TW202430215A - Compositions and methods for delivery of therapeutic agents to bone - Google Patents

Abstract

Macromolecule compositions and related methods that effect targeted delivery of therapeutic agents to effector targets in bone tissue while minimizing or avoiding undesirable delivery to other cells, tissues or organs are provided. Compositions and methods related to macromolecules, such as an ANDbody<SP>TM</SP>, that include an effector target binding domain specific for an effector target, and an address binding domain specific for bone tissue are described.

Description

Compositions and methods for delivering therapeutic agents to bone

Undesirable off-target effects are a problem for desired therapeutic targets that are present in both healthy and diseased tissues.

This disclosure describes macromolecular compositions and related methods that achieve targeted delivery of therapeutic agents to effector targets in bone cells, tissues, or organs while minimizing or avoiding undesired delivery to other cells, tissues, or organs. In general, the compositions described herein include macromolecules, such as ANDbody™, that include an effector target binding domain specific for bone and an address binding domain specific for an address target. The address target is typically sufficiently restricted in a subject to target the macromolecule to the desired bone cell, tissue, or organ. In some embodiments, the effector target binding domain does not affect the effector target in the absence of the address target binding domain. In addition, the address target binding domain does not affect signaling after binding to the address target. However, positioning of the effector target binding domain by the address target binding domain enables the effector target binding domain to fully bind to the effector target to cause an effect on the signaling of the effector target in the target cell or tissue. In addition, the macromolecules described herein can be linked to one or more small molecules. The compositions described herein can be used, for example, to specifically deliver therapeutic agents (e.g., effector target binding domains, small molecules, or both) to a desired location in a subject, such as a bone cell, tissue, or organ, while avoiding undesirable off-target effects (e.g., brain; skin; cardiovascular system, such as undesirable off-target effects in the heart or vascular system) and/or avoiding certain toxicities, such as avoiding cardiovascular diseases, such as stroke, myocardial infarction.

In one aspect, the present disclosure provides a method for localizing a macromolecule at a bone tissue or bone cell of a subject, the method comprising administering to the subject a macromolecule comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target in the bone tissue or bone cell of the subject, and (b) the second binding site is specific for an address target expressed in the bone tissue or bone cell in the subject; wherein (i) the second binding site localizes the first binding site to the address target such that the first binding site affects effector target signaling in the bone tissue or bone cell; (ii) the second binding site does not substantially affect signaling after binding to the address target; and (iii) The first binding site does not substantially affect effector-target signaling when not localized by the second binding site; and allows the macromolecule to localize to the bone tissue or bone cells of the subject.

In some embodiments, at least 25% of the macromolecule detectable in the subject is detected in bone tissue or bone cells at a time point between 1 day and 7 days after administration of the macromolecule to the subject.

In some embodiments, the efficacy of the first binding site at bone tissue or bone cells is significantly increased relative to a reference macromolecule lacking the second binding site.

In some embodiments, the first binding site has a low affinity for the effector target.

In some embodiments, the first binding site has a low affinity for the effector target.

In some embodiments, the affinity of the first binding site for the effector target is lower than the affinity of the second binding site for the address target.

In some embodiments, the affinity of the first binding site for the effector target is lower than the affinity of the second binding site for the address target.

In some embodiments, effector-target signaling by the macromolecule is significantly reduced in non-target tissues or cells of the subject relative to a reference macromolecule lacking the second binding site.

In some embodiments, the address target is expressed regionally in a subject. In some embodiments, the address target is expressed locally in a subject. In some embodiments, the expression of the address target is limited to a cell type in a subject.

In some embodiments, the address target is only expressed by cells in a subject that are in a particular cellular state. In some embodiments, the address target is only expressed by cells in a subject that are in a disease state.

In some embodiments, the first binding site or the second binding site comprises a polypeptide. In some embodiments, the polypeptide is an antibody or an antigen-binding fragment thereof. In some embodiments, the macromolecule is an antibody comprising a first binding site specific for an effector target in a subject and a second binding site specific for an address target.

In some embodiments, the polypeptide is a ligand of an effector target or a ligand of an address target. In some embodiments, (a) the first binding site comprises an antibody or an antigen-binding fragment thereof and the second binding site comprises a ligand of the address target; or (b) the first binding site comprises a ligand of the effector target and the second binding site comprises an antibody or an antigen-binding fragment thereof.

In some embodiments, (a) the first binding site comprises an antibody or an antigen-binding fragment thereof and the second binding site comprises a small molecule that binds an address target; (b) the first binding site comprises a small molecule that binds an effector target and the second binding site comprises an antibody or an antigen-binding fragment thereof.

In some embodiments, the second binding site is specific for chondrogenic adhesin (CHAD). In some embodiments, the second binding site is specific for dentin matrix acidic phosphoprotein 1 (DMP1). In some embodiments, the second binding site is specific for bone sialoprotein (IBSP). In some embodiments, the second binding site is specific for trophoblast glycoprotein (TPBG). In some embodiments, the second binding site is specific for hydroxyapatite. In some embodiments, the second binding site is a bisphosphonate (e.g., alendronate, risenodrate, etidronate, ibandronate, clodronate, tiludronate, pamidronate, or zoledronate).

In some embodiments, the first binding site is specific for sclerostin (SOST). In some embodiments, the first binding site is specific for dickkopf-1 (DKK1).

In some embodiments, the second binding site is specific for interferon-induced transmembrane protein 5 (IFITM5). In some embodiments, the first binding site is specific for SOST.

In some embodiments, the macromolecule is linked to a small molecule. In some embodiments, the small molecule is odanacatib. In some embodiments, the small molecule is a bisphosphonate (e.g., alendronate, risenodrate, etidronate, ibandronate, clodronate, tiludronate, pamidronate, or zoledronate).

In some embodiments, the macromolecule and the small molecule are connected by a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

The details of one or more embodiments of the present invention are described in the following description. Other features or advantages of the present invention will become apparent from the following drawings and detailed description of several embodiments, as well as from the attached patent claims.

Provided herein are ANDbody ^™ molecules comprising a therapeutic effector target binding domain and an address target binding domain, wherein the address target is bone tissue or cells. In some embodiments, the ANDbody molecule is linked to a small molecule or more than one small molecule. The therapeutic effector target on the ANDbody molecule effectively engages its therapeutic effector target only when the address target binding domain also engages the address target on the target tissue or cell to localize the effector target to the target cell or tissue, for example to form an AND-gate type of logical gate. For example, in some embodiments, an ANDbody is a macromolecule comprising at least (a) a first binding site specific for a therapeutic effector target expressed (e.g., ubiquitously expressed) on a mammalian subject, such as on a cell surface; and (b) a second binding site specific for an address target, wherein the address target is bone tissue or cells. In embodiments, expression of the address target is limited to bone tissue or cells in the subject. In some embodiments, binding of the first binding site to the therapeutic effector target is weaker than binding of the second binding site to the address marker. The effector and address target can be located on the same cell, or in different cells or compartments within the same tissue.

In some embodiments, at a time point between 1 day and 7 days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and/or 7 days) after administration of the macromolecule (e.g., ANDbody) to the subject, at least 25% of the macromolecule (e.g., ANDbody) detectable in the subject is detected at the target bone tissue or cells. For example, in some embodiments, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% (e.g., 25%-30%, 30%-35%, 35%-40%, 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100%) of the detectable macromolecule in the subject is detected at the target bone tissue or cells.

Bone effector targets

The ANDbody ^TM of the present invention comprises an effector that modulates a therapeutic effector target in a subject in need thereof (e.g., a mammalian subject, such as a human). As used herein, an "effector target" is a discrete structure of a cell or tissue of a subject (e.g., a cell surface protein, a transmembrane protein, a receptor) to which the therapeutic effector binding domain of the ANDbody can bind and exert a modulatory effect, such as a therapeutic effect, on the subject. The ANDbody described herein has a binding site that is specific to the effector target. When the effector binding domain binds to the effector target, the effector modulates the target bone cell or tissue to produce a biological response, such as a therapeutic effect, on the subject. However, in some embodiments, the effector target binding domain provided herein may not elicit a biological effect unless it is provided in conjunction with an address targeting domain to localize the effector to the desired target site in the target bone cell or tissue. In some embodiments, such therapeutic communication may require multiple macromolecules according to the invention to bind to multiple effector targets.

In some embodiments, the effector target binding domain can produce a small/weak biological effect when provided alone, and provide a greater/stronger biological effect when provided in conjunction with an address targeting domain that localizes and concentrates/aggregates the effector to a desired target site in a targeted bone cell or tissue. In some embodiments, the effector target binding domain can produce an acceptable biological effect when provided alone, and provide an even greater/stronger biological effect when provided in conjunction with an address targeting domain to localize the effector target binding domain to a targeted bone cell or tissue. In some embodiments, the effector target binding domain can produce a strong biological effect when provided alone, and provide strong or stronger targeting in combination with the address targeting domain to localize the effector target binding domain to the targeted bone cells or tissues. In some embodiments, the effector target binding domain can produce a biological effect with undesirable off-target biological effects when provided alone, but can be targeted, concentrated and aggregated to the desired site in the targeted bone cells or tissues to reduce or eliminate undesirable off-target biological effects when provided in combination with the address targeting. Thus, the effector target binding domains of the present technology provide superior therapeutic agents that, when provided in combination with the site target binding domains described herein, provide a more potent on-target biological effect with fewer side effects, including fewer unintended off-target biological effects.

Examples of such therapeutic signaling effects include, but are not limited to: (i)        Blocking signaling pathways that promote or maintain a disease state; (ii)       Activating signaling pathways that reduce or prevent a disease state; (iii)      Promoting antibody-dependent cellular cytotoxicity (ADCC); (iv)      Inducing complement activation on target cells or tissues; (v)       Promoting phagocytosis; (vi)      Blocking or activating signaling pathways that promote cell differentiation; (vii)         Inducing tissue remodeling to reduce or prevent fibrosis.

In some embodiments, a therapeutic effector target is more widely expressed in a subject than an address target. In some embodiments, a therapeutic effector target is expressed systemically, regionally, or locally in an organism. "Systemic expression" of a therapeutic effector target means that the therapeutic effector target is expressed at substantially the same level in most of the subject organism. Systemic expression involves multiple tissues. "Regional expression" of a therapeutic effector target means that the therapeutic target is expressed in an area that is smaller than systemic expression but larger than local expression. Regional expression is not limited to a single tissue, but can occur in multiple different tissues. "Local expression" of a therapeutic effector target means that the therapeutic target is expressed in a single or a few tissue regions. Local manifestations are not limited to a single tissue but can occur in multiple different tissues.

In some embodiments, the effector target binding domain has a low affinity for the effector target. For example, the low affinity can be an affinity greater than 10 nM (e.g., an affinity between 10 nM-1 μM, such as an affinity between 10 nM and 100 nM). In some aspects, the effector target binding domain has an affinity ( _KD ) for the effector target equal to or less than 100 nM, as measured using biolayer interferometry.

In some embodiments, the effector target binding domain has low affinity for the effector target. Non-limiting examples of therapeutic effector targets that can be targeted with the ANDbodies disclosed herein, as well as exemplary functions of the effector targets, are listed in Table 1.

[ Table 1 ] : Exemplary effector targets Effector Target Type of effector binding domain Exemplary effector binding domains Sclerostin (SOST) SOST inhibitor Yu et al., Acta Pharm Sin B , 12(5): 2150-2170, 2022. Romosozumab, blosozumab, setrusumab, SHR-1222, and sclerostin inhibitors. Dickkopf-1 (DKK1) DKK1 inhibitors DKK1 inhibitors provided by Jiang et al., Front Pharmacol , 13: 847387, 2022. Receptor activator of nuclear factor kappa-beta ligand (RANKL) RANKL inhibitors Denoshu monoclonal antibody. Histoplasmase K Histoplasmosis inhibitor Odanacatib. PTHR (GPCR) (SEQ ID NO: U6CS43) PTHR agonists (eg, agonist peptides, eg, intermittent agonist peptides)

In some embodiments, the effector target is sclerostin (SOST) and the effector target binding domain comprises a SOST inhibitor (e.g., Romo monoclonal antibody, Buso group monoclonal antibody, Sesu monoclonal antibody, SHR-1222, or a sclerostin inhibitor or an antigen-binding fragment thereof provided in Yu et al. Acta Pharm Sin B, 12(5): 2150-2170, 2022).

In some embodiments, the SOST inhibitor is an anti-SOST antibody comprising three heavy chain CDRs given by SEQ ID NOs: 245, 246 and 247 of U.S. Patent No. 8,017,120 B2 and three light chain CDRs given by SEQ ID NOs: 78, 79 and 80 of U.S. Patent No. 8,017,120 B2, which are incorporated by reference. In some embodiments, the anti-SOST antibody is a Romo monoclonal antibody. In some embodiments, the anti-SOST antibody is a variant of the aforementioned antibody, such as a variant of the Romomonoclonal antibody, for example, in some embodiments, a variant comprising at least the following: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions relative to the Romomonoclonal antibody, including non-conservative, conservative or highly conservative substitutions (or any combination), optionally wherein one or more substitutions are conservative substitutions in the CDRs, for example, in some embodiments, in CDRs other than hCDR3 and/or other than lCDR1. In some embodiments, the anti-SOST antibodies useful in the present invention comprise substitutions that promote conjugation (e.g., such substitutions relative to the Romomonoclonal antibody, relative to an antibody comprising 6 CDRs of the Romomonoclonal antibody, or relative to a variant of any of the foregoing), such as the S239C substitution.

In some embodiments, the effector target is dickkopf-1 (DKK1) and the effector target binding domain comprises a DKK1 inhibitor (e.g., a DKK1 inhibitor or an antigen-binding fragment thereof provided in Jiang et al., Front Pharmacol , 13: Article No. 847387, 2022).

In some embodiments, the effector target is receptor activator of nuclear factor kappa-beta ligand (RANKL) and the effector target binding domain comprises a RANKL inhibitor (e.g., a Denosumab antibody or an antigen-binding fragment thereof).

In some embodiments, the effector target is cathepsin K and the effector target binding domain comprises a cathepsin K inhibitor (e.g., odanacatib).

In some embodiments, the effector target is PTHR and the effector target binding domain comprises a PTHR agonist (e.g., an agonist peptide, such as a pyruvic agonist peptide), for example to stimulate bone growth in osteoporosis.

Bone site target

ANDbodies of the present invention also include address-target conjugates that bind to an address target to provide targeted delivery of an effector, wherein the address target is bone tissue or cells. As used herein, an "address target" is a structure on a cell or tissue whose expression in an organism is sufficiently restricted to allow it to identify an organ, tissue, cell, or cellular state of interest in an organism (e.g., bone tissue or cells). An address target can be, for example, a cell surface protein or a structure localized to the extracellular matrix. As used herein, "restricted" expression of an address target means that the address target has a difference, such as less extensive in vivo expression, as opposed to systemic expression. In certain embodiments, the address target is expressed in a single bone cell type, tissue, or cell state, for example, in a mammalian subject (e.g., a human subject).

In some embodiments, the currently provided address target binding domain does not substantially affect biological signaling after binding to the address target, for example, does not modulate signaling pathways or other biological responses in target bone cells or tissues. For example, the address target binder can be inert or inactive, wherein it lacks any additional activity (other than binding) after binding to the address target, including lack of catalytic activity. For example, the address target binder binds to a non-signaling site or motif of the address target. "Signal" is used herein to indicate a conformational, enzymatic and/or electrical result that occurs as a result of target binding. Thus, as described herein, the address target binding domain does not emit a signal after address target binding. As used herein, a domain that "substantially" does not affect biological signaling is one that modulates a signaling pathway or other biological response in a target cell or tissue to which it binds by no more than 25% relative to a control condition (e.g., relative to signaling in the absence of the domain). For example, the domain may modulate (e.g., increase or decrease) a signaling pathway or other biological response by less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, or less than 1% (e.g., 20%-25%, 15%-20%, 10%-15%, 5%-10%, 2%-5%, or 1%-2%). For example, in some embodiments, the address target binding domain does not substantially block chondrocyte α2β1 integrin-dependent adhesion, such as a reduction of chondrocyte α2β1 integrin-dependent adhesion by less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, or less than 1% (e.g., 20-25%, 15-20%, 10-15%, 5-10%, 2-5%, or 1-2%). In some embodiments, the address target binding domain does not substantially interfere with the expression (e.g., upregulation) of one or more osteogenic-related genes (e.g., Runx2, ALP, and OCN), such as substantially not interfering with the upregulation of one or more osteogenic-related genes in human osteosarcoma SaOS2 cells that have undergone osteogenic differentiation. In some embodiments, the address target binding domain does not substantially block mineralization of osteoblasts, e.g., blocks mineralization by less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, or less than 1% (e.g., 20-25%, 15-20%, 10-15%, 5-10%, 2-5%, or 1-2%).

Similarly, when the effector target binding domain is not positioned by the address target binding domain, the effector target binding domain may not signal substantially, or may not signal at all. In embodiments, when the effector target binding domain is positioned by the address target binding domain to bone tissue or cells, the effector target binding domain signals with higher potency (e.g., with higher affinity) than when the effector target binding domain is not positioned by the address target binding domain. When the effector target binding domain is positioned to targeted bone cells or tissues by the address target binding domain that is part of the same macromolecule, effector target signaling may be affected as discussed above.

In some embodiments, address targeting is used for organ-specific addressing (e.g., addressing to bone), tissue-specific addressing (e.g., addressing to one or more bone tissues), or cell-specific addressing (e.g., addressing to one or more bone cell types).

The specificity of the cell or tissue address target binding domain can be detected using methods known in the art. In one embodiment, the Gini coefficient (GC) score is used, which is a method for evaluating the expression variation of a specific gene in a data set. (See O’Hagan et al., GeneGini: assessment via the Gini coefficient of reference “housekeeping” genes and diverse human transporter expression profiles. Cell systems 6, 230–244, https://doi.org/10.1016/j. cels.2018.01.003 (2018); Wright Muelas et al., The role and robustness of the Gini coefficient as an unbiased tool for the selection of Gini genes for normalising expression profiling data. Sci Rep 9, 17960 (2019). https://doi.org/10.1038/s41598-019-54288-7). Addressable target binders can be identified using cellular expression data generated for addressable target binders as described herein (Tables 2A and 2B). In some embodiments, addressable target markers exhibit a Gini score greater than 0.4, such as between 0.74 and 1.00. In contrast, non-addressable markers that are more systemically expressed exhibit a Gini score between 0.15 and 0.19.

In one embodiment, a Tau score is used that represents the expression variation of a particular gene in a dataset. The calculation of Tau uses information about the expression of genes in each tissue and their maximum expression across all tissues, while also taking into account the number of tissues in which expression was measured (see Itai Yanai et al. , Genome-wide midrange transcription profiles reveal expression level relationships in human tissue specification, Bioinformatics, vol. 21, no. 5, Mar 1, 2005, pp. 650-659; Kryuchkova-Mostacci N, Robinson-Rechavi M. A benchmark of gene expression tissue-specificity metrics. Brief Bioinform. 2017 Mar 1; 18(2):205-214. doi: 10.1093/bib/bbw008). In some embodiments, the address-targeted markers exhibit a Tau score greater than 0.6, such as between 0.74 and 1.00. In contrast, non-addressed markers that are more systemically expressed exhibit a Tau score less than 0.3, such as between 0.15 and 0.19.

In some embodiments, the specificity of an address-target binding domain for a particular cell or tissue, such as indicated by an appropriate Gini and/or Tau score, is determined by a tissue-based analysis that excludes tissues with natural biological isolation barriers (i.e., the blood-brain barrier). For example, in some embodiments, Gini and/or Tau scores can be calculated without data from, for example, but not limited to, the following tissues: central nervous system, brain, eye, and/or testicular tissue. In some embodiments, the address-targets provided herein identify cell states. As used herein, "cell state" refers to a given physiological condition of a cell. A cell state can be, for example, a disease state (relative to a non-disease state or normal state of a cell or tissue); or an activated state (relative to an inactivated state of a cell). Exemplary disease states include inflammation, infection (e.g., bacterial, viral, or fungal infection), and cancer-related states (e.g., precancerous or cancerous cell states). In some aspects, a cell state reflects the fact that a particular type of cell can exhibit variability in one or more characteristics and/or can exist under a variety of different conditions while retaining its particular cell type characteristics and not acquiring characteristics that would cause them to be classified as a different cell type. The different states or conditions in which a cell can exist can be characteristic of a particular cell type (e.g., can relate to properties or characteristics exhibited only by that cell type and/or relate to functions performed only or primarily by that cell type) or can occur in multiple different cell types. In some embodiments, a cell state reflects the ability of a cell to respond to a particular stimulus or environmental condition (e.g., whether the cell will respond, or the type of response that will be elicited) or the state of the cell is caused by a stimulus or environmental condition. Cells in different cell states can be distinguished from each other in a variety of ways. For example, they may express, produce or secrete one or more different genes, proteins or other molecules ("markers", e.g., address targets provided herein), exhibit differences in protein modifications, e.g., phosphorylation, acetylation, etc., or may exhibit differences in appearance. Thus, a cell state can be a condition of a cell in which the cell expresses, produces or secretes one or more markers, exhibits one or more specific protein modifications, has a specific appearance, and/or will or will not express one or more biological responses to a stimulus or environmental condition.

In some embodiments, the address target is dentin matrix acidic phosphoprotein 1 (DMP1).

In some embodiments, the address target is the integrin bone sialoprotein (IBSP).

In some embodiments, the address target is trophoblast glycoprotein (TPBG).

In some embodiments, the address target is hydroxyapatite.

In some embodiments, the address targets are expressed on osteoblasts, osteoclasts, osteocytes and/or osteocytes. In some embodiments, the address targets are expressed on osteoblasts. In some embodiments, the address targets are expressed on osteoclasts. Exemplary address targets of the present technology are provided in Table 2 below.

[ Table 2 ] : Exemplary bone target Target Uniprot accession number ADAM12 O43184 ADAMTS14 Q8WXS8 ADGRD1 Q6QNK2 ALPL P05186 ANGPT4 Q9Y264 BAMBI Q13145 BGLAP P86546 BMP3 P12645 CD109 Q6YHK3 CD276 Q5ZPR3 CDH15 P55291 CHAD O55226 CHAD O15335 CILP2 Q8IUL8 COL11A1 P12107 COL11A2 P13942 COL12A1 Q99715 COL13A1 Q5TAT6 COL2A1 P02458 COL8A2 P25067 DKK1 O94907 DMP1 O55188 DMP1 Q13316 DSEL Q8IZU8 FAT3 Q8TDW7 FBLN7 Q53RD9 FLRT2 O43155 GALNT5 Q7Z7M9 GJJ5 P36382 GPR1 P46091 GPR88 Q9GZN0 GXYLT2 A0PJZ3 IBSP Q61711 IFITM5 A6NNB3 KCNH1 O95259 KCNK2 O95069 LAMC3 Q9Y6N6 LOXL4 Q96JB6 LRRC15 Q8TF66 LRRC15 Q8TF66 MAGI2 Q86UL8 MEPE Q8K4L6 MEPE Q9NQ76 MGAT3 Q09327 NTM Q9P121 OMD Q99983 P4HA3 Q7Z4N8 PCDH18 Q9HCL0 PCDHGC5 Q9Y5F6 PHEX P78562 RHBDL2 Q9NX52 SCARF2 Q96GP6 SEMA3D O95025 SEMA5A Q13591 SEMA7A O75326 SHISAL1 Q3SXP7 SLC6A15 Q8BG16 SLC8A3 P57103 SSP1 P10451 TGFB2 P61812 THBS2 P35442 TMTC2 Q8N394 TNN Q9UQP3 TPBG Q13641 UNC5B Q8I WIF1 Q9Y5W WIF1 Q9Y5W WNT10B O00744 WNT16 Q9UBV4 WNT5B Q9H1J7 WNT5B Q9H1J7 Small molecules

The macromolecules (e.g., ANDbodies) of the present invention can be linked to small molecules. The macromolecule and small molecule can be linked by a cleavable linker. Alternatively, the macromolecule and small molecule can be linked by a non-cleavable linker. Any useful linker can be used for this purpose.

One or more (e.g., one, two, three, four, five or more) small molecules can be attached to a macromolecule. If multiple small molecules are attached to a macromolecule, the small molecules may be the same. Alternatively, one or more small molecules attached to the macromolecule may be different.

The small molecule linked to the macromolecule can be any desired small molecule. For example, the small molecule can be a therapeutic agent that is targeted or concentrated at a specific site by the macromolecule. In one example, the small molecule can be a therapeutic agent that works with or complements the effector target binding site domain. Alternatively, the small molecule can modulate the effector target binding site domain. In another example, the small molecule can modulate the address target binding site domain.

In some embodiments, the small molecule is a tissue proteinase K inhibitor, such as odenklatin.

In some embodiments, the small molecule is a bisphosphonate. In some embodiments, the bisphosphonate is alendronate, risedronate, etidronate, ibandronate, clodronate, tiludronate, pamidronate, or zoledronic acid. In some embodiments, the small molecule is alendronate.

Other exemplary classes of small molecules that can be linked to macromolecules according to the present invention include those in Table 3.

