Method for bone-specific delivery of polypeptides - Patent Application 20070123633

Abstract

The present disclosure provides methods for treating bone diseases, such as bone cancer, bone metastasis of cancer, or osteoporosis, by administering a bone-targeting polypeptide conjugate, which may be a bone-targeting antibody conjugate.

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of priority to U.S. Provisional Application No. 63 / 138,972, filed January 19, 2021, the entire contents of which are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grants W81XWH-16-1-0073 and W81XWH-21-1-0789 awarded by the Department of Defense. The Government has certain rights in this invention.

[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. [Background technology]

[0004] 2. Related Technologies Antibody-based therapies, including those using monoclonal antibodies, antibody-drug conjugates, bispecific antibodies, checkpoint inhibitors, and others, have recognized clinical potential in terms of their ability to treat a variety of cancers. 1~4 Nevertheless, despite the fact that most therapeutic antibodies have high affinity for their targets, the presence of these same targets in normal tissues can dramatically limit the ability of therapeutic agents to attack their targets without inducing unacceptable "on-target" toxicity in healthy cells. 5~7 Furthermore, the low levels of delivery of therapeutic antibodies to some tissues, such as the brain or bone, can significantly limit their effectiveness in treating diseases in these tissues. 8 Therefore, improving both the antigen and tissue specificity of antibodies is likely to ultimately transform the efficacy of antibody therapy for the clinical treatment of cancer.

[0005] Half of patients initially diagnosed with metastatic breast cancer (BCa) will develop bone metastases 9 Patients with only skeletal metastases usually have a better prognosis than those with significant organ metastases. 9、10 Furthermore, bone metastases are associated with serious complications such as spinal cord compression, pathological fractures, and hypercalcemia. 11 Deep understanding of molecular mechanisms 12、13 Despite this, effective therapies capable of eliminating cancer cells remain lacking. 14 Bone is not the final destination of disseminated metastases; recent genomic analyses have revealed frequent "metastasis-to-metastasis" dissemination. 15~17 More than two-thirds of bone-only metastases subsequently develop secondary metastases to other organs, ultimately resulting in the death of the patient. 9、10 Indeed, some metastases initially identified in organs other than bone are in fact the result of dissemination from asymptomatic bone micrometastases (BMMs). This is apparently the result of cancer cells first reaching bone and then acquiring a more aggressive phenotype that allows them to establish more overt metastases both in bone and at other sites. 18 Thus, there is an unmet need for strategies to prevent BMM from establishing more overt metastases in both bone and non-bone tissues.

[0006] Targeted antibody therapy and immunotherapy are currently emerging as new avenues for treating metastatic breast cancer, but the performance of these agents in patients with bone metastases has been disappointing. For example, trastuzumab (Herceptin) and pertuzumab (Perjeta) antibodies targeting human epidermal growth factor receptor 2 (HER2) have been used to treat patients in the adjuvant and metastatic settings. Although many BCa patients benefit from these therapies, in the majority of BCa patients with bone metastases, the disease progresses within a year, and few patients experience long remissions. 19~22In another phase III clinical trial testing atezolizumab in patients with metastatic triple-negative BCa, progression-free survival was significantly longer in the atezolizumab group than in the placebo group. However, among BCa patients with bone metastases, no significant differences in the risk of progression or death were observed between the atezolizumab-treated group and the placebo group. 23 Therefore, therapies that improve the outcomes of BCa patients with bone metastases are highly desirable. Summary of the Invention

[0007] overview In certain embodiments, the present disclosure provides a method for treating or preventing a bone disease in a subject, comprising administering to the subject an effective amount of a bone-targeting conjugate comprising a bisphosphonate (BP) conjugated to a polypeptide or protein. In one embodiment, the present disclosure provides a method for treating or preventing a bone tumor in a subject, comprising administering to the subject an effective amount of a bone-targeting conjugate comprising a bisphosphonate (BP) conjugated to an antibody.

[0008] 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 loss of mineral content of cortical and / or trabecular bone, improve bone biomechanical resistance, increase bone formation, and / or reduce bone resorption.

[0009] In some embodiments, the subject has bone cancer or bone metastases. In particular 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 certain embodiments, the bone metastases are breast cancer bone metastases. In some embodiments, the breast cancer is triple negative breast cancer. In certain embodiments, the breast cancer is HER2 negative breast cancer. In other embodiments, the breast cancer is HER2 positive breast cancer.

[0010] In certain embodiments, the BP is negatively charged. In some embodiments, the BP is alendronate, zoledronate, pamidronate, risedronate, medronic acid, aminomethylene bisphonic acid, clodronate, etidronate, tiludronate, ibandronate, pomidronate, neridonate, olpadronate, or oxidronate. In certain embodiments, the BP is alendronate (ALN).

[0011] In some embodiments, the BP is conjugated to an adrenergic agonist, an anti-apoptotic factor, an apoptosis inhibitor, a cytokine receptor, a cytokine, a cytotoxin, an erythropoietic agent, 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.

[0012] In some embodiments, 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 embodiments, the antibody is an immune checkpoint inhibitor. In certain 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-15 antibody, an anti-RANKL antibody, or an anti-TGFβ antibody. In certain embodiments, the antibody is an anti-HER2 antibody, such as trastuzumab (Herceptin), pertuzumab (Perjeta), or atezolizumab. In certain embodiments, the antibody is trastuzumab. In some embodiments, the bone-targeting conjugate comprises alendronate conjugated to trastuzumab. In certain embodiments, the antibody is not an anti-M-CSF antibody.

[0013] In certain embodiments, the BP is not conjugated to an N-glycan on the Fc region of the antibody. In some embodiments, the BP is site-specifically conjugated to the antibody using pClick conjugation, NHS-ester chemistry, or cysteine ​​chemistry. In certain embodiments, the BP is site-specifically conjugated to the antibody using pClick conjugation. In some embodiments, the BP is conjugated to the CH2-CH3 junction of the antibody. In some embodiments, the BP is conjugated to the antibody using 4-fluorophenylcarbamate ridine (FPheK). In certain embodiments, the FPheK is bound to a fragment of the B domain of protein A from Staphylococcus aureus (FB protein). In certain embodiments, the pClick conjugation involves the conjugation of an antibody containing an azide functional moiety to a BP functionalized with bicyclo[6.1.0]nonyne (BCN).

[0014] In certain embodiments, the bone-targeting conjugates result in an increased concentration of the therapeutic antibody in the bone tumor niche, inhibiting cancer development in bone and / or limiting secondary metastasis to other organs, hi some embodiments, the bone-targeting conjugates result in a reduction in micrometastasis-induced osteolytic lesions.

[0015] In additional 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. In certain aspects, the additional anti-cancer therapy comprises immunotherapy or chemotherapy.

[0016] A further embodiment provides the use of a bone-targeting conjugate comprising a bisphosphonate (BP) conjugated to an antibody for the treatment or prevention of a bone tumor in a subject with cancer.

[0017] In some embodiments, the subject has bone cancer or bone metastases. In particular 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 certain embodiments, the bone metastases are breast cancer bone metastases. In some embodiments, the breast cancer is triple negative breast cancer. In certain embodiments, the breast cancer is HER2 negative breast cancer. In other embodiments, the breast cancer is HER2 positive breast cancer.

[0018] In certain embodiments, the BP is negatively charged. In some embodiments, the BP is alendronate, zoledronate, pamidronate, risedronate, medronic acid, aminomethylenebisphonic acid, clodronate, etidronate, tiludronate, ibandronate, pomidronate, neridonate, olpadronate, or oxidronate. In certain embodiments, the BP is alendronate (ALN).

[0019] In some embodiments, 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 embodiments, the antibody is an immune checkpoint inhibitor. In certain 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-15 antibody, an anti-RANKL antibody, or an anti-TGFβ antibody. In certain embodiments, the antibody is an anti-HER2 antibody, such as trastuzumab (Herceptin), pertuzumab (Perjeta), or atezolizumab. In certain embodiments, the antibody is trastuzumab. In some embodiments, the bone-targeting conjugate comprises alendronate conjugated to trastuzumab. In certain embodiments, the antibody is not an anti-M-CSF antibody.

[0020] In certain embodiments, the BP is not conjugated to an N-glycan on the Fc region of the antibody. In some embodiments, the BP is site-specifically conjugated to the antibody using pClick conjugation, NHS-ester chemistry, or cysteine ​​chemistry. In certain embodiments, the BP is site-specifically conjugated to the antibody using pClick conjugation. In some embodiments, the BP is conjugated to the CH2-CH3 junction of the antibody. In some embodiments, the BP is conjugated to the antibody using 4-fluorophenylcarbamate ridine (FPheK). In certain embodiments, the FPheK is attached to a fragment of the B domain of protein A from Staphylococcus aureus (FB protein). In certain embodiments, the pClick conjugation involves the conjugation of an antibody containing an azide functional moiety to a BP functionalized with bicyclo[6.1.0]nonyne (BCN).

[0021] In certain embodiments, the bone-targeting conjugates result in an increased concentration of the therapeutic antibody in the bone tumor niche, inhibiting cancer development in bone and / or limiting secondary metastasis to other organs, hi some embodiments, the bone-targeting conjugates result in a reduction in micrometastasis-induced osteolytic lesions.

[0022] In additional 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. In certain aspects, the additional anti-cancer therapy comprises immunotherapy or chemotherapy.

[0023] 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.

[0024] 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]

[0025] 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.

