Compositions and methods for targeted delivery of therapeutic agents

Abstract

Macromolecular compositions and related methods are provided that enable targeted delivery of therapeutic agents to effector targets in desired cells, tissues and / or organs of interest while minimizing or avoiding undesired delivery to other cells, tissues or organs. Compositions and methods related to a macromolecule, such as ANDbodyTM, that includes an effector target binding domain specific for an effector target and an address binding domain specific for an address target are described. The macromolecules are connected with the micromolecules.

Description

[0001] Sequence Listing

[0002] This application contains a Sequence Listing, which has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on October 13, 2023, is named 51666-003WO2_Sequence_Listing_10_13_23 and is 96,536 bytes in size. Background Art

[0003] For therapeutically desirable targets that are present in both healthy and diseased tissues, unwanted off-target effects are a problem. Summary of the Invention

[0004] This disclosure describes, in part, macromolecular compositions and related methods that achieve targeted delivery of a therapeutic agent to an effector target in a desired cell, tissue, and / or organ while minimizing or avoiding unwanted delivery to other cells, tissues, or organs. Generally, the compositions described herein comprise macromolecules, such as ANDbody TM , which comprises an effector target-binding domain specific for an effector target 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 a desired 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. Additionally, the address target-binding domain does not affect signal transduction after binding to the address target. However, the positioning of the effector target-binding domain by the address target-binding domain enables the effector target-binding domain to sufficiently bind the effector target to cause an effect on signal transduction of the effector target in the target cell or tissue. Additionally, the macromolecules described herein are linked to one or more small molecules. The compositions described herein can be used, for example, to specifically deliver a therapeutic agent (e.g., an effector target-binding domain, a small molecule, or both) to a desired location in a subject, such as a cell, tissue, or organ, while avoiding unwanted off-target effects.

[0005] In one aspect, the present disclosure provides a method of localizing a macromolecule at a target tissue or 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 subject, and (b) the second binding site is specific for an address target expressed in the target tissue or 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 target tissue or cell; (ii) the second binding site substantially does not affect signaling after binding to the address target; and (iii) the first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site; and allowing the macromolecule to localize at the target tissue or cell of the subject, wherein the macromolecule is linked to a small molecule.

[0006] In some embodiments, the macromolecule and the small molecule are linked by a linker (e.g., a cleavable linker or a non-cleavable linker).

[0007] One or more small molecules may be linked to the macromolecule.

[0008] In some embodiments, at a time point between 1 day and 7 days after administration of the macromolecule to the subject, at least 25% of the detectable macromolecule in the subject is detected at the target tissue or cell.

[0009] In some embodiments, the potency of the first binding site at the target tissue or cell is significantly increased relative to a reference macromolecule lacking the second binding site.

[0010] In some embodiments, the first binding site has a low affinity for the effector target.

[0011] In some embodiments, the first binding site has a low avidity for the effector target.

[0012] 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.

[0013] In some embodiments, the avidity of the first binding site for the effector target is lower than the avidity of the second binding site for the address target.

[0014] In some embodiments, effector target signaling by the macromolecule in non-target tissues or cells of the subject is significantly reduced relative to a reference macromolecule lacking the second binding site.

[0015] In some embodiments, the address target is expressed in a region 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.

[0016] In some embodiments, the address target is expressed only by cells in a subject that are in a specific cell state.

[0017] In some embodiments, the address target is expressed only by cells in a subject that are in a diseased state.

[0018] In some embodiments, the first binding site or the second binding site comprises a polypeptide.

[0019] In some embodiments, the polypeptide is an antibody or an antigen-binding fragment thereof.

[0020] In some embodiments, the macromolecule is an antibody that comprises a first binding site specific for an effector target in a subject and a second binding site specific for an address target.

[0021] In some embodiments, the polypeptide is a ligand of an effector target or a ligand of an address target.

[0022] 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 an address target; or (b) the first binding site comprises a ligand of an effector target and the second binding site comprises an antibody or an antigen-binding fragment thereof.

[0023] In some embodiments, the target tissue is skin and the second binding site is specific for desmoglein-1 (DSG-1).

[0024] In some embodiments, the target tissue is lung tissue and the second binding site is specific for RAGE.

[0025] In some embodiments, the target tissue is kidney tissue and the second binding site is specific for cadherin 16 (CDH16).

[0026] In some embodiments, the target tissue is intestinal tissue and the second binding site is specific for cadherin 17 (CDH17).

[0027] On the other hand, the present disclosure provides 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 subject, and (b) the second binding site is specific for an address target expressed in a target tissue or 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 target tissue or cell; (ii) the second binding site substantially does not affect signaling after binding to the address target; and (iii) the first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site, wherein the macromolecule is linked to a small molecule.

[0028] On the other hand, the present disclosure provides 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 subject, and (b) the second binding site is specific for an address target expressed in a target tissue or 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 target tissue or cell; (ii) the second binding site substantially does not affect signaling after binding to the address target; and (iii) the first binding site substantially does not affect effector target signaling in the absence of localization 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, wherein the macromolecule is linked to a small molecule.

[0029] On the other hand, the present disclosure provides 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 subject, and (b) the second binding site is specific for an address target expressed in a target tissue or 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 target tissue or cell; (ii) the second binding site substantially does not affect signaling after binding to the address target; and (iii) the first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site; and wherein the localization of the macromolecule to the target tissue or cell is significantly increased relative to the localization of a reference macromolecule lacking the second binding site, wherein the macromolecule is linked to a small molecule.

[0030] On the other hand, the present disclosure provides 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 a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or 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 target tissue or cell; (ii) the second binding site substantially does not affect signaling after binding to the address target; and (iii) the first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site; and wherein at least 25% of the macromolecule administered to the subject is detected at the target tissue or cell at a time point between 1 day and 7 days after administration, wherein the macromolecule is linked to a small molecule.

[0031] On the other hand, the present disclosure provides 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 a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or 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 target tissue or cell; (ii) the second binding site substantially does not affect signaling after binding to the address target; and (iii) the first binding site substantially does not affect effector target signaling in the absence of localization 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, wherein the macromolecule is linked to a small molecule.

[0032] On the other hand, the present disclosure provides 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 a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or 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 target tissue or cell; (ii) the second binding site substantially does not affect signaling after binding to the address target; and (iii) the first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site; and wherein the avidity of the first binding site for the effector target is lower than the avidity of the second binding site for the address target, wherein the macromolecule is linked to a small molecule.

[0033] On the other hand, the present disclosure provides 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 a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or 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 target tissue or cell; (ii) the second binding site substantially does not affect signaling after binding to the address target; and (iii) the first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site; and wherein the potency of the first binding site at the target tissue or cell is significantly increased relative to a reference macromolecule lacking the second binding site, wherein the macromolecule is linked to a small molecule.

[0034] In some embodiments of any of the above aspects, the macromolecule and the small molecule are linked by a linker (e.g., a cleavable linker or a non-cleavable linker).

[0035] One or more small molecules may be linked to the macromolecule.

[0036] In some embodiments, the first binding site has a low affinity for the effector target.

[0037] In some embodiments, the first binding site has a low avidity for the effector target.

[0038] 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.

[0039] In some embodiments, the avidity of the first binding site for the effector target is lower than the avidity of the second binding site for the address target.

[0040] In some embodiments, (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 EC 50 of the first binding site for the effector target is higher than the EC 50 of the second binding site for the address target; or (c) the IC 50 of the first binding site for the effector target is higher than the IC 50 of the second binding site for the address target.

[0041] In some embodiments, the affinity of the first binding site for the effector target is at least about 2-fold, at least about 5-fold, or at least about 10-fold less than the affinity of the second binding site for the address target.

[0042] In some embodiments, the second binding site has a Kd for the address target of greater than about 1 nM, greater than about 2 nM, or greater than about 50 nM.

[0043] In some embodiments, the effector target is a protein, a lipid, or a sugar.

[0044] In some embodiments, the effector target is a cell membrane-associated target.

[0045] In some embodiments, the effector target is a protein. In some embodiments, the effector target is a secreted protein.

[0046] In some embodiments, the effector target is encoded by a gene selected from the group consisting of the genes listed in Table 1.

[0047] In some embodiments, the macromolecule agonizes the effector target.

[0048] In some embodiments, the macromolecule antagonizes the effector target.

[0049] In some embodiments, the address target is a protein, a lipid, or a sugar.

[0050] In some embodiments, the address target is a protein.

[0051] In some embodiments, the expression of the effector target or the address target is the expression of the RNA sequence encoding the effector target or the address target.

[0052] In some embodiments, the expression level of the effector target or the address target is evaluated by using an RNA sequence dataset.

[0053] In some embodiments, the RNA sequence dataset is a Genotype-Tissue Expression (GTEx) dataset or a Human Protein Atlas (HPA) dataset.

[0054] In some embodiments, the expression of the effector target or the address target is protein expression.

[0055] In some embodiments, the effector target is expressed systemically in a subject.

[0056] In some embodiments, the effector target is expressed regionally in a subject.

[0057] In some embodiments, the effector target is expressed locally in a subject.

[0058] In some embodiments, the address target is expressed regionally in a subject.

[0059] In some embodiments, the address target is expressed locally in a subject.

[0060] In some embodiments, the expression of the address target is limited to cell types in a subject.

[0061] In some embodiments, the address target is a soluble protein or an extracellular matrix (ECM)-associated protein and is not present on the cell surface in detectable amounts.

[0062] In some embodiments, the address target is expressed in the ECM and is not present in detectable amounts elsewhere in the subject.

[0063] In some embodiments, the address target is expressed only by cells in a specific cell state in a subject.

[0064] In some embodiments, the address target is expressed only by cells in a diseased state in a subject.

[0065] In some embodiments, the address target is not expressed in tissues where the binding of the effector target to the second binding site is harmful to the subject.

[0066] In some embodiments, the binding site of the address target does not bind to the binding site of the natural ligand of the address target in detectable amounts.

[0067] In some embodiments, the expression of the effector target or the address target includes expression in one or more of the following: minor salivary gland, thyroid, lung, breast, breast tissue, pancreas, adrenal gland, liver, kidney, renal cortex, renal medulla, adipose visceral tissue, omentum, small intestine, terminal ileum, fallopian tube, ovary, uterus, skin, skin not exposed to sunlight, skin over the pubic arch, cervix, endocervix, exocervix, vagina, skin exposed to sunlight, calf skin, anterior cingulate cortex, Brodmann area 24 (BA24), basal ganglia, caudate nucleus, dura mater, nucleus accumbens, hypothalamus, amygdala, hippocampus, cerebellum, cerebellar hemisphere, substantia nigra, pituitary gland, spinal cord, cervical spinal cord, artery, aorta, heart, auricle, coronary artery, left ventricle, esophagus, esophageal mucosa, esophageal muscular layer, gastroesophageal junction, spleen, stomach, colon, transverse colon, sigmoid colon, testis, whole blood cells, EBV-transformed lymphocytes, tibial artery, or tibial nerve tissue.

[0068] In some embodiments, the expression of the effector target or the address target includes expression in skin tissue, lung tissue, kidney tissue, or intestinal tissue. In some embodiments, the expression of the address target is significantly higher in skin tissue, lung tissue, kidney tissue, or intestinal tissue than in any other tissue.

[0069] In some embodiments, the effector target and / or the address target is expressed on the structural tissues of a subject.

[0070] In some embodiments, the effector target and the address target are located on the same cell.

[0071] In some embodiments, the effector target and the address target are on different cells.

[0072] In some embodiments, the effector target and the address target are on different cells of the same cell type.

[0073] In some embodiments, the effector target and the address target are on different cells of different cell types.

[0074] In some embodiments, the effector target and the address target are on different cells in the same tissue.

[0075] In some embodiments, (a) the effector target is on a circulating cell and the address target is on a tissue-restricted cell; or (b) the effector target is on a tissue-restricted cell and the address target is on a circulating cell.

[0076] In some embodiments, the effector target and the address target are on different cells in a subject that are within 100 nm of each other.

[0077] In some embodiments, the effector target or the address target is present on the cell surface.

[0078] In some embodiments, the macromolecule is a DNA polynucleotide.

[0079] In some embodiments, the macromolecule comprises RNA or an RNA-polypeptide conjugate.

[0080] In some embodiments, the macromolecule comprises a polypeptide. In some embodiments, the macromolecule is a polypeptide.

[0081] In some embodiments, the polypeptide is an antibody or an antigen-binding fragment thereof.

[0082] In some embodiments, the first binding site and the second binding site each comprise a VH and / or a VL.

[0083] In some embodiments, the macromolecule is an antibody that comprises a first binding site specific for an effector target in a subject and a second binding site specific for an address target.

[0084] In some embodiments, the macromolecule is an asymmetric antibody or a symmetric antibody.

[0085] In some embodiments, the antibody or antigen-binding fragment thereof comprises scFv, BsIgG, BsAb fragment, BiTE, dual-affinity retargeting 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 quadroma, ImmTac, HSAbody, IgG-IgG, Cov-X-Body, scFv1-PEG-scFv2, additional IgG, DVD-IgG, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, monomer, nanoCLAMP, bis-Fab, Fv, Fab, Fab'-SH, linear antibody, scFv, antibody with only heavy chain (Humobody), ScFab, IgG antibody fragment, single-chain variable region antibody, single-domain heavy chain antibody, bispecific trisbody, BiKE, CrossMAb, dsDb, scDb, tandem dAb / VHH, triple dAb VHH, tetravalent dAb / VHH, Fab-scFv, Fab-Fv, or DART-Fc, adnectin, Kunitz-type inhibitor, or receptor decoy.

[0086] In some embodiments, the polypeptide is a ligand of an effector target or a ligand of an address target.

[0087] In some embodiments, the ligand is a natural ligand, a modified ligand or a synthetic ligand.

[0088] In some embodiments, the effector target or the address target is a receptor and the polypeptide is its ligand.

[0089] In some embodiments, the first binding site comprises an antibody or an antigen-binding fragment thereof and the second binding site comprises a ligand of an address target.

[0090] In some embodiments, the first binding site comprises a ligand of an effector target and the second binding site comprises an antibody or an antigen-binding fragment thereof.

[0091] In some embodiments, 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.

[0092] In some embodiments, the address target has a Gini coefficient higher 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.

[0093] In some embodiments, the address target has a Tau coefficient higher than about 0.67, about 0.75, about 0.8, about 0.85, about 0.90, or about 0.95.

[0094] In some embodiments, the effector target has a Gini coefficient lower than about 0.25, about 0.20, or about 0.15.

[0095] In some embodiments, the effector target has a Tau coefficient lower than about 0.25, about 0.20, or about 0.15.

[0096] In some embodiments, the macromolecule further comprises a third binding site. In some embodiments, the third binding site is the same as the first binding site. In some embodiments, the third binding site is the same as the second binding site.

[0097] In some embodiments, the first binding site and the second binding site are directly connected to each other in the macromolecule.

[0098] In some embodiments, the first binding site and the second binding site in the macromolecule are connected by a stabilizing domain.

[0099] In some embodiments, the effector target is Notch2 and the address target is RAGE.

[0100] In some embodiments, RAGE signaling is not affected by the binding of the second site to the RAGE address target.

[0101] In some embodiments, the effector target is Notch2 and the address target is uromodulin (UMOD).

[0102] In some embodiments, UMOD signaling is not affected by the binding of the second site to the UMOD address target.

[0103] In some embodiments, the effector target is Notch2 and the address target is membrane peptidase A subunit beta (MEP1B).

[0104] In some embodiments, MEP1B signaling is not affected by the binding of the second site to the MEP1B address target.

[0105] In some embodiments, the effector target is IL11Ra and the address target is RAGE. In some embodiments, RAGE signaling is not affected by the binding of the second site to the RAGE address target.

[0106] In some embodiments, the effector target is IL 11Ra and the address target is UMOD. In some embodiments, UMOD signaling is not affected by binding of a second agent to the UMOD address target.

[0107] In some embodiments, the subject is a human.

[0108] On the other hand, the present disclosure provides a method of delivering a moiety to a target tissue or cell in a subject, the method comprising administering to the subject a macromolecule as recited in any one of claims 1-86, wherein the target tissue comprises an address target.

[0109] In some embodiments, the moiety is a molecule.

[0110] In some embodiments, the moiety is not a toxin.

[0111] In some embodiments, the moiety is a cell.

[0112] In some embodiments, the moiety is not a T cell or an NK cell.

[0113] In some embodiments, the target tissue is not a tumor.

[0114] On the other hand, the present disclosure provides a method of modulating an effector target in a target tissue, the method comprising administering to the tissue a macromolecule as recited in any one of claims 1-86, wherein the target tissue comprises an address target and an effector target.

[0115] On the other hand, the present disclosure provides a method of biasing a binder away from binding to an effector target when the effector target is found in the heart or lung, the method comprising administering a macromolecule as recited in any one of claims 1-86, wherein the address target is substantially not expressed in the heart or lung.

[0116] On the other hand, the present disclosure provides a method of modulating a target tissue in a subject, the method comprising administering to the subject a macromolecule as recited in any one of claims 1-86, wherein the target tissue comprises an address target and an effector target.

[0117] On the other hand, the present disclosure provides a method of treating a subject having a disease or disorder associated with an effector target, the method comprising administering to the subject a macromolecule as recited in any one of claims 1-86, wherein a first binding site of the macromolecule binds the effector target.

[0118] On the other hand, the present disclosure provides 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 a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell in 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 target tissue or cell, wherein the first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site, and wherein the second binding site does not bind to the binding site of the natural ligand of the address target, and wherein the macromolecule is linked to a small molecule.

[0119] On the other hand, the present disclosure provides 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 a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell in 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 target tissue or cell, wherein the first binding site substantially does not 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, and wherein the macromolecule is linked to a small molecule.

[0120] On the other hand, the present disclosure provides 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 a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell in 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 target tissue or cell, wherein the first binding site substantially does not 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 linked to each other through a stabilizing domain, and wherein the macromolecule is linked to a small molecule.

[0121] On the other hand, the present disclosure provides 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 a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell in 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 target tissue or cell, wherein the first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site, and wherein the effector target and / or the address target is expressed on the structural tissue of the host, and wherein the macromolecule is linked to a small molecule.

[0122] On the other hand, the present disclosure provides a pharmaceutical composition comprising the macromolecule of any one of the above embodiments.

[0123] On the other hand, the present disclosure provides 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 a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell in 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 target tissue or cell, and wherein the first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site, and wherein the macromolecule is linked to a small molecule.

[0124] In some embodiments, the pharmaceutical composition is an RNA pharmaceutical composition.

[0125] In some embodiments, the pharmaceutical composition further comprises a carrier.

[0126] In some embodiments, the carrier is a lipid nanoparticle.

[0127] In some embodiments, the carrier is a viral vector.

[0128] In some embodiments, the carrier is a membrane-based carrier.

[0129] In some embodiments, the membrane-based carrier is a cell.

[0130] In some embodiments, the membrane-based carrier is a vesicle.

[0131] On the other hand, the present disclosure provides methods for modulating the activity of an effector target in the skin of a subject, the methods 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 subject, and (b) the second binding site is specific for desmoglein-1 (DSG-1), wherein the macromolecule is linked to a small molecule.

[0132] On the other hand, the present disclosure provides methods for modulating the activity of an effector target in the lung of a subject, the methods 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 subject, and (b) the second binding site is specific for RAGE, wherein the macromolecule is linked to a small molecule.

[0133] On the other hand, the present disclosure provides methods for modulating the activity of an effector target in the kidney of a subject, the methods 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 subject, and (b) the second binding site is specific for cadherin 16 (CDH16), wherein the macromolecule is linked to a small molecule.

[0134] On the other hand, the present disclosure provides methods for modulating the activity of an effector target in the intestine of a subject, the methods 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 subject, and (b) the second binding site is specific for cadherin 17 (CDH17), wherein the macromolecule is linked to a small molecule.

[0135] On the other hand, the present disclosure provides a method of localizing a macromolecule at a target tissue or 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 subject, and (b) the second binding site is specific for an address target expressed in the target tissue or 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 target tissue or cell; (ii) the second binding site substantially does not affect signaling after binding to the address target; and (iii) the first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site; and allowing the macromolecule to localize at the target tissue or cell of the subject, wherein the macromolecule is linked to a small molecule.

[0136] On the other hand, the present disclosure provides a method of concentrating a macromolecule in a target tissue or 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 subject, and (b) the second binding site is specific for an address target expressed in the target tissue or 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 target tissue or cell; (ii) the second binding site substantially does not affect signaling after binding to the address target; and (iii) the first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site; and allowing the macromolecule to concentrate at the target tissue or cell of the subject, wherein at a time point between 1 day and 7 days after administering the macromolecule to the subject, at least 25% of the detectable macromolecule in the subject is detected at the target tissue or cell, wherein the macromolecule is linked to a small molecule.

[0137] In some embodiments, the potency of the first binding site at the target tissue or cell is significantly increased relative to a reference macromolecule lacking the second binding site.

[0138] In some embodiments, effector target signaling by the macromolecule in non-target tissues or cells of the subject is significantly reduced relative to a reference macromolecule lacking the second binding site.

[0139] In some embodiments, the macromolecule is the macromolecule of any of the above embodiments.

[0140] Details of one or more embodiments of the present invention are set forth 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 appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0141] Figure 1 is a schematic diagram illustrating an exemplary ANDbody TM molecule and its use as a logic-gated drug. Figure 1 Shows the broad distribution of a therapeutic target (right side) (e.g., effector target) in a human subject without address targeting, and the local and restricted distribution with address targeting (left side), which is provided by the address target-binding domain. Figure 1 Also provided is a representative bipartite structure of an ANDbody having an address target-binding domain linked to an effector target-binding domain, which includes functional portions, such as portions that modulate (e.g., agonize or antagonize) the target effector in the address-targeted cell or tissue. The address target-binding domain directs the ANDbody to the desired location, e.g., the target cell or tissue, thereby allowing the effector target-binding domain to engage the therapeutic effector target in the locally and restricted distribution area. In some embodiments, a high affinity of the effector domain for the target effector may not be required; the positioning of the effector target-binding domain by the address target-binding domain enables the effector target-binding domain to sufficiently bind the effector target to cause an effect on the 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 can alternatively be used to transport a molecule or cellular cargo to the desired address.

[0142] Figure 2 is a schematic diagram showing the activity of an exemplary effector target, and by developing an ANDbody therapeutic agent that includes an effector-targeting domain and an address-targeting domain, the effector target can be restricted to the tissue or cell of interest. According to the prior art, these ANDbody biologics represent effective, address-restricted drugs.

[0143] Figure 3 Exemplary structures of ANDbody biologics that can be engineered according to the present technology are provided, and these ANDbody biologics include (but are not limited to): asymmetric antibodies, dual-affinity retargeting proteins (DARTs), tandem diabodies (TandAbs), diabodies, Fab2, IgG(L,H)-Fv, or BiTE.

[0144] Figure 4 Shows the EC of an exemplary bispecific ANDbody biologic (e.g., di-scFc) having an address target-binding domain and an effector target-binding domain50 (solid line), the EC of an exemplary single effector targeting domain (e.g., a monospecific biologic (such as an scFv) having a single binding domain for an effector target) 50 curve (dashed line), such that the single effector targeting domain (which is typically widely expressed) is targeted / confined to local, address target-specific tissues and / or cells, thereby effectively increasing the affinity of the effector target binding domain for the effector target binding site, as shown by the left shift of the curve (lower EC 50 , higher affinity).

[0145] Figure 5A is a bar graph showing the levels of fluorescence intensity detected in the indicated tissues in mice treated with anti-DSG1 antibody PRO003 conjugated to 800CW. Data are shown as the mean of three mice. To control for differences in labeling efficiency, the strongest signal among the displayed values was set to 1.

[0146] Figure 5B is a bar graph showing the levels of fluorescence intensity detected in the indicated tissues in mice treated with anti-DSG1 antibody PRO004 conjugated to 800CW or with a vehicle control (untreated). Data are shown as the mean of three mice. To control for differences in labeling efficiency, the strongest signal among the displayed values was set to 1.

[0147] Figure 6A is a bar graph showing the levels of fluorescence intensity detected in the indicated tissues in mice treated with anti-RAGE antibody PRO001 conjugated to 800CW or with a vehicle control. Data are shown as the mean of three mice. To control for differences in labeling efficiency, the strongest signal among the displayed values was set to 1.

[0148] Figure 6B is a bar graph showing the levels of fluorescence intensity detected in the indicated tissues in mice treated with anti-RAGE antibody PRO002 conjugated to 800CW or with a vehicle control. Data are shown as the mean of three mice. To control for differences in labeling efficiency, the strongest signal among the displayed values was set to 1.

[0149] Figure 7 is a pair of micrographs showing representative IHC staining of anti-human secondary antibody conjugated to horseradish peroxidase in the lung tissues of Balb / C mice injected via the tail vein with 3 mg / kg of anti-RAGE antibody PRO002 (left sub-panel), compared to untreated mice (right sub-panel). PRO002 contains a human IgG1 backbone.

[0150] Figure 8 is a bar graph showing the levels of fluorescence intensity detected in the indicated tissues in mice treated with anti-RAGE antibody PRO002 conjugated to Bar graph of the fluorescence intensity levels detected in the indicated tissues in mice treated with anti-CDH16 antibody PRO056 conjugated to 800CW or with vehicle control. Data are shown as the mean of three mice. To control for differences in labeling efficiency, the strongest signal among the displayed values was set to 1.

[0151] Figure 9 is a bar graph showing the fluorescence intensity levels detected in the indicated tissues in mice treated with anti-CDH17 antibody PRO061 conjugated to 800CW or with vehicle control. Data are shown as the mean of three mice. To control for differences in labeling efficiency, the strongest signal among the displayed values was set to 1.