[ Table 3 ] : Exemplary small molecule classes Category 1,3 β-glucan synthase (EC 2.4.1.34) inhibitors 11 beta-hydroxysteroid dehydrogenase (corticosteroid 11 beta-dehydrogenase or corticosteroid 11 reductase or HSD11 or EC 1.1.1.146) inhibitors 16S ribosomal RNA (16S rRNA) inhibitors 20S proteasome inhibitor 23S ribosomal RNA (23S rRNA) inhibitors 26S proteasome inhibitors 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMG CoA reductase or hydroxymethylglutaryl-CoA reductase or HMGCR or EC 1.1.1.34) inhibitors 3-hydroxy-5α-steroid-4-dehydrogenase (steroid 5α-reductase or 5α-reductase or EC 1.3.1.22) inhibitors 3-hydroxy-5-alpha steroid-4-dehydrogenase 1 (steroid 5-alpha reductase 1 or SR type 1 or SRD5A1 or EC 1.3.1.22) inhibitors 3-hydroxy 5α-steroid 4-dehydrogenase 2 (5α SR2 or steroid 5α-reductase 2 or type II 5α-reductase or SR type 2 or SRD5A2 or EC 1.3.1.22) inhibitors 30S ribosomal subunit (30S RNA) inhibitors Mitochondrial 4-aminobutyrate transaminase ((S) 3-amino-2-methylpropionic acid transaminase or GABA transaminase or γ-amino-N-butyrate transaminase or L AIBAT or ABAT or EC 2.6.1.22 or EC 2.6.1.19) inhibitors 4-Hydroxyphenylpyruvate dioxygenase (4-Hydroxyphenylpyruvate oxidase or 4HPPD or HPD or EC 1.13.11.27) inhibitors 50S ribosomal subunit (50S RNA) inhibitors 5-hydroxytryptamine receptor 1 (5 HT1 receptor or HTR1) agonists 5-hydroxytryptamine receptor 1A (5 HT1A or G21 or serotonin receptor 1A or HTR1A) agonists 5-hydroxytryptamine receptor 1B (5 HT1B or S12 or serotonin 1D β receptor or serotonin receptor 1B or HTR1B) agonists 5-hydroxytryptamine receptor 1D (5 HT1D or serotonin 1D alpha receptor or serotonin receptor 1D or HTR1D) antagonists 5-hydroxytryptamine receptor 1F (5 HT1F or serotonin receptor 1F or HTR1F) agonists 5-hydroxytryptamine receptor 2 (5 HT2 receptor or HTR2) antagonists 5-hydroxytryptamine receptor 2A (5 HT2A or serotonin receptor 2A or HTR2A) antagonists 5-hydroxytryptamine receptor 2C (5 HT2C or 5-hydroxytryptamine receptor 1C or serotonin receptor 2C or HTR2C) antagonists 5-hydroxytryptamine receptor 3 (5HT3 or 5 HT3 receptor or HTR3) antagonists 5-hydroxytryptamine receptor 4 (5 HT4 or serotonin receptor 4 or HTR4) agonists 5-hydroxytryptamine receptor 7 (5 HT7 or 5 HTX or serotonin receptor 7 or HTR7) antagonists 70S ribosome inhibitor Acetylcholinesterase (acetylcholine hydrolase or Yt blood group or apoptosis-associated acetylcholinesterase or ACHE or EC 3.1.1.7) activators Acetylcholinesterase (acetylcholine hydrolase or Yt or apoptosis-associated acetylcholinesterase or ACHE or EC 3.1.1.7) inhibitors Adenosine deaminase (adenosine aminohydrolase or ADA or EC 3.5.4.4) inhibitors AMP-activated protein kinase ([hydroxymethylglutaryl coenzyme A reductase (NADPH)] kinase or AMPK or EC 2.7.11.31) activators Adenosine receptor (ADORA) antagonists Adenosine receptor A2a (ADORA2A) antagonists Adenosine receptor A2b (ADORA2B) antagonists Adrenocortin receptor (adrenocortin receptor or melanocortin receptor 2 or ACTHR or MC2R) agonists ALK tyrosine kinase receptor (anaplastic lymphoma kinase or CD246 or ALK or EC 2.7.10.1) inhibitors α1 adrenergic receptor (ADRA1) agonists α1 adrenaline receptor (ADRA1) antagonists α1B adrenaline receptor (α1B adrenaline receptor or ADRA1B) antagonists α2 adrenergic receptor (ADRA2) agonists α2 adrenergic receptor (ADRA2) antagonists α2A adrenaline receptor (α2 adrenaline receptor subtype C10 or α2A adrenaline receptor or ADRA2A) agonists α2C adrenaline receptor (α2 adrenaline receptor subtype C4 or α2C adrenaline receptor or ADRA2C) antagonist Alpha adrenergic receptor (ADRA) agonists α-amylase 2B (1,4-αD-glucan glucanohydrolase 2B or carcinoid α-amylase or AMY2B or EC 3.2.1.1) inhibitors α-galactosidase A (αD-galactosidase A or αD-galactoside galactohydrolase or melibiase or agassizase or GLA or EC 3.2.1.22) activator Amine oxidase [flavin] A (monoamine oxidase type A or MAOA or EC 1.4.3.4) inhibitors Androgen receptor (dihydrotestosterone receptor or nuclear receptor subfamily 3 group C member 4 or DHTR or NR3C4 or AR) agonists Androgen receptor (dihydrotestosterone receptor or nuclear receptor subfamily 3 group C member 4 or DHTR or NR3C4 or AR) antagonists Angiopoietin 1 receptor (endothelial tyrosine kinase or intimal endothelial cell kinase or tyrosine kinase 2 with Ig and EGF homology domains or tyrosine protein kinase receptor TEK or tyrosine protein kinase receptor TIE 2 or p140 TEK or CD202b or TIE2 or TEK or EC 2.7.10.1) inhibitors Angiotensin converting enzyme (dipeptidyl carboxypeptidase I or kininase II or CD143 or ACE or EC 3.4.15.1 or EC 3.2.1.) inhibitors Arachidonic acid 5-lipoxygenase (5-lipoxygenase or leukotriene A4 synthase or ALOX5 or EC 1.13.11.34) inhibitors Aromatase (estrogen synthase or cytochrome P 450AROM or cytochrome P450 19A1 or CYP19A1 or EC 1.14.14.14) inhibitors Aryl hydrocarbon receptor (E-basic helix-loop-helix protein 76 or bHLHe76 or AHR) agonists ATP-binding cassette subfamily C member 8 (sulfonylurea receptor 1 or ABCC8) inhibitors ATP citrate synthase (ATP citrate (Pro S) lyase or citrate lyase or ACLY or EC 2.3.3.8) inhibitors ATP-sensitive inward rectifier potassium channel 1 (potassium channel inward rectifier subfamily J member 1 or inward rectifier potassium channel Kir1.1 or KCNJ1) activator B-cell lymphoma 2 (Bcl 2) inhibitors Bacterial cell membrane disruptors C55 isoprenyl pyrophosphate inhibitor Glucocorticoid receptor (GR or nuclear receptor subfamily 3 group C member 1 or NR3C1) agonists Bacterial cell wall disruptors Bcr-Abl tyrosine kinase (EC 2.7.10.2) inhibitor BDNF/NT 3 growth factor receptor (GP145 TrkB or neurotrophic tyrosine kinase receptor type 2 or TrkB tyrosine kinase or tropomyosin-related kinase B or NTRK2 or EC 2.7.10.1) inhibitors β1 adrenergic receptor (β1 adrenergic receptor or ADRB1) agonists β1 adrenergic receptor (β1 adrenergic receptor or ADRB1) agonists β2 adrenergic receptor (β2 adrenergic receptor or ADRB2) agonists β3 adrenergic receptor (β3 adrenergic receptor or ADRB3) agonists β-adrenergic receptor (ADRB) agonists Beta-heme (malaria pigment) inhibitors β-lactamase (EC 3.5.2.6) inhibitors Bile acid receptor (farnesol X-activated receptor or farnesol receptor HRR 1 or nuclear receptor subfamily 1 H group member 4 or retinoid X receptor interacting protein 14 or FXR or NR1H4) agonists Bile acid chelators C5a allergic toxin troponin receptor 1 (CD88 or C5AR1) antagonists Calcium-regulated phosphatase (protein serine/threonine phosphatase 3 or protein phosphatase 3 or EC 3.1.3.16) inhibitors Calcium gene-related peptide type 1 receptor (calcitonin receptor-like receptor or CALCRL) antagonists Calcium-activated potassium channel blockers Calcium channel blockers Calcium chelates Calcium-sensitive receptor agonists Cannabinoid receptor 1 (CB1 or CANN6 or CNR1) agonists Cannabinoid receptor 2 (CB2 or CX5 or CNR2) agonists Mitochondrial (aminoformyl phosphate synthetase I or CPS1 or EC 6.3.4.16) activator Carbonic anhydrase (carbonic dehydratase or carbonic anhydrase or carboxy anhydrase or CA or EC 4.2.1.1) inhibitors Carbonic anhydrase 2 (carbonic anhydrase II or carbonic anhydrase C or CAC or CA2 or EC 4.2.1.1) inhibitors Carbonic anhydrase 4 (carbonic anhydrase IV or CA4 or EC 4.2.1.1) inhibitor; gamma-aminobutyric acid type A receptor subunit (GABA(A) receptor or GABR) agonist Catechol O-methyltransferase (epididymal secretory sperm binding protein Li 98n or COMT or EC 2.1.1.6) inhibitors CC chemokine receptor type 5 (CHEMR13 or HIV 1 fusion co-receptor or CD195 or CCR5) antagonists Cell membrane disruptors Neurosamine glucosyltransferase (glucose ceramide synthase or GLCT 1 or UDP-glucose:N-acylsphingosine D-glucosyltransferase or UDP-glucose ceramide glucosyltransferase or UGCG or EC 2.4.1.80) inhibitors cGMP-specific 3',5'-cyclic phosphodiesterase (cGMP-binding cGMP-specific phosphodiesterase or PDE5 or PDE5A or EC 3.1.4.35) inhibitors Chloride ion channel protein 2 (CLCN2) activators Cholinergic receptor muscarinic (muscarinic acetylcholine receptor or CHRM) antagonists Cholergic receptor muscarinic (muscarinic acetylcholine receptor or CHRM) agonists Cholinergic receptor nicotinic subunit (nicotinic acetylcholine receptor or CHRN) agonists Cholinergic receptor nicotinic subunit (nicotinic acetylcholine receptor or CHRN) antagonists Cholinesterase (butyrylcholinesterase or acetylcholine acyl hydrolase or cholinesterase II or pseudocholesterase or BCHE or EC 3.1.1.8) inhibitors Coagulation factor X (Stuart Prower factor or Stuart factor or F10 or EC 3.4.21.6) inhibitors Copper Chelating Agents Cyclic nucleotide-gated channel (CNG) blockers Cyclic protein-dependent kinase 4 (cell division protein kinase 4 or PSK J3 or CDK4 or EC 2.7.11.22) inhibitors Cyclic protein-dependent kinase 6 (cell division protein kinase 6 or serine/threonine protein kinase PLSTIRE or CDK6 or EC 2.7.11.22) inhibitors Cysteamine leukotriene receptor 1 (cysteamine leukotriene D4 receptor or G protein-coupled receptor HG55 or HMTMF81 or CYSLTR1) antagonists Cysteine leukotriene receptor 2 (G protein-coupled receptor GPCR21 or G protein-coupled receptor HG57 or HPN321 or CYSLTR2) antagonist Cystic fibroblast transmembrane conductance regulator (ATP-binding cassette subfamily C member 7 or channel conductance-controlled ATPase Camp-dependent chloride channel or ABCC7 or CFTR or EC 5.6.1.6) activator Cystine depleting agents Cytidine deaminase (cytidine aminohydrolase or CDA or EC 3.5.4.5) inhibitors Cytochrome bc1 complex (complex III) inhibitors Cytochrome c oxidase (cytochrome a3 or indigophenolase or ferrocytochrome c oxidase or EC 1.9.3.1) inhibitors Mitochondrial (aldosterone synthase or cytochrome P450Aldo or cytochrome P450C18 or steroid 18-hydroxylase or CYP11B2 or EC 1.14.15.4 or EC 1.14.15.5) inhibitors Cytochrome P450 3A4 (1,8-cineole 2-exomonooxygenase or albendazole monooxygenase or albendazole sulfoxidase or cholesterol 25-hydroxylase or cytochrome P450 3A3 or nifedipine oxidase or quinine 3-monooxygenase or taurodeoxycholate 6-alpha-hydroxylase or CYP3A4 or EC 1.14.14. or EC 1.14.14.56 or EC 1.14.14.73 or EC 1.14.14.55) inhibitors Cytochrome P450 3A5 (Cytochrome P450 HLp2 or Cytochrome P450 PCN3 or CYP3A5 or EC 1.14.14.1) inhibitors; HIV 1 integrase (EC 2.7.7.) inhibitors Cytotoxic to cells expressing glutamine carboxypeptidase 2 (folate hydrolase 1 or prostate-specific membrane antigen or PSMA or pteryl poly-gamma-glutamine carboxypeptidase or cell growth inhibitory gene 27 protein or FOLH1 or EC 3.4.17.21) D1A dopamine receptor (dopamine D1 receptor or DRD1) agonists D1B dopamine receptor (D5 dopamine receptor or D1 beta dopamine receptor or dopamine D5 receptor or DRD5) agonist D2 dopamine receptor (dopamine D2 receptor or DRD2) agonists D3 dopamine receptor (dopamine D3 receptor or DRD3) agonists D4 dopamine receptor (D2C dopamine receptor or dopamine D4 receptor or DRD4) agonist Delta opioid receptor (DOR1 or OPRD or OPRD1) antagonists Diacylglycerol O-acyltransferase (diacylglycerol acyltransferase or DGAT or EC 2.3.1.20) inhibitors Dihydrofolate reductase (DHFR or EC 1.5.1.3) inhibitors Mitochondrial dihydroorotate dehydrogenase (quinone) (dihydroorotate oxidase or DHODH or EC 1.3.5.2) inhibitors Dihydropteroate synthase (EC 2.5.1.15) inhibitors Dihydropyrimidinase-related protein 2 (collapse response mediator protein 2 or CRMP2 or N2A3 or Unc 33-like phosphoprotein 2 or DPYSL2) inhibitor Dipeptidyl peptidase 1 (cathepsin C or cathepsin J or dipeptidyl transferase or DPPI or CTSC or EC 3.4.14.1) inhibitors Dipeptidyl peptidase 4 (ADABP or adenosine deaminase complex protein 2 or T cell activation antigen CD26 or TP103 or CD26 or DPP4 or EC 3.4.14.5) inhibitors DNA (cytosine 5) methyltransferase 1 (CXXC-type zinc finger protein 9 or DNA methyltransferase HsaI or MCMT or DNMT1 or EC 2.1.1.37) inhibitors DNA-directed RNA polymerase (POLR2 or EC 2.7.7.6) inhibitors DNA-directed RNA polymerase subunit β (RNA polymerase subunit β or transcriptase subunit β or rpoB or EC 2.7.7.6) inhibitors DNA gyrase (EC 5.99.1.3) inhibitors DNA helicase (EC 3.6.4.12) inhibitors DNA inhibitors DNA ligase (EC 6.5.1.) inhibitors DNA polymerase (EC 2.7.7.7) inhibitors DNA polymerase alpha (POLA or EC 2.7.7.7) inhibitors DNA primase (EC 2.7.7.6) inhibitors DNA synthesis inhibitors DNA topoisomerase I (TOP1 or EC 5.99.1.2) inhibitors DNA topoisomerase II (EC 5.99.1.3) inhibitors DNA topoisomerase IV (EC 5.99.1.3) inhibitors Dopamine receptor (DRD) agonists Dopamine receptor (DRD) antagonists Bispecific mitogen-activated protein kinase kinase 1 (ERK-activated kinase 1 or MAPK/ERK kinase 1 or MAP2K1 or EC 2.7.12.2) inhibitor Bispecific mitogen-activated protein kinase kinase 2 (ERK-activated kinase 2 or MAPK/ERK kinase 2 or MAP2K2 or EC 2.7.12.2) inhibitor Endothelial PAS domain-containing protein 1 (basic helix-loop-helix PAS protein MOP2 or E-class basic helix-loop-helix protein 73 or HIF 1α-like factor or hypoxia-inducing factor 2α or PAS domain-containing protein 2 or EPAS1) inhibitors Endothelin 1 receptor (endothelin A receptor or EDNRA) antagonists Endothelin B receptor (endothelin receptor non-selective or EDNRB) antagonists Enyl[acyl carrier protein] reductase[NADH]FabI (NADH-dependent enyl ACP reductase or enyl ACP reductase or EC 1.3.1.9) inhibitors Epidermal growth factor receptor (oncogene c ErbB 1 or receptor tyrosine protein kinase erbB 1 or HER1 or ERBB1 or EGFR or EC 2.7.10.1) inhibitors Equilibrium nucleoside transporter 1 (equilibrium nitrobenzyl-purine nucleoside-sensitive nucleoside transporter or nucleoside transporter Es type or solute carrier family 29 member 1 or SLC29A1) inhibitors Ergosterol inhibitors Estrogen receptor (ERα or estradiol receptor or nuclear receptor subfamily 3 group A member 1 or NR3A1 or ESR1) agonists Estrogen receptor (ERα or estradiol receptor or nuclear receptor subfamily 3 group A member 1 or NR3A1 or ESR1) antagonists Estrogen receptor (ESR) agonists Estrogen receptor (ESR) antagonists Estrogen receptor (ESR) modulators Estrogen receptor beta (ER beta or nuclear receptor subfamily 3 group A member 2 or NR3A2 or ESR2) agonists Estrogen receptor beta (ER beta or nuclear receptor subfamily 3 group A member 2 or NR3A2 or ESR2) antagonists Exoα sialidase (neuraminase or acetylneuraminase or EC 3.2.1.18) inhibitors Exportin 1 (chromosomal maintenance 1 homolog or XPO1) inhibitor Farnesyl pyrophosphate synthase (farnesyl diphosphate synthase or (2E,6E) farnesyl diphosphate synthase or dimethylallyl transferase or geranyl transferase or FDPS or EC 2.5.1.10 or EC 2.5.1.1) inhibitors Fibroblast growth factor receptor 1 (basic fibroblast growth factor receptor 1 or Fms-like tyrosine kinase 2 or N Sam or proto-oncogene c Fgr or CD331 or FLT2 or FGFR1 or EC 2.7.10.1) inhibitors Fibroblast growth factor receptor 2 (keratinocyte growth factor receptor or K Sam or KGFR or CD332 or FGFR2 or EC 2.7.10.1) inhibitors Fibroblast growth factor receptor 3 (tyrosine kinase JTK4 or aryl protein kinase or CD333 or FGFR3 or EC 2.7.10.1) inhibitors Fibroblast growth factor receptor 4 (CD334 or FGFR4 or EC 2.7.10.1) inhibitors Free Radical Scavengers G protein-coupled receptor 55 (GPR55) antagonists GABA(A) receptor subunit alpha 1 (GABRA1) agonists GABA(A) receptor subunit alpha 3 (GABRA3) agonists GABA(A) receptor subunit gamma 2 (GABRG2) agonists GABA(A) receptor or GABR agonists GABA(A) receptor or GABR antagonists GABA(A) receptor subunit alpha or GABRA agonists GABA B receptor subunit (GABA B receptor or GABBR) agonists Gastric triglyceride lipase (gastric lipase or LIPF or EC 3.1.1.3) inhibitors Geranylgeranyl pyrophosphate synthase (geranylgeranyl diphosphate synthase or dimethylallyl transferase or farnesyl diphosphate synthase or farnesyl transferase or geranyl transferase or GGPS1 or EC 2.5.1.29 or EC 2.5.1.1 or EC 2.5.1.10) inhibitors Glucagon receptor (GL R or GCGR) antagonists Glucocorticoid receptor (GR or nuclear receptor subfamily 3 group C member 1 or NR3C1) agonists Glucocorticoid receptor (GR or nuclear receptor subfamily 3 C group member 1 or NR3C1) antagonists GRI antagonists Glutathione receptor AMPA subunit (AMPA receptor or GRIA) antagonists Glutamine ion receptor kainate subunit (kainate receptor or GRIK) antagonist Glutathione receptor NMDA subunit (NMDAR or GRIN) antagonists GRM agonists Glutamine depletion agent Gonadotropin-releasing hormone receptor (GNRHR) agonists Gonadotropin-releasing hormone receptor (GNRHR) antagonists GTPase KRas (KRas 2 or Ki Ras or c K Ras or KRAS or EC 3.6.5.2) inhibitors Guanylate cyclase (guanylate cyclase or GTP diphosphate lyase (cyclizing) or GC or EC 4.6.1.2) activator Inhibitors of H+ transporting two-part ATPase (F1F0 ATP synthase or ATP synthase or mitochondrial ATPase or bacterial Ca2+/Mg2+ ATPase or EC 7.1.2.2) Hepacivirin (NS3/4A protease or NS3 serine protease or EC 3.4.21.98) inhibitor Hepatitis B virus DNA polymerase (hepatitis B virus reverse transcriptase or hepatitis B virus ribonuclease H or EC 2.7.7.7 or EC 2.7.7.49 or EC 3.1.26.4) inhibitors Hepatocyte growth factor receptor (proto-oncogene c-Met or tyrosine protein kinase Met or HGF/SF receptor or scatter factor receptor or MET or EC 2.7.10.1) inhibitor High affinity neural growth factor receptor (neurotrophic tyrosine kinase receptor type 1 or TRK1 transduced tyrosine kinase protein or tropomyosin-related kinase A or tyrosine kinase receptor or gp140trk or p140 TrkA or NTRK1 or EC 2.7.10.1) inhibitor Histamine H1 receptor (HRH1) antagonists Histamine H2 receptor (gastric receptor I or HRH2) antagonists Histamine H3 receptor (G protein coupled receptor 97 or GPCR97 or HRH3) antagonists Histone deacetylase (HDAC or EC 3.5.1.98) inhibitors Histone deacetylase 1 (HDAC1 or EC 3.5.1.98) inhibitors Histone deacetylase 2 (transcriptional regulator homolog RPD3 or YY1-related factor 1 or HDAC2 or EC 3.5.1.98) inhibitors Histone deacetylase 3 (SMAP45 or RPD3 2 or HDAC3 or EC 3.5.1.98) inhibitors Histone deacetylase 6 (protein phosphatase 1 regulatory subunit 90 or HDAC6 or EC 3.5.1.98) inhibitors Histone lysine N-methyltransferase EZH2 (ENX 1 or Enhancer of Zeste homolog 2 or Lysine N-methyltransferase 6 or EZH2 or EC 2.1.1.43) inhibitors HIV 1 integrase (EC 2.7.7.) inhibitors HIV 1 reverse peptidase (HIV aspartate protease or HIV protease or reverse transpeptidase or Gag protease or HIV aspartate protease or EC 3.4.23.16) inhibitors HIV 2 pepsin (HIV 2 protease or EC 3.4.23.47) inhibitors HIV integrase (EC 2.7.7.) inhibitors Human cytomegalovirus viral terminase inhibitors Hypoxanthine guanine phosphoribosyltransferase (HPRT1 or EC 2.4.2.8) inhibitors Ileal sodium/bile acid cotransporter (apical sodium-dependent bile acid transporter or ASBT or sodium/taurocholate cotransporter or solute carrier family 10 member 2 or SLC10A2) inhibitors Influenza M2 proton channel blockers Inosine monophosphate dehydrogenase (inosinic acid dehydrogenase or IMP oxidoreductase or IMPDH or EC 1.1.1.205) inhibitors Integrin α2b (GPα IIb or platelet glycoprotein IIb or CD41 or ITGA2B) antagonists Integrin αL (CD11 antigen-like family member A or leukocyte adhesion glycoprotein LFA 1 alpha chain or CD11a or ITGAL) antagonists Interferon alpha/beta receptor 1 (interferon receptor class II member 1 or interferon receptor family 2 member 1 or type I interferon receptor 1 or IFNAR1) agonists Interferon alpha/beta receptor 2 (interferon alpha binding protein or type I interferon receptor 2 or IFNAR2) agonists Interleukin-1 receptor-associated kinase 1 (IRAK1 or EC 2.7.11.1) inhibitors Iron chelates Iron substitutes Cytosolic isocitrate dehydrogenase [NADP] (oxalylsuccinate decarboxylase or cytosolic NADP isocitrate dehydrogenase or IDP or NADP (+) specific ICDH or IDH1 or EC 1.1.1.42) inhibitors Mitochondrial isocitrate dehydrogenase [NADP] (NADP (+) specific ICDH or oxalylsuccinate decarboxylase or ICD M or IDP or IDH2 or EC 1.1.1.42) inhibitors Isoleucine tRNA ligase cytoplasmic (Isoleucine tRNA synthetase or IARS or EC 6.1.1.5) inhibitors Kinin-releasing enzyme (KLK or EC 3.4.21.) inhibitors Kappa opioid receptor (KOR1 or OPRK or OPRK1) agonists Kappa opioid receptor (KOR1 or OPRK or OPRK1) antagonists Lanosterol 14α-demethylase (sterol 14α-demethylase or cytochrome P450 14DM or cytochrome P450LI or cytochrome P450 51A1 or CYPLI or CYP51A1 or EC 1.14.14.154) inhibitors Lead chelators Inhibitors of cytosolic leucine tRNA ligase (leucine tRNA synthetase or LeuRS or LARS or EC 6.1.1.4) Leukotriene B4 (LTB4) inhibitors Leukotriene D4 (LTD4) inhibitors Leukotriene inhibitors Lipoxygenase (LOX or EC 1.13.11.) inhibitors Macrophage colony stimulating factor 1 receptor (CSF 1 receptor or proto-oncogene c Fms or CD115 or CSF1R or EC 2.7.10.1) inhibitors Major histocompatibility complex (MHC) inhibitors Maltase-glucoamylase (α-1 4-glucosidase or MGAM or EC 3.2.1.20) inhibitors Mast/stem cell growth factor receptor panel (proto-oncogene c panel or tyrosine protein kinase panel or v panel Hardy Zuckerman 4 feline sarcoma viral oncogene homolog or piebald trait protein or p145 c panel or CD117 or KIT or EC 2.7.10.1) inhibitors Melatonin receptor type 1A (MT1 or MTNR1A) agonists Melatonin receptor type 1B (MT2 or MTNR1B) agonists Mercury chelating agents Microsomal triglyceride transfer protein large subunit (MTTP) inhibitors Nosocomial receptor (nuclear receptor subfamily 3 group C member 2 or Nosocomial receptor delta or Aldosterone receptor or MR or NR3C2) agonists Nosocomial receptor (nuclear receptor subfamily 3 group C member 2 or Nosocomial receptor delta or Aldosterone receptor or MR or NR3C2) antagonists Monoamine oxidase (epinephrine oxidase or adrenal hormone oxidase or MAO or EC 1.4.3.4) inhibitors Mu opioid receptor (MOR1 or Mu opioid receptor or Mu opioid receptor or OPRM1) antagonists Mu opioid receptor (MOR1 or Mu opioid receptor or Mu opioid receptor or OPRM1) agonist Muscicapine acetylcholine receptor M1 (CHRM1) agonists Musculoskeletal acetylcholine receptor M1 (CHRM1) antagonists Muscarinic acetylcholine receptor M3 (CHRM3) agonists Muscarinic acetylcholine receptor M3 (CHRM3) antagonists Musculoskeletal acetylcholine receptor M4 (CHRM4) antagonists Myosin inhibitors Na+/K+ exchanger ATPase (Na+/K+ ATPase or Na+/K+ pump or EC 7.2.2.13) inhibitors NAD+ ADP-ribosyltransferase (poly ADP-ribose polymerase or PARP or EC 2.4.2.30) inhibitors Enkephalinase (neutral endopeptidase 24.11 or cardiopeptidase or common acute lymphoblastic leukemia antigen or skin fibroblast elastase or enkephalinase or CD10 or MME or EC 3.4.24.11) inhibitors Niemann Pick C1-like protein 1 (NPC1L1) inhibitor Non-receptor tyrosine protein kinase TYK2 (TYK2 or EC 2.7.10.2) inhibitors Nonstructural protein 5A (NS5A) inhibitors NS5B (nonstructural protein 5B polymerase or EC 2.7.7.48) inhibitors NT 3 growth factor receptor (TrkC tyrosine kinase or GP145 TrkC or neurotrophic tyrosine kinase receptor type 3 or NTRK3 or EC 2.7.10.1) inhibitors Nuclear factor erythroid 2-related factor 2 (HEBP1 or nuclear factor erythroid-derived 2-like 2 or NFE2L2 or NRF2) activator Orexin receptor type 1 (hypothalamic acid receptor type 1 or HCRTR1) antagonists Orexin receptor type 2 (hypothalamic acid receptor type 2 or HCRTR2) antagonists P2Y purine receptor 12 (ADP glucose receptor or P2Y12 platelet ADP receptor or SP1999 or P2T (AC) or P2Y (AC) or P2Y (cyc) or P2Y12 or P2RY12) antagonist p37 envelope protein (vaccinia virus envelope protein) inhibitor Pancreatic alpha-amylase (1,4αD-glucan glucanohydrolase or AMY2A or EC 3.2.1.1) inhibitors Pancreatic triglyceride lipase (pancreatic lipase or triglyceride hydrolase or PNLIP or EC 3.1.1.3) inhibitors Penicillin Binding Protein (PBP) Inhibitors Penicillin Binding Protein (PBP) Inhibitors; Potassium-Transporting ATPase alpha chain 1 (gastric H(+)/K(+) ATPase subunit alpha or proton pump or ATP4A or EC 7.2.2.19) Inhibitors Penicillin Binding Protein 1A (PBP1A) Inhibitors Penicillin Binding Protein 1B (PBP1B) Inhibitors Penicillin Binding Protein 1C (PBP1C) Inhibitors Penicillin Binding Protein 2a (PBP2a) Inhibitors Penicillin Binding Protein 3 (PBP3) Inhibitors Peptidoglycan (murein) inhibitors Peptidylprolinyl cis-reversible isomerase FKBP1A (12 kDa FK506 binding protein or Calstabin 1 or FK506 binding protein 1A or immunophilin FKBP12 or gyrase or FKBP12 or FKBP1A or EC 5.2.1.8) inhibitors Peroxisome proliferator-activated receptor alpha (nuclear receptor subfamily 1 group C member 1 or NR1C1 or PPARA) agonists Peroxisome proliferator-activated receptor gamma (nuclear receptor subfamily 1 group C member 3 or NR1C3 or PPARG) agonists Phenylalanine 4-hydroxylase (Phe 4-monooxygenase or PAH or EC 1.14.16.1) activator Phosphate binders Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isomer (PI3K-alpha or phosphatidylinositol 4,5-bisphosphate 3-kinase 110 kDa catalytic subunit alpha or phosphoinositide 3-kinase catalytic alpha polypeptide or serine/threonine protein kinase PIK3CA or PIK3CA or EC 2.7.1.137 or EC 2.7.1.153) inhibitors Phosphodiesterase 3 (PDE3 or EC 3.1.4.17) inhibitors Phosphodiesterase 4 (PDE4 or EC 3.1.4.53) inhibitors Fibrolysinogen activator (EC 3.4.21.) inhibitors Platelet-derived growth factor receptor alpha (platelet-derived growth factor receptor type alpha or CD140 antigen-like family member A or platelet-derived growth factor receptor 2 or CD140a or PDGFRA or EC 2.7.10.1) inhibitors Platelet-derived growth factor receptor beta (platelet-derived growth factor receptor type β or CD140 antigen-like family member B or platelet-derived growth factor receptor 1 or CD140b or PDGFRB or EC 2.7.10.1) inhibitors Poly[ADP-ribose] polymerase 1 (ADP-ribosyltransferase diphtheria toxin-like 1 or NAD(+) ADP-ribosyltransferase 1 or poly[ADP-ribose] synthase 1 or PARP1 or EC 2.4.2.30) inhibitors Poly[ADP-ribose] polymerase 2 (ADP-ribosyltransferase diphtheria toxin-like 2 or NAD(+) ADP-ribosyltransferase 2 or poly[ADP-ribose] synthase 2 or PARP2 or EC 2.4.2.30) inhibitors Poly[ADP-ribose] polymerase 3 (ADP-ribosyltransferase diphtheria toxin-like 3 or NAD(+) ADP-ribosyltransferase 3 or poly[ADP-ribose] synthase 3 or PARP3 or EC 2.4.2.30) inhibitors Polymerase acidic protein (RNA-directed RNA polymerase subunit P2 or PA or EC 3.1.) inhibitors Potassium channel (KCN) activators Potassium channel (KCN) blockers Potassium Channel Voltage Gate Control (KCNN) Blockers Potassium depletion agent Potassium-transporting ATPase alpha chain 1 (gastric H(+)/K(+) ATPase subunit alpha or proton pump or ATP4A or EC 7.2.2.19) inhibitors Potassium voltage-gated channel subfamily H member 2 (HERG or voltage-gated potassium channel subunit Kv11.1 or Eag homolog or KCNH2) blocker Progesterone receptor (nuclear receptor subfamily 3 group C member 3 or NR3C3 or PGR) agonists Progesterone receptor (nuclear receptor subfamily 3 group C member 3 or NR3C3 or PGR) antagonists Prostacyclin receptor (prostaglandin I2 receptor or prostaglandin IP receptor or PTGIR) agonists Prostaglandin E2 receptor EP1 subtype (prostaglandin EP1 receptor or PTGER1) agonists Prostaglandin E2 receptor EP1 subtype (prostaglandin EP1 receptor or PTGER1) antagonists Prostaglandin E2 receptor EP2 subtype (prostaglandin EP2 receptor or PTGER2) agonists Prostaglandin E2 receptor EP3 subtype (PGE2 R or prostaglandin EP3 receptor or PTGER3) agonists Prostaglandin F2α receptor (prostaglandin FP receptor or PTGFR) agonists Prostaglandin G/H synthase (cyclooxygenase or COX or PTGS or EC 1.14.99.1) inhibitors Prostaglandin G/H synthase 1 (COX1 or prostaglandin endoperoxide synthase 1 or prostaglandin H2 synthase 1 or PTGS1 or EC 1.14.99.1) inhibitors Prostaglandin G/H synthase 2 (COX2 or prostaglandin endoperoxide synthase 2 or PHS II or prostaglandin H2 synthase 2 or PTGS2 or EC 1.14.99.1) inhibitors Proteasome inhibitors Protein Cereblon (CRBN) Activator Protease activated receptor 1 (PAR1 or coagulation factor II receptor or thrombin receptor or F2R) antagonists Prothrombin (coagulation factor II or F2 or EC 3.4.21.5) inhibitors Proto-oncogene tyrosine kinase receptor Ret (calcified mucin family member 12 or proto-oncogene c Ret or RET or EC 2.7.10.1) inhibitors Proto-oncogene tyrosine protein kinase ROS (proto-oncogene c Ros 1 or receptor tyrosine kinase c Ros oncogene 1 or c Ros receptor tyrosine kinase or ROS1 or EC 2.7.10.1) inhibitors Pyruvate kinase PKLR (pyruvate kinase 1 or erythrocyte/liver pyruvate kinase or R-type/L-type pyruvate kinase or pyruvate kinase isozyme L/R or PKLR or EC 2.7.1.40) activator Pyruvate synthase (pyruvate ferrodoxin oxidoreductase or PFOR or EC 1.2.7.1) inhibitors Receptor type tyrosine protein kinase FLT3 (FMS-like tyrosine kinase 3 or FL interleukin receptor or stem cell tyrosine kinase 1 or fetal liver kinase 2 or CD135 or FLT3 or EC 2.7.10.1) inhibitors Receptor tyrosine protein kinase ERBB 2 (metastatic lymph node gene 19 protein or proto-oncogene Neu or proto-oncogene C ErbB 2 or tyrosine kinase type cell surface receptor HER2 or p185erbB2 or HER2 or CD340 or ERBB2 or EC 2.7.10.1) inhibitors Receptor tyrosine protein kinase ERBB 4 (tyrosine kinase type cell surface receptor HER4 or proto-oncogene-like protein c ErbB 4 or p180erbB4 or HER4 or ERBB4 or EC 2.7.10.1) inhibitors Renin (angiotensinogenase or REN or EC 3.4.23.15) inhibitors Retinoic acid receptor (RAR) agonists Retinoic acid receptor alpha (RARα or nuclear receptor subfamily 1 group B member 1 or NR1B1 or RARA) agonists Retinoic acid receptor beta (RAR beta or HBV activation protein or nuclear receptor subfamily 1 group B member 2 or RAR epsilon or NR1B2 or RARB) agonists Retinoic acid receptor gamma (RAR gamma or nuclear receptor subfamily 1 group B member 3 or NR1B3 or RARG) agonists Retinoic acid receptor RXRα (nuclear receptor subfamily 2 group B member 1 or retinoid X receptor α or NR2B1 or RXRA) agonists Retinoic acid receptor RXR beta (nuclear receptor subfamily 2 group B member 2 or retinoid X receptor beta or NR2B2 or RXRB) agonists Retinoic acid receptor RXR gamma (nuclear receptor subfamily 2 group B member 3 or retinoid X receptor gamma or NR2B3 or RXRG) agonists Retinoid X receptor (RXR) agonists Reverse transcriptase (EC 2.7.7.49) inhibitors Rho-associated protein kinase 2 (Rho kinase 2 or Rho-associated coiled-coil containing protein kinase 2 or p164 ROCK 2 or ROCK2 or EC 2.7.11.1) inhibitors Rho kinase (Rho-associated coiled-coil-forming protein kinase or ROCK or EC 2.7.11.1) inhibitors Ribonucleoside diphosphate reductase (ribonucleotide reductase or RRM or EC 1.17.4.1) inhibitors RNA-directed RNA polymerase (EC 2.7.7.48) inhibitors RNA polymerase (EC 2.7.7.6) inhibitors RNA polymerase II (RNAP II or Pol II or EC 2.7.7.6) inhibitors RNA synthesis inhibitors Rhinoidine receptor 1 (skeletal muscle calcium release channel or skeletal muscle ryanodine receptor or ryanodine receptor type 1 or RYR1) antagonists Serine D-type Ala D Ala carboxypeptidase (D-alanyl D-alanine carboxypeptidase or DD transpeptidase or EC 3.4.16.4) inhibitors Serine/threonine protein kinase B Raf (p94 or proto-oncogene B Raf or v Raf murine sarcoma viral oncogene homolog B1 or BRAF or EC 2.7.11.1) inhibitors Serine/threonine protein kinase mTOR (FK506 binding protein 12 rapamycin complex-associated protein 1 or FKBP12 rapamycin complex-associated protein or mammalian target of rapamycin or mechanistic target of rapamycin or target of rapamycin and FKBP12 1 or target of rapamycin 1 or MTOR or EC 2.7.11.1) inhibitors Serine/threonine protein kinase UL97 (HSRF3 protein or ganciclovir kinase or UL97 or EC 2.7.11.) inhibitors Sigma non-opioid intracellular receptor 1 (senescence-associated gene 8 protein or SR31747 binding protein or sigma type 1 opioid receptor or SIGMAR1) agonist Smoothened homolog (protein Gx or SMO) antagonists Sodium and chloride-dependent GABA transporter 1 (GAT1 or solute carrier family 6 member 1 or SLC6A1) inhibitor Sodium channel blockers Sodium channel protein type 1 subunit alpha (sodium channel protein brain I subunit alpha or voltage-gated sodium channel subunit alpha Nav1.1 or SCN1A) blockers Sodium channel protein type 10 subunit alpha (peripheral nerve sodium channel 3 or voltage-gated sodium channel subunit alpha Nav1.8 or SCN10A) blockers Sodium channel protein type 2 subunit alpha (HBSC II or sodium channel protein brain II subunit alpha or sodium channel protein type II subunit alpha or voltage-gated sodium channel subunit alpha Nav1.2 or SCN2A) blocker Sodium channel protein type 3 subunit alpha (voltage-gated sodium channel subunit III or voltage-gated sodium channel subunit alpha Nav1.3 or SCN3A) blockers Sodium channel protein type 4 subunit alpha (sodium channel protein skeletal muscle subunit alpha or voltage-gated sodium channel subunit alpha Nav1.4 or SCN4A) blockers Sodium channel protein type 5 subunit alpha (sodium channel protein cardiac subunit alpha or voltage-gated sodium channel subunit alpha Nav1.5 or SCN5A) blockers Sodium channel protein type 8 subunit alpha (voltage-gated sodium channel subunit alpha Nav1.6 or SCN8A) blockers Sodium channel protein type 9 subunit alpha (neuroendocrine sodium channel or peripheral sodium channel 1 or voltage-gated sodium channel subunit alpha Nav1.7 or SCN9A) blockers Sodium-dependent dopamine transporter (DA transporter or DAT or solute carrier family 6 member 3 or SLC6A3) inhibitors Sodium-dependent norepinephrine transporter (NET or solute carrier family 6 member 2 or SLC6A2) inhibitors Sodium-dependent serotonin transporter (5HT transporter or 5HTT or solute carrier family 6 member 4 or SLC6A4) inhibitors Sodium/glucose cotransporter 2 (low-affinity sodium-glucose cotransporter or solute carrier family 5 member 2 or SGLT2 or SLC5A2) inhibitors Sodium/hydrogen exchanger 3 (NHE3 or solute carrier family 9 member 3 or SLC9A3) inhibitors Sodium/iodine symporter (sodium-iodide symporter or solute carrier family 5 member 5 or SLC5A5) activators Soluble guanylate cyclase (sGC or EC 4.6.1.2) activator Solute carrier family 12 member 1 (bumetanide-sensitive sodium (potassium) chloride symporter 2 or kidney-specific Na-K-Cl symporter or SLC12A1) inhibitors Solute carrier family 12 member 2 (basolateral Na-K-Cl symporter or bumetanide-sensitive sodium (potassium) chloride symporter 1 or SLC12A2 or NKCC1) inhibitors Solute carrier family 12 member 3 (Na-Cl cotransporter or NCC or Na-Cl symporter or thiazide-sensitive sodium chloride Sphingosine 1-phosphate receptor (S1PR) modulators Sphingosine 1-phosphate receptor 1 (endothelial differentiation G protein-coupled receptor 1 or sphingosine 1 phosphate receptor Edg 1 or CD363 or S1PR1) agonist Sphingosine 1-phosphate receptor 4 (endothelial differentiation G protein-coupled receptor 6 or sphingosine 1-phosphate receptor Edg 6 or S1PR4) agonists Sphingosine 1-phosphate receptor 5 (endothelial differentiation G protein-coupled receptor 8 or sphingosine 1-phosphate receptor Edg 8 or S1PR5) agonists Squalene monooxygenase (squalene epoxidase or SQLE or EC 1.14.14.17) inhibitors Inhibitors of steroid 17α-hydroxylase/17,20-lyase (17α-hydroxyprogesterone aldolase or cytochrome P450 17A1 or cytochrome P450 C17 or steroid 17α-monooxygenase or CYP17 or CYP17A1 or EC 1.14.14.19 or EC 1.14.14.32) Substance P receptor (tachykinin receptor 1 or NK 1 receptor or NK1R or TACR1) antagonists Inhibitors of intestinal sucrase and isomaltase (SI or EC 3.2.1.48 or EC 3.2.1.10) Surface protein gp120 inhibitor Survival motor neuron protein (component of Gems 1 or Gemin 1 or SMN1 or SMN2) activator Synaptic vesicle glycoprotein 2A (SV2A) binder Synaptic vesicular amine transporter (monoamine transporter or solute carrier family 18 member 2 or vesicular amine transporter 2 or VAT2 or SLC18A2) inhibitors Tetrahydrobiopterin (THB or BH4) alternatives Thioredoxin disulfide reductase (NADPH thioredoxin reductase or EC 1.8.1.9) inhibitors Thrombopoietin receptor (myeloproliferative leukemia protein or proto-oncogene c Mpl or CD110 or MPL) agonist Thymidine kinase (TK or EC 2.7.1.21) activators Thymidine phosphorylase (glostatin or platelet-derived endothelial growth factor or TdRPase or TYMP or EC 2.4.2.4) inhibitors Thymidylate synthase (TYMS or EC 2.1.1.45) inhibitors Thyroid hormone receptor agonists Thyroid hormone receptor alpha (nuclear receptor subfamily 1 group A member 1 or VerbA-related protein 7 or c erbA 1 or c erbAα or EAR7 or THRA) agonists Thyroid hormone receptor beta (nuclear receptor subfamily 1 group A member 2 or c erbA 2 or c erbA beta or ERBA2 or THRB) agonists Thyroid peroxidase (thyroid peroxidase or thyroid microsomal antigen or TPO or EC 1.11.1.8) inhibitors Toll-like receptor 7 (TLR7) agonists Toll-like receptor 7 (TLR7) antagonists Toll-like receptor 9 (CD289 or TLR9) antagonists Transferrin receptor (TFR) agonists Transient receptor potential cation channel subfamily V member 1 (capsaicin receptor or vanilloid receptor 1 or TRPV1) inhibitors Transient receptor potential cation channel subfamily V member 1 (capsaicin receptor or vanilloid receptor 1 or TRPV1) activator Thyroxine transporter (ATTR or prealbumin or TBPA or TTR) activator Thyroxine transporter (ATTR or prealbumin or TBPA or TTR) inhibitors Trifunctional purine biosynthetic protein adenosine 3 (phosphoribosylglycerol amine formyl transferase or phosphoribosylglycerol amine ligase or phosphoribosylglycerol amine guanyl ligase or GART or EC 2.1.2.2 or EC 6.3.3.1 or EC 6.3.4.13) inhibitors Tryptophan 5-monooxygenase (tryptophan hydroxylase or TPH or EC 1.14.16.4) inhibitors Tubulin inhibitors Angiotensin II receptor type 1 (AT1AR or AT1BR or angiotensin II type 1 receptor or AGTR1) antagonists Tyrosinase (monophenol monooxygenase or tumor rejection antigen AB or LB24 AB or SK29 AB or TYR or EC 1.14.18.1) inhibitors Tyrosine 3-monooxygenase (Tyrosine 3-hydroxylase or TH or EC 1.14.16.2) inhibitors Tyrosine protein kinase BTK (Brutton's tyrosine kinase or B-cell progenitor kinase or agammaglobulinemia tyrosine kinase or BTK or EC 2.7.10.2) inhibitors Tyrosine protein kinase CSK (C Src kinase or protein tyrosine kinase CYL or CSK or EC 2.7.10.2) inhibitors Tyrosine protein kinase JAK1 (Janus kinase 1 or JAK1 or EC 2.7.10.2) inhibitors Tyrosine protein kinase JAK2 (Janus kinase 2 or JAK2 or EC 2.7.10.2) inhibitors Tyrosine protein kinase JAK3 (Janus kinase 3 or Leukocyte Janus kinase or JAK3 or EC 2.7.10.2) inhibitors Tyrosine protein kinase receptor UFO (AXL oncogene or AXL or EC 2.7.10.1) inhibitor Tyrosine protein kinase SYK (spleen tyrosine kinase or p72 Syk or SYK or EC 2.7.10.2) inhibitors Tyrosine kinase Tec (TEC or EC 2.7.10.2) inhibitors UDP N-acetylglucosamine 1-carboxyvinyl transferase (enylpyruvate transferase or UDP N-acetylglucosamine 1-carboxyvinyl transferase or UDP N-acetylglucosamine enolpyruvate transferase or murA or EC 2.5.1.7) inhibitors Urease (ureamide hydrolase or EC 3.5.1.5) inhibitors Vascular endothelial growth factor receptor 1 (Fms-like tyrosine kinase 1 or tyrosine protein kinase receptor FLT or tyrosine protein kinase FRT or vascular permeability factor receptor or VEGFR1 or FLT1 or EC 2.7.10.1) inhibitors Vascular endothelial growth factor receptor 2 (fetal liver kinase 1 or kinase insert domain receptor or protein tyrosine kinase receptor flk 1 or VEGFR2 or CD309 or KDR or EC 2.7.10.1) inhibitors Vascular endothelial growth factor receptor 3 (Fms-like tyrosine kinase 4 or tyrosine protein kinase receptor FLT4 or VEGFR3 or FLT4 or EC 2.7.10.1) inhibitors Vasopressin V1a receptor (AVPR V1a or vasopressin receptor 1a or vascular/hepatic arginine vasopressin receptor or AVPR1A) antagonists Vasopressin V2 receptor (AVPR V2 or vasopressin receptor or renal arginine vasopressin receptor or ADHR or AVPR2) antagonists Viral mRNA Vitamin D receptor (calcitriol receptor) agonists Vitamin D3 receptor (1,25-dihydroxyvitamin D3 receptor or nuclear receptor subfamily 1 group I member 1 or NR1I1 or VDR) agonists Voltage-dependent calcium channel activators Voltage-dependent calcium channel blockers Voltage-dependent calcium channel subunit α2/δ1 (CACNA2D1) blocker Voltage-dependent calcium channel subunit α2/δ2 (CACNA2D2) blocker Voltage-dependent L-type calcium channel blocker Voltage-dependent L-type calcium channel subunit α1S (voltage-gated calcium channel subunit αCav1.1 or CACNA1S) blockers Voltage-dependent N-type calcium channel subunit α1B (voltage-gated calcium channel subunit αCav2.2 or brain calcium channel III or CACNA1B) blockers Voltage-dependent T-type calcium channel blocker Voltage-dependent T-type calcium channel subunit α1G (voltage-gated calcium channel subunit αCav3.1 or CACNA1G) blockers Voltage-gated sodium channel (SCN) blockers Xanthine dehydrogenase/oxidase (xanthine dehydrogenase or xanthine oxidase or xanthine oxidoreductase or XDH or EC 1.17.1.4 or EC 1.17.3.2) inhibitors