[0026] [Figure 1]Figure 1A-L: (A) Therapeutic antibodies can be delivered site-specifically to bone by pClick conjugation of bisphosphonate molecules that bind to bone hydroxyapatite matrix. (B) SDS-PAGE analysis of Tras, Tras-ALN, and their near-infrared (NIR) fluorophore conjugates under reducing and non-reducing conditions, visualized by Coomassie blue staining (left) and a fluorescent scanner (right). (C) Mass spectrometry analysis of Tras and Tras-ALN. (D) Flow cytometry profiles of Tras and Tras-ALN binding to BT474 (HER2+++), SK-BR-3 (HER2+++), MDA-MB-361 (HER2++), and MDA-MB-468 (HER2) cells. (E-G) In vitro cytotoxicity of Tras and Tras-ALN against BT474, MDA-MB-361, and MDA-MB-468 cells. (H) Differential bone targeting ability of unmodified Tras and Tras-ALN conjugates. Non-demineralized bone sections from C57 / BL6 mice were incubated overnight with 50 μg / mL Tras or Tras-ALN, followed by staining with fluorescein isothiocyanate (FITC)-labeled anti-human IgG and 4 μg / mL xylenol orange (XO, known to label bone) (scale bar, 200 μm). (I-J) Binding kinetics of Tras and Tras-ALN to hydroxyapatite (HA) and native bone. (K) Ex vivo fluorescence images of the lower limbs of athymic nude mice bearing MDA-MB-361 tumors 24 h, 96 h, or 168 h after retro-orbital injection of Cy7.5-labeled Tras and Tras-ALN. The right limbs of nude mice were inoculated with tumor cells via IIA injection. (L) Non-demineralized bone sections from the biodistribution study were stained with FITC-labeled anti-human IgG (green), RFP (red), and DAPI (blue) (scale bar, 100 μm). [Diagram 2]Figure 2A-N: Tras-ALN inhibits breast cancer micrometastases in bone. (A) Nude mice were IIA-injected with MDA-MB-361 cells into the right hind limb and subsequently treated with PBS, ALN (10 μg / kg retro-orbital sinus in PBS, twice weekly), Tras (1 mg / kg retro-orbital sinus in sterile PBS, twice weekly), and Tras-ALN conjugate (same as Tras). Tumor burden was monitored by weekly bioluminescence imaging. (B) Fold change in mean luminescence intensity of MDA-MB-361 tumors in mice treated as described in (A), two-way ANOVA comparing Tras with Tras-ALN. (C) Fold change in individual luminescence intensity of HER2-positive MDA-MB-361 tumors in mice treated as described in (A). (D) Kaplan-Meier plot of time to euthanasia for mice treated as described in (A). The end point was considered when the whole body BLI signal of each individual mouse reached 107 photons-sec-1. (E) Body weight change of tumor-bearing mice over time. (F) MicroCT scans in supine position of PBS, ALN, Tras, or Tras-ALN treated groups 82 days after tumor implantation. (G) Quantitative analysis of bone volume density (BV / TV). (H) Quantitative analysis of trabecular thickness (Tb.Th). (I) Quantitative analysis of trabecular bone mineral density (BMD). (J) Representative longitudinal sagittal plane hematoxylin and eosin (H&E) stained sections of tibia / femur from each group. T: tumor; B: bone; BM: bone marrow. (K) Representative images of HER2 and TRAP staining of bone sections from each group. (L) Number of osteoclasts per image calculated at the tumor-bone interface of each group (pink cells in (K) were considered as osteoclast positive cells). (M) Serum TRAcP 5b levels in mice treated as described in (A). (N) Serum calcium levels in mice treated as described in (A). ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, and ns=P>0.05. [Diagram 3]3A-C: (A) Secondary metastases observed in different organs of mice treated with Tras (top) or Tras-ALN (bottom). (B) Pie charts show the frequency of metastases observed in different organs of mice treated with Tras (1 mg / kg retro-orbital in sterile PBS, twice a week) and Tras-ALN conjugate (same as Tras). (C) Quantification of bioluminescence signal intensity in different organs, including other bones, measuring metastases arising from Tras and Tras-ALN treated mice. p values ​​are based on one-way ANOVA. *P<0.05 and ns=P>0.05. [Figure 4] Figure 4A-E: In vivo comparison of Tras and Tras-ALN in a HER2-negative model. (A) Tumor burden was monitored weekly by bioluminescence imaging and (B) quantified by radiance detected in regions of interest. (C) Fold change in individual luminescence intensity of HER2-negative MCF-7 tumors in mice treated as described in (A). (D) Kaplan-Meier plot of time to killing of mice treated as described in (A). The end point was considered to be when the whole body BLI signal of each individual mouse reached 107 photons sec-1. (E) Body weight change of tumor-bearing mice over time. ****P<0.0001, *P<0.05, and ns=P>0.05. [Diagram 5] ESI-MS spectrum of BCN-ALN. [Figure 6] ESI-MS spectrum of ssFB-FPheK. [Figure 7] ESI-MS spectrum of Tras-azide. [Figure 8] Figure 8A-H: Tras binding to BT474 cells. BT474 cells were incubated with increasing concentrations of Tras, treated as described in the methods, and fluorescence was measured on a flow cytometer. KD was determined as follows: 1 / F = 1 / Fmax + (KD / Fmax) (1 / [Ab]). [Figure 9]Figure 9A-H: Tras-ALN binding to BT474 cells. BT474 cells were incubated with increasing concentrations of Tras-ALN, treated as described in Methods, and fluorescence was measured on a flow cytometer. KD was determined as follows: 1 / F = 1 / Fmax + (KD / Fmax) (1 / [Ab]). [Figure 10] Figure 10A-H: Tras binding to SK-BR-3 cells. SK-BR-3 cells were incubated with increasing concentrations of Tras, treated as described in the Methods, and fluorescence was measured on a flow cytometer. KD was determined as follows: 1 / F = 1 / Fmax + (KD / Fmax) (1 / [Ab]). [Figure 11] Figure 11A-H: Tras-ALN binding to SK-BR-3 cells. SK-BR-3 cells were incubated with increasing concentrations of Tras, treated as described in Methods, and fluorescence was measured on a flow cytometer. KD was determined as follows: 1 / F = 1 / Fmax + (KD / Fmax) (1 / [Ab]). [Figure 12] Binding of Tra-ALN in BT-474, SK-BR-3, and MDA-MB-468 cells visualized by confocal microscopy. Cells were incubated with 30 nM Tras-ALN in medium at 37° C. for 30 min and stained with DilC18 (red fluorescence) and Hoechst nuclear stain (blue fluorescence). [Figure 13-1] Figures 13A-D: Ex vivo fluorescence images of major organs: heart, liver, spleen, lung, kidney, and brain of athymic nude mice bearing MDA-MB-361 tumors 96 hours after retro-orbital injection of Cy7.5-labeled (A) Tras and (B) Tras-ALN. [Figure 13-2] Figures 13A-D: Ex vivo fluorescence images of major organs (C) heart, liver, spleen, lungs, kidneys, and bones of tumor-bearing mice MDA-MB-361 tumors 72 hours after retro-orbital injection of Cy7.5-labeled Tras and Tras-ALN. [Figure 13-3]13A-D: Ex vivo fluorescence images of major organs. (D) Pharmacokinetic profile of Tras and Tras-ALN. Tumor-bearing athymic nude mice (3 months after surgery) were retro-orbitally injected with 1 mg / kg Tras and Tras-ALN in PBS. Antibody concentrations in serum were determined by ELISA (data represent the mean ± SD of three independent replicates). [Figure 14] Ex vivo fluorescence image analysis for bone biodistribution of Tras and Tras-ALN. 24, 96, or 168 hours after retro-orbital injection of Cy7.5-labeled Tras and Tras-ALN. Bones were collected and analyzed. Quantitative data were summarized from Fig. 1K. Tumor-free signals from Tras-treated mice were considered as blanks. Relative signals were calculated as follows: signal from hind limb - signal from tumor-free hind limb (from Tras-treated group). [Figure 15] Tras-ALN inhibits breast cancer micrometastasis in bone. Nude mice were IIA-injected with MDA-MB-361 cells into the right hind limb, followed by treatment with PBS, ALN (10 μg / kg in PBS, retro-orbital sinus, twice weekly), Tras (1 mg / kg in sterile PBS, retro-orbital sinus, twice weekly), and Tras-ALN conjugate (same as Tras). Tumor burden was monitored twice weekly by bioluminescence imaging (selected imaging data from days 6, 20, 33, 48, and 68 are shown in FIG. 2A). [Figure 16] Figures 16A-B: Whole-body BLI quantification. (A) BLI from each treatment group quantified by radiance detected in the whole body. (B) Individual whole-body luminescence intensity of the different treatment groups as described in Figure 2A. ****P<0.0001. [Figure 17]Figure 17A-D: BLI signals in hind paws of Tras and Tras-ALN treatment groups were quantified and shown. (A) Fold change in mean hind paw luminescence intensity in mice treated with Tras and Tras-ALN (as described in Figure 2A), 2-way ANOVA comparing Tras to Tras-ALN. (B) Fold change in individual luminescence intensity of hind paws of Tras and Tras-ALN treatment groups. (C) Mean BLI of hind paws quantified from Tras and Tras-ALN treatment groups. (D) Individual luminescence intensity of Tras and Tras-ALN treatment groups. ****P<0.0001. [Figure 18] 3D rendering of bone based on MicroCT. Cortical bone, image shows extensive cortical bone destruction. Trabecular bone, image shows trabecular destruction. Bottom panel (growth plate), image plate shows bone loss at the growth plate. [Figure 19] TRAP staining of bone sections from each group. [Figure 20] 20A-E: In vivo quantification of secondary metastases. (A) BLI signals in the whole body and hind limbs of mice from various treatment groups were quantified and shown. Secondary metastases were determined as follows: BLI signals in the whole body and hind limbs (indicated by red circles) were quantified. At each time point, animals were imaged twice a week using an IVIS Lumina II (Advanced Molecular Vision) following the recommended procedure and manufacturer settings. For groups where the signal suggested a "saturated luminescence image", scans were performed for a shorter time (indicated below the image). Secondary metastases were calculated as follows: BLI signal intensity in the whole body - BLI signal intensity in the hind limbs. (B) Fold change in the mean luminescence intensity of secondary metastases, two-way ANOVA comparing Tras with Tras-ALN, in mice treated as described in (Figure 2A). (C) Fold change in the individual luminescence intensity of secondary metastases in Tras and Tras-ALN treatment groups. (D) Quantified mean BLI of secondary metastases from Tras and Tras-ALN treatment groups. (E) Individual luminescence intensities of Tras and Tras-ALN treatment groups. ****P<0.0001. [Figure 21]Secondary metastases observed in various organs of mice treated with Tras (top) or Tras-ALN (bottom). [Figure 22] Figure 22A-C: In vivo comparison of Tras and Tras-ALN in a HER2-negative model. Nude mice were IIA-injected with MCF-7 cells into the right hind limb, followed by treatment with Tras (1 mg / kg in sterile PBS, retro-orbital sinus, twice weekly) and Tras-ALN conjugate (same as Tras). (A) Tumor burden was monitored twice weekly by bioluminescence imaging (selected imaging data from days 4, 12, 19, 29, and 42 are shown in Figure 4A). (B) BLI from each treatment group quantified by radiance detected in the whole body. (C) Individual whole body luminescence intensity of Tras and Tras-ALN treatment groups. ****P<0.0001. [Figure 23] 23A-D: BLI signals in the hind paws of MCF-7 models were quantified and shown for Tras and Tras-ALN treatment groups. (A) Fold change in mean hind paw luminescence intensity in mice treated with Tras and Tras-ALN (as described in FIG. 4A), two-way ANOVA comparing Tras with Tras-ALN. (B) Fold change in individual luminescence intensity of hind paws of Tras and Tras-ALN treatment groups. (C) Quantified hind paw mean BLI from Tras and Tras-ALN treatment groups. (D) Individual luminescence intensity of Tras and Tras-ALN treatment groups. ****P<0.0001. [Figure 24] Figures 24A-B: Effect of Tras-ALN on MCF-7 HER2 negative model: Serum TRACP 5b and calcium level analysis. (A) Serum TRACP 5b concentration in Tras and Tras-ALN at the end of the experiment (*P<0.05). (B) Serum calcium concentration in Tras and Tras-ALN groups at the end of the experiment (*P<0.05). [Diagram 25]25A-E: In vivo quantification of secondary metastases. (A) BLI signals in the whole body and hind limbs of mice from various treatment groups were quantified and shown. Secondary metastases were determined as follows: BLI signals in the whole body and hind limbs (indicated by red circles) were quantified. At each time point, animals were imaged twice a week using an IVIS Lumina II (Advanced Molecular Vision) following the recommended procedure and manufacturer settings. For groups where the signal suggested a "saturated luminescence image", scans were performed for a shorter time (indicated below the image). Secondary metastases were calculated as follows: BLI signal intensity in the whole body - BLI signal intensity in the hind limbs. (B) Fold change in the mean luminescence intensity of secondary metastases, two-way ANOVA comparing Tras with Tras-ALN, in mice treated as described in FIG. 22. (C) Fold change in the individual luminescence intensity of secondary metastases in Tras and Tras-ALN treatment groups. (D) Mean BLI of quantified secondary metastases from each treatment group. (E) Individual luminescence intensities of Tras and Tras-ALN treatment groups. ****P<0.0001. [Figure 26] Figures 26A-B: Effect of Tras-ALN on multi-organ metastasis in MCF-7 cell line. (A) Metastasis observed in various organs of mice treated with Tras or Tras-ALN. (B) Pie chart shows frequency of metastasis observed in various organs of Tras and Tras-ALN treatment groups. [Figure 27]Figure 27A-C: Therapeutic effect of Tras-ALN on mice bearing both bone metastases and primary tumors. (A) Luciferase-labeled MDA-MB-361 cells were injected into the right hind limb using IIA injection. Nonluciferase-labeled MDA-MB-361 cells were inoculated into the mammary fat pad of the same mice. Mice were then treated with PBS (n=5), Tras (retro-orbital sinus at 1 mg / kg in sterile PBS, n=7), and Tras-ALN (same as Tras, n=7). Tumor burden in the hind limbs was monitored by bioluminescence imaging (BLI). Tumor burden in the mammary fat pad was measured using a caliper. (B) Two-way ANOVA comparing hind limb tumor fold change in mean luminescence intensity in mice treated as described in (A), hind limb tumors of Tras and Tras-ALN groups. (C) Mammary fat pad tumor fold change in mean luminescence intensity in mice treated as described in (A), two-way ANOVA comparing mammary fat pad tumors in Tras and Tras-ALN groups. *P<0.05, ns indicates P>0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Description of exemplary embodiments Over the past two decades, antibody-based therapies have proven to be of great value in cancer treatment. Despite the clinical success of these biopharmaceuticals, reaching targets in the bone microenvironment has proven difficult, likely due to the relatively low vascularization of bone tissue and the presence of physical barriers that impair drug penetration. Attempts to ensure effective concentrations of therapeutic drugs in bone inevitably result in high concentrations in other tissues as well, often resulting in adverse systemic or side effects that may limit or eliminate the use of the drug. 24、25 In this case, the potential benefits of passive targeting are lost.