[0152] Figure 10 is a set of micrographs showing the staining of Notch2-antagonistic mAbs PRO034, PRO035, and PRO036 and the corresponding RAGE-targeting ANDbodies PRO051, PRO052, and PRO053 on sections of a fresh-frozen healthy mouse tissue microarray (FFTMA). Lung sections are indicated by boxes.

[0153] Figure 11 is a graph showing the concentrations of PRO052, a control antibody that binds RAGE and respiratory syncytial virus (RSV) glycoprotein F (RAGEXT-4 / Motavizumab), and a control antibody that binds Notch2 and RSV glycoprotein F (Notch2-2 / Motavizumab), as detected by sandwich ELISA. Points show the mean of three mice. Error bars show the standard deviation.

[0154] Figure 12 is a set of schematic diagrams showing the designs of PRO023, PRO025, PRO024, PRO027, and PRO026 IL-10 / DSG1 ANDbodies.

[0155] Figure 13A Bar graph showing the levels of tumor necrosis factor α (TNFα) in peripheral blood mononuclear cell (PBMC) cell cultures after pre-stimulation with hrIL-10 followed by treatment with lipopolysaccharide (LPS) for the indicated length of time.

[0156] Figure 13B Bar graph showing the levels of TNFα in PBMC cell cultures after pre-stimulation with an anti-DSG1 monoclonal antibody (mAb) followed by treatment with LPS for the indicated length of time.

[0157] Figure 13CThe bar graph shows the TNFα levels in PBMC cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO024 followed by LPS treatment for a specified length of time.

[0158] Figure 13D The bar graph shows the TNFα levels in PBMC cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO026 followed by LPS treatment for a specified length of time.

[0159] Figure 13E The bar graph shows the TNFα levels in PBMC cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO023 followed by LPS treatment for a specified length of time.

[0160] Figure 13F The bar graph shows the TNFα levels in PBMC cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO025 followed by LPS treatment for a specified length of time.

[0161] Figure 13G The bar graph shows the TNFα levels in PBMC cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO027 followed by LPS treatment for a specified length of time.

[0162] Figure 14A The bar graph shows the TNFα levels in primary macrophage cultures after pre-stimulation with hrIL-10 followed by LPS treatment for a specified length of time.

[0163] Figure 14B The bar graph shows the TNFα levels in primary macrophage cultures after pre-stimulation with anti-DSG1 monoclonal antibody (mAb) PRO003 followed by LPS treatment for a specified length of time.

[0164] Figure 14C The bar graph shows the TNFα levels in primary macrophage cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO024 followed by LPS treatment for a specified length of time.

[0165] Figure 14D The bar graph shows the TNFα levels in primary macrophage cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO026 followed by LPS treatment for a specified length of time.

[0166] Figure 14EThe bar graph shows the TNFα levels in primary macrophage cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO023 followed by treatment with LPS for the specified length of time.

[0167] Figure 14F The bar graph shows the TNFα levels in primary macrophage cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO025 followed by treatment with LPS for the specified length of time.

[0168] Figure 14G The bar graph shows the TNFα levels in primary macrophage cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO027 followed by treatment with LPS for the specified length of time.

[0169] Figure 15 The figure shows the IL-10 signaling levels detected in parental HEK-BLUE TM IL-10 cells or HEK-BLUE TM IL-10 cells (+ DSG1 expression), which were treated overnight with IL-10 / DSG1 ANDbody PRO058 (functionally equivalent to PRO026) or a control antibody containing IL-10 and binding to RSV glycoprotein F (IL-10 / movetizumab) at the specified concentrations. IL-10 was measured using a colorimetric assay to detect the expression of secreted embryonic alkaline phosphatase (SEAP). OD630: Optical density at 630 nm. Curve fitting was performed using GraphPad Prism 9 to fit a four-parameter log (agonist) vs. response.

[0170] Figure 16A The figure shows the concentrations (ng / mL) of PRO003, PRO024, and PRO058 over time in serum samples from BALB / c mice administered 3 mg / kg of the indicated antibody or ANDbody by tail vein injection. The concentration of the circulating molecule was measured by ELISA. Mean concentration and standard deviation are shown. N = 3.

[0171] Figure 16B The figure shows the concentrations (ng target protein / mg total protein) of PRO003, PRO024, and PRO058 (functionally equivalent to PRO026) over time in skin tissue samples from BALB / c mice administered 3 mg / kg of the indicated antibody or ANDbody by tail vein injection. Skin samples were collected at the specified time points and homogenized to extract proteins. The concentration was measured by ELISA. Mean concentration and standard deviation are shown. N = 3.

[0172] Figure 17 These are a set of schematic diagrams showing the design of the PRO070, PRO074, PRO075, and PRO077 TNFα-blocking anti-DSG1 ANDbody.

[0173] Figure 18A The figure shows the IL-10 signaling levels detected in parental HEK-BLUE TM IL-10 cells treated overnight with PRO003, recombinant human IL-10 (rhIL-10), or recombinant human IL-10 fused to the human Fc domain (IL-10-Fc). IL-10 was measured using a colorimetric assay to detect the expression of SEAP. OD630: Optical density at 630 nm. Curve fitting was performed using GraphPad Prism 9 to fit a four-parameter log (agonist) vs. response.

[0174] Figure 18B The figure shows the IL-10 signaling levels detected in parental HEK-BLUE TM IL-10 cells treated overnight with IL-10 / DSG1 and ANDbody PRO023, PRO024, PRO025, PRO026, and PRO027. IL-10 was measured using a colorimetric assay to detect the expression of SEAP. OD630: Optical density at 630 nm. Curve fitting was performed using GraphPad Prism 9 to fit a four-parameter log (agonist) vs. response. Detailed Description

[0175] Provided herein are ANDbody TM molecules that include a therapeutic effector target-binding domain and an address target-binding domain. 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 an address target on the target tissue or cell to localize the effector target to the target cell or tissue, e.g., to form an AND-gate type of logic gate. For example, in some embodiments, the ANDbody is a macromolecule that includes at least (a) a first binding site specific for a therapeutic effector target expressed (e.g., widely expressed) on a mammalian subject, such as on the cell surface; and (b) a second binding site specific for an address target. In embodiments, the expression of the address target is restricted in the subject. In some embodiments, the binding of the first binding site to the therapeutic effector target is weaker than the binding of the second binding site to the address marker. The effector and address targets can be on the same cell or on different cells or compartments within the same tissue.

[0176] 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 a macromolecule (e.g., ANDbody) to a subject, at least 25% of the macromolecule (e.g., ANDbody) detectable in the subject is detected at the target tissue or cell. For example, in some embodiments, at a time point between 1 day and 7 days after administration of the macromolecule to the subject, 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 is detected at the target tissue or cell.

[0177] Effector target

[0178] The ANDbody of the present invention TM 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 (e.g., a cell surface protein, a transmembrane protein, a receptor) of a cell or tissue of a subject to which the therapeutic effector binding domain of the ANDbody can bind and exert a modulating effect, such as a therapeutic effect, on the subject. The ANDbodies described herein have a binding site specific for the effector target. When the effector binding domain binds to the effector target, the effector modulates the target cell or tissue to produce a biological response, such as a therapeutic effect, in the subject. However, in some embodiments, the effector target binding domains provided herein may not elicit a biological effect unless it is provided in combination with an address targeting domain to localize the effector to a desired target address in the target cell or tissue. In some embodiments, such therapeutic signal transduction may require multiple macromolecules according to the present invention to bind to multiple effector targets.

[0179] In some embodiments, an effector target binding domain, when provided alone, may produce a small / weak biological effect and provide a greater / stronger biological effect when provided in combination with an address targeting domain that localizes and concentrates / accumulates the effector to a desired target address in a targeted cell or tissue. In some embodiments, an effector target binding domain, when provided alone, may produce an acceptable biological effect and provide an even greater / stronger biological effect when provided in combination with an address targeting domain to localize the effector target binding domain to a targeted cell or tissue. In some embodiments, an effector target binding domain, when provided alone, may produce a strong biological effect and provide a strong or stronger targeting effect when provided in combination with an address targeting domain to localize the effector target binding domain to a targeted cell or tissue. In some embodiments, an effector target binding domain may produce a biological effect with an undesired off-target biological effect when provided alone, but may be targeted, concentrated, and accumulated to a desired address in a targeted cell or tissue when provided in combination with an address targeting to reduce or eliminate the undesired off-target biological effect. Accordingly, the effector target binding domains of the present technology provide excellent therapeutic agents which, when provided in combination with the address target binding domains described herein, provide stronger targeted biological effects with fewer side effects, including fewer unintended off-target biological effects.

[0180] Examples of such therapeutic signaling effects include, but are not limited to:

[0181] (i) blocking signal transduction pathways that promote or maintain a disease state;

[0182] (ii) activating signal transduction pathways that reduce or prevent a disease state;

[0183] (iii) promoting antibody-dependent cell cytotoxicity (ADCC);

[0184] (iv) inducing complement activation on a target cell or tissue;

[0185] (v) promoting phagocytosis;

[0186] (vi) blocking or activating signal transduction pathways that promote cell differentiation; and

[0187] (vii) inducing tissue remodeling to reduce or prevent fibrosis.

[0188] In some embodiments, the therapeutic effector target is more widely expressed in a subject than the address target. In some embodiments, the 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 a region that is less than systemic expression but greater 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 expression is not limited to a single tissue but can occur in multiple different tissues.

[0189] In some embodiments, the effector target binding domain has 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 and 1 μM, such as an affinity between 10 nM and 100 nM).

[0190] In some embodiments, the effector target binding domain has low avidity for the effector target. Non-limiting examples of therapeutic effector targets that can be targeted by the ANDbody disclosed herein are listed in Table 1, along with exemplary functions of the effector targets.

[0191] Table 1: Exemplary Effector Targets

[0192]

[0193] Address Target

[0194] The ANDbody of the present invention further includes an address target binder that binds to the address target to provide targeted delivery of the effector. As used herein, an "address target" is a structure on a cell or tissue whose expression is sufficiently restricted in an organism to allow identification of a target organ, tissue, cell, or cell state in the organism. The address target can be, for example, a cell surface protein or a structure localized in the extracellular matrix. As used herein, "restricted" expression of an address target means that the address target is differential, e.g., less widespread in vivo expression, as opposed to systemic expression. In certain embodiments, the address target is expressed in a single cell type, tissue, or cell state in, for example, a mammalian subject (e.g., a human subject).

[0195] In some embodiments, the currently provided address target binding domain substantially does not affect biological signal transduction after binding to the address target. For example, it does not modulate signal transduction pathways or other biological responses in the target cell or tissue. For example, the address target binding agent can be inert or inactive, where it lacks any additional activity (other than binding) after binding to the address target, including lack of catalytic activity. For example, the address target binding agent binds to a non-signaling site or motif of the address target. "Signal" is used herein to indicate conformational, enzymatic, and / or electrical results that occur due to 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 signal transduction is a domain that modulates the signal transduction pathway or other biological responses in the target cell or tissue to which it binds by no more than 25% relative to a control condition (e.g., relative to signal transduction in the absence of the domain). For example, the domain can modulate (e.g., increase or decrease) the signal transduction pathway or other biological responses 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%).

[0196] Similarly, when the effector target binding domain is not localized by the address target binding domain, the effector target binding domain can substantially not emit a signal, or can not emit a signal at all. In embodiments, the effector target binding domain emits a signal with greater potency (e.g., with higher affinity) when the effector target binding domain is localized by the address target binding domain compared to when the effector target binding domain is not localized by the address target binding domain. Effector target signal transduction can be affected as discussed above when the effector target binding domain is localized to the targeted cell or tissue by the address target binding domain that is part of the same macromolecule.

[0197] In some embodiments, the address target is used for organ-specific addressing, tissue-specific addressing, or cell-specific addressing.

[0198] The specificity of the address target binding domain of a cell or tissue can be detected using methods known in the art. In one embodiment, the Gini coefficient (GC) score is used, which is a method for assessing the expression variation of a specific gene in a dataset. (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). The address target binder can be identified using the cell expression data generated for the address target binder as described herein (Tables 2A and 2B). In some embodiments, the address target marker exhibits a Gini score greater than 0.4, such as between 0.74 and 1.00. In contrast, non-address markers that are more ubiquitously expressed can exhibit a Gini score between 0.15 and 0.19.

[0199] In one embodiment, Tau scores representative of expression variation of a particular gene in a dataset are used. Tau is calculated using the expression information of the gene in each tissue and its maximum expression across all tissues, while also taking into account the number of tissues in which expression is measured (see Itai Yanai et al., Genome-wide midrange transcription profiles reveal expression level relationships in human tissue specification, Bioinformatics, Vol. 21, No. 5, March 1, 2005, pp. 650-659; Kryuchkova-Mostacci N, Robinson-Rechavi M. A benchmark of gene expression tissue-specificity metrics. Brief Bioinform. March 1, 2017; 18(2):205-214. doi:10.1093 / bib / bbw008). In some embodiments, address target markers exhibit Tau scores greater than 0.6, for example, between 0.74 and 1.00. In contrast, non-address markers that are more ubiquitously expressed may exhibit Tau scores below 0.3, for example, between 0.15 and 0.19.

[0200] In some embodiments, the specificity of the address target binding domain for a particular cell or tissue, such as indicated by an appropriate Gini and / or Tau score, is determined by tissue-based analysis that excludes tissues having a natural biological isolation barrier (i.e., the blood-brain barrier). For example, in some embodiments, the Gini and / or Tau score can be calculated without data from tissues such as (but not limited to) the central nervous system, brain, eye, and / or testicular tissue. In some embodiments, the address target herein identifies a cell state. 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 or normal state of the cell or tissue); or an activation state (relative to a non-activated state of the cell). Exemplary disease states include inflammation, infection (e.g., bacterial, viral, or fungal infection), and cancer-related states (e.g., pre-cancerous or cancerous cell states). In some aspects, cell state reflects the fact that a particular type of cell can exhibit variability in one or more characteristics and / or can exist in a variety of different conditions while retaining its specific cell type characteristics and without 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 involve properties or characteristics exhibited only by that cell type and / or functions performed only or primarily by that cell type) or can occur in a variety of different cell types. In some embodiments, 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 is the condition of the cell caused by a stimulus or environmental condition. Cells in different cell states can be distinguished from one another in a variety of ways. For example, they can express, produce, or secrete one or more different genes, proteins, or other molecules ("markers", such as the address targets provided herein), exhibit differences in protein modification, such as phosphorylation, acetylation, etc., or may exhibit differences in appearance. Thus, a cell state can be the 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 particular appearance, and / or will or will not exhibit one or more biological responses to a stimulus or environmental condition.

[0201] In some embodiments, the address target is CD20 (e.g., the address target binding domain binds to CD20 (e.g., human CD20)). The target cell can be, for example, an immune cell. In some embodiments, the address target binding domain comprises rituximab (DrugBank accession number: DB00073) or a fragment, derivative, or variant thereof. In certain embodiments, the address target binding domain comprises an antigen-binding fragment of rituximab, such as the CDRs (or complete variable domains) disclosed in US 7381560, includingFigure 4 and FIG. 5, which are incorporated by reference. In other embodiments, the address target binding domain comprises a variant of the antigen-binding fragment of rituximab, e.g., a humanized variant or a variant otherwise comprising one or more amino acid substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions, e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% or more different from the rituximab sequence; in certain embodiments, any substitution is in the CDRs, while in other embodiments, any substitution is outside the CDRs, and in other embodiments, substitutions can be in both CDR and non-CDR sequences. In some embodiments, the substitutions can be non-conservative, conservative, highly conservative or combinations thereof, e.g., conservative or highly conservative substitutions in the CDRs (especially in the complementarity-determining residue positions, while in some embodiments, any substitution is outside the complementarity-determining residue positions) and non-conservative, conservative, highly conservative substitutions or combinations thereof in non-CDR residues. In some embodiments, the address target is CD20, and the small molecule is fluocinonide, ibrutinib or tofacitinib.

[0202] In some embodiments, the address target is CD33 (e.g., the address target binding domain binds to CD33 (e.g., human CD33)). The target cell can be, for example, an immune cell. In some embodiments, the address target binding domain comprises gemtuzumab or a fragment, derivative or variant thereof. In certain embodiments, the address target binding domain comprises the antigen-binding fragment of gemtuzumab, e.g., the CDRs (or complete variable domain) disclosed in US5773001, including Figure 1 and Figure 2(SEQ ID No: 2 and 4), which is incorporated by reference. In other embodiments, the address target binding domain comprises a variant of the antigen-binding fragment of gemtuzumab, e.g., a humanized variant or a variant otherwise comprising one or more amino acid substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions, e.g., a degree of difference from the gemtuzumab sequence of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% or more; in certain embodiments, any substitution is in the CDR, while in other embodiments, any substitution is outside the CDR, and in other embodiments, substitutions can be in both the CDR and non-CDR sequences. In some embodiments, the substitutions can be non-conservative, conservative, highly conservative or combinations thereof, e.g., conservative or highly conservative substitutions in the CDR (especially in the complementarity-determining residue, while in some embodiments, any substitution is outside the complementarity-determining residue) and non-conservative, conservative, highly conservative substitutions or combinations thereof in non-CDR residues. In some embodiments, the address target is CD33, and the small molecule is fluocinonide, ibrutinib or tofacitinib

[0203] In some embodiments, the address target is a glucocorticoid address target. In some embodiments, the address target is a glucocorticoid address target, and the target cell is an immune cell.

[0204] In some embodiments, the target cell is a myeloid cell, and the glucocorticoid address target is HRH2, KCNE3, TLR4, MCTP1, CLEC4A, SIRPB1, FGCGRT, RNF130, CSFR2, SIRPB2, TYROBP, TBXAS1, CD300LF, LRRC25, FCER1G, TNFSF13, FES, CHST13, CLEC1B or CD302.

[0205] In some embodiments, the target cell is a T cell, and the glucocorticoid address target is PRKCH, CD2, IL7R, GIMAP5, CD6, KCNA3, CLEC2D, CD3D, CD3G, LCK, CD3E, CD247, SKAP1, LAT, SIRPG, TRAC, TRBV25-1, CD5, TRBC1 or IL23A.

[0206] In some embodiments, the target cells are monocytes or dendritic cells, and the glucocorticoid address targets are OSCAR, SIGLEC9, TNFSF13B, TLR8, FGL2, TLR2, IGSF6, LILRA1, CSF1R, GPBAR1, MS4A4A, UPK3A, CD300C, CCD86, CD68, MPEG1, FCN1, TNFSF13, MS4A6A, or CST3.

[0207] In some embodiments, the target cells are monocytes or dendritic cells or T cells, and the glucocorticoid address targets are GPA33, ALG3, ENO1, IL10RA, STING1, LRRC8C, LPAR6, C1QL3, SIGMAR1, SLC3A2, GIMAP5, GIMAP1, TNFSF8, CACNA2D4, IL27RA, SCL9A6, HCST, CD48, SPN, or ADA.

[0208] In some embodiments, the target cells are monocytes or myeloid dendritic cells, and the glucocorticoid address targets are SEZ6L, NFAM1, CLEC7A, TLR8, S100Z, FGL2, CD300C, CD86, CD68, SLC24A4, GPBAR1, SLCA7, CD300E, SIGLEC9, TLR5, CTSS, FCN1, CD33, MPEG1, or CST3.

[0209] In some embodiments, the target cells are monocytes or myeloid dendritic cells or T cells, and the glucocorticoid address targets are CD44, STING1, LRRC8C, LPAR6, ENO1, CACNA2D4, SERPINB9, IL10RA, IL27RA, IL12RB1, SLC9A6, MYO1G, CD48, S100A4, CRTAM, CELA1, GIMAP5, GIMAP1, TNFSF8, or GPA33.

[0210] Additional exemplary address targets of the present technology are provided in Tables 2A (HPA database analysis) and 2B (Gtex database analysis) below.

[0211] Table 2A: Exemplary Address Targets (HPA Database Analysis)

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265] Table 2B contains address targets based on analysis of the Gtex database:

[0266] Table 2B: Exemplary Address Targets (Gtex Database Analysis)

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320] Small molecule

[0321] The macromolecule (e.g., ANDbody) of the present invention is linked to a small molecule. The macromolecule and the small molecule can be linked by a cleavable linker. Alternatively, the macromolecule and the small molecule can be linked by a non-cleavable linker. Any useful linker can be used for this purpose.

[0322] One or more (e.g., one, two, three, four, five or more) small molecules can be linked to the macromolecule. If multiple small molecules are linked to one macromolecule, the small molecules may be the same. Alternatively, one or more small molecules linked to the macromolecule can be different.

[0323] 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 instance, the small molecule can be a therapeutic agent that acts together with or complements the effector target binding site domain. Alternatively, the small molecule can modulate the effector target binding site domain. In another instance, the small molecule can modulate the address target binding site domain.

[0324] Exemplary classes of small molecules that can be linked to the macromolecule according to the present invention include those in Table 3.

[0325] Table 3: Exemplary small molecule classes

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344] 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. In particular, exemplary glucocorticoid receptor agonists include, but are not limited to, cortisone, dexamethasone, fluticasone, mometasone, fluocinolone acetonide, budesonide, butixocort, 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 cathepsin K inhibitors include, but are not limited to, odanacatib, relacatib, MIV-711, and KGP-207. Exemplary topoisomerase inhibitors include, but are not limited to, irinotecan, doxorubicin, daunorubicin, doxorubicin, epirubicin, etoposide, idarubicin, topotecan, and valrubicin.

[0345] In some embodiments, the small molecule is a steroid. In some embodiments, the small molecule is fluocinolone acetonide (PubChemID: 91488) or a salt, ester, or conjugate thereof.

[0346] In some embodiments, the small molecule is a Bruton's tyrosine kinase (BTK) inhibitor. In some embodiments, the small molecule is ibrutinib (PubChem ID: 23821094) or a salt, ester, or conjugate thereof.

[0347] In some embodiments, the small molecule is a Janus kinase (JAK) inhibitor. In some embodiments, the small molecule is tofacitinib (PubChem ID: 9926791) or a salt, ester, or conjugate thereof.

[0348] Any conjugation technique known in the art can be used to conjugate small molecules to the macromolecules of the present invention. For example, ligation techniques can be used to link small molecule carboxyl, hydroxyl, and amine residues to amine and thiol residues on proteins. Alternatively, any complementary functional groups on the two components can be used to react with each other to form covalent bonds. 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 tetrazine. In addition, any available linker can be used in the present invention, including heterobifunctional linkers that allow the connection of small molecules through, for example, disulfide bonds and amide bonds.

[0349] ANDbody structure

[0350] Generally, an ANDbody can be any macromolecule, such as a polypeptide or protein containing an effector 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.

[0351] In some embodiments, the binding site for the effector target and the binding site for the address target of the ANDbody each comprise an antibody heavy chain and / or light chain domain. In some embodiments, the ANDbody comprises a first antibody variable domain that has binding specificity for an effector target and a second antibody variable domain that has binding specificity for an address target. In other embodiments, the ANDbody comprises a first antigen-binding site of an antibody and a second antigen-binding site of an antibody, wherein the first antigen-binding site has binding specificity for an effector target and the second antigen-binding site has binding specificity for an address target.

[0352] In some embodiments, the 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 is specific for more than one, e.g., 2, 3, 4 antigens. For example, the antibody molecule comprises multiple variable domain sequences, wherein the first variable domain sequence among the multiple variable domain sequences has binding specificity for a first epitope (e.g., an effector target) and the second variable domain sequence among the multiple variable domain sequences has binding specificity for a second epitope (e.g., an address target).

[0353] In some embodiments, the ANDbody is an antibody molecule having an arm or domain that binds to an effector target and an arm or domain that binds to an address target. In an embodiment, the ANDbody is an antibody molecule that comprises a light chain that binds to one of the effector target and the address target and a heavy chain that binds to the other of the effector target and the address target.

[0354] In some embodiments, the ANDbody has the following structures: scFv, BsIgG, BsAb fragment, BiTE, dual-affinity retargeting 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 quadroma, ImmTac, HSAbody, IgG-IgG, Cov-X-Body, scFv1-PEG-scFv2, additional IgG, DVD-IgG, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, monomer, nanoCLAMP, bis-Fab, Fv, Fab, Fab'-SH, linear antibody, scFv, antibody with only heavy chain (Humabody), ScFab, IgG antibody fragment, single-chain variable region antibody, single-domain heavy chain antibody, bispecific trisbody, BiKE, CrossMAb, dsDb, scDb, tandem dAb / VHH, triple dAb VHH, tetravalent dAb / VHH, Fab-scFv, Fab-Fv, or DART-Fc, adnectin, Kunitz-type inhibitor, or receptor decoy.

[0355] The effector target-binding site and the address target-binding site of the 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 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 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.

[0356] 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.

[0357] Binding affinity can be quantified by determining the dissociation constant (Kd) of the polypeptide and its conjugate. A lower Kd indicates a higher affinity for the binding partner. Similarly, the specificity of binding of a polypeptide to its binding partner can be defined in terms of the relative dissociation constant (Kd) of the polypeptide for its binding partner, compared to the dissociation constant of the polypeptide for another non-target molecule.

[0358] The value of the dissociation constant can be determined by known methods. For example, a dual-filter nitrocellulose membrane binding assay can be used to determine the Kd, such as the assay disclosed by Wong & Lohman (Proc. Natl. Acad. Sci. USA 90, 5428 - 5432, 1993). Other standard assays for evaluating the binding ability of a ligand (such as an antibody) to a target are known in the art, including, for example, ELISA, Western blotting, RIA, and flow cytometry analysis. The binding kinetics of an antibody (e.g., binding affinity) can also be evaluated by standard assays known in the art, such as by analysis using a BiacoreTM system.

