EP4673179A1

EP4673179A1 - Bone-directed therapeutics for the treatment of cancer

Info

Publication number: EP4673179A1
Application number: EP24764496.6A
Authority: EP
Inventors: Han XIAO; Julian Berger
Original assignee: Columbia University in the City of New York; William Marsh Rice University
Current assignee: Columbia University in the City of New York; William Marsh Rice University
Priority date: 2023-02-27
Filing date: 2024-02-27
Publication date: 2026-01-07
Also published as: AU2024229931A1; IL322937A; WO2024182454A1

Abstract

The disclosure provides a complex comprising an antigen-recognizing compound, a target agent linked to the antigen-recognizing compound, and a bone -targeting moiety linked to the antigen¬ recognizing compound, wherein the antigen-recognizing compound has binding affinity specific for an antigen expressed in prostate cancer cells. Also provided are methods of delivering the same to a subject, such as a subject with cancer.

Description

BONE-TARGETED THERAPEUTICS FOR THE TREATMENT OF CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims priority to United States Provisional Application Nos. 63/507,584, filed June 12, 2023, and 63/487,151, filed February 27, 2023, the entirety of each of which is incorporated herein by reference.

BACKGROUND

[2] Antibody-based therapies have proved to be of great value in targeted cancer treatment. Despite the clinical success of some of these biopharmaceuticals, reaching targets in the bone microenvironment has proved to be difficult due to the relatively low vascularization of bone tissue and the presence of physical barriers.

INCORPORATION BY REFERENCE

[3] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

SUMMARY

[4] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to a prostate cancer-associated antigen and wherein the antibody or antigen-binding fragment thereof comprises a bone -targeting moiety. In some embodiments, a prostate cancer-associated antigen comprises or is B7-H3, PSMA, STEAP1, STEAP2, PSCA, TROP2, KLK2, TGFb, DLL-3, SSTR2, Epcam, GPC3, FAP, GRPR, ROR1, HER3, or TMEFF2. In some embodiments, a prostate cancer-associated antigen comprises or is PSMA. In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to an osteosarcoma-associated antigen and wherein the antibody or antigen-binding fragment thereof comprises a bone-targeting moiety. In some embodiments, an osteosarcoma-associated antigen comprises or is IFITM5 or LRRC15. In some embodiments, an antibody or antigen-binding fragment thereof comprises or is a monoclonal antibody.

[5] In some embodiments, a bone-targeting moiety comprises a bone-targeting peptide. In some embodiments, a bone-targeting moiety is a negatively charged peptide. In some embodiments, a bonetargeting moiety comprises one or more carboxylic peptides. In some embodiments, a bone -targeting peptide comprises or is Asp³, Asp⁴, Asp⁵, Asp⁶, Asp⁷, Asp⁸, Asp⁹, Asp¹⁰, Asp¹¹, Asp¹², Asp¹³, Asp¹⁴, or Asp¹⁵. In some embodiments, a bone-targeting peptide comprises or is Asp⁶. In some embodiments, a bone-targeting peptide comprises or is Glu³, Glu⁴, Glu⁵, Glu⁶, Glu⁷, Glu⁸, Glu⁹, Glu¹⁰, Glu¹¹, Glu¹², Glu¹³, Glu¹⁴, or Glu¹⁵.

[6] In some embodiments, a bone-targeting peptide is attached to a N-terminus or C-terminus of a light chain or a heavy chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone-targeting peptide is attached to a N-terminus or C-terminus of a light chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone -targeting peptide is attached to an N- terminus of a light chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone-targeting peptide is attached to a C-terminus of a light chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone -targeting peptide is attached to a N-terminus or C- terminus of a heavy chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone-targeting peptide is attached to an N-terminus of a heavy chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone-targeting peptide is attached to a C-terminus of a heavy chain of an antibody or antigen-binding fragment thereof.

[7] In some embodiments, a bone-targeting peptide is inserted at an internal permissive site of an antibody or antigen-binding fragment thereof. In some embodiments, an internal permissive site is an internal permissive site of a light chain or an internal permissive site of a heavy chain. In some embodiments, an internal permissive site is an internal permissive site of a light chain. In some embodiments, an internal permissive site is an internal permissive site of a heavy chain. In some embodiments, an internal permissive site of a light chain is or corresponds to A 153, optionally wherein the bone-targeting peptide is inserted following A 153. In some embodiments, an internal permissive site of a heavy chain is or corresponds to G118, P123, A165, P243, D283, N344, or G361, optionally wherein the bone -targeting peptide is inserted following G118, P123, A165, P243, D283, N344, or

G361. In some embodiments, an internal permissive site of a heavy chain is or corresponds to G118.

In some embodiments, an internal permissive site of a heavy chain is or corresponds to Pl 23. In some embodiments, an internal permissive site of a heavy chain is or corresponds to A 165. In some embodiments, an internal permissive site of a heavy chain is or corresponds to P243. In some embodiments, an internal permissive site of a heavy chain is or corresponds to D283. In some embodiments, an internal permissive site of a heavy chain is or corresponds to N344. In some embodiments, an internal permissive site of a heavy chain is or corresponds to G361.

[8] In some embodiments, 1 to (n + 15) amino-acid residues are deleted from an antibody or antigen-binding fragment thereof following an insertion of a bone -targeting peptide, wherein n is the number of amino-acid residues in the bone -targeting peptide.

[9] In some embodiments, an antibody or antigen-binding fragment thereof comprises a second bone-targeting moiety. In some embodiments, a second bone-targeting moiety is a negatively charged peptide. In some embodiments, a second bone -targeting moiety comprises one or more carboxylic peptides. In some embodiments, a second bone-targeting moiety comprises or is a bone-targeting peptide. In some embodiments, a second bone-targeting peptide comprises or is Asp³, Asp⁴, Asp⁵, Asp⁶, Asp⁷, Asp⁸, Asp⁹, Asp¹⁰, Asp¹¹, Asp¹², Asp¹³, Asp¹⁴, or Asp¹⁵. In some embodiments, a second bonetargeting peptide comprises or is Glu³, Glu⁴, Glu⁵, Glu⁶, Glu⁷, Glu⁸, Glu⁹, Glu¹⁰, Glu¹¹, Glu¹², Glu¹³, Glu¹⁴, or Glu¹⁵. In some embodiments, a second bone-targeting peptide is attached to a N-terminus or C-terminus of a light chain or a heavy chain of an antibody or antigen-binding fragment thereof. In some embodiments, a second bone-targeting peptide is inserted at an internal permissive site of an antibody or antigen-binding fragment thereof. In some embodiments, 1 to (n + 15) amino-acid residues are deleted from an antibody or antigen-binding fragment thereof following an insertion of a second bone-targeting peptide, wherein n is the number of amino-acid residues in the second bone-targeting peptide.

[10] In some embodiments, an antibody or antigen-binding fragment thereof comprises one or more additional bone -targeting moieties. In some embodiments, one or more additional bone-targeting moieties comprise 3 bone-targeting moieties (e.g., 3 inserted bone -targeting moieties). In some embodiments, one or more additional bone -targeting moieties comprise 4 bone-targeting moieties (e.g., 4 inserted bone-targeting moieties). In some embodiments, one or more additional bone -targeting moieties comprise 5 bone-targeting moieties (e.g., 5 inserted bone -targeting moieties). In some embodiments, one or more additional bone -targeting moieties are inserted at one or more permissive internal sites provided herein, e.g., A153 of a light chain or G118, P123, A165, P243, D283, N344, or G361 of a heavy chain.

[11] In some embodiments, an antibody or antigen-binding fragment thereof comprises a light chain comprising a sequence selected from Table 8. In some embodiments, an antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence selected from Table 9.

[12] In some embodiments, an antibody or antigen-binding fragment thereof is associated with a target agent. In some embodiments, an antibody or antigen-binding fragment thereof is conjugated to a target agent. In some embodiments, an antibody or antigen-binding fragment thereof is directly or indirectly linked to a target agent. In some embodiments, an antibody or antigen-binding fragment thereof is directly or indirectly covalently linked to a target agent.

[13] In some embodiments, a target agent comprises an anti-cancer agent, an alkylating agent, an antitumor antibiotic, an antimetabolite, an antimitotic agent, a cytotoxic agent, a hormone, a nitrosourea, a plant alkaloid, a taxane, a radioligand, or any combination thereof. In some embodiments, a radioligand comprises an alpha emitter or a beta emitter. In some embodiments, a radioligand comprises actinium, lutetium, indium, radium, Pb-212, Cu-67, or thorium. In some embodiments, a target agent comprises calicheamicin, deruxtecan, govitecan SN-38, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansinoid,, SG3199, duocarmycin, exatecan, tubulysin, PNU- 159682, TOP2 inhibitor, TLR agonist, STING agonist or any combination or derivative thereof. In some embodiments, a target agent comprises an androgen receptor degrader, integrin inhibitor, EZH2, adenosine receptor inhibitors (CD73), WNT pathway inhibitor, AKT inhibitor, steroidogenesis inhibitor (e.g., ODM-208), PARP inhibitor, immune activator (e.g., TLR3 agonist), BET inhibitor, pyrrolobenzodiazepine (PBD), MYC pathway inhibitor, RAS pathway inhibitor, PI3K inhibitor, NRG1 inhibitor, Amantin or any combination thereof.

[14] In some embodiments, an antibody or antigen-binding fragment thereof comprises one or more bone targeting moieties comprising a combination of Asp and Glu residues.

[15] In some embodiments, the present disclosure provides a method of delivering a target agent to a cancer cell in a system, comprising administering or delivering to the system an effective amount of an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the present disclosure provides a method of delivering a target agent to a cancer cell in a system, comprising administering or delivering to the system an effective amount of an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to a prostate cancer- associated antigen and wherein the antibody or antigen-binding fragment thereof comprises a bonetargeting moiety.

[16] In some embodiments, the present disclosure provides a method of killing a cancer cell in a system, comprising administering or delivering to the system an effective amount of an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the present disclosure provides a method of killing a cancer cell in a system, comprising administering or delivering to the system an effective amount of an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to a prostate cancer-associated antigen and wherein the antibody or antigen-binding fragment thereof comprises a bone-targeting moiety. In some embodiments, an antibody or antigen-binding fragment thereof is delivered using one or more of a cell therapy, extracellular vesicle, mRNA, LNP, or VLP. In some embodiments, a system comprises or is a population of cells. In some embodiments, a system comprises or is a subject. In some embodiments, a system comprises or is an animal. In some embodiments, a system comprises or is a mammal. In some embodiments, a system comprises or is a human. In some embodiments, a system expresses a prostate cancer-associated antigen. In some embodiments, a system expresses one or more of B7-H3, PSMA, STEAP1, STEAP2, PSCA, TROP2, KLK2, TGFb, DLL-3, SSTR2, Epcam, GPC3, FAP, GRPR, R0R1, HER3, or TMEFF2. In some embodiments, a system expresses PSMA. In some embodiments, a system expresses an osteosarcoma-associated antigen. In some embodiments, a system expresses one or both of IFITM5 or LRRC15.

[17] In some embodiments, the present disclosure provides a method of treating a condition, disorder, or disease, comprising administering or delivering to a subject suffering therefrom a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the present disclosure provides a method of treating a condition, disorder, or disease, comprising administering or delivering to a subject suffering therefrom a therapeutically effective amount of an antibody or antigen-binding fragment thereof, wherein the antibody or antigenbinding fragment thereof binds to a prostate cancer-associated antigen and wherein the antibody or antigen-binding fragment thereof comprises a bone -targeting moiety.

[18] In some embodiments, the present disclosure provides methods of treating prostate cancer comprising administering or delivering to a subject suffering therefrom a therapeutically effective amount of an immune stimulating or immune checkpoint inhibiting antibody or antigen-binding fragment thereof. In some embodiments, an antibody or antigen-binding fragment thereof comprises a bone-targeting moiety.

[19] In some embodiments, a prostate cancer-associated antigen is a membrane bound protein expressed in prostate cancer cells. In some embodiments, a prostate cancer-associated antigen is an immune checkpoint inhibitor. In some embodiments, a prostate cancer-associated antigen is a membrane bound protein expressed in prostate cancer cells. In some embodiments, a prostate cancer- associated antigen is a membrane bound protein expressed in prostate tumor stroma. In some embodiments, a prostate cancer-associated antigen is a membrane bound protein encoded by tumorigenic transforming viruses. In some embodiments, a prostate cancer-associated antigen is a membrane bound protein that promotes tumor cell growth. In some embodiments, a prostate cancer- associated antigen is a membrane bound protein whose inhibition. In some embodiments, a prostate cancer-associated antigen is a receptor tyrosine kinase. In some embodiments, a prostate cancer- associated antigen is a receptor protein serine/threonine kinases. In some embodiments, a prostate cancer-associated antigen is an enzyme. In some embodiments, a prostate cancer-associated antigen is a membrane bound protein expressed in prostate cancer cells. In some embodiments, a prostate cancer- associated antigen is a Matrix metalloproteinase. In some embodiments, a prostate cancer-associated antigen is a membrane bound protease. In some embodiments, a prostate cancer-associated antigen is a membrane bound ion channel. In some embodiments, a prostate cancer-associated antigen is a membrane bound solute carrier. In some embodiments, a prostate cancer-associated antigen is a membrane bound ABC transporter. In some embodiments, a prostate cancer-associated antigen is a cytokine. In some embodiments, a prostate cancer-associated antigen is an integrin. In some embodiments, a prostate cancer-associated antigen is a protein that binds to the extra cellular matrix. In some embodiments, a prostate cancer-associated antigen is a growth factor receptor. In some embodiments, a prostate cancer-associated antigen is a growth factor. In some embodiments, a prostate cancer-associated antigen is a cytokine receptor. In some embodiments, a prostate cancer-associated antigen is a membrane bound glycoprotein. In some embodiments, a prostate cancer-associated antigen is a membrane bound protein that undergoes endocytosis. In some embodiments, a prostate cancer- associated antigen that has undergone cancer-dependent changes in glycosylation. In some embodiments, a prostate cancer-associated antigen is a G-protein coupled receptor. In some embodiments, a prostate cancer-associated antigen is an angiogenic factor. In some embodiments, a prostate cancer-associated antigen is an HLA-expressed antigen. In some embodiments, a prostate cancer-associated antigen is an HLA. In some embodiments, a prostate cancer-associated antigen is an immune co-stimulatory protein. In some embodiments, a prostate cancer-associated antigen is an T cell stimulating protein. In some embodiments, a prostate cancer-associated antigen is an NK cell stimulating protein. In some embodiments, a prostate cancer-associated antigen is a neuropeptide receptor. In some embodiments, a prostate cancer-associated antigen is an endocrine hormone receptor. In some embodiments, a prostate cancer-associated antigen is a peptide hormone receptor. In some embodiments, a prostate cancer-associated antigen is a peptide hormone receptor. In some embodiments, a prostate cancer-associated antigen is a membrane bound tumor necrosis factor. In some embodiments, a prostate cancer-associated antigen is a proteoglycan. In some embodiments, a prostate cancer-associated antigen is a cell adhesion molecule.

[20] In some embodiments, a condition, disorder, or disease comprises or is a cancer. In some embodiments, a condition, disorder, or disease comprises or is prostate cancer. In some embodiments, a condition, disorder, or disease comprises or is a bone metastasis. In some embodiments, a condition, disorder, or disease comprises or is a prostate cancer bone metastasis. In some embodiments, a condition, disorder, or disease comprises or is a tumor. In some embodiments, a condition, disorder, or disease comprises or is a prostate tumor. In some embodiments, a condition, disorder, or disease comprises or is a bone tumor.

[21] In some embodiments, a subject is an animal. In some embodiments, a subject is a mammal. In some embodiments, a subject is a human. In some embodiments, a subject is a patient.

[22] In some embodiments, the disclosure provides a complex comprising: an antigen-recognizing compound; a target agent linked to the antigen-recognizing compound; and a bone-targeting moiety linked to the antigen-recognizing compound, wherein the antigen-recognizing compound has binding affinity specific for an antigen expressed in prostate cancer cells.

[23] In some embodiments, the disclosure provides a complex comprising: an antigen-recognizing compound; a target agent linked to the antigen-recognizing compound; and a bone-targeting moiety linked to the antigen-recognizing compound, wherein the antigen-recognizing compound has binding affinity specific for an antigen expressed in osteosarcoma cells. In some embodiments, an osteosarcoma-associated antigen comprises or is IFITM5 or LRRC15.

[24] In some embodiments, the disclosure provides a method of treating a condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of a foregoing complex.

[25] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. 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 the specific examples, while indicating specific embodiments of the 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 FIGURES

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

[27] FIG. 1 illustrates a schematic of a therapeutic complex comprising an antigen-recognizing peptide, a target agent, and a bone-targeting moiety comprising aspartic acid(s) oligomers.

[28] FIG. 2 illustrates a schematic of a therapeutic complex comprising an antigen-recognizing peptide, a target agent, and a bone-targeting moiety comprising bisphosphonate.

[29] FIG. 3 depicts an exemplary Coomassie blue-stained gel following SDS-PAGE of harvested anti-PSMA humanized J591 antibody (WT) and anti-PSMA humanized J591 antibody comprising Asp¹² at the C-terminus of the heavy chain (12D). Both intact samples (-DTT) and reduced samples (+DTT) were analyzed. Molecular weights of bands in the molecular weight ladder (first lane) are denoted at left.

[30] FIG. 4A depicts exemplary results from analysis of anti-PSMA humanized J591 antibody using mass spectrometry (ESI-MS).

[31] FIG. 4B depicts exemplary results from analysis of anti-PSMA humanized J591 antibody comprising Asp¹² at the C-terminus of the heavy chain using mass spectrometry (ESI-MS).

[32] FIG. 5A displays a graph of exemplary results from a flow cytometry experiment using a 22Rvl prostate cancer cell line. The left-most curve on the graph indicates the negative control. The two overlapping curves at right independently indicate anti-PSMA humanized J591 antibody (WT)- bound cells and anti-PSMA humanized J591 antibody comprising Asp¹² at the C-terminus of the heavy chain (12D)-bound cells. The highly similar and largely overlapping curves for WT-bound cells and 12D-bound cells indicates that binding affinity of WT and 12D antibodies for PSMA-expressing 22Rvl cells was comparable. In some embodiments, a modified antibody or antigen-binding fragment thereof can provide similar binding to a target antigen, e.g., PSMA, as an unmodified antibody or antigenbinding fragment thereof. In some embodiments, an antibody or antigen-binding fragment thereof comprising a bone -targeting moiety, e.g., Asp¹², can provide similar binding affinity for a target antigen, e.g., PSMA, as an antibody or antigen-binding fragment thereof which does not comprise a bonetargeting moiety.

[33] FIG. 5B displays a graph of exemplary results from a flow cytometry experiment using a C4- 2b prostate cancer cell line. The left-most curve on the graph indicates the negative control. The two overlapping curves at right independently indicate anti-PSMA humanized J591 antibody (WT)-bound cells and anti-PSMA humanized J591 antibody comprising Asp¹² at the C-terminus of the heavy chain (12D)-bound cells. The highly similar and largely overlapping curves for WT-bound cells and HD- bound cells indicates that binding affinity of WT and 12D antibodies for PSMA-expressing 22Rvl cells was comparable. In some embodiments, a modified antibody or antigen-binding fragment thereof can provide similar binding to a target antigen, e.g., PSMA, as an unmodified antibody or antigen-binding fragment thereof. In some embodiments, an antibody or antigen-binding fragment thereof comprising a bone -targeting moiety, e.g., Asp¹², can provide similar binding affinity for a target antigen, e.g., PSMA, as an antibody or antigen-binding fragment thereof which does not comprise a bone -targeting moiety.

[34] FIG. 5C displays a graph of exemplary results from a flow cytometry experiment using a LNCaP prostate cancer cell line. The left-most curve on the graph indicates the negative control. The two overlapping curves at right independently indicate anti-PSMA humanized J591 antibody (WT)- bound cells and anti-PSMA humanized J591 antibody comprising Asp¹² at the C-terminus of the heavy chain (12D)-bound cells. The highly similar and largely overlapping curves for WT-bound cells and 12D-bound cells indicates that binding affinity of WT and 12D antibodies for PSMA-expressing 22Rvl cells was comparable. In some embodiments, a modified antibody or antigen-binding fragment thereof can provide similar binding to a target antigen, e.g., PSMA, as an unmodified antibody or antigenbinding fragment thereof. In some embodiments, an antibody or antigen-binding fragment thereof comprising a bone -targeting moiety, e.g., Asp¹², can provide similar binding affinity for a target antigen, e.g., PSMA, as an antibody or antigen-binding fragment thereof which does not comprise a bonetargeting moiety.

[35] FIG. 6A depicts exemplary results from analysis of anti-PSMA humanized J591 antibody conjugated (at 10: 1 ratio) with MMAE-NHS-ester, via a PEG linker, using mass spectrometry (ESIMS).

[36] FIG. 6B depicts exemplary results from analysis of anti-PSMA humanized J591 antibody conjugated (at 20: 1 ratio) with MMAE-NHS-ester, via a PEG linker, using mass spectrometry (ESIMS).

[37] FIG. 6C depicts exemplary results from analysis of anti-PSMA humanized J591 antibody comprising Asp¹² at the C-terminus of the heavy chain and conjugated (at 10: 1 ratio) with MMAE- NHS-ester, via a PEG linker, using mass spectrometry (ESI-MS).

[38] FIG. 6D depicts exemplary results from analysis of anti-PSMA humanized J591 antibody comprising Asp¹² at the C-terminus of the heavy chain and conjugated (at 20: 1 ratio) with MMAE- NHS-ester, via a PEG linker, using mass spectrometry (ESI-MS).

[39] FIG. 7A displays a graph of exemplary results from an in vitro cell -killing assay. 22Rvl prostate cancer cells were seeded and incubated for 24 hours in 200 pl media prior to addition of the indicated concentrations of anti-PSMA humanized J591 antibody (PSMA), anti-PSMA humanized J591 antibody comprising Asp¹² at the C-terminus of the heavy chain (PSMA 12D), anti-PSMA humanized J591 antibody conjugated to MMAE (PSMA MMAE), anti-PSMA humanized J591 antibody comprising Asp¹² at the C-terminus of the heavy chain conjugated to MMAE (PSMA 12D MMAE), or MMAE alone (MMAE). After 4 days of further incubation, living cells were quantified by measuring absorbance at 570 nm using a microplate reader. EC50 was calculated for the PSMA MMAE (EC50 = 0.5119 nM), PSMA 12D MMAE (EC50 = 0.1176 nM), and MMAE alone conditions (EC50 = 0.4001 nM). ). The graphed data series, and corresponding trend lines, in order from top of graph to bottom of graph based on positioning of the trend lines at log [concentration] = 1 are: PSMA, PSMA 12D, PSMA MMAE, MMAE, PSMA 12D MMAE.

[40] FIG. 7B displays a graph of exemplary results from an in vitro cell-killing assay. C4-2b prostate cancer cells were seeded and incubated for 24 hours in 200 pl media prior to addition of the indicated concentrations of anti-PSMA humanized J591 antibody (PSMA), anti-PSMA humanized J591 antibody comprising Asp¹² at the C-terminus of the heavy chain (PSMA 12D), anti-PSMA humanized J591 antibody conjugated to MMAE (PSMA MMAE), anti-PSMA humanized J591 antibody comprising Asp¹² at the C-terminus of the heavy chain conjugated to MMAE (PSMA 12D MMAE), or MMAE alone (MMAE). After 4 days of further incubation, living cells were quantified. EC50 was calculated forthe PSMA MMAE (EC50 = 0.04807 nM), PSMA 12D MMAE (EC50 = 0.02156 nM), and MMAE alone conditions (EC50 = 0.1007 nM). In some embodiments, an antibody-drug conjugate or antigen-binding fragment thereof comprising a bone -targeting moiety, e.g., Asp¹², can provide similar or even improved activity on target cell lines expressing a target antigen, e.g., PSMA, as an antibodydrug conjugate or antigen-binding fragment thereof which does not comprise a bone -targeting moiety. . The graphed data series, and corresponding trend lines, in order from top of graph to bottom of graph based on positioning of the trend lines at log [concentration] = -1 are: PSMA, PSMA 12D, MMAE, PSMA MMAE, PSMA 12D MMAE.

[41] FIG. 8 depicts a graph of exemplary results from a hydroxyapatite (HA)-binding assay. Briefly, 1 mg of anti-PSMA humanized J591 antibody (anti-PSMA) or anti-PSMA humanized J591 antibody comprising Asp¹² at the C-terminus of the heavy chain (anti-PSMA 12D) were diluted in 0.5 ml PBS (pH 7.4). HA (20 equivalents, 20 mg) was suspended in 0.5 ml PBS. Antibody solution and HA solution were mixed and vortexed. The resulting suspension was shaken at 220 rpm at 37 °C. At 0.25, 0.5, 1, 2, 3, 6, and 8 hour timepoints, the suspension was centrifuged at 3000 rpm for 3 minutes and the absorbance of the supernatant at 280 nm was measured using a Nanodrop (Thermo Fisher). Percent binding to HA was calculated as [(ODwithout HA - ODwith HA)/(ODwithout HA)] x 100%, wherein OD represents optical density. In some embodiments, an antibody comprising a bone-targeting moiety (e.g., Asp¹²) exhibits an increased affinity for HA as compared to an antibody without a bone-targeting moiety, e.g., the same antibody with one or more bone-targeting moieties absent. In some embodiments, an anti-PSMA antibody comprising a bone -targeting moiety (e.g., Asp¹²) exhibits an increased affinity for HA as compared to an anti-PSMA antibody without a bone -targeting moiety, e.g., the same anti- PSMA antibody with one or more bone -targeting moieties absent.