[0028] Thus, in certain embodiments, the present disclosure provides an innovative bone targeting (BonTarg) technology that allows for the specific delivery of therapeutic polypeptides, such as antibodies, to bone via the conjugation of bone targeting moieties. The resulting bone targeting antibodies can also specifically target the bone metastatic niche to eliminate bone micrometastases and prevent the seeding of multi-organ metastases from bone lesions. Taking advantage of the high mineral concentration inherent to the bone hydroxyapatite matrix, bisphosphonate (BP) conjugation has been used for the selective delivery of small molecule drugs, imaging probes, nuclear pharmaceuticals, and nanoparticles to bone as a means of treating osteoporosis, primary and metastatic bone neoplasms, and other bone disorders. 24、26~30 Negatively charged BPs have a high affinity for mineralized, positively charged bone matrix, such as hydroxyapatite (HA), the major component of hard bone, resulting in preferential binding to bone. Thus, in certain embodiments, the method involves site-specifically conjugating a BP drug, such as alendronate (ALN), to an antibody, such as the HER2-targeting monoclonal antibody trastuzumab (Tras), using pClick conjugation technology. 31 This study demonstrated that in two xenograft models based on intra-iliac artery (IIA) injection, the resulting trastuzumab-alendronate conjugate (Tras-ALN) significantly enhanced the concentration of therapeutic antibodies in the bone metastatic niche, inhibited cancer development in bone, and limited secondary metastasis to other organs. This type of specific delivery of therapeutic antibodies to bone has the potential to improve both the breadth and efficacy of antibody therapy for bone-related diseases.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] "Subject" and "patient" refer to either humans or non-humans, such as primates, mammals, and vertebrates. In certain embodiments, the subject is a human.

[0036] 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.

[0037] II. Bone-targeting antibody conjugates The present disclosure relates to the conjugation of bone-targeting moieties, such as bisphosphonates, to antibodies. In some embodiments, the bone-targeting agent can be conjugated to the antibody at a site remote from the antigen binding site and the Fc receptor binding site, such as the CH2-CH3 junction.

[0038] Bisphosphonates are synthetic compounds that contain two phosphonate groups attached to a central (geminal) carbon (PCP backbone) and are used to prevent bone resorption in several metabolic and tumor-induced bone diseases, including multiple myeloma. Bisphosphonate treatment has been associated with increased patient survival, indicating that these compounds have a direct effect on tumor cells. Bisphosphonates may contain two additional chains attached to the central geminal carbon. The presence of these two side chains allows for a large number of substitutions to the bisphosphonate backbone, thus allowing the development of a variety of analogs with different pharmacological properties. Exemplary bisphosphonates include, but are not limited to, alendronate, zoledronate, pamidronate, risedronate, medronate, aminomethylenebisphonic acid, clodronate, etidronate, tilundronate, or ibandronate.