Specific examples of small molecules that can be linked to the macromolecules of the present invention fall into any of the categories shown, for example, in Table 3. Specifically, exemplary glucocorticoid receptor agonists include, but are not limited to, cortisone, dexamethasone, fluticasone, mometasone, fluocinolone, budesonide, buticot, and betamethasone. Exemplary tyrosine protein kinase BTK inhibitors include, but are not limited to, acalabrutinib, evobrutinib, fenebrutinib, ibrutinib, orelabrutinib, pirtobrutinib, remibrutinib, rilzabrutinib, tolebrutinib, and zanubrutinib. Exemplary PI3K inhibitors include, but are not limited to, alpelisib, idelalisib, copanlisib, and duvelisib. Exemplary JAK inhibitors include, but are not limited to, abrocitinib, baricitinib, delgocitinib, filgotinib, peficitinib, ruxolitinib, tofacitinib, and upadacitinib. Exemplary tissue proteinase K inhibitors include, but are not limited to, odencatib, relacatib, MIV-711, and KGP-207. Exemplary topoisomerase inhibitors include, but are not limited to, irinotecan, doxorubicin, daunorubicin, adriamycin, epirubicin, etanercept, idarubicin, topotecan, and valrubicin.

Any fusion technique known in the art can be used to fusion small molecules to the macromolecules of the present invention. For example, small molecule carboxyl, hydroxyl and amine residues can be connected to amine and alkyl residues on proteins using fusion techniques. Alternatively, any complementary functional groups on the two components can be used to react with each other to form a covalent bond. Examples of complementary reactive functional groups include, but are not limited to, for example, maleimide and cysteine, amine and activated carboxylic acid, thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and alkene and tetrahedron. In addition, any available linker can be used in the present invention, including heterobifunctional linkers that allow small molecules to be connected by, for example, disulfide bonds and amide bonds.

ANDbody Structure

In general, an ANDbody can be any macromolecule, such as a polypeptide or protein containing an antigen target binding site or binding domain and an address target binding site or binding domain. The binding sites can be present on the same polypeptide chain or on different polypeptide chains linked together, for example, by disulfide bonds.

In some embodiments, the binding site for the effector target and the binding site for the address target of the ANDbody each comprises an antibody heavy chain and/or light chain domain. In some embodiments, the ANDbody comprises a first antibody variable domain having binding specificity for the effector target and a second antibody variable domain having binding specificity for the address target. In other embodiments, the ANDbody comprises a first antigen binding site of the antibody and a second antigen binding site of the antibody, the first antigen binding site having binding specificity for the effector target and the second antigen binding site having binding specificity for the address target.

In some embodiments, ANDbody can have the structure of an antibody molecule. As used herein, the term "antibody" includes full-length antibodies and antigen-binding antibody fragments (e.g., scFv). In some embodiments, the antibody molecule has specificity for more than one, e.g., 2, 3, 4 antigens, e.g., the antibody molecule comprises a plurality of variable domain sequences, wherein a first variable domain sequence in the plurality of variable domain sequences has binding specificity for a first epitope (e.g., an effector target), and a second variable domain sequence in the plurality of variable domain sequences has binding specificity for a second epitope (e.g., an address target).

In some embodiments, an ANDbody is an antibody molecule having an arm or domain that binds an effector target and an arm or domain that binds an address target. In embodiments, an ANDbody is an antibody molecule comprising a light chain that binds one of an effector target and an address target and a heavy chain that binds the other of the effector target and the address target.

In some embodiments, ANDbody has the following structures: scFv, BsIgG, BsAb fragment, BiTE, dual affinity redirecting protein (DART), tandem diabody (TandAb), diabody, Fab2, di-scFv, chemically linked F(ab')2, Ig molecule with 2, 3 or 4 different antigen binding sites, DVI-IgG four-in-one, ImmTac, HSAbody, IgG-IgG, Cov-X-Body, scFv1-PEG- _scFv2 , attached IgG, DVD-IgG, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, monomer, nanoCLAMP, bis-Fab, Fv, Fab, Fab'-SH, linear antibody, scFv, heavy chain only antibody (Humabody), ScFab, IgG antibody fragment, single chain variable region antibody, single domain heavy chain antibody, bispecific triabody, BiKE, CrossMAb, dsDb, scDb, tandem dAb/VHH, triple dAb VHH, tetravalent dAb/VHH, Fab-scFv, Fab-Fv, or DART-Fc, fiber-linked protein (adnectin), Kunitz-type inhibitor, or receptor bait.

In some embodiments, the effector target binding site and/or the address target binding site comprises or consists of a small molecule. For example, in some embodiments, the first binding site comprises an antibody or an antigen-binding fragment thereof, and the second binding site comprises a small molecule that binds the address target. In other embodiments, the first binding site comprises a small molecule that binds the effector target, and the second binding site comprises an antibody or an antigen-binding fragment thereof. In aspects where the ANDbody comprises a small molecule and a polypeptide component (e.g., an antibody or a fragment thereof), the small molecule can be fused to the polypeptide (e.g., an antibody or a fragment thereof) using any fusion technique known in the art.

The effector target binding site and the address target binding site of an ANDbody may have different affinities for their respective binding partners. In some embodiments, the affinity of the first binding site for the therapeutic effector target to which it binds is weaker than the affinity of the second binding site for the address target. In some embodiments, the affinity of the first binding site for the therapeutic effector target to which it binds is more than 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold weaker than the affinity of the second binding site for the address target.

The terms "binding affinity" and "binding activity" refer to the tendency of a macromolecule, such as a polypeptide molecule, to bind or not bind to a target. For the purposes of the present invention of combining two binding sites, the relative affinities of the two binding sites can be determined, for example, by measuring their respective affinities when each binding site is present on a common scaffold, such as in the form of a single chain antibody. This comparison allows the affinities of the two binding sites to be compared while eliminating any interference from other binding sites present on the macromolecule of the present invention.

Binding affinity can be quantified by determining the dissociation constant (Kd) of a polypeptide and its binder. A lower Kd indicates a higher affinity for the binding partner. Similarly, the specificity of a polypeptide's binding to its binding partner can be defined based on the relative dissociation constant (Kd) of the polypeptide to its binding partner compared to the dissociation constant of the polypeptide and another non-target molecule.

The value of the dissociation constant can be determined by known methods. For example, Kd can be determined using a dual filter nitrocellulose filter binding assay, such as the assay disclosed by Wong & Lohman (Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 90, 5428-5432, 1993). Other standard assays for evaluating the binding ability of a ligand (e.g., an antibody) to a target are known in the art, including, for example, ELISA, protein blot, RIA, and flow cytometry analysis. The binding kinetics (e.g., binding affinity) of an antibody can also be evaluated by standard assays known in the art, such as by analysis of a Biacore™ system.

As an alternative to Kd, EC ₅₀ or IC ₅₀ can be used to determine relative affinity. In this article, EC ₅₀ represents the concentration at which the polypeptide achieves 50% of its maximum binding to a fixed amount of binding partner. IC ₅₀ represents the concentration at which the polypeptide inhibits 50% of the maximum binding of a fixed amount of competitor to a fixed amount of binding partner. In both cases, lower levels of EC ₅₀ or IC ₅₀ indicate a higher affinity for the target. Both EC ₅₀ and IC ₅₀ values of an ANDbody binding site for its binding partner can be determined by well-known methods such as ELISA.

In some embodiments, the Kd of the therapeutic effector-target binding agent may be higher than about 1 pM, about 10 pM, about 100 pM, about 1 nM, about 10 nM, about 100 nM, about 500 nM, or about 1 uM (e.g., may be between 1 pM and 10 pM, between 10 pM and 100 pM, between 100 pM and 1 nM, between 1 nM and 10 nM, between 10 nM and 100 nM, between 100 nM and 500 nM, or between 500 nM and 1 uM). In some embodiments, the Kd of the addressee target binder can be less than about 1 uM, about 500 nM, about 100 nM, about 10 nM, about 1 nM, about 100 pM, about 10 pM, or about 1 pM (e.g., can be between 1 uM and 500 nM, between 500 nM and 100 nM, between 100 nM and 10 nM, between 10 nM and 1 nM, between 1 nM and 100 pM, between 100 pM and 10 pM, or between 10 pM and 1 pM). In some embodiments, the Kd of the therapeutic effector target binder can be about 6-fold, about 5-fold, about 4-fold, about 3-fold, or about 2-fold higher than the Kd of the addressee target binder.

In some embodiments, the _EC50 of the therapeutic effector-target binder may be higher than about 1 pM, about 10 pM, about 100 pM, about 1 nM, about 10 nM, about 100 nM, about 500 nM, or about 1 uM (e.g., may be between 1 pM and 10 pM, between 10 pM and 100 pM, between 100 pM and 1 nM, between 1 nM and 10 nM, between 10 nM and 100 nM, between 100 nM and 500 nM, or between 500 nM and 1 uM). In some embodiments, the _EC50 of the addressee target binder can be less than about 1 uM, about 500 nM, about 100 nM, about 10 nM, about 1 nM, about 100 pM, about 10 pM, or about 1 pM (e.g., can be between 1 uM and 500 nM, between 500 nM and 100 nM, between 100 nM and 10 nM, between 10 nM and 1 nM, between 1 nM and 100 pM, between 100 pM and 10 pM, or between 10 pM and 1 pM). In some embodiments, the EC50 of the therapeutic effector target binder can be about 6-fold, about 5-fold, about 4-fold, about 3-fold, or about 2-fold higher than the EC50 of the addressee target binder.

In some embodiments, the _IC50 of the therapeutic effector-target binder may be greater than about 1 pM, about 10 pM, about 100 pM, about 1 nM, about 10 nM, about 100 nM, about 500 nM, or about 1 uM (e.g., may be between 1 pM and 10 pM, between 10 pM and 100 pM, between 100 pM and 1 nM, between 1 nM and 10 nM, between 10 nM and 100 nM, between 100 nM and 500 nM, or between 500 nM and 1 uM). In some embodiments, the IC50 of the addressee target binder can be less than about 1 uM, about 500 nM, about 100 nM, about 10 nM, about 1 nM, about 100 pM, about 10 pM, or about 1 pM (e.g., can be between 1 uM and 500 nM, between 500 nM and 100 nM, between 100 nM and 10 nM, between 10 nM and 1 nM, between 1 nM and 100 pM, between 100 pM and 10 pM, or between 10 pM and 1 pM). In some embodiments, the _IC50 of the therapeutic effector target binder can be about 6-fold, about 5-fold, about 4-fold, about 3-fold, or about 2-fold higher than the _IC50 of the addressee target binder.

The cell or tissue density of the effector target and the addressee target bound by the ANDbody may be different. In embodiments, the density of the therapeutic effector target on the cell bound by the effector target binding site of the ANDbody is greater than about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 50-fold, about 100-fold, about 200-fold, about 500-fold, about 1000-fold, about 10,000-fold, about 100,000-fold lower than the density of the addressee target on the cell bound by the addressee binding site.

In some embodiments, the affinity of the first binding site for the therapeutic effector target bound by it is less than about one-half (½) X Kd than the affinity of the second binding site for the address target bound by it, and the density of the therapeutic effector target on the cell bound by the first binding site is less than about one-half (½) X Kd than the density of the address target on the cell bound by the second binding site.

In some embodiments, an ANDbody has affinity and density parameters as described above.

