Compositions and methods for targeted delivery of therapeutic agents

Abstract

[0003] Polymer compositions and related methods are provided that effect 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. [0004] Compositions and methods are described that relate to polymers, e.g., ANDbodies™, that include an effector target-binding domain specific for the effector target and an address-binding domain specific for the address target. The polymer is linked to a small molecule.

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is 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 technology]

[0002] Unwanted off-target effects are a challenge for desirable therapeutic targets that are otherwise present in healthy as well as diseased tissues. Summary of the Invention [Problem to be solved by the invention]

[0003] The present disclosure describes, in part, polymer compositions and related methods for achieving 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. Generally, the compositions described herein include polymers, e.g., ANDbodies™, that include an effector target binding domain specific for the effector target and an address binding domain specific for the address target. The address target is generally sufficiently localized in a subject to target the polymer to the 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. Furthermore, the address target binding domain does not affect signal transduction upon binding to the address target. However, localization of the effector target binding domain by the address target binding domain allows the effector target binding domain to bind to the effector target sufficiently to trigger an effect on signal transduction by the effector target in the target cell or tissue. Additionally, the polymers 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, e.g., a cell, tissue, or organ, in a subject while avoiding undesirable off-target effects. [Means for solving the problem]

[0004] In one aspect, the disclosure provides a method for localizing a macromolecule to 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 of the subject, and (b) the second binding site is specific for an address target expressed in the target tissue or cell of the subject; wherein (i) the second binding site localizes the first binding site to the address target such that the first binding site affects signal transduction of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signal transduction upon binding to the address target; and further, (iii) the first binding site does not substantially affect signal transduction of the effector target in the absence of localization by the second binding site; allowing the macromolecule to localize to the target tissue or cell of the subject, wherein the macromolecule is linked to a small molecule.

[0005] In some embodiments, the polymer and small molecule are linked by a linker (eg, a cleavable linker or a non-cleavable linker).

[0006] One or more small molecules may be linked to a polymer.

[0007] In some embodiments, at least 25% of the macromolecule detectable in a subject is detected in the target tissue or cells 1 to 7 days after administration of the macromolecule to the subject.

[0008] In some embodiments, the efficacy of the first binding moiety in the target tissue or cell is substantially increased compared to a control macromolecule lacking the second binding moiety.

[0009] In some embodiments, the first binding site has a low affinity for the effector target.

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

[0011] 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.

[0012] 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.

[0013] In some embodiments, effector target signaling by the macromolecule in non-target tissues or cells of the subject is substantially reduced compared to a control macromolecule lacking the second binding moiety.

[0014] In some embodiments, the address target is regionally expressed in the subject. In some embodiments, the address target is locally expressed in the subject. In some embodiments, expression of the address target is limited to one cell type in the subject.

[0015] In some embodiments, an address target is expressed by only one cell of a subject when in a particular cellular state.

[0016] In some embodiments, the address target is expressed by only one cell of a subject in a disease state.

[0017] In some embodiments, the first binding site or the second binding site comprises a polypeptide.

[0018] In some embodiments, the polypeptide is an antibody or an antigen-binding fragment thereof.

[0019] In some embodiments, the macromolecule is an antibody comprising a first binding site specific for an effector target in a subject and a second binding site specific for an address target.

[0020] In some embodiments, the polypeptide is a ligand of an effector target or a ligand of an address target.

[0021] In some embodiments, (a) the first binding site comprises an antibody or 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 antigen-binding fragment thereof.

[0022] In some embodiments, the target tissue is skin and the second binding site is specific for desmoglein-1 (DSG-1).

[0023] In some embodiments, the target tissue is lung tissue and the second binding site is specific for RAGE.

[0024] In some embodiments, the target tissue is kidney tissue and the second binding site is specific for cadherin 16 (CDH16).

[0025] In some embodiments, the target tissue is intestinal tissue and the second binding site is specific for cadherin 17 (CDH17).

[0026] In another aspect, the present disclosure provides a polymer comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target of a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell of the subject; wherein (i) the second binding site localizes the first binding site to the address target such that the first binding site affects signaling of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signaling upon binding to the address target; and (iii) the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site, wherein the polymer is linked to a small molecule.

[0027] In another aspect, the present disclosure provides a polymer comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target of a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell of the subject; wherein (i) the second binding site localizes the first binding site to the address target such that the first binding site affects signal transduction of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signal transduction upon binding to the address target; and (iii) the first binding site does not substantially affect signal transduction of the effector target in the absence of localization by the second binding site; in this case, localization of the polymer to a non-target tissue or cell is substantially reduced compared to localization of a control polymer lacking the second binding site, wherein the polymer is linked to a small molecule.

[0028] In another aspect, the present disclosure provides a polymer comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target of a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell of the subject; wherein (i) the second binding site localizes the first binding site to the address target such that the first binding site affects signal transduction of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signal transduction upon binding to the address target; and (iii) the first binding site does not substantially affect signal transduction of the effector target in the absence of localization by the second binding site; in this case, localization of the polymer to the target tissue or cell is substantially increased compared to localization of a control polymer lacking the second binding site, wherein the polymer is linked to a small molecule.

[0029] In another aspect, the present disclosure provides a polymer comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target of a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell of the subject; wherein (i) the second binding site localizes the first binding site to the address target such that the first binding site affects signaling of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signaling upon binding to the address target; and (iii) the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; wherein at least 25% of the polymer administered to the subject is detected in the target tissue or cell 1 to 7 days after administration, wherein the polymer is linked to a small molecule.

[0030] In another aspect, the present disclosure provides a polymer comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target of a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell of the subject; wherein (i) the second binding site localizes the first binding site to the address target such that the first binding site affects signaling of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signaling upon binding to the address target; and (iii) the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; 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 polymer is linked to a small molecule.

[0031] In another aspect, the present disclosure provides a polymer comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target of a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell of the subject; wherein (i) the second binding site localizes the first binding site to the address target such that the first binding site affects signal transduction of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signal transduction upon binding to the address target; and (iii) the first binding site does not substantially affect signal transduction of the effector target in the absence of localization by the second binding site; wherein the binding activity of the first binding site for the effector target is lower than the binding activity of the second binding site for the address target, and wherein the polymer is linked to a small molecule.

[0032] In another aspect, the present disclosure provides a polymer comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target of a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell of the subject; wherein (i) the second binding site localizes the first binding site to the address target such that the first binding site affects signaling of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signaling upon binding to the address target; and (iii) the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; in this case, the efficacy of the first binding site in the target tissue or cell is substantially increased compared to a control polymer lacking the second binding site, wherein the polymer is linked to a small molecule.