[0039] In certain embodiments, bone-targeting agents can be conjugated to antibodies by site-specific conjugation methods. In one method, antibodies are conjugated by cysteine ​​chemistry, which includes engineered cysteine ​​substitutions at positions on the light and heavy chains that provide reactive thiol groups and do not interfere with immunoglobulin folding and assembly or alter antigen binding (Junutula et al., 2008, incorporated herein by reference in its entirety). In another method, the conjugation method involves the site-specific introduction of aldehyde groups into recombinant proteins using a six-amino acid consensus sequence recognized by formylglycine-generating enzyme (Carrico et al., 2007, incorporated herein by reference in its entirety). This genetically encoded "aldehyde tag" is no more than a His6 tag and can be utilized for a number of protein labeling applications. In some embodiments, the site-specific conjugation method involves remodeling the Fc N-glycan of the antibody using mutant glycosyltransferases, such as mutant beta 1,4-galactosyltransferases (Boeggeman et al., 2009, incorporated herein by reference in its entirety) or transglutaminase-mediated site-specific conjugation. In certain embodiments, the site-specific conjugation involves the use of disulfide bridges (Zhang et al., 2016, incorporated herein by reference in its entirety). In certain embodiments, the site-specific conjugation method involves the incorporation of non-canonical amino acids (Leisle et al., 2015, incorporated herein by reference in its entirety). For example, bone-targeting agents can be conjugated to antibodies using pClick technology, which involves proximity-induced site-specific conjugation using affinity compounds (WO2019 / 217900, incorporated herein by reference in its entirety). pClick is a site-specific technology that does not require antibody engineering and any chemical or enzymatic treatment. The pClick method can allow for site-specific covalent bond formation between the bone-targeting moiety and the antibody. In certain embodiments, the bone-targeting antibody conjugate does not include a polymer backbone.In certain embodiments, the bone-targeting moiety is not conjugated to an N-glycan of the Fc domain of the antibody.

[0040] Specifically, the method may involve proximity-induced reactivity between an ncAA and a nearby antibody residue, such as a lysine or cysteine. pClick may enable covalent bond formation between a bone-targeting moiety and a defined residue, such as a lysine, of an antibody without performing antibody engineering.

[0041] The conjugated antibody may be further conjugated to an imaging or diagnostic agent.

[0042] 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.

[0043] 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.

[0044] The therapeutic or diagnostic agent can be a small molecule, peptide, protein, polypeptide, antibody, antibody fragment, DNA, or RNA.

[0045] A. Formulation and Administration The present disclosure provides pharmaceutical compositions comprising an antibody conjugated to a bone-targeting agent such as a bisphosphonate. 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 the 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] B. Hyperproliferative disorders Hyperproliferative diseases 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 apoptotic 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 antibody conjugates can be used to treat various types of cancer, such as bone cancer and cancer that metastasizes to bone.

[0052] 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. ;

[0053] C. Treatment Methods In particular, compositions are disclosed herein that can be used to treat cancer in subjects (e.g., human subjects). The compositions are preferably administered to mammals (e.g., rodents, humans, non-human primates, dogs, cows, sheep, horses, cats, etc.) in effective amounts, i.e., amounts capable of producing the desired results 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 specific composition administered, the time and route of administration, general health, clinical symptoms of infection or cancer, and other drugs administered simultaneously. The compositions described herein are typically administered at dosages that inhibit bacterial cell growth or proliferation, inhibit biofilm growth, or induce cancerous cell death (e.g., induce apoptosis of cancer cells), as assayed by hematological parameters (complete blood count (CBC)) or by identifying a reduction in cancer cell growth or proliferation.

[0054] The therapeutic methods (including prophylactic treatments) of the present disclosure generally involve 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 preferably 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.

[0055] 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.

[0056] D. Additional Therapies In certain embodiments, the compositions and methods of the present invention include bone-targeting antibody conjugates 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.

[0057] In certain embodiments, the compositions and methods of the present invention include a bone-targeting antibody conjugate 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] Various combinations can be used. In the following example, the bone-targeting antibody conjugate is "A" and the anti-cancer therapy is "B": TIFF2024503480000001.tif18128

[0064] 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.

[0065] 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.

[0066] 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;

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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).

[0080] 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.

[0081] 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).

[0082] 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.

[0083] 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.

[0084] 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, conjugated 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, which are containers that will not react with the components of the kit. The containers may be made of sterilizable materials, such as plastic or glass.

[0085] 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

[0086] 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.

[0087] Example 1 - Antibodies for bone metastases Development of the first bone-targeting antibody using BonTarg To investigate the possibility of specifically delivering therapeutic antibodies to bone via conjugation to BP molecules, a model was designed using the HER2-targeting antibody trastuzumab (Tras) and the BP drug alendronate (ALN). ALN is a second-generation BP drug used as a bone-targeting agent and as a regimen to treat osteoporosis and bone metastases. 31 To ensure that ALN conjugation does not impair the therapeutic efficacy of the antibody, we used a novel proximity-triggered antibody conjugation strategy called pClick. 31 pClick technology allows site-specific conjugation of payloads to native antibodies under mild conditions, thus minimizing disruption of binding to antigen receptors or FcγRIII receptors, the receptors responsible for activating antibody-dependent cell-mediated cytotoxicity (ADCC). pClick technology does not rely on antibody engineering, nor on UV / chemical / enzymatic treatments that characterize the generation of most therapeutic antibodies. To prepare trastuzumab-alendronate conjugates (Tras-ALN), pClick was used to generate Tras containing an azide functional moiety, which was subsequently reacted with bicyclo[6.1.0]nonyne (BCN)-functionalized ALN (Figure 1A, and Figures 5-8). The resulting Tras-ALN was further purified on a desalting column and fully characterized by SDS-PAGE and ESI-MS (Figure 1B, Figure 1C). No unconjugated heavy chain or degradation products were evident on SDS-PAGE, indicating a coupling efficiency of over 95%. ESI-MS analysis also revealed that more than 95% of the heavy chains were conjugated to ALN molecules.

[0088] Antibody conjugation to ALN retains antigen binding and specificity To investigate the effect of ALN conjugation on antigen binding affinity and specificity, the binding affinity of Tras and Tras-ALN was evaluated by flow cytometry analysis of HER2-positive and HER2-negative cell lines. Figure 1D reveals that both Tras and Tras-ALN have strong binding affinity to the HER2-expressing cell lines BT474, SK-BR-3, and MDA-MB-361, but not to the HER2-negative cell line MDA-MB-468, suggesting that antibody specificity was not altered by ALN conjugation (Table 1). The Kd values ​​for binding to HER2-positive cells were in a similar range for Tras and Tras-ALN (BT474, 3.0 vs. 3.8 nM; SK-BR-3, 2.3 vs. 3.0 nM, respectively), indicating that ALN conjugation does not affect the strength of antigen binding (Figures 8-11). Confocal fluorescence imaging further confirms that Tras-ALN retains antigen binding and specificity (Figure 12). HER2-positive BT474 and SK-BR-3 cells, and HER2-negative MDA-MB-468 cells were incubated with fluorescein isothiocyanate (FITC)-labeled Tras-ALN for 30 minutes. Confocal imaging shows that cell surface-associated fluorescence is only exhibited by HER2-positive BT474 and SK-BR-3 cells, but not by HER2-negative MDA-MB-468 cells (Figure 12). Thus, ALN modification of Tras does not affect its antigen-binding affinity and specificity. Next, the Tras-ALN conjugate was tested for selective cytotoxicity against HER2-expressing and HER2-negative breast cancer cells. As shown in Figure 1E, Figure 1F, Figure 1G, and Table 1, the Tras-ALN conjugate inhibited HER2-positive BT-474 cells (EC of 2.3 ± 0.7 μg / ml). 50 ) and MDA-MB-361 (EC of 78 ± 21 μg / ml 50 ) and showed cytotoxic activity against Tras (EC of 1.4 ± 0.9 μg / ml). 50 and an EC of 57 ± 10 μg / ml. 50 ) were indistinguishable from those of HER2-negative MDA-MB-468 cells. 50>500 μg / ml). These results demonstrate that conjugation of the negatively charged moiety ALN maintains the antigen binding and in vitro antitumor cell activity of the Tras antibody.

[0089] Enhanced bone metastatic niche targeting by Tras-ALN in vitro and in vivo Next, we investigated the ability of the Tras-ALN conjugate to target bone tissue. Non-demineralized bone sections from C57BL / 6 mice were incubated with 50 μg / mL of Tras or Tras-ALN conjugates overnight at 4 °C, followed by labeling with FITC-conjugated anti-human IgG. These bone sections were further stained with 4 μg / mL of xylenol orange (XO, known to label bone) for 30 min before imaging via confocal laser scanning microscopy. FITC signal was observed in sections stained with the Tras-ALN conjugates, but not in sections stained with unmodified Tras (Figure 1H). Furthermore, the localization of the Tras-ALN signal correlated well with the XO signal, confirming the specific targeting of bone by Tras-ALN. To quantify the difference in affinity between the binding of the Tras-ALN conjugates and that of unmodified Tras, Tras-ALN and Tras were incubated with hydroxyapatite or natural bone. As shown in Figure 1I and Figure 1J, unmodified Tras exhibited only weak binding to HA or native bone. Even with increasing incubation time, the binding affinity of Tras did not change significantly. In contrast, approximately 80%-90% of Tras-ALN bound to HA and native bone after 2 and 10 hours, respectively.