DETAILED DESCRIPTION

[42] The present disclosure provides compositions, methods, and systems for directing cancer killing, agent-antigen-recognizing peptide conjugates to bone metastasis and/or tumors resulting from cancer, for example, prostate cancer and osteosarcoma. In some embodiments, the disclosure provides compositions, methods, and systems for binding to hydroxyapatite in the bone. In some embodiments, the disclosure provides compositions, methods, and systems for synthesizing cancer killing agentantigen-recognizing peptide conjugates using proximity induced, site-specific antibody conjugation (pClick). In some embodiments, the disclosure provides compositions, methods, and systems for cancer killing agent and antigen-recognizing peptide conjugates recognizing prostate and/or osteosarcoma bone metastasis(es).

Definitions

[43] Administering: As used herein, the term “administering” or “administration” typically refers to the administration of a composition to a subject to achieve delivery of an agent that is, or is included in, a composition to a target site or a site to be treated. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be parenteral. In some embodiments, administration may be intravenous. In some embodiments, administration may be oral. In some embodiments, administration may be via injection. In some embodiments, administration may be systemic. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., infusion, perfusion) for at least a selected period of time.

[44] Agent: In general, the term “agent”, as used herein, is used to refer to an entity (e.g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or complex, combination, mixture or system (e.g., cell, tissue, organism) thereof), or phenomenon (e.g., heat, electric current or field, magnetic force or field, etc.). In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and/or is obtained from nature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and/or produced through action of the hand of man and/or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. In some cases, the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer and/or is substantially free of any polymer and/or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.

[45] Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the nonhuman animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish and/or worms. In some embodiments, an animal may be a transgenic animal, a genetically engineered animal and/or a clone.

[46] Cancer: The term “cancer” is used herein to generally refer to a disease or condition in which cells of a tissue of interest exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, cancer may comprise cells that are precancerous (e.g., benign), malignant, pre -metastatic, metastatic, and/or non-metastatic.

[47] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g., animal, plant and/or microbe).

[48] In vivo: As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant and/or microbe).

[49] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, an active agent is present in unit dose amounts appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[50] Pharmaceutically acceptable: As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[51] Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid fdler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[52] Subject: As used herein, the term “subject” or “test subject” refers to any organism to which a compound (e.g., an oligonucleotide) or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject is a human. In some embodiments, a subject may be suffering from and/or susceptible to a disease, disorder, and/or condition. In some embodiments, a subject may be suffering from and/or susceptible to a cancer. In some embodiments, a subject displays one or more symptoms of a disease, disorder, and/or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered. [53] Suffering from: An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with and/or displays one or more symptoms of a disease, disorder, and/or condition.

[54] Susceptible to: An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

[55] Therapeutic agent: As used herein, the term “therapeutic agent” in general refers to any agent that elicits a desired effect (e.g., a desired biological, clinical, or pharmacological effect) when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, an appropriate population is a population of subjects suffering from and/or susceptible to a disease, disorder or condition. In some embodiments, an appropriate population is a population of model organisms. In some embodiments, an appropriate population may be defined by one or more criterion such as age group, gender, genetic background, preexisting clinical conditions, prior exposure to therapy. In some embodiments, a therapeutic agent is a substance that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms or features of a disease, disorder, and/or condition in a subject when administered to the subject in an effective amount. In some embodiments, a “therapeutic agent” is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a “therapeutic agent” is an agent for which a medical prescription is required for administration to humans. In some embodiments, a therapeutic agent is a provided compound, composition, or complex.

[56] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and/or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and/or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[57] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who is suffering from a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who is suffering from a cancer.

Cancers

[58] Various conditions, disorders, and diseases can be treated with provided technologies (e.g., antigen-recognizing peptides, antibodies, conjugates, complexes, compositions, methods). In some embodiments, a condition, disorder, or disease is a neoplastic condition, disorder, or disease. In some embodiments, a condition, disorder, or disease is a tumor. In some embodiments, a condition, disorder, or disease is a cancer. In some embodiments, a condition, disorder, or disease is a metastasis.

[59] In some embodiments, a cancer is a solid tumor, a hematological cancer, a metastatic cancer, a soft tissue tumor, or a combination thereof. In some embodiments, a cancer is a solid tumor, and wherein the solid tumor is selected from a group consisting of melanoma, pancreatic cancer, breast cancer, colorectal cancer, lung cancer, skin cancer, ovarian cancer, prostate cancer, osteosarcoma, liver cancer, and a combination thereof. In some embodiments, a cancer is a hematological cancer, and wherein the hematological cancer is selected from a group consisting of Hodgkin’s lymphoma, NonHodgkin’s lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, multiple myeloma, T-cell lymphoma, acute lymphocytic leukemia, and a combination thereof. In some embodiments, a Non-Hodgkin’s lymphoma is selected from a group consisting of B cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (B-CLL), mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, and a combination thereof. In some embodiments, a T-cell lymphoma is peripheral T-cell lymphoma. In some embodiments, a cancer is a primary cancer. In some embodiments, a cancer is a secondary cancer. In some embodiments, a secondary cancer is a metastasis of the primary cancer.

[60] Non-limiting examples of cancers that can be treated with compounds of the present disclosure include cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testis, tongue, cervix, or uterus. Non-limiting examples of cancer histological types include neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infdtrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra- mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fiingoides; other specified non-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid 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 some embodiments, a tumor may comprise an osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing sarcoma, glioblastoma, neuroblastoma, leukemia, prostate acinar adenocarcinoma, prostate ductal adenocarcinoma, prostate squamous cell carcinoma, or small cell prostate cancer.

[61] In some embodiments, a tumor is a PSMA-expressing tumor. In some embodiments, a cancer is a PSMA-expressing cancer. In some embodiments, a metastasis is a PSMA-expressing metastasis.

Antigen-Recognizing Peptide

[62] Among other things, the present disclosure provides a composition, method, and system comprising an antigen-recognizing peptide or an antigen-recognizing fragment thereof wherein the antigen-recognizing peptide binds to antigens on cancer cells, such as prostate cancer cells and osteosarcoma cells.

[63] In some embodiments, an antigen-recognizing peptide comprises an antibody or antigenbinding fragment thereof, a growth factor, a hormone, a peptide, a polypeptide, a protein, an aptamer, a small molecule, an imaging agent, a cofactor, or a cytokine. In some embodiments, an antigenrecognizing peptide is an antibody, a growth factor, a hormone, a peptide, a polypeptide, a protein, an aptamer, a small molecule, an imaging agent, a cofactor, or a cytokine. In some embodiments, an antigen-recognizing peptide comprises one or more of an antibody or antigen-binding fragment thereof, a growth factor, a hormone, a peptide, a polypeptide, a protein, an aptamer, a small molecule, an imaging agent, a cofactor, or a cytokine. In some embodiments, an antigen-recognizing peptide is one or more of an antibody or antigen-binding fragment thereof, a growth factor, a hormone, a peptide, a polypeptide, a protein, an aptamer, a small molecule, an imaging agent, a cofactor, or a cytokine.

[64] In some embodiments, an antigen-recognizing peptide comprises an antibody or antigenbinding fragment thereof. In some embodiments, an antigen-recognizing peptide is an antibody or antigen-binding fragment thereof. An antigen-recognizing fragment may also be referred to as an antigen-binding fragment. In some embodiments, an antigen-recognizing peptide or an antigenrecognizing fragment thereof is an antibody or an antigen-recognizing fragment thereof. Non-limiting examples of an antibody or an antigen-recognizing fragment thereof can be a monoclonal antibody, for example, a chimeric antibody wherein a portion of the heavy and/or light chain is identical to or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, or an antigen-recognizing fragment of such an antibody.

[65] In some embodiments, a therapeutic complex of the present disclosure can be used in conjugates with an antibody or an antigen-binding fragment thereof for a specific antigen that is expressed by a cancer cell but not in normal tissues (e.g., STEAP1, STEAP2, PSCA, TROP2, KLK2, TGFb, DLL-3, SSTR2, Epcam, GPC3, FAP, GRPR, R0R1, HER3, PSMA, LRRC15, and IFITM5). In some embodiments, an antigen is expressed by normal, e.g., noncancerous, tissues or cells, but is expressed at an increased level in cancerous tissues or cells. An antigen-recognizing peptide can target factors in an extracellular environment. For example, an antigen-recognizing peptide can bind to a prostate cancer cell, such as a DU145 cell, or an osteosarcoma cell, such as a Saos-2 cell.

[66] In some embodiments, an antigen is preferentially associated with a cancer cell. In some embodiments, an antigen is preferentially associated with a prostate cancer cell. In some embodiments, an antigen is preferentially associated with a tumor cell. In some embodiments, an antigen is preferentially associated with a bone tumor cell. In some embodiments, an antigen is preferentially associated with a cell of a metastasis. In some embodiments, an antigen is preferentially associated with a cell of a bone metastasis. In some embodiments, an antigen is preferentially associated with a cell of a prostate cancer bone metastasis.

[67] In some embodiments, an antigen is expressed by a cell, e.g., a cancer cell, e.g., a prostate cancer cell. In some embodiments, an antigen is present in a cell, e.g., a cancer cell, e.g., a prostate cancer cell. In some embodiments, an antigen is present on a cell, e.g., a cancer cell, e.g., a prostate cancer cell. In some embodiments, an antigen is present in an extracellular environment associated with a cell, e.g., a cancer cell, e.g., a prostate cancer cell.

[68] In some embodiments, an antigen is PSMA. In some embodiments, an antigen is STEAP1. In some embodiments, an antigen is STEAP2. In some embodiments, an antigen is LRRC15. In some embodiments, an antigen is IFITM5. In some embodiments, an antigen is TMEFF2. In some embodiments, an antigen is B7-H3. In some embodiments, an antigen is PSCA. In some embodiments, an antigen is KLK2. In some embodiments, an antigen is TGFb. In some embodiments, an antigen is DLL-3. In some embodiments, an antigen is SSTR2. In some embodiments, an antigen is Epcam. In some embodiments, an antigen is GPC3. In some embodiments, an antigen is FAP. In some embodiments, an antigen is GRPR. In some embodiments, an antigen is ROR1. In some embodiments, an antigen is HER3.

[69] An antibody fragment or antigen-recognizing fragment can comprise a portion of an antibody, for example, the antigen-binding or variable region of the intact antibody. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, dimers and trimers of Fab conjugates, Fv, scFv, minibodies, dia-, tria-, and tetrabodies, and linear antibodies. Fab and Fab' are antigen-binding fragments that can comprise the VH and CHI domains of the heavy chain linked to the VL and CL domains of the light chain via a disulfide bond. A F(ab')2 can comprise two Fab or Fab' that are joined by disulfide bonds. A Fv can comprise the VH and VL domains held together by non-covalent interactions. A scFv (single-chain variable fragment) is a fusion protein that can comprise the VH and VL domains connected by a peptide linker. Manipulation of the orientation of the VH and VL domains and the linker length can be used to create different forms of molecules that can be monomeric, dimeric (diabody), trimeric (triabody), or tetrameric (tetrabody). Minibodies are scFv-CH3 fusion proteins that assemble into bivalent dimers.

[70] A monoclonal antibody can be obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. In contrast to polyclonal antibody preparations, which include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope.

[71] In some embodiments, the antibody or antibody fragment is an immune checkpoint inhibitor.

[72] In some embodiments, an antigen-recognizing peptide or antigen-recognizing fragment thereof can bind to an antigen presented by a tumor cell. In some embodiments, an antigen-recognizing peptide or antigen-recognizing fragment thereof can bind to an antigen present by a cancer cell. In some embodiments, an antigen-recognizing peptide or antigen-recognizing fragment thereof can bind to a cancer antigen presented by a tumor cell. In some embodiments, an antigen-recognizing peptide or antigen-recognizing fragment thereof can bind to a cancer antigen presented by a cancer cell. In some embodiments, an antibody or antigen-binding fragment thereof can bind to an antigen presented by a tumor cell. In some embodiments, an antibody or antigen-binding fragment thereof can bind to an antigen presented by a cancer cell. In some embodiments, an antibody or antigen-binding fragment thereof can bind to a cancer antigen presented by a tumor cell. In some embodiments, an antibody or antigen-binding fragment thereof can bind to a cancer antigen presented by a cancer cell. In some embodiments, an antigen-recognizing peptide or antigen-recognizing fragment thereof binds to one or more antigens. In some embodiments, an antibody or antigen-binding fragment thereof binds to one or more antigens. In some embodiments, an antigen-recognizing peptide or antigen-recognizing fragment thereof binds to one or more cancer antigens. In some embodiments, an antibody or antigen-binding fragment thereof binds to one or more cancer antigens. [73] In some embodiments, a cancer antigen is a tumor antigen, stromal antigen, or a hematological antigen. In some embodiments, an antigen is selected from a group consisting of PSMA, DUPA, STEAP1, STEAP2, CD3, HER-2, TROP2, CEACAM5, BCMA, LRRC15, GPNMB, AXL, Nectin-4, HER-3, MET, DLL3, VEGFR, EGFR, Mesothelin, Claudin 18.2, Claudin 18.11, cMET, ENPP3, TIM- 1, CD70, CA9, CDH6, uPARAP, PD-1, PD-L1, CTLA-4, LAG3, SIGLEC, TIGIT, and CD47. In some embodiments, an antigen comprises or is PSMA. In some embodiments, a cancer antigen comprises or is PSMA. In some embodiments, a prostate cancer antigen comprises or is PSMA.

[74] In some embodiments, an antigen-recognizing peptide or antigen-recognizing fragment thereof is an anti-STEAPl antibody, anti-STEAP2 antibody, anti-PSMA antibody (e.g., J591, PSMA617), anti- PSCA antibody, anti-KLK2 antibody, anti-SSTR2 antibody, anti-FAP antibody, anti-RORl antibody, anti-GRPR antibody, anti-GPC3 antibody, anti-LRRC15 antibody (e.g., Samrotamab), anti -GPNMB, anti-IFITM5 antibody, anti -AR antibody, anti-CD99 antibody, anti -IGF -IR antibody, anti-PD-Ll, anti- PD-1 antibody, anti-CTLA4-antibody, anti-Siglec-2 antibody, anti-Siglec-3 antibody, anti-Siglec-5 antibody, anti-Siglec-6 antibody, anti-Siglec-7 antibody, anti-Siglec- 8 antibody, anti-Siglec9 antibody, anti-Siglec-10 antibody, anti-Siglec- 11 antibody, anti-Siglec- 15 antibody, anti-RANKL antibody, anti- EGFR antibody, anti-VEGFR antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD52 antibody, anti-TROP-2 antibody, anti-CD30 antibody, anti-CD152 antibody, anti-IL-6R antibody, anti-GD2 antibody, anti -DUPA antibody, anti-PSMA617 antibody, anti-CD3 antibody, anti -HERZ antibody, anti- TROP antibody, anti-CEACAM5 antibody, anti-BCMA antibody, anti-AXL antibody, anti-Nectin-4 antibody, anti-HER3 antibody, anti-MET antibody, anti-DLL3 antibody, anti-VEGFR antibody, anti- EGFR antibody, anti -Mesothelin antibody, anti-Claudin 18.2 antibody, anti-Claudin 18.11 antibody, anti-cMET antibody, anti-ENPP3 antibody, anti -TIM 1 antibody, anti-CD70 antibody, anti-CA9 antibody, anti-CDH6 antibody, anti-uP ARAP antibody, anti-LAG3 antibody, anti-TIGIT antibody, anti-CD47 antibody, or anti-TGFB antibody (e.g., BioXcell BE0057, BCA101).

[75] In some embodiments, an antigen-recognizing peptide or antigen-recognizing fragment thereof comprises an anti-PSMA antibody or antigen-binding fragment thereof. In some embodiments, an antigen-recognizing peptide or antigen-recognizing fragment thereof is an anti-PSMA antibody or antigen-binding fragment thereof. Various anti-PSMA antibodies are known in the art. In some embodiments, an anti-PSMA antibody or antigen-binding fragment thereof is J591 or fragment thereof. In some embodiments, an anti-PSMA antibody is humanized J591 or fragment thereof. Additional anti- PSMA antibodies include, but are not limited to those described in WO 2019/191728, WO 2021/000018, and/or WO 2023/088966.

[76] In some embodiments, an antigen-recognizing peptide or antigen-recognizing fragment thereof is a TGFB inhibitor. In some embodiments, a TGFB inhibitor comprises an extracellular domain of human TGFp receptor II (TGFpRII). Non-limiting examples of TGFB inhibitors include Vactosertib (TEW-7197), Galunisertib (LY2157299), Galunisertib, and SB-431542. [77] Non-limiting examples of TGFB antibodies include Bintrafusp alfa, Fresolimumab (GC1008), LY3022859, PF-03446962, Trabedersen, and Belagenpumatucel-L.

[78] In some embodiments, an antigen-recognizing peptide or antigen-recognizing peptide fragment can be a multifunctional or multispecific molecule. A multifunctional or a multispecific molecule can be, for example, a molecule, e.g., a polypeptide, that has two or more functionalities, e.g., two or more binding specificities. In some embodiments, the functionalities can include one or more immune cell engagers, one or more tumor binding molecules, one or more cytokine molecules, one or more stromal modifiers, and other moieties described herein. In some embodiments, the multispecific molecule is a multispecific antibody molecule, e.g., a bispecific antibody molecule. In some embodiments, a multispecific antibody, e.g., a bispecific antibody, comprises or is a T-cell engager, e.g., a bi-specific T-cell engager. In some embodiments, a multispecific antibody, e.g., a bispecific antibody, binds to a prostate cancer-associated antigen and an effector antigen comprising CD3, CD28, CD137, CTLA-4, PD-1, ICOS, Integrin Beta 6, Integrin Beta 1, Integrin Beta 3, an IL-18 activator, an IL23 activator, or Jagged- 1.

[79] In some embodiments, an antigen-recognizing peptide or antigen-recognizing peptide fragment is a small molecule. In some embodiments, a small molecule is DUPA or PSMA-617.

Bone-Targeting Moieties

[80] A therapeutic complex disclosed herein can comprise a number of moieties that associate the complex with bone, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 such moieties. Association between a moiety and bone can be, for example, permanent, transient, reversible, irreversible, or for sufficient time to exert a therapeutic effect. An association can be by attachment, for example, by covalent bond. An association can be non-covalent, for example, by Coulombic interactions, ionic interactions, hydrogen bonds, van Der Waals interactions, London forces, hydrophobic effects, and biological recognition. A bone -targeting moiety may be called a bone -directing moiety, and a bone -directing moiety may be called a bone -targeting moiety.

[81] Bones are composed in part of hydroxyapatite (HA) crystals, which are insoluble salts of calcium and phosphate. Hydroxyapatite crystals reportedly have surfaces with varying levels of crystallinity. Surfaces with higher levels of crystallinity are characterized by the presence of bone resorption surfaces and are known as the osteolytic bone metastatic niche. In some embodiments, a bone-targeting moiety can bind to bone at an osteolytic bone metastatic niche. In some embodiments, a bone-targeting peptide can bind to bone at an osteolytic bone metastatic niche. In some embodiments, an oligomer can bind to bone at an osteolytic bone metastatic niche. In some embodiments, a bonetargeting moiety preferentially binds to bone at an osteolytic bone metastatic niche. In some embodiments, a bone-targeting peptide preferentially binds to bone at an osteolytic bone metastatic niche. In some embodiments, an oligomer preferentially binds to bone at an osteolytic bone metastatic niche. Osteolytic bone metastases are reportedly common in, e.g., breast cancers (Kozlow and Guise, J Mammary Gland Biol Neoplasia. 2005 Apr; 10(2): 169-80). In contrast, bone formation surfaces, e.g., osteoblastic bone, reportedly exhibits newly formed hydroxyapatite that is less mature, less crystalline, and less ordered as compared to bone resorption surfaces, e.g., osteolytic bone (Blair et al., Tissue Eng Part B Rev. 2017 Jun;23(3):268-280). Prostate cancers are reportedly most commonly associated with osteoblastic bone metastases (Goode et al., Oncol Lett. 2023 Mar 8;25(4): 163). In some embodiments, a bone-targeting moiety can bind to bone at an osteoblastic bone metastatic niche. In some embodiments, a bone -targeting peptide can bind to bone at an osteoblastic bone metastatic niche. In some embodiments, an oligomer can bind to bone at an osteoblastic bone metastatic niche. In some embodiments, a bone-targeting moiety preferentially binds to bone at an osteoblastic bone metastatic niche. In some embodiments, a bone -targeting peptide preferentially binds to bone at an osteoblastic bone metastatic niche. In some embodiments, an oligomer preferentially binds to bone at an osteoblastic bone metastatic niche. Furthermore, osteolytic bone metastases and osteoblastic bone metastases reportedly exhibit opposing environments related to activation of different cell types (e.g., osteoclasts or osteoblasts), different intracellular pathways (e.g., RUNX2 or NFATcl), and different secreted regulatory ligands (e.g., WNT, NOTCH, FGF, and TGFB or RankL). Such differing environments can provide a challenge for implementation and successful therapeutic usage of any compound or composition in both an osteolytic environment and an osteoblastic environment.

[82] Accordingly, bone-targeting moieties may encounter different bone structure, e.g., different hydroxyapatite structure, based on the type of bone metastases present, e.g., in a subject. Previous reports have focused on usage of bone -targeting moieties with osteolytic bone, e.g., in conditions, disorders, or diseases characterized by bone resorption and/or osteolytic bone metastases, e.g., breast cancer bone metastases. In some embodiments, the present disclosure provides technologies (e.g., antigen-recognizing peptides, antibodies, conjugates, complexes, compositions, methods) for targeting bone metastases. In some embodiments, a bone-targeted antigen-recognizing peptide, e.g., a bone- targeted antibody, can be targeted to a bone metastasis. In some embodiments, a bone metastasis is an osteolytic bone metastasis. In some embodiments, a bone metastasis is an osteoblastic bone metastasis. In some embodiments, a bone metastasis is a prostate cancer bone metastasis.

[83] Formation of osteolytic bone lesions can be driven by paracrine crosstalk among cancer cells, osteoblasts, and osteoclasts. Cancer cells secrete molecules, such as parathyroid hormone-related protein (PTHrP) and interleukin 8, that stimulate osteoclast formation directly or indirectly by acting to modulate expression of osteoblast genes, such as receptor activator of nuclear factor-KB ligand (RANKL) and osteoprotegerin (OPG). Consequent increase in bone resorption reportedly leads to release of growth factors (e.g., IGF1) that reciprocally stimulate tumor growth. Cancer cells can also secrete cancer-specific surface markers, such as STEAP1, STEAP2, PSMA, LRRC15, B7-H3, PSCA, TROP2, KLK2, TGFb, DLL-3, SSTR2, Epcam, GPC3, FAP, GRPR, ROR1, HER3, and IFITM5. [84] Formation of osteoblastic bone lesions can reportedly be driven by secretion of various factors by cancer cells, e.g., prostate cancer cells. Potential factors that may upregulate osteoblast activity include BMPs, TGFB, ET-1, and CXCL1, among others (Lin et al., Curr Osteoporos Rep. 2018 Dec; 16(6):642-647). Moreover, osteoblasts within osteoblastic bone areas may also produce additional factors, e.g., osteocrines, which can modulate gene expression in cancer cells, e.g., prostate cancer cells. Such osteocrine-based modulation of cancer cell gene expression may reportedly lead to increased therapy resistance by said cancer cells. Accordingly, effective targeting of bone metastases, particularly, e.g., osteoblastic bone metastases, remains an important aspect of various cancers, e.g., prostate cancers, to be therapeutically addressed.

[85] In some embodiments, a bone -targeting moiety is conjugated to a target agent, such as an anticancer agent, an adrenergic agonist, an anti-apoptosis factor, an apoptosis inhibitor, a cytokine receptor, a cytokine, a cytotoxin, an erythropoietic agent, a glutamic acid decarboxylase, a glycoprotein, a growth factor, a growth factor receptor, a hormone, a hormone receptor, an immune response potentiator, 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 radioligand, a semaphorin, a semaphorin receptor, a serotonin transport protein, a serotonin uptake inhibitor, a serotonin receptor, a serpin, a serpin receptor, a small molecule inhibitor, a toxin, or a tumor suppressor.

[86] In some embodiments, a bone-targeting moiety is an oligomer of species that are electrically charged at physiological pH. In some embodiments, a bone-targeting moiety is an oligomer of species that are negatively charged, positively charged, or Zwitterionic at physiological pH. In some embodiments, a bone-targeting moiety is an oligomer of amino acid residues, charged amino acid residues, negatively charged amino acid residues, or amino acid residues with acidic side chains.

[87] In some embodiments, a bone -targeting moiety comprises or is a bone-targeting peptide. In some embodiments, a bone-targeting moiety is an oligomer of carboxylic peptides (i.e., aspartic acid, glutamic acid, or carboxyglutamic acid).

