolic bone disease, parathyroid disorder, steroid-induced osteoporosis, chemotherapy-induced bone loss, premenopausal bone loss, fragility and recurrent fractures, renal osteodystrophy, bone infection, or Paget's disease.

68. The pharmaceutical composition according to claim 60, wherein the bone-targeted composition results in an increase in the concentration of a therapeutic polypeptide in the bone tumor niche, inhibits cancer development in the bone, and / or limits secondary metastasis to other organs.

69. The pharmaceutical composition according to claim 60, wherein the bone-targeted composition results in a decrease in micro-metastasis-induced osteolytic lesions.

70. The pharmaceutical composition according to claim 60, further administered with additional anti-cancer therapy.

71. The pharmaceutical composition according to claim 70, wherein the additional anti-cancer therapy includes surgery, chemotherapy, radiotherapy, hormone therapy, immunotherapy, or cytokine therapy.

72. The pharmaceutical composition according to claim 70, wherein the additional anti-cancer therapy includes immunotherapy or chemotherapy.

73. Use of the bone-targeted composition according to any one of claims 45 to 59 in the manufacture of a pharmaceutical composition for treating or preventing bone tumors in a subject having cancer.

74. The use according to claim 73, wherein the subject has bone cancer or bone metastasis.

75. The use according to claim 74, wherein the bone cancer is Ewing's sarcoma, osteosarcoma, or chondrosarcoma.

76. The use according to claim 74, wherein the bone metastasis is from breast cancer, myeloma, renal cancer, lung cancer, prostate cancer, thyroid cancer, or bladder cancer.

77. A method for manipulating a bone-targeted composition according to any one of claims 45 to 59, the method comprising the step of inserting at least one bone-homing peptide into an acceptable internal site of the polypeptide.

78. The method according to claim 77, wherein the polypeptide comprises two, three, or four bone-homing peptides.

79. wherein the bone homing peptide is L-Asp ６ The method according to claim 77, wherein the method is as described above. ６

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