[0033] In some embodiments of any of the above aspects, the polymer and small molecule are linked by a linker (e.g., a cleavable linker or a non-cleavable linker).

[0034] One or more small molecules may be linked to a polymer.

[0035] In some embodiments, the first binding site has a low affinity for the effector target.

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

[0037] 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.

[0038] 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.

[0039] 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; and (b) the EC 50 addresses the EC of the second binding site to the target 50 (c) IC of the first binding site for the effector target 50 is the IC of the second binding site to the address target 50 Higher.

[0040] 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.

[0041] In some embodiments, the affinity of the second binding site for the address target has a Kd of greater than about 1 nM, greater than about 2 nM, or greater than about 50 nM.

[0042] In some embodiments, the effector target is a protein, lipid, or sugar.

[0043] In some embodiments, the effector target is a cell membrane-associated target.

[0044] In some embodiments, the effector target is a protein, hi some embodiments, the effector target is a secreted protein.

[0045] In some embodiments, the effector target is encoded by a gene selected from the group consisting of the genes listed in Table 1.

[0046] In some embodiments, the macromolecule agonizes the effector target.

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

[0048] In some embodiments, the address target is a protein, lipid, or sugar.

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

[0050] In some embodiments, expression of an effector target or address target is expression of an RNA sequence encoding the effector target or address target.

[0051] In some embodiments, the expression levels of effector targets or address targets are assessed using an RNA-seq dataset.

[0052] In some embodiments, the RNA-seq dataset is a Genotype-Tissue Expression (GTEx) dataset or a Human Protein Atlas (HPA) dataset.

[0053] In some embodiments, expression of an effector target or address target is protein expression.

[0054] In some embodiments, the effector target is systemically expressed in the subject.

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

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

[0057] In some embodiments, the address target is regionally expressed in the subject.

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

[0059] In some embodiments, expression of the address target is restricted to one cell type of interest.

[0060] In some embodiments, the address target is a soluble protein or an extracellular matrix (ECM) associated protein that is not present in detectable amounts on the cell surface.

[0061] In some embodiments, the address target is expressed in the ECM and is not present in detectable amounts elsewhere in the subject.

[0062] In some embodiments, the address target is only expressed in cells of interest when in a particular cellular state.

[0063] In some embodiments, the address target is expressed only in cells of the subject when in a disease state.

[0064] In some embodiments, the address target is not expressed in tissues where binding of the second binding moiety to the effector target is deleterious to the subject.

[0065] In some embodiments, the binding site for an address target does not detectably bind to the binding site of the address target's natural ligand.

[0066] In some embodiments, expression of the effector target or address target is in the following: minor salivary glands, thyroid, lung, breast, mammary tissue, pancreas, adrenal gland, liver, kidney, renal cortex, renal medulla, adipose visceral tissue, omentum, small intestine, terminal ileum, fallopian tube, ovary, uterus, skin, non-sun-exposed skin, suprapubic skin, cervix, endocervix, cervicovaginal region, vagina, sun-exposed skin, calf skin, anterior cingulate cortex, cortex), Brodmann area 24 (BA24), basal ganglia, caudate nucleus, putamen, nucleus accumbens, hypothalamus, amygdala, hippocampus, cerebellum, cerebellar hemispheres, substantia nigra, pituitary gland, spinal cord, cervical spinal cord, artery, aorta, heart, atrial appendage, coronary artery, left ventricle, esophagus, esophageal mucosa, esophageal muscularis, gastroesophageal junction, spleen, stomach, colon, transverse colon, sigmoid colon, testis, whole blood cells, EBV-transformed lymphocytes, tibial artery, or tibial nerve tissue.

[0067] In some embodiments, expression of an effector target or address target comprises expression in skin tissue, lung tissue, kidney tissue, or intestinal tissue, hi some embodiments, expression of an address target is substantially higher in skin tissue, lung tissue, kidney tissue, or intestinal tissue than in any other tissue.

[0068] In some embodiments, the effector target and / or address target is expressed on a structural tissue of the subject.

[0069] In some embodiments, the effector target and the address target are on the same cell.

[0070] In some embodiments, the effector target and the address target are on different cells.

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

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

[0073] In some embodiments, the effector target and the address target are on different cells within the same tissue.

[0074] 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.

[0075] In some embodiments, the effector target and the address target are on different cells located within 100 nm of each other in the subject.

[0076] In some embodiments, either the effector target or the address target is present on the cell surface.

[0077] In some embodiments, the macromolecule is a DNA polynucleotide.

[0078] In some embodiments, the macromolecule comprises an RNA or an RNA-polypeptide conjugate.

[0079] In some embodiments, the polymer comprises a polypeptide. In some embodiments, the polymer is a polypeptide.

[0080] In some embodiments, the polypeptide is an antibody or an antigen-binding fragment thereof.

[0081] In some embodiments, the first binding site and the second binding site comprise a VH and / or a VL, respectively.

[0082] In some embodiments, the macromolecule is an antibody comprising a first binding site specific for an effector target in a subject and a second binding site specific for an address target.

[0083] In some embodiments, the macromolecule is an asymmetric or symmetric antibody.

[0084] In some embodiments, the antibody or antigen-binding fragment thereof comprises: an scFv, a BsIgG, a BsAb fragment, a BiTE, a dual-affinity re-targeting protein (DART), a tandem diabody (TandAb), a diabody, a Fab2, a bivalent scFv (di-scFv), a chemically conjugated F(ab')2, an Ig molecule with three or four different antigen-binding sites, a DVI-IgG four-in-one (DVI-IgG four-in-one), ImmTac, HSAbody, IgG-IgG, Cov-X-Body, scFv1-PEG-scFv2, conjugated IgG, DVD-IgG, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, monobody, nanoCLAMP, bis-Fab, Fv, Fab'-SH, linear antibody, scFv, antibody having only a heavy chain (Humabody), ScFab, IgG antibody fragment, single-chain variable region antibody, single-domain heavy chain antibody, bispecific triple body, BiKE, CrossMAb, dsDb, scDb, tandem dAb / VHH, triple dAb VHH, tetravalent dAb / VHH, Fab-scFv, Fab-Fv, or DART-Fc, adectin, Kunitz inhibitor, or receptor decoy.

[0085] In some embodiments, the polypeptide is a ligand of an effector target or a ligand of an address target.

[0086] In some embodiments, the ligand is a natural ligand, a modified ligand, or a synthetic ligand.

[0087] In some embodiments, the effector target or address target is a receptor and the polypeptide is its ligand.

[0088] In some embodiments, the first binding site comprises an antibody or antigen-binding fragment thereof, and the second binding site comprises a ligand that addresses the target.