In some embodiments, the first binding site and the second binding site in the ANDbody are directly linked to each other. Directly linked means that the first binding site encoding sequence is adjacent to the second binding site encoding sequence and there is no sequence derived from other sequences (e.g., linkers). In some embodiments, the first binding site and the second binding site in the ANDbody are not directly linked to each other.

ANDbodies as disclosed herein can also be linked to one or more additional moieties, e.g., extracellular components, intracellular components, soluble factors (e.g., enzymes, hormones, interleukins, growth factors, toxins, venoms, pollutants, etc.), or transmembrane proteins (e.g., cell surface receptors). In some embodiments, ANDbodies are linked to small molecules and one or more additional moieties.

In some cases, the effector target sequence and/or the address target sequence comprises a full-length protein sequence and/or an Fc fusion sequence with or without a signal peptide region. In some embodiments, the ANDbody of the present technology includes a binding domain that binds to a bone address target protein or an effector target protein. In embodiments, the binding domain of the ANDbody of the present invention can bind to a protein sequence including a signal peptide. In other embodiments, the binding domain of the ANDbody of the present invention can bind to a protein lacking a signal protein. In some embodiments, the binding domain of the ANDbody of the present invention can bind to a full-length protein. In other embodiments, the binding domain of the ANDbody of the present invention can bind to a protein fusion fused to other proteins (e.g., an Fc sequence), such as a full-length protein sequence or a peptide fragment thereof with or without a signal peptide region. In other embodiments, the binding domain of an ANDbody of the invention can bind to a protein comprising less than the full-length protein sequence, such as a peptide fragment of an address target or effector target.

In some embodiments, an ANDbody of the invention comprises (a) a first binding site (effector-target binding site) specific for sclerostin (SOST) and (b) a second binding site (addressor-target binding site) specific for chondrocyte adhesin (CHAD).

In some embodiments, an ANDbody of the invention comprises (a) a first binding site (effector target binding site) specific for sclerostin (SOST) and (b) a second binding site (addressor target binding site) specific for interferon-induced transmembrane protein 5 (IFITM5).

In some embodiments, an ANDbody of the invention comprises (a) a first binding site (effector-target binding site) specific for Dickkopf-1 (DKK1) and (b) a second binding site (addressor-target binding site) specific for chondrocyte adhesion molecule (CHAD).

In some embodiments, an ANDbody of the invention comprises (a) a first binding site (effector-target binding site) specific for sclerostin (SOST) and (b) a second binding site (addressor-target binding site) specific for dentin matrix acidic phosphoprotein 1 (DMP1).

In some embodiments, an ANDbody of the invention comprises (a) a first binding site (effector-target binding site) specific for sclerostin (SOST) and (b) a second binding site (addressor-target binding site) specific for bone sialoprotein (IBSP).

In some embodiments, an ANDbody of the invention comprises (a) a first binding site (effector-target binding site) specific for sclerostin (SOST) and (b) a second binding site (addressor-target binding site) specific for trophoblast glycoprotein (TPBG).

In some embodiments, an ANDbody of the invention comprises (a) a first binding site specific for sclerostin (SOST) (effector-target binding site) and (b) a second binding site specific for hydroxyapatite (addressor-target binding site).

ANDbody Production of components

ANDbody Production of Peptides

The ANDbody polypeptides of the present invention can be produced by any suitable method. For example, all or part of an ANDbody can be expressed by a host cell comprising nucleotides encoding the ANDbody. Such methods for preparing therapeutic polypeptides are routine in the art. Generally, see Smales and James (eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar and Meibohm (eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).

Methods for producing ANDbodies may involve expression in mammalian cells, although recombinant proteins may also be produced using insect cells, yeast, bacteria, or other cells under the control of an appropriate promoter. Mammalian expression vectors may contain non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, for example, the SV40 origin, early promoter, enhancer, splice and polyadenylation sites can be used to provide other genetic elements required for expression of heterologous DNA sequences. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (4th ed.), Cold Spring Harbor Laboratory Press (2012).

A variety of mammalian cell culture systems can be used to express and manufacture the ANDbodies described herein. Examples of mammalian expression systems include, but are not limited to, CHO cells, COS cells, HeLA, and BHK cell lines. Processes for host cell culture for the production of protein therapeutics are described in, e.g., Zhou and Kantardjieff (eds.), Mammalian Cell Cultures for Biologics Manufacturing (Advances in Biochemical Engineering/Biotechnology), Springer (2014). Purification of protein therapeutics is described in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013) and Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010). Formulation of protein therapeutics is described in Meyer (ed.), Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).

Antibody production techniques are known. See, for example, Zhiqiang (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic. 1st ed. Wiley 2009; Greenfield (ed.) Antibodies: A Laboratory Manual. (Second edition) Cold Spring Harbor Laboratory Press 2013; Ferrara et al. 2012. Using Phage and Yeast Display to Select Hundreds of Monoclonal Antibodies: Application to Antigen 85, a Tuberculosis Biomarker. PLoS ONE 7(11): e49535. Methods for making recombinant antibodies, including antibody engineering, use of degenerate oligonucleotides, 5'-RACE, phage display, and mutagenesis; antibody testing and characterization; antibody pharmacokinetic and pharmacodynamics; antibody purification and storage; and screening and labeling techniques.

ANDbody RNA Production

In some embodiments, ANDbody RNA can be produced, for example, for delivery to a subject. Generally, therapeutic mRNA is produced by in vitro transcription. Modifications (e.g., incorporation of modified bases, 5' cap analogs, and poly(A) tails) can optimize activity and function. For example, translation and stability of mRNA can be achieved by cap and poly(A) tail modifications. For example, incorporation of cap analogs such as ARCA (anti-reverse cap analog) and a 100-200 bp poly(A) tail into in vitro transcribed (IVT) mRNA can improve expression and stability (Kaczmarek et al. Genome Medicine (2017) 9:60). Novel cap analogs, such as 1,2-dithiodiphosphate-modified caps, can further improve translation efficiency (Strenkowska et al. Nucleic Acids Res. 2016;44:9578–90). Codon optimization can also improve the efficiency of protein synthesis and limit mRNA instability caused by rare codons (Presnyak et al. Cell. 2015;160:1111–24. 93; Thess et al. Mol Ther. 2015;23:1456–64). Modification of the 3' and 5' untranslated regions (UTRs) containing sequences responsible for recruiting RNA-binding proteins (RBPs) and miRNAs can increase the level of protein production (Kaczmarek). In addition, UTRs can be modified to encode regulatory elements (e.g., K-turn motifs and miRNA binding sites) to control RNA expression in a cell-specific manner (Wroblewska et al. Nat Biotechnol. 2015;33:839–41). RNA base modifications (e.g., incorporation of pseudouridines such as N1-methyl-pseudouridine into mRNA) can help mask mRNA immunostimulatory activity and increase mRNA translation by enhancing translation initiation (Andries et al. J Control Release. 2015;217:337–44; Svitkin et al. Nucleic Acids Res. 2017;45:6023–36). mRNA compositions and methods for making them are known and disclosed, for example, in WO 2016011306; WO 2016014846; WO 2016022914; WO 2016077123; WO 2016164762; WO 2016201377; WO 2017049275; US 9937233; US 8710200; US 10022425; US 9878056; US 9572897; Jemielity et al. RNA. 2003;9:1108–22.90; Mockey et al. Biochem Biophys Res Commun. 2006;340:1062–8. 91; Strenkowska et al. Nucleic Acids Res. [Nucleic Acids Res] 2016;44:9578–90. 92; Presnyak et al. Cell. [Cell] 2015;160:1111–24. 93; Kaczmarek et al. Genome Medicine [Genome Medicine] (2017) 9:60.

Affinity changes ANDbody Production

ANDbodies with altered binding sites can be prepared using methods known in the art, for example, ANDbodies can be engineered to have a target binding site with reduced affinity for an effector target. See, for example, U.S. Patent No. 10,654,928. In general, ANDBody can be modified to alter the affinity of an effector target binding site to its effector target or to alter the affinity of a site target binding site to its site target. Modifications can increase or decrease the affinity of a binding partner to a binding site.

Target and site assessment

Expression of a therapeutic target can be assessed at the RNA or protein level using methods known in the art. In embodiments, expression of a therapeutic target is assessed by measuring RNA expression, for example using an RNA sequence dataset as a proxy for protein expression levels. RNA datasets include those of the Genotype Tissue Expression (GTEx) dataset (see, e.g., https://www.genome.gov/Funded-Programs-Projects/Genotype-Tissue-Expression-Project) or the Human Protein Atlas (HPA) dataset (https://www.proteinatlas.org/).

A non-limiting list of tissues that can be assessed for expression of therapeutic targets includes, for example, bone tissue and/or bone cells, minor salivary glands, thyroid glands, lungs, breast (breast tissue), pancreas, adrenal glands, liver, kidney (cortex), kidney (medullary), fatty viscera (omentum), small intestine-terminal ileum, fallopian tubes, ovaries, uterus, skin not exposed to the sun (suprapis); cervix-endocervix, cervix-ectocervix, vagina, skin exposed to the sun (calf), anterior cingulate cortex cells (BA24), caudate nucleus (basal ganglia), putamen (basal ganglia), nucleus accumbens (basal ganglia), hypothalamus, amygdala, hippocampus, cerebellum/cerebellar hemispheres, substantia nigra, pituitary, spinal cord (cervical), artery-aorta, heart-auricle, artery-coronary artery-heart, left ventricle, esophagus-mucosa, esophagus-muscle layer, esophagus-gastroesophageal junction, spleen, stomach, transverse colon, sigmoid colon, testis, whole blood, cells (EBV-transformed lymphocytes, artery-tibia or nerve-tibia tissue. In some embodiments, the expression of the address target is significantly higher in bone tissue than in any other tissue. In some embodiments, the expression of the address target is limited to bone tissue.

Address markers can be assessed using methods well known in the art, for example, gene expression can be assessed at the mRNA level using Northern blots, cDNA or oligonucleotide microarrays or sequencing (e.g., RNA-Seq), or at the protein expression level using protein microarrays, Western blots, flow cytometry, immunohistochemistry, etc. For example, modifications can be assessed using antibodies specific for a particular modified form of the protein (e.g., phospho-specific antibodies) or mass spectrometry.

ANDbody Purpose

The ANDbodies and pharmaceutical compositions provided herein are suitable for administration to a subject in need thereof, wherein the subject is a human or non-human animal, e.g., suitable for human therapy or veterinary use.

Veterinary uses include use in the treatment of mammals, including commercially related mammals, such as pet and livestock animals, such as cattle, pigs, horses, sheep, goats, cats, dogs, mice and/or rats; and/or birds, including commercially related birds, such as parrots, poultry, chickens, ducks, geese, hens or roosters and/or turkeys; zoo animals, such as felines; non-mammalian animals, such as reptiles, fish, amphibians, etc.

The present invention also relates to a subject or subject cell comprising an ANDbody composition described herein. In some embodiments, the subject or subject cell is a plant, insect, bacterium, fungus, vertebrate, mammal ( e.g. , human) or other organism or cell.

In some embodiments, a subject or subject cell is contacted with (e.g., delivered or administered) an ANDbody composition. In some embodiments, the subject is a mammal (e.g., a human). The amount of the ANDbody composition, expression product, or both in the subject can be measured at any time after administration.

In some embodiments, the subject to whom an ANDbody or ANDbody composition provided herein is administered has a bone disease, disorder, or condition. For example, in some embodiments, the subject has a bone density disease (e.g., low bone density, osteopenia, or osteoporosis), a kidney disease associated with calcium filtration in bone (e.g., renal bone disease, chronic kidney disease (CKD) (e.g., CKD associated with mineral and bone disease, or renal osteodystrophy), osteoarthritis, rheumatoid arthritis, osteogenesis imperfecta, Paget's disease, fibrodysplasia, osteomalacia, rickets, osteonecrosis, bone cancer (e.g., osteosarcoma or Ewing's sarcoma), osteomyelitis, or a bone infection.

Drug composition

Polypeptide drug composition

The ANDbody compositions described herein (e.g., ANDbody polypeptide or RNA compositions) can be administered to a subject in need thereof. The present invention includes pharmaceutical compositions comprising an ANDbody composition in combination with one or more pharmaceutically acceptable excipients.

Formulation of protein therapeutics is routine. See, e.g., Ribeiro et al., Insights on the Formulation of Recombinant Proteins. Adv Biochem Eng Biotechnol. 2020; 171:23-54. doi: 10.1007/10_2019_119. PMID: 31844925.

RNA Drug composition

A nucleic acid (e.g., RNA) encoding an ANDBody may alternatively or additionally be administered to a subject. Generally, therapeutic mRNA is produced by in vitro transcription. Modifications (e.g., incorporation of modified bases, 5' cap analogs, and poly(A) tails) can optimize activity and function. For example, translation and stability of mRNA can be achieved by cap and poly(A) tail modifications. For example, incorporation of cap analogs such as ARCA (anti-reverse cap analog) and a 100-200 bp poly(A) tail into in vitro transcribed (IVT) mRNA improves expression and stability (Kaczmarek et al. Genome Medicine (2017) 9:60). Novel cap analogs, such as 1,2-dithiodiphosphate-modified caps, can further improve translation efficiency (Strenkowska et al. Nucleic Acids Res. 2016;44:9578–90). Codon optimization can also improve the efficiency of protein synthesis and limit mRNA instability caused by rare codons (Presnyak et al. Cell. 2015;160:1111–24. 93; Thess et al. Mol Ther. 2015;23:1456–64). Modification of the 3' and 5' untranslated regions (UTRs) containing sequences responsible for recruiting RNA-binding proteins (RBPs) and miRNAs can increase the level of protein production (Kaczmarek). In addition, UTRs can be modified to encode regulatory elements (e.g., K-turn motifs and miRNA binding sites) to control RNA expression in a cell-specific manner (Wroblewska et al. Nat Biotechnol. 2015;33:839–41). RNA base modifications (e.g., incorporation of pseudouridines such as N1-methyl-pseudouridine into mRNA) can help mask mRNA immunostimulatory activity and increase mRNA translation by enhancing translation initiation (Andries et al. J Control Release. 2015;217:337–44; Svitkin et al. Nucleic Acids Res. 2017;45:6023–36). mRNA compositions and methods for making them are known and disclosed, for example, in WO 2016011306; WO 2016014846; WO 2016022914; WO 2016077123; WO 2016164762; WO 2016201377; WO 2017049275; US 9937233; US 8710200; US 10022425; US 9878056; US 9572897; Jemielity et al. RNA. 2003; 9:1108–22.90; Mockey et al. Biochem Biophys Res Commun. 2006; 340:1062–8.91; Strenkowska et al. Nucleic Acids Res. [Nucleic Acids Research] 2016; 44:9578–90.92; Presnyak et al. Cell. [Cell] 2015; 160:1111–24.93; Kaczmarek et al. Genome Medicine [Genome Medicine] (2017) 9:60.

In embodiments, the RNA is a circular RNA. See, e.g., WO 2019118919, describing the expression of therapeutic RNA, such as antibody RNA, from a circular RNA. In some embodiments, the present invention includes a circular polyribonucleotide comprising (a) an internal ribosome entry site (IRES), (b) an expression sequence encoding an ANDbody described herein and lacking a poly-A sequence, and (c) a termination element. The circular RNA encoding an ANDbody described herein can be delivered naked (i.e., not formulated with a carrier) or delivered with a carrier.

Combination therapy

In some embodiments, an ANDbody or ANDbody composition provided herein is administered in combination with one or more additional therapeutic agents, e.g., one or more additional bone therapeutic agents. For example, an ANDbody or ANDbody composition can be administered in combination with an agent for treating a bone density disease (e.g., low bone density, osteopenia, or osteoporosis), a kidney disease associated with calcium filtration in bone (e.g., renal bone disease, CKD (e.g., CKD associated with mineral and bone disease), or renal osteodystrophy), osteoarthritis, rheumatoid arthritis, osteogenesis imperfecta, Paget's disease, fibrodysplasia, osteomalacia, rickets, osteonecrosis, bone cancer (e.g., osteosarcoma or Ewing's sarcoma), osteomyelitis, or a bone infection.

In some embodiments, the additional bone therapeutic agent is an agent used to treat osteoporosis, such as a bisphosphonate (e.g., alendronate, ibandronate, risedronate, or zoledronic acid).

In some embodiments, the additional bone therapeutic agent is a sclerostin (SOST) inhibitor (e.g., romone monoclonal antibody, busulfan monoclonal antibody, sersu monoclonal antibody, SHR-1222 or SOST inhibitor. See, e.g., Yu et al., Acta Pharm Sin B , 12(5): 2150-2170, 2022), a dickkopf-1 (DKK1) inhibitor (see, e.g., Jiang et al., Front Pharmacol , 13: Article No. 847387, 2022), a receptor activator of nuclear factor kappa-beta ligand (RANKL) inhibitor (e.g., dinosul monoclonal antibody) or a cathepsin K inhibitor (e.g., odanacatib).

Carrier

Lipid Nanoparticles

Formulations of the compositions described herein (e.g., polypeptide or RNA ANDbody compositions) for in vivo delivery with a carrier include lipid nanoparticle (LNP) formulations. See, e.g., U.S. Pat. No. 9,764,036; U.S. Pat. No. 9,682,139; Kauffman et al. Nano Lett. 2015; 15: 7300–6. 37; Fenton et al. Adv Mater. 2016; 28: 2939–43). In some embodiments, the LNP comprises one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic or zwitterionic lipids); one or more fused lipids (such as PEG-fused lipids or lipids fused to polymers described in Table 5 of WO 2019217941; which is incorporated herein by reference in its entirety); one or more sterols (e.g., cholesterol); and, optionally, one or more targeting molecules (e.g., fused receptors, receptor ligands, antibodies); or a combination of the foregoing.

Lipids that can be used for nanoparticle formation (e.g., lipid nanoparticles) include, for example, those described in Table 4 of WO 2019217941, which is incorporated herein by reference—for example, the lipid-containing nanoparticles may include one or more lipids in Table 4 of WO 2019217941. The lipid nanoparticles may include additional elements, such as polymers, such as the polymers described in Table 5 of WO 2019217941, which is incorporated by reference.

In some embodiments, the conjugated lipid, when present, may include one or more of the following: PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristyl glycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), polyethylene glycol phospholipid ethanolamine (PEG-PE), PEG succinic acid diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearyl-sn-glycero-3-phosphoethanolamine sodium salt, and in WO Those described in Table 2 of 2019051289 (incorporated by reference) and combinations of the foregoing.

In some embodiments, the sterol that can be incorporated into the lipid nanoparticles includes one or more of cholesterol or cholesterol derivatives, such as those in WO2009/127060 or US 2010/0130588, which are incorporated by reference. Additional exemplary sterols include plant sterols, including those described in Eygeris et al. (2020), dx.doi.org/10.1021/acs.nanolett.0c01386, which are incorporated herein by reference.

In some embodiments, the lipid particles include ionizable lipids, non-cationic lipids, ligands that inhibit particle aggregation, and sterols. The amounts of these components can be varied independently to obtain desired properties. For example, in some embodiments, the lipid nanoparticles include: ionizable lipids in an amount of about 20 mol% to about 90 mol% of the total lipids (in other embodiments, it can be 20-70% (mol), 30-60% (mol) or 40-50% (mol) of the total lipids present in the lipid nanoparticles; about 50 mol% to about 90 mol%); non-cationic lipids in an amount of about 5 mol% to about 30 mol% of the total lipids; fused lipids in an amount of about 0.5 mol% to about 20 mol% of the total lipids, and sterols in an amount of about 20 mol% to about 50 mol% of the total lipids. The ratio of total lipids to nucleic acids can be varied as desired. For example, the ratio of total lipids to nucleic acids (mass or weight) can be about 10: 1 to about 30: 1.

In some embodiments, the ratio of lipid to nucleic acid (mass/mass ratio; w/w ratio) can be in the following ranges: about 1: 1 to about 25: 1, about 10: 1 to about 14: 1, about 3: 1 to about 15: 1, about 4: 1 to about 10: 1, about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1. The amount of lipid and nucleic acid can be adjusted to provide a desired N/P ratio, such as 3, 4, 5, 6, 7, 8, 9, 10 or higher N/P ratios. Typically, the total lipid content of the lipid nanoparticle formulation can be in the range of about 5 mg/ml to about 30 mg/mL.

Some non-limiting examples of lipid compounds that can be used (e.g., in combination with other lipid components) to form lipid nanoparticles for delivery of compositions described herein, such as nucleic acids (e.g., RNA) described herein, include: (i) .

In some embodiments, a LNP comprising formula (i) is used to deliver an ANDbody RNA composition described herein to the liver and/or hepatocytes, (ii) .

In some embodiments, LNPs comprising formula (ii) are used to deliver an ANDbody RNA composition described herein to the liver and/or hepatocytes, (iii) .

In some embodiments, LNPs comprising formula (iii) are used to deliver an ANDbody RNA composition described herein to the liver and/or hepatocytes, (iv) (v) .

In some embodiments, LNPs comprising formula (v) are used to deliver an ANDbody RNA composition described herein to the liver and/or hepatocytes, (vi) .

In some embodiments, a LNP comprising formula (vi) is used to deliver an ANDbody RNA composition described herein to the liver and/or hepatocytes, (vii) (viii) .

In some embodiments, LNPs comprising formula (viii) are used to deliver the ANDbody RNA compositions described herein to the liver and/or hepatocytes, (ix) .

In some embodiments, LNPs comprising formula (ix) are used to deliver an ANDbody RNA composition described herein to the liver and/or hepatocytes, (x) wherein ^X1 is O, ^NR1 or a direct bond, ^X2 is C2-5 alkylene, ^X3 is C(=O) or a direct bond, ^R1 is H or Me, ^R3 is C1-3 alkylene, ^R2 is C1-3 alkylene, or ^R2 together with the nitrogen atom to which it is attached and 1-3 carbon atoms of ^X2 form a 4-, 5- or 6-membered ring, or ^X1 is ^NR1 , ^R1 and ^R2 together with the nitrogen atom to which they are attached form a 5- or 6-membered ring, or ^R2 and ^R3 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered ring, ^Y1 is C2-12 alkylene, and ^Y2 is selected from (in either orientation), (in either orientation), (in any orientation), n is 0 to 3, R ⁴ is C1-15 alkyl, Z ¹ is C1-6 alkylene or a direct bond, (in either orientation) or absent, provided that if Z ¹ is a direct bond, then Z ² is absent; R ⁵ is C 5-9 alkyl or C 6-10 alkoxy, R ⁶ is C 5-9 alkyl or C 6-10 alkoxy, W is methylene or a direct bond, and R ⁷ is H or Me, or a salt thereof, provided that if R ³ and R ² are C 2 alkyl, X ¹ is O, X ² is a linear C 3 alkylene radical, X ³ is C(=0), Y ¹ is a linear C 3 alkylene radical, (Y ² )nR ⁴ is , R ⁴ is a linear C5 alkyl group, Z ¹ is a C2 alkylene group, Z ² is absent, W is a methylene group, and R ⁷ is H, then R ⁵ and R ⁶ are not Cx alkoxy groups.

In some embodiments, LNPs comprising formula (xii) are used to deliver an ANDbody RNA composition described herein to the liver and/or hepatocytes, .

In some embodiments, LNPs comprising formula (xi) are used to deliver an ANDbody RNA composition described herein to the liver and/or hepatocytes. Where R = (xii) (xiii) (xiv) .

In some embodiments, the LNP comprises a compound of formula (xiii) and a compound of formula (xiv), (xv) .

In some embodiments, LNPs comprising formula (xv) are used to deliver an ANDbody RNA composition described herein to the liver and/or hepatocytes, (xvi) .

In some embodiments, LNPs comprising the formulation of formula (xvi) are used to deliver the ANDbody RNA compositions described herein to lung endothelial cells, (xvii) Where X = (xviii) (a) (xviii)(b) (xix).

In some embodiments, lipid compounds used to form lipid nanoparticles for delivery of compositions described herein, such as nucleic acids (e.g., RNA) described herein, are made by one of the following reactions: + (xx) (a) + (xx)(b).

In some embodiments, a composition described herein (e.g., a nucleic acid or protein) is provided in an LNP comprising an ionizable lipid. In some embodiments, the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); for example, as described in Example 1 of US 9,867,888 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadec-9,12-dienoate (LP01), for example, as synthesized in Example 13 of WO 2015/095340 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is di((Z)-non-2-en-1-yl) 9-((4-dimethylamino)butyryl)oxy)heptadecanedioate (L319), for example, as synthesized in Example 7, Example 8, or Example 9 of US 2012/0027803 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperidin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), for example, as synthesized in Examples 14 and 16 of WO 2010/053572 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is an imidazolyl cholesterol ester (ICE) lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-(1H-imidazol-4-yl)propanoate, such as structure (I) from WO 2020/106946 (incorporated herein by reference in its entirety).

In some embodiments, the ionizable lipid can be a cationic lipid, an ionizable cationic lipid, such as a cationic lipid that can exist in a positively charged form or a neutral form according to pH, or an amine-containing lipid that can be easily protonated. In some embodiments, the cationic lipid is, for example, a lipid that can be positively charged under physiological conditions. Exemplary cationic lipids include one or more positively charged amine groups. In some embodiments, the lipid particles include cationic lipids formulated with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (such as sterols), PEG, cholesterol, and polymer-conjugated lipids. In some embodiments, the cationic lipid can be an ionizable cationic lipid. Exemplary cationic lipids as disclosed herein can have an effective pKa of more than 6.0. In embodiments, the lipid nanoparticles can include a second cationic lipid having an effective pKa different from that of the first cationic lipid (e.g., greater than the first effective pKa). The lipid nanoparticles can include 40 mol% to 60 mol% of cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and therapeutic agents encapsulated in or conjugated to the lipid nanoparticles, such as nucleic acids (e.g., RNA) described herein. In some embodiments, the nucleic acids are co-formulated with the cationic lipids. Nucleic acids can be adsorbed to the surface of LNPs (e.g., LNPs comprising cationic lipids). In some embodiments, nucleic acids can be encapsulated in LNPs (e.g., LNPs comprising cationic lipids). In some embodiments, lipid nanoparticles can include targeting moieties, such as targeting moieties coated with targeting agents. In embodiments, LNP formulations are biodegradable. In some embodiments, lipid nanoparticles comprising one or more lipids described herein (e.g., formula (i), (ii), (vii), and/or (ix)) encapsulate at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or 100% of RNA molecules.

Exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, but are not limited to, those listed in Table 1 of WO 2019051289, which is incorporated herein by reference. Additional exemplary lipids include, but are not limited to, one or more of the following formulae: X of US 2016/0311759; I of US 20150376115 or US 2016/0376224; I, II, or III of US 20160151284; I, IA, II, or IIA of US 20170210967; I-c of US 20150140070; A of US 2013/0178541; I of US 2013/0303587 or US 2013/0123338; I of US 2015/0141678; II, III, IV, or V of US 2015/0239926; I of US 2017/0119904; I or II of US 2017/117528; A of US 2012/0149894; A of US 2015/0057373; A of WO 2013/116126; A of US 2013/0090372; A of US 2013/0274523; A of US 2013/0274504; A of US 2013/0053572; A of WO2013/016058; A of WO2012/162210; I of US 2008/042973; I, II, III or IV of US 2012/01287670; I or II of US 2014/0200257; I, II or III of US 2015/0203446; I or III of US 2015/0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID or III-XXIV of US 2014/0308304; I, II, III or IV of WO2009/132131; A of US 2012/01011478; I or XXXV of US 2012/0027796; XIV or XVII of US 2012/0058144; I of US 2013/0323269; I of US 2011/0117125; I, II or III of US 2011/0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US 2012/0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV or XVI of US 2011/0076335; I or II of US 2006/008378; I of US 2013/0123338; I or X-A-Y-Z of US 2015/0064242; XVI, XVII or XVIII of US 2013/0022649; I, II or III of US 2013/0116307; I, II or III of US 2013/0116307; I or II of US 2010/0062967; I-X of US 2013/0189351; I of US 2014/0039032; V of US 2018/0028664; I of US 2016/0317458; I of US 2013/0195920; 5, 6 or 10 of US 10,221,127; III-3 of WO 2018/081480; I-5 or I-8 of WO 2020/081938; 18 or 25 of US 9,867,888; A of US 2019/0136231; II of WO 2020/219876; 1 of US 2012/0027803; OF-02 of US 2019/0240349; US 23 of 10,086,013; cKK-E12/A6 of Miao et al. (2020); C12-200 of WO 2010/053572; 7C1 of Dahlman et al. (2017); 304-O13 or 503-O13 of Whitehead et al.; TS-P4C2 of US 9,708,628; I of WO 2020/106946; I of WO 2020/106946.