[0359] As an alternative to Kd, EC 50 or IC 50 can be used to determine relative affinity. Here, EC 50 represents the concentration at which the polypeptide reaches 50% of its maximum binding to a fixed amount of the binding partner. IC 50 represents the concentration at which the polypeptide inhibits 50% of the maximum binding of a fixed amount of a competitor to a fixed amount of the binding partner. In both cases, a lower level of EC 50 or IC 50 indicates a higher affinity for the target. The EC 50 and IC 50 values of the ANDbody binding site for its binding partner can both be determined by well-known methods such as ELISA.

[0360] In some embodiments, the Kd of the therapeutic effector target binder can 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 μM (e.g., it can 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 μM). In some embodiments, the Kd of the address target binder can be less than about 1 μM, about 500 nM, about 100 nM, about 10 nM, about 1 nM, about 100 pM, about 10 pM, or about 1 pM (e.g., it can be between 1 μM 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 address target binder.

[0361] In some embodiments, the EC of the therapeutic effector target binder 50 can 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 μM (e.g., it can 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 μM). In some embodiments, the EC of the address target binder 50 can be less than about 1 μM, about 500 nM, about 100 nM, about 10 nM, about 1 nM, about 100 pM, about 10 pM, or about 1 pM (e.g., it can be between 1 μM 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 EC of the therapeutic effector target binder 50 can be about 6-fold, about 5-fold, about 4-fold, about 3-fold, or about 2-fold that of the EC of the address target binder 50

[0362] In some embodiments, the IC of the therapeutic effector target binder 50It can 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 μM (e.g., it can 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 μM). In some embodiments, the IC50 of the address target binder can be less than about 1 μM, about 500 nM, about 100 nM, about 10 nM, about 1 nM, about 100 pM, about 10 pM or about 1 pM (e.g., it can be between 1 μM 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 IC 50 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 IC 50 of the address target binder.

[0363] The cellular or tissue density of the effector target and the address target bound by the ANDbody may be different. In an embodiment, the density of the therapeutic effector target on the cells bound by the effector target binding site of the ANDbody is more 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 address target on the cells bound by the address target binding site.

[0364] In some embodiments, the affinity of the first binding site for the therapeutic effector target it binds is about half (1 / 2) X Kd lower than the affinity of the second binding site for the address target it binds, and the density of the therapeutic effector target on the cells bound by the first binding site is about half (1 / 2) X Kd smaller than the density of the address target on the cells bound by the second binding site.

[0365] In some embodiments, the ANDbody has the affinity and density parameters as described above.

[0366] In some embodiments, the first binding site and the second binding site in the ANDbody are directly connected to each other. Direct connection means that the first binding site coding sequence is adjacent to the second binding site coding sequence and there is no sequence derived from other sequences (such as a linker). In some embodiments, the first binding site and the second binding site in the ANDbody are not directly connected to each other.

[0367] In addition to small molecules, ANDbodies as disclosed herein can also be linked to one or more additional moieties, such as, for example, extracellular components, intracellular components, soluble factors (such as enzymes, hormones, cytokines, growth factors, toxins, venoms, pollutants, etc.) or transmembrane proteins (such as cell surface receptors).

[0368] Exemplary effector targets and address target sequences to which the ANDbodies of the present technology may have affinity are provided in Table 4 and the Sequence Listing. In some cases, the sequences comprise full-length protein sequences and / or Fc fusion sequences with or without signal peptide regions. In some embodiments, the ANDbodies of the present technology include binding domains that bind to address target proteins or effector target proteins. In embodiments, the binding domain of the ANDbody of the present invention can bind to a protein sequence that includes 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, such as a full-length protein sequence or a peptide fragment thereof with or without a signal peptide region, that is fused to another protein (such as an Fc sequence). In other embodiments, the binding domain of the ANDbody of the present invention can bind to a protein that comprises less than a full-length protein sequence, such as a peptide fragment of an address target or effector target.

[0369] Table 4: Exemplary Effector Targets and Address Target Sequences

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388] Production of the ANDbody composition

[0389] Production of ANDbody polypeptide

[0390] The polypeptide component of the macromolecule (ANDbody) of the present invention can be produced by any suitable method. For example, all or part of the ANDbody can be expressed by a host cell comprising one or more nucleotides encoding the ANDbody. Thus, in some embodiments, the present invention provides one or more nucleic acids encoding any one of the macromolecules provided herein or one or more of its subunits. The nucleic acid can be, for example, a linear nucleic acid, a circular nucleic acid, and / or a modified nucleic acid. The present invention further provides one or more vectors comprising one or more nucleic acids encoding any one of the macromolecules provided herein or one or more of its subunits.

[0391] The present invention also provides one or more host cells (e.g., mammalian host cells) that comprise one or more nucleic acids encoding any one of the macromolecules provided herein or one or more of its subunits (e.g., comprising a vector containing the one or more nucleic acids). The present invention further provides a method for producing any one of the macromolecules provided herein, the method comprising culturing one or more host cells in a medium. The method can further comprise recovering the macromolecule from the one or more host cells or the medium.

[0392] Such methods for preparing therapeutic polypeptides are conventional 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).

[0393] Methods for producing ANDbody may involve expression in mammalian cells, although recombinant proteins can also be produced using insect cells, yeast, bacteria, or other cells under the control of appropriate promoters. Mammalian expression vectors may contain non-transcribed elements such as origins of replication, appropriate promoters and enhancers, as well as other 5' or 3' flanking non-transcribed sequences, and 5' or 3' untranslated 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 the 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 Edition), Cold Spring Harbor Laboratory Press (2012).

[0394] A variety of mammalian cell culture systems can be employed to express and manufacture the ANDbodies described herein. Examples of mammalian expression systems include, but are not limited to, CHO cells, COS cells, the HeLA and BHK cell lines. The processes for host cell culture for the production of protein therapeutics are described in the following literature: for example, Zhou and Kantardjieff (eds.), Mammalian Cell Cultures for Biologics Manufacturing, Advances in Biochemical Engineering / Biotechnology, Springer (2014). The purification of protein therapeutics is described in the following literature: Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and Cutler, Protein Purification Protocols, Methods in Molecular Biology, Humana Press (2010). The formulations of protein therapeutics are described in the following literature: Meyer (ed.), Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).

[0395] Antibody production techniques are known. See, e.g., Zhiqiang (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic. 1st ed. Wiley 2009; Greenfield (ed.), Antibodies: A Laboratory Manual. (2nd ed.) 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, for methods of making recombinant antibodies, including antibody engineering, use of degenerate oligonucleotides, 5'-RACE, phage display, and mutagenesis; antibody testing and characterization; antibody pharmacokinetics and pharmacodynamics; antibody purification and storage; and screening and labeling techniques.

[0396] Production of ANDbody RNA

[0397] In some embodiments, ANDbody RNAs can be produced, for example, for delivery to a subject. Generally, therapeutic mRNAs are produced by in vitro transcription. Modifications (e.g., incorporation of modified bases, 5’ cap analogs, and polyA tails) can optimize activity and function. For example, translation and stability of mRNAs can be achieved through cap and polyA tail modifications. For example, incorporation of cap analogs such as ARCA (anti-reverse cap analog) and poly(A) tails of 100 - 200 bp into in vitro transcribed (IVT) mRNAs can enhance expression and stability (Kaczmarek et al. Genome Medicine (2017) 9:60). Novel cap analogs, such as 1,2-dithiodiphosphate-modified caps, can further enhance 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). Modifying the 3' and 5' untranslated regions (UTRs) containing sequences responsible for recruiting RNA-binding proteins (RBPs) and miRNAs can increase the level of protein product (Kaczmarek). In addition, UTRs can be modified to encode regulatory elements (e.g., K-turn motifs and miRNA binding sites) in order 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 pseudouridine into mRNAs, such as N1-methyl-pseudouridine) help to mask the immunostimulatory activity of mRNAs 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 their manufacture are known and disclosed, for example, in the following: WO 2016011306; WO 2016014846; WO 2016022914; WO2016077123; WO 2016164762; WO 2016201377; WO 2017049275; US9937233; US 8710200; US10022425; US 9878056; US 9572897; Jemielity et al. RNA. 2003; 9:1108–22.90; Mockey et al. Biochem Biophys Res Commun. [Biochemical and Biophysical Research Communications] 2006; 340:1062–8.91; Strenkowska, Nucleic Acids Res. [Nucleic Acids Research] 2016; 44:9578–90.92; Presnyak et al. Cell. 2015; 160:1111–24.93; Kaczmarek et al. Genome Medicine (2017) 9:60.

[0398] Production of ANDbody with altered affinity

[0399] ANDbodies with binding sites of altered affinity can be prepared using methods known in the art. For example, an ANDbody 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. Generally, an ANDbody can be modified to alter the affinity of the effector target binding site for its effector target or to alter the affinity of the address target binding site for its address target. The modification can increase or decrease the affinity of the binding partner for the binding site.

[0400] Target and Address Evaluation

[0401] The expression of a therapeutic target can be evaluated at the RNA or protein level using methods known in the art. In an embodiment, the expression of a therapeutic target is evaluated by measuring RNA expression, for example, using an RNA sequence dataset as a representative of the protein expression level. RNA datasets include those Genotype-Tissue Expression (GTEx) datasets (see, for example, https: / / www.genome.gov / Funded-Programs-Projects / Genotype-Tissue-Expression-Project) or Human Protein Atlas (HPA) datasets (https: / / www.proteinatlas.org / ).

[0402] A non-limiting list of tissues in which the expression of a therapeutic target can be evaluated includes, for example, minor salivary glands, thyroid, lung, breast (mammary tissue), pancreas, adrenal gland, liver, kidney (cortex), kidney (medulla), adipose viscera (omentum), small intestine - terminal ileum, fallopian tube, ovary, uterus, skin not exposed to sunlight (suprapubic), cervix - endocervix, cervix - ectocervix, vagina, skin exposed to sunlight (calf), anterior cingulate cortex cells (BA24), caudate nucleus (basal ganglia), putamen (basal ganglia), nucleus accumbens (basal ganglia), hypothalamus, amygdala, hippocampus, cerebellum / cerebellar hemisphere, substantia nigra, pituitary gland, spinal cord (cervical), artery - aorta, heart - auricle, artery - coronary artery - heart, left ventricle, esophagus - mucosa, esophagus - muscular layer, esophagus - gastroesophageal junction, spleen, stomach, transverse colon, sigmoid colon, testis, whole blood, cells (EBV - transformed lymphocytes, artery - tibial or nerve - tibial tissue).

[0403] Address markers can be evaluated using methods well - known in the art. For example, gene expression can be evaluated at the mRNA level using Northern blotting, cDNA or oligonucleotide microarrays or sequencing (such as RNA - Seq), or at the protein expression level using protein microarrays, Western blotting, flow cytometry, immunohistochemistry, etc. For example, antibodies specific for a particular modified form of a protein (such as phospho - specific antibodies) or mass spectrometry can be used to evaluate modifications.

[0404] Use of ANDbody

[0405] The ANDbodies and pharmaceutical compositions provided herein are suitable for administration to a subject in need thereof, where the subject is a human or non - human animal, for example, suitable for human therapy or veterinary use. Thus, in some aspects, the present disclosure provides a method that includes administering to a subject in need a therapeutically effective amount of any of the macromolecules (i.e., ANDbodies) provided herein, a composition comprising the macromolecule, or one or more nucleic acids encoding the macromolecule.

[0406] Veterinary uses include treating mammals, including commercially relevant mammals such as pets and livestock animals, such as cattle, pigs, horses, sheep, goats, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant 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.

[0407] The present invention also relates to a subject or a subject cell comprising the ANDbody composition described herein. In some embodiments, the subject or subject cell is a plant, an insect, a bacterium, a fungus, a vertebrate, a mammal (e.g., a human), or other organism or cell.

[0408] In some embodiments, the subject or subject cell is contacted (e.g., delivered or administered) with the ANDbody composition. In some embodiments, the subject is a mammal (such as a human). The amount of the ANDbody composition, the expression product, or both, in the subject can be measured at any time after administration.

[0409] In some aspects, provided herein are methods of enhancing the activation of a signaling pathway in a tissue or cell, the method comprising contacting the tissue or cell with a macromolecule provided herein, wherein the first binding site and the second binding site substantially do not activate the signaling pathway in the absence of the second binding site being positioned; and wherein when the tissue or cell is contacted with the macromolecule, the activation of the signaling pathway in the tissue or cell by the first binding site is significantly increased relative to a reference macromolecule lacking the second binding site.

[0410] Drug composition

[0411] Polypeptide pharmaceutical composition

[0412] The ANDbody compositions (e.g., ANDbody polypeptides or RNA compositions) described herein can be administered to a subject in need thereof. The present invention includes drug compositions comprising the macromolecules (i.e., ANDbodies) provided herein, e.g., in combination with one or more pharmaceutically acceptable carriers or excipients. In some embodiments, the composition is a unit dosage form suitable for administration to a human subject (e.g., intravenously, orally, or subcutaneously).

[0413] The 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.

[0414] RNA pharmaceutical composition

[0415] Nucleic acids (such as RNA) encoding ANDbody can be alternatively or additionally administered to a subject. Generally, therapeutic mRNA is produced by in vitro transcription. Modifications (such as incorporation of modified bases, 5’ cap analogs, and polyA tails) can optimize activity and function. For example, translation and stability of mRNA can be achieved by cap and polyA tail modifications. For example, incorporation of cap analogs such as ARCA (anti-reverse cap analog) and a poly(A) tail of 100-200bp 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). Modifying the 3' and 5' untranslated regions (UTRs) containing sequences responsible for recruiting RNA-binding proteins (RBPs) and miRNAs can increase the level of the protein product (Kaczmarek). In addition, UTRs can be modified to encode regulatory elements (such as K-turn motifs and miRNA binding sites) in order to control RNA expression in a cell-specific manner (Wroblewska et al. Nat Biotechnol. 2015;33:839–41). RNA base modifications (such as incorporation of pseudouridine into mRNA, such as N1-methyl-pseudouridine) help to mask the immunostimulatory activity of mRNA 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 of making them are known and disclosed, for example, in the following: WO 2016011306; WO 2016014846; WO2016022914; WO 2016077123; WO 2016164762; WO 2016201377; WO 2017049275; US9937233; US 8710200; US10022425; US 9878056; US 9572897; Jemielity et al. RNA. 2003; 9:1108–22.90; Mockey et al. Biochem Biophys Res Commun. [Biochemical and Biophysical Research Communications] 2006; 340:1062–8.91; Strenkowska, Nucleic Acids Res. [Nucleic Acids Research] 2016; 44:9578–90.92; Presnyak et al. Cell. 2015; 160:1111–24.93; Kaczmarek et al. Genome Medicine (2017) 9:60.

[0416] In embodiments, the RNA is circular RNA. See, for example, WO 2019118919, which describes the expression of therapeutic RNAs, such as antibody RNAs, from circular RNAs. In some embodiments, the invention includes circular polynucleotides that comprise (a) an internal ribosome entry site (IRES), (b) an expression sequence encoding an ANDbody as described herein and lacking a poly-A sequence, and (c) a termination element. The circular RNA encoding an ANDbody as described herein can be delivered naked (i.e., not formulated with a carrier) or together with a carrier.

[0417] Combination therapy

[0418] In some embodiments, the ANDbody or ANDbody composition provided herein is administered in combination with one or more additional therapeutic agents.

[0419] Carrier

[0420] Lipid nanoparticles

[0421] Formulations of the compositions described herein for in vivo delivery with a carrier (e.g., a polypeptide or RNA ANDbody composition) include lipid nanoparticle (LNP) formulations. See, e.g., U.S. Patent 9,764,036; U.S. Patent 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 ionizable lipids, such as non-cationic lipids (e.g., neutral or anionic or zwitterionic lipids); one or more conjugated lipids (such as the PEG-conjugated lipids or lipids conjugated to a polymer described in Table 5 of WO2019217941; 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., conjugated receptors, receptor ligands, antibodies); or combinations of the foregoing.

[0422] Lipids useful for nanoparticle formation (e.g., lipid nanoparticles) include, for example, those described in Table 4 of WO 2019217941, which is incorporated herein by reference—e.g., lipid-containing nanoparticles can include one or more lipids from Table 4 of WO 2019217941. Lipid nanoparticles can include additional elements, such as polymers, as described in Table 5 of WO 2019217941, which is incorporated by reference.

[0423] In some embodiments, the conjugated lipid, when present, can include one or more of the following: PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), polyethylene glycolated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, sodium N-(carbonyloxy-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and those described in Table 2 of WO 2019051289 (incorporated by reference) and combinations of the foregoing.

[0424] In some embodiments, the sterols that can be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in W02009 / 127060 or US2010 / 0130588 incorporated by reference. Additional exemplary sterols include phytosterols, such as those described in Eygeris et al. (2020), dx.doi.org / 10.1021 / acs.nanolett.0c01386 incorporated by reference herein.

[0425] In some embodiments, the lipid particles comprise an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits particle aggregation, and a sterol. The amounts of these components can vary independently to obtain the desired properties. For example, in some embodiments, the lipid nanoparticles comprise: an ionizable lipid in an amount of about 20 mol% to about 90 mol% of the total lipid (in other embodiments, it can be 20 - 70% (mol), 30 - 60% (mol), or 40 - 50% (mol) of the total lipid present in the lipid nanoparticles; about 50 mol% to about 90 mol%); a non-cationic lipid in an amount of about 5 mol% to about 30 mol% of the total lipid; a conjugated lipid in an amount of about 0.5 mol% to about 20 mol% of the total lipid, and a sterol in an amount of about 20 mol% to about 50 mol% of the total lipid. The ratio of total lipid to nucleic acid can vary as needed. For example, the ratio of total lipid to nucleic acid (mass or weight) can be about 10:1 to about 30:1.

[0426] In some embodiments, the ratio of lipid to nucleic acid (mass / mass ratio; w / w ratio) can be in the range of: 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 amounts of lipid and nucleic acid can be adjusted to provide the desired N / P ratio, such as an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Generally, the total lipid content of the lipid nanoparticle formulation can be in the range of about 5 mg / ml to about 30 mg / mL.

[0427] Some non-limiting examples of lipid compounds that can be used (e.g., in combination with other lipid components) to form lipid nanoparticles for delivering the compositions described herein, such as the nucleic acids (e.g., RNA) described herein, include:

[0428] (i)

[0429] In some embodiments, the LNP comprising formula (i) is used to deliver the ANDbody RNA composition described herein to the liver and / or hepatocytes.

[0430] (ii)

[0431] In some embodiments, the LNPs of formula (ii) are used to deliver the ANDbody RNA compositions described herein to the liver and / or hepatocytes.

[0432] (iii)

[0433] In some embodiments, the LNPs of formula (iii) are used to deliver the ANDbody RNA compositions described herein to the liver and / or hepatocytes.

[0434] (iv)

[0435]

[0436] (v)

[0437] In some embodiments, the LNPs of formula (v) are used to deliver the ANDbody RNA compositions described herein to the liver and / or hepatocytes.

[0438] (vi)

[0439] In some embodiments, the LNPs of formula (vi) are used to deliver the ANDbody RNA compositions described herein to the liver and / or hepatocytes.

[0440] (vii)

[0441] (viii)

[0442] In some embodiments, the LNPs of formula (viii) are used to deliver the ANDbody RNA compositions described herein to the liver and / or hepatocytes.

[0443] (ix)

[0444] In some embodiments, the LNPs of formula (ix) are used to deliver the ANDbody RNA compositions described herein to the liver and / or hepatocytes.

[0445] (x)

[0446] wherein

[0447] X1 is O, NR1 or a direct bond, X2 is a C2-5 alkylene group, X3 is C(=O) or a direct bond, R1 is H or Me, R3 is a C1-3 alkyl group, R2 is a C1-3 alkyl group, or R2 together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X2 forms a 4-membered, 5-membered or 6-membered ring, or X1 is NR1, R1 and R2 together with the nitrogen atom to which they are attached form a 5-membered or 6-membered ring, or R2 together with R3 and the nitrogen atom to which they are attached forms a 5-membered, 6-membered or 7-membered ring, Y1 is a C2-12 alkylene group, Y2 is selected from (in any orientation), (in any orientation), (in any orientation),

[0448] n is from 0 to 3, R4 is a C1-15 alkyl group, Z1 is a C1-6 alkylene group or a direct bond,

[0449] Z 2 is (in any orientation) or absent, provided that if Z1 is a direct bond, then Z2 is absent;

[0450] R5 is a C5-9 alkyl group or a C6-10 alkoxy group, R6 is a C5-9 alkyl group or a C6-10 alkoxy group, W is a methylene group or a direct bond, and R7 is H or Me, or a salt thereof, provided that if R3 and R2 are C2 alkyl groups, X1 is O, X2 is a straight-chain C3 alkylene group, X3 is C(=O), Y1 is a straight-chain C6 alkylene group, (Y2)n-R4 is

[0451]

[0452] R4 is a straight-chain C5 alkyl group, Z1 is a C2 alkylene group, Z2 is absent, W is a methylene group, and R7 is H, then R5 and R6 are not Cx alkoxy groups.

[0453] In some embodiments, the LNPs comprising formula (xii) are used to deliver the ANDbody RNA compositions described herein to the liver and / or hepatocytes.

[0454] (xi)

[0455] In some embodiments, the LNPs comprising formula (xi) are used to deliver the ANDbody RNA compositions described herein to the liver and / or hepatocytes.

[0456] wherein R = (xii)

[0457] (xiii)

[0458] (xiv)

[0459] In some embodiments, the LNP comprises the compound of formula (xiii) and the compound of formula (xiv).

[0460] (xv)

[0461] In some embodiments, the LNP comprising the formula (xv) is used to deliver the ANDbody RNA composition described herein to the liver and / or hepatocytes.

[0462] (xvi)

[0463] In some embodiments, the LNP of the formulation comprising the formula (xvi) is used to deliver the ANDbody RNA composition described herein to lung endothelial cells.

[0464] (xvii)

[0465] where X = (xviii)

[0466] (a)

[0467] (xviii) (b)

[0468] (xix)

[0469] In some embodiments, the lipid compounds for forming lipid nanoparticles for delivering the compositions described herein, such as the nucleic acids (e.g., RNA) described herein, are made by one of the following reactions:

[0470] (xx)(a)

[0471] (xx)(b)

[0472] In some embodiments, the compositions described herein (e.g., nucleic acids or proteins) are provided in LNPs comprising ionizable lipids. In some embodiments, the ionizable lipid is 17-(nonan-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)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (LP01), for example, synthesized as described in Example 13 of WO 2015 / 095340 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is bis((Z)-non-2-en-1-yl) 9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), for example, synthesized as described in Examples 7, 8, or 9 of US2012 / 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)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), for example, synthesized as described in Examples 14 and 16 of WO 2010 / 053572 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is an imidazole cholesterol ester (ICE) lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propionate, for example, Structure (I) from WO 2020 / 106946 (incorporated herein by reference in its entirety).

[0473] 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 depending on the pH, or an amine-containing lipid that can be easily protonated. In some embodiments, the cationic lipid is a lipid that can be positively charged under physiological conditions. Exemplary cationic lipids include one or more amine groups with a positive charge. In some embodiments, the lipid particle comprises a cationic lipid formulated with one or more of a neutral lipid, an ionizable amine-containing lipid, a biodegradable alkyne lipid, a steroid, a phospholipid including a polyunsaturated lipid, a structural lipid (such as a sterol), PEG, cholesterol, and a polymer-conjugated lipid. In some embodiments, the cationic lipid can be an ionizable cationic lipid. The exemplary cationic lipids disclosed herein can have an effective pKa greater than 6.0. In an embodiment, the lipid nanoparticle can comprise a second cationic lipid having an effective pKa different from (e.g., greater than) the first cationic lipid. The lipid nanoparticle can comprise 40 mol% to 60 mol% of a cationic lipid, a neutral lipid, a steroid, a polymer-conjugated lipid, and a therapeutic agent, such as a nucleic acid (e.g., RNA) as described herein, encapsulated within or associated with the lipid nanoparticle. In some embodiments, the nucleic acid is co-formulated with the cationic lipid. The nucleic acid can be adsorbed onto the surface of an LNP (e.g., an LNP comprising a cationic lipid). In some embodiments, the nucleic acid can be encapsulated within an LNP (e.g., an LNP comprising a cationic lipid). In some embodiments, the lipid nanoparticle can comprise a targeting moiety, such as a targeting moiety coated with a targeting agent. In an embodiment, the LNP formulation is biodegradable. In some embodiments, a lipid nanoparticle comprising one or more of the lipids described herein (e.g., of formula (i), (ii), (ii), (vii), and / or (ix)) encapsulates 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 the RNA molecules.