[0090] Encouraged by the in vitro bone targeting ability of ALN-conjugated Tras, in vivo biodistribution studies with Tras-ALN conjugates were performed using tumor xenograft models. To facilitate in vivo antibody detection, Tras and Tras-ALN were first conjugated with cyanine 7.5 (Cy7.5)-hydroxysuccinimide (NHS) ester. The resulting Cy7.5-labeled conjugates were analyzed using SDS-PAGE. As expected, fluorescence was associated only with the Cy7.5-labeled conjugates (Figure 1B). An important feature of BPs is that the uptake of bisphosphonates into bone metastases is much higher than that in healthy bone tissue due to the relatively low pH of the bone metastatic microenvironment. 32~35 To investigate whether ALN-Tras can specifically target bone metastases and therefore minimize on-target toxicity to normal bone tissue, the targeting properties of ALN-Tras were evaluated in a bone tumor model. A bone micrometastasis model was created by using intra-internal iliac artery (IIA) injection of luciferase and red fluorescent protein (RFP)-labeled MDA-MB-361 cells into the right hind limb of nude mice. IIA injection is a novel technique recently developed to establish bone micrometastases. The method allows the selective delivery of cancer cells to the hind limb bones without causing tissue damage. 36~38This technique allows sufficient time for some low-malignant cells to eventually colonize bone and for a large number of cancer cells to specifically colonize bone, thereby enriching micrometastases at an early stage. This allows for rapid detection and robust quantification of micrometastases. Following establishment of micrometastases, they were treated with Tras or Tras-ALN (1 mg / kg). 24, 96, or 168 hours after administration of the antibody or antibody conjugate, major organs including heart, liver, spleen, kidney, lung, and bone were removed and analyzed using a Caliper IVIS Lumina II imager (Figure 1K and Figure 13). Notably, ex vivo fluorescence images 96 hours after injection of the antibody confirmed a clear accumulation of Cy7.5-labeled Tras-ALN in bone compared to Cy7.5-labeled Tras (Figure 1K and Figure 14). Moreover, uptake of Tras-ALN in cancer-bearing bone was significantly higher than that in healthy bone tissue. This is consistent with previous observations that BP molecules preferentially bind to bone matrix in acidic tumor environments. 39 In a separate study, unlabeled Tras-ALN (1 mg / kg) was administered to nude mice bearing MDA-MB-361 tumors in the right hind leg. Bone sections from this study were also stained with FITC-labeled anti-human IgG, RFP, and DAPI. FITC signal was observed only in sections from the right leg bearing MDA-MB-361 tumors. No FITC signal was detected in the tumor-free left leg (Figure 1L). Notably, the FITC signal correlated well with the red fluorescence of MDA-MB-361 cells, suggesting that the Tras-ALN conjugate selectively targets bone metastatic sites, but not healthy bone. These results demonstrate that ALN conjugation can significantly improve the delivery and concentration of therapeutic antibodies at bone metastatic sites.

[0091] Next, the effect of ALN conjugation on antibody pharmacokinetics and FcRn binding was evaluated. A single dose of 1 mg / kg Tras and Tras-ALN in PBS was injected retro-orbitally, and serum was collected periodically for 7 days and analyzed by Trastuzumab ELISA kit. The serum concentrations of both Tras and Tras-ALN decreased and showed no significant difference (Figure 13). Next, the effect of ALN conjugation on FcRn binding was determined. It was found that ALN conjugation had no obvious effect on FcRn binding at pH 6.0 (Table 4).

[0092] Improving the therapeutic efficacy of Tras-ALN for bone micrometastases To determine whether bone-targeted trastuzumab represents a novel therapeutic approach for treating breast cancer micrometastases in bone, xenograft studies were performed in nude mice. Using intra-iliac artery (IIA) injection, 5 × 10 firefly luciferase-labeled trastuzumab were injected into the right hind limb of nude mice. 5MDA-MB-361 cells were inoculated into the mice. Five days after IIA injection, the mice were treated with phosphate-buffered saline (PBS), ALN (10 μg / kg), Tras (1 mg / kg), or Tras-ALN (1 mg / kg) via retro-orbital injection. As shown in Figure 2A and Figure 15, micrometastases in PBS- and ALN-treated mice accumulated rapidly, whereas lesion development in Tras- and Tras-ALN-treated mice was delayed. Whole-body bioluminescence imaging (BLI) signals suggested that treatment with Tras-ALN caused a more pronounced inhibition of micrometastasis progression compared to that seen in Tras-treated mice (Figures 16A and 16B). The increase in BLI from day 6 to day 87 showed that the Tras-ALN treatment group had a smaller fold increase in tumor size compared to the Tras treatment group (Tras vs. Tras-ALN: 1965.1±798.3 vs. 42.6±23.4, FIG. 2B and FIG. 2C). As bone metastases were established in the hind limbs, the effect of Tras-ALN on the BLI signal in the hind limbs was also quantified. Similar to the whole-body BLI signal, the Tras-ALN treatment group had a lower BLI signal intensity and a smaller fold increase in the hind limbs (FIG. 17). Furthermore, the survival rate of Tras-ALN-treated mice was significantly improved compared to that of PBS, ALN, and Tras-treated mice, demonstrating the effectiveness of Tras-ALN against HER2-positive cells in vivo (FIG. 2D). Furthermore, weight loss as a sign of ill health was not observed in any of the treated mice, suggesting the absence of toxicity associated with bone-targeting antibodies (FIG. 2E).

[0093] These results were further confirmed by microcomputed tomography (microCT) data and histology, which highlight the finding that bone-targeting antibodies can reduce both the number and extent of osteolytic lesions. As shown in Figure 2F and Figure 18, femurs from the PBS, ALN, and Tras treatment groups exhibited a significant reduction in bone mass, whereas bone loss in the Tras-ALN treatment group was significantly reduced. Quantitative analysis showed that compared to the Tras treatment group, the Tras-ALN treatment group had significantly higher bone volume (Figure 2G, 6B: BV / TV (%), 35.08 ± 2.65 vs. 56.67 ± 1.02, p = 0.0005), thicker trabecular bone (Figure 2H, Tb.Th (mm), 0.061 ± 0.003 vs. 0.094 ± 0.002, p = 0.003), and higher trabecular bone mineral density (Figure 2I, BMD (mg / mm 3 ), 101.16±12.24 vs 165.94±12.84, p=0.035).

[0094] Tumor size was also analyzed by histometric analysis of bone sections. Tibias and femurs from the PBS- and ALN-treated groups had high tumor burden (Figure 2J). Tras treatment slightly reduced tumor burden, but the reduction was not statistically significant. In contrast, a significant reduction in tumor burden was observed in the Tras-ALN-treated group. Generally, bone matrix is ​​destroyed in bones with high tumor burden, but bones with less tumor burden in the Tras-ALN-treated group exhibited intact bone matrix, as revealed by histological examination of bone samples from various treatment groups. Reduction in tumor burden was also confirmed by HER2 immunohistochemistry (IHC). As shown in Figure 2K, the number of HER2-positive breast cancer cells was dramatically reduced in Tras-ALN-treated mice, even though HER2 expression by individual tumor cells was unchanged. This suggests that long-term treatment with Tras-ALN has no effect on HER2 expression by MDA-MB-361 cells.

[0095] To examine Tras-ALN inhibition of tumor-induced osteolytic bone destruction, we examined bone-resorbing, tartrate-resistant, acid phosphatase-positive multinucleated osteoclasts in bone samples (Figure 2K). Tartrate-resistant acid phosphatase (TRAP) staining identified a reduced number of osteoclasts (pink cells) enveloping eroded bone surfaces in Tras-ALN-treated mice compared with Tras-treated mice (Figures 2K, 2L, and 19). Serum TRAcP 5b and calcium levels, indicators of bone resorption, were also measured at the experimental endpoint. A significantly greater reduction in bone resorption was observed in the Tras-ALN-treated group (Figures 2M and 2N). Taken together, these results indicate that bisphosphonate modification of a therapeutic antibody significantly improved its ability to delay the development of micrometastasis-induced osteolytic lesions (Table 2).

[0096] To further evaluate the therapeutic efficacy of Tras-ALN in the presence of both primary and secondary tumors, xenograft studies were performed in nude mice using both mammary fat pad and IIA injections. Cells inoculated into the right hind limb contained luciferase-labeled MDA-MB-361 cells (2 × 10 5 For mammary fat pad injections, unlabeled MDA-MB-361 cells (1 × 10 6 ) were injected into the mice. Six days after injection, the mice were treated with Tras (1 mg / kg) and Tras-ALN (1 mg / kg). Primary tumor progression and bone metastasis were monitored by tumor size measurement and bioluminescence, respectively. Compared with the Tras treatment group, Tras-ALN had a remarkable effect on preventing tumor growth in the hind limbs (Figure 27A-B). However, there was no significant growth difference in the mammary fat pad tumor (Figure 27C). These results suggested that compared with wild-type Tras, Tras-ALN has a better therapeutic effect on bone metastasis but a similar effect on primary tumors.