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

[0090] 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.

[0091] In some embodiments, the address target has a Gini coefficient greater than about 0.4, about 0.5, about 0.57, about 0.65, about 0.7, about 0.85, about 0.90, or about 0.95.

[0092] In some embodiments, the address target has a Tau coefficient greater than about 0.67, about 0.75, about 0.8, about 0.85, about 0.90, or about 0.95.

[0093] In some embodiments, the effector target has a Gini coefficient of less than about 0.25, about 0.20, or about 0.15.

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

[0095] In some embodiments, the polymer 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.

[0096] In some embodiments, the first binding site and the second binding site are directly linked to each other in the polymer.

[0097] In some embodiments, the first binding site and the second binding site in the macromolecule are connected by a stable domain.

[0098] In some embodiments, the effector target is Notch2 and the address target is RAGE.

[0099] In some embodiments, RAGE signaling is not affected by the second moiety binding to the RAGE address target.

[0100] In some embodiments, the effector target is Notch2 and the address target is uromodulin (UMOD).

[0101] In some embodiments, UMOD signaling is not affected by the second moiety binding to the UMOD address target.

[0102] In some embodiments, the effector target is Notch2 and the address target is meprin A subunit beta (MEP1B).

[0103] In some embodiments, MEP1B signaling is not affected by the second moiety binding to the MEP1B address target.

[0104] In some embodiments, the effector target is IL11Ra and the address target is RAGE. In some embodiments, RAGE signaling is not affected by the second moiety binding to the RAGE address target.

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

[0106] In some embodiments, the subject is a human.

[0107] In another aspect, the present disclosure provides a method for delivering a moiety to a target tissue or cell in a subject, comprising administering to the subject a macromolecule described in any one of claims 1-86, wherein the target tissue comprises an address target.

[0108] In some embodiments, the moiety is a molecule.

[0109] In some embodiments, the moiety is not a toxin.

[0110] In some embodiments, the moiety is a cell.

[0111] In some embodiments, the moiety is not a T cell or an NK cell.

[0112] In some embodiments, the target tissue is not a tumor.

[0113] In another aspect, the present disclosure provides a method of modulating an effector target in a target tissue, comprising administering to the tissue a macromolecule described in any one of claims 1-86, wherein the target tissue comprises an address target and an effector target.

[0114] In another aspect, the present disclosure provides a method of biasing a binding agent away from binding to an effector target when the effector target is found in the heart or lung, comprising administering a macromolecule of any one of claims 1-86, wherein the address target is not substantially expressed in the heart or lung.

[0115] In another aspect, the present disclosure provides a method for modulating a target tissue in a subject, comprising administering to the subject a macromolecule described in any one of claims 1 to 86, wherein the target tissue comprises an address target and an effector target.

[0116] In another aspect, the disclosure provides a method of treating a subject having a disease or condition associated with an effector target, comprising administering to the subject a macromolecule of any one of claims 1-86, wherein a first binding moiety of the macromolecule binds to the effector target.

[0117] In another aspect, the disclosure provides a polymer comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target of a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell of the subject; wherein the second binding site localizes the first binding site to the address target such that the first binding site affects signaling of the effector target in the target tissue or cell, wherein the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site, and further, the second binding site does not bind to a binding site of a natural ligand of the address target, wherein the polymer is linked to a small molecule.

[0118] In another aspect, the present disclosure provides a polymer comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target of a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell of the subject; wherein the second binding site localizes the first binding site to the address target such that the first binding site affects signaling of the effector target in the target tissue or cell, wherein the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; and further wherein the first binding site and the second binding site are directly linked to each other in the polymer, wherein the polymer is linked to a small molecule.

[0119] In another aspect, the present disclosure provides a polymer comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target of a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell of the subject; wherein the second binding site localizes the first binding site to the address target such that the first binding site affects signaling of the effector target in the target tissue or cell, wherein the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site, and further wherein the first binding site and the second binding site are linked to each other by a stable domain, and wherein the polymer is linked to a small molecule.

[0120] In another aspect, the disclosure provides a polymer comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target of a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell of the subject; wherein the second binding site localizes the first binding site to the address target such that the first binding site affects signaling of the effector target in the target tissue or cell, wherein the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site, and further wherein the effector target and / or the address target are expressed on a structural tissue of a host, and wherein the polymer is linked to a small molecule.

[0121] In another aspect, the present disclosure provides a pharmaceutical composition comprising the macromolecule of any one of the preceding embodiments.

[0122] In another aspect, the disclosure provides a pharmaceutical composition comprising a polymer and one or more pharmaceutically acceptable excipients, wherein the polymer comprises a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target of a subject, and (b) the second binding site is specific for an address target expressed in a target tissue or cell of the subject; wherein the second binding site localizes the first binding site to the address target such that the first binding site affects signaling of the effector target in the target tissue or cell, and wherein the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site, and wherein the polymer is linked to a small molecule.

[0123] In some embodiments, the pharmaceutical composition is an RNA pharmaceutical composition.

[0124] In some embodiments, the pharmaceutical composition further comprises a carrier.

[0125] In some embodiments, the carrier is a lipid nanoparticle.

[0126] In some embodiments, the carrier is a viral vector.

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

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

[0129] In some embodiments, the membrane carrier is a vesicle.

[0130] In another aspect, the present disclosure provides 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 (a) the first binding site is specific for an effector target of the subject, and (b) the second binding site is specific for desmoglein-1 (DSG-1), and wherein the macromolecule is linked to a small molecule.

[0131] In another aspect, the disclosure provides 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 (a) the first binding site is specific for an effector target of the subject, and (b) the second binding site is specific for RAGE, and wherein the macromolecule is linked to a small molecule.

[0132] In another aspect, the disclosure provides a method of 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 (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), and wherein the macromolecule is linked to a small molecule.

[0133] In another aspect, the disclosure provides a method of 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 (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), and wherein the macromolecule is linked to a small molecule.

[0134] In another aspect, the disclosure provides a method for localizing a macromolecule to 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 of the subject, and (b) the second binding site is specific for an address target expressed in the target tissue or cell of the subject; wherein (i) the second binding site localizes the first binding site to the address target such that the first binding site affects signal transduction of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signal transduction upon binding to the address target; and further, (iii) the first binding site does not substantially affect signal transduction of the effector target in the absence of localization by the second binding site; allowing the macromolecule to localize to the target tissue or cell of the subject, wherein the macromolecule is linked to a small molecule.