[0474] 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 formulas: X of US2016 / 0311759; I of US20150376115 or US2016 / 0376224; I, II, or III of US20160151284; I, IA, II, or IIA of US20170210967; I-c of US20150140070; A of US2013 / 0178541; I of US2013 / 0303587 or US2013 / 0123338; I of US 2015 / 0141678; II, III, IV, or V of US2015 / 0239926; I of US2017 / 0119904; I or II of WO 2017 / 117528; A of US2012 / 0149894; A of US2015 / 0057373; A of WO 2013 / 116126; A of US2013 / 0090372; A of US2013 / 0274523; A of US2013 / 0274504; A of US 2013 / 0053572; A of W02013 / 016058; A of W02012 / 162210; I of US2008 / 042973; I, II, III, or IV of US2012 / 01287670; I or II of US2014 / 0200257; I, II, or III of US2015 / 0203446; I or III of US2015 / 0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of US2014 / 0308304; of US2013 / 0338210; I, II, III, or IV of W02009 / 132131; A of US2012 / 01011478; I or XXXV of US2012 / 0027796; XIV or XVII of US2012 / 0058144; of US2013 / 0323269; I of US2011 / 0117125; I, II, or III of US2011 / 0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US2012 / 0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US2011 / 0076335; I or II of US2006 / 008378; I of US2013 / 0123338; I or X-A-Y-Z of US2015 / 0064242;XVI, XVII, or XVIII of US2013 / 0022649; I, II, or III of US2013 / 0116307; I, II, or III of US2013 / 0116307; I or II of US2010 / 0062967; I-X of US2013 / 0189351; I of US2014 / 0039032; V of US2018 / 0028664; I of US2016 / 0317458; I of US 2013 / 0195920; 5, 6, or 10 of US10,221,127; III-3 of WO2018 / 081480; I-5 or I-8 of WO 2020 / 081938; 18 or 25 of US9,867,888; A of US2019 / 0136231; II of WO 2020 / 219876; 1 of US2012 / 0027803; OF-02 of US2019 / 0240349; 23 of US10,086,013; 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;

[0475] In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,3lZ)-heptatriaconta-6,9,28,3l-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO 2019051289 A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is lipid ATX-002, e.g., as described in Example 10 of WO 2019051289 A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is (l3Z,l6Z)-A,A-dimethyl-3-nonyldocosa-13,16-dien-1-amine (Compound 32), e.g., 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, e.g., as described in Example 12 of WO 2019051289A9 (incorporated herein by reference in its entirety).

[0476] Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), l8-l-trans PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dierucoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, or mixtures thereof. It should be understood that other diacyl phosphatidylcholines and diacyl phosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having a C10-C24 carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. 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 (such as DGTS) that have been found to improve hepatic transfection with mRNA.

[0477] Other examples of non-cationic lipids suitable for use in lipid nanoparticles include, but are not limited to, non-phospholipids such as stearylamine, dodecylamine, cetylamine, acetyl palmitate, glyceryl ricinoleate, cetyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfates, polyethoxylated fatty acid amides, didodecyldimethylammonium bromide, ceramides, sphingomyelin, and the like. Other non-cationic lipids are described in WO2017 / 099823 or U.S. Patent Publication US2018 / 0028664, the contents of which are incorporated herein by reference in their entirety.

[0478] In some embodiments, the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US 2018 / 0028664 incorporated herein by reference in its entirety. The non-cationic lipid can be, for example, 0 - 30% (mol) of the total lipids present in the lipid nanoparticle. In some embodiments, the non-cationic lipid content is 5% - 20% (mol) or 10% - 15% (mol) of the total lipids present in the lipid nanoparticle. In an embodiment, the molar ratio of ionizable lipid to neutral lipid is from 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).

[0479] In some embodiments, the lipid nanoparticle does not contain any phospholipids.

[0480] In some aspects, the lipid nanoparticle can further comprise a component such as a sterol to provide membrane integrity. An exemplary sterol useful in lipid nanoparticles is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog, e.g., cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT Publication WO2009 / 127060 and U.S. Patent Publication US2010 / 0130588, each of which is incorporated herein by reference in its entirety.

[0481] In some embodiments, a component that provides membrane integrity, such as a sterol, can be 0 - 50% (mol) of the total lipids present in the lipid nanoparticle (e.g., 0 - 10%, 10% - 20%, 20% - 30%, 30% - 40%, or 40% - 50%). In some embodiments, such a component is 20% - 50% (mol), 30% - 40% (mol) of the total lipid content of the lipid nanoparticle.

[0482] In some embodiments, the lipid nanoparticles may comprise polyethylene glycol (PEG) or conjugated lipid molecules. Generally, these are used to inhibit aggregation of the lipid nanoparticles and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, such as (methoxypolyethylene glycol) conjugated lipid.

[0483] Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), polyethylene glycolated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, sodium N-(carbonyl-methoxypolyethylene glycol 2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine, or mixtures thereof. Additional exemplary PEG-lipid conjugates are described, for example, in US 5,885,6l3, US 6,287,59l,

[0484] Described in US2003 / 0077829, US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US 2011 / 0117125, US2010 / 0130588, US2016 / 0376224, US2017 / 0119904, and US / 099823, the content of all of which is incorporated herein by reference in its entirety. In some embodiments, the PEG-lipid is a compound of Formula III, III-a-I, III-a-2, III-b-1, III-b-2, or V of US2018 / 0028664, the content of which is incorporated herein by reference in its entirety. In some embodiments, the PEG-lipid has Formula II of US20150376115 or US 2016 / 0376224, the content of both of which is incorporated herein by reference in its entirety. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauroxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of the following: PEG-DMG, PEG-dilauroylglycerol, PEG-dipalmitoyl glycerol, PEG-distearoyl glycerol, PEG-dilauroyl glycerol amide, PEG-dimyristoyl glycerol amide, PEG-dipalmitoyl glycerol amide, PEG-distearoyl glycerol amide, PEG-cholesterol (l-[8'-(cholest-5-en-3[β]-yloxy)formamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-ditetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG and 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:

[0485] and

[0486] In some embodiments, lipids conjugated to molecules other than PEG can also be used in place of the PEG-lipid. 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 the PEG-lipid or in combination with the PEG-lipid.

[0487] Exemplary conjugated lipids, namely 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, the contents of all of which are incorporated herein by reference in their entirety.

[0488] In some embodiments, PEG or conjugated lipids can account for 0-20% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the content of PEG or conjugated lipids is 0.5%-10% or 2%-5% (mol) of the total lipids present in the lipid nanoparticles. The molar ratios of ionizable lipids, non-cationic lipids, sterols, and PEG / conjugated lipids can vary as needed. For example, the lipid particles can contain 30%-70% of ionizable lipids by mole or total weight of the composition, 0-60% of cholesterol by mole or total weight of the composition, 0-30% of non-cationic lipids by mole or total weight of the composition, and 1%-10% of conjugated lipids by mole or total weight of the composition. Preferably, the composition contains 30%-40% of ionizable lipids by mole or total weight of the composition, 40%-50% of cholesterol by mole or total weight of the composition, and 10%-20% of non-cationic lipids by mole or total weight of the composition. In some other embodiments, the composition is 50%-75% of ionizable lipids by mole or total weight of the composition, 20%-40% of cholesterol by mole or total weight of the composition, 5% to 10% of non-cationic lipids by mole or total weight of the composition, and 1%-10% of conjugated lipids by mole or total weight of the composition. The composition can contain 60%-70% of ionizable lipids by mole or total weight of the composition, 25%-35% of cholesterol by mole or total weight of the composition, and 5%-10% of non-cationic lipids by mole or total weight of the composition. The composition can also contain up to 90% of ionizable lipids by mole or total weight of the composition and 2% to 15% of non-cationic lipids by mole or total weight of the composition.The formulation can also be a lipid nanoparticle formulation, for example, containing 8%-30% ionizable lipid, 5%-30% non-cationic lipid, and 0-20% cholesterol, by mole or total weight of the composition; 4%-25% ionizable lipid, 4%-25% non-cationic lipid, 2% to 25% cholesterol, 10% to 35% conjugated lipid, and 5% cholesterol, by mole or total weight of the composition; or 2%-30% ionizable lipid, 2%-30% non-cationic lipid, 1% to 15% cholesterol, 2% to 35% conjugated lipid, and 1%-20% cholesterol, by mole or total weight of the composition; or even up to 90% ionizable lipid and 2%-10% non-cationic lipid, by mole or total weight of the composition, or even 100% cationic lipid, by mole or total weight of the composition. In some embodiments, the lipid particle formulation contains an ionizable lipid, a phospholipid, cholesterol, and a polyethylene glycolated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation contains an ionizable lipid, cholesterol, and a polyethylene glycolated lipid in a molar ratio of 60:38.5:1.5.

[0489] In some embodiments, the lipid particle contains an ionizable lipid, a non-cationic lipid (such as a phospholipid), a sterol (such as cholesterol), and a polyethylene glycolated lipid, wherein the lipid molar ratio of the ionizable lipid ranges from 20 to 70 mol%, with a target of 40-60 mol%, the molar percentage of the non-cationic lipid ranges from 0 to 30 mol%, with a target of 0 to 15 mol%, the molar percentage of the sterol ranges from 20 to 70 mol%, with a target of 30 to 50 mol%, and the molar percentage of the polyethylene glycolated lipid ranges from 1 to 6 mol%, with a target of 2 to 5 mol%.

[0490] In some embodiments, the lipid particle contains an ionizable lipid / non-cationic lipid / sterol / conjugated lipid in a molar ratio of 50:10:38.5:1.5.

[0491] In one aspect, the present disclosure provides a lipid nanoparticle formulation comprising a phospholipid, lecithin, phosphatidylcholine, and phosphatidylethanolamine.

[0492] In some embodiments, one or more additional compounds may also be included. Those compounds may be administered alone, or the additional compounds may be included in the lipid nanoparticles of the present invention. In other words, in addition to the nucleic acid or at least the second nucleic acid, the lipid nanoparticles may contain other compounds different from the first nucleic acid. Non-limitingly, the 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, their peptide analogs and derivatives, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made of biomaterials, or any combination thereof.

[0493] In some embodiments, LNPs are directed to specific tissues by adding an LNP targeting domain. For example, a biologic ligand can be displayed on the surface of the LNP to enhance interaction with cells that display the cognate receptor, thereby facilitating association with the tissue expressing the receptor on the cell and delivery of the cargo therein. In some embodiments, the biologic ligand can be a ligand that drives delivery to the liver, such as an LNP displaying GalNAc that promotes delivery of a nucleic acid cargo to hepatocytes that display the asialoglycoprotein receptor (ASGPR). The work of Akinc et al., Mol Ther [Molecular Therapy] 18(7):1357-1364 (2010) teaches conjugating a trivalent GalNAc ligand to a PEG-lipid (GalNAc-PEG-DSG) to produce an ASGPR-dependent LNP to obtain an observable LNP cargo effect (see, e.g., FIG. 6 of Akinc et al., 2010, ibid).Other LNP formulations that display ligands, such as formulations incorporating folic acid, transferrin, or antibodies, are discussed in WO 2017223135, which is hereby incorporated by reference in its entirety, as well as the references used therein, which are also incorporated herein: 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.

[0494] In some embodiments, LNPs are selected for tissue-specific activity by adding a Selective ORgan Targeting (SORT) molecule to a formulation comprising conventional components such as ionizable cationic lipids, amphiphilic phospholipids, cholesterol, and poly(ethylene glycol) (PEG). The teachings of Cheng et al., Nat Nanotechnol 15(4):313-320 (2020) demonstrate that the addition of a supplemental "SORT" component can precisely alter the in vivo RNA delivery profile and mediate tissue-specific (e.g., lung, liver, spleen) gene delivery and editing, depending on the percentage and biophysical properties of the SORT molecule.

[0495] In some embodiments, the LNP comprises a biodegradable ionizable lipid. In some embodiments, the LNP comprises (9Z,12Z)-3-(((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also known as 3-(((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-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 lipids of the references provided therein. 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 the pH.

[0496] In some embodiments, the average LNP diameter of the LNP formulation can be between tens of nm and hundreds of nm, e.g., as measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation can be from 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 can be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation can be from about 70 nm to about 100 nm. In certain embodiments, the average LNP diameter of the LNP formulation can 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 range of the LNP formulation is from about 1 mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, or from about 38 mm to about 42 mm.

[0497] In some cases, the LNP can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of the LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The polydispersity index of the LNP can be from 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 LNP can be from about 0.10 to about 0.20.

[0498] The ζ potential of the LNP can be used to indicate the electrokinetic potential of the composition. In some embodiments, the ζ potential can describe the surface charge of the LNP. Lipid nanoparticles with relatively low charge (positive or negative) are generally desirable because more highly charged substances may undesirably interact with cells, tissues, and other elements in the body. In some embodiments, the ζ potential of the LNP can be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0499] The encapsulation efficiency of the protein and / or nucleic acid describes the amount of protein and / or nucleic acid that is encapsulated or otherwise associated with the LNP after preparation relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing lipid nanoparticles before and after disrupting the lipid nanoparticles with one or more organic solvents or detergents. Anion exchange resins can be used to measure the amount of free protein or nucleic acid (e.g., RNA) in the solution. Fluorescence can be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in the solution. For the lipid nanoparticles described herein, the encapsulation efficiency of the protein and / or nucleic acid 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%.

[0500] The LNP can optionally include one or more coatings. In some embodiments, the LNP can be formulated in a capsule, film, or tablet having a coating. Capsules, films, or tablets containing the compositions described herein can have any available size, tensile strength, hardness, or density.

[0501] Additional exemplary lipids, formulations, methods, and LNP characterizations are taught by WO 2020061457, which is incorporated herein by reference in its entirety.

[0502] In some embodiments, in vitro or ex vivo cell liposome transfection is performed using Lipofectamine MessengerMax (ThermoFisher) or TransIT-mRNA transfection reagent (Mirus Bio). In certain embodiments, LNPs are formulated using the GenVoy_ILM ionizable lipid mixture (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or dilinoleyldimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl [Angewandte Chemie International Edition in English] 51(34):8529-8533 (2012), which is incorporated herein by reference in its entirety.

[0503] Optimized LNP formulations for delivering CRISPR-Cas systems (such as Cas9-gRNA RNP, gRNA, Cas9 mRNA) are described in WO 2019067992 and WO 2019067910, both of which are incorporated by reference.

[0504] Additional specific LNP formulations useful for delivering nucleic acids are described in US 8158601 and US 8168775, both of which are incorporated by reference, which include the formulation sold under the name ONPATTRO used in patisiran.

[0505] Exemplary doses of LNPs containing the 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 administrations of AAVs containing nucleic acids encoding one or more components of the system may include an MOI of about 1011, 1012, 1013, and 1014 vg / kg.

[0506] In some embodiments, the invention includes lipid nanoparticles (LNPs) that contain an ANDbody polypeptide (or RNA encoding it) as described herein, a nucleic acid molecule, or DNA encoding an ANDbody as described herein. In embodiments, the LNP contains a cationic lipid. In some embodiments, the LNP further contains 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 a polyethylene glycolylated lipid, such as PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or a PEG dialkoxypropyl carbamate. In some embodiments, the cationic lipid of the LNP has a structure according to the following:

[0507] (i),

[0508] (ii), or

[0509] (iii).

[0510] For a review of LNPs, see also Li et al. 2017, Nanomaterials 7, 122; doi:10.3390 / nano7060122.

[0511] Other carriers

[0512] Viral vector

[0513] 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.

[0514] Examples of viral vectors include retroviruses (e.g., retroviridae viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses (such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus)), positive-strand RNA viruses (such as picornaviruses and alphaviruses), and double-stranded DNA viruses (including adenoviruses, herpesviruses (e.g., herpes simplex virus type 1 and 2, Epstein-Barr virus, cytomegalovirus, replication-deficient herpesviruses), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, 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-type virus, mammalian C-type virus, B-type virus, D-type virus, oncovirus, HTLV-BLV group, lentivirus, alpha-retrovirus, gamma-retrovirus, foamy virus (Coffin, J.M., Retroviridae: The viruses and their replication, Virology (Third Edition) 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 leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in U.S. Patent No. 5,801,030, which is incorporated herein by reference.

[0515] Circovirus vectors can also be used to deliver the ANDbody compositions described herein. Circovirus 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 circovirus vector composition comprises a genomic element comprising a promoter operably linked to a nucleic acid sequence encoding an ANDbody as described herein, the genetic element being externally encapsulated by a protein comprising the circovirus ORF1, such as the circovirus capsid protein.

[0516] Cell- and vesicle-based carriers

[0517] The compositions described herein (e.g., polypeptide or RNA ANDbody compositions) described herein can be administered to cells, vesicles, or cells in 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 single or multiple lipid bilayers surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be anionic, neutral, or cationic. Liposomes are biocompatible, non-toxic, can deliver hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood-brain barrier (BBB) (for a review, see, e.g., Spuch and Navarro, Journal of Drug Delivery, Volume 2011, Article ID 469679, page 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 vesicle lipids are known in the art (see, e.g., U.S. Patent No. 6,693,086, the teachings of which regarding the preparation of multilamellar vesicle lipids are incorporated herein by reference). Although vesicle formation can be spontaneous when lipid membranes are mixed with aqueous solutions, it can also be accelerated by applying force in the form of oscillations via the use of a homogenizer, sonicator, or extrusion device (for a review, see, e.g., Spuch and Navarro, Journal of Drug Delivery, Volume 2011, Article ID 469679, page 12, 2011. doi:10.1155 / 2011 / 469679). Extruded lipids can be prepared by extrusion through a filter with a reduced size, as described in Templeton et al., Nature Biotech, 15:647-652, 1997, the teachings of which regarding the preparation of extruded lipids are incorporated herein by reference).

[0518] Exosomes can also be used as drug delivery mediators for the compositions and systems described herein. For a review, see Ha et al. July 2016. Acta Pharmaceutica Sinica B, Volume 6, Issue 4, pages 287-296; https: / / doi.org / 10.1016 / j.apsb.2016.02.001.

[0519] In vitro differentiated red blood cells can also be used as carriers for the agents (e.g., inhibitors) described herein, such as antibodies or nucleic acids described herein. See, e.g., WO 2015073587; WO 2017123646; WO 2017123644; WO2018102740; WO 2016183482; WO 2015153102; WO 2018151829; WO 2018009838; Shi et al. 2014.Proc NatlAcad Sci USA. [Proceedings of the National Academy of Sciences of the United States of America] 111(28):10131–10136; U.S. Patent 9,644,180; Huang et al. 2017.Nature Communications [Nature Communications] 8:423; Shi et al. 2014.ProcNatlAcad Sci USA. [Proceedings of the National Academy of Sciences of the United States of America] 111(28):10131–10136.

[0520] Fusion body compositions, e.g., as described in WO 2018208728, can also be used as carriers to deliver the [agents] or formulations described herein.

[0521] Plant nanovesicles and plant messenger packages (PMPs), e.g., as described in WO 2011097480, WO 2013070324, WO 2017004526, or WO 2020041784, can also be used as carriers to deliver the compositions described herein.

[0522] Without further elaboration, it is believed that one of ordinary skill in the art can, to the fullest extent, utilize the present invention based on the above description. Accordingly, the following specific examples will be interpreted as merely illustrative and in no way limit the remainder of the disclosure. For purposes of citation or subject matter herein, all publications and sections thereof cited herein are hereby incorporated by reference.

[0523] Examples

[0524] The present invention will be further illustrated in the following non-limiting examples.

[0525] Table of Contents

[0526] Example 1 ANDBODY binds to murine and human RAGE and NOTCH2 Example 2 ANDBODY binds to murine and human UMOD and NOTCH2 Example 3 ANDBODY binds to murine and human MEP1B and NOTCH2 Example 4 ANDBODY binds to murine and human RAGE and IL11RA Example 5 ANDBODY binds to murine and human UMOD and IL11RA Example 6 ANDBODY binds to murine and human MEP1B and IL11RA Example 7 Exemplary skin address-restricted binder Example 8 Exemplary lung address-restricted binder Example 9 Exemplary kidney address-restricted binder Example 10 Exemplary intestine address-restricted binder Example 11 Predicted address tissue restriction Example 12 Production and use of ANDBODY Example 13 Production and use of ANDBODY Example 14 TNFA-blocking molecule conjugated with DSG1 targeting moiety

[0527] Example 1. ANDBODY Binds to Mouse and Human RAGE and NOTCH2

[0528] 1.1 Vaccination to Generate Anti-RAGE Antibodies

[0529] Antibodies against the extracellular domain of human RAGE (an exemplary addressed target of the present technology) were generated by immunization. The extracellular domain of human RAGE (NCBI protein accession number Q15109 positions N24 - A344) (huRAGE) fused to the human IgG1 Fc region (UniProt ID P01857 positions P100 - K330) was expressed in HEK293F cells. Briefly, the DNA sequence was codon - optimized for mammalian expression and ordered in the pcDNA3.4 - TOPO expression vector (ThermoFisher Scientific). The protein was transiently transfected into HEK293 cells and purified using rProtein A Sepharose Fast Flow resin according to the manufacturer's instructions (GE Healthcare) similar to the prior art method (Rothschilds et al. 2019). Female BALB / c mice were immunized intraperitoneally with 50 μg of huRAGE - Fc fusion protein in CFA / IFA adjuvants (Millipore Sigma, catalog numbers F5881 - 10ML and F5506 - 10ML). Subsequently, hybridomas were generated (Listek et al. 2020). Clones with specific IgG reactivity against the huRAGE - Fc fusion protein used for immunization were first screened in ELISA format, and then flow cytometry studies were performed using cells stably (CHO) or transiently (HEK293F) transfected with full - length huRAGE. Next, anti - RAGE hybridoma clones were evaluated for murine cross - reactivity. Flow cytometry studies were performed using cells stably (CHO) or transiently (HEK293F) transfected with full - length murine RAGE (mRAGE), and clones that bound to mRAGE were selected. Then, positive clones expressing anti - RAGE mAbs with cross - reactivity between humans and mice were further purified by limiting dilution cloning. Hybridomas were grown in DMEM / 2% ultra - low IgG serum and the mAbs were purified by protein G chromatography according to the manufacturer's instructions (Millipore Sigma, P3296 - 1ML).

[0530] 1.2 Selection of inert anti - RAGE antibodies

[0531] The address target binding site of the present technology is designed not to affect signal transduction after binding to an address target (e.g., an exemplary RAGE address target). Thus, anti-RAGE hybridoma clones generated as described above that do not block RAGE ligand binding are further evaluated. Human RAGE ligands tested included HMGB1 (full-length, from Creative BioMart catalog number HMGB1-29332TH), advanced glycation end products (fused to bovine serum albumin, Millipore Sigma catalog number 121800-10MG-M), S100A12 (full-length, from R&D Systems catalog number 1052-ER-050), S100A1 (full-length, from R&D Systems catalog number 9705-S1-100), S100A4 (R&D Systems catalog number 4137-S4-050), S100A10 (full-length, from Creative BioMart catalog number S100A10-157H), S100A11 (R&D Systems catalog number 9015-S11-050), S100A13 (R&D Systems catalog number 4327-SA-050), S100B (R&D Systems catalog number 1820-SB-050), amyloid-β-peptide (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA, from NM_000484.2, Millipore Sigma catalog number AG912-1MG), and Mac-1 (F17-N1105 from NP_001139280 and Q23-N700 from UniProt P05107, R&D Systems catalog number 4047-AM-050). ELISA was used to quantify the binding ability of the ligands in the presence of anti-RAGE antibodies. HuRAGE-Fc was adsorbed onto ELISA plates and then, after blocking, the plates were incubated with anti-RAGE antibody clones from the hybridomas at concentrations ranging from 10 fM to 10 μM (one condition per concentration and per clone). After washing the plates, each ligand was biotinylated according to the manufacturer's instructions (ThermoFisher product catalog number 21435) and then incubated on the plates at concentrations ranging from 10 fM to 10 μM. After washing, SA-conjugated HRP secondary antibody was added, followed by TMB substrate and colorimetric readings were quantified by absorbance. Ligand binding within a given ligand was compared in the presence of anti-RAGE antibodies versus in the absence of anti-RAGE antibodies to isolate anti-RAGE clones that are inert and do not affect the binding of one or more ligands.

[0532] The ability of anti-RAGE antibodies to inhibit IFNα-induced gene signatures was also evaluated, and anti-RAGE hybridoma clones that do not alter cell signaling were selected based on IFNα-induced gene signature assays. PBMCs from healthy human donors were stimulated with 50% serum from SLE patients for 4 hours. The assays were done in the presence of anti-RAGE antibodies or an irrelevant (negative) isotype control human IgG1 antibody (Bio X Cell catalog number BE0297) at antibody concentrations ranging from 10 fM to 10 μM. In addition, a huRAGE-Fc fusion molecule was used as a positive control. Total RNA was purified and the expression of type I IFN-induced genes (including DDX58, G1P2, MX1, OAS3, RSAD2, IFIT1, IFI35) was measured by real-time qRT-PCR analysis as described in the prior art method (WO 2008 / 137552 A2, https: / / patentimages.storage.googleapis.com / 94 / 26 / c8 / 7b9f27f693c4b6 / WO2008137552A2.pdf). The inhibition values of gene expression were normalized against the negative control Ab.

[0533] 1.3 Vaccination to generate anti-Notch2 antibodies

[0534] Antibodies against the extracellular domain of human Notch2 (huNotch2), which is fused to the Fc region of human IgG1 (an exemplary effector target of the present technology), were generated by immunization similar to that of RAGE described above. After immunization and hybridoma generation, screening of the clones was performed in exactly the same manner as above, but binding to full-length human and mouse Notch2 (instead of RAGE). Then the positive clones expressing anti-Notch2 mAbs with cross-reactivity between human and mouse were further purified by limited dilution cloning. The hybridomas were grown in DMEM / 2% ultra-low IgG serum and the mAbs were purified by protein G chromatography.

[0535] 1.4 Select active anti-Notch2 antibodies in a wide IC 50 range

[0536] The effector target binding sites of the present technology, such as Notch2, are designed to not affect signal transduction after binding to the effector target, unless they are targeted to the target tissue through an address target binding site, such as RAGE. Thus, the binding affinity of the effector target (e.g., Notch2) binding site was analyzed. In particular, flow cytometry was used to evaluate the IC 50 of Notch2 antibodies for the ligand human Jagged-2-Fc fusion protein (Creative BioMart, JAG2-382H) that binds to surface Notch2, to select the IC50 Antibodies in the range of less than 1 nM to 5 μM. According to the manufacturer's instructions (Thermo Fisher Scientific, A20186) and the method described previously (Tzeng et al., 2015), Jagged-2-Fc was labeled with Alexa flor 647 (AF647).