[0097] Tras-ALN inhibits multiple organ metastasis from bone lesions In more than two-thirds of cases, bone metastases are not limited to the skeleton but rather lead to subsequent metastases to other organs 9、10To investigate early bone colonization, IIA injections were used, but as these bone lesions progress over 8–12 weeks, metastases begin to appear in other organs, including additional bones, lung, liver, kidney, and brain. Therefore, we investigated the ability of Tras-ALN to reduce metastasis of HER2-positive MDA-MB-361 cancer cells to other organs. As before, 5 × 10 5 MDA-MB-361 cells were introduced into the right hind limb of nude mice via IIA injection, followed by treatment with Tras (1 mg / kg) and Tras-ALN (1 mg / kg). Mice were then subjected to whole-body BLI twice a week after tumor cell injection. Whole-body and hind limb BLI signals were quantified and shown in Figure 20A. Secondary metastasis in various organs was calculated as follows: BLI signal in whole body - BLI signal in hind limb. As shown in Figure 20, in the Tras-treated group, 10 6 Photon second -1 There was a time-dependent increase in organ BLI signals up to 10 min. There was also a significant inhibition in BLI signal accumulation in the organs of the Tras-ALN treatment group (P<0.0001). At the study endpoint, mice were euthanized and organs were harvested for bioluminescence imaging. Much higher levels of right hind limb (100%), heart (20%), liver (80%), spleen (40%), lung (60%), kidney (60%), and brain metastasis (40%) were observed in the Tras treatment group compared with right hind limb (42.9%) and liver (14.3%, Figure 3A, Figure 3B, and Figure 21) in the Tras-ALN group. There was no metastasis in other organs such as lung, spleen, kidney, and brain in Tras-ALN treated mice. Our data showed that the bone-targeted antibody can significantly inhibit multi-organ metastasis resulting from the spread of early bone micrometastases compared with the unmodified antibody. Mice treated with Tras-ALN exhibited fewer metastases to other organs than mice in other treatment groups, establishing the ability of the bone-targeted antibody to inhibit "recurrent seeding of metastases."

[0098] Table 1. Efficacy and cell surface reactivity of Tras and Tras-ALN against breast cancer epithelial cell lines. TIFF2024503480000002.tif58143Abbreviations: MFI, median fluorescence intensity. Binding was determined as the mean fold increase in median fluorescence over PBS control.

[0099] Table 2. Comparison of different treatment groups across multiple assays (MDA-MB-361 model) TIFF2024503480000003.tif43170Abbreviations: ANOVA, analysis of variance; BLI, bioluminescence imaging; TRAP, tartrate-resistant acid phosphatase. a Whole-body BLI signal intensity over the course of the experiment. b Signal intensity fold increase of BLI after treatment (BLI on day 87 / BLI on day 6). C Osteoclast number measurements from TRAP stained tibia / femur 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 to have reached the endpoint. a、b were statistically analyzed 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.

[0100] Table 3 Comparison of Tras and Tras-ALN groups in multiple assays (MCF7 model) TIFF2024503480000004.tif19158Abbreviations: ANOVA, analysis of variance; BLI, bioluminescence imaging; TRAP, tartrate-resistant acid phosphatase. a Whole-body BLI signal intensity over the course of the experiment. b Signal intensity fold increase of BLI after treatment (BLI on day 68 / BLI on day 6). cSerum calcium concentrations at the end of the experiment. d Serum TRACP 5b concentrations at the end of the experiment. e Weight progression over the course of the experiment. f 10 7 A mouse BLI intensity of > 100 was considered to have reached 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 were analyzed by using one-way analysis of variance followed by Tukey's multiple comparison test. f were analyzed by using the log-rank test. ****P<0.0001, *P<0.05, NS indicates P>0.05.

[0101] Table 4. Binding to human FcRn at pH=6 TIFF2024503480000005.tif35128

[0102] Improved therapeutic efficacy of Tras-ALN in HER2-negative models Nonetheless, previous reports have shown that a substantial portion of the minimal residual disease seen in HER2-negative patients can be attributed to HER2 signaling. 40、41 It has also been reported that HER2 signaling can mediate stem cell properties in a subpopulation of HER2-negative cells, raising the possibility that anti-HER2 therapy may be capable of eradicating bone metastases from both HER2-positive and HER2-negative breast cancers. 42 Therefore, we evaluated the therapeutic effect of Tras-ALN using breast cancer cells that are not HER2 positive but exhibit HER2 upregulation specifically in bone. We used intrailiac artery (IIA) injection to deliver MCF-7 (HER2-, ER+) cancer cells to the hindlimb bones. 36、38, followed by treatment with Tras or Tras-ALN (7 mice per group, 1 mg / kg). Mice were imaged twice weekly and signal intensity was quantified in the whole body and hind limbs. As shown in Figure 4, Figure 22, and Figure 23, treatment with Tras-ALN resulted in a more significant inhibition of tumor growth than seen in Tras-treated mice, demonstrating the efficacy of Tras-ALN against HER2-negative cells in vivo (p<0.005). Meanwhile, a significant reduction in serum TRACP 5b (4.41±1.12 U / L, p<0.05) and serum calcium (10.36±0.53 mg / dL, p<0.05) levels was observed in the Tras-ALN treatment group (Figure 24). Similar to the HER2+ model, secondary metastases in various organs also exhibited a significant reduction (P<0.0001) in BLI signal over the course of the study (Figure 25). Next, the ability of Tras-ALN to inhibit multi-organ metastasis from bone lesions was also evaluated ex vivo. On day 68, metastatic cells were observed in the right hind limb (83.4%), liver (33.4%), lung (83.4%), and brain (66.7%) in the Tras-treated group, compared with the values ​​found in the right hind limb (50%), lung (50%), and brain (50%, FIG. 26) of Tras-ALN-treated mice. These data suggest that bone-targeted Tras-ALN conjugates may be useful in preventing the progression of HER2-negative bone micrometastases to overt bone metastases, as well as blocking secondary metastasis of HER2-negative cells to other organs (Table 3).

[0103] Thus, it has been shown that conjugation of bone-targeting moieties can be used to develop innovative bone-targeting (BonTarg) technologies that allow the preparation of antibodies with both antigen and bone specificity. Data show that modification of the therapeutic HER2 antibody trastuzumab (Tras) with the bone-targeting bisphosphonate molecule alendronate (ALN) results in improved conjugate localization within the bone metastatic niche compared to other tissues, raising the intriguing possibility that bone-targeted antibodies may offer improved targeted therapy to patients with bone metastases. The affinity of ALN for bone tissue helps to overcome physical and biological barriers in the bone microenvironment that impede the delivery of therapeutic antibodies, thereby enriching and retaining Tras in bone. Tras-ALN conjugates can reach higher concentrations in the bone metastatic niche compared to healthy bone tissue due to the low pH of the bone tumor site. 50 .

[0104] Through the use of site-specific modification with bone-targeting moieties, the BonTarg technology represents an innovative platform for the specific delivery of therapeutic antibodies to bone metastatic sites. The resulting bone-targeted antibodies exhibit improved in vivo therapeutic efficacy in the treatment of breast cancer micrometastases and in the prevention of secondary metastatic spread from the initial bone lesion. This type of precise delivery of biological drugs to the bone niche represents a promising route for treating bone-related diseases. The improved therapeutic profile of our bone-targeted HER2 antibody in the treatment of microscopic BCa bone metastases will herald the extension of the BonTarg strategy to the treatment of other metastatic cancers and bone diseases.

[0105] Example 2 - Materials and Methods Construction of Tras-ALN conjugate The non-canonical amino acid azido-Lys was incorporated at the C-terminus of the ssFB-FPheK peptide via solid-phase peptide synthesis (Figure 6). After HPLC purification, the peptide was denatured with 6 M urea and dialyzed stepwise to remove the urea and allow the peptide to refold. After buffer exchange into PBS (pH 8.5), 32 equivalents of ssFB-azido peptide were co-incubated with Tras (BS046D from Syd labs) in PBS (pH 8.5) buffer at 37 °C for 2 days. The Tras-azido conjugate was then purified via a PD-10 desalting column to remove excess ssFB-azido. The Tras-azido conjugate was characterized by ESI-MS. ESI-MS: expected 53564, observed: 53558 (Figure 7). 10 equivalents of BCN-ALN were added to the solution overnight at room temperature to selectively react with the azide groups on the conjugate. Finally, the ALN-labeled antibody conjugate was purified through a PD-10 desalting column to remove excess ALN-BCN. The conjugate was characterized by ESI-MS. ESI-MS: expected 53988, observed: 53984 (Figure 1C).

[0106] cell line MDA-MB-361, MCF-7, BT474, SK-BR-3, and MDA-MB-468 cell lines were cultured according to ATCC instructions. Firefly luciferase and GFP-labeled MDA-MB-361 and MCF7 cell lines were generated as previously described. 51 .

[0107] HA binding assay Briefly, Tras or ALN-Tras was diluted in 1 mL of PBS in an Eppendorf tube. Hydroxyapatite (15 equivalents, 15 mg) was added and the resulting suspension was shaken at 37°C and 220 rpm. As a control, a sample without hydroxyapatite was used. After 0.25, 0.5, 1, 2, 4, and 8 hours, the suspension was centrifuged (3000 rpm, 3 min) and the absorbance of the supernatant was measured at 280 nm by Nanodrop. The percentage of binding to HA was calculated as follows: where OD stands for optical density. [(ODHAを含まない -OD HAを含む ) / (OD HAを含まない )] × 100%.

[0108] Native bone binding assay Mouse long bones were cut into small fragments, washed with distilled H2O and absolute ethanol, and then dried overnight at room temperature. For binding studies, Tras or ALN-Tras was diluted in 1 mL of PBS in an Eppendorf tube. 30 mg of dried bone fragments were added to the tube, and the resulting suspension was shaken at 37°C and 220 rpm. As a control, a sample without bone fragments was used. After 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 hours, the suspension was centrifuged (3000 rpm, 5 min) and the absorbance of the supernatant at 280 nm was measured by Nanodrop. The percentage of binding to native bone was calculated according to the following formula: where OD stands for optical density. [(OD 天然骨を含まない -OD 天然骨を含む ) / (OD 天然骨を含まない )] × 100%.