[0135] In another aspect, the disclosure provides 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 (a) the first binding site is specific for an effector target of the subject, and (b) the second binding site is specific for an address target expressed in the target tissue or cell of the subject; wherein (i) the second binding site binds to the first binding site such that the first binding site affects signaling of the effector target in the target tissue or cell. to the address target; (ii) the second binding moiety does not substantially affect signal transduction upon binding to the address target; and (iii) the first binding moiety does not substantially affect signal transduction of the effector target in the absence of localization by the second binding moiety; and enables the macromolecule to concentrate in the target tissue or cell of the subject, wherein at least 25% of the macromolecule detectable in the subject is detected in the target tissue or cell 1 to 7 days after administration of the macromolecule to the subject, wherein the macromolecule is linked to a small molecule.

[0136] In some embodiments, the efficacy of the first binding moiety in the target tissue or cell is substantially increased compared to a control macromolecule lacking the second binding moiety.

[0137] In some embodiments, effector target signaling by the macromolecule in non-target tissues or cells of the subject is substantially reduced compared to a control macromolecule lacking the second binding moiety.

[0138] In some embodiments, the polymer is the polymer of any one of the preceding embodiments.

[0139] The details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, as well as from the appended claims. [Brief explanation of the drawings]

[0140] [Figure 1]Figure 1 is a schematic diagram illustrating an exemplary ANDbody™ molecule and its use as a logic gate pharmaceutical. Figure 1 shows the broad distribution of a therapeutic target, e.g., an effector target, in a human subject without address targeting (right side) and the localized and restricted distribution due to address targeting achieved by an address target binding domain (left side). Figure 1 also provides a representative bipartite structure of an ANDbody having an address target binding domain linked to an effector target binding domain, which contains a functional moiety, e.g., a moiety that modulates, e.g., agonizes or antagonizes, the target effector in the address-targeted cell or tissue. The address target binding domain directs the ANDbody to a desired location, such as a target cell or tissue, and enables the effector target binding domain to bind to the therapeutic effector target in a localized and restricted distribution area. In some embodiments, high affinity of the effector domain for the target effector may not be required; localization of the effector target binding domain by the address target binding domain allows the effector target binding domain to bind sufficiently to the effector target to induce signaling effects by the effector target in the target cell or tissue, despite the low affinity of the effector domain for the effector target. Alternatively, the address target binding domain can be used to transport molecular or cellular cargo to a desired address. [Figure 2] Figure 2 is a schematic map showing the activity of exemplary effector targets that can be restricted to tissues or cells of interest by developing ANDbody therapeutics consisting of an effector targeting domain and an address targeting domain. These ANDbody biologics provide potent, address-restricted medicines through this technology. [Figure 3] FIG. 3 provides exemplary structures of ANDbody biologics that can be designed in accordance with the present technology, including, but not limited to, the following: asymmetric antibodies, dual affinity retargeting proteins (DARTs), tandem diabodies (TandAbs), diabodies, Fab2s, IgG(L,H)-Fvs, or BiTEs. [Figure 4] FIG. 4 shows the EC50 curves for an exemplary single effector targeting domain (dashed line), such as a monospecific biologic (e.g., scFv) having a single binding domain to an effector target, compared to the EC50 (solid line) for an exemplary bispecific ANDbody biologic (e.g., di-scFc) comprising an address target binding domain and an effector target binding domain, whereby the single effector targeting domain (typically broadly expressed) is targeted / restricted to local, address target-specific tissues and / or cells, effectively increasing the affinity of the effector target binding domain for the effector target binding site, as evidenced by a shift of the curve to the left (lower EC50, higher affinity). [Figure 5A] Figure 5A is a bar graph showing the level of fluorescence intensity detected in the indicated tissues in mice treated with anti-DSG1 antibody PRO003 conjugated to IRDYE® 800CW. Data are presented as the average of three mice. To adjust for differences in labeling efficiency, values ​​are presented with the most intense signal set to 1. [Figure 5B] Figure 5B is a bar graph showing the level of fluorescence intensity detected in the indicated tissues in mice treated with anti-DSG1 antibody PRO004 conjugated to IRDYE® 800CW or mice treated with vehicle control (untreated). Data are presented as the average of three mice. To adjust for differences in labeling efficiency, values ​​are presented with the most intense signal set to 1. [Figure 6A] Figure 6A is a bar graph showing the level of fluorescence intensity detected in the indicated tissues in mice treated with the anti-RAGE antibody PRO001 conjugated to IRDYE® 800CW or with vehicle control. Data are presented as the mean value of three mice. To adjust for differences in labeling efficiency, values ​​are presented with the most intense signal set to 1. [Figure 6B]Figure 6B is a bar graph showing the levels of fluorescence intensity detected in the indicated tissues in mice treated with the anti-RAGE antibody PRO002 conjugated to IRDYE® 800CW or vehicle control. Data are shown as the mean value of three mice. To adjust for differences in labeling efficiency, values ​​are shown with the most intense signal set to 1. [Figure 7] Figure 7 is a pair of photomicrographs showing representative IHC staining of an anti-human secondary antibody conjugated to horseradish peroxidase in lung tissue from a Balb / C mouse treated by tail vein injection of 3 mg / kg of the anti-RAGE antibody PRO002 (left panel) compared to an untreated mouse (right panel). PRO002 contains a human IgG1 backbone. [Figure 8] Figure 8 is a bar graph showing the level of fluorescence intensity detected in the indicated tissues in mice treated with the anti-CDH16 antibody PRO056 conjugated to IRDYE® 800CW or with vehicle control. Data are presented as the mean value of three mice. To adjust for differences in labeling efficiency, values ​​are presented with the most intense signal set to 1. [Figure 9] 9 is a bar graph showing the level of fluorescence intensity detected in the indicated tissues in mice treated with the anti-CDH17 antibody PRO061 conjugated to IRDYE® 800CW or with vehicle control. Data are presented as the mean value of three mice. To adjust for differences in labeling efficiency, values ​​are presented with the most intense signal set to 1. [Figure 10] Figure 10 is a series of photomicrographs showing staining of Notch2 antagonist mAbs: PRO034, PRO035, PRO036, and the corresponding RAGE-targeting ANDbodies: PRO051, PRO052, PRO053, in fresh frozen healthy mouse tissue microarray (FF TMA) sections. Lung sections are indicated by boxes. [Figure 11]11 is a plot showing the concentrations of PRO052, a control antibody that binds RAGE and respiratory syncytial virus (RSV) glycoprotein F (RAGE XT-4 / motavizumab), and a control antibody that binds Notch2 and RSV glycoprotein F (Notch2-2 / motavizumab) as detected by sandwich ELISA. Points represent the average of three mice. Error bars represent standard deviation. [Figure 12] FIG. 12 is a series of schematic diagrams showing the design of the PRO023, PRO025, PRO024, PRO027, and PRO026 IL-10 / DSG1 ANDbodies. [Figures 13A-13D] Figure 13A is a bar graph showing tumor necrosis factor alpha (TNFα) levels in peripheral blood mononuclear cell (PBMC) cell cultures after pre-stimulation with hrIL-10 followed by treatment with lipopolysaccharide (LPS) for the indicated times. Figure 13B is a bar graph showing TNFα levels in PBMC cell cultures after pre-stimulation with anti-DSG1 monoclonal antibody (mAb) followed by treatment with LPS for the indicated times. Figure 13C is a bar graph showing TNFα levels in PBMC cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO024 followed by treatment with LPS for the indicated times. Figure 13D is a bar graph showing TNFα levels in PBMC cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO026 followed by treatment with LPS for the indicated times. [Figures 13E-13G] Figure 13E is a bar graph showing the levels of TNFα in PBMC cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO023 followed by treatment with LPS for the indicated times. Figure 13F is a bar graph showing the levels of TNFα in PBMC cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO025 followed by treatment with LPS for the indicated times. Figure 13G is a bar graph showing the levels of TNFα in PBMC cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO027 followed by treatment with LPS for the indicated times. [Figures 14A-14D]Figure 14A is a bar graph showing the levels of TNFα in primary macrophage cell cultures after pre-stimulation with hrIL-10 followed by treatment with LPS for the indicated times. Figure 14B is a bar graph showing the levels of TNFα in primary macrophage cell cultures after pre-stimulation with anti-DSG1 monoclonal antibody (mAb) PRO003 followed by treatment with LPS for the indicated times. Figure 14C is a bar graph showing the levels of TNFα in primary macrophage cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO024 followed by treatment with LPS for the indicated times. Figure 14D is a bar graph showing the levels of TNFα in primary macrophage cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO026 followed by treatment with LPS for the indicated times. [Figures 14E-14G] Figure 14E is a bar graph showing levels of TNFα in primary macrophage cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO023 followed by treatment with LPS for the indicated times. Figure 14F is a bar graph showing levels of TNFα in primary macrophage cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO025 followed by treatment with LPS for the indicated times. Figure 14G is a bar graph showing levels of TNFα in primary macrophage cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO027 followed by treatment with LPS for the indicated times. [Figure 15]Figure 15 is a plot showing the levels of IL-10 signaling detected in parental HEK-BLUE™ IL-10 cells or HEK-BLUE™ IL-10 cells stably expressing DSG1 (+DSG1 expressing) 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 / motavizumab) at the indicated concentrations. IL-10 was measured using a colorimetric assay to detect 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. [Figures 16A-16B] Figure 16A is a plot showing the concentration (ng / mL) of PRO003, PRO024, and PRO058 over time in serum samples from BALB / c mice administered 3 mg / kg of the indicated antibodies or ANDbodies via tail vein injection. The concentrations of circulating molecules were measured by ELISA. Mean concentrations and standard deviations are shown. N=3. Figure 16B is a plot showing the concentration (ng of target protein per mg of 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 antibodies or ANDbodies via tail vein injection. Skin samples were collected at the indicated time points, homogenized, and protein was extracted. Concentrations were measured by ELISA. Mean concentrations and standard deviations are shown. N=3. [Figure 17] FIG. 17 is a series of schematic diagrams showing the design of the PRO070, PRO074, PRO075, and PRO077 TNFα-blocking anti-DSG1 ANDbodies. [Figures 18A-18B]Figure 18A is a plot showing the level of IL-10 signaling detected in parental HEK-BLUE™ 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 SEAP expression. OD630: optical density at 630 nm. Curve fitting was performed using GraphPad Prism 9 to fit a four-parameter log(agonist) vs. response. Figure 18B is a plot showing the level of IL-10 signaling detected in parental HEK-BLUE™ IL-10 cells treated overnight with IL-10 / DSG1 ANDbodies PRO023, PRO024, PRO025, PRO026, and PRO027. IL-10 was measured using a colorimetric assay to detect SEAP expression. 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 OF THE INVENTION