[88] In some embodiments, a moiety that associates with bone (e.g., aspartic acid oligomer(s), glutamic acid oligomer(s), bisphosphonate) is attached to a complex, for example, an antibody complex, at a site where insertion of a moiety is minimally disruptive to the native IgG structure and function of an antibody or antigen-binding fragment thereof and allows retention of a moiety’s high affinity for bone matrix, for example, a light chain (LC), heavy chain (CHI), and/or C-terminus (CT) of an antibody or antigen-binding fragment thereof. To screen for such sites, flow cytometry is used. Binding affinity of a bone -targeting antibody or antigen-binding fragment thereof can be evaluated using a HA binding assay. For example, an antibody or antigen-binding fragment thereof can be an anti-STEAPl antibody, an anti-STEAP2 antibody, anti-PSMA antibody (e.g., J591 or a humanized variant thereof), anti-LRRC15 antibody (e.g., Samrotamab), anti-IFITM5 antibody, an anti-PSCA antibody, an anti- TR0P2 antibody, an anti-KLK2 antibody, an anti-TGFb antibody, an anti-DLL-3 antibody, an anti- SSTR2 antibody, an anti-Epcam antibody, an anti-GPC3 antibody, an anti-FAP antibody, an anti-GRPR antibody, an anti-RORl antibody, an anti-HER3 antibody, or antigen-binding fragment of any of the foregoing.

[89] In some embodiments, a bone-targeting moiety is attached to an antibody or antigen-binding fragment thereof. In some embodiments, a bone -targeting moiety is inserted in an antibody or antigenbinding fragment thereof. In some embodiments, a bone-targeting moiety is directly covalently linked to an antibody or an antigen-binding fragment thereof. In some embodiments, a bone -targeting moiety is linked via a linker to an antibody or antigen-binding fragment thereof.

[90] In some embodiments, a bone -targeting moiety is attached to a light chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone-targeting moiety is attached to a heavy chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone-targeting moiety is inserted in a light chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone-targeting moiety is inserted in a heavy chain of an antibody or antigen-binding fragment thereof.

[91] In some embodiments, a bone targeting moiety is attached to one or both of a N-terminus or a C-terminus of a light chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone-targeting moiety is attached to an N-terminus of a light chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone -targeting moiety is attached to a C-terminus of a light chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone-targeting moiety is directly covalently linked to a N-terminus of a light chain of an antibody or an antigen-binding fragment thereof. In some embodiments, a bone -targeting moiety is directly covalently linked to a C- terminus of a light chain of an antibody or an antigen-binding fragment thereof. In some embodiments, a bone-targeting moiety is attached via a linker to a N-terminus of a light chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone-targeting moiety is attached via a linker to a C-terminus of a light chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone-targeting moiety is attached to a light chain of an antibody or antigen-binding fragment thereof as described in WO 2018/136698. In some embodiments, a bone-targeting moiety is attached via a linker to a light chain of an antibody or antigen-binding fragment thereof as described in WO 2018/136698.

[92] In some embodiments, a bone targeting moiety is attached to one or both of a N-terminus or a C-terminus of a heavy chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone -targeting moiety is attached to a N-terminus of a heavy chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone -targeting moiety is attached to a C-terminus of a heavy chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone-targeting moiety is directly covalently linked to a N-terminus of a heavy chain of an antibody or an antigenbinding fragment thereof. In some embodiments, a bone-targeting moiety is directly covalently linked to a C-terminus of a heavy chain of an antibody or an antigen-binding fragment thereof. In some embodiments, a bone -targeting moiety is attached via a linker to a N-terminus of a heavy chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone -targeting moiety is attached via a linker to a C-terminus of a heavy chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone-targeting moiety is attached to a heavy chain of an antibody or antigen-binding fragment thereof as described in WO 2018/136698. In some embodiments, a bonetargeting moiety is attached via a linker to a heavy chain of an antibody or antigen-binding fragment thereof as described in WO 2018/136698. In some embodiments, a bone-targeting moiety is attached to a C-terminus of a heavy chain of an antibody or an antigen-binding fragment thereof as described in WO 2023/004348. In some embodiments, a bone -targeting moiety is attached to a C-terminus of a heavy chain of an antibody or an antigen-binding fragment thereof, wherein the C-terminus is or corresponds to residue G449.

[93] In some embodiments, a bone -targeting moiety is attached to an internal permissive site of an antibody or antigen-binding fragment thereof. In some embodiments, a bone-targeting moiety is inserted at an internal permissive site of an antibody or antigen-binding fragment thereof.

[94] In some embodiments, a bone-targeting moiety is attached to an internal permissive site of a light chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone-targeting moiety is inserted at an internal permissive site of a light chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bone -targeting moiety is inserted at an internal permissive site of a light chain of an antibody or antigen-binding fragment thereof, wherein the internal permissive site is a site or corresponds to a site described in WO 2012/097333, WO 2015/187428, or WO 2023/004348. In some embodiments, a bone -targeting moiety is inserted at an internal permissive site of a light chain of an antibody or antigen-binding fragment thereof, wherein the internal permissive site is or corresponds to A153 optionally wherein the bone-targeting moiety is inserted following the identified residue, e.g., A153.

[95] In some embodiments, a bone-targeting moiety is attached to an internal permissive site of a heavy chain of an antibody or antigen-binding fragment thereof. In some embodiments, a bonetargeting moiety is inserted at an internal permissive site of a heavy chain of an antibody or antigenbinding fragment thereof. In some embodiments, a bone -targeting moiety is inserted at an internal permissive site of a heavy chain of an antibody or antigen-binding fragment thereof, wherein the internal permissive site is a site or corresponds to a site described in WO 2012/097333, WO 2015/187428, or WO 2023/004348. In some embodiments, a bone-targeting moiety is inserted at an internal permissive site of a heavy chain of an antibody or antigen-binding fragment thereof, wherein the internal permissive site is or corresponds to G118, P123, A165, P243, D283, N344, or G361, optionally wherein the bonetargeting moiety is inserted following the identified residue, e.g., G118, P123, A165, P243, D283, N344, or G361. In some embodiments, a bone-targeting moiety is inserted at an internal permissive site of a heavy chain of an antibody or antigen-binding fragment thereof, wherein the Internal permissive site is or corresponds to G118. In some embodiments, a bone-targeting moiety is inserted at an internal permissive site of a heavy chain of an antibody or antigen-binding fragment thereof, wherein the internal permissive site is or corresponds to P123. In some embodiments, a bone -targeting moiety is inserted at an internal permissive site of a heavy chain of an antibody or antigen-binding fragment thereof, wherein the internal permissive site is or corresponds to A 165. In some embodiments, a bone-targeting moiety is inserted at an internal permissive site of a heavy chain of an antibody or antigen-binding fragment thereof, wherein the internal permissive site is or corresponds to P243. In some embodiments, a bone-targeting moiety is inserted at an internal permissive site of a heavy chain of an antibody or antigen-binding fragment thereof, wherein the internal permissive site is or corresponds to D283. In some embodiments, a bone -targeting moiety is inserted at an internal permissive site of a heavy chain of an antibody or antigen-binding fragment thereof, wherein the internal permissive site is or corresponds to N344. In some embodiments, a bone-targeting moiety is inserted at an internal permissive site of a heavy chain of an antibody or antigen-binding fragment thereof, wherein the internal permissive site is or corresponds to G361.

[96] As understood by those skilled in the art, the internal permissive sites disclosed herein, e.g., of a light chain (e.g., A153) or a heavy chain (e.g., G118, P123, A165, P243, D283, N344, or G361) are referenced according to standardized numbering, e.g., Kabat numbering. Accordingly, such residues may be numbered differently based on the particular antibody or antigen-binding fragment thereof wherein a bone-targeted moiety, e.g., a bone-targeted peptide is inserted. In some embodiments, A153 corresponds to A 153 in a sequence in Table 8. In some embodiments, G118 corresponds to G 120 in a sequence in Table 9. In some embodiments, P123 corresponds to P126 in a sequence in Table 9. In some embodiments, A 165 corresponds to A 160 in a sequence in Table 9. In some embodiments, P243 corresponds to P228 in a sequence in Table 9. In some embodiments, D283 corresponds to D267 in a sequence in Table 9. In some embodiments, N344 corresponds to N323 in a sequence in Table 9. In some embodiments, G361 corresponds to G339 in a sequence in Table 9.

[97] In some embodiments, a bone -targeting moiety is attached via a linker to an antibody or antigen-binding fragment thereof. In some embodiments, a linker comprises or is an amino-acid linker, e.g., a peptide linker or a polypeptide linker. In some embodiments, a linker comprises or is a nonamino-acid linker. In some embodiments, a linker comprises or is a peptide comprising glycine residues and/or serine residues. In some embodiments, a linker comprises or is a peptide comprising one or more repeats of glycine residues and/or serine residues. In some embodiments, a linker comprises or is a peptide linker comprising an amino-acid sequence of (G_nS_m)x, wherein n is 1-10, m is 1-10, and x is 1-10. In some embodiments, a linker comprises or is a peptide comprising one or more repeats of an amino-acid sequence of GGGGS. In some embodiments, a linker comprises or is a peptide comprising an amino-acid sequence of (6481)1, i.e., GGGGS. In some embodiments, a linker comprises or is a peptide comprising an amino-acid sequence of (6481)2, i.e., GGGGSGGGGS. In some embodiments, a linker comprises or is a linker described in WO 2018/136698.

Aspartic Acid

[98] In some embodiments, a bone-targeting moiety is an oligomer of aspartic acid residues, L- aspartic acid residues, or D-aspartic acid residues. In some embodiments, a bone-targeting moiety comprises or is a bone -targeting peptide comprising an oligomer of aspartic acid residues, e.g., L- aspartic acid residues or D-aspartic acid residues. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, comprises at least three aspartic acid residues, which are each independently and optionally L-aspartic acid residues or D-aspartic acid residues. In some embodiments, a bonetargeting moiety, e.g., a bone-targeting peptide, comprises at least four aspartic acid residues, which are each independently and optionally L-aspartic acid residues or D-aspartic acid residues. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, comprises at least five aspartic acid residues, which are each independently and optionally L-aspartic acid residues or D-aspartic acid residues. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, comprises at least six aspartic acid residues, which are each independently and optionally L-aspartic acid residues or D-aspartic acid residues. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, comprises at least seven aspartic acid residues, which are each independently and optionally L-aspartic acid residues or D-aspartic acid residues. In some embodiments, a bone -targeting moiety, e.g., a bonetargeting peptide, comprises at least eight aspartic acid residues, which are each independently and optionally L-aspartic acid residues or D-aspartic acid residues. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, comprises at least nine aspartic acid residues, which are each independently and optionally L-aspartic acid residues or D-aspartic acid residues. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, comprises at least ten aspartic acid residues which are each independently and optionally L-aspartic acid residues or D-aspartic acid residues. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, comprises at least eleven aspartic acid residues which are each independently and optionally L-aspartic acid residues or D-aspartic acid residues. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, comprises at least twelve aspartic acid residues which are each independently and optionally L-aspartic acid residues or D-aspartic acid residues. In some embodiments, a bone -targeting moiety, e.g., a bone -targeting peptide, comprises at least thirteen aspartic acid residues which are each independently and optionally L-aspartic acid residues or D-aspartic acid residues. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, comprises at least fourteen aspartic acid residues which are each independently and optionally L-aspartic acid residues or D- aspartic acid residues. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, comprises at least fifteen aspartic acid residues which are each independently and optionally L-aspartic acid residues or D-aspartic acid residues.

[99] In some embodiments, a bone -targeting moiety, e.g., a bone -targeting peptide, is represented as Aspⁿ, wherein n represents the number of aspartic acid residues forming the bone-targeting moiety, e.g., Asp⁶ represents a bone-targeting moiety, e.g., a bone -targeting peptide, comprising six aspartic acid residues, i.e., Asp-Asp-Asp-Asp-Asp-Asp. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, is Asp³, Asp⁴, Asp⁵, Asp⁶, Asp⁷, Asp⁸, Asp⁹, Asp¹⁰, Asp¹¹, Asp¹², Asp¹³, Asp¹⁴, or Asp¹⁵. In some embodiments, a bone targeting moiety is L-Asp³, L-Asp⁴, L-Asp⁵, L-Asp⁶, L-Asp⁷, L-Asp⁸, L-Asp⁹, L-Asp¹⁰, L-Asp¹¹, L-Asp¹², L-Asp¹³, L-Asp¹⁴, or L-Asp¹⁵. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is L-Asp³. In some embodiments, a bonetargeting moiety, e.g., a bone-targeting peptide, is L-Asp⁴. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is L-Asp⁵. In some embodiments, a bone -targeting moiety, e.g., a bone -targeting peptide, is L-Asp⁶. In some embodiments, a bone -targeting moiety, e.g., a bonetargeting peptide, is L-Asp⁷. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, is L-Asp⁸. In some embodiments, a bone -targeting moiety, e.g., a bone -targeting peptide, is L- Asp⁹. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is L-Asp¹⁰. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is L-Asp¹¹. some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is L-Asp¹². some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is L-Asp¹³. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is L-Asp¹⁴. some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is L-Asp¹⁵. In some embodiments, a bone -targeting moiety, e.g., a bone -targeting peptide, is D-Asp³, D-Asp⁴, D-Asp⁵, D- Asp⁶, D-Asp⁷, D-Asp⁸, D-Asp⁹, D-Asp¹⁰, D-Asp¹¹, D-Asp¹², D-Asp¹³, D-Asp¹⁴, or D-Asp¹⁵. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is D-Asp³. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is D-Asp⁴. In some embodiments, a bonetargeting moiety, e.g., a bone -targeting peptide, is D-Asp⁵. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, is D-Asp⁶. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is D-Asp⁷. In some embodiments, a bone -targeting moiety, e.g., a bonetargeting peptide, is D-Asp⁸. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is D-Asp⁹. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is D-Asp¹⁰. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is D-Asp¹¹. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is D-Asp¹². In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is D-Asp¹³. some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is D-Asp¹⁴. some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is D-Asp¹⁵. Glutamic Acid

[100] In some embodiments, a bone -targeting moiety is an oligomer of glutamic acid residues, L- glutamic acid residues, or D-glutamic acid residues. In some embodiments, a bone -targeting moiety comprises or is a bone-targeting peptide comprising an oligomer of glutamic acid residues, e.g., L- glutamic acid residues or D-glutamic acid residues. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, comprises at least three glutamic acid residues, which are each independently and optionally L-glutamic acid residues or D-glutamic acid residues. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, comprises at least four glutamic acid residues, which are each independently and optionally L-glutamic acid residues or D-glutamic acid residues. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, comprises at least five glutamic acid residues, which are each independently and optionally L-glutamic acid residues or D-glutamic acid residues. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, comprises at least six glutamic acid residues, which are each independently and optionally L- glutamic acid residues or D-glutamic acid residues. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, comprises at least seven glutamic acid residues, which are each independently and optionally L-glutamic acid residues or D-glutamic acid residues. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, comprises at least eight glutamic acid residues, which are each independently and optionally L-glutamic acid residues or D-glutamic acid residues. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, comprises at least nine glutamic acid residues, which are each independently and optionally L-glutamic acid residues or D-glutamic acid residues. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, comprises at least ten glutamic acid residues which are each independently and optionally L- glutamic acid residues or D-glutamic acid residues.

[101] In some embodiments, a bone -targeting moiety, e.g., a bone -targeting peptide, is represented as Gluⁿ, wherein n represents the number of glutamic acid residues forming the bone-targeting moiety, e.g., Glu⁶ represents a bone -targeting moiety, e.g., a bone-targeting peptide, comprising six glutamic acid residues, i.e., Glu-Glu-Glu-Glu-Glu-Glu. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is Glu³, Glu⁴, Glu⁵, Glu⁶, Glu⁷, Glu⁸, Glu⁹, Glu¹⁰, Glu¹¹, Glu¹², Glu¹³, Glu¹⁴, or Glu¹⁵. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is Glu³. In some embodiments, a bone -targeting moiety, e.g., a bone -targeting peptide, is Glu⁴. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is Glu⁵. In some embodiments, a bone -targeting moiety, e.g., a bone -targeting peptide, is Glu⁶. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is Glu⁷. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is Glu⁸. In some embodiments, a bone -targeting moiety, e.g., a bone -targeting peptide, is Glu⁹. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is Glu¹⁰. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is Glu¹¹. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is Glu¹². In some embodiments, a bone -targeting moiety, e.g., a bone -targeting peptide, is Glu¹³. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is Glu¹⁴. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is Glu¹⁵. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, is L- Glu³, L-Glu⁴, L-Glu⁵, L-Glu⁶, L-Glu⁷, L-Glu⁸, L-Glu⁹, L-Glu¹⁰, L-Glu¹¹, L-Glu¹², L-Glu¹³, L-Glu¹⁴, or L-Glu¹⁵. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is L-Glu³. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is L-Glu⁴. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, is L-Glu⁵. In some embodiments, a bone -targeting moiety, e.g., a bone -targeting peptide, is L-Glu⁶. In some embodiments, a bonetargeting moiety, e.g., a bone -targeting peptide, is L-Glu⁷. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, is L-Glu⁸. In some embodiments, a bone -targeting moiety, e.g., a bone -targeting peptide, is L-Glu⁹. In some embodiments, a bone -targeting moiety, e.g., a bonetargeting peptide, is L-Glu¹⁰. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is L-Glu¹¹. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, is L-Glu¹². In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is L-Glu¹³. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, is L-Glu¹⁴. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is L-Glu¹⁵. In some embodiments, the moiety is D-Glu³, D-Glu⁴, D-Glu⁵, D-Glu⁶, D-Glu⁷, D-Glu⁸, D-Glu⁹, D-Glu¹⁰, D- Glu¹¹, D-Glu¹², D-Glu¹³, D-Glu¹⁴, or D-Glu¹⁵. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is D-Glu³. In some embodiments, a bone -targeting moiety, e.g., a bonetargeting peptide, is D-Glu⁴. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, is D-Glu⁵. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is D-Glu⁶. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is D-Glu⁷. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, is D-Glu⁸. In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is D-Glu⁹. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, is D-Glu¹⁰. In some embodiments, a bonetargeting moiety, e.g., a bone -targeting peptide, is D-Glu¹¹. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, is D-Glu¹². In some embodiments, a bone-targeting moiety, e.g., a bone -targeting peptide, is D-Glu¹³. In some embodiments, a bone -targeting moiety, e.g., a bonetargeting peptide, is D-Glu¹⁴. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, is D-Glu¹⁵.

Other Amino Acids

[102] In some embodiments, a bone-targeting moiety is an oligomer of two amino acids. In some embodiments, a bone-targeting moiety comprises or is a bone -targeting peptide comprising an oligomer of two amino acids. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is an oligomer of two amino acids, one with a negatively charged sidechain and one with a neutral, polar side chain. In some embodiments, a bone -targeting moiety, e.g., a bone-targeting peptide, is an oligomer of two amino acids, one with a carboxylate sidechain and one with a hydroxyl side chain. In some embodiments, a bone-targeting moiety, e.g., a bone-targeting peptide, is an oligomer of aspartic acid and serine. Non-limiting examples include Ser-Asp-Ser-Ser-Asp and (Asp-Ser-Ser)_x, wherein x is 1-100, for example, 1-10, for example, (Asp-Ser-Ser)i, (Asp-Ser-Ser)2, (Asp-Ser-Ser)s, (Asp-Ser-Ser)4, (Asp-Ser-Ser)5, (Asp-Ser-Ser)e, (Asp-Ser-Ser)?, (Asp-Ser-Ser)s, (Asp-Ser-Ser)g, and (Asp-Ser-Ser)io. Bisphosphonate

[103] In some embodiments, a bone-target moiety that associates a therapeutic complex disclosed herein with bone is a bisphosphonate (BP). Bisphosphonates are synthetic compounds containing two phosphonate groups bound to a central, or geminal, carbon (the P-C-P backbone) that are used to prevent bone resorption in a number of metabolic and tumor-induced bone diseases, such as multiple myeloma. Presence of these two side chains allows numerous substitutions to the bisphosphonate backbone. Negatively-charged bisphosphonate has a high affinity for mineralized, positively charged bone matrix, such as hydroxyapatite (HA).

[104] In some embodiments, a bisphosphonate is negatively-charged. In some embodiments, a bisphosphonate is alendronate, zoledronate, pamidronate, risedronate, medronic acid, aminomethylene bisphonic acid, clodronate, etidronate, tiludronate, ibandronate pomidronate, neridonate, olpadronate, or oxidronate.

[105] In some embodiments, a bisphosphonate is alendronate (ALN). In some embodiments, the bisphosphonate is conjugated to a target agent, such as an adrenergic agonist, an anti-apoptosis factor, an apoptosis inhibitor, a cytokine receptor, a cytokine, a cytotoxin, an erythropoietic agent, a glutamic acid decarboxylase, a glycoprotein, a growth factor, a growth factor receptor, a hormone, a hormone receptor, an immune response potentiator, 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 radioligand, a semaphorin, a semaphorin receptor, a serotonin transport protein, a serotonin uptake inhibitor, a serotonin receptor, a serpin, a serpin receptor, a small molecule inhibitor, a toxin, or a tumor suppressor.

[106] Various bisphosphonates have been described in the art. In some embodiments, a bisphosphonate is a bisphosphonate described in WO 2022/159492, the entirety of which is incorporated herein by reference. In some embodiments, a bisphosphonate is attached to a complex using a method described in WO 2022/159492. In some embodiments, a bisphosphonate is attached to a complex at a location described in WO 2022/159492.

Exemplary Bone-Targeted Antibodies [107] In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises or is an antibody or antigen-binding fragment thereof provided herein. In some embodiments, a bone- targeted antibody or antigen-binding fragment thereof comprises or is an antibody or antigen-binding fragment thereof provided herein and modified to comprise one or more bone-targeting moieties as provided herein. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises or is an anti-PSMA antibody or antigen-binding fragment thereof comprising one or more bone-targeting moieties. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises or is bone-targeted J591 antibody. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises or is bone-targeted humanized J591 antibody. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises or is an antibody described in WO 2019/191728 and modified to comprise one or more bone -targeting moieties as provided herein.

[108] In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a light chain comprising a sequence provided herein. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a light chain comprising an amino-acid sequence comprising a sequence provided in Table 8. In some embodiments, a bone-targeted antibody or antigenbinding fragment thereof comprises a light chain comprising an amino-acid sequence comprising any one of SEQ ID NOs: 14-21 or 49-56. In some embodiments, a bone-targeted antibody or antigenbinding fragment thereof comprises a light chain comprising an amino-acid sequence comprising SEQ ID NO: 14. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a light chain comprising an amino-acid sequence comprising SEQ ID NO: 15. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a light chain comprising an amino-acid sequence comprising SEQ ID NO: 16. In some embodiments, a bone- targeted antibody or antigen-binding fragment thereof comprises a light chain comprising an aminoacid sequence comprising SEQ ID NO: 17. In some embodiments, a bone-targeted or antigen-binding fragment thereof antibody comprises a light chain comprising an amino-acid sequence comprising SEQ ID NO: 18. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a light chain comprising an amino-acid sequence comprising SEQ ID NO: 19. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a light chain comprising an amino-acid sequence comprising SEQ ID NO: 20. In some embodiments, a bone- targeted antibody or antigen-binding fragment thereof comprises a light chain comprising an aminoacid sequence comprising SEQ ID NO: 21. In some embodiments, a bone-targeted antibody or antigenbinding fragment thereof comprises a light chain comprising an amino-acid sequence comprising SEQ ID NO: 49. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a light chain comprising an amino-acid sequence comprising SEQ ID NO: 50. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a light chain comprising an amino-acid sequence comprising SEQ ID NO: 51. In some embodiments, a bone- targeted antibody or antigen-binding fragment thereof comprises a light chain comprising an aminoacid sequence comprising SEQ ID NO: 52. In some embodiments, a bone-targeted antibody or antigenbinding fragment thereof comprises a light chain comprising an amino-acid sequence comprising SEQ ID NO: 53. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a light chain comprising an amino-acid sequence comprising SEQ ID NO: 54. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a light chain comprising an amino-acid sequence comprising SEQ ID NO: 55. In some embodiments, a bone- targeted antibody or antigen-binding fragment thereof comprises a light chain comprising an aminoacid sequence comprising SEQ ID NO: 56. [109] In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence provided herein. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising a sequence provided in Table 9. In some embodiments, a bone-targeted antibody or antigenbinding fragment thereof comprises a light chain comprising an amino-acid sequence comprising any one of SEQ ID NOs: 22-48 or 57-64. In some embodiments, a bone-targeted antibody or antigenbinding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 22. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 23. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 24. In some embodiments, a bone- targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an aminoacid sequence comprising SEQ ID NO: 25. In some embodiments, a bone-targeted antibody or antigenbinding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 26. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 27. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 28. In some embodiments, a bone- targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an aminoacid sequence comprising SEQ ID NO: 29. In some embodiments, a bone-targeted antibody or antigenbinding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 30. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 31. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 32. In some embodiments, a bone- targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an aminoacid sequence comprising SEQ ID NO: 33. In some embodiments, a bone-targeted antibody or antigenbinding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 34. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 35. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 36. In some embodiments, a bone- targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an aminoacid sequence comprising SEQ ID NO: 37. In some embodiments, a bone-targeted antibody or antigenbinding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 38. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 39. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 40. In some embodiments, a bone- targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an aminoacid sequence comprising SEQ ID NO: 41. In some embodiments, a bone-targeted antibody or antigenbinding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 42. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 43. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 44. In some embodiments, a bone- targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an aminoacid sequence comprising SEQ ID NO: 45. In some embodiments, a bone-targeted antibody or antigenbinding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 46. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 47. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 48. In some embodiments, a bone- targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an aminoacid sequence comprising SEQ ID NO: 57. In some embodiments, a bone-targeted antibody or antigenbinding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 58. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 59. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 60. In some embodiments, a bone- targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an aminoacid sequence comprising SEQ ID NO: 61. In some embodiments, a bone-targeted antibody or antigenbinding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 62. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 63. In some embodiments, a bone-targeted antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 64.