In some embodiments, the ionizable lipid is MC3 (6Z, 9Z, 28Z, 31Z)-heptatriacontane-6, 9, 28, 31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA or MC3), for example, as described in Example 9 of WO 2019051289A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is lipid ATX-002, for example, as described in Example 10 of WO 2019051289 A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is (13Z, 16Z)-A, A-dimethyl-3-nonyldidocosa-13, 16-dien-1-amine (Compound 32), for example, as described in Example 11 of WO 2019051289 A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is Compound 6 or Compound 22, for example, as described in Example 12 of WO 2019051289 A9 (incorporated herein by reference in its entirety).

Exemplary non-cationic lipids include, but are not limited to, distearyl-sn-glycero-phosphoethanolamine, distearylphospholipid acyl choline (DSPC), dioleoylphospholipid acyl choline (DOPC), dimalmitoylphospholipid acyl choline (DPPC), dioleoylphospholipid acyl choline (DOPG), dimalmitoylphospholipid acyl glycerol (DPPG), dioleoylphospholipid acyl ethanolamine (DOPE), palmitoylphospholipid acyl choline (POPC), palmitoylphospholipid acyl choline (POPC), palmitoylphospholipid acyl glycerol (DPPG), Phosphatidylethanolamine (POPE), dioleyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dimalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16 -O-dimethyl PE), l8-l-trans PE, l-stearyl-2-oleyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phospholipid acylcholine (HSPC), phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearylphosphatidylglycerol (DSPG), dimustine Acylphospholipid acyl choline (DEPC), palm acyl phospholipid acyl glycerol (POPG), dioleyl-phosphatidyl ethanolamine (DEPE), lecithin, phosphatidyl ethanolamine, lysophosphatidyl choline, lysophosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dihexadecyl phosphate, lysophosphatidyl choline, dilinoleyl phosphatidyl choline or mixtures thereof. It should be understood that other diacyl phosphatidyl choline and diacyl phosphatidyl ethanolamine phospholipids can also be used. The acyl groups in the lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, such as lauryl, myristyl, palmityl, stearyl or oleyl. In certain embodiments, additional exemplary lipids include, but are not limited to, those described in Kim et al. (2020) dx.doi.org/10.1021/acs.nanolett.0c01386, which is incorporated herein by reference. In some embodiments, such lipids include plant lipids (e.g., DGTS) that have been found to improve liver transfection with mRNA.

Other examples of non-cationic lipids suitable for use in lipid nanoparticles include, but are not limited to, non-phospholipids, such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, ricinoleic acid glyceryl, hexadecyl stearate, isopropyl myristate, amphoteric acrylic acid polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfates, polyethoxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, etc. Other non-cationic lipids are described in WO 2017/099823 or U.S. Patent Publication No. US 2018/0028664, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the non-cationic lipid is oleic acid or a compound of formula I, II or IV of US 2018/0028664, which is incorporated by reference in its entirety. The non-cationic lipid can account for, for example, 0-30% (mol) of the total lipid present in the lipid nanoparticles. In some embodiments, the non-cationic lipid content is 5%-20% (mol) or 10%-15% (mol) of the total lipid present in the lipid nanoparticles. In embodiments, the molar ratio of ionizable lipid to neutral lipid is about 2: 1 to about 8: 1 (e.g., about 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1 or 8: 1).

In some embodiments, the lipid nanoparticles do not contain any phospholipids.

In some aspects, the lipid nanoparticles may further comprise components such as sterols to provide membrane integrity. An exemplary sterol that can be used in lipid nanoparticles is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs such as 5a-cholestanol, 53-naphthalene stanol, cholesteryl-(2,-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether and 6-ketocholestanol; non-polar analogs such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone and cholesterol decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog, for example, cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT Publication No. WO 2009/127060 and U.S. Patent Publication No. US 2010/0130588, each of which is incorporated herein by reference in its entirety.

In some embodiments, components that provide membrane integrity, such as sterols, may comprise 0-50% (mol) of the total lipid present in the lipid nanoparticles (e.g., 0-10%, 10%-20%, 20%-30%, 30%-40%, or 40%-50%). In some embodiments, such components are 20%-50% (mol), 30%-40% (mol) of the total lipid content of the lipid nanoparticles.

In some embodiments, the lipid nanoparticles may contain polyethylene glycol (PEG) or tethered lipid molecules. Typically, these are used to inhibit the aggregation of lipid nanoparticles and/or provide spatial stability. Exemplary tethered lipids include, but are not limited to, PEG-lipid tethers, polyoxazoline (POZ)-lipid tethers, polyamide-lipid tethers (such as ATTA-lipid tethers), cationic polymer lipids (CPL) tethers, and mixtures thereof. In some embodiments, the tethered lipid molecule is a PEG-lipid tether, such as a (methoxypolyethylene glycol) tethered lipid.

Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), PEGylated phospholipid ethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAA), and PEG-phospholipid esters. G) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG dialkoxypropylcarbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearyl-sn-glycero-3-phosphoethanolamine sodium salt or a mixture thereof. Additional exemplary PEG-lipid conjugates are described, for example, in US 5,885,613, US 6,287,591, US 2003/0077829, US 2003/0077829, US 2005/0175682, US 2008/0020058, US 2011/0117125, US 2010/0130588, US 2016/0376224, US 2017/0119904 and US/099823, all of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-lipid is US 2018/0028664, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-lipid has US 20150376115 or US 2016/0376224, both of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmitoyloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of the following: PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-distearylglycerol, PEG-dilaurylglyceramide, PEG-dimyristylglyceramide, PEG-dipalmitoylglyceramide, PEG-distearylglycerol In some embodiments, the PEG-lipid comprises PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises a structure selected from the following: ,

In some embodiments, lipids conjugated to molecules other than PEG can also be used in place of PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic polymer lipid (GPL) conjugates can be used in place of PEG-lipids or in combination with PEG-lipids.

Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids are described in the PCT and LIS patent applications listed in Table 2 of WO 2019051289 A9, all of which are incorporated herein by reference in their entirety.

In some embodiments, PEG or tethered lipids may account for 0-20% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the content of PEG or tethered lipids is 0.5%-10% or 2%-5% (mol) of the total lipids present in the lipid nanoparticles. The molar ratio of ionizable lipids, non-cationic lipids, sterols and PEG/tethered lipids may vary as needed. For example, the lipid particles may contain 30%-70% ionizable lipids by mole or total weight of the composition, 0-60% cholesterol by mole or total weight of the composition, 0-30% non-cationic lipids by mole or total weight of the composition, and 1%-10% tethered lipids by mole or total weight of the composition. Preferably, the composition comprises 30%-40% ionizable lipids by mole or total weight of the composition, 40%-50% cholesterol by mole or total weight of the composition, and 10%-20% non-cationic lipids by mole or total weight of the composition. In some other embodiments, the composition comprises 50%-75% ionizable lipids by mole or total weight of the composition, 20%-40% cholesterol by mole or total weight of the composition, 5% to 10% non-cationic lipids by mole or total weight of the composition, and 1%-10% hydrating lipids by mole or total weight of the composition. The composition may contain 60%-70% ionizable lipids by mole or total weight of the composition, 25%-35% cholesterol by mole or total weight of the composition, and 5%-10% non-cationic lipids by mole or total weight of the composition. The composition may also contain up to 90% ionizable lipids by mole or total weight of the composition and 2% to 15% non-cationic lipids by mole or total weight of the composition. The formulation can also be a lipid nanoparticle formulation, for example comprising 8%-30% ionizable lipid by mole or total weight of the composition, 5%-30% non-cationic lipid by mole or total weight of the composition, and 0-20% cholesterol by mole or total weight of the composition; 4%-25% ionizable lipid by mole or total weight of the composition, 4%-25% non-cationic lipid by mole or total weight of the composition, 2% to 25% cholesterol by mole or total weight of the composition, 10% to 35% fused lipid by mole or total weight of the composition, and 0% to 20% cholesterol by mole or total weight of the composition; or 2% to 30% by mole or total weight of ionizable lipids, 2% to 30% by mole or total weight of non-cationic lipids, 1% to 15% by mole or total weight of cholesterol, 2% to 35% by mole or total weight of fused lipids, and 1% to 20% by mole or total weight of cholesterol; or even up to 90% by mole or total weight of ionizable lipids and 2% to 10% by mole or total weight of non-cationic lipids, or even 100% by mole or total weight of cationic lipids. In some embodiments, the lipid particle formulation comprises an ionizable lipid, a phospholipid, cholesterol, and a PEGylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation comprises an ionizable lipid, cholesterol, and a PEGylated lipid in a molar ratio of 60:38.5:1.5.

In some embodiments, the lipid particles comprise ionizable lipids, non-cationic lipids (e.g., phospholipids), sterols (e.g., cholesterol), and pegylated lipids, wherein the lipid molar ratio of the ionizable lipids is in the range of 20 to 70 mol%, with a target of 40-60 mol%, the molar percentage of the non-cationic lipids is in the range of 0 to 30 mol%, with a target of 0 to 15 mol%, the molar percentage of the sterols is in the range of 20 to 70 mol%, with a target of 30 to 50 mol%, and the molar percentage of the pegylated lipids is in the range of 1 to 6 mol%, with a target of 2 to 5 mol%.

In some embodiments, the lipid particles contain ionizable lipid/non-cationic lipid/sterol/conjugated lipid in a molar ratio of 50:10:38.5:1.5.

In one aspect, the present disclosure provides a lipid nanoparticle formulation comprising phospholipids, phosphatidylcholine, phosphatidylcholine and phosphatidylethanolamine.

In some embodiments, one or more additional compounds may also be included. Those compounds may be administered alone, or additional compounds may be included in the lipid nanoparticles of the present invention. In other words, in addition to nucleic acids or at least second nucleic acids, lipid nanoparticles may contain other compounds different from the first nucleic acid. Non-limitingly, other additional compounds may be selected from the group consisting of: small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptide mimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, or any combination thereof.

In some embodiments, LNPs are directed to specific tissues by adding LNP targeting domains. For example, biological ligands can be displayed on the surface of LNPs to enhance interaction with cells displaying cognate receptors, thereby promoting association with tissues where cells express the receptors and cargo delivery therein. In some embodiments, the biological ligand can be a ligand that drives delivery to the liver, for example, LNPs displaying GalNAc promote delivery of nucleic acid cargo to liver cells displaying asialoglycoprotein receptors (ASGPR). The work of Akinc et al. Mol Ther [Molecular Ther] 18(7):1357-1364 (2010) taught that conjugating trivalent GalNAc ligands to PEG-lipid (GalNAc-PEG-DSG) to generate ASGPR-dependent LNPs resulted in observable LNP loading effects (see, e.g., Figure 6 of Akinc et al. 2010, supra). Other ligand-displaying LNP formulations, such as those incorporating folic acid, transferrin, or antibodies, are discussed in WO 2017223135, which is incorporated herein by reference in its entirety, along with the references used therein: namely, Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release. 20:63–68; Peer et al., Proc Natl Acad Sci U S A. 2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630; and Peer and Lieberman, Gene Ther. 2011 18:1127-1133.

In some embodiments, LNPs are selected for tissue-specific activity by adding Selective ORgan Targeting (SORT) molecules to a formulation comprising traditional components such as ionizable cationic lipids, amphiphilic phospholipids, cholesterol, and poly(ethylene glycol) (PEG). Cheng et al. Nat Nanotechnol 15(4):313-320 (2020) teach that the addition of supplemental "SORT" components can precisely alter the in vivo RNA delivery profile and mediate tissue-specific (e.g., lung, liver, spleen) gene delivery and editing based on the percentage and biophysical properties of SORT molecules.

In some embodiments, LNPs contain biodegradable ionizable lipids. In some embodiments, LNPs contain (9Z, 12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadec-9,12-dienoate, also known as 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadec-9,12-dienoate) or another ionizable lipid. See, e.g., WO 2019/067992, WO/2017/173054, WO 2015/095340, and WO 2014/136086, and the references provided therein for lipids. In some embodiments, the terms cationic and ionizable are interchangeable in the context of LNP lipids, e.g., where the ionizable lipid is cationic depending on pH.

In some embodiments, the average LNP diameter of the LNP formulation can be between tens of nm and hundreds of nm, for example, as measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation can be about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the LNP formulation may be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation may be about 70 nm to about 100 nm. In a specific embodiment, the average LNP diameter of the LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of the LNP formulation can be about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation ranges from about 1 mm to about 500 mm, about 5 mm to about 200 mm, about 10 mm to about 100 mm, about 20 mm to about 80 mm, about 25 mm to about 60 mm, about 30 mm to about 55 mm, about 35 mm to about 50 mm, or about 38 mm to about 42 mm.

In some cases, LNPs can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of LNPs, such as the particle size distribution of lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The polydispersity index of LNPs can be about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the LNPs may be from about 0.10 to about 0.20.

The zeta potential of LNP can be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential can describe the surface charge of the LNP. Lipid nanoparticles with relatively low charge (positive or negative) are generally desired because higher charged substances may not interact ideally with cells, tissues and other elements in the body. In some embodiments, the zeta potential of the LNP can be between about -10 mV and about +20 mV, about -10 mV and about +15 mV, about -10 mV and about +10 mV, about -10 mV and about +5 mV, about -10 mV and about 0 mV, about -10 mV and about -5 mV, about -5 mV and about +20 mV, about -5 mV and about +15 mV, about -5 mV and about +10 mV, about -5 mV and about +5 mV, about -5 mV and about 0 mV, about 0 mV and about +20 mV, about 0 mV and about +15 mV, about 0 mV and about +10 mV, about 0 mV and about +5 mV, about +5 mV and about +20 mV, about +5 mV and about +15 mV, or about +5 mV and about +10 mV.

The encapsulation efficiency of proteins and/or nucleic acids describes the amount of proteins and/or nucleic acids that are encapsulated or otherwise associated with LNPs after preparation relative to the initial amount provided. Encapsulation efficiency is ideally high (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of proteins or nucleic acids in a solution containing lipid nanoparticles before and after the lipid nanoparticles are broken with one or more organic solvents or detergents. Anion exchange resins can be used to measure the amount of free proteins or nucleic acids (e.g., RNA) in a solution. Fluorescence can be used to measure the amount of free proteins and/or nucleic acids (e.g., RNA) in a solution. For lipid nanoparticles described herein, the encapsulation efficiency of proteins and/or nucleic acids can be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In some embodiments, the encapsulation efficiency can be at least 90%. In some embodiments, the encapsulation efficiency can be at least 95%.

The LNP may optionally include one or more coatings. In some embodiments, the LNP may be formulated in a capsule, film, or tablet having a coating. The capsule, film, or tablet comprising the composition described herein may have any useful size, tensile strength, hardness, or density.

Additional exemplary lipids, formulations, methods, and LNP characterizations are taught by WO 2020061457, which is incorporated herein by reference in its entirety.

In some embodiments, in vitro or ex vivo cellular liposomal transfection is performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA transfection reagent (Mirus Bio). In certain embodiments, LNPs are formulated using GenVoy_ILM ionizable lipid mixtures (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl [German Applied Chemistry] 51(34):8529-8533 (2012), which is incorporated herein by reference in its entirety.

LNP formulations optimized for delivery of CRISPR-Cas systems (e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA) are described in WO 2019067992 and WO 2019067910, both of which are incorporated by reference.

Additional specific LNP formulations useful for delivery of nucleic acids are described in US 8,158,601 and US 8,168,775, both of which are incorporated by reference, including the formulation sold under the name ONPATTRO used in patisiran.

Exemplary dosages of LNPs comprising RNA compositions described herein may include about 0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, or 100 mg/kg (RNA). Exemplary dosing of AAVs comprising nucleic acids encoding one or more components of the system may include MOIs of about 1011, 1012, 1013, and 1014 vg/kg.

In some embodiments, the present invention includes lipid nanoparticles (LNPs) comprising an ANDbody polypeptide described herein (or RNA encoding it), a nucleic acid molecule, or a DNA encoding an ANDbody described herein. In embodiments, the LNP comprises a cationic lipid. In some embodiments, the LNP further comprises one or more neutral lipids, such as DSPC, DPPC, DMPC, DOPC, POPC, DOPE, SM, a steroid, such as cholesterol, and/or one or more polymer-conjugated lipids, such as PEGylated lipids, such as PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkoxypropylcarbamate. In some embodiments, the cationic lipid of the LNP has a structure according to the following: (i) (ii) or (iii).

For a review of LNPs, see also Li et al. 2017, Nanomaterials 7, 122; doi:10.3390/nano7060122.

Other carriers

Viral vector

The compositions described herein (e.g., polypeptide or RNA ANDbody compositions) can be delivered by viral vectors (e.g., viral vectors expressing RNA). The viral vectors can be administered to cells or subjects (e.g., human subjects or non-human animals). The viral vectors can be administered locally or systemically.

Examples of viral vectors include retroviruses (e.g., Retroviridae virus vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-stranded RNA viruses (such as orthomyxoviruses (e.g., influenza virus), rebactoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus)), positive-stranded RNA viruses (such as picornaviruses and alphaviruses), and double-stranded DNA viruses (including adenoviruses, herpes viruses (e.g., herpes simplex virus type 1 and type 2, Espionage-Barr virus, cytomegalovirus, replication-deficient herpes virus), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), chickenpox, and canarypox)). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepatitis virus, human papillomavirus, human foamy virus and hepatitis virus. Examples of retroviruses include: avian leukosis sarcoma, avian C virus, mammalian C virus, B virus, D virus, oncorretrovirus, HTLV-BLV group, lentivirus, alpha retrovirus, gamma retrovirus, foamy virus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology (3rd ed.) Lippincott-Raven, Philadelphia, 1996). Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, monkey sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in U.S. Patent No. 5,801,030, the teachings of which are incorporated herein by reference.

Anellovirus vectors can also be used to deliver the ANDbody compositions described herein. Anellovirus vectors are known in the art and are described, for example, in WO 2020123773, WO 2020123816, WO 2018232017, and WO 2020123773. In certain embodiments, the anellovirus vector composition comprises a genomic element comprising a promoter operably linked to a nucleic acid sequence encoding an ANDbody described herein, the genetic element being encapsidated by a protein exterior comprising anellovirus ORF1, such as anellovirus capsid protein.

Cell- and vesicle-based delivery vehicles

The compositions described herein (e.g., polypeptide or RNA ANDbody compositions) described herein can be administered to cells in cells, vesicles, or other membrane-based carriers. In one embodiment, the compositions and systems described herein can be formulated in liposomes or other similar vesicles. Liposomes are spherical vesicle structures composed of a monolayer or multilayer lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral, or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their cargo across biological membranes and the blood-brain barrier (BBB) (for a review, see, e.g., Spuch and Navarro, Journal of Drug Delivery, Vol. 2011, Article ID 469679, p. 12, 2011. doi:10.1155/2011/469679). Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparing multilamellar vesicular lipids are known in the art (see, e.g., U.S. Patent No. 6,693,086, which is incorporated herein by reference for its teachings on the preparation of multilamellar vesicular lipids). Although vesicle formation can be spontaneous when the lipid membrane is mixed with an aqueous solution, vesicle formation can also be accelerated by applying force in the form of oscillations through the use of a homogenizer, sonicator, or extrusion equipment (for a review, see, e.g., Spuch and Navarro, Journal of Drug Delivery, Vol. 2011, Article ID 469679, p. 12, 2011. doi:10.1155/2011/469679). Extruded lipids can be prepared by extrusion through a filter of decreasing size as described in Templeton et al., Nature Biotech, 15:647-652, 1997, which is incorporated herein by reference for its teachings on the preparation of extruded lipids.

Exosomes can also be used as drug delivery vehicles for the compositions and systems described herein. For a review, see Ha et al. July 2016. Acta Pharmaceutica Sinica B [Pharmaceutica Sinica] Vol. 6, No. 4, pp. 287-296; https://doi.org/10.1016/j.apsb.2016.02.001.

Ex vivo differentiated erythrocytes can also be used as carriers for agents described herein (e.g., inhibitory agents), such as antibodies or nucleic acids described herein. See, e.g., WO 2015073587; WO 2017123646; WO 2017123644; WO 2018102740; WO 2016183482; WO 2015153102; WO 2018151829; WO 2018009838; Shi et al. 2014. Proc Natl Acad Sci USA. 111(28): 10131–10136; U.S. Pat. No. 9,644,180; Huang et al. 2017. Nature Communications 8: 423; Shi et al. 2014. Proc Natl Acad Sci USA. 111(28): 10131–10136. 10131–10136.

Fusion compositions, for example, as described in WO 2018208728, can also be used as carriers for delivering the [agents] or formulations described herein.

Plant nanovesicles and plant messenger packets (PMPs), such as those described in WO 2011097480, WO 2013070324, WO 2017004526, or WO 2020041784, can also be used as carriers for delivering the compositions described herein.

Without further elaboration, it is believed that one skilled in the art can utilize the present invention to its fullest extent based on the above description. The following specific embodiments are therefore to be interpreted as merely illustrative and not limiting in any way the remainder of the present disclosure. All publications and sections cited herein are incorporated herein by reference for the purpose or subject matter of the present invention.

Examples

The present invention will be further illustrated in the following non-limiting examples.

contents Examples 1 Design of ANDbody binding to chondroadherin (CHAD) address and sclerostin (SOST) target Examples 2 Generation and characterization of ANDbodies that bind to CHAD addresses and SOST targets Examples 3 Design of ANDbody that binds to interferon-induced transmembrane protein 5 (IFITM5) address and SOST target Examples 4 Generation and characterization of ANDbodies that bind to both IFITM5 and SOST targets Examples 5 Design of ANDbody binding CHAD and Dickkopf-1 (DKK1) Examples 6 Pharmacokinetics and tissue distribution of bone-targeted ANDbody Examples 7 In vivo effects of bone-targeted ANDbody Examples 8 Generation and characterization of ANDbody constructs that bind to the DMP1 site Examples 9 Design of ANDbody combining DMP1 address and SOST target Examples 10 Verification of the extracellular matrix address of DMP1 in bone cells Examples 11 Characterization of ANDbodies that bind to both the DMP1 site and the SOST target Examples 12 Design of ANDbody combining IBSP address and SOST target Examples 13 Generation and characterization of ANDbodies that bind to IBSP addresses and SOST targets Examples 14 Design of ANDbody combining TPBG address and SOST target Examples 15 Evaluation of anti-TPBG antibodies that bind to cell surface TPBG Examples 16 Generation and characterization of ANDbodies binding to the TPBG address and SOST target Examples 17 Generation of alendronate-conjugated ANDbody molecules that bind to the target SOST Examples 18 Bisphosphonate-conjugated anti-SOST ANDbody blocks sclerostin in cell-based assays Examples 19 Binding of alendronate-conjugated ANDbody molecules to hydroxyapatite Examples 20 Pharmacokinetics and distribution of bone-targeted ANDbody Examples twenty one In vivo effects of bone-targeted ANDbody: P1NP changes in mice after administration of bone-targeted ANDbody constructs Examples twenty two Design of an ANDbody construct combining Dickkopf-1 (DKK1) and SOST as a bispecific actuator fused to a diphosphate skeletal locator targeting hydroxyapatite sites Examples twenty three Evaluation of anti-DKK1 and anti-DKK1 anti-SOST ANDbody in in vitro cell-based assays

Examples 1 ：Binding chondrocyte adherin ( CHAD ) site and sclerostin ( SOST ) Target ANDbody Design

a. Vaccination to produce immunity CHAD antibody

Antibodies against the extracellular domain of human chondroadherin (CHAD) are generated by immunization. The extracellular domain of human CHAD (NCBI Protein Accession No. NP_001258 position A22-H359) (huCHAD) fused to the human IgG1 Fc region (UniProt ID P01857 position P100-K330) is expressed in HEK293F cells. Briefly, the DNA sequence is codon-optimized for mammalian expression and placed in an expression vector. The protein is transiently transfected into cells (e.g., HEK293 cells) and purified using protein A affinity purification as per conventional methods. 50 µg of huCHAD-Fc fusion protein was used to immunize female BALB/c mice by intraperitoneal (ip) injection with complete Freund's adjuvant or incomplete Freund's adjuvant (CFA/IFA adjuvant). Subsequently, fusion tumors were generated (Listek et al., Scientific Reports, 10: 1664, 2020). Clones with specific IgG reactivity to the huCHAD-Fc fusion protein used for immunization were first screened by ELISA format, followed by flow cytometry studies using cells stably (CHO) or transiently (HEK293F) transfected with full-length huCHAD. Anti-CHAD fusion tumor clones were then evaluated for mouse cross-reactivity. Flow cytometry studies were performed using cells stably (CHO) or transiently (HEK293F) transfected with full-length mouse CHAD (mCHAD), and clones were selected for binding to mCHAD. Positive clones expressing anti-CHAD mAbs cross-reactive between human and mouse were then further purified by limiting dilution selection. Fusion tumors were grown in DMEM/2% ultra-low IgG serum, and mAbs were purified by protein G chromatography according to conventional methods.

b. Choose inert resistance CHAD antibody

The address target binding site was designed not to significantly modulate the function of the address target. Therefore, the anti-CHAD fusion clone generated as described above was further evaluated based on its inability to block α2β1 integrin-dependent adhesion of chondrocytes. Recombinant human CHAD protein (R&D Systems catalog number 8218-CH-050) was used to coat 24-well tissue culture plates (5 µg/ml using 4 M guanidine hydrochloride under denaturing conditions). After overnight incubation, the plates were washed with phosphate-buffered saline (PBS) and nonspecific binding was blocked using 1% bovine serum albumin (BSA) in PBS for 1 hour at room temperature.

The ability of the murine chondrocyte cell line ATCD5 (Sigma catalog #99072806) to bind to the wells in the presence of 10 fM to 10 µM of an anti-CHAD antibody clone from a fusion tumor was assessed (one condition per concentration and per clone). 80,000 ATCD5 cells were plated in tissue culture medium for 1 hour in the presence of anti-CHAD antibody and unbound cells were washed with PBS. The number of adherent cells was enumerated using the VYBRANT™ Cell Adhesion Assay Kit (ThermoFisher, catalog #V13181) according to the manufacturer's instructions. An isotype-independent (negative) human IgG1 antibody was used as a control in this assay.

c. Identification of antibodies that specifically bind to mouse bone CHAD antibody

The address binding targets of the present technology, such as the exemplary CHAD address binding targets, are designed to specifically bind to a target tissue or cell type while minimizing binding to other tissues. Therefore, the anti-CHAD fusion clones generated as described above were further evaluated for their specificity for bone. This was achieved in two steps, both using immunohistochemistry (IHC). First, binding to the bone matrix was determined by incubating sections of decalcified human bone tissue fixed on a slide with anti-CHAD antibodies. Antibodies bound to bone tissue were detected by incubating a horseradish peroxidase (HRP)-conjugated secondary antibody that reacts with the mouse antibody. The location and intensity of binding are determined by adding the HRP matrix 3,3'diaminobenzidine (DAB), which produces a brown color at the primary antibody binding site, proportional to the abundance of the accumulated antibody. Nuclei were counterstained with hematoxylin to produce blue color. Second, the specificity of the anti-CHAD antibodies was determined by performing IHC on fresh frozen human tissue microarray (TMA) sections mounted on glass slides (as described above for bone tissue). The TMA includes non-bone tissues representing major tissue components from other parts of the body (e.g., spleen, heart, kidney (medullary and cortical), lung (upper and lower respiratory tract), skin, liver, pancreas, large intestine (ascending, descending, and transverse colon), small intestine (duodenum, jejunum, and ileum), stomach, heart, skeletal muscle, and brain). The specificity of the antibody is determined by the relative reactivity to bone compared to cells in any of the cells on the TMA (excluding bone).

d. Vaccination to produce immunity SOST antibody

The sclerostin (SOST) protein (huSOST) is an exemplary effector target. Human SOST protein (NCBI Protein Accession No. NP_079513, positions Q24-Y213) fused to the Fc region of human IgG1 was generated by immunization using a process similar to the CHAD process described above. Following immunization and hybridoma generation, clones were screened as described above but for binding to full-length human and mouse SOST (rather than CHAD). Positive clones expressing anti-SOST mAbs that were cross-reactive between human and mouse were then further purified by limiting dilution selection. Hybridomas were grown in DMEM/2% ultra-low IgG serum and mAbs were purified by protein G chromatography.

e. Select antibodies with a wide range of inhibitory concentrations and in vitro functionality SOST antibody

The effector target binding antibodies of the present technology, such as SOST, are designed to have little or no pharmacological effect unless they are localized to the target binding site, such as the CHAD protein-positive bone matrix. Therefore, the effector target binding antibodies are selected so that binding to the local target is necessary to achieve sustained pharmacologically active concentrations.