[0109] In vitro cytotoxicity of Tras and Tras-ALN SK-BR-3, BT474, and MDA-MB-468 cells were cultured at 2 × 10 3 Cells were seeded in 96-well plates at a density of 1000 cells / well and incubated overnight to allow attachment. The culture medium was then removed and replaced with different concentrations of Tras and Tras-ALN dissolved in culture medium, then incubated for 4 days. 20 μL of MTT solution (5 mg / mL) was then added to each well and incubated for another 4 hours. The medium was aspirated and 150 μL of DMSO was added to each well. Viable cells were quantified by measuring the absorbance at 570 nm with a microplate reader (Infinite M Plex by Tecan).

[0110] Flow cytometry Cancer cells (3 × 10 3) were resuspended and stained with 30 μg / mL Tras and Tras-ALN for 30 min at 4° C. After staining, cells were washed twice with PBS and then further incubated 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).

[0111] Determination of Kd value The functional affinity of Tras-ALN for HER2 was determined as reported 52 In short, 2×10 5 SK-BR-3, BT474, MDA-MB-361, or MDA-MB-468 cells were incubated for 4 h on ice with increasing concentrations of Tra and Tras-ALN. After washing away unbound material, bound antibody was detected using fluorescein (FITC) AffiniPure goat anti-human IgG (H+L) (Jackson Immunology). After propidium iodide (Molecular Probes, Eugene, OR) staining, cells were analyzed for fluorescence intensity. The linear portion of the saturation curve was used to calculate the dissociation constant, KD, using the Lineweaver-Burk method of plotting the reciprocal of the median fluorescence as an inverse function of antibody concentration. KD was determined as follows: 1 / F=1 / Fmax+(KD / Fmax)(1 / [Ab]), where F corresponds to the background-subtracted median fluorescence, and Fmax was calculated from the plot.

[0112] Confocal imaging Cancer cells were grown to approximately 80% confluency in 8-well confocal imaging chamber plates. Cells were incubated with 30 nM Tras-FITC for 30 min, then fixed with 4% paraformaldehyde for 15 min. Cells were washed three times with PBS (pH 7.4), then incubated with DiIC18(3) (Marker Gene Technologies, Inc.) for 20 min and Hoechst33342 (Cat. No. H1399, Life Technologies™) for 5 min. Cells were then washed three times with PBS (pH 7.4) and used for confocal imaging. Confocal fluorescent images of cells were obtained using a Nikon A1R-si Laser Scanning Confocal Microscope (Japan) equipped with 405 / 488 / 561 / 638 nm lasers.

[0113] Binding to bone frozen sections Non-decalcified long bone sections from C57BL / 6 mice were incubated with 50 μg / mL of Tras or Tras-ALN and 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, dilution 1:500, 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, dilution 1:2000) for 10 min. Slides were then washed with PBS, air-dried, and sealed with Prolong™ gold antifade mounting medium (from ThermoFisher).

[0114] In vivo evaluation of Tras-ALN Internal iliac artery injection and IVIS imaging were performed as previously described. 53Five days after injection, animals were randomized into four groups: PBS-treated control, ALN (representative of those without BP, retro-orbital injection of 10 μg / kg in PBS, twice weekly), Tras (retro-orbital injection of 1.0 mg / kg in sterile PBS, twice weekly), and Tras-ALN conjugate (same as Tras). After injection, animals were imaged twice weekly using an IVIS Lumina II (Advanced Molecular Vision) following recommended procedures and manufacturer settings. On day 110, mice were anesthetized and blood was collected by cardiac puncture, then euthanized. Tumor-bearing tibias, hearts, livers, spleens, lungs, brains, and kidneys were collected for further testing.

[0115] Repeated analysis of ex vivo metastasis Mice were anesthetized with 2.5% isoflurane in oxygen and injected retro-orbitally with luciferin. Mice were then euthanized and the heart, liver, spleen, lungs, kidneys, brain, and tibia were collected. Ex vivo bioluminescence and fluorescence imaging of these organs was immediately performed on an IVIS Lumina.

[0116] Bone histology and immunohistochemistry Harvested long bones were fixed in 10% formalin for 1 week and then decalcified in 12% EDTA for 2 weeks at 4°C. Specimens were embedded in paraffin using standard procedures. 5 μm sections were cut from these blocks and collected on glass slides. Sections were dried in an oven overnight (37°C) and then deparaffinized in xylene solution for 10 min. Hematoxylin and eosin (H&E) staining was performed via conventional methods. Immunohistochemistry analysis was performed on decalcified paraffin-embedded tissue sections using the HRP / DAB ABC IHC kit (abcam) according to the manufacturer's protocol.

[0117] Radiographic analysis Tibiae were dissected, fixed, and scanned by micro-computed tomography (micro-CT, Skyscan1272, Aartselaar, Belgium) at a resolution of 6.64 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 thickness (Tb.Th), bone volume fraction (BV / TV), bone mineral density (BMD), and BS / BV (bone surface / bone volume ratio).

[0118] Biodistribution MDA-MB-361 cells were introduced into female athymic nude mice (body weight = 13-15 g) via intra-iliac artery injection. Three months later, tumor-bearing nude mice were administered Cy7.5-labeled Tras and Tras-ALN (1 mg / kg) via retro-orbital injection. 24 h, 96 h, or 168 h after injection, major organs including heart, liver, spleen, kidney, lung, and bone tumor tissue were removed. Fluorescence intensity in organs and bone tumor tissue was semiquantitatively determined by using a Caliper IVIS Lumina in vivo imager (Caliper Life Science, Boston, MA, USA). Bones from Tras-ALN-treated mice were fixed and sectioned to further evaluate biodistribution.

[0119] In a separate study, unlabeled Tras-ALN (1 mg / kg) was administered to nude mice bearing MDA-MB-361 tumors in the right hind limb via retro-orbital injection. After 48 hours, long bones from Tras-ALN-treated mice were isolated and immediately sectioned without decalcification. The bone sections were then fixed and incubated with anti-RFP (rabbit) antibody (1:200, purchased from Rockland) overnight at 4°C, followed by staining with fluorescein isothiocyanate (FITC)-labeled anti-human IgG (1:100, purchased from Jackson Immunology) and Alexa Fluor® 555 AffiniPure donkey anti-rabbit IgG (H+L) (1:200, purchased from Thermo Fisher) for 120 minutes at room temperature. The sections were mounted using Prolong™ gold antifade mounting medium with DAPI (from Thermo Fisher), sealed with a cover slip, and then used for confocal imaging.

[0120] Pharmacokinetic analysis and FcRn binding assay A single dose of 1 mg / kg Tras and Tras-ALN in PBS was injected retro-orbitally into athymic nude mice, and serum was collected periodically for 7 days and analyzed by Trastuzumab ELISA kit (Lab Bioreagents).FcRn binding was determined using the LUMIT™ FcRn Binding Immunoassay Kit (Promega) according to the manual.

[0121] Quantification of TRAP and calcium levels in serum At the end of the day, 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 a mouse ACP5 / TRAP ELISA kit (catalog number IT5180, GBiosciences). Serum calcium levels were measured colorimetrically using a calcium detection kit (catalog number DICA-500, Bioassays).

[0122] 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.

[0123] Synthesis of BCN-ALN BCN-PNP(ENDO) (31.5 mg) and DIPEA (38.7 mg) were dissolved in 1 mL of dimethyl sulfoxide (DMSO), followed by dropwise addition of 27.4 mg of ALN (dissolved in 0.3 mL of deionized water) to the mixture. The resulting mixture was stirred for 4 h. Ethyl acetate (1 mL) was added to the reaction solution, and the resulting precipitate was filtered and rinsed three times with ethyl acetate. The product was purified by reversed-phase column chromatography. The structure of BCN-ALN was confirmed by MS. ESI-MS [MH]-: calcd for CHNOP2 424.1, found: 424.1 (Figure 5). TIFF2024503480000006.tif21158

[0124] Solid-phase synthesis of ssFB-FPheK peptide The ssFB peptide was synthesized according to the protocol of Fmoc-based peptide synthesis. Amino acid sequence: FNKEQQNAFYEILHLPNLNXEQRNAFIQSLKDDPS-AzK (X=MMT-Lys) (SEQ ID NO: 1). Rink Amide MBHA resin was used as the solid support. The Fmoc protecting group was removed using 25% piperidine in DMF. After the product was washed five times with DMF, the pre-activated HATU / Fmoc-amino acid mixture in DMF was added to the reaction vessel for amide bond formation. Then, the next amino acid was coupled to the beads via the same reaction cycle. Once the peptide synthesis was completed, the N-terminus was capped by acetic anhydride. To incorporate FPheK into the peptide, the MMT protecting group was first selectively removed using 10% acetic acid (AcOH:TFE:DCM=1:2:7). Then, 2 equivalents of 4-fluorophenyl chloroformate and 4 equivalents of DIEA were added to the reaction vessel to react with the exposed free amine on the Lys side chain to form FPheK. Once the reaction was complete, the appropriate amount of TFA and scavengers (water, anisole, triisopropylsilane) were added to the vessel to cleave the peptide from the resin and quench the removal of all protecting groups. The peptide was then precipitated by the addition of ice-cold ether, purified by HPLC, and lyophilized. ESI-MS [M+H]+: calculated 4524, found: 4524 (Figure 6).

[0125] All methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present 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.