[0537] HEK293F cells were transiently transfected with full-length huNotch2. The cells were incubated with anti-Notch2 antibody at concentrations increasing from 1 pM to 50 μM. Subsequently (without washing the cells), the cells were incubated with a constant concentration of AF647-labeled Jagged-2-Fc at 4 °C for 1 hour, with concentration ranges (for different IC 50 assays) from 1 pM to 50 μM. For each IC 50 assay, a constant concentration of AF647 Jagged-2-Fc and different anti-Notch2 were selected. The binding of AF647 Jagged-2-Fc to increasing concentrations of anti-Notch2 antibody on the cells was quantified by flow cytometry using Thermo Fisher Scientific AttuneNxT (B2R3Y3V6).

[0538] 1.5 Expression and purification of ANDbody as a bispecific

[0539] Using In-Fusion HD cloning (Takara Bio, catalog number 638911), the DNA sequences of 10 RAGE antibodies and 10 Notch2 antibodies in the range of IC 50 (from <1 nM to 5 μM) were cloned into the human IgG1 framework (with a single matching point mutation in the Fc region of the CH3 domain) according to the "controlled Fab-arm exchange" (cFAE) method (Labrijn et al., 2014). After expressing the antibodies separately from transient HEK293 expression and purifying each antibody using protein A affinity resin, the parental antibodies (a combination of 1 RAGE antibody and 1 Notch2 antibody) were made into bispecific RAGE / Notch2 ANDbody according to the cFAE method. Briefly, the parental antibodies were mixed under permissive redox conditions to achieve recombination of half-molecules. Subsequently, the reducing agent was removed to allow reoxidation of interchain disulfide bonds. Finally, the exchange efficiency was quantified using chromatography-based or mass spectrometry-based methods. Approximately 100 variants of RAGExNotch2 ANDbody were made.

[0540] 1.6 Affinity of ANDbody variants

[0541] To identify ANDbody that meets the desired effector target and meets the address target binding affinity criteria TMVariants, SPR-based affinity measurements were performed at 25 °C on a BIAcore model 2000 or T100 (Biacore / GE Healthcare, Piscataway, NJ) using HBS-EP+ buffer (Cytiva catalog number BR100669) containing 0.1 mg / ml BSA (Millipore Sigma catalog number A9418) as the running buffer. Sensor chip Protein A (Cytiva catalog number 29127557) was used to capture mouse RAGE-Fc, human RAGE-Fc, mouse Notch2-Fc or human Notch2-Fc. ANDbody was injected in a 3-fold dilution series from 60 to 0.74 nM, and the dissociation of all proteins was monitored for 10 minutes. Kinetic analysis was done by simultaneously fitting the association and dissociation phases of the sensorgrams using a 1:1 Langmuir binding model in the BIAevaluation software (Biacore) provided by the manufacturer. A dual reference was applied in each analysis to subtract the background responses of the reference surface and the buffer-only control.

[0542] This assay can quantitatively evaluate the affinity of each ANDbody variant for RAGE and Notch2. ANDbody variants with higher affinity for RAGE than for Notch2, as well as variants with no affinity difference or higher affinity for Notch2, were used for subsequent in vitro and in vivo experiments.

[0543] 1.7 In vitro assay of Notch2 antagonism on cells with or without RAGE expression

[0544] To analyze ANDbody characteristics, in vitro, HEK293F cells were transiently transfected with full-length huRAGE (R+), full-length huNotch2 (N+), or co-transfected with both (RN+). According to the manufacturer's instructions (Thermo Fisher Scientific, A20186) and previously described methods (Tzeng et al. 2015), many variant RAGExNotch2 ANDbodies were fluorophore-labeled with Alexa flor 647 (AF647). Subsequently, the ANDbodies were incubated with R+, N+, RN+, or a combination of R+ plus N+ cells at ANDbody concentrations ranging from 10 fM to 10 μM. According to the manufacturer's instructions (Thermo Fisher Scientific, 53027), the parental anti-Notch2 monospecific antibody (one for each variant) was fluorophore-labeled with FITC. Under certain conditions, the parental anti-Notch2 FITC-labeled antibody was incubated with cells pre-bound with AF647 RAGExNotch2 (matching Notch2 variant) at concentrations ranging from 10 fM to 10 μM to saturate the remaining binding sites of Notch2. Flow cytometry was used to quantify the binding EC 50 of the AF647-labeled RAGExNotch2 ANDbody variants and the parental FITC-labeled parental anti-Notch2 antibody. When the FITC Notch2 antibody was added after AF647 RAGExNotch2, the FITC signal from these cells was subtracted from the FITC signal of FITC Notch2 alone on the cells (and then normalized against the signal of Notch2 alone) to quantify the % Notch2 bound by RAGExNotch2. These numbers at different concentrations of the RAGExNotch2 ANDbody were used to create an EC 50 curve. The assay was also performed using an AF647-labeled anti-Notch2 antibody in place of the AF647-labeled ANDbody.

[0545] The difference in the EC 50 observed on N+ cells compared to RN+ cells indicates enhanced Notch2 blockade when RAGE is present and identifies cells expressing the RAGExNotch2 ANDbody.

[0546] 1.8 Biodistribution of ANDbody and parental antibodies (in vivo)

[0547] To analyze ANDbody distribution, in vivo, the biodistribution of the RAGExNotch2 ANDbody and each parental antibody (anti-Notch2 or anti-RAGE used for cFAE of the ANDbody) was quantified in female Balb / c and C57BL / 6 mice.

[0548] For quantitative cell biodistribution, proteins (ANDbody and antibody) were labeled individually with AF647 according to the manufacturer's instructions (Thermo Fisher Scientific, A20186) and the method described previously (Tzeng et al., 2015). Then, each labeled antibody was injected individually at doses of 10 μg, 100 μg, and 500 μg IV (tail vein). An equal volume of saline (PBS) was also injected as a control.

[0549] For cell biodistribution, at time points of 12 hours, 1 day, 2 days, 3 days, 7 days, and 14 days after injection, mice were euthanized with CO2, and tissues including heart, lung, spleen, blood, kidney, liver, and intestine were processed into single-cell suspensions according to the method described previously (Tzeng et al., 2015). Briefly, blood was collected into EDTA-treated tubes (BD catalog number 365974) by cardiac puncture, and the other tissues were harvested, weighed, mechanically dissociated between frosted glass slides, and filtered through a 70-μm mesh (Millipore Sigma, catalog number CLS431751-50EA) to make single-cell suspensions. Spleen cells, 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 according to a previous method (Covarrubias et al., 2019). Flow cytometry of immune cells was performed as described previously (Tzeng et al., 2015) 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 cells (CD326+CD31-CD45-), endothelial cells (CD326-CD31+CD45-), and hematopoietic lineages (CD326-CD31-CD45+) were also analyzed according to previous definitions (Singer et al., 2016). Antibodies were purchased from Biolegend, and flow cytometry was run on a Thermo Fisher Scientific Attune NxT (B2R3Y3V6). The presence of ANDbody or other labeled antibodies on the cell surface was defined by the fluorescence of AF647 (and this fluorophore was avoided in the flow group).

[0550] For quantitative tissue biodistribution, proteins (ANDbody and antibody) were first labeled separately with NHS-5 / 6-FAM (Thermo Fisher Scientific, catalog number 46409) according to the manufacturer's instructions.

[0551] For tissue biodistribution, at time points of 12 hours, 1 day, 2 days, 3 days, 7 days, and 14 days post-injection, mice were euthanized with CO2, and tissues including lung, spleen, blood, kidney, liver, and intestine were harvested, weighed, and imaged on an IVIS Spectrum imaging system (Caliper Life Sciences; excitation, 500 nm; emission, 540 nm). The images were analyzed using Living Image software.

[0552] 1.9 Quantitative in vivo bioactivity Gene expression changes

[0553] To analyze ANDbody activity, in vivo, female Balb / c and C57BL / 6 mice were used to quantify bioactivity. To quantify bioactivity across tissues, RAGExNotch2 ANDbody or each of its respective parental antibodies (anti-Notch2 or anti-RAGE used for cFAE against ANDbody) were injected at doses of 10 μg, 100 μg, and 500 μg IV (tail vein). An equal volume of saline (PBS) was also injected as a control.

[0554] At time points of 12 hours, 1 day, 2 days, 3 days, 7 days, and 14 days post-injection, tissues such as lung, spleen, blood, kidney, liver, heart, and intestine were processed into single-cell suspensions according to the method described previously (Tzeng et al., 2015). Briefly, blood was collected by cardiac puncture into EDTA-treated tubes (BD catalog number 365974), and other tissues were harvested, weighed, mechanically separated between frosted glass slides, and filtered through a 70-μm mesh (Millipore Sigma, catalog number CLS431751-50EA) to make single-cell suspensions. Spleen cells and whole blood were treated with ammonium chloride potassium (ACK) lysis buffer (Thermo Fisher Scientific catalog number A1049201).

[0555] qRT-PCR was performed using a previously described method (Nandagopal et al., 2018). RNA was prepared using the RNeasy kit (QIAGEN). cDNA was prepared from 500 ng of RNA using the iScript cDNA Synthesis kit (Bio-Rad). 0.5 μL of cDNA was used per 10 μL RT-qPCR reaction mixture, which contained 1X iQ SYBR Green Supermix (Bio-Rad) and 450 nM total forward and reverse primers. Reactions were carried out on a Bio-Rad CFX Real-Time PCR Detection System using a two-step amplification protocol with the following thermal cycling parameters: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s (melting) and 55 °C for 30 s (annealing + extension). All reactions were performed in duplicate.

[0556] Genes associated with Notch2 signaling were mouse Hes1, Hey1, and HeyL, and the reference gene was SdhA. Primers used for amplification were the previously described (Nandagopal et al., 2018) mouse Hes1 primer set (forward, 5'-CAACACGACACCGGACAAAC-3' and reverse, 5'-AAGAATAAATGAAAGTCTAAGCCAA-3'), mouse Hey1 primer set (forward, 5'-GCCGAAGTTG CCCGTTATCT-3' and reverse, 5′-CGCTGGGATG CGTAGTTGTT-3′), mouse HeyL primer set (forward, 5′-GAGCTGAC TTCCCACAACCA-3′ and reverse, 5′-GAGAGGTGCCTTTGCGTAGA-3′), and mouse SdhA primer set (forward, 5′-AGTGGGCTGTCTTCCTTAAC-3' and reverse, 5'-GGATTGCTTCT GTTTGCTTGG-3'). All primers were purchased from IDT DNA.

[0557] Hes1, Hey1, and HeyL gene expression was measured in ANDbody-treated mice, untreated mice, and anti-Notch2-treated mice (including in the lungs).

[0558] 1.10 In vivo bioactivity using weight and histology

[0559] Female Balb / c and C57BL / 6 mice were used for histological examination of organs such as spleen, kidney, liver, heart, intestine, teeth, and lung to compare pathology with ANDbody treatment, anti-Notch2 alone or saline (PBS) treatment. Starting at 8 weeks of age, 10 μg, 100 μg, and 500 μg of ANDbody or the corresponding Notch2 antibody (before cFAE) were injected IV (tail vein) 1x or 2x per week. Equal volumes of 1x or 2x saline (PBS) were also injected weekly as a control. Mice were weighed 2x per week starting before the first treatment. After 2, 4, and 6 weeks of treatment, the mice were euthanized and the organs were processed histologically.

[0560] Unless otherwise stated, the organs were transferred to cassettes and then placed directly into 10% neutral buffered formalin (Sigma-Aldrich) for 12 - 24 hours and then embedded in paraffin. The lungs were perfused with 10% neutral buffered formalin before being placed in cassettes and soaked in neutral buffered formalin. The intestine was rinsed thoroughly and then placed in cassettes and soaked in 10% neutral buffered formalin. Paraffin sections (1 - 2 μm) were cut and deparaffinized before histochemical staining. The sections were stained with hematoxylin / eosin (H&E; Merck, Darmstadt, Germany) and blindly scored based on immune infiltration and tissue morphology.

[0561] In addition to lung morphology, weight loss (or not) of ANDbody-treated mice during the treatment was compared with weight loss (or not) of anti-Notch2-treated mice and weight loss (or not) of untreated mice.

[0562] Example 2. ANDbody Binding to Mouse and Human UMOD and NOTCH2

[0563] 2.1 Yeast Surface Display to Generate Anti-UMOD Antibodies

[0564] Yeast surface display (Chao et al., 2006) was used to engineer antibodies against murine UMOD (Creative BioMart, catalog number UMOD-17835M, untagged) (an exemplary address target of the present technology). This was accomplished by using the method described previously (Angelini et al., 2015) and summarized below. Yeast display began with a synthetic antibody library from the Sidhu laboratory, which was based on the natural framework library "G" (Van Deventer et al., 2015). ScFvs displayed on the yeast surface were selected for binding to murine UMOD. Subsequently, sorting could be performed against the human UMOD antigen (Creative BioMart, catalog number UMOD-001H, untagged) such that the binders could cross-react between human and murine forms. To increase the affinity of the scFv binders, affinity maturation was performed using error-prone PCR as described previously (Angelini et al., 2015), and the resulting library was re-sorted for binding to murine and human UMOD. After engineering, many scFvs with multispecies cross-reactivity were cloned back into the human IgG1 antibody form.

[0565] 2.2 Selection of inert anti-UMOD antibodies

[0566] The address target binding site of the present technology was designed not to affect signal transduction after binding to the address target (e.g., the exemplary UMOD address target). Thus, in assays such as those described above for RAGE antibodies or in vivo assays, anti-UMOD antibodies were further evaluated based on their inability to block UMOD ligand binding. Inert UMOD antibodies could be identified in such assays, whereby the screened UMOD antibodies would not affect or alter renal structure.

[0567] 2.3 Vaccination to generate anti-Notch2 antibodies

[0568] Antibodies cross-reactive with murine and human Notch2 were created, cloned into the human IgG1 framework, and expressed according to the prior art methods described above.

[0569] 2.4 Selection of active Notch2 antibodies in a wide IC 50 range

[0570] As described above, antibodies against Notch2 will be selected in a wide IC 50 range.

[0571] 2.5 Expression and purification of ANDbody as a bispecific

[0572] As described above, from the IC 50DNA sequences of 10 UMOD antibodies and 10 Notch2 antibodies in the range of (from 1 nM to 5 μM) were used to make approximately 100 variant ANDbodies.

[0573] 2.6 Affinity of ANDbody variants for UMOD and Notch2

[0574] The affinity of ANDbody for UMOD and Notch2 was evaluated similarly using BIAcore as described above (as above). In this case, His-tagged UMOD of human and mouse versions was immobilized on the sensor chip NTA (Cytiva catalog number BR100034). Human and mouse Notch2-Fc were immobilized as described above.

[0575] 2.7 Biodistribution of ANDbody and parental antibodies (in vivo)

[0576] Cell and tissue biodistribution studies were performed using the method described above (as above). However, the ANDbody used in this case was UMODxNotch2, and the parental antibodies corresponded to anti-UMOD and anti-Notch2.

[0577] 2.8 Quantitative gene expression changes of in vivo biological activity

[0578] The in vivo biological activity of UMODxNotch2 ANDbody was quantified using the gene expression method described above.

[0579] 2.9 In vivo biological activity using weight and histology

[0580] The in vivo biological activity of UMODxNotch2 ANDbody was quantified using the weight and histology methods described above.

[0581] Example 3. ANDbody binds mouse and human MEP1B and NOTCH2

[0582] 3.1 Yeast surface display to generate anti-MEP1B antibodies

[0583] Yeast surface display (Chao et al., 2006) was used to engineer antibodies against mouse MEP1B (Cusabio, CSB-MP730755MO) (an exemplary target of this technology). Yeast display was performed as described above (2.1) to obtain cross-reactive mouse / human MEP1B binders (human MEP1B, Cusabio, CSB-MP618098HU). After engineering, many scFvs cross-reactive with mouse and human MEP1B were cloned into human IgG1, transiently transfected into HEK293F cells, and purified using the floor A resin as described above.

[0584] 3.2 Inoculate with a vaccine to produce anti-Notch2 antibodies

[0585] Antibodies that cross-react with murine and human Notch2 are created, cloned into the human IgG1 framework, and expressed according to the prior art methods described above.

[0586] 3.3 Select active Notch2 antibodies within a wide IC 50 range

[0587] As described above, antibodies against Notch2 are selected within a wide IC 50 range.

[0588] 3.4 Express and purify ANDbody as a bispecific

[0589] As described above, DNA sequences of 10 MEP1B antibodies and 10 Notch2 antibodies from within the IC 50 (from <1 nM to 5 μM) are made into approximately 100 variant ANDbodies.

[0590] 3.5 Affinity of ANDbody variants for MEP1B and Notch2

[0591] The affinity of ANDbody for MEP1B and Notch2 is evaluated using BIAcore similarly to the above. In this case, the His-tagged MEP1B of human and murine versions is immobilized on the sensor chip NTA (GE Healthcare catalog number BR100034). Human and murine notch2-Fc are immobilized as described above.

[0592] 3.6 Biodistribution of ANDbody and parental antibodies (in vivo)

[0593] Cell and tissue biodistribution studies are performed using the methods described above. However, the ANDbody used in this case is MEP1BxNotch2, and the parental antibodies correspond to anti-MEP1B and anti-Notch2.

[0594] 3.7 Quantitative gene expression changes of in vivo biological activity

[0595] The in vivo biological activity of MEP1BxNotch2 ANDbody is quantified using the gene expression method described above.

[0596] 3.8 In vivo biological activity using weight and histology

[0597] The in vivo biological activity of MEP1BxNotch2 ANDbody is quantified using the weight and histology methods described above.

[0598] Example 4. ANDBODY combination of murine and human RAGE and IL11RA

[0599] 4.1 Vaccination to generate anti-RAGE antibodies

[0600] The method of vaccination to generate murine / human cross-reactive anti-RAGE antibodies was described above.

[0601] 4.2 Selection of inert anti-RAGE antibodies

[0602] The method of selecting inert anti-RAGE antibodies was described above.

[0603] 4.3 Yeast surface display to generate anti-IL11Ra antibodies

[0604] Similar to above, yeast surface display was used to generate antibodies of different affinities that cross-react with murine and human IL11Ra (exemplary effector targets of the present technology). The DNA sequences encoding the extracellular domain of murine IL11Ra (positions 24 - 372 of UniProt ID Q64385) and the extracellular domain of human IL11Ra (positions 24 - 370 of UniProt ID Q14626) were codon-optimized for mammalian expression and ordered with a C-terminal His tag in a pcDNA3.4-TOPO expression vector (Thermo Fisher Scientific). The proteins were transiently transfected into HEK293F cells and purified using metal affinity resin similar to the prior art method (Rothschilds et al. 2019). These soluble recombinant murine and human IL11Ra were used as antigens for yeast surface display.

[0605] ScFvs that cross-react with murine and human IL11Ra were cloned into human IgG1, transiently transfected into HEK293F cells, and purified using the Floor A resin as described above.

[0606] 4.4 Selection of active anti-IL11Ra antibodies in a wide IC 50 range

[0607] As described above, flow cytometry was used to evaluate the IC 50 of IL11Ra antibodies against the ligand human IL11 (R&D Systems, catalog number 218-IL-025 / CF) that binds to surface IL11Ra, to select the IC 50Antibodies in the range of less than 1 nM to 5 μM. In the current example, full-length human IL11Ra was transiently transfected onto the surface of HEK293F cells. Using the method in the previous example, the IL11Ra antibody was replaced with the Notch2 antibody, and IL11 was replaced with Jagged-2-Fc.

[0608] 4.5 Expression and purification of ANDbody as a bispecific

[0609] As described above, DNA sequences of 10 RAGE antibodies and 10 IL11Ra antibodies in the range of <1 nM to 5 μM were made into approximately 100 variant ANDbodies. 50 (from <1 nM to 5 μM)

[0610] 4.6 Affinity of ANDbody variants for RAGE and IL11Ra

[0611] The affinity of ANDbody for RAGE and IL11Ra was evaluated similarly using BIAcore as described above. In this case, the His-tagged IL11Ra of human and mouse versions was immobilized on the sensor chip NTA (Cytiva catalog number BR100034), and the RAGE-Fc of human and mouse versions was captured on the sensor chip Protein A (Cytiva catalog number 29127557).

[0612] 4.7 In vitro assay of IL11Ra antagonism on cells with or without RAGE expression

[0613] This assay was performed as described above, except that full-length human IL11Ra was used instead of Notch2, and the anti-IL11Ra antibody was used instead of the anti-Notch antibody. The corresponding RAGExIL11Ra ANDbody was also used.

[0614] 4.8 Biodistribution of ANDbody and parental antibodies (in vivo)

[0615] Cell and tissue biodistribution studies were performed using the method described above. However, the ANDbody used in this case was RAGExIL11Ra, and the parental antibodies corresponded to anti-RAGE and anti-IL11Ra.

[0616] 4.9 In vivo biological activity of RAGExIL11Ra ANDbody

[0617] In response to treating mice with murine IL11, the collagen content in the ventricle and kidney both increased (Schafer et al., 2017). Therefore, the collagen content was measured to quantify the amount of IL11Ra biological activity after ANDbody treatment.

[0618] Similar to the prior art method (Schafer et al., 2017), male C57BL / 6 mice at 10 weeks of age were subcutaneously injected with 2 μg of murine IL11 or the same volume of saline daily for 21 days. Murine IL11 was recombinantly prepared by: synthesizing codon-optimized DNA using the murine IL11 sequence with a C-terminal His tag (UniProt ID P47873), performing transient transfection of HEK293F, and purifying the His-tagged IL11 with TALON resin as described above. Starting 3 days before the first IL11 injection, and then 2x weekly, IL11- and saline-treated mice received therapeutic IP injections consisting of 250 μg of ANDbody RAGExIL11Ra, the parental anti-IL11Ra alone, or an equal volume of saline (PBS).

[0619] At the end of 21 days of IL11 treatment, the mice were euthanized, and the total collagen amount in the lung, spleen, blood, kidney, liver, heart, and intestine was quantified using the Quickzyme total collagen assay kit (Quickzyme Biosciences) and a hydroxyproline-based colorimetric detection similar to the prior art method (Schafer et al., 2017).

[0620] Example 5. ANDbody Binding to Mouse and Human UMOD and IL11RA

[0621] 5.1 Yeast Surface Display to Generate Anti-UMOD Antibodies

[0622] Anti-UMOD (e.g., address target) antibodies were selected as described above and cloned into human IgG1.

[0623] 5.2 Selection of Inert Anti-UMOD Antibodies

[0624] In an assay such as described above, anti-UMOD antibodies were further evaluated based on their inability to block UMOD ligand binding.

[0625] 5.3 Yeast Surface Display to Generate Anti-IL11Ra Antibodies

[0626] As described above, the same IL11Ra antibodies generated in yeast surface display above were used here. The scFv with cross-reactivity to mouse and human IL11Ra was cloned into human IgG1, transiently transfected into HEK293F cells, and purified using the floor A resin as described above.

[0627] 5.4 Selection of Active IL11Ra Antibodies in a Wide IC 50 Range

[0628] The above method was used to select IL11Ra antibodies with different IC 50 values.

[0629] 5.5 Expression and purification of ANDbody as a bispecific

[0630] As described above, DNA sequences of 10 UMOD antibodies and 10 IL11Ra antibodies in the range of 50 (from <1 nM to 5 μM) were made into approximately 100 variant ANDbodies.

[0631] 5.6 Affinity of ANDbody variants for UMOD and IL11Ra

[0632] The affinity of ANDbody for UMOD and IL11Ra was evaluated using BIAcore in a similar manner as above. In this case, human and mouse versions of His-tagged UMOD and His-tagged IL11Ra were immobilized on the sensor chip NTA (Cytiva catalog number BR100034). Although all variants were tested in future assays, some ANDbody variants were expected to have a higher affinity for UMOD than for IL11Ra.

[0633] 5.7 Biodistribution of ANDbody and parental antibodies (in vivo)

[0634] Cell and tissue biodistribution studies were conducted using the method described above. However, the ANDbody used in this example was UMODxIL11Ra, and the parental antibodies corresponded to anti-UMOD and anti-IL11Ra.

[0635] 5.8 In vivo biological activity of UMODxIL11Ra ANDbody

[0636] The in vivo biological activity of UMODxIL11Ra ANDbody was quantified using the same method as above.

[0637] Example 6. ANDbody binds to mouse and human MEP1B and IL11RA

[0638] 6.1 Yeast surface display to generate anti-MEP1B antibodies

[0639] Anti-MEP1B (e.g., address target) antibodies were selected and cloned into human IgG1 as described above.

[0640] 6.2 Yeast surface display to generate anti-IL11Ra antibodies

[0641] The same IL11Ra (e.g., effector target) antibodies generated above in yeast surface display will be used here. ScFvs that cross-react with murine and human IL11Ra were cloned into human IgG2, transiently transfected into HEK293F cells, and purified using the Floor A resin as described above.

[0642] 6.3 Selection of active anti-IL11Ra antibodies with a broad IC 50 range

[0643] The above method was used to select IL11Ra antibodies with different IC 50 values.