When assessing the ability to block SOST activity in vitro, anti-SOST antibodies are selected with a range of inhibitory concentrations ( _IC50 ), e.g., <1 nM to 1 µM. SOST blocking activity is measured by β-catenin-dependent Wnt reporter gene assays. For these assays, HEK293 cell lines containing a T-cytokine/lymphoid enhancer factor (TCF/LEF) transgenic reporter gene that drives luciferase expression to measure activation of the Wnt signaling pathway are used (BPS Bioscience Catalog No. 60501). 35,000 cells are plated overnight in 100 µL of medium containing 10 mM lithium chloride (LiCl) in a 96-well plate. After 18 hours, cells were treated with soluble recombinant mouse Wnt1 (37 ng/mL) (R&D Systems Catalog No. 9765-WN-010) or human Wnt3a (111.1 ng/mL) (R&D Systems Catalog No. 5036-WN) together with SOST (1 µg/mL) and serial dilutions of anti-SOST antibody (10 µg/mL and 1:3 dilutions). After 5 hours at 37°C, cells were lysed in 100 µL of luciferase assay buffer (Component A) containing 1 µL of luciferase assay medium (Component B) (BPS Biosciences Catalog No. 60690) and added to the wells. Luminescence was assessed using a SPECTRAMAX® i3x Multi-Mode Microplate Analyzer. Wnt activation of reporter cells results in luciferase activity that is blocked by SOST. Blockade of SOST activity by the antibody is assessed as an increase in luciferase signaling.

Functional in vitro assays were performed using MC3T3 osteoblasts (ATCC catalog number CRL-2593). To assess mineralization, 10,000 MC3T3 cells were plated in 24-well plates and allowed to reach confluence. Mineralization medium containing 50 µg/mL vitamin C, 10 mM β-glycerophosphate, and 20 ng/mL bone morphogenic protein-2 (BMP-2) was added. 400 ng/mL of SOST and serial dilutions of anti-SOST antibodies (10 µg/mL and 1:3 dilutions) were added to the wells, and the medium containing the corresponding supplements was replaced every 2-3 days. After 8 days, cells were washed and fixed in 10% formalin for 10 min, washed 3 times with water, and incubated with 500 µl of 40 mM alizarin solution (Millipore Sigma catalog number TMS-008-C). After 15 min, cells were washed 3 times with water. For calcium quantification, 500 µl of 10% acetic acid (v/v) was added to the cells for 30 min at room temperature (RT). Cells were scraped and transferred to an Eppendorf tube, vortexed for 30 sec, and incubated at 85°C for 15 min. Samples were centrifuged at 20,000 x g for 15 min, and 250 μl of supernatant was transferred to another Eppendorf tube. 100 μl of 10% NH ₄ OH (v/v) was added to the sample and mixed. The absorbance was measured at 405 nm using a SPECTRAMAX® i3x Multi-Plate Analyzer.

f. Reduce resistance SOST Antibody affinity

Effector-binding antibodies may need to have their binding affinity reduced to meet the design requirements of the ANDbody effector-target binding domain. In the exemplary anti-SOST antibody, the affinity for SOST may be too strong compared to the address target binder. In this case, dematuration is performed before generating the corresponding ANDbody. This provides a series of antibodies that are advantageous for targeting bone-addressed targets where the pharmacodynamic effect is locally targeted (e.g., _KD > 100 nM). Affinity dematuration is performed in selected residues of each complementation determining region (CDR) of the anti-SOST antibody variable light chain (VL) by alanine scanning-induced mutagenesis (Phase 1).

To assess the binding affinity of anti-SOST and address antibodies, the dissociation constant ( _KD ), association rate ( _kon ), and dissociation rate ( _koff ) of antibody binding to antigen were measured using biolayer interferometry (GatorPrime). Polypropylene 96-well black F-bottom plates (RATIOLAB®) were used for this experiment. Briefly, pre-equilibrated anti-human IgG Fc capture biosensor tips (Gator Bio) were baselined in 1 × K buffer (Gator Bio), and antibodies were loaded onto anti-human IgG Fc capture tips at a concentration of 2 μg/mL. An additional baseline step was performed in 1 × K buffer, followed by an association step using different concentrations of antigen (0–300 nM). To analyze and determine the K _D for each antigen/antibody pair, a global fit was performed using all antigen concentrations for which the association response was greater than 0.1 and dissociation resulted in a measurable dissociation rate. A 1:1 model was used for all curve fits. Data were analyzed using GATOR ^™ GatorOne analysis software (Gator Bio).

If changes in single VL CDR residues do not result in an affinity equal to or less than the 100 nM range, multiple combinatorial alanine substitutions are generated and the newly synthesized constructs are assessed for affinity changes (Phase 2). If there is no reduction in affinity after Phases 1 and 2, alanine scanning mutagenesis is then performed in the variable heavy chain (VH) sequence (Phase 3) and the antibody affinity for SOST is assessed by BLI.

Examples 2 : Combine CHAD Address and SOST Target ANDbody The generation and expression of

a. ANDbody The production

DNA sequences encoding 10 anti-CHAD and 10 anti-SOST antibodies with _IC50 ranging from <1 nM to 5 µM were cloned into a human IgG1 framework (with a single matched point mutation in the Fc region of the CH3 domain) using IN-FUSION® HD cloning (Takara Bio, catalog number 638911) according to the “controlled Fab-arm exchange” (cFAE) method (Labrijn et al., Nature Protocols , 9(10): 2450-2463, 2014). After expressing each antibody individually from transient HEK293 expression and purifying each antibody using protein A affinity resin, the parental antibodies (a combination of one anti-CHAD antibody and one anti-SOST antibody) were made into anti-CHAD/SOST ANDbodies according to the cFAE method. Briefly, the parental antibodies are mixed under permissive redox conditions to achieve reconstitution of the half-molecules. Subsequently, the reducing agent is removed to allow reoxidation of the interchain disulfide bonds. Finally, the exchange efficiency is quantified using chromatography-based or mass spectrometry-based methods. Approximately 100 variant ANDbodies against CHAD/SOST were generated.

b. In vitro simultaneous binding CHAD and SOST

To identify ANDbody variants that met the desired effector target and resolved the target binding affinity criteria, dual binding assays for two different antigens were performed using the BLI assay as described above with the following modifications. Briefly, ANDbody candidates were loaded onto sensor tips (anti-human IgG Fc) pre-equilibrated with K buffer, followed by a baseline step. The loaded tips were dipped into wells containing purified soluble CHAD (first association step) and then into wells containing purified soluble SOST (second association step), followed by a dissociation step. BLI-based affinity measurements were performed as described above. ANDbody variants with higher affinity for CHAD than SOST and variants with no difference in affinity or higher affinity for SOST were used for subsequent in vitro and in vivo experiments.

c. ANDbody In vitro assay for sclerostin blocking activity

ANDbodies of this technology must retain the functional properties of the parent effector target binding antibody, and the binding affinity must be such that pharmacology can be localized by binding to the address target. In the case of anti-CHAD/SOST ANDbodies, the inhibitory potency ( _IC50 ) against SOST was determined by measuring the relief of SOST inhibition in a Wnt signaling assay using TCF/LEF reporter cells and by measuring the relief of mineralization inhibition in MC3T3 osteoblasts.

d. anti- CHAD/SOST AND body Tissue-specific binding

ANDbodies of the present technology retain tissue and/or cell binding properties that are partially similar to their parental localizers (address target binding). To identify ANDbody variants that meet this criterion, binding to mouse bone was assessed by IHC, tissue on mouse tissue microarrays (as described above, mouse tissue was substituted for human tissue) and anti-human IgG1 secondary antibodies were substituted for anti-mouse secondary antibodies for molecules with a range of affinities (such as the anti-CHAD/SOST variants described above).

Examples 3 : Binding interferon-induced transmembrane protein 5 （ IFITM5 ) address and SOST Target ANDbody Design

a. Vaccination to produce immunity IFITM5 antibody

Antibodies binding to interferon-induced transmembrane protein 5 (IFITM5) were generated as described above for CHAD, except that the extracellular domain of human IFITM5 (NCBI Protein Accession No. NP_001020466, positions M1-H36) was fused to human IgG1 and used for immunization. Human and equivalent mouse protein sequences were used to identify anti-IFITM5 antibodies.

b. Choose inert resistance IFITM5 antibody

Anti-IFITM5 fusion clones generated as described above were further evaluated based on their inability to block mineralization in osteoblastic cell lines. To this end, MC3T3 cells were subjected to a mineralization assay as described above in the presence of 1, 2, 5, or 10 µg/mL of the selected anti-IFITM5 antibody or antibody control. Antibodies that did not interfere with the mineralization process were selected for their ability to block SOST activity in vitro, as measured in the β-catenin-dependent Wnt reporter gene assay as described above.

Additionally, human osteosarcoma SaOS2 cells (ATCC catalog no. HTB-85) were transfected with human IFITM5 cDNA cloned into pCDNA3.1 using Saos-2 CELL AVALANCHE™ Transfection Reagent (EZBIOSYSTEMS ^TM catalog no. EZT-SAOS-1) according to the manufacturer's protocol. For this, 1 x 10 ⁴ SaOS2 cells were plated in 96-well plates 24 hours prior to transfection. Two days after transfection, cells were subjected to osteogenic differentiation by supplementing the culture medium with 10 ^-8 M dexamethasone, 50 µg/mL vitamin C, and 10 mM β-glycerophosphate in the presence of 1, 2, 5, or 10 ug/mL of the selected anti-IFITM5 antibody, ANDbody, or antibody control. Three days later, cells were harvested and mRNA was extracted (RNeasy Micro Kit, Qiagen, catalog number 74004) and reverse transcribed into cDNA (first strand cDNA synthesis, Roche, catalog number 11483188001). qPCR was performed using the APPLIED BIOSYSTEMS ^TM 7500 Real-Time PCR System (APPLIED BIOSYSTEMSTM) (POWERUP™ SYBR™ Green Master Mix, Applied Biosystems, catalog number A25741). Osteogenesis-related genes evaluated included Runx2, ALP, and OCN. Antibodies that did not interfere with the upregulation of the above osteogenic markers were selected for use in ANDbody.

c. Identification of antibodies that specifically bind to mouse bone IFITM5 antibody

As described above, IHC analysis of human bone tissue and tissue microarrays was used to assess the binding strength and specificity of anti-IFITM5 antibodies.

Examples 4 : Combine IFITM5 Address and SOST Target ANDbody The generation and expression of

a. will ANDbody Expression and purification as bispecific antibodies

ANDbodies containing bispecific antibodies binding to IFITM5 and SOST were expressed and purified as described above.

b. In vitro simultaneous binding IFITM5 and SOST

The ability of ANDbody to simultaneously bind to IFITM5 and SOST was determined, and the binding affinity of IFITM5 and SOST was measured using BLI as described above.

c. ANDbody In vitro assay for sclerostin blocking activity

As described above, mineralization of TCF/LEF Wnt reporter cells and MC3T3 osteoblasts was used to determine the ability of ANDbodies targeting IFITM5 and SOST to inhibit SOST activity.

d. anti- IFITM5/SOST ANDbody Tissue-specific binding

The ability of ANDbodies targeting IFITM5 and SOST to specifically bind to bone tissue and inhibit SOST activity was assessed using IHC to test binding to murine bone, tissue on murine tissue microarrays (as described above, with murine tissue substituted for human tissue), and with an anti-human IgG1 secondary antibody substituted for the anti-mouse secondary antibody.

Examples 5 : Combine CHAD and Dickkopf-1 （ DKK1 )of ANDbody Design

a. DKK1 antibody

Antibodies against Dickkopf-1 (DKK1) protein (huDKK1) are exemplary effector targets of the present technology. Human DKK1 protein (NCBI Protein Accession No. NP_036374, position T32-H266) fused to the Fc region of human IgG1 was produced by immunization similar to the CHAD protocol described above. After immunization and fusion tumor generation, strains were screened as described above but for binding to full-length human and mouse DKK1 (rather than CHAD). Positive strains expressing anti-DKK1 mAbs with cross-reactivity between human and mouse were then further purified by limiting dilution selection. Fusion tumors were grown in DMEM/2% ultra-low IgG serum and mAbs were purified by protein G chromatography. b. Selection of anti- DKK1 antibodies with a wide range of IC ₅₀

When assessing the ability to block DKK1 activity in vitro, anti-DKK1 antibodies are selected with a range of inhibitory concentrations ( _IC50 ), e.g., <1 nM to 1 µM. DKK1 blocking activity is measured by β-catenin-dependent Wnt reporter gene assays. For these assays, HEK (human embryonic kidney) 293 cell lines containing a TCF/LEF transgenic reporter gene that drives luciferase expression to measure activation of the Wnt signaling pathway are used (BPS Bioscience catalog #60501). 35,000 cells are plated overnight in 100 µl of medium containing 10 mM LiCl in a 96-well plate. After 18 hours, cells were treated with soluble recombinant mouse Wnt1 (37 ng/mL) (R&D Systems Catalog No. 9765-WN-010) or human Wnt3a (111.1 ng/mL) (R&D Systems Catalog No. 5036-WN) together with DKK1 (1 µg/mL) and serial dilutions of anti-DKK1 antibody (10 µg/mL and 1:3 dilutions). After 5 hours at 37°C, cells were lysed in 100 µl of luciferase assay buffer (Component A) containing 1 µl of luciferase assay medium (Component B) (BPS Biosciences Catalog No. 60690) and added to the wells. The plate was gently rocked for 15 minutes at room temperature and luminescence was assessed using a SPECTRAMAX® i3x Multi-Mode Microplate Analyzer. Wnt activation in reporter cells results in the blockade of luciferase activity by DKK1.

Functional in vitro assays were performed using MC3T3 osteoblasts (ATCC catalog number CRL-2593). To assess mineralization, 10,000 MC3T3 cells were plated in 24-well plates and allowed to reach confluence. Mineralization medium containing 50 µg/mL vitamin C, 10 mM β-glycerophosphate, and 20 ng/mL bone morphogenic protein-2 (BMP-2) was added. 400 ng/mL of DKK1 and serial dilutions of anti-DKK1 antibodies (10 µg/mL and 1:3 dilutions) were added to the wells, and the medium containing the corresponding supplements was replaced every 2-3 days. After 8 days, cells were washed and fixed in 10% formalin for 10 min, washed 3 times with water, and incubated with 500 µl of 40 mM alizarin solution. After 15 min, cells were washed 3 times with water. For calcium quantification, 500 µl of 10% acetic acid (v/v) was added to the cells and left at RT for 30 min. Cells were scraped and transferred to Eppendorf tubes, vortexed for 30 sec, and incubated at 85°C for 15 min. Samples were centrifuged at 20,000 xg for 15 min, and 250 μl of supernatant was transferred to another Eppendorf tube. 100 μl of 10% NH ₄ OH (v/v) was added to the samples and mixed. Absorbance was measured at 405 nm using a SPECTRAMAX® i3x Multi-Plate Analyzer.

c. CHAD and DKK1 ANDbody Expression, purification and representation of

ANDbodies comprising bispecific antibodies that bind CHAD and DKK1 were expressed and purified as described above for anti-CHAD/SOST ANDbodies. The ability of ANDbodies targeting CHAD and DKK1 to simultaneously engage CHAD and DKK1 was determined, and the binding affinity to CHAD and DKK1 was measured using BLI as described above. The ability of ANDbodies targeting CHAD and DKK1 to inhibit DKK1 activity was determined using mineralization of TCF/LEF Wnt reporter cells and MC3T3 osteoblasts as described above. The ability of ANDbodies targeting CHAD and DKK1 to specifically bind to bone tissue and inhibit DKK1 activity was assessed using IHC to test binding to murine bone, tissue on murine tissue microarrays (as described above, with murine tissue substituted for human tissue), and with an anti-human IgG1 secondary antibody substituted for the anti-mouse secondary antibody.

Examples 6 : Bone Targeting ANDbody Pharmacokinetics and tissue distribution of

To analyze the in vivo distribution of ANDbody, exemplary ANDbody (e.g., anti-CHAD/SOST, anti-IFITM5/SOST, or anti-CHAD/DKK1) and each parental antibody (anti-CHAD, anti-IFITM5, anti-SOST, or anti-DKK1 used for cFAE of ANDbody) were quantified in female Balb/c and C57BL/6 mice.

To quantify tissue biodistribution, each antibody and ANDbody was injected individually at doses of 1 mg/kg, 3 mg/kg, and 10 mg/kg IV (tail vein). An equal volume of saline (PBS) was also injected as a control. At 12 hours, 1 day, 2 days, 3 days, 7 days, and 14 days after injection, mice were euthanized using CO ₂ and tissues (including bone matrix, heart, lung, spleen, blood, kidney, liver, and intestine) were processed into homogenates. The total protein concentration in each sample was measured using the PIERCE™ Rapid Gold BCA Protein Assay Kit (ThermoFisher Scientific, A53225). ELISA assays were used to determine the concentration of each ANDbody or antibody in a homogenate containing 0.5 mg of protein.

To quantify cellular biodistribution, ANDbody and antibody were first individually labeled with Alexa Fluor 647 (AF647) using conventional methods according to the manufacturer's instructions (Thermo Fisher Scientific, A20186).

To determine cell biodistribution, mice were euthanized using CO ₂ at 12 h, 1 day, 2 days, 3 days, 7 days, and 14 days after injection, and tissues (including bone matrix, heart, lung, spleen, blood, kidney, liver, and intestine) were processed into single-cell suspensions according to previously described methods. Briefly, blood was collected by cardiac puncture into EDTA-treated tubes (BD Catalog No. 365974), and other tissues were harvested, weighed, mechanically separated between ground glass slides, and filtered through a 70 μm mesh (Millipore Sigma, Catalog No. CLS431751-50EA) to make single-cell suspensions. Splenocytes, whole blood, and lungs were treated with ammonium chloride potassium (ACK) lysis buffer (Thermo Fisher Scientific, catalog number A1049201). Hearts were digested with collagenase and processed into single-cell suspensions as previously described (Covarrubias et al. 2019 Am J Physiol Heart Circ Physiol. 317(3):H658-H666). Flow cytometry of immune cells was performed using conventional methods using markers for CD8 T cells (CD3e+ CD8+), CD4 T cells (CD3e+ CD4+ Foxp3-), regulatory T cells (CD4+ CD25+ FOXP3+), monocytes/macrophages (CD3e- CD11b+ CD11c-/lo NK1.1− Ly6G− SSClo), dendritic cells (CD3e- CD11chi), NK cells (NK1.1+ CD3e−), and NKT cells (NK1.1+ CD3e+). Lung cells including epithelial (CD326+CD31−CD45−), endothelial (CD326−CD31+CD45−), and hematopoietic lineages (CD326−CD31−CD45+) were also analyzed.

To quantify tissue biodistribution by IVIS, proteins (ANDbody and antibody) were first individually labeled with NHS-5/6-FAM (Thermo Fisher Scientific, Catalog No. 46409) according to the manufacturer's instructions. Each ANDbody or antibody was administered to mice as described above. At 12 hours, 1 day, 2 days, 3 days, 7 days, and 14 days after injection, mice were euthanized using CO ₂ , and tissues including bone, lung, spleen, blood, kidney, liver, and intestine were harvested, weighed, and imaged on the IVIS® In Vivo Spectroscopic Imaging System (Caliper Life Sciences; excitation, 500 nm; emission, 540 nm). Images were analyzed using LIVING IMAGE® software (PERKINELMER®).

Examples 7 : Bone Targeting ANDbody Effects in vivo

a. Testing bone-targeted antibodies in a rat model of osteoporosis

Sixteen-week-old nulliparous female Sprague Dawley rats (Harlan) were sham-operated (Sham) or ovariectomized (OVX) and left untreated for 8 weeks to allow bone loss to occur. OVX rats were treated with a range of concentrations (e.g., 5, 10, 20 mg/kg, subcutaneously weekly) of control antibody or ANDbody for 1, 2, or 4 months. Body weight was recorded, and areal bone mineral density (BMD) was measured in anesthetized rats using dual-energy X-ray absorptiometry (DXA) (Hologic FAXITRON® DXA x-ray cabinet). BMD records and blood samples were collected every 3 weeks for 9 weeks until necropsy. Two weeks and 4 days before necropsy, rats were injected subcutaneously with calcein (20 mg/kg; Sigma-Aldrich). Lumbar vertebrae, tibiae, and femurs were collected for further analysis, including dynamic tissue morphometry, microcomputed tomography, and biomechanical analysis.

For bone tissue morphometry analysis, femurs and lumbar vertebrae were dissected using a slow-speed diamond saw, dehydrated in isopropyl alcohol, and embedded in methyl methacrylate (MMA). The cured blocks were sectioned using a microtome (Leica, Wetzlar, Germany), and 5-µm sections were examined under a fluorescent microscope to track calcixopyrin labeling. Images were analyzed using ImageJ software. Static and dynamic parameters evaluated were cortical thickness, endosteal thickness, endosteal and periosteal mineralized surface/bone surface, as well as mineral deposition and bone formation rate.

Biochemical analysis of bone turnover at necropsy may include assessment of serum levels of osteocalcin (rat osteocalcin ELISA, Novus Biologicals, catalog number NBP2-68153), P1NP (rat procollagen type 1 N-terminal propeptide ELISA, Novus Biologicals, catalog number NBP2-76467), and rat TRACP-5b (tartrate-resistant acid phosphatase 5b) ELISA, Elabscience, catalog number E-EL-R0939).

For μCT analysis, the lumbar spine, femoral neck, and trunk were examined using a desktop μCT scanner (GE eXplore Locus Micro CT scanner), and the regions of interest were analyzed using a software algorithm.

b. Testing bone-targeted antibodies in a serum-induced metastatic arthritis model

Arthritis serum was harvested from 10-week-old arthritic K/BxN mice. Male wild-type mice aged 12 weeks were injected intravenously with 150 μL of arthritis serum and different concentrations (e.g., 2, 10, and 20 mg/kg subcutaneously) of ANDbody to induce arthritis on days 0, 2, and 6. Mice were sacrificed on day 14 and the forepaws were fixed in 70% ethanol for imaging. For histopathological analysis, the knees and ankles were fixed in 4% paraformaldehyde, decalcified in 15% EDTA, and then paraffin-embedded. 4 μm knee and ankle sections were obtained for standard histopathological analysis. H&E images of various sites of periosteal bone formation in the knee and ankle were captured, and the average area of each site was calculated. This value was multiplied by the distance through the total cross-section obtained to calculate the total volume of each site. The total periosteal bone volume for each knee or ankle was determined by summing the volume of bone formation at all sites. Microcomputed tomography imaging was also used to quantify periosteal bone formation as described above.

Examples 8 : Combine DMP1 Address ANDbody The creation and representation of structures

a. Design, expression and purification of bispecific ANDbody for fusion

To generate dentin matrix acidic phosphoprotein 1 (DMP1) binding sites for subsequent ligation into bispecific structures (e.g., ANDbody constructs), EXPI293F ^™ (Gibco) cells were transiently transfected with polyethylenimine (PEI) at a 1:2 mass ratio of heavy chain:light chain plasmids encoding the site-directed anti-DMP1 controlled Fab arm exchange (cFAE) F405L monoclonal antibody (mAb) and maintained according to the manufacturer's instructions (37°C, 8% CO ₂ on a shaking platform). Cultures were fed 4-24 hours after transfection to a final concentration of 5% v/v Gibco Feed B, 1% v/v L-alanine-glutamine, and 4 mM valproic acid. After 4-7 days, supernatants were harvested by 0.22 μm filtration. Filtered supernatants were purified by protein A affinity chromatography (Cytiva 5 mL MABSELECT ^™ PrismA columns) equilibrated in PBS pH 7.4 running buffer and 0.1 M sodium citrate pH 3.0 elution buffer. The eluted protein was immediately neutralized with 10% v/v 1 M sodium acetate (pH 6.0) or 10% v/v 1 M Tris (pH 8.0). The protein was further purified by size exclusion chromatography equilibrated in PBS pH 7.4.

To generate SOST binding effector sets of ANDbody constructs for subsequent synergism of bispecific structures (e.g., ANDbody constructs), effector anti-SOST cFAE K409R FC star (H435R, Y436F) mAb constructs were generated to eliminate binding to protein A. EXPI293F ^™ cells were transiently transfected with heavy chain:light chain plasmids at a 1:2 mass ratio using PEI and maintained according to the manufacturer's instructions (37°C, 8% CO ₂ on a shaking platform). Cultures were fed 4-24 hours after transfection to a final concentration of 5% v/v Gibco feed B, 1% v/v L-alanine-glutamine, and 4 mM valproic acid. After 4-7 days, harvest the supernatant by 0.22 μm filtration. Purify the filtered supernatant by protein G affinity chromatography (5 mL HITRAP® Protein G HP columns from Stervan) in PBS 7.4 running buffer and 0.1 M glycine pH 2.7 elution buffer. Immediately neutralize the eluted protein with 10% v/v 1 M Tris pH 7.5. Buffer exchange the protein into PBS for conjugation.

Anti-DMP1 and anti-SOST mAbs were conjugated to bispecific ANDbodies by mixing the above products in equimolar ratios with 75 mM 2-MEA in PBS pH 7.4. The solution was incubated at 31°C for 5 hours, and then the buffer was exchanged to PBS to remove all 2-MEA. After removing 2-MEA, the solution was incubated at 4°C for 16 hours. The bispecific ANDbody was purified from the solution using a gradient elution from 40 mM sodium acetate pH 6, 500 mM NaCl to 100% 40 mM sodium acetate pH 3, 500 mM NaCl to separate the correctly conjugated bispecific ANDbody from the parental mAb.

b. Verification of binding to target

Binding kinetics studies were performed on a GATOR® Plus biosensor based on biolayer interferometry (BLI) at 25°C in a buffer containing 10 mM HEPES, 150 mM NaCl, 1 mg/ml BSA, 0.04% NaN3, 0.05% Tween20, pH 7.4 (HBS-BNT). ANDbody molecules (e.g., fused or unfused ANDbody) were captured by immersing anti-human Fc BLI biosensors (Crocodile Bio - hFC) in wells containing 2 µg/mL antibody for 90 seconds. The biosensor was then immersed in wells containing different concentrations of the relevant antigen across a range of values for 120 seconds and then dissociated in HBS-BNT buffer for 300 seconds. Between each step, all sensors were washed in HBS-BNT buffer.

To compensate for any drift in the baseline, ANDbody-captured biosensors were immersed in wells containing HBS-BNT buffer and the observed raw data were subtracted from the raw data obtained for antigen binding. This single reference-subtracted data was then globally fit using a Langmuir 1:1 binding model and the binding kinetic parameters were determined.

Examples 9 : Combine DMP1 Address and SOST Target ANDbody Design

a. Vaccination to produce anti- DMP1 antibodies

Antibodies binding to dentin matrix acidic phosphoprotein 1 (DMP1) were generated as described for CHAD in Example 1, except that the full-length sequence of human DMP1 (protein accession number NP_004398.1) was fused to human IgG1 and used for immunization. Human and equivalent mouse protein sequences were used to identify anti-DMP1 antibodies.

b. Selection of inert anti- DMP1 antibodies

The anti-DMP1 fusion clones generated as described above were further evaluated based on their inability to block mineralization in MC3T3 osteoblasts, as described above (see, e.g., Example 3(b)).

Anti-DMP1 antibodies that do not interfere with mineralization were selected for ANDbody generation.

c. Identification of antibodies that specifically bind to mouse bone DMP1 antibody

As described above, IHC analysis of human bone tissue and tissue microarrays was used to assess the binding strength and specificity of anti-DMP1 antibodies (see, e.g., Example 1(c)).

d. Anti- SOST targeted group

As described in Example 1(d) above, anti-DMP1 antibodies were paired with anti-SOST targeting panels to generate ANDbodies that bind to both the DMP1 site and the SOST target.

Examples 10 ：Osteocyte extracellular matrix address DMP1 Verification

Immunohistochemistry using a peroxidase-based chromogen detection system was used to evaluate DMP1 as a bone site and to characterize the targeting specificity of the anti-DMP1 antibodies (Fig. 5).

Binding of anti-DMP1 mouse IgG1k monoclonal antibody Ab349 to bone tissue was tested using 5 μm formalin-fixed paraffin-embedded (FFPE) decalcified mouse femur sections on standard histology microscope slides. After standard dewaxing and rehydration, the samples were immersed in excess Tris EDTA solution at pH 9 (Vector Laboratories, #H-3301-250) and incubated overnight in a 37°C water bath as a mild modification to the typical heat-induced epitope retrieval (HIER). The next day, samples were treated with a commercial blocking reagent against endogenous tissue peroxidase (BLOXALL®, Vector Laboratories #SP-6000-100). To prevent nonspecific binding of the commercial horseradish peroxidase (HRP) polymer-conjugated anti-mouse IgG detection reagent to endogenous mouse IgG, tissue sections were subsequently treated with an additional blocking reagent (Blocking + Detection Reagent: Mouse to Mouse Polymer IHC Kit, Abcam #ab269452). After washing several times with PBS, samples were washed with 20 μg/ml of anti-DMP1 antibody at room temperature for 2 hours, followed by several washes with PBS. A brown color development pigment dependent on the presence of bound detection reagent/anti-DMP1 antibody/bone DMP1 complex was developed using the mouse-to-mouse polymer IHC kit (Abcam #ab269452). Signal intensity was compared to that of a photographic neighbor slide that had not been incubated with primary antibody ( The color development time was determined by rapid quenching of the matrix mixture with distilled water and final immersion in excess distilled water for 5 minutes at room temperature. The duration of color development for all slides was carefully controlled to reduce variability between slides. Finally, sections were counterstained with Mount Hematoxylin QS (Mount Laboratories #H-3404) and non-water mounted for brightfield imaging.