[0141] Detailed Description of the Invention Provided herein are ANDbody™ molecules comprising a therapeutic effector target binding domain and an address target binding domain. The ANDbody molecule is linked to one small molecule or two or more small molecules. A therapeutic effector target on an ANDbody molecule productively binds to its therapeutic effector target only if the address target binding domain also binds to an address target on the target tissue or cell, localizing the effector target to the target cell or tissue, e.g., forming an AND gate-type logic gate. For example, in some embodiments, an ANDbody is a macromolecule comprising at least (a) a first binding site specific for a therapeutic effector target expressed, e.g., ubiquitously expressed, in a mammalian subject, e.g., on a cell surface; and (b) a second binding site specific for the address target. In some embodiments, expression of the address target is restricted in vivo in the subject. In some embodiments, binding of the first binding site to the therapeutic effector target is weaker than binding of the second binding site to the address marker. The effector target and address target may be on the same cell or may be in different cells or compartments within the same tissue.

[0142] In some embodiments, at least 25% of the macromolecule (e.g., ANDbody) detectable in a subject is detectable in the target tissue or cell 1 to 7 days (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and / or 7 days) after administration of the macromolecule (e.g., ANDbody) to the subject. For example, in some embodiments, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% (e.g., 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-100%) of the detectable macromolecules in a subject are detectable in target tissues or cells 1-7 days after administration of the macromolecule to the subject.

[0143] Effector Target The ANDbodies™ of the present invention comprise 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) in a cell or tissue of a subject to which a therapeutic effector binding domain of an 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, resulting in 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 provided with an address targeting domain to localize the effector to a desired target address in the targeted cell or tissue. In some embodiments, such therapeutic signaling may require binding of multiple effector targets by multiple macromolecules of the invention.

[0144] In some embodiments, an effector target binding domain may produce a small / weak biological effect when provided alone, but may provide a greater / stronger biological effect when provided together with an address targeting domain that localizes and concentrates / focuses the effector to a desired target address in a targeted cell or tissue. In some embodiments, an effector target binding domain may produce an acceptable biological effect when provided alone, but may provide a greater / stronger biological effect when provided together with an address targeting domain that localizes the effector target binding domain to a targeted cell or tissue. In some embodiments, an effector target binding domain may produce a strong biological effect when provided alone, but may provide a strong or stronger target effect when provided together with an address targeting domain that localizes the effector target binding domain to a targeted cell or tissue. In some embodiments, an effector target binding domain, when provided alone, may produce biological effects, including undesired off-target biological effects, but when provided together with an address targeting domain, may be targeted, concentrated, and concentrated at a desired address in a targeted cell or tissue to reduce or eliminate the undesired off-target biological effects. Thus, the effector target binding domains of the present technology, when provided together with an address target binding domain as described herein, provide superior therapeutic agents that produce more potent targeted biological effects with reduced side effects, including reduced unintended off-target biological effects.