[0644] 6.4 Expression and purification of ANDbody as a fusion protein with human IgG2

[0645] The 10 highest-affinity MEP1B scFvs and 10 IL11Ra antibodies with an affinity range (from <1 nM to 5 μM) were made into ANDbodies with the human IgG2 Fc region. For this, the scFv sequences from the MEP1B variants were cloned separately onto the IgG2 IL11Ra antibody variants. The MEP1B scFv was separated from the N or C terminus of the IL11Ra antibody light or heavy chain by a flexible linker (3xGGGGS) (2 MEP1B scFvs per ANDbody). Variants with a total of 4 MEP1B scFvs per ANDbody were prepared by cloning the MEP1B scFv before the N terminus and after the C terminus of the heavy or light chain, respectively (always separated by a linker). Other variants with 4 or more MEP1B scFvs per IL11Ra antibody on the MEP1BxIL11Ra ANDbody were prepared by mixing and matching the positions of the scFv on the following IL11Ra antibodies: at the N terminus of the heavy and light chains; at the C terminus of the heavy and light chains; at the N terminus of the heavy chain and the C terminus of the light chain; at the C terminus of the heavy chain and the N terminus of the light chain; and other variants with scFv at 3 or 4 different positions (yielding a total of 6 or 8 scFvs per ANDbody, respectively).

[0646] 6.5 Affinity of ANDbody variants for MEP1B and IL11Ra

[0647] The affinity of the ANDbody for MEP1B and IL11Ra was evaluated using BIAcore similarly as above. In this case, the His-tagged MEP1B and His-tagged IL11Ra of the human and murine versions were immobilized on the sensor chip NTA (Cytiva catalog number BR100034).

[0648] 6.6 Biodistribution (in vivo) of ANDbody and parental antibodies

[0649] The above method was used for cell and tissue biodistribution studies. However, the ANDbody used in this case was MEP1BxIL11Ra, and the parental antibodies corresponded to anti-MEP1B and anti-IL11Ra.

[0650] 6.7 In vivo biological activity of MEP1BxIL11Ra ANDbody

[0651] The in vivo biological activity of MEP1BxIL11Ra ANDbody was quantified using the same method as above.

[0652] Example 7. Exemplary skin address-restricted binder

[0653] This example demonstrates the restricted expression of anti-DSG1 antibody (address conjugate) in the skin.

[0654] 7.1 Expression and purification of anti-DSG1 monoclonal antibody

[0655] The sequences encoding the variable heavy chain regions of two desmoglein-1 (anti-DSG1) antibodies named 3-09*5 and 3-07 / 1e (HC: SEQ ID NO:24 and SEQ ID NO:26, see Table 5) (Yamagami et al., J Immunol. [Journal of Immunology], 183(9):5615 - 5621, 2009) were fused to the human IgG 1 (huIgG1) backbone with effector-inactivating mutations L234A, L235A, and P329G (LALA-PG) and cloned into PCDNA3.4 TM vector (Thermo Fisher Scientific). The variable light chain regions (SEQ ID NO:25 and SEQ ID NO:27) were fused with the constant κ light chain (for 3-09*5) (SEQ ID NO:22) or the constant λ light chain (for 3-07 / 1e) (SEQ ID NO:23) and cloned into PCDNA3.4 TM vector.

[0656] To express and purify the antibody, the heavy chain and light chain DNAs were transfected into EXPI293F at a 1:1 ratio using the EXPIFECTAMINE TM 293 transfection kit (Thermo Fisher Scientific) following the manufacturer's recommendations. TMIn cells (Thermo Fisher Scientific). Five days after transfection, the transiently expressed antibody was purified from the conditioned medium by filtering out the transfected cells. The conditioned medium was incubated with protein A agarose beads for 1 hour. The bound beads were washed with phosphate-buffered saline (PBS) pH 7.4 and then the bound antibody was eluted with 0.1 M glycine pH 2.5 and neutralized with 1 / 10 volume of Tris pH 8.5. The neutralized eluate was buffer-exchanged into PBS. The resulting mAbs were named PRO003 (3-09*5HC) (heavy chain sequence: SEQ ID NO:28; light chain sequence: SEQ ID NO:29) and PRO004 (3-07 / 1e HC) (heavy chain sequence: SEQ ID NO:30; light chain sequence: SEQ ID NO:31).

[0657] The monodispersity of the purified mAbs was analyzed by analytical size exclusion chromatography (SEC) and the purity was analyzed by SDS-PAGE.

[0658] Table 5. PRO003 and PRO004 sequences

[0659]

[0660]

[0661] 7.2 Anti-DSG1 antibodies PRO003 and PRO004 bind to murine DSG1 expressed on cells

[0662] According to the manufacturer's protocol, murine DSG1 (NCBI accession number NP_034209.2) with a c-Myc epitope tag at the protein C-terminus was transiently expressed in RAW264.7 cells using LIPOFECTAMINE TM 3000 (Thermo Fisher Scientific). The expression of DSG1 was confirmed by fixing and permeabilizing the cells and then staining with an anti-c-Myc antibody (Life Technologies A-21281) and analyzing by flow cytometry. PRO003 and PRO004 specifically bind to cells transfected with murine DSG1, confirming their expected binding specificity and suitability for studies in mice.

[0663] 7.3 Anti-DSG1 antibodies injected into mice preferentially accumulate in the skin

[0664] To demonstrate that binding to skin address results in antibody accumulation in the skin, anti-DSG1 antibodies PRO003 and PRO004 were conjugated to a near-infrared (IR) dye according to the manufacturer's instructions (LI- 928-38044). 800CW Chemical Conjugation.

[0665] The labeled antibodies were administered to mice via tail vein injection at a dose level of 3 mg / kg each. Each antibody was administered to two groups of 3 mice each, and the mice were euthanized on day 3 and day 7 after dosing. After euthanasia, 9 organs (heart, lung, pancreas, kidney, small intestine, large intestine, skin, liver, stomach) were collected and imager was used to measure the near-IR fluorescence of each tissue. To image the skin, a patch of skin was shaved and approximately 1 cm 2 was collected for imaging. Samples from each mouse were arranged in a standard format and the total fluorescence intensity was measured. The fluorescence intensity of each organ was quantified and averaged by measuring the total signal and subtracting the local background. High background signals were observed in the livers of all treated mice, so the liver was excluded from the analysis. Without wishing to be bound by theory, it is believed that the liver can take up the fluorescent dye independently of antibody targeting. Similarly, background signals were observed in the stomachs of all groups, including mice not treated with any antibody. Fluorescence was observed from the food fed to the mice, so the stomach was excluded from the analysis.

[0666] Figure 5A and 5B show the fluorescence signals of PRO003 ( Figure 5A ) and PRO004 ( Figure 5B ) across tissues. The distribution of both antibodies strongly favored the skin. These data suggest that DSG1 antibodies can be used as addresses for ANDbody preferential skin targeting.

[0667] Example 8. Exemplary Lung Address-Restricted Binding Agents

[0668] This example demonstrates the restricted expression of anti-RAGE antibodies (address conjugates) in the lung.

[0669] 8.1 Expression and Purification of Anti-RAGE Monoclonal Antibodies

[0670] The sequences encoding the variable heavy chain regions of two anti-RAGE mAbs named h11E6.8 and XT-M4 (Creative Biolabs) (SEQ ID NO:32 and SEQ ID NO:34, shown in Table 6) were fused to a huIgG1 backbone with effector null mutations L234A, L235A, and P329G (LALA-PG) and cloned into the PCDNA3.4 TM vector (Thermo Fisher Scientific). The sequences encoding the variable light chain regions (SEQ ID NO:33 and SEQ ID NO:35) were fused to the constant κ light chain and cloned into PCDNA3.4 TM .

[0671] For expression and purification, EXPIFECTAMINE TM 293 transfection kit (Thermo Fisher Scientific) was used to transfect heavy and light chain DNAs at a 1:1 ratio into EXPI293F TM cells (Thermo Fisher Scientific) following the manufacturer's recommendations. Five days after transfection, transiently expressed antibodies were purified from the conditioned medium by filtering the transfected cells. The conditioned medium was incubated with protein A agarose beads for 1 hour. The bound beads were washed with phosphate-buffered saline (PBS) pH 7.4 and then the bound antibodies were eluted with 0.1 M glycine pH 2.5 and neutralized with 1 / 10 volume of Tris pH 8.5. The neutralized eluate was buffer-exchanged into PBS. The resulting mAbs were named PRO001 (h11E6.8) (heavy chain sequence: SEQ ID NO:36; light chain sequence: SEQ ID NO:37) and PRO002 (XT-M4) (heavy chain sequence: SEQ ID NO:38; light chain sequence: SEQ ID NO:39).

[0672] The monodispersity of the purified mAbs was analyzed by analytical size-exclusion chromatography and the purity was analyzed by SDS-PAGE. PRO001 and PRO002 were highly monodisperse in expression and resolved at the expected molecular weights on SDS-PAGE. Binding studies confirmed binding to the RAGE antigen (not shown).

[0673] Table 6. PRO001 and PRO002 sequences

[0674]

[0675]

[0676] To test the binding of the anti-RAGE antibodies by ELISA, recombinant His-tagged murine RAGE protein (ab276858 from Abcam) was coated at a concentration of 1 μg / mL on NUNC-IMMUNO TM MAXISORP TMOvernight on an ELISA plate. The next day, the coated antigen was removed and the wells were blocked with 1% IgG-free bovine serum albumin (BSA), then incubated with 11 four-fold serial dilutions (starting concentration 20 nM) of anti-RAGE antibodies (PRO001 and PRO002). Bound antibodies were detected using a peroxidase-conjugated anti-human IgG antibody along with tetramethylbenzidine (TMB) and an acid termination reagent. By ELISA, both PRO001 and PRO002 bound to murine RAGE antigen with similar affinities, with an apparent affinity of approximately 90 pM, indicating that both antibodies are tight binders.

[0677] 8.2 Binding of anti-RAGE antibodies PRO001 and PRO002 to murine RAGE expressed on cells

[0678] The binding of PRO001 and PRO002 to murine RAGE was tested in cell culture. To confirm the binding activity and specificity of both antibodies, EXPIFECTAMINE TM (Thermo Fisher Scientific) was used to transiently express murine RAGE (NCBI accession number NP_031451.2) with a c-Myc epitope tag at the C-terminus of the protein in EXPI293 TM cells (Thermo Fisher Scientific). Expression of RAGE was confirmed by fixing and permeabilizing the cells, then staining with an anti-c-Myc antibody (Life Technologies A-21281) and analyzing by flow cytometry. Both antibodies specifically bound to murine RAGE expressed on the cells, confirming their expected binding specificity and suitability for studies in mice.

[0679] 8.3 Anti-RAGE antibodies injected into mice preferentially accumulate in the lungs

[0680] To demonstrate that binding to a lung address can result in antibody accumulation in the lungs, the anti-RAGE antibodies PRO001 and PRO002 were chemically conjugated to a near-IR dye as described in Example 7.

[0681] As described in Example 7, the labeled antibodies were administered to mice by tail vein injection and imaged. Figure 6A and 6B Fluorescence signals measured from tissues of mice treated with both antibodies are shown, with each antibody normalized so that the brightest signal equals 1. A group of three untreated mice was included as a negative control for autofluorescence. Compared to other antibodies tested, the distribution of these two antibodies was strongly biased towards the lungs. These data indicate that the two antibodies that bind to RAGE preferentially accumulate in the lungs, demonstrating that they can be used as addresses for ANDbody preferential lung targeting.

[0682] 8.4 Anti-RAGE antibody specifically accumulates on alveolar cells

[0683] Single-cell expression analysis showed that RAGE was specifically expressed in type I alveolar cells and was expressed at a lower level in type II alveolar cells. To test the hypothesis that an antibody can address a specific cell type, three Balb / C mice were treated with 3 mg / kg of PRO002 by tail vein injection. Three untreated mice were used as negative controls. Three days after dosing, the mice were euthanized, the lungs and other tissues were collected, and all tissues were fixed in formalin. Sections of each tissue were analyzed by immunohistochemistry (IHC) using an anti-human secondary antibody conjugated to horseradish peroxidase. Figure 7 Representative staining of treated and untreated mice is shown. Strong staining was observed in the alveolar tissue of mice treated with PRO002, but not in the adjacent airways or under negative control conditions. This result indicates that conjugates with specific addresses for specific cell types can be used to direct antibodies to those cells within a larger tissue.

[0684] Example 9. Exemplary renal address-restricted binder

[0685] This example demonstrates the restricted expression of an anti-CDH16 antibody (address binder) in the kidney.

[0686] 9.1 Expression and purification of anti-CDH16 monoclonal antibody

[0687] The sequence encoding the variable heavy chain region of anti-cadherin 16 (anti-CDH16) mAb Ab270263 (Abcam) (SEQ ID NO: 40; shown in Table 7) was fused to a huIgG1 backbone with effector null mutations L234A, L235A, and P329G (LALA-PG) and cloned into the PCDNA3.4 TM vector (Thermo Fisher Scientific). The sequence encoding the variable light chain region (SEQ ID NO: 41) was fused to the constant κ light chain and cloned into PCDNA3.4 TM vector.

[0688] For expression and purification, the heavy chain and light chain DNAs were transfected into EXPI293F at a 1:1 ratio using the EXPIFECTAMINE TM Transfection Kit (Thermo Fisher Scientific) following the manufacturer's recommendations. TMIn cells (Thermo Fisher Scientific). Five days after transfection, the transiently expressed antibody was purified from the conditioned medium by filtering out the transfected cells. The conditioned medium was incubated with protein A agarose beads for 1 hour. The bound beads were washed with phosphate buffered saline (PBS) pH 7.4 and then the bound antibody was eluted with 0.1 M glycine pH 2.5 and neutralized with 1 / 10 volume of Tris pH 8.5. The neutralized eluate was buffer exchanged into PBS. The resulting mAb was named PRO056 (heavy chain sequence: SEQ ID NO:42; light chain sequence: SEQ ID NO:43).

[0689] The monodispersity of the purified mAb was analyzed by analytical size exclusion chromatography and the purity was analyzed by SDS-PAGE. PRO056 was highly monodisperse and resolved at the expected molecular weight on SDS-PAGE.

[0690] Table 7. PRO056 sequence

[0691]

[0692]

[0693] To test the binding of the anti-CDH16 antibody by ELISA, internally expressed and purified recombinant His-tagged murine CDH16 protein was coated on NUNC-IMMUNO TM MAXISORP TM ELISA plates overnight. The next day, the coated antigen was removed, the wells were blocked with 1% IgG-free BSA and then incubated with 11 triplicate serial dilutions (starting concentration 533 nM) of the anti-CDH16 antibody (PRO056). Bound antibody was detected using a peroxidase-conjugated anti-human IgG antibody along with TMB and acid stop reagent. By ELISA, PRO056 bound to murine CDH16 antigen with an affinity of 200 pM.

[0694] 9.2 Anti-CDH16 antibody preferentially accumulates in the kidney

[0695] The anti-CDH16 antibody PRO056 was chemically conjugated to a near-IR fluorescent dye as described in Example 7. The labeled antibody was administered to mice by tail vein injection at a dose level of 3 mg / kg. Two groups of 3 mice each were euthanized on days 3 and 7 after dosing. After euthanasia, organs were collected and the near-IR fluorescence of each tissue was measured on a imager as described above. The fluorescence intensity of each organ was quantified and averaged by measuring the total signal and subtracting the local background.Figure 8 Shows fluorescence signals measured from tissues of mice treated with PRO056. Each signal was normalized so that the brightest signal equals 1. A group of three untreated mice was included as a negative control for autofluorescence.

[0696] When compared to the antibodies provided herein that target addresses in the skin, lung, or kidney, the distribution is strongly biased towards the kidney. These data suggest that antibodies that bind to CDH16 preferentially accumulate in the kidney, indicating that they can be used as the address bidding domain for ANDbody preferential kidney targeting.

[0697] Example 10. Exemplary intestinal address-restricted binder

[0698] This example demonstrates the restricted expression of an anti-CDH17 antibody (address binder) in the intestine.

[0699] 10.1 Expression and purification of anti-CDH17 monoclonal antibody

[0700] The sequence encoding the variable heavy chain region of anti-cadherin 17 (anti-CDH17) mAb MAB8524 (R&D Systems) (SEQ ID NO: 44; shown in Table 8) was fused to a huIgG1 backbone with effector null mutations L234A, L235A, and P329G (LALA-PG) and cloned into PCDNA3.4 TM vector (Thermo Fisher Scientific). The sequence encoding the variable light chain region (SEQ ID NO: 45) was fused to the constant κ light chain and cloned into PCDNA3.4 TM into.

[0701] For expression and purification, the heavy chain and light chain DNA were transfected into EXPI293F TM cells (Thermo Fisher Scientific) at a 1:1 ratio using the EXPIFECTAMINE TM 293 transfection kit (Thermo Fisher Scientific) following the manufacturer's recommendations. Five days after transfection, the transiently expressed antibody was purified from the conditioned medium by filtering out the transfected cells. The conditioned medium was incubated with protein A agarose beads for 1 hour. The bound beads were washed with phosphate-buffered saline (PBS) pH 7.4 and then the bound antibody was eluted with 0.1 M glycine pH 2.5 and neutralized with 1 / 10 volume of Tris pH 8.5. The neutralized eluate was buffer-exchanged into PBS. The resulting mAb was named PRO061 (heavy chain sequence: SEQ ID NO: 46; light chain sequence: SEQ ID NO: 47).

[0702] The monodispersity of the purified mAb was analyzed by analytical size exclusion chromatography, and the purity was analyzed by SDS-PAGE. PRO061 was highly monodisperse and resolved at the expected molecular weight on SDS-PAGE.

[0703] Table 8. PRO061 sequence

[0704]

[0705]

[0706] To confirm the binding activity and specificity, according to the manufacturer's protocol, mouse CDH17 (NCBI accession number NP_062727.1) with a c-Myc epitope tag at the protein C-terminus was transiently expressed in RAW 264.7 cells using LIPOFECTAMINE TM 3000 (Thermo Fisher Scientific). The expression of CDH17 was confirmed by fixing and permeabilizing the cells, then staining with an anti-c-Myc antibody (Life Technologies A-21281) and analyzing by flow cytometry. PRO061 specifically bound to the cells transfected with mouse CDH17, confirming that it had the expected binding specificity and was suitable for studies in mice.

[0707] 10.2 The anti-CDH17 antibody injected into mice preferentially accumulated in the intestine

[0708] PRO061 was chemically conjugated to a near-IR fluorescent dye as described in Example 7. The labeled antibody was administered to mice by tail vein injection at a dose level of 3 mg / kg. Two groups of three mice each were used and euthanized on days 3 and 7 after dosing. After euthanasia, the organs were collected and the near-IR fluorescence of each tissue was measured on a imager as described above. The fluorescence intensity of each organ was quantified and averaged by measuring the total signal and subtracting the local background. Figure 9 The fluorescence signals measured from the tissues of mice treated with PRO061 are shown. Each signal was normalized so that the brightest signal was equal to 1. A group of three untreated mice was included as a negative control for autofluorescence. The distribution was strongly biased towards the intestine, indicating that the antibody that binds to CDH17 preferentially accumulated in the intestine and could be used as an address targeting domain for ANDbody preferential intestinal targeting.

[0709] Example 11. Tissue restriction of the predicted address

[0710] Immunohistochemistry (IHC) was performed on fresh frozen (FF) healthy mouse tissue microarray (TMA) sections mounted on glass slides to analyze whether the predicted organ-specific or preferentially expressed addresses actually had the highest abundance in the intended organs and to determine which monoclonal antibody clone (mAb) most preferentially bound to the desired organ.

[0711] Slides coated with FF TMA were generated by first assembling fresh frozen tissue microarray blocks. To enable the formation of TMA blocks, individual organs from freshly sacrificed C57BL / 6 mice were embedded in optimal cutting temperature (OCT) medium in separate freezing molds and frozen. Then, cylindrical tissue cores were removed from each block and placed into a block to create the final FF TMA. Layers of the TMA were then cut using a cryostat, loaded onto positively charged microscope slides, and stored at -80 °C until staining.

[0712] Addresses were verified by directly binding slides coated with FF TMA to polyclonal antibodies or mAbs generated against the relevant addresses. These address-specific antibodies were detected with horseradish peroxidase (HRP)-conjugated antibodies specific for the IgG of the host that produced the primary address-specific antibody. The location and intensity of binding were determined by adding the HRP substrate 3,3'-diaminobenzidine (DAB), which produced a brown color at the primary antibody binding sites, proportional to the abundance of the deposited antibody. Nuclei were counterstained with hematoxylin to produce a blue color. For each respective address, the tissue specificity of a range of different mAb clones was analyzed, and their tissue binding patterns were evaluated by performing IHC on the same FF TMA as described above.

[0713] Table 9 summarizes the binding of the tested antibodies. All of the tested antibodies reacted primarily with the expected target tissues, with varying degrees of weaker reactivity to other tissues. Without wishing to be bound by theory, much of the off-tissue reactivity may reflect non-specific binding of the antibody. For example, all four antibodies tested for binding to RAGE showed binding to the lung, but three antibodies showed variable low levels of binding to other tissues.

[0714] Table 9. Candidate addresses evaluated on mouse tissue microarrays by IHC

[0715]

[0716] Example 12. Production and use of ANDbody

[0717] This example demonstrates the generation of an exemplary ANDbody that blocks Notch2 and binds to RAGE as an address.

[0718] 12.1 Expression and purification of anti-Notch2 monoclonal antibody

[0719] The sequences encoding the variable heavy chain regions of four anti-Notch2 mAbs (Wu et al., Nature, 464:1052–1057, 2010) (SEQ ID NOs: 48, 50, 52, and 54; shown in Table 10) were fused to the huIgG1 backbone with effector null mutations L234A, L235A, and P329G (LALA-PG) and cloned into the PCDNA3.4 TM vector (Thermo Fisher Scientific). The sequences encoding the corresponding variable light chain regions (SEQ ID NOs: 49, 51, 53, and 55) were fused to the constant κ light chain and cloned into PCDNA3.4 TM vector.

[0720] For expression and purification, the heavy and light chain DNAs were transfected into EXPI293F TM cells (Thermo Fisher Scientific) at a 1:1 ratio using the EXPIFECTAMINE TM 293 transfection kit (Thermo Fisher Scientific) following the manufacturer's recommendations. Five days after transfection, the transiently expressed antibodies were purified from the conditioned medium by filtering the transfected cells. The conditioned medium was incubated with protein A agarose beads for 1 hour. The bound beads were washed with phosphate-buffered saline (PBS) pH 7.4 and then the bound antibodies were eluted with 0.1 M glycine pH 2.5 and neutralized with 1 / 10 volume of Tris pH 8.5. The neutralized eluate was buffer-exchanged into PBS. The resulting mAbs were named PRO034 (heavy chain sequence: SEQ ID NO: 56; light chain sequence: SEQ ID NO: 57), PRO035 (heavy chain sequence: SEQ ID NO: 58; light chain sequence: SEQ ID NO: 59), PRO036 (heavy chain sequence: SEQ ID NO: 60; light chain sequence: SEQ ID NO: 61), and PRO037 (heavy chain sequence: SEQ ID NO: 62; light chain sequence: SEQ ID NO: 63).

[0721] Table 10. Sequences of PRO034, PRO035, PRO036, and PRO037

[0722]

[0723]

[0724]

[0725]

[0726] 12.2 Anti-Notch2 Binding and Affinity Assays

[0727] To test the binding of anti-Notch2 antibodies by ELISA, internally expressed and purified recombinant His-tagged human and murine Notch2 NRR domains were coated onto NUNC-IMMUNO TM MAXISORP TM ELISA plates at a concentration of 1 μg / mL overnight. The next day, the coated antigen was removed, the wells were blocked with 1% IgG-free BSA, and then incubated with anti-Notch2 antibodies (PRO034, PRO035, PRO036, and PRO037) at 11 three-fold serial dilutions (starting concentrations of 200 nM for PRO035 and PRO037, and 666 nM for PRO034 and PRO036). Bound antibodies were detected using peroxidase-conjugated anti-human IgG antibody along with TMB and acid termination reagent. Each antibody bound to the human and murine Notch2 NRR domains with an affinity between 47 pM and 140 nM.

[0728] To test the binding affinity of anti-Notch2 antibodies by Biolayer Interferometry (BLI), PRO034, PRO035, PRO036, and PRO037 were each immobilized on an anti-human IgG Fc biosensor and dipped into recombinant His-tagged murine and human Notch2 NRR proteins at different concentrations ranging from 1000 nM to 31 nM to measure the association rate with the antigen. The dissociation rate was then measured by dipping the biosensor into buffer. The binding affinity was calculated as the ratio of the dissociation rate to the association rate. The intrinsic affinities determined by BLI assays were all in the nM range, indicating that the affinity could be improved in the ELISA format.

[0729] 12.3 Design and Production of Notch2 / RAGE ANDbody

[0730] Generate an ANDbody (Labrijn et al., Proc Natl Acad Sci U.S.A., 110(13):5145-5150, 2013) using a controlled Fab-arm exchange (cFAE) reaction that contains the first fragment antigen-binding (Fab) arm of the above anti-Notch2 antibodies (PRO034, PRO035, and PRO036) and the second Fab arm of the anti-RAGE antibody PRO002 (Example 8). Site-directed mutagenesis was performed to introduce an F405L amino acid substitution mutation in the Fc fragment of the anti-RAGE antibody (PRO002) and a K409R amino acid substitution mutation in the Fc fragment of each anti-Notch2 antibody (PRO034, PRO035, and PRO036). The expression and purification of these antibodies were as described previously for the parental antibodies. Then, the individual monoclonal antibodies were mixed in equimolar ratios in a controlled reduction and reoxidation reaction that drives the recombination of the bispecific antibody guided by the matching point mutations (F405L-K409R). The formation of the ANDbody was analyzed by analytical chromatography and SDS-PAGE. The resulting ANDbodies were designated PRO051, PRO052, and PRO053 (containing PRO034, PRO035, and PRO036, respectively).