[HO] In some embodiments, a bone-targeted antibody comprises a light chain comprising an aminoacid sequence comprising a sequence provided in Table 8 and a heavy chain comprising an amino-acid sequence comprising a sequence provided in Table 9. In some embodiments, a bone-targeted antibody comprises a light chain comprising an amino-acid sequence comprising any one of SEQ ID NOs: 14- 21 or 49-56 and a heavy chain comprising an amino-acid sequence comprising any one of SEQ ID NOs: 22-48 or 57-64. In some embodiments, a bone-targeted antibody comprises a light chain comprising an amino-acid sequence comprising SEQ ID NO: 15 and a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 22. In some embodiments, a bone-targeted antibody comprises a light chain comprising an amino-acid sequence comprising SEQ ID NO: 16 or 17 and a heavy chain comprising an amino-acid sequence comprising any one of SEQ ID NOs: 25-47 or 59-64. In some embodiments, a bone-targeted antibody comprises a light chain comprising an amino-acid sequence comprising SEQ ID NO: 16 and a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 29. In some embodiments, a bo— targeted antibody comprises a light chain comprising an aminoacid sequence comprising SEQ ID NO: 17 and a heavy chain comprising an amino-acid sequence comprising SEQ ID NO: 29.

[Hl] In some embodiments, a bone-targeted antibody comprises a light chain comprising an aminoacid sequence with about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to a sequence in Table 8. In some embodiments, a bone-targeted antibody comprises a heavy chain comprising an amino-acid sequence with about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to a sequence in Table 9. In some embodiments, a bone-targeted antibody comprises a light chain comprising an amino-acid sequence with about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to a sequence in Table 8 and a heavy chain comprising an amino-acid sequence with about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to a sequence in Table 9.

Target Agent

[112] The therapeutic complex disclosed herein can comprise a target agent, such as a therapeutic agent, cell-targeting agent and/or cancer-killing agent.

[113] Non-limiting examples of target agents that can be conjugated to the complex include exogenous materials that do not exist naturally in virions, such as nucleic acid molecules such as DNA (e.g., nuclear DNA and mitochondrial DNA), RNA such as mRNA, tRNA, miRNA, and siRNA, aptamers and other nucleic acid-containing molecules, peptides, proteins, ribozymes, carbohydrates, polymers, therapeutics, and small molecules. In some embodiments, the heterologous sequence can be a peptide, nucleic acid, antibody, or fragment thereof. The nucleic acid can be an inhibitory nucleic acid, such as siRNA, shRNA, or miRNA.

[114] In some embodiments, a target agent is an anti-cancer agent. Various suitable anti-cancer agents are known in the art, and can belong to any of various classes of compounds including, but not limited to, small molecules, peptides, saccharides, steroids, antibodies, fusion proteins, antisense polynucleotides, ribozymes, small interfering RNAs, peptidomimetics, and the like. Similarly, suitable anti-cancer agents can be found among any of a variety of classes of anti-cancer agents including, but not limited to, alkylating agents, anti-metabolite drugs, anti-mitotic antibiotics, alkaloidal anti-cancer agents, hormones and anti-hormones, interferons, non-steroidal anti-inflammatory drugs, and various other anti -cancer agents. In some embodiments, a target agent is an anti -cancer agent selected from the group consisting of alkylating drugs (e.g., mechlorethamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide), antimetabolites (e.g., methotrexate), purine antagonists and pyrimidine antagonists (e.g., 6-mercaptopurine, 5 -fluorouracil, cytarabile, gemcitabine), spindle poisons (e.g., vinblastine, vincristine, vinorelbine, paclitaxel), podophyllotoxins (e.g., etoposide, irinotecan, topotecan), antibiotics (e.g., doxorubicin, bleomycin, mitomycin), nitrosoureas (e.g., carmustine, lomustine), inorganic ions (e.g., cisplatin, carboplatin), enzymes (e.g., asparaginase), and hormones (e.g., tamoxifen, leuprolide, flutamide, and megestrol).

[115] In some embodiments, a target agent is a member of the group including radioisotopes, enzymes, prodrug activating enzymes, radiosensitizers, nucleic acid molecules, interfering RNAs, superantigens, anti-angiogenic agents, alkylating agents, purine antagonists, pyrimidine antagonists, plant alkaloids, intercalating antibiotics, aromatase inhibitors, anti-metabolites, mitotic inhibitors, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones and anti -androgens. In certain embodiments, the nucleic acid molecules are or include DNA, enzymatic RNA, RNA:DNA hybrid, triplexed DNA, ssRNA, dsRNA, tRNA, mRNA, rRNA, or any combination thereof.

[116] In some embodiments, a target agent is a detectable moiety. Suitable detectable moieties include, but are not limited to: various ligands, radionuclides; fluorescent dyes; chemiluminescent agents (such as, for example, acridinum esters, stabilized dioxetanes, and the like); biolumine scent agents; spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots); microparticles; metal nanoparticles (e.g., gold, silver, copper, platinum, etc.); nanoclusters; paramagnetic metal ions; enzymes; colorimetric labels (such as, for example, dyes, colloidal gold, and the like); biotin; dioxigenin; haptens; and proteins for which antisera or monoclonal antibodies are available. In some embodiments, a target agent is a radioactive and/or paramagnetic isotope or ion.

[117] In some embodiments, a target agent is an adrenergic agonist, an anti-apoptosis factor, an apoptosis inhibitor, a cytokine receptor, a cytokine, a cytotoxin, an erythropoietic agent, a glutamic acid decarboxylase, a glycoprotein, a growth factor, a growth factor receptor, a hormone, a hormone receptor, an immune response potentiator, 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 radioligand, a semaphorin, a semaphorin receptor, a serotonin transport protein, a serotonin uptake inhibitor, a serotonin receptor, a serpin, a serpin receptor, a small molecule inhibitor, a toxin, or a tumor suppressor.

[118] In some embodiments, a toxin is monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), ozogamicin, tiuxetan, vedotin, pasudotox-tdfx. mafodotin, calicheamicin, maytansine, or deruxtecan, or derivatives or combinations thereof.

[119] In some embodiments, a radioligand is astatine-211, carbon-14, chromium-51, chlorine-36, cobalt-57, cobalt-58, copper-67, europium-152, gallium-67, hydrogen-3, iodine-123, iodine-125, iodine-131, indium-i l l, iron-59, lutetium-177, phosphorus-32, rhenium-186, rhenium-188, selenium- 75, sulphur-35, technicium-99m, or yttrium-90. in some embodiments, a radioligand is an alpha emitter or a beta emitter. In some embodiments, a radioligand is actinium, lutetium, indium, radium, Pb-212, Cu-67, or thorium.

[120] In some embodiments, an immune response potentiator is an anti-CD3 antibody, STING agonist, or TLR agonist.

[121] In some embodiments, a small molecule inhibitor is an androgen receptor (AR) inhibitor (e.g., Enzalutamide).

[122] In some embodiments, a target agent is a chemotherapeutic (e.g., alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, or nitrosoureas) or radiotherapeutic. A wide variety of chemotherapeutic agents may be used in accordance with the present disclosure. The term “chemotherapy” refers to the use of drugs to treat cancer. A “chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

[123] Non-limiting examples of chemotherapeutic agents include alkylating agents (e.g., thiotepa and cyclosphosphamide), alkyl sulfonates (e.g., busulfan, improsulfan, and piposulfan), aziridines (e.g., such as benzodopa, carboquone, meturedopa, and uredopa), ethylenimines and methylamelamines (e.g., altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine), acetogenins (e.g., bullatacin and bullatacinone), camptothecin (e.g., topotecan); bryostatin; callystatin, CC-1065 (e.g., adozelesin, carzelesin and bizelesin), cryptophycins (e.g., cryptophy cin 1 and cryptophy cin 8), dolastatin, duocarmycin (e.g., KW-2189 and CB1-TM1); eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustards (e.g., chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard) nitrosureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (e.g., morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin, anti-metabolites (e.g., methotrexate and 5- fluorouracil (5-FU)), folic acid analogues (e.g., denopterin, pteropterin, and trimetrexate), purine analogs (e.g., fludarabine, 6- mercaptopurine, thiamiprine, and thioguanine) pyrimidine analogs (e.g., ancitabine, azacitidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine); androgens (e.g., calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone), anti-adrenals (e.g., as mitotane and trilostane), folic acid replenisher (e.g., frolinic acid; aceglatone; aldophosphamide glycoside), aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatrexate, defofamine, demecolcine, diaziquone, elformithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids (e.g., such as maytansine and ansamitocins), mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, PSKpoly saccharide complex, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2, 2’, 2”- trichlorotriethylamine, trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine), urethan, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside (e.g., Ara-C), cyclophosphamide; toxoids (e.g., paclitaxel and docetaxel), gemcitabine, 6-thioguanine, mercaptopurine, platinum coordination complexes (cisplatin, oxaliplatin, and carboplatin), vinblastine, platinum, etoposide (e.g., VP-16), ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan (e.g., CPT-11), topoisomerase inhibitor, difluorometlhylomithine (DMFO), retinoids (retinoic acid), capecitabine, carboplatin, procarbazine, and plicamycin. Pharmaceutically acceptable salts, acids, or derivatives of any of the above are also chemotherapeutic agents described herein.

[124] In some embodiments, a target agent comprises a radiotherapy. Other factors that cause DNA damage and have been used extensively include what are commonly known as y-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287, each of which are hereby incorporated by reference in their entirety), and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

[125] In some embodiments, a target agent can be an amino acid sequence up to 200 amino acids, such as up to 50 amino acids. In some embodiments, a target agent comprises or is a peptide. In some embodiments, a peptide has a length of about 5 to about 10, about 10 to about 20 amino acids, about 20 to about 30, about 30 to about 40, or about 40— about 50 residues.

Conjugation

[126] The therapeutic complex disclosed herein comprising an antigen-recognizing peptide, a bonedirecting moiety, and a target agent can be conjugated using proximity induced, site-specific antibody conjugation (pClick), NHS-ester chemistry, or cysteine chemistry.

[127] Provided compounds or compositions comprising an antigen-comprising peptide, an antibody or antigen-binding fragment thereof, a bone-targeting moiety, a target agent, or a combination thereof provided herein can be conjugated using various conjugation methods known in the art. In some embodiments, conjugation comprises chemical conjugation. In some embodiments, conjugation comprises lysine amide coupling. In some embodiments, conjugation comprises cysteine coupling. In some embodiments, conjugation comprises incorporation of non-natural amino acids into, e.g., an antigen-recognizing peptide, e.g., an antibody or antigen-binding fragment thereof . In some embodiments, conjugation comprises enzymatic conjugation. In some embodiments, conjugation comprises site-specific enzymatic conjugation. Various enzymatic conjugation methods are known in the art, including sortase-mediated conjugation, transglutamaniase-mediated conjugation, GalT- mediated conjugation, SialT-mediated conjugation, etc.

[128] In some embodiments, the therapeutic complex disclosed herein comprises an antigenrecognizing peptide, a bone -directing moiety, a target agent, or a combination thereof. In some embodiments, the therapeutic complex disclosed herein does not comprise an antigen-recognizing peptide.

[129] The present disclosure provides methods comprising proximity-induced reactivity between a non-canonical amino acid (ncAA) and a nearby residue, such as a lysine or cysteine. pClick provides covalent bond formation between functional moieties and a defined residue, such as lysine, of an antigen-recognizing peptide without or with minimal antibody engineering. [130] The present disclosure also provides methods for proximity-induced, site-specific conjugation (pClick) of a target agent to an antigen-recognizing peptide comprising an affinity compound having a proximity-reactive motif, wherein the affinity compound is conjugated to the target agent. pClick can bring the affinity compound into proximity of the antigen-recognizing peptide for a sufficient period of time to link the affinity compound covalently to the antigen-recognizing peptide.

[131] In some embodiments, the proximity-reactive motif comprises a ncAA.

[132] In some embodiments, the affinity compound is a small molecule, DNA, RNA, peptide, protein, or a derivative thereof. In some embodiments, the RNA is an RNA aptamer. In some embodiments, the affinity compound is produced by solid-phase synthesis or recombinant expression. In some embodiments, the affinity compound is an antibody-binding compound comprising a proximity reaction motif.

[133] In some embodiments, the ncAA can crosslink with an amino acid residue of an antigenrecognizing peptide. In some embodiments, the amino acid residue is histidine, serine, threonine, tryptophan, tyrosine, lysine or cysteine. In some embodiments, the ncAA has a reactive halide, aryl ketone, Michael acceptor, aryl isothiocyanate, or aryl carbamate side chain. In some embodiments, the ncAA contains a 4-fluorophenyl, acryloyl, fluorosulfate, sulfonyl fluoride, or reactive halide side chain(s). In some embodiments, the ncAA is 4-fluorophenyl carbamate lysine (fPheK), phenyl carbamate lysine (PheK), N-acryloyl-lysine (AcrK), 2-amino-6-(6-bromohexanamido)hexanoic acid (BrC6K), fluorosulfate-L-tyrosine (FSY), 2-amino-3-(4-(3 -bromopropoxy )phenyl)propanoic acid (BprY), sulfonyl fluoride phenylalanine, or N-fluoroacetyllysine (fAcK). In some embodiments, the ncAA is fPheK.

[134] In some embodiments, the affinity compound exhibits binding for the fragment crystallizable (Fc) region, an antigen-binding (Fab) region, or hinge region of an antibody. In some embodiments, the affinity compound exhibits binding for the CH2 or CH3 region of the antigen-recognizing peptide. In some embodiments, the affinity compound exhibits binding for the CH2-CH3 junction of the antigenrecognizing peptide.

[135] In some embodiments, the affinity compound is a peptide derived from protein A (e.g., Z domain), protein G, or antibody-binding peptides evolved via phage display (e.g., FcIII). In some embodiments, the affinity compound is the B domain of protein A (FB protein) from Staphylococcus aureus, such as SEQ ID NO: 1. In some embodiments, the ncAA is inserted at residue 25 of the FB protein. In some embodiments, fPheK is inserted at residue 25 of the FB protein (FB-E25fPheK). In some embodiments, the affinity compound is a peptide of SEQ ID NO: 1 (e.g., a peptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1) or a fragment thereof, such as a peptide of 66, 65, 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, or fewer amino acid residues in length. In some embodiments, the affinity compound is a peptide of SEQ ID NO: 2. In some embodiments, the affinity compound is synthesized via Fmoc-based solid-phase peptide synthesis, such as ssFB.

[136] In some embodiments, the affinity peptide has a length of about 10 to about 60 amino acids residues, such as about 30— about 60 amino acid residues, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or more amino acid residues.

[137] In some embodiments, the covalent linking has an efficiency of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. In some embodiments, the covalent linking does not comprise enzymatic treatment. In some embodiments, the covalent linking of the affinity peptide occurs without the use of other agents or the application of additional treatments.

[138] In some embodiments, molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light. In some embodiments, 2- and 8-azido analogues of purine nucleotides can be used as site-directed photoprobes to identify nucleotide binding proteins in crude cell lysates. The 2- and 8-azido nucleotides can be used to map nucleotide binding domains of purified proteins and can be used as antibody binding agents.

[139] The present disclosure also provides a composition comprising an ncAA linker conjugated to a target agent. In some embodiments, the composition further comprises an antigen-recognizing peptide.

[140] In some embodiments, more than one target agent is conjugated to the antigen-recognizing peptide. In some embodiments, 2, 3, 4, or 5 target agents are conjugated to the antigen-recognizing peptide. In some embodiments, the target agents are conjugated to the antigen-recognizing peptide at different sites.

[141] In some embodiments, the affinity compound can bind to the fragment crystallizable (Fc) region, an antigen-binding (Fab) region, or hinge region of the antigen-recognizing peptide. In some embodiments, the affinity compound exhibits binding for the CH2 or CH3 region of the antigenrecognizing peptide. In some embodiments, the affinity compound exhibits binding for the CH2-CH3 junction of antigen-recognizing peptide.

[142] In some embodiments, the affinity compound is a peptide derived from protein A (e.g., Z domain), protein G, or antibody-binding peptides evolved via phage display (e.g., FcIII). In some embodiments, the affinity compound is the B domain of protein A (FB protein) from Staphylococcus aureus. In some embodiments, the ncAA is inserted at a residue of the FB protein with the highest affinity to the antigen-recognizing peptide. In some embodiments, fPheK is inserted at said residue of the FB protein (FB -fPheK).

[143] The present disclose also provides a method for producing a fPheK-labeled FB affinity peptide comprising synthesizing a truncated FB peptide with a monomethoxytrityl (MMT) protecting group using Fmoc-based solid-phase peptide synthesis; selectively removing the MMT protecting group using acetic acid; and reacting the truncated FB peptide with 4-fluorophenyl chloroformate, thereby producing the fPheK-labeled FB affinity peptide. In some embodiments, the MMT protection is at the residue of the truncated FB peptide with the highest affinity to the antigen-recognizing peptide. In some embodiments, solid-phase synthesis comprises stepwise synthesis starting from rink amide resin. In some embodiments, the truncated FB peptide is N-terminal acetylated. In some embodiments, the acetic acid is 10% acetic acid. In some embodiments, the truncated FB peptide comprises SEQ ID NO:2. In some embodiments, the method further comprises lyophilizing the fPheK-labeled FB affinity peptide. In some embodiments, the method further comprises denaturing the fPheK-labeled FB affinity peptide using urea.

[144] In some embodiments, the bone -directing moiety such as BP can be site-specifically conjugated to an antigen-recognizing peptide or a fragment thereof such as antibody or antibody fragment using pClick. The BP can be conjugated to the Fc receptor binding site, such as the CH2-CH3 junction of the antibody. The BP can be conjugated to the antibody using 4-fluorophenyl carbamate lysine (fPheK). The fPheK can be attached to a fragment of the B domain of protein A (FB protein) from Staphylococcus aureus. In some embodiments, pClick conjugation comprises conjugation of an antibody with an azide functional moiety with BP functionalized with bicyclo [6. 1.0] nonyne (BCN). The bone -targeting conjugate can result in increased concentration of therapeutic antibody at the bone tumor niche, inhibited cancer development in the bone, and/or limited secondary metastases to other organs. The bone -targeting conjugate can result in decreased micrometastasis-induced osteolytic lesions.

[145] In some embodiments, a conjugation method is a site-specific conjugation comprising cysteine chemistry comprising engineered cysteine substitutions at positions on the light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly or alter antigen binding.

[146] In some embodiments, a conjugation method comprises site-specific introduction of aldehyde groups into recombinant proteins using the 6-amino-acid consensus sequence recognized by the formylglycine -generating enzyme (aldehyde tag). The aldehyde tag is no larger than a His6 tag.

[147] In some embodiments, a conjugation method comprises remodeled Fc N-glycans of antibodies using mutant glycosyltransferases, such as mutant betal,4-galactosyltransferase or transglutaminase- mediated site-specific conjugation. In some embodiments, the conjugation method comprises use of disulfide bridges.

[148] TABLE 1 shows amino acid sequences of affinity compounds.

Homology

[149] Those skilled in the art, reading the present disclosure, will observe reference to various biological polymers - e.g., polypeptides and/or polynucleotides. Those skilled in the art are aware that typically identical sequences are not required to have reasonably comparable activity. One of skill in the art is aware of how to define characteristic sequence elements and an appropriate degree of homology.

[150] As used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and/or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution. Typical amino acid categorizations are summarized in Table 0 below:

[151] As will be understood by those skilled in the art, a variety of algorithms are available that permit comparison of sequences in order to determine their degree of homology, including by permitting gaps of designated length in one sequence relative to another when considering which residues “correspond” to one another in different sequences. Calculation of the percent homology between two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-corresponding sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position; when a position in the first sequence is occupied by a similar nucleotide as the corresponding position in the second sequence, then the molecules are similar at that position. The percent homology between the two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Representative algorithms and computer programs useful in determining the percent homology between two nucleotide sequences include, for example, the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent homology between two nucleotide sequences can, alternatively, be determined for example using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[152] As used herein, the term “identity” refers to overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In some embodiments, a length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of length of a reference sequence; residues at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as a corresponding position in the second sequence, then the two molecules (i.e., first and second) are identical at that position. Percent identity between two sequences is a function of the number of identical positions shared by the two sequences being compared, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17, which is herein incorporated by reference in its entirety), which has been incorporated into the ALIGN program (version 2.0). In some embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[153] A sequence of a therapeutic complex of the present disclosure can have at least about 70% homology, at least about 71% homology, at least about 72% homology, at least about 73% homology, at least about 74% homology, at least about 75% homology, at least about 76% homology, at least about 77% homology, at least about 78% homology, at least about 79% homology, at least about 80% homology, at least about 81% homology, at least about 82% homology, at least about 83% homology, at least about 84% homology, at least about 85% homology, at least about 86% homology, at least about 87% homology, at least about 88% homology, at least about 89% homology, at least about 90% homology, at least about 91% homology, at least about 92% homology, at least about 93% homology, at least about 94% homology, at least about 95% homology, at least about 96% homology, at least about 97% homology, at least about 98% homology, at least about 99% homology, at least about 99.1% homology, at least about 99.2% homology, at least about 99.3% homology, at least about 99.4% homology, at least about 99.5% homology, at least about 99.6% homology, at least about 99.7% homology, at least about 99.8% homology, at least about 99.9% homology, at least about 99.91% homology, at least about 99.92% homology, at least about 99.93% homology, at least about 99.94% homology, at least about 99.95% homology, at least about 99.96% homology, at least about 99.97% homology, at least about 99.98% homology, or at least about 99.99% homology to a sequence of the target antigen described herein (e.g., antigens on cancer cells).

[154] Various methods and software programs can be used to determine the homology between two or sequences, such as NCBI BLAST, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, or another suitable method or algorithm. Pharmaceutical Compositions

[155] A pharmaceutical composition of the present disclosure can comprise an antigen-recognizing peptide, an antibody, a bone -targeting moiety, a target agent, or a composition thereof provided herein. A pharmaceutical composition of the disclosure can comprise a therapeutic complex of the present disclosure. A pharmaceutical composition can be a combination of any therapeutic complexes described herein with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the therapeutic complex to an organism.

[156] Pharmaceutical formulations for administration can include aqueous solutions of the active compound in water-soluble form. Suspensions of the active compound can be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. The active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[157] The pharmaceutical compositions can include at least one pharmaceutically acceptable carrier, diluent, or excipient and compounds described herein as free-base or pharmaceutically acceptable salt form.

[158] Non-limiting examples of pharmaceuticall -acceptable excipients suitable for use in the disclosure include binding agents, disintegrating agents, anti-adherents, anti-static agents, surfactants, antioxidants, coating agents, coloring agents, plasticizers, preservatives, suspending agents, emulsifying agents, anti-microbial agents, spheronization agents, and any combination thereof.

[159] Non-limiting examples of pharmaceutically-acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.

[160] A therapeutic complex described herein can be conveniently formulated into pharmaceutical compositions composed of one or more pharmaceutically acceptable carriers. See e.g., Remington’s Pharmaceutical Sciences, latest edition, by E.W. Martin Mack Pub. Co., Easton, PA, incorporated by reference in its entirety, which discloses typical carriers and conventional methods of preparing pharmaceutical compositions. Such carriers can be carriers for administration of compositions to humans and non-humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. Pharmaceutical compositions can also include one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents, and anesthetics.

[161] Non-limiting examples of pharmaceutically acceptable carriers include saline, Ringer’s solution, and dextrose solution. In some embodiments, the pH of the solution can be from about 5 to about 8, and can be from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic complex. The matrices can be in the form of shaped articles, for example, fdms, liposomes, microparticles, or microcapsules.

[162] Non-limiting examples of pharmaceutically active agents suitable for combination with compositions of the disclosure include anti-infectives, i.e., aminoglycosides, antiviral agents, antimicrobials, anti-cholinergics/anti-spasmotics, antidiabetic agents, antihypertensive agents, anti- neoplastics, cardiovascular agents, central nervous system agents, coagulation modifiers, hormones, immunologic agents, immunosuppressive agents, and ophthalmic preparations.

[163] In some embodiments, the pharmaceutical composition provided herein comprises a therapeutically effective amount of a therapeutic complex herein in admixture with a pharmaceutically acceptable carrier and/or excipient, for example, saline, phosphate buffered saline, phosphate and amino acids, polymers, polyols, sugar, buffers, preservatives, and other proteins. Illustrative agents include octylphenoxy polyethoxy ethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringe”s and Han”s solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycol.

[164] In some embodiments, a pharmaceutical formulation disclosed herein can comprise: (i) a therapeutic complex disclosed herein; (ii) a buffer; (iii) a non-ionic detergent; (iv) a tonicity agent; and (v) a stabilizer. In some embodiments, the pharmaceutical formulation disclosed herein is a stable liquid pharmaceutical formulation.

[165] In some embodiments, a pharmaceutical formulation disclosed herein is a liquid formulation that can comprise about 5 mg/mL to about 150 mg/mL of the therapeutic complex, about 7.5 mg/mL to about 140 mg/mL of the therapeutic complex, about 10 mg/mL to about 130 mg/mL of the therapeutic complex, about 10 mg/mL to about 100 mg/mL of the therapeutic complex, about 20 mg/mL to about 80 mg/mL of the therapeutic complex, or about 30 mg/mL to about 70 mg/mL of the therapeutic complex. For example, a formulation of the present disclosure can comprise about 5 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg/mL, about 80 mg/mL, about 90 mg/mL, about 100 mg/mL, about 120 mg/mL, about 140 mg/mL, or about 150 mg/mL of a therapeutic complex described herein.