[0145] Examples of such therapeutic signaling effects include, but are not limited to, the following: (i) Blocking signaling pathways that promote or maintain the disease state; (ii) activation of signaling pathways that alleviate or prevent a disease state; (iii) promotion of antibody-dependent cellular cytotoxicity (ADCC); (iv) induction of complement activation in target cells or tissues; (v) promotion of phagocytosis; (vi) blocking or activating signaling pathways that promote cell differentiation; and (vii) Induction of tissue remodeling to reduce or prevent fibrosis.

[0146] In some embodiments, a therapeutic effector target is expressed more broadly in a subject than an address target. In some embodiments, a therapeutic effector target is expressed systemically, regionally, or locally in an organism. "Systemic expression" of a therapeutic effector target means that the therapeutic effector target is expressed at substantially the same level in most parts of the subject organism's body. Systemic expression includes multiple tissues. "Regional expression" of a therapeutic effector target means that the therapeutic target is expressed in an area that is less than systemic expression but more 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 areas. Local expression is not limited to a single tissue but can occur in multiple different tissues.

[0147] In some embodiments, the effector target binding domain has low affinity for the effector target. For example, low affinity can be an affinity of greater than 10 nM (e.g., an affinity of 10 nM to 1 μM, e.g., an affinity of 10 nM to 100 nM).

[0148] In some embodiments, the effector target binding domain has low binding activity for the effector target. Non-limiting examples of therapeutic effector targets that can be targeted using the ANDbodies disclosed herein, along with exemplary functions of the effector targets, are listed in Table 1.

[0149] [Table 1]

[0150] Address Target The ANDbodies of the present invention also include address target binders that bind to address targets to achieve targeted delivery of effectors. 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 an organ, tissue, cell, or cellular state of interest in the organism. An address target may 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 has 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 cellular state in a mammalian subject, such as a human subject.

[0151] In some embodiments, the presently provided address target binding domains do not substantially affect biological signaling upon binding to an address target, e.g., do not modulate signaling pathways or other biological responses in target cells or tissues. For example, an address target binder can be inactive or inert, in that it has no additional activity (other than binding) after binding to an address target, including a lack of catalytic activity. For example, an address target binder binds to a non-signaling site or motif of an address target. "Signal" is used herein to refer to a conformational, enzymatic, and / or electrical result that occurs as a result of target binding. Thus, as described herein, an address target binding domain does not signal upon binding to an address target. As used herein, a domain that "substantially" does not affect biological signaling is a domain that modulates a signaling pathway or other biological response in a target cell or tissue to which it binds by no more than 25% compared to a control condition, e.g., signaling in the absence of the domain. For example, the domain may modulate (e.g., increase or decrease) a signaling pathway or other biological response by less than 20%, less than 15%, less than 10%, less than 5%, less than 2%, or less than 1% (e.g., 20-25%, 15-20%, 10-15%, 5-10%, 2-5%, or 1-2%).

[0152] Similarly, an effector target binding domain may signal substantially no, or no, signal at all, when not localized by an address target binding domain. In some embodiments, an effector target binding domain signals with increased efficacy (e.g., has higher binding activity) when localized by an address target binding domain compared to signaling when not localized by an address target binding domain. When an effector target binding domain is localized to a targeted cell or tissue by an address target binding domain as part of the same macromolecule, signaling of the effector target can be affected as described above.

[0153] In some embodiments, the address target is used for organ-specific addressing, tissue-specific addressing, or cell-specific addressing.

[0154] The specificity of the address target binding domain for 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 changes in the expression of specific genes in a dataset. (See, for example, 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 normalizing expression profiling data. Sci Rep 9, 17960 (2019). https: / / doi.org / 10.1038 / s41598-019-54288-7). Address target binders can be identified using cellular expression data generated for address target binders as described herein (Tables 2A and 2B). In some embodiments, address target markers exhibit a Gini score of greater than 0.4, such as 0.74 to 1.00. Conversely, non-address markers, which are more systematically expressed, may exhibit a Gini score of 0.15 to 0.19.

[0155] In one embodiment, a tau score is used to represent the expression changes of a particular gene in a dataset. The tau score is calculated using information about the gene's expression in each tissue and its maximum expression across all tissues, taking into account the number of tissues in which expression is measured (see, for example, Itai Yanai, et al., Genome-wide midrange transcription profiles reveal expression level relationships in human tissue specification, Bioinformatics, Volume 21, Issue 5, 1 March 2005, Pages 650-659; Kryuchkova-Mostacci N, Robinson-Rechavi MA benchmark of gene expression tissue-specificity metrics. Brief Bioinform. 2017 Mar 1;18(2):205-214. doi:10.1093 / bib / bbw008). In some embodiments, addressable target markers exhibit a tau score of greater than 0.6, such as between 0.74 and 1.00. Conversely, non-addressable markers that are more systematically expressed may exhibit tau scores below 0.3, such as 0.15-0.19.

[0156] In some embodiments, the specificity of an address target binding domain for a particular cell or tissue, as indicated by an appropriate Gini score and / or Tau score, is determined using a tissue-based analysis that does not include tissues with a natural biological separation barrier (i.e., the blood-brain barrier). For example, in some embodiments, the Gini score and / or Tau score can be calculated without using data from tissues such as, but not limited to, central nervous system, brain, eye, and / or testicular tissue. In some embodiments, an address target as provided herein reveals a cellular state. As used herein, "cellular state" refers to a given physiological state of a cell. A cellular state can be, for example, a disease state (as opposed to a non-disease or normal state of a cell or tissue); or an activated state (as opposed to a non-activated state of a cell). Exemplary disease states include inflammation, infection (e.g., bacterial infection, viral infection, fungal infection), and cancer-related conditions (e.g., a pre-cancerous or cancerous cell state). In some aspects, a cell state refers to the fact that cells of a particular type retain the characteristics of that particular cell type and do not acquire characteristics that would classify them as a different cell type, but may exhibit variability with respect to one or more characteristics and / or may exist in a variety of different conditions. The various states or conditions that a cell can exist in may be unique to a particular cell type (e.g., may include characteristics or features exhibited only by that cell type and / or may include functions performed exclusively or primarily by that cell type), or may occur in multiple different cell types. In some embodiments, a cell state reflects the ability of a cell to respond to a particular stimulus or environmental condition (e.g., whether the cell responds or the type of response elicited), or is the state of the cell brought about by the stimulus or environmental condition. Cells in different cell states can be distinguished from one another in various ways. For example, they may express, produce, or secrete one or more different genes, proteins, or other molecules (such as "markers," such as address targets provided herein), exhibit differences in protein modifications such as phosphorylation, acetylation, or exhibit different appearances.Thus, a cellular state may be one in which the cell expresses, produces, or secretes one or more markers, exhibits particular protein modifications, has a particular appearance, and / or exhibits or does not exhibit one or more biological responses to a stimulus or environmental condition.