[0731] SDS-PAGE and analytical size exclusion chromatography showed that the major product formed after the cFAE reaction had a molecular weight typical of IgG1 (150 kDa), indicating complete reoxidation. Analytical hydrophobic interaction chromatography indicated the formation of a new product, the desired heterodimeric antibody.

[0732] 12.4 BLI shows that the Notch2 / RAGE ANDbody binds Notch2 and RAGE simultaneously

[0733] To test the simultaneous dual-antigen binding of the BLI to the Notch2 / RAGE ANDbody, PRO051, PRO052, and PRO053, as well as the monovalent parental antibody controls, were immobilized on an anti-human IgG Fc biosensor and immersed in 150 nM of recombinant His-tagged murine RAGE protein, followed by a second association step into a well containing 150 nM recombinant murine Notch2 NRR to measure dual-antigen binding. The dissociation rate was then measured by immersing the biosensor in buffer.

[0734] The sensorgram shows that ANDbodies PRO051, PRO052, and PRO053 are capable of simultaneously binding to both the RAGE and Notch2 antigens, while the monovalent parental antibodies bind to only one of the RAGE and Notch2 NRR. This supports the conclusion that the ANDbody is the correct composition and is functional in simultaneously binding two antigens.

[0735] 12.5 Immunohistochemistry shows that the Notch2 / RAGE ANDbody preferentially binds to human lung tissue

[0736] Immunohistochemistry (IHC) on sections of a fresh frozen healthy mouse tissue microarray (FF TMA) mounted on slides was used to evaluate the tissue binding of the ANDbody containing the above-mentioned Notch2 inhibitory antibody. The TMA was constructed and stained as described in Example 11. Figure 10 Staining of the mouse TMA with three anti-Notch2 antibodies PRO034, PRO035, and PRO036, as well as the Notch2 / RAGE ANDbodies PRO051, PRO052, and PRO053, is shown. In each case, the binding to lung tissue in the RAGE-targeted ANDbodies was significantly enhanced. These data indicate that combining a receptor-targeting conjugate with an addressing conjugate in the form of an antibody can confer the tissue specificity of the addressing arm to the ANDbody.

[0737] 12.6 The Notch2 / RAGE ANDbody preferentially distributes to the lung compared to a matched non-targeting anti-Notch2 antibody

[0738] To evaluate the in vivo performance of the ANDbody targeting Notch2 and RAGE, mice were treated with an IV administration of 3 mg / kg of the PRO051, PRO052, and PRO053 antibodies. All groups contained 3 mice. Tissues were collected from each mouse on days 3, 7, 14, and 21 after dosing. The accumulation of each antibody in the lung was measured by homogenizing a fixed amount of lung tissue, normalizing each sample to a fixed amount of extracted protein, and then detecting the human antibody by sandwich ELISA.

[0739] Figure 11Shows the accumulation of PRO052 in the lung compared to a matching antibody that binds RAGE and the control target respiratory syncytial virus (RSV) glycoprotein F (RAGE XT-M4 / Morvexizumab) and a matching antibody that binds Notch2 and RSV glycoprotein F (Notch2-2 / Morvexizumab). Antibodies against RSV glycoprotein F and Notch2 were not detected in the lung at any time point. In contrast, the Notch2 / RAGE ANDbody was clearly detectable in the lung for at least two weeks. The overall accumulation of PRO052 was lower than that of the bispecific antibody that binds RAGE and RSV glycoprotein F, indicating that the overall specificity of PRO052 was independently intermediate between the two arms. These results suggest that the addressing arm in the ANDbody can significantly redirect the binding specificity of the target-binding arm.

[0740] Example 13. Production and Use of ANDbody

[0741] This example describes the production of an exemplary ANDbody that (i) contains a ligand effector targeting the IL-10 pathway and (ii) binds DSG1 as an address.

[0742] 13.1 Description, Design, and Production of IL-10 / DSG1 ANDbody

[0743] To test the form of the IL-10 / anti-DSG1 ANDbody, three parameters were explored: the valence of IL-10 (1 or 2 IL-10 moieties), the valence of the anti-DSG1 arm (1 or 2 Fab arms), and two versions of IL-10 (dimeric or monomeric IL-10). Forms representing different combinations of IL-10 molecule, IL-10 valence, and antibody valence were evaluated. For the bivalent form, wild-type (WT) IL-10 (accession number P22301), an engineered monomeric IL-10 sequence (Josephson et al., J Biol Chem. [Journal of Biological Chemistry], 275(18):13552-7, 2000), and an engineered dimeric IL-10 sequence (Minshawi et al., Front Immunol. [Frontiers in Immunology], 11:1794, 2020) were fused to the C-terminus of the heavy chain with the PRO003 sequence (Example 7). For the monovalent form, monomeric and dimeric IL-10 were fused to the N-terminus of Fc and co-expressed with PRO003. The monovalent form was asymmetric, and mutations in the Fc domain (chain A: S364K / K409S; chain B: K370S / F405K (WO 2017 / 106462 A1)) were used to effect asymmetric pairing.

[0744] To express and purify the antibody, EXPIFECTAMINE was usedTM The 293 transfection kit (Thermo Fisher Scientific) was used to transfect heavy chain to light chain DNA at a 1:1 ratio or heavy chain to light chain to IL-10-Fc (as listed below) at a 1:1:1 ratio into EXPI293F TM cells (Thermo Fisher Scientific) according to the manufacturer's recommendations. Five days after transfection, the transiently expressed antibody was purified from the conditioned medium by filtering the transfected cells. The conditioned medium was incubated with protein A agarose beads for 1 hour. The bound beads were washed with phosphate-buffered saline (PBS) pH 7.4 and then the bound antibody was eluted with 0.1 M glycine pH 2.5 and neutralized with 1 / 10 volume of Tris pH 8.5. The neutralized eluate was buffer-exchanged into PBS.

[0745] The resulting mAbs were named PRO023, PRO024, PRO025, PRO026, and PRO027( Figure 12 ).

[0746] PRO023 contains (a) a heavy chain sequence (SEQ ID NO:67) that contains the heavy chain sequence of PRO003 (SEQ ID NO:28) and the wild-type human IL-10 sequence (SEQ ID NO:64) and (b) the light chain sequence of PRO003 (SEQ ID NO:29).

[0747] PRO024 contains (a) a heavy chain sequence (SEQ ID NO:68) that contains the heavy chain sequence of PRO003 (SEQ ID NO:28) and the monomeric human IL-10 sequence (SEQ ID NO:64) and (b) the light chain sequence of PRO003 (SEQ ID NO:29).

[0748] PRO025 contains (a) a heavy chain sequence (SEQ ID NO:69) that contains the heavy chain sequence of PRO003 (SEQ ID NO:28) and the dimeric human IL-10 sequence (SEQ ID NO:66) and (b) the light chain sequence of PRO003 (SEQ ID NO:29).

[0749] PRO026 contains (a) the heavy chain sequence of PRO003 that further contains a mutation in the Fc domain to effect asymmetric pairing (SEQ ID NO:70), (b) the light chain sequence of PRO003 (SEQ ID NO:29), and (c) an IL-10-Fc fusion protein (SEQ ID NO:72) that contains an Fc region with a mutation to effect asymmetric pairing (SEQ ID NO:71) and the monomeric human IL-10 sequence (SEQ ID NO:64).

[0750] PRO027 comprises (a) the heavy chain sequence of PRO003, which further comprises a mutation in the Fc domain to effect asymmetric pairing (SEQ ID NO:70), (b) the light chain sequence of PRO003 (SEQ ID NO:29), and (c) an IL-10-Fc fusion protein (SEQ ID NO:73), which comprises an Fc region containing a mutation to effect asymmetric pairing (SEQ ID NO:71) and a dimeric human IL-10 sequence (SEQ ID NO:66).

[0751] The monodispersity of the purified ANDbody was analyzed by analytical size exclusion chromatography, and the purity was analyzed by SDS-PAGE. After one-step purification, PRO024 and PRO026 had the highest yields and monodispersity. PRO023 and PRO027 had moderate yields and a monodispersity of approximately 70%. PRO025 had a lower yield and a monodispersity of approximately 89%.

[0752] Table 11. Sequences of PRO023, PRO024, PRO025, PRO026, PRO058, and PRO027

[0753]

[0754]

[0755]

[0756]

[0757] 13.2 IL-10 / DSG1 ANDbody binds to two IL-10 receptors

[0758] To test the binding of the IL-10 / anti-DSG1 ANDbody to IL-10 receptor α (IL-10Ra) by ELISA, recombinant His-tagged human IL-10Ra (Creative Biomart) was coated onto NUNC-IMMUNO TM MAXISORP TM ELISA plates at a concentration of 1 μg / mL overnight. The next day, the coated antigen was removed, the wells were blocked with 1% IgG-free BSA, and then incubated with 11 triplicate serial dilutions (starting concentration of 30 μg / mL) of the anti-IL-10 / anti-DSG1 ANDbody (as described above). Bound antibody was detected using a peroxidase-conjugated anti-human IgG antibody along with TMB and acid stop reagent.

[0759] Among the tested molecules, only the molecules containing the IL-10 moiety (PRO023, PRO024, PRO025, PRO026, PRO027 and the positive control IL-10Fc) (IL10-326H from Creative Biomart) bound to IL-10Ra, demonstrating that the binding was driven by IL-10 rather than the negative control anti-DSG1 antibody (PRO003), and that the IL-10 moiety was functional in binding to its receptor.

[0760] 13.3 IL-10 / DSG1 ANDbody activates the IL-10 signaling pathway

[0761] To show that IL-10 retained its biological activity as part of the various ANDbodies described above, the ability of each IL-10 / DSG1 ANDbody to activate the IL-10 signaling pathway was tested. HEK-BLUE TM IL-10 cells (InvivoGen) were used to evaluate the signaling activity and relative potency of each molecule. These cells express all components of the IL-10 signaling pathway, including the IL-10-inducible gene encoding secreted embryonic alkaline phosphatase (SEAP). When the IL-10 signaling in these cells is activated, they express SEAP and secrete it into the cell culture medium. By adding QUANTI-BLUE TM solution colorimetric reagent (InvivoGen) to the cell culture medium and then reading the absorbance at 630 nm to measure the extent of IL-10 signaling.

[0762] In this experiment, PRO023, PRO024, PRO025, PRO026 and PRO027 were each titrated from 1 pM to >1 nM in cell culture overnight. Figure 18A and 18B Representative activity data for each IL-10 / DSG1 ANDbody and the positive and negative controls described in Table 12 are shown. Table 12 shows HEK-BLUE TMThe EC50 of the cells' responses to three control molecules ((1) recombinant human IL-10 (Boqi Co., #573204) (rhIL-10), (2) recombinant human IL-10 fused to the human Fc domain (hIL-10Fc fusion), and (3) parental anti-DSG1 antibody (PRO003)) and each of the IL-10 / DSG1 ANDbody described above. These data confirm that all five IL-10 / DSG1 ANDbody retain the signaling activity of human IL-10. They show a range of potencies, indicating that the relative biological potency of the ANDbody can be adjusted by changes in molecular form, the structure of the bioactive portion, and the valence of the active portion.

[0763] Table 12. Potency of IL-10 / DSG1 ANDbody in HEK-BLUE TM Potency in the IL-10 Signaling Assay

[0764]

[0765] 13.4 IL-10 / DSG1 ANDbody Inhibits the Inflammatory Response of Primary Mouse Macrophages

[0766] To demonstrate that the IL-10 / DSG1 ANDbody can inhibit the inflammatory immune response, the effects of the IL-10 / DSG1 ANDbody on mouse peripheral blood mononuclear cells (PBMC) and macrophages treated with lipopolysaccharide (LPS) as an inflammatory stimulant were evaluated. In these experiments, PBMC were isolated from the blood of Balb / C mice by magnetic bead negative enrichment (Miltenyi Biotech #130-110-434), and macrophages were isolated from their spleens. Macrophage activation was determined by measuring the level of the TNFα cytokine present in the culture medium 3 hours and 5 - 6 hours after LPS stimulation.

[0767] Figure 13A - 13G The levels of tumor necrosis factor α (TNFα) in PBMC cell cultures are shown after pre-stimulation with the indicated IL-10 / DSG1 ANDbody or control molecule, followed by treatment with LPS for the indicated length of time. Figure 14A - 14G The TNFα levels in primary macrophage cultures are shown after pre-stimulation with the indicated IL-10 / DSG1 ANDbody or control molecule, followed by treatment with LPS for the indicated length of time. Since these groups represent experiments run over multiple days, the data between groups are not comparable. These data indicate that all five IL-10 / DSG1 ANDbody are able to inhibit the inflammatory stimulus in primary macrophages.

[0768] 13.5 Address targeting of the effector enhances the activity / potency of the ANDbody effector function

[0769] The combination of addressing (e.g., using an address targeting domain) with a bioactive molecule has the potential to enhance bioactivity in a variety of ways. One exemplary enhancement is by increasing the potency of the effector moiety towards specific cells that also bear the address target.

[0770] To test whether the signaling potency of an effector targeting domain can be enhanced by the presence of an address targeting domain, lentivirus was used to stably express human DSG1 on HEK-BLUE TM IL-10 cells (the stably expressing cells are referred to as HEKBLUE TM IL-10 / DSG1). The DSG1 gene (NP_034209.2) was cloned into a suitable lentiviral plasmid backbone and packaged into viral particles using the VIRAPOWER TM Lentiviral Packaging Mix (Thermo Fisher Scientific) and transduced according to the manufacturer's instructions. Expression of DSG1 was confirmed by qPCR.

[0771] The potencies of recombinant human IL-10, an ANDbody (where monomeric IL-10 replaces one Fab of an anti-DSG1 mAb (PRO058, functionally equivalent to PRO026)), and a matching control (where the antibody sequence contains moxetumomab as a negative control) were evaluated. Activity was tested on HEK-BLUE TM IL-10 cells and HEK-BLUE stably expressing DSG1 TM IL-10 cells. Figure 15 Representative signaling responses of each molecule in parental HEK-BLUE TM IL-10 cells and HEK-BLUE TM IL-10 / DSG1 cells are shown. The two cell lines responded similarly to recombinant IL-10, confirming that DSG1 expression had little effect on their sensitivity to IL-10. When DSG1 was expressed on the target cells, the DSG1 / IL-10 ANDbody showed an approximately 15-fold increase in potency. No effect on the potency of the matched IL-10 / moxetumomab protein was found, confirming that the effect was mediated through binding to DSG1. This indicates that the ANDbody design provided herein enables address-mediated enhancement of biological potency, which allows the use of a less potent target-binding moiety in the ANDbody to further reduce unwanted off-target effects.

[0772] 13.6 The IL-10 / DSG1 ANDbody retains the pharmacokinetic and tissue distribution properties of the parental anti-DSG1 antibody

[0773] In some ANDbodies, the targeting moiety is designed to confer tissue or cell targeting of the parental mAb or other targeting molecule to a bioactive moiety that would otherwise have undesirable pharmacokinetics or tissue distribution.

[0774] The IL-10 / DSG1 ANDbody is designed to direct IL-10 activity to the skin. In Example 7, it was shown that the anti-DSG1 antibody preferentially distributes to the skin of mice. In contrast, IL-10 has been reported to be cleared from the human circulation with a half-life of approximately 2 hours (Radwanski et al., Pharm Res. [Drug Research] December 1998; 15(12):1895-901). Accordingly, we evaluated whether the IL-10 / DSG1 ANDbody retains the skin targeting ability of the parental antibody.

[0775] BALB / c mice were dosed by tail vein injection with 3 mg / kg of PRO003, PRO024, or PRO058. PRO058 has the same function as PRO026, except that substitutions were made in the Fc domain to improve purification of the recombinant protein. PRO058 comprises (a) the heavy chain sequence of PRO0026 (SEQ ID NO:70), (b) the light chain sequence of PRO003 (SEQ ID NO:29), and (c) an IL-10-Fc fusion protein (SEQ ID NO:75) that comprises an Fc region (SEQ ID NO:74) and a monomeric human IL-10 sequence (SEQ ID NO:64).

[0776] Serum samples were collected at time points from 1 hour to 48 hours. Tissue samples were collected at 1, 2, 4, and 7 days after dosing. The amount of anti-DSG1 or ANDbody in each serum or tissue sample was measured by ELISA. Figure 16A and 16B It was shown that the IL-10 / anti-DSG1 ANDbodies PRO024 and PRO058 have PK properties similar to those of the parental anti-DSG1 antibody PRO003 in skin and serum. These data demonstrate that antibody-cytokine fusions containing recombinant IL-10 can retain the pharmacokinetic properties of the parental antibody.

[0777] Example 14. TNFα-blocking molecule conjugated to DSG1 targeting moiety

[0778] This example describes the generation of an exemplary ANDbody that blocks TNFα and binds DSG1 as an address.

[0779] 14.1 Expression and purification of anti-TNFα monoclonal antibody

[0780] Anti-TNFα antibodies were generated with VH and VL sequences from commercial antibodies fused to the huIgG1 backbone with effector null mutations L234A, L235A, P329G (LALA-PG), and their binding and affinity to TNFα were characterized as described above. The resulting mAbs were named PRO076 and PRO078.

[0781] 14.2 Anti-TNFα–DSG1 ANDbody Design, Expression, and Purification

[0782] The ANDbody was designed by combining PRO004 (Example 7) with previously reported dominant-negative TNFα (Steed et al. Science. 26 September 2003; 301(5641)) or the clinically validated anti-TNFα antibodies listed below, which were intended to locally downregulate TNFα in the extracellular environment of inflamed skin cells. TNFα-blocking anti-DSG1 ANDbody design explored various formats and valences, including cytokine / antibody and TNF receptor 2 (TNFR2) / antibody fusions.

[0783] To express and purify the antibodies, the EXPIFECTAMINE TM 293 transfection kit (Thermo Fisher Scientific) was used to transfect heavy-chain and light-chain DNA into EXPI293F TM cells (Thermo Fisher Scientific) at a 1:1 ratio following the manufacturer's recommendations. Five days after transfection, the transiently expressed antibodies were purified from the conditioned medium by filtering out the transfected cells. The conditioned medium was incubated with protein A agarose beads for 1 hour. The bound beads were washed with phosphate-buffered saline (PBS) pH 7.4 and then the bound antibodies were eluted with 0.1 M glycine pH 2.5 and neutralized with 1 / 10 volume of Tris pH 8.5. The neutralized eluate was buffer-exchanged into PBS. The resulting mAbs were named PRO070, PRO074, PRO075, and PRO077 ( Figure 17 ).

[0784] The monodispersity of the purified ANDbody was analyzed by analytical size-exclusion chromatography and the purity was analyzed by SDS-PAGE. Standard additional purification steps were performed to remove aggregates. The final yield of PRO077 was the highest, followed by PRO074 and PRO075, while the final yield of PRO070 was the lowest. As shown by SDS-PAGE, the purified ANDbody was pure and had the correct composition.

[0785] 14.3 Anti-TNFα–DSG1 ANDbody Binding and Affinity Testing

[0786] ELISA binding assays showed that all constructs were active in binding human and murine TNFα with different affinities. PRO074 and PRO075 had similar affinities for human and murine TNFα, within two- to three-fold of the parental antibody. Compared to the parental antibody, the binding affinities of PRO077 for human and murine TNFα were reduced by 5- and 12-fold, respectively. This reduction in affinity may be due to the change in format from Fab to single-chain variable fragment (scFv).

[0787] 14.4 Anti-TNFα–DSG1 ANDbody in vitro activity assay

[0788] Using HEK-BLUE TM TNFα cells (Invitrogen) were used to evaluate the ability of each anti-TNFα / anti-DSG1 ANDbody to inhibit TNFα signaling. These cells were engineered to express secreted embryonic alkaline phosphatase (SEAP) in response to signaling through TNFα. TNFα was measured according to the manufacturer's instructions. To evaluate inhibitory activity, the concentration of TNFα was fixed at 225 pM (approx. the EC 80 ) of recombinant human TNFα in this assay, and the cells were pre-incubated with TNFα-blocking molecules at 10 nM to approximately 10 pM. Table 13 shows the IC 50 of each anti-TNFα / DSG1 ANDbody and the matching parental antibody used as a positive control. These data indicate that the ANDbody retained TNFα-blocking activity compared to the original anti-TNFα parental antibody.

[0789] Table 13. IC 50

[0790] Protein <![CDATA[IC 50 (nM, repeated 1)]]> <![CDATA[IC 50 (nM, repeated 2)]]> Adalimumab (PRO076) 0.05 0.03 Etanercept (PRO078) 0.06 0.07 PRO070 No inhibition No inhibition PRO074 0.3 0.2 PRO075 0.2 0.1 PRO077 0.04 0.04

[0791] VII. Other embodiments

[0792] Some embodiments of the techniques described herein may be defined according to any of the following numbered embodiments:

[0793] 1. A macromolecule comprising a first binding site and a second binding site, wherein:

[0794] (a) the first binding site is specific for an effector target in a subject, and

[0795] (b) the second binding site is specific for an address target expressed in a target tissue or cell in the subject; wherein:

[0796] (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 target tissue or cell;

[0797] (ii) The second binding site substantially does not affect signaling after binding to the address target; and

[0798] (iii) The first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site, wherein the macromolecule is linked to a small molecule.

[0799] 2. A macromolecule comprising a first binding site and a second binding site, wherein:

[0800] (a) The first binding site is specific for an effector target in a subject, and

[0801] (b) The second binding site is specific for an address target expressed in a target tissue or cell in the subject; wherein:

[0802] (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 target tissue or cell;

[0803] (ii) The second binding site substantially does not affect signaling after binding to the address target; and

[0804] (iii) The first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site;

[0805] 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, wherein the macromolecule is linked to a small molecule.

[0806] 3. A macromolecule comprising a first binding site and a second binding site, wherein:

[0807] (a) The first binding site is specific for an effector target in a subject, and

[0808] (b) The second binding site is specific for an address target expressed in a target tissue or cell in the subject; wherein:

[0809] (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 target tissue or cell;

[0810] (ii) The second binding site substantially does not affect signal transduction after binding to the address target; and

[0811] (iii) The first binding site substantially does not affect effector target signal transduction in the absence of being localized by the second binding site;

[0812] And wherein the localization of the macromolecule to the target tissue or cell is significantly increased relative to the localization of a reference macromolecule lacking the second binding site, wherein the macromolecule is linked to a small molecule.

[0813] 4. A macromolecule comprising a first binding site and a second binding site, wherein:

[0814] (a) The first binding site is specific for an effector target in a subject, and

[0815] (b) The second binding site is specific for an address target expressed in the target tissue or cell in the subject; wherein:

[0816] (i) The second binding site localizes the first binding site to the address target such that the first binding site affects effector target signal transduction in the target tissue or cell;

[0817] (ii) The second binding site substantially does not affect signal transduction after binding to the address target; and

[0818] (iii) The first binding site substantially does not affect effector target signal transduction in the absence of being localized by the second binding site;

[0819] And wherein at least 25% of the macromolecule administered to the subject is detected at the target tissue or cell at a time point between 1 day and 7 days after administration, wherein the macromolecule is linked to a small molecule.

[0820] 5. A macromolecule comprising a first binding site and a second binding site, wherein:

[0821] (a) The first binding site is specific for an effector target in a subject, and

[0822] (b) The second binding site is specific for an address target expressed in the target tissue or cell in the subject; wherein:

[0823] (i) The second binding site localizes the first binding site to the address target such that the first binding site affects effector target signal transduction in the target tissue or cell;

[0824] (ii) After binding to the address target, the second binding site substantially does not affect signal transduction; and

[0825] (iii) In the absence of being localized by the second binding site, the first binding site substantially does not affect effector target signal transduction;

[0826] 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, and wherein the macromolecule is linked to a small molecule.

[0827] 6. A macromolecule comprising a first binding site and a second binding site, wherein:

[0828] (a) The first binding site is specific for an effector target in a subject, and

[0829] (b) The second binding site is specific for an address target expressed in a target tissue or cell in the subject; wherein:

[0830] (i) The second binding site localizes the first binding site to the address target such that the first binding site affects effector target signal transduction in the target tissue or cell;

[0831] (ii) After binding to the address target, the second binding site substantially does not affect signal transduction; and

[0832] (iii) In the absence of being localized by the second binding site, the first binding site substantially does not affect effector target signal transduction;

[0833] 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, and wherein the macromolecule is linked to a small molecule.

[0834] 7. A macromolecule comprising a first binding site and a second binding site, wherein:

[0835] (a) The first binding site is specific for an effector target in a subject, and

[0836] (b) The second binding site is specific for an address target expressed in a target tissue or cell in the subject; wherein:

[0837] (i) The second binding site localizes the first binding site to the address target such that the first binding site affects effector target signal transduction in the target tissue or cell;

[0838] (ii) The second binding site substantially does not affect signal transduction after binding to the address target; and

[0839] (iii) The first binding site substantially does not affect effector target signal transduction in the absence of being localized by the second binding site;

[0840] and wherein, relative to a reference macromolecule lacking the second binding site, the potency of the first binding site at the target tissue or cell is significantly increased, wherein the macromolecule is linked to a small molecule.

[0841] 8. The macromolecule according to any one of Examples 1 - 7, wherein the first binding site has a low affinity for the effector target.

[0842] 9. The macromolecule according to any one of Examples 1 - 7, wherein the first binding site has a low avidity for the effector target.

[0843] 10. The macromolecule according to any one of Examples 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.

[0844] 11. The macromolecule according to any one of Examples 1 - 10, wherein the avidity of the first binding site for the effector target is lower than the avidity of the second binding site for the address target.