[166] In some embodiments, a pharmaceutical formulation disclosed herein can comprise a buffer. In some embodiments, the buffer serves to maintain a stable pH and to help stabilize a therapeutic complex disclosed herein. In some embodiments, the buffer or buffer system comprises at least one buffer that has a buffering range that overlaps fully or in part the range of pH 5.5-7.4. In some embodiments, the buffer has a pKa of about 6.2±0.5. In some embodiments, the buffer comprises a sodium phosphate buffer. In some embodiments, the sodium phosphate is present at a concentration of about 5 mM to about 15 mM, about 6 mM to about 14 mM, about 7 mM to about 13 mM, about 8 mM to about 12 mM, about 9 mM to about 11 mM, or about 10 mM. In certain embodiments, the buffer system comprises sodium phosphate at 10 mM, at a pH of 6.2±0.3 or 6.1±0.3.

[167] The pH of the disclosed composition can range from about 3 to about 12. The pH of the composition can be, for example, from about 3 to about 4, from about 4 to about 5, from about 5 to about 6, from about 6 to about 7, from about 7 to about 8, from about 8 to about 9, from about 9 to about 10, from about 10 to about 11, or from about 11 to about 12 pH units. The pH of the composition can be, for example, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 pH units. The pH of the composition can be, for example, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or at least 12 pH units. The pH of the composition can be, for example, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, or at most 12 pH units. A pharmaceutical formulation disclosed herein can have a pH of from about 5.5 to about 6.5. For example, a formulation of the present disclosure can have a pH of about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, or about 6.5. In some embodiments, the pH is 6.2±0.3, 6.2±0.2, 6.2±0.1, about 6.2, or 6.2.

[168] If the pH is outside the range desired by the formulator, the pH can be adjusted by using sufficient pharmaceutically acceptable acids and bases.

[169] In some embodiments, a pharmaceutical formulation disclosed herein can comprise a non-ionic detergent. In some embodiments, the non-ionic detergent is a nonionic polymer containing a polyoxyethylene moiety. In some embodiments, the non-ionic detergent is any one or more of polysorbate 20, poloxamer 188 or polyethylene glycol 3350. In some embodiments, the non-ionic detergent is polysorbate 20. In some embodiments, the non-ionic detergent is polysorbate 80. In some embodiments, a pharmaceutical formulation disclosed herein can contain about 0.01% to about 1% non- ionic detergent. For example, a formulation of the present disclosure can comprise about 0.0085%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.11%, about 0.12%, about 0.13%, about 0.14%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.20%, about 0.21%, about 0.22%, about 0.23%, about 0.24%, about 0.25%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.15%, about 1.2%, about 1.25%, about 1.3%, about 1.35%, about 1.4%, about 1.45%, about 1.5%, about 1.55%, about 1.6%, about 1.65%, about 1.7%, about 1.75%, about 1.8%, about 1.85%, about 1.9%, about 1.95%, or about 2% polysorbate 20, polysorbate 80 or poloxamer 188.

[170] In some embodiments, a pharmaceutical formulation disclosed herein can comprise a tonicity agent. In some embodiments, the tonicity agent is sodium chloride or potassium chloride. In some embodiments, the tonicity agent is sodium chloride. In some embodiments, the sodium chloride is present at a concentration of about 5 mM to about 100 mM, about 10 mM to about 50 mM, or about 40 mM.

[171] In some embodiments, a pharmaceutical formulation disclosed herein can comprise a stabilizer. In some embodiments, the stabilizer is a thermal stabilizer that can stabilize a therapeutic complex disclosed herein under conditions of thermal stress. In some embodiments, the stabilizer maintains greater than about 93% of the therapeutic complex in a native conformation when the solution containing the therapeutic complex and the thermal stabilizer is kept at about 45 °C for up to about 28 days. In some embodiments, the stabilizer prevents aggregation of the therapeutic complex and less than 4% of the therapeutic complex is aggregated when the solution containing the therapeutic complex and the thermal stabilizer is kept at about 45 °C for up to about 28 days. In some embodiments, the stabilizer maintains greater than about 96% of the therapeutic complex in a native conformation when the solution containing the therapeutic complex and the thermal stabilizer is kept at about 37 °C for up to about 28 days. In some embodiments, the stabilizer prevents aggregation of the therapeutic complex and less than about 2% of the therapeutic complex is aggregated when the solution containing the therapeutic complex and the thermal stabilizer is kept at about 37 °C for up to about 28 days.

[172] In some embodiments, the thermal stabilizer is a sugar or sugar alcohol, for example, sucrose, sorbitol, glycerol, trehalose, or mannitol, or any combination thereof. In some embodiments, the stabilizer is a sugar. In some embodiments, the sugar is sucrose, mannitol or trehalose. In some embodiments, the stabilizer is sucrose. In some embodiments, a pharmaceutical formulation or ophthalmic formulation disclosed herein can comprise about 1% to about 20% sugar or sugar alcohol, about 2% to about 18% sugar or sugar alcohol, about 3% to about 15% sugar or sugar alcohol, about 4% to about 10% sugar or sugar alcohol, or about 5% sugar or sugar alcohol. For example, a pharmaceutical formulation or ophthalmic formulation of the present disclosure can comprise about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, or about 14% sugar or sugar alcohol (e.g., sucrose, trehalose or mannitol). In some embodiments, the stabilizer is at a concentration of from about 1% w/v to about 20% w/v. In some embodiments, the stabilizer is sucrose at a concentration of from about 1% w/v to about 15% w/v, or from about 1% w/v to about 10% w/v. In some embodiments, the stabilizer is sucrose at a concentration of 5% w/v or about 5% w/v. In some embodiments, the stabilizer is sucrose at a concentration of 7.5% w/v or about 7.5% w/v. In some embodiments, the stabilizer is sucrose at a concentration of 10% w/v or about 10% w/v. In some embodiments, the stabilizer is sucrose at a concentration of 12.5% w/v or about 12.5% w/v. In some embodiments, the stabilizer is sucrose at a concentration of 15% w/v or about 15% w/v. In some embodiments, the stabilizer is sucrose at a concentration of 20% w/v or about 20% w/v.

[173] A therapeutic complex of the disclosure can be, for example, an immediate release form or a controlled release formulation. An immediate release formulation can be formulated to allow the therapeutic complex to act rapidly. Non-limiting examples of immediate release formulations include readily dissolvable formulations. A controlled release formulation can be a pharmaceutical formulation that has been adapted such that release rates and release profdes of the active agent can be matched to physiological and chronotherapeutic requirements, or has been formulated to effect release of an active agent at a programmed rate. Non-limiting examples of controlled release formulations include granules, delayed release granules, hydrogels (e.g., of synthetic or natural origin), other gelling agents (e.g., gelforming dietary fibers), matrix-based formulations (e.g., formulations comprising a polymeric material having at least one active ingredient dispersed through), granules within a matrix, polymeric mixtures, and granular masses.

[174] In some embodiments, a controlled release formulation is a delayed release form. A delayed release form can be formulated to delay a therapeutic complex’s action for an extended period of time. A delayed release form can be formulated to delay the release of an effective dose of one or more therapeutic complexes, for example, for about 4, about 8, about 12, about 16, or about 24 hours.

[175] A controlled release formulation can be a sustained release form. A sustained release form can be formulated to sustain, for example, the therapeutic complex’s action over an extended period of time. A sustained release form can be formulated to provide an effective dose of any therapeutic complex described herein (e.g., provide a physiologically effective blood profde) over about 4, about 8, about 12, about 16, or about 24 hours.

[176] A therapeutic complex disclosed herein can be produced by various methods in any quantity. For example, a therapeutic complex disclosed herein can be produced in an amount of about 1 microgram, about 1 milligram, about 1 gram, about 1 kilogram, or more. Non-limiting examples of production methods include in vitro transcription methods, polymerase chain transcription (PCT), recombinant overexpression (e.g., in E. coli, R. sulfidophilum, or other in vitro systems), transfer RNA (tRNA) scaffold methods, enzymatic methods, chemical methods, solid-phase oligonucleotide synthesis, solid-phase chemical synthesis, ribozyme cleavage methods, T4 ligation methods, position- selective labeling of RNA (PLOR), T7 RNA polymerase in vitro methods, T3 RNA polymerase in vitro methods, SP6 RNA polymerase in vitro methods, phosphoramidite chemistry, cell-free nucleic acid expression methods, or a combination thereof. Non-limiting examples of purification methods include precipitation and solvent extraction, ultracentrifugation, polyacrylamide gel electrophoresis (PAGE), liquid chromatography (e.g., reversed-phase ion-pairing HPLC (RP-IP-HPLC), ion-exchange HPLC (IE-HPLC), ion-exchange fast-performance liquid chromatography (IE-FPLC), affinity chromatography (e.g., systematic evolution of ligands by exponential enrichment (SELEX), and sizeexclusion chromatography (SEC)), or a combination thereof. Purification methods can be used to achieve varying degrees of purity of a therapeutic complex disclosed herein, e.g., at least 80% purity, at least 85% purity, at least 90% purity, at least 91% purity, at least 92% purity, at least 93% purity, at least 94% purity, at least 95% purity, at least 96% purity, at least 97% purity, at least 98% purity, at least 99% purity, or at least 99.99%.

[177] Activity of the therapeutic complex disclosed herein can be detected with various protein activity assays, such as western blot, flow cytometry, immunofluorescence, immunoprecipitation, ELISA, and the like. In some embodiments, an antibody such as an antibody-HRP conjugate or antibody fluorophore conjugate is used for the protein activity assays.

[178] Methods for the preparation of compositions comprising the therapeutic complex described herein include formulating the therapeutic complex with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid, or liquid composition. Solid compositions include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Liquid compositions include, for example, solutions in which a therapeutic complex is dissolved, emulsions comprising a therapeutic complex, or a solution containing liposomes, micelles, nanoparticles, vesicles, microvesicles, or nanovesicles comprising the therapeutic complex as disclosed herein. Semi-solid compositions include, for example, gels, suspensions, and creams. The compositions can be in liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or as emulsions. These compositions can also contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically acceptable additives.

[179] Compositions of the present disclosure can be packaged as a kit. In some embodiments, the present disclosure provides a kit comprising a therapeutic complex disclosed herein, or a pharmaceutically acceptable salt thereof, and written instructions on use of the kit in the treatment of a condition described herein.

Characterization and Assessment

[180] In some embodiments, properties and/or activities of provided agents, complexes, compounds, and compositions thereof can be characterized and/or assessed using various technologies available to those skilled in the art, e.g., biochemical assays, cell-based assays, animal models, clinical trials, etc. Certain useful technologies are described in the Examples. Those skilled in the art reading the present disclosure will readily appreciate that other technologies, e.g., in vitro models (e.g., cell lines) for various conditions, disorders, or diseases (e.g., cancer), animal models for various conditions, disorders, or diseases (e.g., cancer), clinical trials, etc. may be designed and/or utilized to assess provided technologies (e.g., agents, complexes, compounds, and compositions thereof, methods, etc.) in accordance with the present disclosure. Biological Applications

[181] As appreciated by those skilled in the art, antigen-recognizing peptides, antibodies or antigenbinding fragments thereof, conjugates, complexes, and compositions provided herein are useful for many purposes. In some embodiments, provided technologies (e.g., antigen-recognizing peptides, antibodies, conjugates, complexes, compositions, methods) provided herein are useful for treating various conditions, disorders, or diseases, e.g., cancer, e.g., prostate cancer, in a subject. In some embodiments, technologies provided herein are useful for treating one or more bone metastases. In some embodiments, provided technologies provided herein are useful for targeting antibodies (e.g., anti-PSMA antibodies), conjugates (e.g., antibody-drug conjugates), complexes, or compositions thereof to bone. In some embodiments, provided technologies provided herein are useful for killing cancer cells, e.g., prostate cancer cells, in a system, e.g., a subject.

[182] In some embodiments, the present disclosure provides a method for administering or delivering an effective amount of an antigen-recognizing peptide, antibody, conjugate, complex, or composition thereof to a system. In some embodiments, the present disclosure provides a method for killing a cancer cell in a system, comprising administering or delivering an effective amount of an antigen-recognizing peptide, antibody, conjugate, complex, or composition thereof to the system.

[183] In some embodiments, a system comprises PSMA. In some embodiments, a system expresses PSMA, e.g., PSMA polypeptides.

[184] In some embodiments, a system comprises or is an in vitro system. In some embodiments, a system comprises or is an in vivo system.

[185] In some embodiments, a system comprises or is a cell. In some embodiments, a system comprises or is a population of cells. In some embodiments, a cell is a tumor cell. In some embodiments, a cell is a cancer cell. In some embodiments, a cell is a prostate cancer cell. In some embodiments, a cell comprises PSMA. In some embodiments, a cell is a PSMA-expressing cell.

[186] In some embodiments, a system comprises or is a tissue. In some embodiments, a system comprises or is an organ. In some embodiments, a system comprises or is an organism. In some embodiments, a system comprises or is a subject. In some embodiments, a system comprises or is an animal. In some embodiments, a system comprises or is a mammal, e.g., a mouse, rat, monkey, etc. In some embodiments, a system is a human.

[187] Various conditions, disorders, or diseases may be treated with provided technologies (e.g., antigen-recognizing peptides, antibodies, complexes, compositions, and methods). In some embodiments, a condition, disorder, or disease is a tumor. In some embodiments, a condition, disorder, or disease is a cancer. In some embodiments, a condition, disorder, or disease is a cancer described herein. In some embodiments, a condition, disorder, or disease is a prostate cancer. In some embodiments, a condition, disorder, or disease is a cancer associated with expression of PSMA. In some embodiments, a condition, disorder, or disease is a bone metastasis. [188] In some embodiments, a condition, disorder, or disease that can be treated in accordance with the present disclosure is described in WO 2019/191728.

[189] In some embodiments, the present disclosure provides methods for treating a condition, disorder, or disease in a subject comprising administering to the subject a therapeutically effective amount of an antibody, conjugate, complex, or composition provided herein. In some embodiments, the present disclosure provides methods for treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of an antibody, conjugate, complex, or composition provided herein. In some embodiments, the present disclosure provides methods for treating a tumor in a subject comprising administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof, conjugate, complex, or compositions provided herein.

[190] In some embodiments, the present disclosure provides methods for treating bone metastasis and/or tumors resulting from a cancer or a histological type of cancer in a subject comprising administering to the subject a therapeutically effective amount of a bone-targeting conjugate comprising bisphosphonate (BP) attached to a polypeptide or protein. In some embodiments, the present disclosure provides methods for treating bone metastasis and/or tumors resulting from a cancer or a histological type of cancer in a subject comprising administering to the subject a therapeutically effective amount of a bone-targeting conjugate comprising bisphosphonate (BP) conjugated to a polypeptide or protein. In some embodiments, the present disclosure provides methods for treating bone metastasis and/or tumors in a subject comprising administering to the subject a therapeutically effective amount of a bonetargeting conjugate comprising aspartic acid(s) attached to a polypeptide or protein. In some embodiments, the present disclosure provides methods for treating bone metastasis and/or tumors in a subject comprising administering to the subject a therapeutically effective amount of a bone-targeting conjugate comprising aspartic acid(s) conjugated to a polypeptide or protein.

[191] In some embodiments, the present disclosure provides a method for treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a bone-targeted antigenrecognizing peptide or antigen-recognizing fragment thereof. In some embodiments, the present disclosure provides a method for treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a bone-targeted antibody or antigen-binding fragment thereof.

[192] In some embodiments, the present disclosure provides a method for treating a tumor in a subject comprising administering to the subject a therapeutically effective amount of a bone-targeted antigenrecognizing peptide or antigen-recognizing fragment thereof. In some embodiments, the present disclosure provides a method for treating a tumor in a subject comprising administering to the subject a therapeutically effective amount of a bone-targeted antibody or antigen-binding fragment thereof.

[193] In some embodiments, the present disclosure provides a method for treating bone metastasis in a subject comprising administering to the subject a therapeutically effective amount of a bone-targeted antigen-recognizing peptide or antigen-recognizing fragment thereof. In some embodiments, the present disclosure provides a method for treating bone metastasis in a subject comprising administering to the subject a therapeutically effective amount of a bone-targeted antibody or antigen-binding fragment thereof.

[194] In some embodiments, an antigen-recognizing peptide, antibody, conjugate, complex, or composition provided herein may be utilized in combination with another therapy, e.g., an additional therapeutic agent.

[195] In some embodiments, a method for treating provided herein further comprises administering to a subject a therapeutically effective amount of one or more additional therapeutic agents. In some embodiments, one or more additional therapeutic agents comprise one or more therapeutic agents described herein. In some embodiments, one or more additional therapeutic agents comprise one or more chemotherapeutic agents described herein.

Administration

[196] In practicing the methods of treatment or use provided herein, therapeutically effective amounts of the therapeutic complex described herein are administered in pharmaceutical compositions to a subject having a disease or condition to be treated. In some embodiments, the subject is a mammal such as a human. Non-limiting examples of possible subjects for administration include the following. Subjects can be humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; and laboratory animals including rats, mice, and guinea pigs. A subject can be of any age. Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants, and neonates.

[197] A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic complex used, and other factors.

[198] A pharmaceutical composition disclosed herein can be administered in a therapeutically- effective amount by various forms and routes including, for example, parenteral, intravenous injection, intravenous infusion, subcutaneous injection, subcutaneous infusion, intramuscular injection, intramuscular infusion, intradermal injection, intradermal infusion, intraperitoneal injection, intraperitoneal infusion, intracerebral injection, intracerebral infusion, subarachnoid injection, subarachnoid infusion, intraocular injection, intraspinal injection, intrastemal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, rectal administration, intraarterial administration, intrathecal administration, inhalation, intralesional administration, intradermal administration, epidural administration, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa), intracapsular administration, subcapsular administration, intracardiac administration, transtracheal administration, subcuticular administration, subarachnoid administration, subcapsular administration, intraspinal administration, or intrastemal administration. [199] A pharmaceutical composition can be administered in a local manner, for example, via injection of the therapeutic complex directly into an organ, optionally in a depot or sustained release formulation or implant. A pharmaceutical composition can be provided in the form of a rapid release formulation, in the form of an extended-release formulation, or in the form of an intermediate release formulation. A rapid release form can provide an immediate release. An extended-release formulation can provide a controlled release or a sustained delayed release.

[200] A therapeutic complex described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the composition containing a therapeutic complex can vary. For example, a therapeutic complex can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to lessen or reduce a likelihood of the occurrence of the disease or condition. A therapeutic complex and composition can be administered to a subject during or as soon as possible after the onset of the symptoms. The administration of a therapeutic complex can be initiated within the first 48 hours of the onset of the symptoms, within the first 24 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, or within 3 hours of the onset of the symptoms. The initial administration can be via any route practical, such as by any route described herein using any formulation described herein.

[201] A therapeutic complex can be administered as soon as is practical after the onset of a disease or condition is detected or suspected, and for a length of time necessary for the treatment of the disease, such as, for example, from about 1 month to about 3 months. In some embodiments, the length of time a therapeutic complex can be administered can be about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 3 months, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 4 months, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 5 months, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months about 23 months, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, about 12 years, about 13 years, about 14 years, about 15 years, about 16 years, about 17 years, about 18 years, about 19 years, about 20 years, about 21 years, about 22 years, about 23 years, about 24 years, or about 25 years.

[202] A therapeutic complex described herein can be administered at any interval desired. The administration of the therapeutic complex can have regular or irregular dosing schedules to accommodate either the person administering the therapeutic complex or the subject receiving the therapeutic complex. For example, the therapeutic complex can be administered twice a day, once a day, five times a week, four times a week, three times a week, two times a week, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every five weeks, once every six weeks, once every eight weeks, once every two months, once every twelve weeks, once every three months, once every four months, once every six months, once a year, or less frequently. In some embodiments, administration is every other week.

[203] The amount administered can be of the same amount in each dose or the dosage can vary between doses. For example, a first amount can be administered in the morning and a second amount can be administered in the evening.

[204] Multiple therapeutic complexes disclosed herein can be administered in any order or simultaneously. If simultaneously, the therapeutic complexes can be provided in a single, unified form, or in multiple forms, for example, as multiple separate injections or infusions. The therapeutic complexes can be packed together or separately, in a single package or in a plurality of packages. One or all of the therapeutic complexes can be given in multiple doses. If not simultaneous, the timing between the multiple doses can vary to as much as about a month.

[205] The length of treatment can vary for each subj ect. Amounts effective for this use can vary based on the severity and course of the disease or condition, previous therapy, the subject’s health status, weight, and response to the drugs, and the judgment of the treating physician.

Dosing

[206] A therapeutic complex disclosed herein can be administered via subcutaneous or intravenous injection. The volume of an injection can be about 0. 1 mb, about 0.2 mb, about 0.3 mb, about 0.4 mb, about 0.5 mb, about 0.6 mb, about 0.7 mb, about 0.8 mb, about 0.9 mb, about 1 mb, about 1.1 mb, about 1.2 mb, about 1.3 mb, about 1.4 mb, about 1.5 mb, about 1.6 mb, about 1.7 mb, about 1.8 mb, about 1.9 mb, about 2 mb, about 2.1 mb, about 2.2 mb, about 2.3 mb, about 2.4 mb, about 2.5 mb, about 2.6 mb, about 2.7 mb, about 2.8 mb, about 2.9 mb, or about 3 mb.

[207] A therapeutic complex disclosed herein can be administered at a dosage of about 0.0001 mg/kg to about 1000 mg/kg, about 0.001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 20 mg/kg, about 0.02 mg/kg to about 7 mg/kg, about 0.03 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 3 mg/kg, about 0.1 mg/kg to about 50 mg/kg, about 0.1 mg/kg to about 0.5 mg/kg, about 0.2 mg/kg to about 0.6 mg/kg, about 0.3 mg/kg to about 0.7 mg/kg, about 0.4 mg/kg to about 0.8 mg/kg, about 0.1 mg/kg to about 0.9 mg/kg, about 0.01 mg/kg to about 50 mg/kg, about 0. 1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, or about 3 mg/kg to about 7 mg/kg by mass of the subject.

[208] A therapeutic complex described herein can be administered in any amount necessary or convenient. For example, a therapeutic complex described herein can be administered in an amount from about 0.05 mg to about 300 mg, about 0.1 mg to about 300 mg, about 0.1 mg to about 200 mg, about 0.1 mg to about 100 mg, about 0.05 mg to about 1.5 mg, about 0.1 mg to about 1.5 mg, about 0.05 mg to about 1 mg, about 1 mg to about 1.5 mg, about 0.5 mg to about 6 mg, about 1 mg to about 4 mg, about 2 mg to about 10 mg, about 10 mg to about 30 mg, about 30 mg to about 50 mg, about 50 mg to about 70 mg, about 70 mg to about 100 mg, or about 0. 1 mg to about 1 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.11 mg, about 0.12 mg, about 0.13 mg, about 0.14 mg, about 0.15 mg, about 0.16 mg, about 0.17, mg, about 0.18 mg, about 0.19 mg, about 0.2 mg, about 0.21 mg, about 0.22 mg, about 0.23 mg, about 0.24 mg, about 0.25 mg, about 0.26 mg, about 0.27, mg, about 0.28 mg, about 0.29 mg, about 0.3 mg, about 0.31 mg, about 0.32 mg, about 0.33 mg, about 0.34 mg, about 0.35 mg, about 0.36 mg, about 0.37, mg, about 0.38 mg, about 0.39 mg, about 0.4 mg, about 0.41 mg, about 0.42 mg, about 0.43 mg, about 0.44 mg, about 0.45 mg, about 0.46 mg, about 0.47, mg, about 0.48 mg, about 0.49 mg, about 0.5 mg, about 0.51 mg, about 0.52 mg, about 0.53 mg, about 0.54 mg, about 0.55 mg, about 0.56 mg, about 0.57, mg, about 0.58 mg, about 0.59 mg, about 0.6 mg, about 0.61 mg, about 0.62 mg, about 0.63 mg, about 0.64 mg, about 0.65 mg, about 0.66 mg, about 0.67, mg, about 0.68 mg, about 0.69 mg, about 0.7 mg, about 0.71 mg, about 0.72 mg, about 0.73 mg, about 0.74 mg, about 0.75 mg, about 0.76 mg, about 0.77, mg, about 0.78 mg, about 0.79 mg, about 0.8 mg, about 0.81 mg, about 0.82 mg, about 0.83 mg, about 0.84 mg, about 0.85 mg, about 0.86 mg, about 0.87, mg, about 0.88 mg, about 0.89 mg, about 0.9 mg, about 0.91 mg, about 0.92 mg, about 0.93 mg, about 0.94 mg, about 0.95 mg, about 0.96 mg, about 0.97, mg, about 0.98 mg, about 0.99 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, or about 300 mg per dose for a subject by any route of administration.