Figure 5 shows the longitudinal interface of the bone marrow and trabecular region (femoral “cap”) in a mouse femur section stained with anti-DMP1 monoclonal antibody (mAb), as compared to a control. The anti-DMP1 mAb exhibits strong immunoreactivity in murine bone tissue.

Examples 11 : Combine DMP1 Address and SOST Target ANDbody Signs of

a. ANDbody In vitro assay for sclerostin blocking activity

As described above, mineralization of TCF/LEF Wnt reporter cells and MC3T3 osteoblasts was used to determine the ability of ANDbodies targeting DMP1 and SOST to inhibit SOST activity (see, e.g., Example 1(e)).

b. Tissue-specific binding of anti -DMP1/SOST ANDbody

The ability of ANDbodies targeting DMP1 and SOST to specifically bind to bone tissue and inhibit SOST activity was assessed using IHC to test binding to murine bone, tissue on murine tissue microarrays (as described above, with murine tissue substituted for human tissue), and with an anti-human IgG1 secondary antibody substituted for the anti-mouse secondary antibody, as described above (see, e.g., Example 1(c)).

Examples 12 : Combine IBSP Address and SOST Target ANDbody Design

a. Vaccination to produce anti- IBSP antibodies

Antibodies binding to integrin bone sialoprotein (IBSP) were generated as described for CHAD in Example 1, except that the full-length sequence of human IBSP (protein accession number NP_004958.2) was fused to human IgG1 and used for immunization. Human and equivalent mouse protein sequences were used to identify IBSP antibodies.

b. Selection of inert anti -IBSP antibodies

The anti-IBSP fusion clones generated as described above were further evaluated based on their inability to block mineralization in MC3T3 osteoblasts, as described above (see, e.g., Example 3(b)).

Anti-IBSP antibodies that do not interfere with mineralization were selected for use in ANDbody.

c. Identification of anti -IBSP antibodies that specifically bind to mouse bone

As described above, IHC analysis of human bone tissue and tissue microarrays was used to assess the binding strength and specificity of anti-IBSP antibodies (see, e.g., Example 1(c)).

Examples 13 : Combine IBSP Address and SOST Target ANDbody The generation and expression of

a. Expression and purification of ANDbody as a bispecific antibody

ANDbodies comprising bispecific antibodies that bind IBSP and SOST were expressed and purified as described above (see, e.g., Examples 2A and 8A).

b. In vitro simultaneous combination of IBSP and SOST

The ability of ANDbody to simultaneously bind to IBSP and SOST was determined, and the binding affinity of IBSP and SOST was measured using BLI as described above (see, e.g., Example 8B).

c. In vitro determination of sclerostin blocking activity in ANDbody

As described above, mineralization of TCF/LEF Wnt reporter cells and MC3T3 osteoblasts was used to determine the ability of ANDbodies targeting IBSP and SOST to inhibit SOST activity (see, e.g., Example 1(e)).

d. Tissue-specific binding of anti -IBSP/SOST ANDbody

The ability of ANDbodies targeting IBSP and SOST to specifically bind to bone tissue and inhibit SOST activity was assessed using IHC to test binding to murine bone, tissue on murine tissue microarrays (as described above, with murine tissue substituted for human tissue), and with an anti-human IgG1 secondary antibody substituted for the anti-mouse secondary antibody, as described above (see, e.g., Example 1(c)).

Examples 14 : Combine TPBG Address and SOST Target ANDbody Design

a. Vaccination to produce anti- TPBG antibodies

Antibodies binding to trophoblast glycoprotein (TPBG) were generated as described in Example 1 for CHAD, except that the extracellular domain of human TPBG (NCBI Protein Accession No. NP_001363851.1, positions M1-H355) was fused to human IgG1 and used for immunization. Human and equivalent mouse protein sequences were used to identify anti-TPBG antibodies.

b. Select inert anti- TPBG antibodies

The anti-TPBG fusion clones generated as described above were further evaluated based on their inability to block mineralization in osteoblast cell lines, as described above (see, e.g., Example 3(b)).

Anti-TPBG antibodies that do not interfere with mineralization or upregulation of osteogenic markers were selected for use in ANDbody.

c. Identification of anti -TPBG antibodies that specifically bind to mouse bone

As described above, IHC analysis of human bone tissue and tissue microarrays was used to assess the binding strength and specificity of anti-TPBG antibodies (see, e.g., Example 1(c)).

Examples 15 ：Binding to cell surface TPBG Anti TPBG Antibody Assessment

Commercially available antibodies recognizing human and mouse TPBG were tested as candidate ANDbody locators (addressing the target binding domain) in a cell binding assay using the primary osteoblast cell line MC3T3.

Anti-TPBG antibody MA5-24228 (Thermo Fisher Scientific) was further characterized for live cell binding using human and mouse transient overexpression flow cytometry and immunofluorescence in HEK293T cells and human MC3T3 osteoblast-like cell lines, respectively.

Full-length human and mouse TPBG were transiently expressed in HEK 293T cells, and the binding of anti-TPBG antibodies to TPBG was then quantified by flow cytometry. Custom expression plasmids encoding the common domain consensus sequence of human and mouse TPBG sequences were obtained from Genscript based on their commercial expression vectors, which contain a C-terminal intracellular affinity epitope (FLAG tag) in the same open reading frame as the inserted TPBG sequence. Human or mouse TPBG plasmids were transfected into healthy 293T cells seeded in 96-well plates by complexing with LIPOFECTAMINE ^™ 3000 according to the manufacturer's recommendations (Invitrogen #L3000001). After 48 hours of putative TPBG expression, cells were gently harvested using the non-disruptive EDTA-based VERSENE ^™ Cell Dissociation Reagent (Gibco) and stained by flow cytometry with a defined serial dilution range of selected commercial anti-TPBG monoclonal or polyclonal antibodies or anti-FLAG monoclonal antibodies to represent the total amount of recombinant TPBG-FLAG protein present at harvest. After incubation with the primary antibody, cells were stained using the fluorophore-conjugated antibody anti-mouse AF647. For intracellular staining of FLAG-tagged sequences, cells were permeabilized using PERM/WASH ^™ buffer (BD Bioscience #544723) according to the manufacturer's protocol and then stained with rabbit anti-FLAG-AF647 primary antibody. After staining, cells were washed, fixed in 1% PFA, and analyzed by flow cytometry. ZOMBIE AQUA ^™ was used as a viability dye. The median fluorescence intensity of the evaluated antibodies at each antibody concentration in live cells (non-dead cells), single cells (double discrimination) was obtained by FACS analysis using FlowJo software to generate dose-response curves and _EC50 values.

As shown in Figure 6, anti-TPBG monoclonal antibody (mAb) MA5-24228 binds to both mouse membrane-bound TPBG and human membrane-bound TPBG. Untransfected cells were stained and incubated with secondary antibody alone as a negative control. The expression level of FLAG in cells transfected with mouse and human TPBG is comparable, facilitating the comparison of antibody binding to mouse and human TPBG.

To evaluate live cell binding of anti-TPBG antibody MA5-24228 to endogenous TPBG, immunofluorescence analysis was performed using osteoblast MC3T3-E1 subclone 4 cell line (ATCC #CRL-2593). 6000 cells were plated in 100 µl complete medium in 96-well glass bottom wells for 9 days. Cells were incubated with anti-TPBG MA5-24228 primary antibody on ice, washed and incubated with secondary antibody goat anti-mouse AF647 (A21235) on ice, washed and lightly fixed in 1% PFA. Qualitative images were acquired using the EVOS ^TM M7000 imaging system. As shown in FIG7 , anti-TPBG antibody MA5-24228 bound to the cell surface of cells.

Examples 16 : Combine TPBG Address and SOST Target ANDbody The generation and expression of

a. Expression and purification of ANDbody as a bispecific antibody

ANDbodies comprising bispecific antibodies that bind TPBG and SOST were expressed and purified as described above (see, e.g., Examples 2A and 8A).

b . In vitro simultaneous binding of TPBG and SOST

The ability of ANDbody to simultaneously bind to TPBG and SOST was determined, and the binding affinity of TPBG and SOST was measured using BLI as described above (see, e.g., Example 8B).

c . In vitro determination of sclerostin blocking activity in ANDbody

As described above, mineralization of TCF/LEF Wnt reporter cells and MC3T3 osteoblasts was used to determine the ability of ANDbodies targeting TPBG and SOST to inhibit SOST activity (see, e.g., Example 1(e)).

d . Tissue-specific binding of anti -TPBG/SOST ANDbody

The ability of ANDbodies targeting TPBG and SOST to specifically bind to bone tissue and inhibit SOST activity was assessed using IHC to test binding to murine bone, tissue on murine tissue microarrays (as described above, with murine tissue instead of human tissue), and with an anti-human IgG1 secondary antibody instead of an anti-mouse secondary antibody (see, e.g., Example 1(c)).

Examples 17 : Binding target SOST Aldronate yoke ANDbody Molecular formation

To generate antibodies conjugated to small molecules for conjugation to linkers, CHO cells were transiently transfected with S239C FC variant heavy chain:light chain plasmids at a mass ratio of 2:3 using PEI and maintained according to the manufacturer's instructions (37°C, 5% _CO2 , on a shaking platform). Cultures were fed 4-24 hours after transfection to a final concentration of 5% v/v Gibco feed B, 1% v/v L-alanyl-glutamine, and 4 mM valproic acid. After 4-7 days, supernatants were harvested by 0.22 μm filtration. The filtered supernatant was purified by protein A affinity chromatography (Cytiva 5 mL MABSELECT ^™ PrismA column) equilibrated in PBS pH 7.4 running buffer and 0.1 M sodium citrate pH 3.0 elution buffer. The eluted protein was immediately neutralized with 10% v/v 1 M sodium acetate, pH 6.0. The protein was further purified by size exclusion chromatography equilibrated in 20 mM His-HAC, 150 mM NaCl, pH 5.5 hydration buffer.

The antibody constructs binding to SOST and DKK1 targets were conjugated to bisphosphonate molecules with different binding potencies to the bone mineral hydroxyapatite (including alendronate, risedronate, etidronate, ibandronate, clodronate, tiludronate, pamidronate and zoledronic acid) through a two-step process to generate ANDbody.

To generate BIS-L1 (allendronate molecule), first, anti-SOST mAb (Romo mAb, containing S239C amino acid substitution mutation) was conjugated to BCN-PEG4-maleimide at the S239C site by thiol chemistry. Then, the bisphosphonate molecule was conjugated to this site by click chemistry to generate the final drug (ANDbody containing BIS-L1) (Figure 8).

a. Bisphosphonate yoke ANDbody of the characteristics (purity, drug - Antibody ratio)

To determine the aggregation state of the antibody-drug conjugate (ADC) material (M5-D7: ANDbody containing anti-SOST antibody conjugated to alendronate), 20 µL of sample was injected by HPLC onto a 5/150 SUPERDEX ^TM Plus 200 pg column (Sterofam). The column was washed with 1.2 column volumes of formulation buffer to determine the elution time of ADC and aggregates.

To determine whether the drug-antibody ratio (DAR) of the conjugated ANDbody material was within the target range of 2.0 ± 0.2, hydrophobic interaction chromatography (HIC) and liquid chromatography-mass spectrometry (LC-MS) were employed. For LC-MS, 50 µg of ADC was prepared at a concentration of 1 mg/mL using 50 mmol/L DTT and incubated at 37°C to reduce the ANDbody material. The sample was injected onto a PLRP-S 1000Å, 5um, 2.1 × 50 mm (Agilent) column equilibrated with mobile phase buffer A (100% HPLC grade water containing 0.1% formic acid and 0.025% trifluoroacetic acid) and eluted with a 27%-49%-95% gradient of mobile phase buffer B (100% acetonitrile containing 0.1% formic acid and 0.025% trifluoroacetic acid) at 70°C. The LC data were deconvoluted to calculate the drug-antibody ratio (DAR) for each peak.

To determine DAR by HIC, samples were diluted into HIC buffer A (25 mM KP pH 7, 1.5 M NH4SO4) and loaded onto a HIC column (HIC column, TSKgel Butyl NPR, 4.8 mm x 3.5 cm). The column was washed with 0-100% HIC buffer B (25 mM KP pH 7) at a flow rate of 1 mL/min for 12 min. The eluted peak was analyzed against the parent ANDbody HIC elution to determine DAR.

To determine endotoxin levels, 25 µL of ANDbody and ADC material were loaded into each well of a Nexgen multi-cartridge PTS system (Charles River Laboratories) following the manufacturer’s instructions.

The results are shown in Table 4. Anti-SOST antibody and alendronate were successfully conjugated to generate ANDbody:DAR within an acceptable range, with no significant change in aggregation state and endotoxin levels low enough for in vivo experiments.

[ Table 4 ] : ANDbody characterization data Antibody components Conjugated bisphosphonate component ANDbody (ADC) Concentration (mg/mL) Restored MS-DAR Drug-free (%mol/mol) ASEC (% POI) Endotoxin M5: anti-SOST mAb (Romo mAb containing the S239C amino acid substitution mutation) Alendronate (BIS-L1) M5-D7: Anti-SOST antibody M5 conjugated to alendronate 2.59 1.92 ＜ 2（＜ 0.266 µM） 98.96% ＜ 0.093

b. BLI Alpha-alendronate ANDbody Kinetic measurements of binding of molecules to target proteins

In vitro characterization of the M5-D7 ANDbody and control antibodies was performed by BLI as described previously (Table 5). The parental antibodies PRO136 (humanized anti-SOST mAb) and PRO236 (murine parental version of PRO136 before humanization) were used as control antibodies. The molecules were evaluated for binding to human SOST (hSOST) and mouse SOST (mSOST). Conjugation of alendronate did not disrupt or alter the kinetic parameters of the parental antibodies.

[ Table 5 ] : Kinetic parameters of anti- SOST bisphosphonate yokes Antibodies tested Antigen tested kon（1/Ms） koff（1/s） KD (M) M5-D7 hSOST 2.91E+06 3.83E-04 1.32E-10 M5-D7 mSOST 1.51E+06 1.20E-03 7.95E-10 PRO136 hSOST 4.15E+06 2.19E-04 5.28E-11 PRO136 mSOST 2.07E+06 6.45E-04 3.12E-10 PRO236 hSOST 3.23E+06 2.74E-04 8.49E-11 PRO236 mSOST 5.54E+06 4.93E-03 8.90E-10 PRO236 rSOST 3.61E+06 9.43E-03 2.61E-09

Examples 18 : Bisphosphonate-conjugated anti- SOST AND body Blocking sclerostin in cell-based assays

The bisphosphonate-conjugated anti-SOST ANDbody (M5-D7; also referred to herein as anti-SOST-BIS-L1(2), where "2" represents a drug-antibody ratio of two alendronate molecules per anti-SOST mAb molecule) was tested for its ability to block sclerostin and restore Wnt1 signaling in vitro in a cell-based assay using a commercial HEK293 reporter cell line (BPS Biosciences #60501) . Prior to testing, the EC ₅₀ concentration of Wnt1 (R&D Systems #R&D 9765-WN-010) and the IC ₅₀ of SOST (Acro Biosystems #HST-H5245) relative to the Wnt1 EC ₅₀ were determined according to the assay protocol recommended by the supplier. This included plating and treating 35,000 cells per 96-well plate with 10 mM LiCl, followed by Wnt1 stimulation for 6 hours the next day to induce luciferase expression, followed by endpoint cell lysis and luminescence quantification (ONE-STEP ^™ Luciferase Assay System, BPS Biosciences #60690-2). After assay calibration, a dose-response analysis was performed in the same assay format using increasing amounts of bisphosphonate _- conjugated anti-SOST ANDbody (M5-D7) by adding a pre-incubation step in which the ANDbody was mixed with the _IC50 concentration of SOST, followed by mixing with the Wnt1 EC50 and stimulating reporter cells for 6 hours. The results showed that the bisphosphonate-conjugated anti-SOST ANDbody retained the ability to block SOST with the same potency as the unconjugated anti-SOST parental mAb (Figure 9).

Examples 19 ：Alendronate-coupled ANDbody Rapid in vitro binding of molecules to hydroxyapatite

The bone homing ability of the bisphosphonate-conjugated anti-SOST ANDbody (M5-D7) was evaluated in a binding assay using hydroxyapatite (Hap) crystals, a mineral component of bone tissue.

Physiologically relevant concentrations (approximately 1 nM) of anti-SOST (PRO136) or anti-SOST-BIS-L1(2) (M5-D7) were incubated with excess hydroxyapatite powder (Sigma 900204, mean particle size, 5 µm) to simulate the ability to bind to bone matrix in vivo. Samples were mixed in a 5 ml starting total volume in 5 ml Protein LoBind tubes (EPPENDORF®) and kept rotating in a dry 37°C incubator. Periodically, unbound mAb remaining in solution was collected by separating the hydroxyapatite crystals with bound hIgG using a 0.2 μm spin filter and stored at 4°C until all time points were collected. The percentage of mAb binding over time was calculated by sandwich ELISA by measuring the free (unbound) human IgG remaining in the solution and comparing it to the solution incubated with hIgG in the absence of hydroxyapatite. The exact concentration was determined by a standard curve using the same PRO136 hIgG antibody (capture and detection antibodies: Jackson ImmunoResearch #109-005-170, BIO-RAD #STAR127P, TMB matrix: Thermo Fisher Scientific #34028). Anti-SOST-BIS-L1(2) was completely depleted from solution within 2 h of incubation with hydroxyapatite, indicating that the bisphosphonate has a high affinity for bone mineral material compared to the unconjugated anti-SOST(PRO136) antibody (Figure 10).

Examples 20. Bone Targeting ANDbody Pharmacokinetics and distribution of

Mice were injected subcutaneously (SQ) with 20 mg/kg body weight (mpk) of bisphosphonate-conjugated anti-SOST ANDbody (M5-D7), unconjugated anti-SOST antibody, or control anti-RSV antibody. Femora were collected on days 1, 3, and 8 and fixed, decalcified, and embedded in paraffin blocks. Femoral sections were stained with anti-human IgG and visualized using VECTOR® Red Matrix (Vector Laboratories). Sections were imaged using the EVOS ^TM M7000i Cell Imaging System and analyzed using the HALO® Image Analysis Platform. Bisphosphonate conjugation resulted in significant accumulation in the bone cortex (i.e., enrichment in the bone compartment), which was easily observed on day 1 and increased over time compared to the control antibody molecule (Figures 11A and 11B).

Examples twenty one : Bone Targeting ANDbody In vivo effects: bone-targeted administration ANDbody After constructing P1NP Changes

Mice were injected (SQ) with 20 mpk of bisphosphonate-conjugated anti-SOST ANDbody (M5-D7), unconjugated anti-SOST antibody (PRO136), or control anti-RSV antibody (PRO022). Serum samples were obtained on days 1, 3, and 8, and serum levels of the bone turnover marker procollagen type 1 N-terminal propeptide (P1NP) were assessed using ELISA (Abcam # ab210579). An increase in P1NP levels was observed with the bisphosphonate-conjugated anti-SOST ANDbody at a similar rate to the unconjugated anti-SOST antibody, demonstrating the efficacy of the M5-D7 ANDbody construct (Figure 12).

Examples twenty two ：Design combined Dickkopf-1 （ DKK1 )and SOST of ANDbody Constructs as bispecific actuators fused to biphosphate bone locators targeting hydroxyapatite sites

a. anti- DKK1 and Anti DKK1 anti- SOST Generation of bispecific antibody constructs

Anti-DKK1 antibody constructs and anti-DKK1 anti-SOST bispecific antibodies were generated using the protocols provided herein.

b. Antibiotics conjugated to bisphosphonates DKK1 and Anti DKK1 anti- SOST Generation of bispecific antibody constructs

The anti-DKK1 antibody and the anti-DKK1 anti-SOST bispecific antibody constructs were fused to the bisphosphonate molecule as described above (see, e.g., Example 17), thereby generating (i) an ANDbody comprising: an anti-DKK1 antibody fused to a bisphosphonate molecule; and (ii) an ANDbody comprising an anti-DKK1 anti-SOST bispecific antibody fused to a bisphosphonate molecule.

c. anti- DKK1 anti- SOST In vitro characterization of bispecific antibody constructs

In vitro characterization of anti -DKK1 antibodies and anti- DKK1 anti- SOST bispecific antibodies was performed (Table 6). PRO136 and PRO236 were synthesized as described previously and served as control antibodies. The molecules were evaluated for binding to human DKK1 (hDKK1) and human SOST (hSOST).

[ Table 6 ] : Kinetic parameters of anti -DKK1 mAb and anti -DKK1 anti- SOST bispecific antibody Antibodies tested Antigen tested kon（1/Ms） koff（1/s） KD (M) PRO243 (anti-DKK1) HkDK1 6.83E+05 1.00E-04 1.46E-10 PRO244 (anti-DKK1) HkDK1 5.94E+05 1.00E-04 1.68E-10 PRO243 hSOST No binding No binding No binding PRO244 hSOST 1.89E+06 4.62E-04 2.45E-10 PRO396 (anti-DKK1 x anti-SOST) HkDK1 2.72E+06 1.00E-04 3.68E-11 PRO397 (anti-DKK1 x anti-SOST) HkDK1 7.26E+05 2.26E-04 3.12E-10 PRO396 hSOST 2.40E+06 3.76E-04 1.56E-10 PRO397 hSOST 1.61E+06 7.20E-04 4.48E-10

Examples twenty three : Evaluation of Antibodies in In Vitro Cell-Based Assays DKK1 and Anti DKK1 anti- SOST AND body

Purified ANDbody constructs, mAbs and bispecific antibodies were tested for their ability to block DKK1 function in an in vitro cell-based assay using HEK293T cells as described above, see e.g. (Example 1(e)).

VII. Other implementation methods

Some implementations of the technology described herein may be defined according to any of the following numbered implementations:

1.     A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target in bone tissue or bone cells of a subject, and (b) the second binding site is specific for an address target expressed in bone tissue or bone cells of the subject; wherein: (i) the second binding site localizes the first binding site to the address target so that the first binding site affects effector target signaling in the bone tissue or bone cells; (ii) the second binding site does not substantially affect signaling after binding to the address target; and (iii) the first binding site does not substantially affect effector target signaling when not localized by the second binding site.

2.     A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target in bone tissue or bone cells of a subject, and (b) the second binding site is specific for an address target expressed in bone tissue or bone cells of the subject; wherein: (i) the second binding site positions the first binding site at the address target so that the first binding site affects effector target signaling in the bone tissue or bone cells; (ii) the second binding site does not substantially affect signaling after binding to the address target; and (iii) the first binding site does not substantially affect effector target signaling when not positioned by the second binding site; And wherein the localization of the macromolecule to non-target tissues or cells is significantly reduced relative to the localization of a reference macromolecule lacking the second binding site.

3.     A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target in bone tissue or bone cells of a subject, and (b) the second binding site is specific for an address target expressed in bone tissue or bone cells of the subject; wherein: (i) the second binding site positions the first binding site at the address target so that the first binding site affects effector target signaling in the bone tissue or bone cells; (ii) the second binding site does not substantially affect signaling after binding to the address target; and (iii) the first binding site does not substantially affect effector target signaling when not positioned by the second binding site; And wherein the localization of the macromolecule to the bone tissue or bone cells is significantly increased relative to the localization of a reference macromolecule lacking the second binding site.

4.     A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target in bone tissue or bone cells of a subject, and (b) the second binding site is specific for an address target expressed in bone tissue or bone cells of the subject; wherein: (i) the second binding site localizes the first binding site to the address target so that the first binding site affects effector target signaling in the bone tissue or bone cells; (ii) the second binding site does not substantially affect signaling after binding to the address target; and (iii) the first binding site does not substantially affect effector target signaling when not localized by the second binding site; And wherein at least 25% of the macromolecule administered to the subject is detected in the bone tissue or bone cells at a time point between 1 day and 7 days after administration.

5.     A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target in bone tissue or bone cells of a subject, and (b) the second binding site is specific for an address target expressed in bone tissue or bone cells of the subject; wherein: (i) the second binding site localizes the first binding site to the address target so that the first binding site affects effector target signaling in the bone tissue or bone cells; (ii) the second binding site does not substantially affect signaling after binding to the address target; and (iii) the first binding site does not substantially affect effector target signaling when not localized by the second binding site; And wherein the affinity of the first binding site for the effector target is lower than the affinity of the second binding site for the address target.

6.     A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target in bone tissue or bone cells of a subject, and (b) the second binding site is specific for an address target expressed in bone tissue or bone cells of the subject; wherein: (i) the second binding site localizes the first binding site to the address target so that the first binding site affects effector target signaling in the bone tissue or bone cells; (ii) the second binding site does not substantially affect signaling after binding to the address target; and (iii) the first binding site does not substantially affect effector target signaling when not localized by the second binding site; And wherein the affinity of the first binding site for the effector target is lower than the affinity of the second binding site for the address target.

7.     A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target in bone tissue or bone cells of a subject, and (b) the second binding site is specific for an address target expressed in bone tissue or bone cells of the subject; wherein: (i) the second binding site localizes the first binding site to the address target so that the first binding site affects effector-target signaling in the target tissue or cell; (ii) the second binding site does not substantially affect signaling after binding to the address target; and (iii) the first binding site does not substantially affect effector-target signaling when not localized by the second binding site; And wherein the efficacy of the first binding site at the bone tissue or cell is significantly increased relative to a reference macromolecule lacking the second binding site.

8.     The macromolecule as described in any of embodiments 1-7, wherein the first binding site has a low affinity for the effector target.

9.     A macromolecule as described in any of embodiments 1-7, wherein the first binding site has a low affinity for the effector target.

10. A macromolecule as described in any of embodiments 1-4 and 6-9, wherein the affinity of the first binding site for the effector target is lower than the affinity of the second binding site for the address target.

11. A macromolecule as described in any of embodiments 1-10, wherein the affinity of the first binding site for the effector target is lower than the affinity of the second binding site for the address target.

12. A macromolecule as described in any of embodiments 1-11, wherein: (a) the Kd of the first binding site for the effector target is higher than the Kd of the second binding site for the address target; (b) the _EC50 of the first binding site for the effector target is higher than the _EC50 of the second binding site for the address target; or (c) the _IC50 of the first binding site for the effector target is higher than the _IC50 of the second binding site for the address target.

13. A macromolecule as described in any of embodiments 1-12, wherein the affinity of the first binding site for the effector target is at least about 2 times, at least about 5 times, or at least about 10 times less than the affinity of the second binding site for the address target.

14. A macromolecule as described in any of embodiments 1-13, wherein the affinity of the second binding site for the address target has a Kd greater than about 1 nM, greater than about 2 nM or greater than about 50 nm.

15. A macromolecule as described in any of embodiments 1-14, wherein the effector target is a protein, lipid or sugar.

16. A macromolecule as described in any of embodiments 1-15, wherein the effector target is a cell membrane-associated target.

17. A macromolecule as described in embodiment 15 or 16, wherein the effector target is a protein.

18. A macromolecule as described in embodiment 17, wherein the effector target is a secreted protein.

19. A macromolecule as described in any of embodiments 1-18, wherein the macromolecule promotes the effector target.

20. A macromolecule as described in any of embodiments 1-18, wherein the macromolecule antagonizes the effector target.

21. A macromolecule as described in any of embodiments 1-20, wherein the address target is a protein, a lipid or a sugar.

22. A macromolecule as described in embodiment 21, wherein the address target is a protein.

23. A macromolecule as described in any of embodiments 17-22, wherein the expression of the effector target or the address target is the expression of an RNA sequence encoding the effector target or the address target.

24. A macromolecule as described in embodiment 23, wherein the expression level of the effector target or the address target is assessed by using an RNA sequence dataset.

25. The macromolecule as described in embodiment 24, wherein the RNA sequence dataset is a genotype-tissue expression (GTEx) dataset or a human protein atlas (HPA) dataset.

26. A macromolecule as described in embodiment 22, wherein the expression of the effector target or the address target is protein expression.

27. A macromolecule as described in any of embodiments 1-26, wherein the effector target is expressed systemically in the subject.

28. A macromolecule as described in any of embodiments 1-26, wherein the effector target is regionally expressed in the subject.

29. A macromolecule as described in any of embodiments 1-26, wherein the effector target is locally expressed in the subject.