[0845] 12. The macromolecule according to any one of Examples 1 - 11, wherein:

[0846] (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;

[0847] (b) The EC 50 of the first binding site for the effector target is higher than the EC 50 of the second binding site for the address target; or

[0848] (c) The IC 50 of the first binding site for the effector target is higher than the IC 50 of the second binding site for the address target.

[0849] 13. The macromolecule according to any one of Examples 1 - 12, wherein the affinity of the first binding site for the effector target is at least about 2 - fold, at least about 5 - fold, or at least about 10 - fold less than the affinity of the second binding site for the address target.

[0850] 14. The macromolecule according to any one of embodiments 1-13, wherein the second binding site has a Kd for the address target with an affinity greater than about 1 nM, greater than about 2 nM, or greater than about 50 nM.

[0851] 15. The macromolecule according to any one of embodiments 1-14, wherein the effector target is a protein, a lipid, or a sugar.

[0852] 16. The macromolecule according to any one of embodiments 1-15, wherein the effector target is a cell membrane-associated target.

[0853] 17. The macromolecule according to embodiment 15 or 16, wherein the effector target is a protein.

[0854] 18. The macromolecule according to embodiment 17, wherein the effector target is a secreted protein.

[0855] 19. The macromolecule according to embodiment 17 or 18, wherein the effector target is encoded by a gene selected from the group consisting of the genes listed in Table 1.

[0856] 20. The macromolecule according to any one of embodiments 1-19, wherein the macromolecule agonizes the effector target.

[0857] 21. The macromolecule according to any one of embodiments 1-19, wherein the macromolecule antagonizes the effector target.

[0858] 22. The macromolecule according to any one of embodiments 1-21, wherein the address target is a protein, a lipid, or a sugar.

[0859] 23. The macromolecule according to embodiment 22, wherein the address target is a protein.

[0860] 24. The macromolecule according to any one of embodiments 17-23, 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.

[0861] 25. The macromolecule according to embodiment 24, wherein the expression level of the effector target or the address target is evaluated by using an RNA sequence dataset.

[0862] 26. The macromolecule according to embodiment 25, wherein the RNA sequence dataset is a Genotype-Tissue Expression (GTEx) dataset or a Human Protein Atlas (HPA) dataset.

[0863] 27. The macromolecule according to embodiment 23, wherein the expression of the effector target or the address target is protein expression.

[0864] 28. The macromolecule according to any one of embodiments 1-27, wherein the effector target is expressed systemically in the subject.

[0865] 29. The macromolecule according to any one of embodiments 1-27, wherein the effector target is expressed regionally in the subject.

[0866] 30. The macromolecule according to any one of embodiments 1-27, wherein the effector target is expressed locally in the subject.

[0867] 31. The macromolecule according to any one of embodiments 1-30, wherein the address target is expressed regionally in the subject.

[0868] 32. The macromolecule according to any one of embodiments 1-30, wherein the address target is expressed locally in the subject.

[0869] 33. The macromolecule according to any one of embodiments 1-30, wherein the expression of the address target is limited to cell types in the subject.

[0870] 34. The macromolecule according to any one of embodiments 1-33, wherein the address target is a soluble protein or an extracellular matrix (ECM)-associated protein and is not present on the cell surface in detectable amounts.

[0871] 35. The macromolecule according to embodiment 34, wherein the address target is expressed in the ECM and is not present elsewhere in the subject in detectable amounts.

[0872] 36. The macromolecule according to any one of embodiments 1-35, wherein the address target is expressed only by cells in a specific cell state in the subject.

[0873] 37. The macromolecule according to any one of embodiments 1-36, wherein the address target is expressed only by cells in a diseased state in the subject.

[0874] 38. The macromolecule according to any one of embodiments 1-37, wherein the address target is not expressed in tissues where binding of the second binding site to the effector target is harmful to the subject.

[0875] 39. The macromolecule according to any one of embodiments 1-38, wherein the binding site of the address target does not bind to the binding site of the natural ligand of the address target in detectable amounts.

[0876] 40. The macromolecule according to any one of Examples 1-39, wherein the expression of the effector target or the address target comprises expression in one or more of the following: minor salivary glands, thyroid gland, lung, mammary gland, mammary tissue, pancreas, adrenal gland, liver, kidney, renal cortex, renal medulla, adipose visceral tissue, omentum, small intestine, terminal ileum, fallopian tube, ovary, uterus, skin, skin not exposed to sunlight, skin on the pubic arch, cervix, endocervix, exocervix, vagina, skin exposed to sunlight, calf skin, anterior cingulate cortex, Brodmann area 24 (BA24), basal ganglia, caudate nucleus, dura mater, nucleus accumbens, hypothalamus, amygdala, hippocampus, cerebellum, cerebellar hemisphere, substantia nigra, pituitary gland, spinal cord, cervical spinal cord, artery, aorta, heart, auricle, coronary artery, left ventricle, esophagus, esophageal mucosa, esophageal muscular layer, gastroesophageal junction, spleen, stomach, colon, transverse colon, sigmoid colon, testis, whole blood cells, EBV-transformed lymphocytes, tibial artery, or tibial nerve tissue.

[0877] 41. The macromolecule according to Example 40, wherein the expression of the effector target or the address target comprises expression in skin tissue, lung tissue, kidney tissue, or intestinal tissue.

[0878] 42. The macromolecule according to Example 41, wherein the expression of the address target is significantly higher in skin tissue, lung tissue, kidney tissue, or intestinal tissue than in any other tissue.

[0879] 43. The macromolecule according to any one of Examples 1-42, wherein the effector target and / or the address target is expressed on the structural tissue of the subject.

[0880] 44. The macromolecule according to any one of Examples 1-43, wherein the effector target and the address target are located on the same cell.

[0881] 45. The macromolecule according to any one of Examples 1-43, wherein the effector target and the address target are located on different cells.

[0882] 46. The macromolecule according to Example 45, wherein the effector target and the address target are located on different cells of the same cell type.

[0883] 47. The macromolecule according to Example 45, wherein the effector target and the address target are located on different cells of different cell types.

[0884] 48. The macromolecule according to Example 45, wherein the effector target and the address target are located on different cells in the same tissue.

[0885] 49. The macromolecule according to any one of Examples 45, 47, and 48, wherein:

[0886] (a) The effector target is on the circulating cells and the address target is on the tissue-restricted cells; or

[0887] (b) The effector target is on the tissue-restricted cells and the address target is on the circulating cells.

[0888] 50. The macromolecule according to any one of Examples 45 - 49, wherein the effector target and the address target are on different cells in the subject that are within 100 nm of each other.

[0889] 51. The macromolecule according to any one of Examples 45 - 49, wherein the effector target or the address target is present on the cell surface.

[0890] 52. The macromolecule according to any one of Examples 1 - 51, wherein the macromolecule is a DNA polynucleotide.

[0891] 53. The macromolecule according to any one of Examples 1 - 51, wherein the macromolecule comprises RNA or an RNA - polypeptide conjugate.

[0892] 54. The macromolecule according to any one of Examples 1 - 51 and 53, wherein the macromolecule comprises a polypeptide.

[0893] 55. The macromolecule according to any one of Examples 1 - 51, wherein the macromolecule is a polypeptide.

[0894] 56. The macromolecule according to Example 54 or 55, wherein the polypeptide is an antibody or an antigen - binding fragment thereof.

[0895] 57. The macromolecule according to Example 56, wherein the first binding site and the second binding site each comprise a VH and / or a VL.

[0896] 58. The macromolecule according to Example 57, wherein the macromolecule is an antibody that comprises a first binding site specific for the effector target in the subject and a second binding site specific for the address target.

[0897] 59. The macromolecule according to Example 57 or 58, wherein the macromolecule is an asymmetric antibody or a symmetric antibody.

[0898] 60. The macromolecule according to any one of embodiments 56-59, wherein the antibody or antigen-binding fragment thereof comprises scFv, BsIgG, BsAb fragment, BiTE, dual-affinity retargeting 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 quadroma, ImmTac, HSAbody, IgG-IgG, Cov-X-Body, scFv1-PEG-scFv2, additional IgG, DVD-IgG, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, monomer, nanoCLAMP, bis-Fab, Fv, Fab, Fab'-SH, linear antibody, scFv, antibody with only heavy chain (Humabody), ScFab, IgG antibody fragment, single-chain variable region antibody, single-domain heavy-chain antibody, bispecific trisbody, BiKE, CrossMAb, dsDb, scDb, tandem dAb / VHH, triple dAb VHH, tetravalent dAb / VHH, Fab-scFv, Fab-Fv, or DART-Fc, adnectin, Kunitz-type inhibitor, or receptor decoy.

[0899] 61. The macromolecule according to embodiment 54, wherein the polypeptide is a ligand of the effector target or a ligand of the address target.

[0900] 62. The macromolecule according to embodiment 61, wherein the ligand is a natural ligand, a modified ligand or a synthetic ligand.

[0901] 63. The macromolecule according to embodiment 61 or 62, wherein the effector target or address target is a receptor and the polypeptide is its ligand.

[0902] 64. The macromolecule according to any one of embodiments 61-63, wherein the first binding site comprises an antibody or antigen-binding fragment thereof and the second binding site comprises a ligand of the address target.

[0903] 65. The macromolecule according to any one of embodiments 61-63, wherein the first binding site comprises a ligand of the effector target and the second binding site comprises an antibody or antigen-binding fragment thereof.

[0904] 66. A macromolecule as described in any one of Examples 1 - 51 and 54 - 65, 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.

[0905] 67. A macromolecule as described in any one of Examples 1 - 66, wherein the address target has a Gini coefficient higher 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.

[0906] 68. A macromolecule as described in any one of Examples 1 - 67, wherein the address target has a Tau coefficient higher than about 0.67, about 0.75, about 0.8, about 0.85, about 0.90 or about 0.95.

[0907] 69. A macromolecule as described in any one of Examples 1 - 68, wherein the effector target has a Gini coefficient lower than about 0.25, about 0.20 or about 0.15.

[0908] 70. A macromolecule as described in any one of Examples 1 - 69, wherein the effector target has a Tau coefficient lower than about 0.25, about 0.20 or about 0.15.

[0909] 71. A macromolecule as described in any one of Examples 1 - 70, which further comprises a third binding site.

[0910] 72. The macromolecule as described in Example 71, wherein the third binding site is the same as the first binding site.

[0911] 73. The macromolecule as described in Example 71, wherein the third binding site is the same as the second binding site.

[0912] 74. A macromolecule as described in any one of Examples 1 - 73, wherein the first binding site and the second binding site are directly connected to each other in the macromolecule.

[0913] 75. A macromolecule as described in any one of Examples 1 - 73, wherein the first binding site and the second binding site in the macromolecule are connected by a stabilizing domain.

[0914] 76. A macromolecule as described in any one of Examples 1 - 75, wherein the effector target is Notch2 and the address target is RAGE.

[0915] 77. The macromolecule as described in Example 76, wherein the RAGE signaling is not affected by the second site binding to the RAGE address target.

[0916] 78. A macromolecule as described in any one of Examples 1 - 75, wherein the effector target is Notch2 and the address target is uromodulin (UMOD).

[0917] 79. The macromolecule as described in Example 78, wherein the UMOD signal transduction is not affected by the binding of the second site to the UMOD address target.

[0918] 80. A macromolecule as described in any one of Examples 1 - 75, wherein the effector target is Notch2 and the address target is membrane metalloendopeptidase subunit beta (MEP1B).

[0919] 81. The macromolecule as described in Example 80, wherein the MEP1B signal transduction is not affected by the binding of the second site to the MEP1B address target.

[0920] 82. A macromolecule as described in any one of Examples 1 - 75, wherein the effector target is IL11Ra and the address target is RAGE.

[0921] 83. The macromolecule as described in Example 82, wherein the RAGE signal transduction is not affected by the binding of the second site to the RAGE address target.

[0922] 84. A macromolecule as described in any one of Examples 1 - 75, wherein the effector target is IL 11Ra and the address target is UMOD.

[0923] 85. The macromolecule as described in Example 84, wherein the UMOD signal transduction is not affected by the binding of the second site to the UMOD address target.

[0924] 86. The macromolecule as described in any one of Examples 1 - 85, wherein the subject is a human.

[0925] 87. A method of delivering a moiety to a target tissue or cell in a subject, the method comprising administering to the subject a macromolecule as described in any one of Examples 1 - 86, wherein the target tissue comprises an address target.

[0926] 88. The method as described in Example 87, wherein the moiety is a molecule.

[0927] 89. The method as described in Example 87 or 88, wherein the moiety is not a toxin.

[0928] 90. The method as described in Example 87, wherein the moiety is a cell.

[0929] 91. The method according to embodiment 90, wherein the portion is not a T cell or an NK cell.

[0930] 92. The method according to any one of embodiments 87 - 91, wherein the target tissue is not a tumor.

[0931] 93. A method of modulating an effector target in a target tissue, the method comprising administering to the tissue a macromolecule according to any one of embodiments 1 - 86, wherein the target tissue comprises an address target and an effector target.

[0932] 94. A method of biasing a binding agent to not bind to an effector target when the effector target is found in the heart or lung, the method comprising administering a macromolecule according to any one of embodiments 1 - 86, wherein the address target is substantially not expressed in the heart or lung.

[0933] 95. A method of modulating a target tissue in a subject, the method comprising administering to the subject a macromolecule according to any one of embodiments 1 - 86, wherein the target tissue comprises an address target and an effector target.

[0934] 96. A method of treating a subject suffering from a disease or disorder associated with an effector target, the method comprising administering to the subject a macromolecule according to any one of embodiments 1 - 86, wherein a first binding site of the macromolecule binds the effector target.

[0935] 97. A macromolecule comprising a first binding site and a second binding site, wherein:

[0936] (a) the first binding site is specific for an effector target in a subject, and

[0937] (b) the second binding site is specific for an address target expressed in the target tissue or cell in the subject;

[0938] wherein the second binding site localizes the first binding site to the address target such that the first binding site affects effector target signal transduction in the target tissue or cell,

[0939] wherein the first binding site substantially does not affect effector target signal transduction in the absence of being localized by the second binding site, and

[0940] wherein the second binding site does not bind to the binding site of the natural ligand of the address target, and wherein the macromolecule is linked to a small molecule.

[0941] 98. A macromolecule comprising a first binding site and a second binding site, wherein:

[0942] (a) The first binding site is specific for an effector target in a subject, and

[0943] (b) The second binding site is specific for an address target expressed in a target tissue or cell in the subject;

[0944] 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 target tissue or cell,

[0945] wherein the first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site, and

[0946] wherein the first binding site and the second binding site are directly connected to each other in the macromolecule, and the macromolecule is linked to a small molecule.

[0947] 99. A macromolecule comprising a first binding site and a second binding site, wherein:

[0948] (a) The first binding site is specific for an effector target in a subject, and

[0949] (b) The second binding site is specific for an address target expressed in a target tissue or cell in the subject;

[0950] 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 target tissue or cell,

[0951] wherein the first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site, and

[0952] wherein the first binding site and the second binding site are connected to each other through a stabilizing domain, and the macromolecule is linked to a small molecule.

[0953] 100. A macromolecule comprising a first binding site and a second binding site, wherein:

[0954] (a) The first binding site is specific for an effector target in a subject, and

[0955] (b) The second binding site is specific for an address target expressed in a target tissue or cell in the subject;

[0956] 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 target tissue or cell,

[0957] wherein the first binding site substantially does not affect effector target signal transduction in the absence of being localized by the second binding site, and

[0958] wherein the effector target and / or the address target are expressed on the structural organization of the host, and wherein the macromolecule is linked to a small molecule.

[0959] 101. A pharmaceutical composition comprising a macromolecule as described in any one of Examples 1-86.

[0960] 102. A pharmaceutical composition comprising a macromolecule and one or more pharmaceutically acceptable excipients,

[0961] wherein the macromolecule comprises a first binding site and a second binding site, wherein:

[0962] (a) the first binding site is specific for an effector target in a subject, and

[0963] (b) the second binding site is specific for an address target expressed in a target tissue or cell in the subject;

[0964] wherein the second binding site localizes the first binding site to the address target such that the first binding site affects effector target signal transduction in the target tissue or cell, and

[0965] wherein the first binding site substantially does not affect effector target signal transduction in the absence of being localized by the second binding site, and wherein the macromolecule is linked to a small molecule.

[0966] 103. The pharmaceutical composition according to Example 101 or 102, wherein the pharmaceutical composition is an RNA pharmaceutical composition.

[0967] 104. The pharmaceutical composition according to any one of Examples 101-103, further comprising a carrier.

[0968] 105. The pharmaceutical composition according to Example 104, wherein the carrier is a lipid nanoparticle.

[0969] 106. The pharmaceutical composition according to Example 104, wherein the carrier is a viral vector.

[0970] 107. The pharmaceutical composition according to Example 104, wherein the carrier is a membrane-based carrier.

[0971] 108. The pharmaceutical composition according to Example 107, wherein the membrane-based carrier is a cell.

[0972] 109. The pharmaceutical composition according to embodiment 107, wherein the membrane-based carrier is a vesicle.

[0973] 110. A method for modulating the activity of an effector target in the skin of a subject, the method comprising administering to the subject a macromolecule comprising a first binding site and a second binding site, wherein:

[0974] (a) the first binding site is specific for the effector target in the subject, and

[0975] (b) the second binding site is specific for desmoglein-1 (DSG-1), wherein the macromolecule is linked to a small molecule.

[0976] 111. A method for modulating the activity of an effector target in the lung of a subject, the method comprising administering to the subject a macromolecule comprising a first binding site and a second binding site, wherein:

[0977] (a) the first binding site is specific for the effector target in the subject, and

[0978] (b) the second binding site is specific for RAGE, wherein the macromolecule is linked to a small molecule.

[0979] 112. A method for modulating the activity of an effector target in the kidney of a subject, the method comprising administering to the subject a macromolecule comprising a first binding site and a second binding site, wherein:

[0980] (a) the first binding site is specific for the effector target in the subject, and

[0981] (b) the second binding site is specific for cadherin 16 (CDH16), wherein the macromolecule is linked to a small molecule.

[0982] 113. A method for modulating the activity of an effector target in the intestine of a subject, the method comprising administering to the subject a macromolecule comprising a first binding site and a second binding site, wherein:

[0983] (a) the first binding site is specific for the effector target in the subject, and

[0984] (b) the second binding site is specific for cadherin 17 (CDH17), wherein the macromolecule is linked to a small molecule.

[0985] 114. A method for localizing a macromolecule at a target tissue or cell of a subject, the method comprising administering to the subject a macromolecule comprising a first binding site and a second binding site, wherein:

[0986] (a) the first binding site is specific for an effector target in the subject, and

[0987] (b) the second binding site is specific for an address target expressed in the target tissue or cell in the subject; wherein:

[0988] (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 target tissue or cell;

[0989] (ii) the second binding site substantially does not affect signaling after binding to the address target; and

[0990] (iii) the first binding site substantially does not affect effector target signaling in the absence of being localized by the second binding site; and

[0991] allowing the macromolecule to localize at the target tissue or cell of the subject, wherein the macromolecule is linked to a small molecule.

[0992] 115. A method for concentrating a macromolecule in a target tissue or cell of a subject, the method comprising administering to the subject a macromolecule comprising a first binding site and a second binding site, wherein:

[0993] (a) the first binding site is specific for an effector target in the subject, and

[0994] (b) the second binding site is specific for an address target expressed in the target tissue or cell in the subject; wherein:

[0995] (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 target tissue or cell;

[0996] (ii) the second binding site substantially does not affect signaling after binding to the address target; and

[0997] (iii) the first binding site substantially does not affect effector target signaling in the absence of being localized by the second binding site;

[0998] and allowing the macromolecule to concentrate at the target tissue or cells of the subject, wherein at a time point between 1 day and 7 days after administering the macromolecule to the subject, at least 25% of the detectable macromolecule in the subject is detected at the target tissue or cells, wherein the macromolecule is linked to a small molecule.

[0999] 116. The method according to embodiment 114 or 115, wherein the potency of the first binding site at the target tissue or cells is significantly increased relative to a reference macromolecule lacking the second binding site.

[1000] 117. The method according to embodiment 114 or 115, wherein effector target signaling by the macromolecule in non-target tissues or cells of the subject is significantly reduced relative to a reference macromolecule lacking the second binding site.

[1001] 118. The method according to embodiments 110-117, wherein the macromolecule is a macromolecule according to any one of embodiments 1-86.

[1002] 119. The macromolecule, method or pharmaceutical composition according to any one of claims 1-118, wherein the macromolecule and the small molecule are linked by a linker.

[1003] 120. The macromolecule, method or pharmaceutical composition according to claim 119, wherein the linker is a cleavable linker.

[1004] 121. The macromolecule, method or pharmaceutical composition according to claim 119, wherein the linker is a non-cleavable linker.

[1005] 122. The macromolecule, method or pharmaceutical composition according to any one of claims 1-121, wherein the first binding site, the second binding site or both are polypeptides.

[1006] 123. The macromolecule, method or pharmaceutical composition according to any one of claims 1-122, wherein a single small molecule is linked to the macromolecule.

[1007] 124. The macromolecule, method or pharmaceutical composition according to any one of claims 1-122, wherein multiple small molecules are linked to the macromolecule.

[1008] 125. The macromolecule, method or pharmaceutical composition according to claim 124, wherein each of the small molecules is the same.

[1009] 126. The macromolecule, method or pharmaceutical composition according to claim 124, wherein at least two of the small molecules are different from each other.

[1010] 127. The macromolecule, method or pharmaceutical composition according to any one of Examples 1 - 126, wherein the target cell is an immune cell and the second binding site is specific for CD20.

[1011] 128. The macromolecule, method or pharmaceutical composition according to any one of Examples 1 - 126, wherein the target cell is an immune cell and the second binding site is specific for CD33.

[1012] 129. The macromolecule, method or pharmaceutical composition according to any one of Examples 1 - 128, wherein the small molecule is fluocinonide.

[1013] 130. The macromolecule, method or pharmaceutical composition according to any one of Examples 1 - 128, wherein the small molecule is ibrutinib.

[1014] 131. The macromolecule, method or pharmaceutical composition according to any one of Examples 1 - 128, wherein the small molecule is tofacitinib.

[1015] 132. The macromolecule, method or pharmaceutical composition according to any one of Examples 1 - 128, wherein the macromolecule linked to the small molecule exhibits increased internalization relative to a control macromolecule not linked to the small molecule.

[1016] 133. The macromolecule, method or pharmaceutical composition according to any one of Examples 1 - 128, wherein the macromolecule substantially does not induce cell death.

[1017] 134. The macromolecule, method or pharmaceutical composition according to any one of Examples 1 - 128, wherein the macromolecule modulates the function of immune cells such as B cells or myeloid cells.

[1018] Although the foregoing invention has been described in detail for purposes of clarity of understanding by way of illustration and example, the description and examples should not be construed as limiting the scope of the invention.

Claims

1. A method of localizing a macromolecule at a target tissue or 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 subject, and (b) the second binding site is specific for an address target expressed in the target tissue or 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 target tissue or cell; (ii) the second binding site substantially does not affect signaling after binding to the address target; and (iii) the first binding site substantially does not affect effector target signaling in the absence of localization by the second binding site; and allowing the macromolecule to localize at the target tissue or cell of the subject, wherein the macromolecule is linked to a small molecule.

2. The method of claim 1, wherein the macromolecule and the small molecule are linked by a linker.

3. The method of claim 2, wherein the linker is a cleavable linker.

4. The method of claim 2, wherein the linker is a non-cleavable linker.

5. The method of claim 1, wherein at a time point between 1 day and 7 days after administering the macromolecule to the subject, at least 25% of the detectable macromolecule in the subject is detected at the target tissue or cell.

6. The method of claim 1, wherein the potency of the first binding site at the target tissue or cell is significantly increased relative to a reference macromolecule lacking the second binding site.

7. The method of claim 6, wherein the first binding site has a low affinity for the effector target.

8. The method of claim 6, wherein the first binding site has a low avidity for the effector target.

9. 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.

10. The method of claim 1, wherein the avidity of the first binding site for the effector target is lower than the avidity of the second binding site for the address target.

11. The method of claim 1, wherein effector target signaling by the macromolecule in non-target tissues or cells of the subject is significantly reduced relative to a reference macromolecule lacking the second binding site.

12. The method of claim 1, wherein the address target is regionally expressed in the subject.

13. The method of claim 1, wherein the address target is locally expressed in the subject.

14. The method of claim 1, wherein the expression of the address target is limited to a cell type in the subject.

15. The method of claim 1, wherein the address target is expressed only by cells in a specific cell state in the subject.

16. The method according to claim 1, wherein the address target is expressed only by cells in a diseased state in the subject.

17. The method according to claim 1, wherein the first binding site or the second binding site comprises a polypeptide.

18. The method according to claim 17, wherein the polypeptide is an antibody or an antigen-binding fragment thereof.

19. The method according to claim 18, wherein the macromolecule is an antibody, and the antibody comprises a first binding site specific for the effector target in the subject and a second binding site specific for the address target.

20. The method according to claim 17, wherein the polypeptide is a ligand of the effector target or a ligand of the address target.

21. The method according to claim 20, 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.

22. The method according to claim 1, wherein the target tissue is skin and the second binding site is specific for desmoglein-1 (DSG-1).

23. The method according to claim 1, wherein the target tissue is lung tissue and the second binding site is specific for RAGE.

24. The method according to claim 1, wherein the target tissue is kidney tissue and the second binding site is specific for cadherin 16 (CDH16).

25. The method according to claim 1, wherein the target tissue is intestinal tissue and the second binding site is specific for cadherin 17 (CDH17).

26. The method according to claim 1, wherein the target cell is an immune cell and the second binding site is specific for CD20.

27. The method according to claim 1, wherein the target cell is an immune cell and the second binding site is specific for CD33.

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