[0157] 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 may be, for example, an immune cell. In some embodiments, the address target binding domain comprises rituximab (DrugBank Accession No.: DB00073) or a fragment, derivative, or variant thereof. In certain embodiments, the address target binding domain comprises an antigen-binding fragment of rituximab, e.g., the CDRs (or entire variable domains) disclosed in U.S. Pat. No. 7,381,560 (incorporated by reference), including Figures 4 and 5. In other embodiments, the address target binding domain comprises a variant, e.g., a humanized variant, or otherwise, of an antigen-binding fragment of rituximab with one or more amino acid substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions, e.g., a variant having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% or more divergence from the rituximab sequence; in certain embodiments, any substitutions are within the CDRs, while in other embodiments, any substitutions are outside the CDRs, and in other embodiments, substitutions can be in both CDR and non-CDR sequences. In some embodiments, substitutions may be non-conservative, conservative, highly conservative, or a combination thereof, e.g., conservative or highly conservative substitutions in the CDRs (particularly in the case of paratope residues, in some embodiments, any substitutions are outside of the paratope residues), and non-conservative, conservative, highly conservative, or a combination thereof in non-CDR residues. In some embodiments, the address target is CD20 and the small molecule is fluocinolone, ibrutinib, or tofacitinib.

[0158] 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 may 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 an antigen-binding fragment of gemtuzumab, e.g., the CDRs (or entire variable domains) disclosed in U.S. Pat. No. 5,773,001, including Figures 1 and 2 (SEQ ID NOs: 2 and 4). In other embodiments, the address target binding domain comprises a variant, e.g., a humanized variant, or otherwise, of an antigen-binding fragment of gemtuzumab, with one or more amino acid substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions, e.g., a variant having about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% or more divergence from the gemtuzumab sequence; in certain embodiments, any substitutions are within the CDRs, while in other embodiments, any substitutions are outside the CDRs, and in other embodiments, substitutions can be present in both the CDR and non-CDR sequences. In some embodiments, substitutions may be non-conservative, conservative, highly conservative, or a combination thereof, e.g., conservative or highly conservative substitutions in the CDRs (particularly in the case of paratope residues, in some embodiments, any substitutions are outside of the paratope residues), and non-conservative, conservative, highly conservative, or a combination thereof in non-CDR residues. In some embodiments, the address target is CD33 and the small molecule is fluocinolone, ibrutinib, or tofacitinib.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] In some embodiments, the target cell is a monocyte or a dendritic cell and the glucocorticoid address target is OSCAR, SIGLEC9, TNFSF13B, TLR8, FGL2, TLR2, IGSF6, LILRA1, CSF1R, GPBAR1, MS4A4A, UPK3A, CD300C, CCD86, CD68, MPEG1, FCN1, TNFSF13, MS4A6A, or CST3.

[0163] In some embodiments, the target cell is a monocyte or a dendritic cell or a T cell, and the glucocorticoid target address is GPA33, ALG3, ENO1, IL10RA, STING1, LRRC8C, LPAR6, C1QL3, SIGMAR1, SLC3A2, GIMAP5, GIMAP1, TNFSF8, CACNA2D4, IL27RA, SCL9A6, HCST, CD48, SPN, or ADA.

[0164] In some embodiments, the target cell is a monocyte or myeloid dendritic cell and the glucocorticoid address target is SEZ6L, NFAM1, CLEC7A, TLR8, S100Z, FGL2, CD300C, CD86, CD68, SLC24A4, GPBAR1, SLCA7, CD300E, SIGLEC9, TLR5, CTSS, FCN1, CD33, MPEG1, or CST3.

[0165] In some embodiments, the target cell is a monocyte or myeloid dendritic cell or a T cell, and the glucocorticoid address target is CD44, STING1, LRRC8C, LPAR6, ENO1, CACNA2D4, SERPINB9, IL10RA, IL27RA, IL12RB1, SLC9A6, MYO1G, CD48, S100A4, CRTAM, CELA1, GIMAP5, GIMAP1, TNFSF8, or GPA33.

[0166] Further exemplary address targets of the present technology are listed below in Table 2A (HPA database analysis) and Table 2B (Gtex database analysis).

[0167] [Table 2A-1]

[0168] [Table 2A-2]

[0169] [Table 2A-3]

[0170] [Table 2A-4]

[0171] [Table 2A-5]

[0172]

Table 2A-6

[0173]

Table 2A-7

[0174]

Table 2A-8

[0175]

Table 2A-9

[0176]

Table 2A-10

[0177]

Table 2A-11

[0178]

Table 2A-12

[0179]

Table 2A-13

[0180]

Table 2A-14

[0181]

Table 2A-15

[0182]

Table 2A-16

[0183]

Table 2A-17

[0184]

Table 2A-18

[0185]

Table 2A-19

[0186]

Table 2A-20

[0187]

Table 2A-21

[0188]

Table 2A-22

[0189]

Table 2A-23

[0190]

Table 2A-24

[0191]

Table 2A-25

[0192]

Table 2A-26

[0193]

Table 2A-27

[0194]

Table 2A-28

[0195]

Table 2A-29

[0196]

Table 2A-30

[0197]

Table 2A-31

[0198]

Table 2A-32

[0199]

Table 2A-33

[0200]

Table 2A-34

[0201]

Table 2A-35

[0202] [Table 2A-36]

[0203] Table 2B contains address targets based on Gtex database analysis.

[0204] [Table 2B-1]

[0205] [Table 2B-2]

[0206] [Table 2B-3]

[0207] [Table 2B-4]

[0208] [Table 2B-5]

[0209] [Table 2B-6]

[0210] [Table 2B-7]

[0211] [Table 2B-8]

[0212] [Table 2B-9]

[0213]

Table 2B-10

[0214]

Table 2B-11

[0215]

Table 2B-12

[0216]

Table 2B-13

[0217]

Table 2B-14

[0218]

Table 2B-15

[0219]

Table 2B-16

[0220]

Table 2B-17

[0221]

Table 2B-18

[0222]

Table 2B-19

[0223]

Table 2B-20

[0224]

Table 2B-21

[0225]

Table 2B-22

[0226]

Table 2B-23

[0227]

Table 2B-24

[0228]

Table 2B-25

[0229]

Table 2B-26

[0230]

Table 2B-27

[0231]

Table 2B-28

[0232]

Table 2B-29

[0233] [Table 2B-30]

[0234] low molecule The polymers (e.g., ANDbodies) of the invention are linked to small molecules. The polymers and small molecules may be linked by a cleavable linker. Alternatively, the polymers and small molecules may be linked by a non-cleavable linker. Any useful linker can be used for this purpose.