30. A macromolecule as described in any of embodiments 1-29, wherein the address target is expressed regionally in the subject.

31. A macromolecule as described in any of embodiments 1-29, wherein the address target is locally expressed in the subject.

32. A macromolecule as described in any of embodiments 1-29, wherein the expression of the address target is limited to a cell type in the subject.

33. A macromolecule as described in any of embodiments 1-32, wherein the address target is a soluble protein or an extracellular matrix (ECM)-associated protein and is not present in detectable amounts on the cell surface.

34. The macromolecule of embodiment 33, wherein the address target is expressed in the ECM and is not present in detectable amounts elsewhere in the subject.

35. A macromolecule as described in any of embodiments 1-34, wherein the address target is expressed only by cells in a specific cellular state in the subject.

36. A macromolecule as described in any of embodiments 1-35, wherein the address target is expressed only by cells in the subject that are in a disease state.

37. A macromolecule as described in any of embodiments 1-36, wherein the address target is not expressed in a tissue that is harmful to the subject by binding of the second binding site to the effector target.

38. A macromolecule as described in any of embodiments 1-37, wherein the binding site of the address target is not bound to the binding site of the natural ligand of the address target in a detectable amount.

39. A macromolecule as described in any of embodiments 1-38, wherein the expression of the address target is significantly higher in bone tissue or bone cells than in any other tissue or cell type.

40. A macromolecule as described in any of embodiments 1-39, wherein the effector target and the address target are located on the same cell.

41. A macromolecule as described in any of embodiments 1-39, wherein the effector target and the address target are located on different cells.

42. A macromolecule as described in embodiment 41, wherein the effector target and the address target are located on different cells of the same cell type.

43. A macromolecule as described in embodiment 41, wherein the effector target and the address target are located on different cells of different cell types.

44. A macromolecule as described in any of embodiments 40-43, wherein the effector target and the address target are located on different cells within 100 nm of each other in the subject.

45. A macromolecule as described in any of embodiments 40-43, wherein the effector target or the address target is present on the cell surface.

46. A macromolecule as described in any one of embodiments 1-45, wherein the macromolecule is a DNA polynucleotide.

47. A macromolecule as described in any one of embodiments 1-45, wherein the macromolecule comprises RNA or an RNA-polypeptide conjugate.

48. A macromolecule as described in any of embodiments 1-45 and 47, wherein the macromolecule comprises a polypeptide.

49. A macromolecule as described in any one of embodiments 1-45, wherein the macromolecule is a polypeptide.

50. The macromolecule as described in embodiment 48 or 49, wherein the polypeptide is an antibody or an antigen-binding fragment thereof.

51. A macromolecule as described in embodiment 50, wherein the first binding site and the second binding site each contain VH and/or VL.

52. A macromolecule as described in embodiment 51, wherein the macromolecule is an antibody, which comprises a first binding site specific for the effector target in the subject and a second binding site specific for the address target.

53. The macromolecule as described in embodiment 51 or 52, wherein the macromolecule is an asymmetric antibody or a symmetric antibody.

54. The macromolecule according to any one of embodiments 50-53, wherein the antibody or antigen-binding fragment thereof comprises scFv, BsIgG, BsAb fragment, BiTE, dual affinity redirecting protein (DART), tandem bispecific antibody (TandAb), bispecific antibody, Fab2, di-scFv, chemically linked F(ab')2, Ig molecules with 2, 3 or 4 different antigen-binding sites, DVI-IgG 4-in-1, ImmTac, HSAbody, IgG-IgG, Cov-X-Body, scFv1-PEG- _scFv2 , attached IgG, DVD-IgG, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, monomer, nanoCLAMP, bis-Fab, Fv, Fab, Fab'-SH, linear antibody, scFv, heavy chain only antibody (Humabody), ScFab, IgG antibody fragment, single chain variable region antibody, single domain heavy chain antibody, bispecific triabody, BiKE, CrossMAb, dsDb, scDb, tandem dAb/VHH, triple dAb VHH, tetravalent dAb/VHH, Fab-scFv, Fab-Fv, or DART-Fc, fiber-linked protein (adnectin), Kunitz-type inhibitor, or receptor bait.

55. The macromolecule as described in embodiment 48, wherein the polypeptide is a ligand of the effector target or a ligand of the address target.

56. A macromolecule as described in embodiment 55, wherein the ligand is a natural ligand, a modified ligand or a synthetic ligand.

57. A macromolecule as described in embodiment 55 or 56, wherein the effector target or address target is a receptor and the polypeptide is its ligand.

58. A macromolecule as described in any of embodiments 55-57, wherein the first binding site comprises an antibody or an antigen-binding fragment thereof and the second binding site comprises a ligand of the address target.

59. A macromolecule as described in any of embodiments 55-57, wherein the first binding site comprises a ligand of the effector target and the second binding site comprises an antibody or an antigen-binding fragment thereof.

60.   The macromolecule of any one of embodiments 1-45 and 48-59, wherein the amino acid sequences of the first and second binding sites are at least about 10% identical, at least about 20% identical, at least about 30% identical, at least about 40% identical, at least about 50% identical, at least about 60% identical or at least about 70% identical.

61. A macromolecule as described in any of embodiments 1-48, wherein the first binding site comprises an antibody or an antigen-binding fragment thereof, and the second binding site comprises a small molecule that binds to the address target.

62. A macromolecule as described in any of embodiments 1-48, wherein the first binding site comprises a small molecule that binds to the effector target, and the second binding site comprises an antibody or an antigen-binding fragment thereof.

63. A macromolecule as described in any of embodiments 1-62, wherein the address target has a Gini coefficient greater than about 0.4, about 0.5, about 0.57, about 0.65, about 0.7, about 0.85, about 0.90 or about 0.95.

64. A macromolecule as described in any of embodiments 1-63, wherein the address target has a Tau coefficient greater than about 0.67, about 0.75, about 0.8, about 0.85, about 0.90 or about 0.95.

65. A macromolecule as described in any of embodiments 1-64, wherein the effector target has a Gini coefficient of less than about 0.25, about 0.20 or about 0.15.

66. A macromolecule as described in any of embodiments 1-65, wherein the effector target has a Tau coefficient of less than about 0.25, about 0.20 or about 0.15.

67.   The macromolecule as described in any one of embodiments 1-66 further comprises a third binding site.

68. A macromolecule as described in embodiment 67, wherein the third binding site is the same as the first binding site.

69. A macromolecule as described in embodiment 67, wherein the third binding site is the same as the second binding site.

70. A macromolecule as described in any of embodiments 1-69, wherein the first binding site and the second binding site are directly connected to each other in the macromolecule.

71. A macromolecule as described in any one of embodiments 1-70, wherein the first binding site and the second binding site in the macromolecule are connected by a stabilizing domain.

72. A macromolecule as described in any of embodiments 1-71, wherein the address target is encoded by a gene selected from the group of genes listed in Table 2.

73. A macromolecule as described in any of embodiments 1-72, wherein the address target is chondrocyte adhesion molecule (CHAD).

74. A macromolecule as described in embodiment 73, wherein the second binding site is an anti-CHAD antibody or an antigen-binding fragment thereof.

75. The macromolecule as described in embodiment 74, wherein the anti-CHAD antibody or its antigen-binding fragment does not substantially block chondrocyte α2β1 integrin-dependent adhesion.

76. A macromolecule as described in any one of embodiments 1-72, wherein the address target is dentin matrix acidic phosphoprotein 1 (DMP1).

77. A macromolecule as described in embodiment 76, wherein the second binding site is an anti-DMP1 antibody or an antigen-binding fragment thereof.

78. The macromolecule as described in embodiment 77, wherein the anti-DMP1 antibody or its antigen-binding fragment does not substantially block the mineralization of osteoblasts.

79. A macromolecule as described in any of embodiments 1-72, wherein the address target is bone sialoprotein (IBSP).

80. The macromolecule as described in embodiment 79, wherein the second binding site is an anti-IBSP antibody or an antigen-binding fragment thereof.

81. The macromolecule of embodiment 80, wherein the anti-IBSP antibody or antigen-binding fragment thereof does not substantially block the mineralization of osteoblasts.

82. A macromolecule as described in any of embodiments 1-72, wherein the address target is trophoblast glycoprotein (TPBG).

83. The macromolecule as described in embodiment 82, wherein the second binding site is an anti-TPBG antibody or an antigen-binding fragment thereof.

84. The macromolecule as described in embodiment 83, wherein the anti-TPBG antibody or its antigen-binding fragment does not substantially block the mineralization of osteoblasts.

85. A polymer as described in any of embodiments 1-72, wherein the address target is hydroxyapatite.

86.   The macromolecule as described in embodiment 85, wherein the second binding site is a bisphosphonate, and optionally the bisphosphonate is alendronate, risedronate, etidronate, ibandronate, clodronate, tiludronate, pamidronate or zoledronic acid.

87. A macromolecule as described in any of embodiments 1-86, wherein the address target is interferon-induced transmembrane protein 5 (IFITM5).

88. The macromolecule as described in embodiment 87, wherein the second binding site is an anti-IFITM5 antibody or an antigen-binding fragment thereof.

89. The macromolecule as described in embodiment 88, wherein the anti-IFITM5 antibody or its antigen-binding fragment does not substantially interfere with the expression of one or more osteogenesis-related genes.

90. The macromolecule as described in embodiment 89, wherein the one or more osteogenesis-related genes include one or more of Runx2, ALP and OCN.

91. A macromolecule as described in any of embodiments 87-90, wherein the anti-IFITM5 antibody or its antigen-binding fragment does not substantially block the mineralization of osteoblasts.

92. A macromolecule as described in any of embodiments 1-91, wherein the effector target is sclerostin (SOST).

93. The macromolecule as described in embodiment 92, wherein the first binding site is an anti-SOST antibody or an antigen-binding fragment thereof.

94.   The macromolecule as described in embodiment 92, wherein the first binding site comprises a SOST inhibitor.

95. The macromolecule as described in embodiment 94, wherein the SOST inhibitor is Romo monoclonal antibody, Buso group monoclonal antibody, Sesu monoclonal antibody or SHR-1222 or an antigen-binding fragment thereof.

96. A macromolecule as described in any of embodiments 1-86, wherein the effector target is dickkopf-1 (DKK1).

97. The macromolecule as described in embodiment 96, wherein the first binding site is an anti-DKK1 antibody or an antigen-binding fragment thereof.

98. A macromolecule as described in embodiment 96 or 97, wherein the first binding site comprises a DKK1 inhibitor.

99.   A macromolecule as described in any of embodiments 1-72, wherein the first binding site comprises a receptor activator of nuclear factor κ-β ligand (RANKL) inhibitor.

100. The macromolecule as described in embodiment 99, wherein the RANKL inhibitor is a Denoshu monoclonal antibody or an antigen-binding fragment thereof.

101. A macromolecule as described in any one of embodiments 1-72, wherein the first binding site comprises a tissue proteinase K inhibitor.

102. The macromolecule according to embodiment 101, wherein the tissue proteinase K inhibitor is odanacatib.

103. The macromolecule according to any one of embodiments 1-102, wherein the subject is a human.

104. A method of delivering a moiety to bone tissue or bone cells in a subject, the method comprising administering to the subject a macromolecule as described in any one of embodiments 1-103, wherein the bone tissue comprises an address target.

105. The method of embodiment 104, wherein the moiety is a molecule.

106. The method of embodiment 104 or 105, wherein the moiety is not a toxin.

107. The method of embodiment 104, wherein the part is a cell.

108. The method of embodiment 107, wherein the portion is not a T cell or a NK cell.

109. A method of modulating an effector target in bone tissue, the method comprising administering to the tissue a macromolecule as described in any one of embodiments 1-103, wherein the bone tissue comprises a site target and an effector target.

110. A method of modulating bone tissue in a subject, the method comprising administering to the subject a macromolecule as described in any one of embodiments 1-103, wherein the bone tissue comprises an address target and an effector target.

111. A method for treating a subject suffering from a disease or condition associated with an effector target, the method comprising administering to the subject a macromolecule as described in any one of embodiments 1-104, wherein the first binding site of the macromolecule binds to the effector target.

112. A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target in bone tissue or bone cells of a subject, and (b) the second binding site is specific for an address target expressed in bone tissue or bone cells of the subject; wherein the second binding site localizes the first binding site to the address target such that the first binding site affects effector target signaling in the bone tissue or bone cells, wherein the first binding site does not substantially affect effector target signaling in the absence of localization by the second binding site, and wherein the second binding site does not bind to a binding site for a natural ligand of the address target.

113. A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target in bone tissue or bone cells of a subject, and (b) the second binding site is specific for an address target expressed in bone tissue or bone cells of the subject; wherein the second binding site localizes the first binding site to the address target such that the first binding site affects effector target signaling in the bone tissue or bone cells, wherein the first binding site does not substantially affect effector target signaling in the absence of localization by the second binding site, and wherein the first binding site and the second binding site are directly linked to each other in the macromolecule.

114. A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target in bone tissue or bone cells of a subject, and (b) the second binding site is specific for an address target expressed in bone tissue or bone cells of the subject; wherein the second binding site localizes the first binding site to the address target such that the first binding site affects effector target signaling in the bone tissue or bone cells, wherein the first binding site does not substantially affect effector target signaling in the absence of localization by the second binding site, and wherein the first binding site and the second binding site are connected to each other by a stabilizing domain.

115. A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target in bone tissue or bone cells of a subject, and (b) the second binding site is specific for an address target expressed in bone tissue or bone cells of the subject; wherein the second binding site localizes the first binding site to the address target such that the first binding site affects effector target signaling in the bone tissue or bone cells, wherein the first binding site does not substantially affect effector target signaling in the absence of localization by the second binding site, and wherein the effector target and/or the address target are expressed on a host's structural tissue.

116. A pharmaceutical composition comprising the macromolecule as described in any one of embodiments 1-103 and 112-115.

117. A pharmaceutical composition comprising a macromolecule and one or more pharmaceutically acceptable excipients, wherein the macromolecule comprises a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target in bone tissue or bone cells of a subject, and (b) the second binding site is specific for an address target expressed in bone tissue or bone cells of the subject; wherein the second binding site localizes the first binding site to the address target such that the first binding site affects effector target signaling in the bone tissue or bone cells, and wherein the first binding site does not substantially affect effector target signaling in the absence of localization by the second binding site.

118. The drug composition according to embodiment 116 or 117, wherein the drug composition is an RNA drug composition.

119. The pharmaceutical composition according to any one of embodiments 116-118, further comprising a carrier.

120. The pharmaceutical composition according to embodiment 119, wherein the carrier is lipid nanoparticles.

121. The pharmaceutical composition according to embodiment 119, wherein the carrier is a viral vector.

122. The pharmaceutical composition of embodiment 119, wherein the carrier is a membrane-based carrier.

123. The pharmaceutical composition of embodiment 119, wherein the membrane-based carrier is a cell.

124. The pharmaceutical composition of embodiment 119, wherein the membrane-based carrier is a vesicle.

125. A method for modulating the activity of an effector target in bone of a subject, the method comprising administering to the subject a macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for the effector target in the subject, and (b) the second binding site is specific for CHAD.

126. A method for modulating the activity of an effector target in bone of a subject, the method comprising administering to the subject a macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for the effector target in the subject, and (b) the second binding site is specific for IFITM5.

127. A method for localizing a macromolecule to bone tissue or cells of a subject, the method comprising administering to the subject a macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target in the bone tissue or bone cells of the subject, and (b) the second binding site is specific for an address target expressed in the bone tissue or bone cells of the subject; wherein: (i) the second binding site localizes the first binding site to the address target such that the first binding site affects effector target signaling in the bone tissue or bone cells; (ii) the second binding site does not substantially affect signaling after binding to the address target; and (iii) The first binding site does not substantially affect effector-target signaling when not localized by the second binding site; and allows the macromolecule to localize to the bone tissue or bone cells of the subject.

128. A method for concentrating a macromolecule in bone tissue or cells of a subject, the method comprising administering to the subject a macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target in the bone tissue or bone cells of the subject, and (b) the second binding site is specific for an address target expressed in the bone tissue or bone cells of the subject; wherein: (i) the second binding site localizes the first binding site to the address target such that the first binding site affects effector target signaling in the bone tissue or bone cells; (ii) the second binding site does not substantially affect signaling after binding to the address target; and (iii) The first binding site does not substantially affect effector-target signaling in the absence of localization by the second binding site; and allows the macromolecule to concentrate at the bone tissue or bone cells of the subject, wherein at least 25% of the macromolecule detectable in the subject is detected at the bone tissue or bone cells at a time point between 1 day and 7 days after administration of the macromolecule to the subject.

129. The method of embodiment 127 or 128, wherein the efficacy of the first binding site at the bone tissue or bone cells is significantly increased relative to a reference macromolecule lacking the second binding site.

130. The method of embodiment 127 or 128, wherein effector-target signaling by the macromolecule is significantly reduced in non-target tissues or cells of the subject relative to a reference macromolecule lacking the second binding site.

131. The method of any one of embodiments 125-130, wherein the macromolecule is a macromolecule of any one of embodiments 1-103.

132. The macromolecule of any one of embodiments 1-103 and 112-115, wherein the macromolecule is linked to a small molecule.

133. The macromolecule, method or pharmaceutical composition according to any one of embodiments 1-132, wherein the macromolecule and the small molecule are linked via a linker.

134. The macromolecule, method or pharmaceutical composition of embodiment 133, wherein the linker is a cleavable linker.

135. The macromolecule, method or pharmaceutical composition of embodiment 133, wherein the linker is a non-cleavable linker.

136. The macromolecule, method or pharmaceutical composition of any one of embodiments 1-135, wherein a single small molecule is linked to the macromolecule.

137. The method of the macromolecule or drug composition as described in any one of embodiments 1-135, wherein a plurality of small molecules are linked to the macromolecule.

138. The macromolecule, method or pharmaceutical composition of embodiment 137, wherein each of said small molecules is identical.

139. The macromolecule, method or pharmaceutical composition of embodiment 137, wherein at least two of the small molecules are different from each other.

140. The macromolecule, method or pharmaceutical composition of any one of embodiments 132-139, wherein the small molecule is odanacatib.

141. The macromolecule, method or pharmaceutical composition of any one of embodiments 132-139, wherein the small molecule is a bisphosphonate.

142. The macromolecule, method or pharmaceutical composition of embodiment 141, wherein the bisphosphonate is alendronate, risedronate, etidronate, ibandronate, clodronate, tiludronate, pamidronate or zoledronic acid.

143. A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for sclerostin (SOST), and (b) the second binding site is specific for chondrocyte adhesion protein (CHAD).

144. A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for sclerostin (SOST), and (b) the second binding site is specific for interferon-induced transmembrane protein 5 (IFITM5).

145. A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for Dickkopf-1 (DKK1), and (b) the second binding site is specific for chondrocyte adhesion molecule (CHAD).

146. A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for sclerostin (SOST), and (b) the second binding site is specific for dentin matrix acidic phosphoprotein 1 (DMP1).

147. A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for sclerostin (SOST), and (b) the second binding site is specific for integrin bone sialoprotein (IBSP).

148. A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for sclerostin (SOST), and (b) the second binding site is specific for trophoblast glycoprotein (TPBG).

149. A macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for sclerostin (SOST), and (b) the second binding site is specific for hydroxyapatite.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and example should not be construed as limiting the scope of the invention.

without

[Figure 1] is a schematic diagram illustrating exemplary ANDbody ^TM molecules and their use as logic-gated drugs. Figure 1 shows the widespread distribution of a therapeutic target (right side) (e.g., effector target) in a human subject without address targeting, and the localized and restricted distribution with address targeting (left side), which is provided by the address target binding domain. Figure 1 also provides a representative bipartite structure of an ANDbody having an address target binding domain connected to an effector target binding domain, which includes a functional portion, such as a portion that modulates (e.g., agonists or antagonists) a target effector in an address-targeted cell or tissue. The address target binding domain directs the ANDbody to a desired location, such as a target cell or tissue, thereby allowing the effector target binding domain to engage a therapeutic effector target in a localized and restricted distribution area. In some embodiments, a high affinity of the effector domain for the target effector may not be required; positioning of the effector target binding domain by the address target binding domain enables the effector target binding domain to bind the effector target sufficiently to cause an effect on signaling of the effector target in the target cell or tissue, despite the low affinity of the effector domain for the effector target. The address target binding domain may alternatively be used to transport molecules or cellular cargo to a desired address.

[Figure 2] is a schematic diagram showing the activity of exemplary effector targets. By developing ANDbody therapeutics comprising an effector targeting domain and an address targeting domain, the effector targets can be restricted to target tissues or cells. According to the prior art, these ANDbody biologics represent effective, address-restricted drugs.

[ FIG. 3 ] provides exemplary structures of ANDbody biopharmaceuticals that can be engineered according to the present technology, including but not limited to: asymmetric antibodies, dual affinity retargeting proteins (DARTs), tandem bispecific antibodies (TandAbs), bispecific antibodies, Fab2s, IgG(L,H)-Fvs, or BiTEs.

[Figure 4] shows the EC ₅₀ curve (dashed line) of an exemplary single effector targeting domain (e.g., a monospecific biologic (e.g., scFv) having a single binding domain directed to an effector target, compared to the EC ₅₀ (solid line) of an exemplary bispecific ANDbody biologic (e.g., di-scFc) having an addressee target binding domain and an effector target binding domain, such that the single effector targeting domain (usually ubiquitously expressed) is targeted/restricted to localized, addressee target-specific tissues and/or cells, thereby effectively increasing the affinity of the effector target binding domain for the effector target binding site, as indicated by a leftward shift in the curve (lower EC ₅₀ , higher affinity).

[Figure 5] is a representative set of images showing the longitudinal interface of the bone marrow and trabecular regions (femoral “cap”) in mouse femoral sections stained with anti-dentine matrix acidic phosphoprotein 1 monoclonal antibody (anti-DMP1 mAb) (right) or incubated with horseradish peroxidase (HRP) secondary (2°) reagent alone (control; left). HRP 2° reagent was from the Mouse on Mouse Polymer IHC kit (Abcam ab269452).

[Figure 6] is a graph showing the levels of anti-TPBG monoclonal antibodies (Abs) detected on HEK293T cells transiently transfected to express full-length mouse or human TPBG constructs tagged with an intracellular FLAG peptide (C’FLAG) at the C-terminus. Cells were incubated with serially diluted anti-TPBG monoclonal antibodies and then detected with a fluorophore-conjugated secondary antibody. Levels are expressed as mean fluorescence intensity (MFI).

[Figure 7] is a representative set of micrographs showing the binding of anti-TPBG mAb (left) to MC3T3 osteoblasts grown in 96-well plates, as indicated by the fluorescent signal from the fluorophore secondary antibody. Binding of control mouse IgG at the same concentration is shown as a control (right).

[Figure 8] is a diagram showing the structure of BIS-L1.

[ FIG. 9 ] is a graph showing the results of Wnt1 luciferase reporter gene assay (measured as relative light units (RLU) of luciferase) in HEK293 reporter cells treated with the indicated amounts of anti-SOST antibody (PRO136) or bisphosphonate-conjugated anti-SOST ANDbody (anti-SOST-BIS-L1(2)).

[ FIG. 10 ] is a graph showing the proportion of anti-SOST-BIS-L1(2) or non-conjugated anti-SOST ANDBody PRO136 bound to hydroxyapatite (HA) in an in vitro assay at the indicated time points.

[FIG. 11A] Images of femoral sections from decalcified mice collected eight days after subcutaneous administration of a bisphosphonate-conjugated anti-SOST ANDbody (anti-SOST-BIS-L1(2)). The ANDbody was detected using an alkaline phosphatase-conjugated anti-human IgG antibody developed using VECTOR® Red matrix.

[FIG. 11B] Images of femoral sections from decalcified mice collected eight days after subcutaneous administration of 20 mg/kg body weight (mpk) of anti-SOST ANDbody (anti-SOST-PRO136). ANDbody was detected using an alkaline phosphatase-conjugated anti-human IgG antibody developed using VECTOR® Red matrix.

[Figure 12] shows the level of type 1 procollagen N-terminal propeptide (P1NP) in mouse serum at the indicated time points after a single subcutaneous injection of 20 mpk of bisphosphonate-conjugated anti-SOST ANDbody (anti-SOST-BIS-L1(2)), anti-SOST antibody (anti-SOST-PRO136) (positive control), anti-RSV antibody (PRO022) (negative control) or vehicle control (PBS). D8: Day 8.

without

Claims (37)

A method for localizing a macromolecule to bone tissue or bone cells of a subject, the method comprising administering to the subject a macromolecule comprising a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target in the bone tissue or bone cells of the subject, and (b) the second binding site is specific for an address target expressed in the bone tissue or bone cells of the subject; wherein: (i) the second binding site localizes the first binding site to the address target such that the first binding site affects effector target signaling in the bone tissue or bone cells; (ii) the second binding site does not substantially affect signaling after binding to the address target; and (iii) The first binding site does not substantially affect effector-target signaling when not localized by the second binding site; and allows the macromolecule to localize to the bone tissue or bone cells of the subject. The method of claim 1, wherein at least 25% of the macromolecule detectable in the subject is detected in the bone tissue or bone cells at a time point between 1 day and 7 days after administration of the macromolecule to the subject. The method of claim 1, wherein the efficacy of the first binding site at the bone tissue or bone cells is significantly increased relative to a reference macromolecule lacking the second binding site. The method of claim 3, wherein the first binding site has a low affinity for the effector target. The method of claim 3, wherein the first binding site has a low affinity for the effector target. The method of claim 1, wherein the affinity of the first binding site for the effector target is lower than the affinity of the second binding site for the site target. The method of claim 1, wherein the affinity of the first binding site for the effector target is lower than the affinity of the second binding site for the address target. The method of claim 1, wherein effector-target signaling by the macromolecule is significantly reduced in non-target tissues or cells of the subject relative to a reference macromolecule lacking the second binding site. The method of claim 1, wherein the address target is regionally expressed in the subject. The method of claim 1, wherein the address target is locally expressed in the subject. The method of claim 1, wherein expression of the address target is restricted to a cell type in the subject. The method of claim 1, wherein the address target is expressed only by cells in the subject that are in a particular cellular state. The method of claim 1, wherein the address target is expressed only by cells in the subject that are in a disease state. The method of claim 1, wherein the first binding site or the second binding site comprises a polypeptide. The method of claim 14, wherein the polypeptide is an antibody or an antigen-binding fragment thereof. The method of claim 15, wherein the macromolecule is an antibody comprising a first binding site specific for the effector target in the subject and a second binding site specific for the site target. The method of claim 14, wherein the polypeptide is a ligand of the effector target or a ligand of the address target. The method of claim 17, wherein: (a) the first binding site comprises an antibody or an antigen-binding fragment thereof and the second binding site comprises a ligand of the address target; or (b) the first binding site comprises a ligand of the effector target and the second binding site comprises an antibody or an antigen-binding fragment thereof. The method of claim 15, wherein: (a) the first binding site comprises an antibody or an antigen-binding fragment thereof, and the second binding site comprises a small molecule that binds to the address target; or (b) the first binding site comprises a small molecule that binds to the effector target, and the second binding site comprises an antibody or an antigen-binding fragment thereof. The method of claim 1, wherein the second binding site is specific for chondrogenic adherin (CHAD). The method of claim 1, wherein the second binding site is specific for dentin matrix acidic phosphoprotein 1 (DMP1). The method of claim 1, wherein the second binding site is specific for the integrin bone sialoprotein (IBSP). The method of claim 1, wherein the second binding site is specific for trophoblast glycoprotein (TPBG). The method of claim 1, wherein the second binding site is specific for hydroxyapatite. The method of claim 24, wherein the second binding site is a bisphosphonate. The method of claim 25, wherein the bisphosphonate is alendronate, risedronate, etidronate, ibandronate, clodronate, tiludronate, pamidronate or zoledronic acid. The method of any one of claims 20-26, wherein the first binding site is specific for sclerostin (SOST). The method of any one of claims 20-26, wherein the first binding site is specific for dickkopf-1 (DKK1). The method of claim 1, wherein the second binding site is specific for interferon-induced transmembrane protein 5 (IFITM5). The method of claim 29, wherein the first binding site is specific for SOST. The method of any one of claims 1-30, wherein the macromolecule is linked to a small molecule. The method of claim 31, wherein the small molecule is odanacatib. The method of claim 31, wherein the small molecule is a bisphosphonate. The method of claim 33, wherein the bisphosphonate is alendronate, risedronate, etidronate, ibandronate, clodronate, tiludronate, pamidronate or zoledronic acid. The method of any one of claims 30-34, wherein the macromolecule and the small molecule are linked via a linker. The method of claim 35, wherein the linker is a cleavable linker. The method of claim 35, wherein the linker is a non-cleavable linker.