[209] Pharmaceutical compositions described herein can be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more therapeutic complexes. The unit dosage can be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged injectables, vials, or ampoules. Aqueous suspension compositions can be packaged in singledose non-reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with or without a preservative. Formulations for parenteral injection can be presented in unit dosage form, for example, in ampoules, or in multi -dose containers with a preservative. [210] In some embodiments, the disclosure provides a complex comprising: an antigen-recognizing compound; a target agent linked to the antigen-recognizing compound; and a bone-targeting moiety linked to the antigen-recognizing compound, wherein the antigen-recognizing compound has binding affinity specific for an antigen expressed in prostate cancer cells. In some embodiments, the target agent is linked to the antigen-recognizing compound by a covalent bond and the bone-targeting moiety is linked to the antigen-recognizing compound by a covalent bond. In some embodiments, the antigenrecognizing compound comprises an antigen-recognizing peptide. In some embodiments, the antigenrecognizing compound comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-recognizing compound comprises a small molecule. In some embodiments, the antigen-recognizing compound comprises a molecule that binds to PSMA. In some embodiments, the antigen-recognizing compound comprises an inhibitor of PSMA. In some embodiments, the antigen-recognizing compound comprises DUPA. In some embodiments, the antigen-recognizing compound comprises PSMA-617. In some embodiments, the antigen-recognizing compound comprises an antibody against PSMA. In some embodiments, the antigen-recognizing compound comprises J591. In some embodiments, the antigen-recognizing compound comprises an antibody against STEAP1. In some embodiments, the antigen-recognizing compound comprises an antibody against STEAP2. In some embodiments, the target agent comprises an anti-cancer agent. In some embodiments, the target agent comprises a small molecule. In some embodiments, the target agent comprises an antibody. In some embodiments, the target agent comprises an antibody against CD3. In some embodiments, the target agent comprises an antibody that suppresses recruitment of T Cells. In some embodiments, the target agent comprises an immune stimulant. In some embodiments, the target agent comprises a STING agonist. In some embodiments, the target agent comprises a TLR agonist. In some embodiments, the target agent comprises a cytotoxic agent. In some embodiments, the target agent comprises MMAE. In some embodiments, the target agent comprises MMAF. In some embodiments, the target agent comprises deruxtecan. In some embodiments, the target agent comprises a radioligand. In some embodiments, the target agent comprises an androgen receptor inhibitor. In some embodiments, the target agent comprises Enzalutamide. In some embodiments, the bonetargeting moiety comprises a bisphosphonate moiety. In some embodiments, the bone -targeting moiety comprises an alendronate moiety. In some embodiments, the bone-targeting moiety comprises an oligomer of species that are negatively charged at physiological pH. In some embodiments, the bonetargeting moiety comprises an oligomer of amino acid residues. In some embodiments, the bonetargeting moiety comprises an oligomer of aspartic acid residues. In some embodiments, the bonetargeting moiety comprises sequentially at least three aspartic acid residues. In some embodiments, the bone-targeting moiety comprises sequentially at least six aspartic acid residues. In some embodiments, the bone -targeting moiety is L-Asp⁶. In some embodiments the disclosure provides a method of treating a condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of any complex above. In some embodiments, the condition is a cancer. In some embodiments, the condition is a bone tumor. In some embodiments, the condition is a bone metastasis. In some embodiments, the condition is a bone metastasis from a prostate cancer.

[211] In some embodiments, the disclosure provides a complex comprising: an antigen-recognizing compound; a target agent linked to the antigen-recognizing compound; and a bone-targeting moiety linked to the antigen-recognizing compound, wherein the antigen-recognizing compound has binding affinity specific for an antigen expressed in osteosarcoma cells. In some embodiments, the target agent is linked to the antigen-recognizing compound by a covalent bond and the bone-targeting moiety is linked to the antigen-recognizing compound by a covalent bond. In some embodiments, the antigenrecognizing compound comprises an antigen-recognizing peptide. In some embodiments, the antigenrecognizing compound comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-recognizing compound comprises a small molecule. In some embodiments, the antigen-recognizing compound comprises an antibody against LRRC15. In some embodiments, the antigen-recognizing compound comprises Samrotamab. In some embodiments, the antigenrecognizing compound comprises an antibody against IFITM5. In some embodiments, the target agent comprises an anti-cancer agent. In some embodiments, the target agent comprises a small molecule. In some embodiments, the target agent comprises an antibody. In some embodiments, the target agent comprises an antibody against CD3. In some embodiments, the target agent comprises an antibody that suppresses recruitment of T Cells. In some embodiments, the target agent comprises an immune stimulant. In some embodiments, the target agent comprises a STING agonist. In some embodiments, the target agent comprises a TLR agonist. In some embodiments, the target agent comprises a cytotoxic agent. In some embodiments, the target agent comprises MMAE. In some embodiments, the target agent comprises MMAF. In some embodiments, the target agent comprises deruxtecan. In some embodiments, the target agent comprises a radioligand. In some embodiments, the target agent comprises lutetium 177. In some embodiments, the target agent comprises an androgen receptor inhibitor. In some embodiments, the target agent comprises Enzalutamide. In some embodiments, the bone-targeting moiety comprises a bisphosphonate moiety. In some embodiments, the bone -targeting moiety comprises an alendronate moiety. In some embodiments, the bone-targeting moiety comprises an oligomer of species that are negatively charged at physiological pH. In some embodiments, the bone-targeting moiety comprises an oligomer of amino acid residues. In some embodiments, the bonetargeting moiety comprises an oligomer of aspartic acid residues. In some embodiments, the bonetargeting moiety comprises sequentially at least three aspartic acid residues. In some embodiments, the bone-targeting moiety comprises sequentially at least six aspartic acid residues. In some embodiments, the bone-targeting moiety is L-Asp⁶. In some embodiments, the disclosure provides a method of treating a condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of any complex above. In some embodiments, the condition is a cancer. In some embodiments, the condition is a bone tumor. In some embodiments, the condition is a bone metastasis. In some embodiments, the condition is a bone metastasis from an osteosarcoma.

Exemplary Embodiments

[212] Among other things, the present disclosure provides the following Exemplary Embodiments:

1. A complex comprising: an antigen-recognizing compound; a target agent linked to the antigenrecognizing compound; and a bone-targeting moiety linked to the antigen-recognizing compound, wherein the antigen-recognizing compound has binding affinity specific for an antigen expressed in prostate cancer cells.

2. The complex of embodiment 1, wherein the target agent is linked to the antigen-recognizing compound by a covalent bond and the bone-targeting moiety is linked to the antigen-recognizing compound by a covalent bond.

3. The complex of any one of the preceding embodiments, wherein the antigen-recognizing compound comprises an antigen-recognizing peptide.

4. The complex of any one of the preceding embodiments, wherein the antigen-recognizing compound comprises an antibody or an antigen-binding fragment thereof.

5. The complex of any one of the preceding embodiments, wherein the antigen-recognizing compound comprises a small molecule.

6. The complex of any one of the preceding embodiments, wherein the antigen-recognizing compound comprises a molecule that binds to PSMA.

7. The complex of any one of the preceding embodiments, wherein the antigen-recognizing compound comprises an inhibitor of PSMA.

8. The complex of any one of the preceding embodiments, wherein the antigen-recognizing compound comprises DUPA.

9. The complex of any one of the preceding embodiments, wherein the antigen-recognizing compound comprises PSMA-617.

10. The complex of any one of the preceding embodiments, wherein the antigen-recognizing compound comprises an antibody against PSMA.

11. The complex of any one of the preceding embodiments, wherein the antigen-recognizing compound comprises J591.

12. The complex of any one of the preceding embodiments, wherein the antigen-recognizing compound comprises an antibody against STEAP1.

13. The complex of any one of the preceding embodiments, wherein the antigen-recognizing compound comprises an antibody against STEAP2.

14. The complex of any one of the preceding embodiments, wherein the target agent comprises an anti -cancer agent. 15. The complex of any one of the preceding embodiments, wherein the target agent comprises a small molecule.

16. The complex of any one of the preceding embodiments, wherein the target agent comprises an antibody.

17. The complex of any one of the preceding embodiments, wherein the target agent comprises an antibody against CD3.

18. The complex of any one of the preceding embodiments, wherein the target agent comprises an antibody that suppresses recruitment of T Cells.

19. The complex of any one of the preceding embodiments, wherein the target agent comprises an immune stimulant.

20. The complex of any one of the preceding embodiments, wherein the target agent comprises a STING agonist.

21. The complex of any one of the preceding embodiments, wherein the target agent comprises a TLR agonist.

22. The complex of any one of the preceding embodiments, wherein the target agent comprises a cytotoxic agent.

23. The complex of any one of the preceding embodiments, wherein the target agent comprises

MMAE.

24. The complex of any one of the preceding embodiments, wherein the target agent comprises

MMAF.

25. The complex of any one of the preceding embodiments, wherein the target agent comprises deruxtecan.

26. The complex of any one of the preceding embodiments, wherein the target agent comprises a radioligand.

27. The complex of any one of the preceding embodiments, wherein the target agent comprises lutetium 177.

28. The complex of any one of the preceding embodiments, wherein the target agent comprises an androgen receptor inhibitor.

29. The complex of any one of the preceding embodiments, wherein the target agent comprises Enzalutamide.

30. The complex of any one of the preceding embodiments, wherein the target agent comprises a TGFB antibody.

31. The complex of embodiment 30, wherein the TGFB antibody is a bispecific TGFB antibody.

32. The complex of any one of the preceding embodiments, wherein the target agent comprises a TGFB inhibitor. 33. The complex of any one of the preceding embodiments, wherein the bone-targeting moiety comprises a bisphosphonate moiety.

34. The complex of any one of the preceding embodiments, wherein the bone-targeting moiety comprises an alendronate moiety.

35. The complex of any one of the preceding embodiments, wherein the bone-targeting moiety comprises an oligomer of species that are negatively charged at physiological pH.

36. The complex of any one of the preceding embodiments, wherein the bone-targeting moiety comprises an oligomer of amino acid residues.

37. The complex of any one of the preceding embodiments, wherein the bone-targeting moiety comprises an oligomer of aspartic acid residues.

38. The complex of any one of the preceding embodiments, wherein the bone-targeting moiety comprises sequentially at least three aspartic acid residues.

39. The complex of any one of the preceding embodiments, wherein the bone-targeting moiety comprises sequentially at least six aspartic acid residues.

40. The complex of any one of the preceding embodiments, wherein the bone-targeting moiety is L-Asp⁶.

41. A method of treating a condition, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a complex of any one of embodiments 1-40.

42. The method of embodiment 41, wherein the condition is a cancer.

43. The method of embodiment 41, wherein the condition is a bone tumor.

44. The method of embodiment 41, wherein the condition is a bone metastasis.

45. The method of embodiment 41, wherein the condition is a bone metastasis from a prostate cancer.

46. A complex comprising: an antigen-recognizing compound; a target agent linked to the antigenrecognizing compound; and a bone-targeting moiety linked to the antigen-recognizing compound, wherein the antigen-recognizing compound has binding affinity specific for an antigen expressed in osteosarcoma cells.

47. The complex of embodiment 46, wherein the target agent is linked to the antigen-recognizing compound by a covalent bond and the bone-targeting moiety is linked to the antigen-recognizing compound by a covalent bond.

48. The complex of any one of embodiments 46-47, wherein the antigen-recognizing compound comprises an antigen-recognizing peptide.

49. The complex of any one of embodiments 46-48, wherein the antigen-recognizing compound comprises an antibody or an antigen-binding fragment thereof.

50. The complex of any one of embodiments 46-49, wherein the antigen-recognizing compound comprises a small molecule. 51. The complex of any one of embodiments 46-50, wherein the antigen-recognizing compound comprises an antibody against LRRC15.

52. The complex of any one of embodiments 46-51, wherein the antigen-recognizing compound comprises Samrotamab.

53. The complex of any one of embodiments 46-52, wherein the antigen-recognizing compound comprises an antibody against IFITM5.

54. The complex of any one of embodiments 46-53, wherein the target agent comprises an anticancer agent.

55. The complex of any one of embodiments 46-54, wherein the target agent comprises a small molecule.

56. The complex of any one of embodiments 46-55, wherein the target agent comprises an antibody.

57. The complex of any one of embodiments 46-56, wherein the target agent comprises an antibody against CD3.

58. The complex of any one of embodiments 46-57, wherein the target agent comprises an antibody that suppresses recruitment of T Cells.

59. The complex of any one of embodiments 46-58, wherein the target agent comprises an immune stimulant.

60. The complex of any one of embodiments 46-59, wherein the target agent comprises a STING agonist.

61. The complex of any one of embodiments 46-60, wherein the target agent comprises a TLR agonist.

62. The complex of any one of embodiments 46-61, wherein the target agent comprises a cytotoxic agent.

63. The complex of any one of embodiments 46-62, wherein the target agent comprises MMAE.

64. The complex of any one of embodiments 46-63, wherein the target agent comprises MMAF.

65. The complex of any one of embodiments 46-64, wherein the target agent comprises deruxtecan.

66. The complex of any one of embodiments 46-65, wherein the target agent comprises a radioligand.

67. The complex of any one of embodiments 46-66, wherein the target agent comprises lutetium 177.

68. The complex of any one of embodiments 46-67, wherein the target agent comprises an androgen receptor inhibitor.

69. The complex of any one of embodiments 46-68, wherein the target agent comprises

Enzalutamide. 70. The complex of any one of embodiments 46-69, wherein the target agent comprises a TGFB antibody.

71. The complex of embodiment 70, wherein the TGFB antibody is a bispecific TGFB antibody.

72. The complex of any one of embodiments 46-71, wherein the target agent comprises a TGFB inhibitor.

73. The complex of any one of embodiments 46-72, wherein the bone -targeting moiety comprises a bisphosphonate moiety.

74. The complex of any one of embodiments 46-73, wherein the bone-targeting moiety comprises an alendronate moiety.

75. The complex of any one of embodiments 46-74, wherein the bone-targeting moiety comprises an oligomer of species that are negatively charged at physiological pH.

76. The complex of any one of embodiments 46-75, wherein the bone-targeting moiety comprises an oligomer of amino acid residues.

77. The complex of any one of embodiments 46-76, wherein the bone-targeting moiety comprises an oligomer of aspartic acid residues.

78. The complex of any one of embodiments 46-77, wherein the bone-targeting moiety comprises sequentially at least three aspartic acid residues.

79. The complex of any one of embodiments 46-78, wherein the bone-targeting moiety comprises sequentially at least six aspartic acid residues.

80. The complex of any one of embodiments 46-79, wherein the bone -targeting moiety is L-Asp⁶.

81. A method of treating a condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of a complex of any one of embodiments 46-79.

82. The method of embodiment 81, wherein the condition is a cancer.

83. The method of embodiment 81 , wherein the condition is a bone tumor.

84. The method of embodiment 81, wherein the condition is a bone metastasis.

85. The method of embodiment 81, wherein the condition is a bone metastasis from an osteosarcoma.

86. An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to a prostate cancer-associated antigen, and the antibody or antigen-binding fragment thereof comprises a bone-targeting moiety.

87. The antibody or antigen-binding fragment thereof of embodiment 86, wherein the prostate cancer-associated antigen comprises or is KLK2, PSCA, TROP2, B7-H3, PSMA, STEAP1, STEAP2, TGFb, DLL-3, SSTR2, Epcam, GPC3, FAP, GRPR, ROR1, HER3, or TMEFF2.

88. The antibody or antigen-binding fragment thereof of embodiment 87, wherein the prostate cancer-associated antigen comprises or is PSMA. 89. The antibody or antigen-binding fragment thereof of any one of embodiments 86-88, wherein the antibody or antigen-binding fragment thereof comprises or is a monoclonal antibody or antigenbinding fragment thereof.

90. The antibody or antigen-binding fragment thereof of any one of embodiments 86-89, wherein the bone -targeting moiety comprises or is a bone -targeting peptide, optionally wherein the bonetargeting moiety is a negatively charged peptide and/or comprises one or more carboxylic peptides.

91. The antibody or antigen-binding fragment thereof of embodiment 90, wherein the bonetargeting peptide comprises or is Asp³, Asp⁴, Asp⁵, Asp⁶, Asp⁷, Asp⁸, Asp⁹, Asp¹⁰, Asp¹¹, Asp¹², Asp¹³, Asp¹⁴, or Asp¹⁵.

92. The antibody or antigen-binding fragment thereof of embodiment 90, wherein the bonetargeting peptide comprises or is Glu³, Glu⁴, Glu⁵, Glu⁶, Glu⁷, Glu⁸, Glu⁹, Glu¹⁰, Glu¹¹, Glu¹², Glu¹³, Glu¹⁴, or Glu¹⁵.

93. The antibody or antigen-binding fragment thereof of any one of embodiments 90-92, wherein the bone-targeting peptide is attached to a N-terminus or C-terminus of a light chain or a heavy chain of the antibody or antigen-binding fragment thereof.

94. The antibody or antigen-binding fragment thereof of any one of embodiments 90-92, wherein the bone -targeting peptide is inserted at an internal permissive site of the antibody or antigen-binding fragment thereof.

95. The antibody or antigen-binding fragment thereof of embodiment 94, wherein the internal permissive site is an internal permissive site of a light chain or an internal permissive site of a heavy chain.

96. The antibody or antigen-binding fragment thereof of embodiment 95, wherein the internal permissive site of the light chain is or corresponds to A153, optionally wherein the bone -targeting peptide is inserted following A 153.

97. The antibody or antigen-binding fragment thereof of embodiment 95, wherein the internal permissive site of the heavy chain is or corresponds to G118, P123, A165, P243, D283, N344, or G361, optionally wherein the bone-targeting peptide is inserted following G118, P123, A165, P243, D283, N344, or G361.

98. The antibody or antigen-binding fragment thereof of any one of embodiments 94-97, wherein 1 to (n + 15) amino-acid residues are deleted from the antibody or antigen-binding fragment thereof following the insertion of the bone-targeting peptide, wherein n is the number of amino-acid residues in the bone -targeting peptide.

99. The antibody or antigen-binding fragment thereof of any one of embodiments 86-98, wherein the antibody or antigen-binding fragment thereof comprises a second bone -targeting moiety. 100. The antibody or antigen-binding fragment thereof of embodiment 99, wherein the second bonetargeting moiety comprises or is a second bone -targeting peptide, optionally wherein the second bonetargeting moiety is a negatively charged peptide and/or comprises one or more carboxylic peptides.

101. The antibody or antigen-binding fragment thereof of embodiment 100, wherein the second bone-targeting peptide comprises or is Asp³, Asp⁴, Asp⁵, Asp⁶, Asp⁷, Asp⁸, Asp⁹, Asp¹⁰, Asp¹¹, Asp¹², Asp¹³, Asp¹⁴, or Asp¹⁵.

102. The antibody or antigen-binding fragment thereof of embodiment 100, wherein the second bone-targeting peptide comprises or is Glu³, Glu⁴, Glu⁵, Glu⁶, Glu⁷, Glu⁸, Glu⁹, Glu¹⁰, Glu¹¹, Glu¹², Glu¹³, Glu¹⁴, or Glu¹⁵.

103. The antibody or antigen-binding fragment thereof of any one of embodiments 99-102, wherein the second bone-targeting peptide is attached to a N-terminus or C-terminus of a light chain or a heavy chain of the antibody or antigen-binding fragment thereof.

104. The antibody or antigen-binding fragment thereof of any one of embodiments 99-102, wherein the second bone-targeting peptide is inserted at an internal permissive site of the antibody or antigenbinding fragment thereof.

105. The antibody or antigen-binding fragment thereof of embodiment 104, wherein the internal permissive site is an internal permissive site of a light chain or an internal permissive site of a heavy chain.

106. The antibody or antigen-binding fragment thereof of embodiment 105, wherein the internal permissive site of the light chain is or corresponds to A153, optionally wherein the bone -targeting peptide is inserted following A 153.

107. The antibody or antigen-binding fragment thereof of embodiment 105, wherein the internal permissive site of the heavy chain is or corresponds to G118, P123, A165, P243, D283, N344, or G361, optionally wherein the bone -targeting peptide is inserted following G118, P123, A165, P243, D283, N344, or G361.

108. The antibody or antigen-binding fragment thereof of any one of embodiments 104-107, wherein 1 to (n + 15) amino-acid residues are deleted from the antibody or antigen-binding fragment thereof following the insertion of the second bone -targeting peptide, wherein n is the number of amino-acid residues in the second bone-targeting peptide.

109. The antibody or antigen-binding fragment thereof of any one of embodiments 99-108, wherein the antibody or antigen-binding fragment thereof comprises one or more additional bone -targeting moieties, optionally wherein the one or more additional bone -targeting moieties comprise 3, 4, or 5 inserted bone -targeting moieties.

110. The antibody or antigen-binding fragment thereof of any one of embodiments 86-109, wherein the antibody or antigen-binding fragment thereof comprises a light chain comprising a sequence selected from Table 8. 111. The antibody or antigen-binding fragment thereof of any one of embodiments 86-110, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence selected from Table 9.

112. The antibody or antigen-binding fragment thereof of any one of embodiments 86-111, wherein the antibody or antigen-binding fragment thereof is associated with a target agent.

113. The antibody or antigen-binding fragment thereof of embodiment 112, wherein the antibody or antigen-binding fragment thereof is conjugated to the target agent.

114. The antibody or antigen-binding fragment thereof of embodiment 112 or l l3, wherein the target agent comprises an anti -cancer agent, an alkylating agent, an antitumor antibiotic, an antimetabolite, an antimitotic agent, a cytotoxic agent, a hormone, a nitrosourea, a plant alkaloid, a taxane, a radioligand, a chemotherapeutic agent, or any combination thereof.

115. The antibody or antigen-binding fragment thereof of embodiment 114, wherein the radioligand comprises an alpha emitter or a beta emitter.

116. The antibody or antigen-binding fragment thereof of embodiment 114, wherein the radioligand comprises actinium, lutetium, indium, radium, Pb-212, Cu-67, or thorium.

117. The antibody or antigen-binding fragment thereof of embodiment 114, wherein the target agent comprises calicheamicin, dertuxtecan, govitecan SN-38, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansinoid, SG3199, duocarmycin, exatecan, tubulysin, PNU- 159682, TOP2 inhibitor, TLR agonist, STING agonist, or any combination or derivative thereof.

118. The antibody or antigen-binding fragment thereof of embodiment 114, wherein the target agent comprises an androgen receptor degrader, integrin inhibitor, EZH2, adenosine receptor inhibitors (CD73), WNT pathway inhibitor, AKT inhibitor, steroidogenesis inhibitor (e.g., ODM-208), PARP inhibitor, immune activator (e.g., TLR3 agonist), BET inhibitor, pyrrolobenzodiazepine (PBD), MYC pathway inhibitor, RAS pathway inhibitor, PI3K inhibitor, NRG1 inhibitor, Amantin or any combination thereof.

119. The antibody or antigen-binding fragment thereof of any one of embodiments 86-118, wherein the antibody or antigen-binding fragment thereof comprises one or more bone targeting moieties comprising a combination of Asp and Glu residues.

120. A method of delivering a target agent to a cancer cell in a system, comprising administering or delivering to the system an effective amount of the antibody or antigen-binding fragment thereof of any one of embodiments 112-119.

121. A method of killing a cancer cell in a system, comprising administering or delivering to the system an effective amount of the antibody or antigen-binding fragment thereof of any one of embodiments 86-119.

122. The method of embodiment 120 or 121, wherein the system comprises or is a population of cells. 123. The method of embodiment 120 or 121, wherein the system comprises or is an animal.

124. The method of embodiment 120 or 121, wherein the system comprises or is a human.

125. A method of treating a condition, disorder, or disease, comprising administering or delivering to a subject suffering therefrom a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of embodiments 86-110.

126. The method of embodiment 125, wherein the condition, disorder, or disease comprises or is a cancer.

127. The method of embodiment 126, wherein the cancer comprises or is prostate cancer.

128. The method of any one of embodiments 125-127, wherein the condition, disorder, or disease comprises or is a bone metastasis.

129. The method of embodiment 125, wherein the condition, disorder, or disease comprises or is a tumor.

130. The method of embodiment 129, wherein the tumor is a prostate tumor.

131. The method of embodiment 129, wherein the tumor is a bone tumor.

132. The method of any one of embodiments 125-131, wherein the subject is a human.

133. The method of any one of embodiments 120-132, wherein the antibody or antigen-binding fragment thereof is delivered using one or more of a cell therapy, mRNA, LNP, or VLP.

134. A method of treating prostate cancer comprising administering or delivering to a subject suffering therefrom a therapeutically effective amount of an immune stimulating or immune checkpoint inhibiting antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a bone -targeting moiety.

135. The method of embodiment 134, wherein the antibody or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof of any one of embodiments 86-119.

EXAMPLES

[213] The following examples are included to demonstrate particular embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute particular modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

EXAMPLE 1: Proximity-induced, Site-specific Antibody Conjugation (pClick)

[214] To react selectively with an adjacent, native amino acid residue side chain such as that of cysteine or lysine, non-canonical amino acids (ncAAs) having side chains such as reactive halide, aryl ketone, Michael acceptor, aryl isothiocyanate, or aryl carbamate are developed. ncAAs are genetically incorporated into various proteins to enhance reactivity between proteins and small molecules or to capture transient protein-protein interactions. ncAAs allowing crosslinking to a proximal lysine residue are introduced into specific sites of peptides that have binding sites at the fragment crystallizable (Fc) or antigen-binding (Fab) fragment of antibodies. Upon binding to the antibody, the crosslinking ncAA- containing peptide allows proximity-induced covalent attachment of the crosslinking ncAA to the nearby lysine residue of the antibody. This method can be used to attach a number of functional reagents (e.g., antibody-drug conjugates, small molecule inhibitors, immune potentiators, STING agonist, and TLR agonist) covalently to any suitable antibodies (e.g., anti-PSMA, anti-STEAPl, anti-STEAP2, anti- LRRC15, an anti-PSCA antibody, an anti-TROP2 antibody, an anti-KLK2 antibody, an anti-TGFb antibody, an anti-DLL-3 antibody, an anti-SSTR2 antibody, an anti-Epcam antibody, an anti-GPC3 antibody, an anti-FAP antibody, an anti-GRPR antibody, an anti-RORl antibody, an anti-HER3 antibody, and anti-IFITM5).