[0126] 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. TIFF2024503480000007.tif187160TIFF2024503480000008.tif245160TIFF2024503480000009.tif238160TIFF20245034800 00010.tif238160TIFF2024503480000011.tif232160TIFF2024503480000012.tif231160TIFF2024503480000013.tif240160

Claims

1. 1. A pharmaceutical composition for treating or preventing a bone tumor in a subject, comprising an effective amount of a bone-targeting conjugate comprising a bisphosphonate (BP) conjugated to an antibody.

2. The pharmaceutical composition of claim 1 , wherein the subject has bone cancer or bone metastasis.

3. The pharmaceutical composition of claim 2 , wherein the bone cancer is Ewing's sarcoma, osteosarcoma, or chondrosarcoma.

4. 3. The pharmaceutical composition of claim 2, wherein the bone metastasis is from breast cancer, myeloma, renal cancer, lung cancer, prostate cancer, thyroid cancer, or bladder cancer.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the BP is negatively charged.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the BP is alendronate, zoledronate, pamidronate, risedronate, medronic acid, aminomethylene bisponic acid, clodronate, etidronate, tiludronate, ibandronate pomidronate, neridonate, olpadronate, or oxidronate.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the BP is alendronate (ALN).

8. The pharmaceutical composition according to any one of claims 4 to 7, wherein the breast cancer is triple-negative breast cancer.

9. The pharmaceutical composition according to any one of claims 4 to 8, wherein the breast cancer is HER2-negative breast cancer.

10. The pharmaceutical composition according to any one of claims 4 to 8, wherein the breast cancer is HER2-positive breast cancer.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein 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.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the antibody is an immune checkpoint inhibitor.

13. The pharmaceutical composition of any one of claims 1 to 12, wherein 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-15 antibody, an anti-RANKL antibody, or an anti-TGFβ antibody.

14. The pharmaceutical composition according to any one of claims 1 to 11, wherein the antibody is an anti-HER2 antibody.

15. 15. The pharmaceutical composition of any one of claims 1 to 14, wherein the antibody is trastuzumab (Herceptin), pertuzumab (Perjeta), or atezolizumab.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the antibody is trastuzumab.

17. The pharmaceutical composition of any one of claims 1 to 16, wherein the bone-targeting conjugate comprises alendronate conjugated to trastuzumab.

18. The pharmaceutical composition of any one of claims 1 to 17, wherein the antibody is not an anti-M-CSF antibody.

19. The pharmaceutical composition of any one of claims 1 to 18, wherein the BP is not conjugated to an N-glycan on the Fc region of the antibody.

20. The pharmaceutical composition of any one of claims 1 to 19, wherein the BP is site-specifically conjugated to the antibody using pClick conjugation, NHS-ester chemistry, or cysteine ​​chemistry.

21. The pharmaceutical composition of any one of claims 1 to 19, wherein the BP is site-specifically conjugated to the antibody using pClick conjugation.

22. The pharmaceutical composition according to any one of claims 1 to 21, wherein said BP is conjugated to the CH2-CH3 junction of said antibody.

23. The pharmaceutical composition of any one of claims 1 to 22, wherein the BP is conjugated to the antibody using 4-fluorophenylcarbamate (FPheK).

24. 24. The pharmaceutical composition of claim 23, wherein FPheK is conjugated to a fragment of the B domain of protein A from Staphylococcus aureus (FB protein).

25. 25. The pharmaceutical composition of any one of claims 21 to 24, wherein the pClick conjugation comprises the conjugation of an antibody containing an azide functional moiety with a BP functionalized with bicyclo[6.1.0]nonyne (BCN).

26. 26. The pharmaceutical composition of any one of claims 1 to 25, wherein said bone-targeting conjugate leads to an increased concentration of a therapeutic antibody in the bone tumor niche, inhibiting cancer development in said bone and / or limiting secondary metastasis to other organs.

27. The pharmaceutical composition of any one of claims 1 to 26, wherein said bone-targeting conjugate results in a reduction of micrometastasis-induced osteolytic lesions.

28. The pharmaceutical composition of any one of claims 1 to 27, further comprising an additional anti-cancer therapy.

29. 29. The pharmaceutical composition of claim 28, wherein the additional anti-cancer therapy comprises surgery, chemotherapy, radiation therapy, hormone therapy, immunotherapy, or cytokine therapy.

30. 29. The pharmaceutical composition of claim 28, wherein the additional anti-cancer therapy comprises immunotherapy or chemotherapy.

31. 1. A pharmaceutical composition for treating or preventing a bone disease in a subject, comprising an effective amount of a bone-targeting conjugate comprising a bisphosphonate (BP) conjugated to one or more polypeptides.

32. 32. The pharmaceutical composition of claim 31, wherein 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.

33. 32. The pharmaceutical composition of claim 31, wherein the bone disease is bone cancer or bone metastasis.

34. 34. The pharmaceutical composition of claim 33, wherein the bone cancer is Ewing's sarcoma, osteosarcoma, or chondrosarcoma.

35. 34. The pharmaceutical composition of claim 33, wherein the bone metastasis is from breast cancer, myeloma, renal cancer, lung cancer, prostate cancer, thyroid cancer, or bladder cancer.

36. The pharmaceutical composition according to any one of claims 31 to 35, wherein the BP is negatively charged.

37. 37. The pharmaceutical composition of any one of claims 31 to 36, wherein the BP is alendronate, zoledronate, pamidronate, risedronate, medronic acid, aminomethylenebisphonic acid, clodronate, etidronate, tiludronate, ibandronate pomidronate, neridonate, olpadronate, or oxidronate.

38. The pharmaceutical composition according to any one of claims 31 to 37, wherein the BP is alendronate (ALN).

39. The pharmaceutical composition of any one of claims 35 to 38, wherein the breast cancer is triple-negative breast cancer.

40. The pharmaceutical composition of any one of claims 35 to 39, wherein the breast cancer is a HER2-negative breast cancer.

41. The pharmaceutical composition of any one of claims 35 to 39, wherein the breast cancer is HER2-positive breast cancer.

42. 42. The pharmaceutical composition of any one of claims 31-41, 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 erythropoietic agent, 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.

43. The pharmaceutical composition of any one of claims 31 to 41, wherein the one or more polypeptides comprise an antibody.

44. The pharmaceutical composition of claim 43, wherein the antibody is a monoclonal antibody, a bispecific antibody, a Fab', an F(ab')2, an F(ab')3, a monovalent scFv, a bivalent scFv, a single domain antibody, or a nanobody.

45. The pharmaceutical composition of any one of claims 43 to 44, wherein the antibody is an immune checkpoint inhibitor.

46. The pharmaceutical composition of any one of claims 43 to 45, wherein 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-15 antibody, an anti-RANKL antibody, or an anti-TGFβ antibody.

47. The pharmaceutical composition of any one of claims 43 to 44, wherein the antibody is an anti-HER2 antibody.

48. 48. The pharmaceutical composition of any one of claims 43 to 47, wherein the antibody is trastuzumab (Herceptin), pertuzumab (Perjeta), or atezolizumab.

49. The pharmaceutical composition of any one of claims 43 to 48, wherein the antibody is trastuzumab.

50. 50. The pharmaceutical composition of any one of claims 31 to 49, wherein the bone-targeting conjugate comprises alendronate conjugated to trastuzumab.

51. The pharmaceutical composition of any one of claims 43 to 50, wherein the antibody is not an anti-M-CSF antibody.

52. The pharmaceutical composition of any one of claims 43 to 51, wherein the BP is not conjugated to an N-glycan on the Fc region of the antibody.

53. 53. The pharmaceutical composition of any one of claims 31 to 52, wherein the BP is site-specifically conjugated to the one or more polypeptides using pClick conjugation, NHS-ester chemistry, or cysteine ​​chemistry.

54. The pharmaceutical composition of any one of claims 31 to 52, wherein the BP is site-specifically conjugated to the one or more polypeptides using pClick conjugation.

55. The pharmaceutical composition of any one of claims 43 to 54, wherein the BP is conjugated to the CH2-CH3 junction of the antibody.

56. 56. The pharmaceutical composition of any one of claims 31 to 55, wherein the BP is conjugated to the one or more polypeptides using 4-fluorophenylcarbamate (FPheK).

57. 57. The pharmaceutical composition of claim 56, wherein FPheK is conjugated to a fragment of the B domain of protein A from Staphylococcus aureus (FB protein).

58. 58. The pharmaceutical composition of any one of claims 54 to 57, wherein the pClick conjugation comprises the conjugation of an antibody comprising an azide functional moiety with a BP functionalized with bicyclo[6.1.0]nonyne (BCN).

59. 59. The pharmaceutical composition of any one of claims 43 to 58, wherein said bone-targeting conjugate leads to an increased concentration of a therapeutic antibody in the bone tumor niche, inhibiting cancer development in said bone and / or limiting secondary metastasis to other organs.

60. 60. The pharmaceutical composition of any one of claims 33 to 59, wherein said bone-targeting conjugate results in a reduction of micrometastasis-induced osteolytic lesions.

61. The pharmaceutical composition of any one of claims 33 to 60, further comprising an additional anti-cancer therapy.

62. 62. The pharmaceutical composition of claim 61, wherein the additional anti-cancer therapy comprises surgery, chemotherapy, radiation therapy, hormone therapy, immunotherapy, or cytokine therapy.

63. 62. The pharmaceutical composition of claim 61, wherein the additional anti-cancer therapy comprises immunotherapy or chemotherapy.

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