[215] The B domain of protein A (FB protein) from Staphylococcus aureus is used to bind to the CH2-CH3 junction of the immunoglobulin G (IgG) antibody. An electrophilic ncAA-containing FB domain is used to label antibodies site-specifically.

[216] 4-fluorophenyl carbamate lysine (fPheK), an ncAA that can react with a proximal lysine side chain to form a stable crosslink, is site-specifically incorporated into the Leul8, Hisl9, Glu25, Glu26, Arg28, and Asn29 residues of the FB protein using a suitable ncAA technology. FB mutants containing fPheK (FB-fPheK) are purified by Ni-NTA chromatography and characterized by SDS-PAGE and ESIMS.

[217] PSMA-MMAE, a synthetic antineoplastic agent linked to a PSMA antibody selectively targeting prostate cancer cells, is incubated with 8 equivalents of each FB mutant separately in PBS buffer (pH 8.5) at 37 ° C for 48 h. Variant(s) with the highest crosslinking to PSMA-MMAE are selected. Reducing SDS-PAGE analysis of PSMA-MMAE incubated with the FB containing a mutation at the high crosslinking residue (e.g., FB-Leul8fPheK) is used to confirm the formation of the FB- heavy chain complex.

[218] Similar FB-fPheK complex(es) is generated comprising a ncAA with a functional target agent comprising:

(1) an antibody selectively targeting prostate cancer cells (e.g., anti-PSMA, PSMA617, anti- STEAPl, DUPA) or osteosarcoma cells (anti-LRRC15, Samrotamab, anti-IFITM5), and

(2) an anti -cancer agent, such as a toxin (e.g., MMAE, deruxtecan), a small molecule inhibitor, a radioligand (e.g., lutetium 177) or an immune response potentiator (e.g., anti-CD3, STING agonist, TLR agonist).

[219] Generality of the conjugation method developed above is determined by the conjugation efficiency and specificity to IgGs from different species and subclasses, including human IgGl, human IgG2, mouse IgGl, mouse IgG2a, and mouse IgG2b. [220] To allow intraoperative imaging and pharmacokinetic characterization of therapeutic agents, the FB-fPheK protein is functionalized with an Alexa Fluor™ 488 succinimidyl ester by nonspecific conjugation to lysine residues. After an overnight reaction, the resulting conjugate is buffer-exchanged into pH 8.5 PBS buffer and added to the native PSMA-MMAE antibody. The resulting Alexa Fluor™ 488-labeled PSMA-MMAE (PSMA-MMAE-488) is further purified by a protein-L column. The conjugation yield of PSMA-MMAE-488 is determined by SDS-PAGE analysis. UV transillumination is used to assess incorporation of the fluorophore.

[221] Antigen-binding ability of site-specific antibody modified with a functionalized FB-fPheK protein is determined with prostate cancer cell lines (e.g., DU145, PC3, and LNCaP). Prostate cancer cells are treated for 30 min with PSMA-MMAE-488 prepared above. Confocal fluorescent imaging is used to indicate cell-surface-association to PSMA-MMAE-488 by prostate cancer cells. PSMA- negative cells are used as negative control. The results indicate whether antibodies site -specifically modified with a functionalized FB-E25fPheK protein retain antigen-binding ability. Similar assays are conducted with site-specific antibody modified with a functionalized FB-fPheK protein targeting osteosarcoma cell lines (e.g., Saos-2).

EXAMPLE 2: Synthesis of ncAAs

[222] Synthesis of 4-fluorophenyl carbamate lysine (fPheK). Na-Boc-L-lysine (4 g, 16.2 mmol) is dissolved in 50 mb anhydrous DCM and then TEA (5.6 mb, 2.5 equiv.) is added at 0 °C. After stirring for 10 min, 4-fluorophenyl chloroformate (2.2 mb, 1.05 equiv.) is added dropwise, and the mixture is stirred at room temperature under a nitrogen atmosphere overnight. The mixture is poured into 50 mb H2O, and the pH is adjusted to 3 with 2 M aq. AcOH. The organic phase is separated and dried with Na2SC>4. The solvent is evaporated, and the intermediate is purified by flash column chromatography on silica (DCM:MeOH, 10: 1) and obtained. 40 mb fresh DCM is used to dissolve the intermediate followed by addition of 10 mb TFA. The solution is stirred at room temperature for 5 h. The solvent is evaporated. The crude product is dissolved in methanol and precipitated in Et2O. The product is dried under vacuum and obtained.

[223] Synthesis ofN-acryloyl-lysine (AcrK). Na-Boc-L-lysine (2.46 g, 10.0 mmol) and Na2COs (2.12 g, 20.0 mmol) is dissolved in 200 mb ethyl acetate/H2O (1: 1) at 0 °C. Acryloyl chloride (0.9 g, 11.0 mmol) is added dropwise over 10 min. The solution is stirred at room temperature under a nitrogen atmosphere overnight. 2 M aq. AcOH is then added to adjust the pH to 3. The mixture is extracted with ethyl acetate (200 mb, 3 times). The organic phase is separated and dried with Na2SO4. 20 mb fresh DCM is used to dissolve the intermediate followed by addition of 5 mb TFA. The solution is stirred at room temperature for 5 h. The solvent is evaporated. The crude product is dissolved in methanol and precipitated in Et2O. The product is dried under vacuum and obtained.

[224] Synthesis of 2-amino-6-(6-bromohexanamido)hexanoic acid (BrC6K). Na-Boc-E-lysine (2.46g, 10.0 mmol) is dissolved in 200 mb mixture solvent of THF/DCM (1: 1) and DIEA (12.0 mmol, 2.1 mL) at 0 °C. 6-bromohexanoyl chloride (13 mmol, 2.0 mL) is added. The solution is stirred at room temperature under a nitrogen atmosphere overnight. 2 M aq. AcOH is then added to adjust the pH to 3. The mixture is extracted with ethyl acetate (200 mL, 3 times). The organic phase is separated and dried with Na2SC>4. The crude material is purified by flash silica gel chromatography using eluent solvent with DCM:MeOH (10: 1). The product is isolated. The pure product is dissolved in dichloromethane (15 mL) followed by addition of 5 mL TFA. The reaction mixture is stirred for 5 h. After the reaction is complete, the solvent is concentrated under reduced pressure. The residue is dissolved in methanol and precipitated in Et2O. The precipitate is washed with Et2O to give the final product.

EXAMPLE 3: Plasmid construction and expression, and purification of FB protein

[225] To generate the fPheK plasmid, the FPheKRS gene is generated by PCR using primers CY012 and CY013 (SEQ ID NO: 9-10) and inserted into the pUltra-MbPylRS plasmid using restriction enzyme Notl, creating pUltra-FPheKRS. The plasmids to express FB mutants are generated by site- directed mutagenesis using primers CY031, CY038, CY039, CY040, CY041 and CY042 (SEQ ID NO: 3-8), using pET22b-T5-FB as the template with QuickChange Lightning Multi Site-Directed Mutagenesis Kit.

[226] The pET22b-T5-FB-E25TAG and pUltra-FPheKRS plasmids are co-transformed into E. coli DH10B strains. Cells are grown in LB media, supplemented with ampicillin (50 ug/mL), spectinomycin (25 ug/mL) and 1 mM fPheK at 37 °C. When the OD reaches 0.6, 1 mM IPTG is added to the culture, and the culture is grown overnight at 30 °C. The cells are harvested by centrifugation at 4,700xg for 10 min and the proteins are purified on Ni-NTA resin.

[227] Site-specific antibody-FB protein conjugation is generated by co-incubating any antibody disclosed in Example 1 with eight equiv of FB mutants in pH 8.5 PBS buffer for 2 days. The resulting antibody conjugates are purified by protein-L column. The conjugation efficiency is analyzed using Image Quant TL.

[228] Alexa Fluor™ 488-labeled PSMA-MMAE conjugate and other site-specific antibody conjugates is generated by reacting FB-fPheK protein (20 uL, 2.3 mg/mL in DPBS buffer with Ca²⁺ and Mg²⁺, pH 8.5) with 10 equivalents of Alexa Fluor 488 carboxylic acid, succinimidyl ester at 37 °C for 12 hours. The resulting Alexa Fluor 488-labeled FB protein is then purified by Ni-NTA chromatography and buffer-exchanged to PBS buffer (pH 8.5) using an Amicon 3,000 molecular-weight-cutoff concentrator. The purified Alexa Fluor™488-labeled FB protein is reacted with PSMA-MMAE at 37 °C. for 2 days. The resulting conjugate is then purified by Protein-L column followed by adjusting pH to 7.0. The isolated protein is characterized by SDS-PAGE analysis followed by Coomassie staining. Protein concentration is measured using Coomassie Plus Protein Assay kit. Confocal fluorescent imaging of living cells is performed using Nikon AlR-si Laser Scanning Confocal Microscope (Japan), equipped with lasers of 405/488/561/638 nm. DiIC18 and Hoechst 33342 are prepared as 2 mM DMSO stock solution and 10 mg/mL water solution, respectively. The stock solution is diluted to the working concentration in complete medium (10 pM and 10 pg/mL, respectively). Cancer cells are incubated in complete medium at 37 °C in atmosphere containing 5% CO2.

[229] TABLE 2 shows DNA sequences of oligonucleotides.

EXAMPLE 4: Proximity-induced, Site-specific Antibody Conjugation (pClick) with Solid-Phase Peptide Synthesis

[230] The fPheK-labeled FB peptide from EXAMPLE 1 is prepared using genetic code expansion technology. Alternatively, the “pClick” technology can be used to generate proximity-induced sitespecific antibody conjugation antibody. A functional fragment of FB (SEQ ID NO: 2) (ssFB) is synthesized using Fmoc -based solid-phase peptide synthesis. Starting from rink amide resin, ssFB with N-terminal acetylation is stepwise synthesized with all the amino acid side chains protected. After the whole peptide is synthesized, the MMT (monomethoxytrityl) protection group is selectively removed with 10% AcOH solution (AcOH:TFEDCM=l:2:7). The exposed free amine is then reacted with 4- fluorophenyl chloroformate under mild basic conditions for fPheK formation.

[231] TFA and scavengers are used to cleave the peptide from the resin, and to remove and to quench all protections at the same time. The peptide is precipitated by ice-cold ether and further purified with HPLC and characterized by ESI-MS. To functionalize the peptide, the peptide is lyophilized and denatured with 8 M urea solution. A stepwise dialysis protocol is used to remove urea and refold the peptide. 32 equiv of ssFB peptide is then mixed with PSMA-MMAE for 2 days at 37 °C in PBS (pH 8.5) buffer. Reducing SDS-PAGE analysis is used to determine PSMA-MMAE-ssFB conjugate formation.

[232] Crystal structure of the antibody:FB complex is obtained. To provide selective introduction of fPheK to the ssFB peptide at residue position with highest crosslinking to PSMA-MMAE, a lysine residue protected with 4-methoxytrityl (Mmt) is first introduced to the residue, while the other lysine residues are orthogonally protected with tert-butyloxycarbonyl (Boc) groups. The resulting, full-length protected peptide is then coupled to an azido-lysine at the N-terminus, followed by selective deprotection of the residue. After site-specific, on-resin deprotection, the free residue is reacted with 4- fluorophenyl chloroformate to yield fPheK at the residue, followed by resin washing to remove excess 4-fluorophenyl chloroformate. Trifluoroacetic acid is then used to free the peptide from the resin to yield the final azide-labeled FB containing the fPheK residue mutation (AzFB).

[233] The antibody conjugation efficiency of the AzFB mutant is examined by incubating with 16 equivalents of AzFB for 48 h to yield PSMA-MMAE with an azide functional moiety (PSMA-MMAE- AzFB). Reducing SDS-PAGE analysis is used to confirm PSMA-MMAE-AzFB conjugate. Strain- promoted alkyne-azide cycloaddition (SPAAC) reaction is used to demonstrate that PSMA-MMAE- AzFB conjugate is reactive with various bicyclo[6.1.0]nonyne (BCN) reagents.

[234] Solid-phase synthesis is used to generate similar AzFB complex(es) comprising an azide- labeled FB with a functional agent comprising:

(1) an antibody selectively targeting prostate cancer cells (e.g., anti-PSMA, PSMA617, anti- STEAP1, DUPA) or osteosarcoma cells (anti-LRRC15, Samrotamab, anti-IFITM5), and

EXAMPLE 5: Generation of Bone-Directed, Site-Specific Antibody Conjugation using Aspartic Acid Oligomer

[235] To direct the proximity-induced, site-specific antibody conjugation generated in EXAMPLE 1 and/or EXAMPLE 4 to bone-metastasis, the antibody is modified to include a hydroxyapatite (HA)- binding peptide (TABLE 3, FIG. 1). Aspartic acid (Asp) peptides can favor binding to HA surface with higher levels of crystallinity. The HA-binding peptide comprises aspartic acids, such as at least four, five, six, seven, eight, nine, or ten aspartic acid residues (e.g., L-Asp⁶). L-Asp⁶ is inserted into permissive internal sites in the PSMA-MMAE (e.g., light chain, heavy chain, C-terminus, N-terminus). The number of L-Asp⁶ peptide sequences inserted can vary (e.g., two L-Asp⁶ peptides, three L-Asp⁶ peptides). The resulting PSMA-MMAE-Asp construct is expressed in ExpiCHO-S cells by transient transfection, followed by purification of immunoglobulins using protein G chromatography and analysis of expressed proteins by SDS-PAGE.

[236] Similar bone -directing modifications are made with other FB-fPheK and/or AzFB complexes disclosed in EXAMPLE 1 and EXAMPLE 4.

[237] TABLE 3 shows amino acid sequences of HA-binding peptides.

EXAMPLE 6: Generation of Bone-Directed Site-Specific Antibody Conjugation using bisphosphonate

[238] To generate bone-directing antibodies using conjugation to bisphosphonate (BP) molecules, a model is designed using the PSMA-MMAE and a BP drug Alendronate (ALN) (FIG. 2). pClick described in EXAMPLE 4 is used to generate PSMA-MMAE-ALN conjugates. PSMA-MMAE AzFB generated by pClick in EXAMPLE 4 is reacted with bicyclo[6.1.0]nonyne (BCN)-functionalized ALN. The resulting PSMA-MMAE-ALN is further purified on a desalting column and is characterized by SDS-PAGE and ESI-MS. Similar BP conjugates are generated with complexes disclosed in EXAMPLE 4.

EXAMPLE 7: Construction of Site-Specific Antibody- ALN conjugates

[239] A non-canonical amino acid azide-Lys is incorporated at the C terminus of the ssFB-fPheK peptide via solid-phase peptide synthesis described in EXAMPLE 4. After HPLC purification, the peptide is denatured with 6 M urea and stepwise dialyzed to remove urea and allow peptide refolding. After buffer-exchange into PBS (pH 8.5), 32 equiv of ssFB-azide peptide is co-incubated with PSMA- MMAE in PBS (pH 8.5) buffer at 37 °C for two days. The PSMA-MMAE-azide conjugate is then purified via a PD-10 desalting column to remove excess ssFB-azide. The PSMA-MMAE-azide conjugate is characterized by ESI-MS. 10 equiv of BCN-ALN is added to the solution at RT overnight to react selectively with the azide group on the conjugate. Finally, the ALN labeled antibody conjugate is purified via a PD-10 desalting column to remove excess ALN-BCN. The conjugate is characterized by ESI-MS. Similar antibody-ALN conjugates are generated with antibodies disclosed in EXAMPLE 4.

EXAMPLE 8: Antigen Binding and Specificity of Bone-Directed Site-Specific Antibody Conjugates

[240] To investigate the specificity of bone-directed, site-specific antibody conjugates generated in EXAMPLE 5, EXAMPLE 6, and EXAMPLE 7 (e.g., PSMA-MMAE-ALN, PSMA-MMAE-Asp, PSMA-ALN), flow cytometry analysis is performed with prostate cancer cell lines (e.g., LNCaP) and negative cell lines (e.g., PSMA-negative cells). Prostate cancer cells are resuspended and stained with site-specific antibody conjugates for 30 minutes at 4 °C. After staining, the cells are washed twice with PBS and incubated with Fluorescein (FITC) AffiniPure Goat Anti-Human IgG (H+L) for 30 minutes at 4 °C. Fluorescence intensity is determined with flow cytometry, with high intensity suggesting antibody specificity. Confocal fluorescent imaging is performed to confirm the antigen binding and specificity of the site-specific antibody conjugates. Prostate cancer cell lines (e.g., LNCaP) and negative cell lines are incubated for 30 minutes with fluorescein isothiocyanate (FITC)-labeled site specific antibody conjugates (e.g., PSMA-MMAE-ALN, PSMA-MMAE-Asp, PSMA-ALN) and are fixed by 4% paraformaldehyde for 15 minutes before imaging.

[241] In addition, site specific antibody conjugates (e.g., PSMA-MMAE-ALN, PSMA-MMAE-Asp, PSMA-ALN) are tested for selective cytotoxicity against prostate cancer cell lines (e.g., LNCaP) and negative cells (e.g., PSMA-negative cells). Cells are seeded in culture medium into 96-well plates at a density of 2 x 10³ cell/well and incubated overnight to allow attachment. Culture medium is removed and replaced by different concentrations of an antibody selectively targeting prostate cancer cells (e.g., anti-PSMA, PSMA617, anti-STEAPl, DUPA) or a site-specific antibody conjugate dissolved in culture medium and incubated for several days (e.g., 4 days). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H- tetrazolium bromide (MTT solution) is added to each well and incubated for 4 hours. Medium is aspirated and dimethylsufoxide (DMSO) is added to each well. The absorbance at 570 nm is measured by microplate reader to quantify living cells, and to see whether treatment with the site-specific antibody conjugate exhibits higher cytotoxic activity against prostate cancer cells than negative cells. The results indicate whether antibody modification with bone-targeting module (e.g., ALN, Asp) preserves the antigen binding and in vitro anti-tumor cell activity of the antibody.

[242] Similar assays are conducted with site-specific antibody conjugates modified with antibody selectively targeting osteosarcoma cells (e.g., anti-LRRC15, Samrotamab, anti-IFIM5), and osteosarcoma cells (e.g., Saos-2).

EXAMPLE 9: Evaluation of Site-Specific Antibody Conjugates to Target Bone in vitro [243] Ability for site-specific antibody conjugates (e.g., PSMA-MMAE-ALN, PSMA-MMAE-Asp, PSMA-ALN) to target bone is evaluated in vitro. Non-decalcified bone sections from C57BL/6 mice are incubated overnight at 4 °C with an antibody selectively targeting prostate cancer cells (e.g., anti- PSMA, PSMA617, anti-STEAPl, DUPA) or a site-specific antibody conjugate (e.g., PSMA-MMAE- ALN, PSMA-MMAE-Asp, PSMA-ALN). Next, FITC-labeled anti -human IgG is added. Before imaging with confocal laser scanning microscopy, the bone sections are further stained with xylenol orange to label bone. Confocal laser scanning microscopy is used to see whether a FITC signal is observed in sections stained with the site-specific antibody conjugate versus sections stained with the antibody only. Co-expression of xylenol orange with FITC is also observed to confirm the specific targeting of bone by the site-specific antibody conjugate compared to the unmodified antibody.

[244] To quantify the difference in affinity between binding of the site-specific antibody conjugate and unmodified antibody, the site-specific antibody conjugate or the antibody is incubated with hydroxyapatite or native bones of mice for 0.25, 0.5, 1, 2, 4, and 8 hours before centrifugation. Next, the absorbance of the supernatant is measured to calculate the percent binding to hydroxyapatite or native bones. Samples without hydroxyapatite or bone fragments are used as controls.

[245] Similar experiments are conducted with site-specific antibody conjugates modified with antibody selectively targeting osteosarcoma cells (e.g., anti-LRRC15, Samrotamab, anti-IFIM5), and osteosarcoma cells (e.g., Saos-2) for its ability to target bone in vitro.

EXAMPLE 10: Evaluation of Site-Specific Antibody Conjugates to Target Bone Metastases in vivo

[246] The ability for site-specific antibody conjugates (e.g., PSMA-MMAE-ALN, PSMA-MMAE- Asp, PSMA-ALN) to target bone metastases is evaluated in vivo. Intra-iliac artery (IIA) injection of prostate cancer cells (e.g., LNCaP) labeled with luciferase and red fluorescent protein (RFP) is performed into the right hind limbs of nude mice to establish bone micrometastases. Following micrometastases, the mice are treated with an antibody or agent selectively targeting prostate cancer cells (e.g., anti-PSMA, PSMA-617, anti-STEAPl, anti-STEAP2, DUPA) or a site-specific antibody conjugate (e g., PSMA-MMAE-ALN, PSMA-MMAE-Asp, PSMA-ALN, PSMA-MMAF-Asp) labeled with Cyanine 7.5 (Cy7.5)-hydroxysuccinimide ester. After administration (e.g., 24, 96, 168 hours) of the antibody or site-specific antibody conjugate, bones are harvested for ex vivo fluorescence imaging. Uptake of the antibody versus site-specific antibody conjugate into cancer-bearing bones are compared and evaluated via imaging.

[247] Alternatively, unlabeled site-specific antibody conjugate is administered into nude mice bearing cancer tumor in the right hind limb. Bones are harvested and sectioned. Bone sections are stained with FITC-labeled anti-human IgG, RFP, and DAPI to see whether the FITC signal correlates with the red fluorescence of cancer cells. A correlation suggests that site-specific antibody selectively targets the bone metastatic site but not healthy bone. [248] Next, the effect of site-specific antibody conjugates on the pharmacokinetics and neonatal Fc receptor (FcRn) binding of antibodies is evaluated. An antibody or agent selectively targeting prostate cancer cells (e.g., anti-PSMA, PSMA-617, anti-STEAPl, anti-STEAP2,DUPA) or a site-specific antibody conjugate (e g., PSMA-MMAE-ALN, PSMA-MMAE-Asp, PSMA-ALN, PSMA-MMAF- Asp) in PBS is injected retro-orbitally in mice. Serum is collected at regular intervals for 7 days and analyzed by antibody ELISA kit to measure the pharmacokinetics. FcRn binding is also evaluated using FcRn binding immunoassay kit.

[249] Similar experiments are conducted with site-specific antibody conjugates modified with antibody selectively targeting osteosarcoma cells (e.g., anti-LRRC15, Samrotamab, anti-IFITM5), and osteosarcoma cells (e.g., Saos-2).

EXAMPLE 11: Enhanced Therapeutic Efficacy of bone-directed, site-specific antibody conjugates against bone micrometastases

[250] To determine whether bone-directed, site-specific antibody conjugates (e.g., PSMA-MMAE- ALN, PSMA-MMAE-Asp, PSMA-ALN, PSMA-MMAF-Asp) represents a therapeutic approach for treating micrometastases of prostate cancer in the bone, a xenograft study is carried out in nude mice. Right hind limbs of nude mice are inoculated with prostate cancer cells (e.g., LNCaP) labeled with firefly luciferase by using intra-iliac artery (IIA) injection. After IIA injections (e.g., after five days), mice are treated with phosphate-buffered saline (PBS), bone targeting module (e.g., ALN, Asp), an antibody or agent selectively targeting prostate cancer cells (e.g., anti-PSMA, PSMA-617, anti- STEAPl, anti-STEAP2, DUPA), or a site-specific antibody conjugate (e.g., PSMA-MMAE-ALN, PSMA-MMAE-Asp, PSMA-ALN, PSMA-MMAF-Asp) via retro-orbital injection. Whole-body bioluminescence imaging and imaging of hind limbs are next performed to see whether mice treated with a site-specific antibody conjugate exhibit less increase in tumor sizes compared to those treated with a bone targeting module or antibody alone. Micro-computed tomography (microCT) and histology of bones are further conducted to evaluate whether a site-specific antibody conjugate reduces the number and extent of osteolytic lesions. Tumor size is analyzed by histomorphometric analysis of the bone sections, and reduction of tumor burden is confirmed by immunohistochemistry with cancer markers (e.g., PMSA). Bone degradation and bone resorption are measured by tartrate-resistant acid phosphatase (TRAP) staining and measurement of serum TRAcP 5b or calcium levels, respectively.

[251] To evaluate the therapeutic efficacy of site-directed antibody conjugates in the presence of both primary and secondary tumors, a xenograft study is carried out in nude mice, using both mammary fat pad and IIA injections. Luciferase-labeled cancer cells (e.g., LNCaP) are inoculated in the right hind limbs and non-labeled cancer cells are used for mammary fat pad injection. After six days of injection, mice are treated with either an antibody or agent selectively targeting prostate cancer cells (e.g., anti- PSMA, PSMA-617, anti-STEAPl, anti-STEAP2DUPA) or a site-specific antibody conjugate (e.g., PSMA-MMAE-ALN, PSMA-MMAE-Asp, PSMA-ALN, PSMA-MMAF-Asp). Tumor processions of primary and bone metastasis are monitored by tumor size measurement and bioluminescence, respectively, to determine whether treatment with a site-specific antibody conjugate has better therapeutic effect on bone metastases or primary tumor compared to treatment with unmodified antibody.

[252] Similar experiments are conducted with site-specific antibody conjugates modified with antibody selectively targeting osteosarcoma cells (e.g., anti-LRRC15, Samrotamab, anti-IFITM5), and osteosarcoma cells (e.g., Saos-2).

EXAMPLE 12: Evaluation of Site-Specific Antibody Conjugates on multi-organ metastases from bone lesions

[253] The ability of site-specific antibody conjugates (e.g., PSMA-MMAE-ALN, PSMA-MMAE- Asp, PSMA-ALN, PSMA-MMAF-Asp) to reduce metastasis of prostate cancer cells to other organs is investigated. Prostate cancer cells (e.g., LNCaP) labeled with firefly luciferase are introduced into the right hind limbs of nude mice via intra-iliac artery (IIA) injection, followed by treatment with an antibody or agent selectively targeting prostate cancer cells (e.g., anti-PSMA, PSMA617, anti-STEAPl, anti-STEAP2, DUPA) or a site-specific antibody conjugate (e.g., PSMA-MMAE-ALN, PSMA- MMAE-Asp, PSMA-ALN, PSMA-MMAF-Asp). Mice are subjected to whole-body bioluminescence imaging twice a week following tumor-cell injection for several days (e.g., 29 days, 33 days, 48 days, 68 days, 80 days). Secondary metastases in various organs are calculated by subtracting bioluminescence imaging signal in hind limbs from bioluminescence imaging signal in whole body. Lastly, mice are euthanized and organs (e.g., bone, right hind limb, heart, liver, spleen, lung, kidney, brain) are harvested for bioluminescence imaging to see degree of metastases in mice treated with a site-specific antibody conjugate versus mice treated with unmodified antibody.

[254] Similar experiments are conducted with site-specific antibody conjugates modified with antibody selectively targeting osteosarcoma cells (e.g., anti-LRRC15, Samrotamab, anti-IFIM5), and osteosarcoma cells (e.g., Saos-2).

EXAMPLE 13: Illustrative Compounds

[255] Non-limiting examples of bone-directed prostate cancer therapeutic conjugates of FIG. 1 and FIG. 2 include the following.

[256] Non-limiting examples of bone-directed osteosarcoma therapeutic conjugates of FIG. 1 and FIG. 2 include the following.

[257] Non-limiting examples of bone-directed cancer therapeutic conjugates of FIG. 1 and FIG. 2 include the following.

EXAMPLE 14: Generation and Assessment of a Bone-Targeted Anti-PSMA Antibody

[258] An anti-PSMA humanized J591 antibody comprising Asp¹² at the C-terminus of the heavy chain was designed. The light chain of the anti-PSMA humanized J591 antibody comprising Asp¹² comprised the amino-acid sequence of SEQ ID NO: 15 as shown in the table below; the heavy chain of the anti-PSMA humanized J591 antibody comprising Asp¹² comprised the amino-acid sequence of SEQ ID NO: 22 as shown in the table below.

[259] Anti-PSMA humanized J591 antibody (“humanized J591”) and anti-PSMA humanized J591 antibody comprising Asp¹² at the C-terminus of the heavy chain (“bone -targeted humanized J591 ”) were expressed by ExpiCHO-S cells (Thermo Fisher) following the manufacturer’s protocol. In brief, cells were grown and subcultured in a 37 °C incubator with >80% relative humidity and 8% CO2 on an orbital shaker platform (125 rpm) until cultures reached a density of 4 x 10⁶ viable cells/ml. Before transfection, complexes of ExpiFectamine CHO (Thermo Fisher) and plasmid DNA encoding either J591 or bone-targeted humanized J591 were prepared and incubated at room temperature for 5 min, and then slowly added to the cell cultures. At 18-22 hours post-transfection, ExpiFectamine CHO Enhancer (Thermo Fisher) and ExpiCHO Feed (Thermo Fisher) were added to the cell cultures. After 12 days of expression, secreted antibodies were harvested by centrifugation at 9000 rpm for 30 min and purified on Protein G resin following manufacturer’s instructions. Each antibody sample was buffer-exchanged into PBS via a PD-10 desalt column, and the concentration was measured using a NanoDrop Lite (Thermo Fisher).

[260] To characterize the produced humanized J591 and bone-targeted humanized J591, samples of each antibody were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE). Intact (unreduced; “-DTT”) and reduced (“+DTT”) samples of each antibody were analyzed using a NuPAGE 4-12% Bis-Tris gel (Invitrogen). For each sample, 20 pl of 0.2 mg/ml antibody was mixed with 4 pl of sample loading dye before loading into the gel. The gel was run under 160 V in MES buffer for 35 minutes and stained using Coomassie blue buffer afterwards. Images of the stained gel were captured using an Amersham Imager 600 and analyzed using ImageQuanTL software. As seen in an exemplary image of the stained gel in Figure 3, the resulting visualized bands match the expected approximate sizes (23430 Da for humanized J591 light chain; 48645 Da for humanized J591 wild-type (WT) heavy chain; 50027 Da for humanized J591 heavy chain comprising Asp¹² at the C- terminus (12D)) for both the intact and reduced samples. No immediate breakdown of bone-targeted humanized J591 was visualized nor were any species of an unexpected size were visualized - indicating that bone-targeted humanized J591 is comparably stable as unmodified humanized J591. Bone-targeted humanized J591 was expressed and purified comparably to unmodified humanized J591.

[261] Mass spectrometry (electrospray ionization mass spectrometry; ESI-MS) was used to characterize the produced humanized J591 and bone-targeted humanized J591 antibodies. In brief, a single quadrupole mass spectrometer (Agilent) coupled with a 1260 Infinity II Quarternary Pump (Agilent) was used to perform analysis of humanized J591 and bone-targeted humanized J591 antibodies. Elution conditions for the antibodies were as follows: Mobile Phase A = 0. 1% formic acid in water; Mobile Phase B= 0.1% formic acid in acetonitrile; gradient 0-0.1 min, 10-15% B; 0.1-8 min, 15-50% B; 8-8.1 min, 50-10% B; flow rate= 0.5 mL/min. Absorbance was measured at 280 nm. Resulting data was automatically processed using MassHunter BioConfirm software (Agilent) to analyze the intact and reduced spectra. Exemplary resulting spectra are shown in Figure 4A and Figure 4B and corresponding expected and observed masses are shown in Table 5 below. Mass spectrometry of bone-targeted humanized J591 confirmed the expected size of the modified antibody. Further, no notable degradation or breakdown of bone-targeted humanized J591 was observed.

[262] The humanized J591 and the bone-targeted humanized J591 were both assessed for binding of PSMA-expressing prostate cancer cell lines, e.g., 22Rvl, C4-2b, and LNCaP. In brief, 3 x 10⁵ cells were incubated with 30 ug/mL of either humanized J591 or bone-targeted humanized J591 for 30 min at 4 °C. Following the incubation time, cells were washed with PBS to remove any unbound antibodies. Bound antibodies were then detected by adding fluorescein (FITC) AffiniPure goat anti-human IgG (H+L) (Jackson Immunology) for 30 min at 4 °C. Fluorescence intensity was subsequently determined using a FACSVerse flow cytometer (BD Biosciences). A negative control was also assayed. Exemplary resulting data is shown in Figure 5A for binding to 22Rvl cells, Figure 5B for binding to C4-2b cells, and Figure 5C for binding to LNCaP cells. As can be seen from the results presented in the aforementioned figures, the humanized J591 and bone-targeted humanized J591 exhibited similar fluorescence when analyzed, as visualized by the two overlapping curves on the right side of each figure. These results indicate that binding affinity for PSMA was unchanged by addition of the bonetargeting moiety, e.g., Asp¹².

[263] Without wishing to be bound by any particular theory, the incorporation of a bone-targeting moiety with, e.g., aspartate repeats, e.g., Asp¹², may lower the isoelectric point (pl) of an antibody as compared to the same antibody without a bone-targeting moiety. Such lowering of the pl of an antibody may result in a bone-targeted antibody having a pl outside of, e.g., lower than, the reported optimal range for therapeutic antibodies (Goyon et al., J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Oct 15: 1065-1066: 119-128). An antibody with a pl outside of, e.g., lower than, the optimal range can exhibit decreased expression, decreased stability, decreased antigen binding, etc. Humanized J591, and other select anti-PSMA antibodies reportedly have a lower pl than other certain antibodies, e.g., trastuzumab. This lower starting pl of an unmodified humanized J591 antibody may increase risk of disrupting normal antibody structure and functionality when incorporating a bone-targeting moiety, e.g., a bone-targeting peptide comprising poly-aspartate, e.g., Asp¹². Other factors that may negatively affect antibody expression, stability, antigen binding, etc. include the presence of a patch of negative residues, e.g., aspartate repeats, e.g., Asp¹², in an antibody and/or isomerization of aspartate residues in a bone -targeting moiety, e.g., Asp¹². Based on the SDS-PAGE and mass spectrometry-based analyses described above, the bone-targeted humanized J591 antibody assessed herein demonstrates no observable decrease in expression nor stability as compared to the humanized J591 antibody without a bone-targeting moiety. Further, the bone-targeted humanized J591 antibody assessed herein demonstrates no observable decrease in binding affinity for PSMA as compared to the humanized J591 antibody without a bone -targeting moiety. In some embodiments, a bone-targeted anti-PSMA antibody exhibits a similar level of expression as compared to an anti-PSMA antibody without a bone -targeting moiety, e.g., the same anti-PSMA antibody with one or more bone -targeting moieties absent. In some embodiments, a bone-targeted anti-PSMA antibody exhibits similar stability as compared to an anti- PSMA antibody without a bone -targeting moiety, e.g., the same anti-PSMA antibody with one or more bone-targeting moieties absent. In some embodiments, a bone-targeted anti-PSMA antibody has similar binding affinity for PSMA as compared to an anti-PSMA antibody without a bone -targeting moiety, e.g., the same anti-PSMA antibody with one or more bone-targeting moieties absent. In some embodiments, a bone-targeted humanized J591 antibody exhibits a similar level of expression as compared to a humanized J591 antibody without a bone-targeting moiety, e.g., the same humanized J591 antibody with one or more bone-targeting moieties absent. In some embodiments, a bone-targeted humanized J591 antibody exhibits similar stability as compared to a humanized J591 antibody without a bone-targeting moiety, e.g., the same humanized J591 antibody with one or more bone -targeting moieties absent. In some embodiments, a bone-targeted humanized J591 antibody has similar binding affinity for PSMA as compared to a humanized J591 antibody without a bone -targeting moiety, e.g., the same humanized J591 antibody with one or more bone -targeting moieties absent. Without wishing to be bound by any particular theory, an antibody conjugated to a target agent and comprising a bonetargeting moiety (e.g., comprising one or more bone targeting moieties comprising one or more Asp and/or one or more Glu residues) may have a normal or similar pK as compared to the same antibody conjugated to a target agent without a bone-targeting moiety. Such features may be critical for clinical use of an antibody described herein in targeting cancer lesions located outside of bone.

EXAMPLE 15: Generation and Assessment of a Bone-Targeted Anti-PSMA Antibody-Drug Conjugate [264] Using the humanized J591 antibody and bone-targeted humanized J591 antibody from Example 14, antibody-drug-conjugates (ADCs) were constructed. In brief, 0.5 mg/ml of either humanized J591 or bone-targeted humanized J591 was mixed with either 10 or 20 equivalents of ssFB-Ns peptide at 37 °C for two days. The resulting antibody-FB-Ns conjugate was purified using a PD-10 desalt column. Purified antibody-FB-Ns conjugate was mixed with 40 equivalents of DBCO-(PEG)4-MMAE at room temperature (RT) for overnight strain-promoted alkyneazide cycloaddition (SPAAC) reaction. The resulting antibody-MMAE conjugates, e.g., humanized J591-MMAE or bone-targeted J591-MMAE, were purified via a PD-10 desalt column.

[265] Mass spectrometry (electrospray ionization mass spectrometry; ESI-MS) was used to characterize the resulting humanized J591-MMAE and bone-targeted, humanized J591-MMAE ADCs. Mass spectrometry analysis was performed as described in Example 14. Resulting exemplary spectra are displayed in Figure 6A (for humanized J591-MMAE produced with 10: 1 ratio of MMAE-NHS- cstcrantibody). Figure 6B (for humanized J591-MMAE produced with 20: 1 ratio of MMAE-NHS- cstcrantibody). Figure 6C (for bone-targeted humanized J591-MMAE produced with 10: 1 ratio of MMAE-NHS-ester:antibody), and Figure 6D (for bone-targeted humanized J591-MMAE produced with 20: 1 ratio of MMAE-NHS-ester:antibody). Calculated drug to antibody ratios (DARs) for each ADC are displayed in Table 6 below. These results indicate that bone-targeted humanized J591 can be efficiently conjugated with MMAE via a linker to form an ADC.

[266] Without wishing to be bound by any particular theory, incorporation of a bone -targeting moiety with, e.g., aspartate repeats, e.g., Asp¹², may lower the isoelectric point (pl) of an antibody as compared to the same antibody without a bone-targeting moiety. Such lowering of the pl of an antibody may result in decreased efficiency of conjugation reactions. Furthermore, presence of a patch of negative residues, e.g., aspartate repeats, e.g., Asp¹², in an antibody may also interfere with conjugation reaction efficiency. Isomerization of aspartate residues in a bone-targeting moiety, e.g., Asp¹² may also impact conjugation reaction efficiency. In some embodiments, a bone-targeted anti-PSMA antibody can be conjugated to a drug at a similar rate to an anti-PSMA antibody without a bone-targeting moiety, e.g., the same anti-PSMA antibody with one or more bone -targeting moieties absent. In some embodiments, a bone-targeted humanized J591 antibody can be conjugated to a drug at a similar rate to a humanized J591 antibody without a bone -targeting moiety, e.g., the same humanized J591 antibody with one or more bone -targeting moieties absent.

[267] To assess the ability of the humanized J591-MMAE and bone-targeted humanized J591- MMAE ADCs to kill prostate cancer cells, in vitro cell killing assays using 22Rvl and C4-2b prostate cancer cell lines were performed. In brief, cells were seeded in 200 pl of culture media in 96-well plates at a density of 2 x 10³ cells per well. Following 24 hr of incubation, cells were treated with various concentrations of (i) humanized J591, (ii) humanized J591-MMAE, (iii) bone-targeted humanized J591, (iv) bone-targeted humanized J591-MMAE, or (v) MMAE alone and incubated for an additional 4 days. Following the 4 day incubation, 20 pl of 5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) solution was then added to each well and incubated for an additional 4 hrs. Media was then aspirated and 150 pl DMSO was added to each well. Living cells were then quantified by measuring absorbance at 570 run using a microplate reader. Exemplary results from the cell killing assays are displayed in Figure 7A (for 22Rvl cells) and Figure 7B (for C4-2b cells). Table 7 below displays calculated EC50 for treatment with an ADC or MMAE alone.

[268] Without wishing to be bound by any particular theory, and as discussed herein, incorporation of a bone -targeting moiety with, e.g., aspartate repeats, e.g., Asp¹², may lower the isoelectric point (pl) of an antibody as compared to the same antibody without a bone -targeting moiety and likewise impact expression, stability, antigen binding, etc. An antibody with apl outside of, e.g., lowerthan, the optimal range; a negative patch of negative residues, e.g., aspartate repeats, e.g., Asp¹²; and/or numerous potentially isomerization-prone aspartate residues in a bone-targeting moiety, e.g., Asp¹², may exhibit decreased expression, decreased stability, decreased antigen binding, etc. Moreover, a decreased pl of an antibody may reportedly obstruct uptake of a corresponding ADC via fluid-phase endocytosis (Boswell, et al., Bioconjug Chem. 2010 Dec 15;21(12):2153-63). Decreased binding affinity for an antibody to an antigen, e.g., PSMA, and/or decreased uptake of an ADC due to the aforementioned biochemical risks may in turn affect, e.g., decrease, cytotoxicity of such an ADC.

[269] In contrast, and as shown in Figure 7A, Figure 7B, and Table 7, bone-targeted humanized J591- MMAE can provide similar or greater killing of prostate cancer cells, e.g., 22Rvl cells, C4-2b cells, as compared to humanized J591-MMAE without a bone -targeting moiety. In some embodiments, an ADC comprising a bone-targeted anti-PSMA antibody can provide similar or greater killing of cancer cells as compared to an ADC comprising an anti-PSMA antibody without a bone-targeting moiety, e.g., the same anti-PSMA antibody with one or more bone-targeting moieties absent. In some embodiments, an ADC comprising a bone-targeted humanized J591 antibody can provide similar or greater killing of cancer cells as compared to an ADC comprising a humanized J591 antibody without a bone -targeting moiety, e.g., the same humanized J591 antibody with one or more bone -targeting moieties absent. In some embodiments, an ADC comprising a bone-targeted anti-PSMA antibody can provide similar or greater cytotoxicity as compared to an ADC comprising an anti-PSMA antibody without a bonetargeting moiety, e.g., the same anti-PSMA antibody with one or more bone-targeting moieties absent. In some embodiments, an ADC comprising a bone-targeted humanized J591 antibody can provide similar or greater cytotoxicity as compared to an ADC comprising a humanized J591 antibody without a bone-targeting moiety, e.g., the same humanized J591 antibody with one or more bone -targeting moieties absent.

[270] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof, wherein: the antibody or antigen-binding fragment thereof binds to a prostate cancer-associated antigen, and the antibody or antigen-binding fragment thereof comprises a bone -targeting moiety.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the prostate cancer- associated antigen comprises or is KLK2, PSCA, TR0P2, B7-H3, PSMA, STEAP1, STEAP2, TGFb, DLL-3, SSTR2, Epcam, GPC3, FAP, GRPR, R0R1, HER3, or TMEFF2.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the prostate cancer- associated antigen comprises or is PSMA.

4. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises or is a monoclonal antibody or antigenbinding fragment thereof.

5. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the bone -targeting moiety comprises or is a bone -targeting peptide, optionally wherein the bone targeting moiety is a negatively charged peptide and/or comprises one or more carboxylic peptides.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the bone-targeting peptide comprises or is Asp³, Asp⁴, Asp⁵, Asp⁶, Asp⁷, Asp⁸, Asp⁹, Asp¹⁰, Asp¹¹, Asp¹², Asp¹³, Asp¹⁴, or Asp¹⁵.

7. The antibody or antigen-binding fragment thereof of claim 5, wherein the bone-targeting peptide comprises or is Glu³, Glu⁴, Glu⁵, Glu⁶, Glu⁷, Glu⁸, Glu⁹, Glu¹⁰, Glu¹¹, Glu¹², Glu¹³, Glu¹⁴, or Glu¹⁵.

8. The antibody or antigen-binding fragment thereof of any one of claims 5-7, wherein the bonetargeting peptide is attached to a N-terminus or C-terminus of a light chain or a heavy chain of the antibody or antigen-binding fragment thereof.

9. The antibody or antigen-binding fragment thereof of any one of claims 5-7, wherein the bonetargeting peptide is inserted at an internal permissive site of the antibody or antigen-binding fragment thereof.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the internal permissive site is an internal permissive site of a light chain or an internal permissive site of a heavy chain.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the internal permissive site of the light chain is or corresponds to A153, optionally wherein the bone-targeting peptide is inserted following A 153.

12. The antibody or antigen-binding fragment thereof of claim 10, wherein the internal permissive site of the heavy chain is or corresponds to G118, P123, A165, P243, D283, N344, or G361, optionally wherein the bone -targeting peptide is inserted following G118, P123, A165, P243, D283, N344, or G361.

13. The antibody or antigen-binding fragment thereof of any one of claims 9-12, wherein 1 to (n + 15) amino-acid residues are deleted from the antibody or antigen-binding fragment thereof following the insertion of the bone-targeting peptide, wherein n is the number of amino-acid residues in the bone targeting peptide.

14. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a second bone-targeting moiety.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein the second bonetargeting moiety comprises or is a second bone -targeting peptide, optionally wherein the second bonetargeting moiety is a negatively charged peptide and/or comprises one or more carboxylic peptides.

16. The antibody or antigen-binding fragment thereof of claim 15, wherein the second bonetargeting peptide comprises or is Asp³, Asp⁴, Asp⁵, Asp⁶, Asp⁷, Asp⁸, Asp⁹, Asp¹⁰, Asp¹¹, Asp¹², Asp¹³, Asp¹⁴, or Asp¹⁵.

17. The antibody or antigen-binding fragment thereof of claim 15, wherein the second bonetargeting peptide comprises or is Glu³, Glu⁴, Glu⁵, Glu⁶, Glu⁷, Glu⁸, Glu⁹, Glu¹⁰, Glu¹¹, Glu¹², Glu¹³, Glu¹⁴, or Glu¹⁵.

18. The antibody or antigen-binding fragment thereof of any one of claims 15-17, wherein the second bone-targeting peptide is attached to a N-terminus or C-terminus of a light chain or a heavy chain of the antibody or antigen-binding fragment thereof.

19. The antibody or antigen-binding fragment thereof of any one of claims 15-17, wherein the second bone -targeting peptide is inserted at an internal permissive site of the antibody or antigenbinding fragment thereof.

20. The antibody or antigen-binding fragment thereof of claim 19, wherein the internal permissive site is an internal permissive site of a light chain or an internal permissive site of a heavy chain.

21. The antibody or antigen-binding fragment thereof of claim 20, wherein the internal permissive site of the light chain is or corresponds to A 153, wherein the bone -targeting peptide is inserted following A153.

22. The antibody or antigen-binding fragment thereof of claim 20, wherein the internal permissive site of the heavy chain is or corresponds to G118, P123, A165, P243, D283, N344, or G361, optionally wherein the bone -targeting peptide is inserted following G118, P123, A165, P243, D283, N344, or G361.

23. The antibody or antigen-binding fragment thereof of any one of claims 19-22, wherein 1 to (n + 15) amino-acid residues are deleted from the antibody or antigen-binding fragment thereof following the insertion of the second bone-targeting peptide, wherein n is the number of amino-acid residues in the second bone -targeting peptide.

24. The antibody or antigen-binding fragment thereof of any one of claims 14-23, wherein the antibody or antigen-binding fragment thereof comprises one or more additional bone -targeting moieties, optionally wherein the one or more additional bone-targeting moieties comprise 3, 4, or 5 inserted bonetargeting moieties.

25. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a light chain comprising a sequence selected from Table 8.

26. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence selected from Table 9.

27. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is associated with a target agent.

28. The antibody or antigen-binding fragment thereof of claim 27, wherein the antibody or antigenbinding fragment thereof is conjugated to the target agent.

29. The antibody or antigen-binding fragment thereof of claim 27 or 28, wherein the target agent comprises an anti-cancer agent, an alkylating agent, an antitumor antibiotic, an antimetabolite, an antimitotic agent, a cytotoxic agent, a hormone, a nitrosourea, a plant alkaloid, a taxane, a radioligand, a chemotherapeutic agent, or any combination thereof.

30. The antibody or antigen-binding fragment thereof of claim 29, wherein the radioligand comprises an alpha emitter or a beta emitter.

31. The antibody or antigen-binding fragment thereof of claim 29, wherein the radioligand comprises actinium, lutetium, indium, radium, Pb-212, Cu-67, or thorium.

32. The antibody or antigen-binding fragment thereof of claim 29, wherein the target agent comprises calicheamicin, deruxtecan, govitecan SN-38, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansinoid, SG3199, duocarmycin, exatecan, tubulysin, PNU- 159682, TOP2 inhibitor, TLR agonist, STING agonist, or any combination or derivative thereof.

33. The antibody or antigen-binding fragment thereof of claim 29, wherein the target agent comprises an androgen receptor degrader, integrin inhibitor, EZH2, adenosine receptor inhibitors (CD73), WNT pathway inhibitor, AKT inhibitor, steroidogenesis inhibitor (e.g., ODM-208), PARP inhibitor, immune activator (e.g., TLR3 agonist), BET inhibitor, pyrrolobenzodiazepine (PBD), MYC pathway inhibitor, RAS pathway inhibitor, PI3K inhibitor, NRG1 inhibitor, Amantin or any combination thereof.

34. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises one or more bone targeting moieties comprising a combination of one or more Asp resides and one or more Glu residues.

35. A method of delivering a target agent to a cancer cell in a system, comprising administering or delivering to the system an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 27-34.

36. A method of killing a cancer cell in a system, comprising administering or delivering to the system an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1- 34.

37. The method of claim 35 or 36, wherein the system comprises or is a population of cells.

38. The method of claim 35 or 36, wherein the system comprises or is an animal.

39. The method of claim 35 or 36, wherein the system comprises or is a human.

40. A method of treating a condition, disorder, or disease, comprising administering or delivering to a subject suffering therefrom a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-34.

41. The method of claim 40, wherein the condition, disorder, or disease comprises or is a cancer.

42. The method of claim 41, wherein the cancer comprises or is prostate cancer.

43. The method of any one of claims 40-42, wherein the condition, disorder, or disease comprises or is a bone metastasis.

44. The method of claim 43, wherein the condition, disorder, or disease comprises or is a tumor.

45. The method of claim 44, wherein the tumor is a prostate tumor.

46. The method of claim 44, wherein the tumor is a bone tumor.

47. The method of any one of claims 40-46, wherein the subject is a human.

48. The method of any one of claims 35-47, wherein the antibody or antigen-binding fragment thereof is delivered using one or more of a cell therapy, mRNA, LNP, or VLP.

49. A method of treating prostate cancer comprising administering or delivering to a subject suffering therefrom a therapeutically effective amount of an immune stimulating or immune checkpoint inhibiting antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a bone-targeting moiety.

50. The method of claim 49, wherein the antibody or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof of any one of claims 1-34.