[0235] A polymer may have one or more (e.g., one, two, three, four, five, or more) small molecules attached to it. When multiple small molecules are attached to a polymer, the small molecules may be the same. Alternatively, one or more of the small molecules attached to a polymer may be different.

[0236] The small molecule linked to the polymer can be any desired small molecule. For example, the small molecule can be a therapeutic agent that is intended to be localized or concentrated at a specific site by the polymer. In one example, the small molecule can be a therapeutic agent that acts in conjunction with or complements the effector target binding site domain. Alternatively, the small molecule can modulate the effector target binding site domain. In another example, the small molecule can modulate the address target binding site domain.

[0237] Exemplary classes of small molecules that can be linked to macromolecules according to the present invention include those listed in Table 3.

[0238] [Table 3-1]

[0239] [Table 3-2]

[0240]

Table 3-3

[0241]

Table 3-4

[0242]

Table 3-5

[0243]

Table 3-6

[0244]

Table 3-7

[0245]

Table 3-8

[0246]

Table 3-9

[0247]

Table 3-10

[0248]

Table 3-11

[0249]

Table 3-12

[0250] Specific examples of small molecules that can be linked to the polymers of the present invention include, for example, those classified into any of the classes shown in Table 3. In particular, exemplary glucocorticoid receptor agonists include, but are not limited to, cortisone, dexamethasone, fluticasone, mometasone, fluocinolone, budesonide, butixicort, and betamethasone. Exemplary tyrosine protein kinase BTK inhibitors include, but are not limited to, acalabrutinib, evobrutinib, fenebrutinib, ibrutinib, orelabrutinib, pirtobrutinib, remibrutinib, rilzabrutinib, trebrutinib, 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, erence, etoposide, idarubicin, topotecan, and barbican.

[0251] In some embodiments, the small molecule is a steroid. In some embodiments, the small molecule is fluocinolone (PubChem ID: 91488) or a salt, ester, or conjugate thereof.

[0252] 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.

[0253] 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.

[0254] Small molecules may be conjugated to the polymers of the invention using any conjugation method known in the art. For example, small molecule carboxyl, hydroxyl, and amine residues may be attached to amine and sulfhydryl residues on proteins using linking methods. Alternatively, any complementary functional groups on two components can be used to form a covalent bond by reacting them with each other. Examples of complementary reactive functional groups include, but are not limited to, 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. Furthermore, any available linker can be used in the present invention, including, for example, heterobifunctional linkers that allow for the attachment of small molecules via disulfide and amide bonds.

[0255] ANDbody structure In general, an ANDbody can be any macromolecule, e.g., a polypeptide or protein, that contains both an effector target binding site or domain and an address target binding site or domain. The binding sites can be on the same polypeptide chain or on different polypeptide chains that are linked to each other, e.g., via disulfide bonds.

[0256] In some embodiments, the binding site for the effector target and the binding site for the address target of an ANDbody comprise antibody heavy and / or light chain domains, respectively. In some embodiments, an ANDbody comprises a first antibody variable domain that has binding specificity for the effector target and a second antibody variable domain that has binding specificity for the address target. In other embodiments, an ANDbody comprises a first antigen-binding site of an antibody, which first antigen-binding site has binding specificity for the effector target, and a second antigen-binding site of an antibody, which second antigen-binding site has binding specificity for the address target.

[0257] In some embodiments, an ANDbody may have the structure of an antibody molecule. As used herein, the term "antibody" includes full-length antibodies and antigen-binding antibody fragments (e.g., scFvs). In some embodiments, an antibody molecule has specificity for two or more antigens, e.g., two, three, or four antigens; e.g., the antibody molecule comprises a plurality of variable domain sequences, wherein a first variable domain sequence of the plurality has binding specificity for a first epitope (e.g., an effector target) and a second variable domain sequence of the plurality has binding specificity for a second epitope (e.g., an address target).

[0258] In some embodiments, an 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 embodiments, an ANDbody is an antibody molecule comprising a light chain that binds to either the effector target or the address target and a heavy chain that binds to the other of the effector target and the address target.

[0259] In some embodiments, an ANDbody has the following structures: scFv, BsIgG, BsAb fragment, BiTE, dual-affinity re-targeting protein (DART), tandem diabody (TandAb), diabody, Fab2, bivalent scFv (di-scFv), chemically linked F(ab')2, Ig ​​molecule with three or four different antigen binding sites, DVI-IgG four-in-one (DVI-IgG four-in-one), ImmTac, HSAbody, IgG-IgG, Cov-X-Body, scFv1-PEG-scFv2, conjugated IgG, DVD-IgG, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, monobody, nanoCLAMP, bis-Fab, Fv, Fab'-SH, linear antibody, scFv, antibody having only a heavy chain (Humabody), ScFab, IgG antibody fragment, single-chain variable region antibody, single-domain heavy chain antibody, bispecific triple body, BiKE, CrossMAb, dsDb, scDb, tandem dAb / VHH, triple dAb VHH, tetravalent dAb / VHH, Fab-scFv, Fab-Fv, or DART-Fc, adectin, Kunitz inhibitor, or receptor decoy.

[0260] 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 to which it binds is weaker than the affinity of the second binding site for the address target. In some embodiments, the affinity of the first binding site for the therapeutic effector target to which it binds is 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold weaker than the affinity of the second binding site for the address target.

[0261] The terms "binding affinity" and "binding activity" refer to the tendency of a macromolecule, e.g., a polypeptide molecule, to bind or not bind to a target. For purposes of the present invention, which combine two binding sites, the relative affinity of the two binding sites can be determined, for example, by measuring the affinity of each binding site when they are present on a common scaffold, such as in the form of a single-chain antibody. Such a comparison allows the affinities of the two binding sites to be compared while excluding any interference from other binding sites present on the macromolecule of the present invention.

[0262] Binding affinity can be quantified by determining the dissociation constant (Kd) of the polypeptide and its binder. A lower Kd indicates a higher affinity for the binding partner. Similarly, the specificity of binding between a polypeptide and its binding partner can be defined as the relative dissociation constant (Kd) of the polypeptide for its binding partner compared to the dissociation constant for the polypeptide and another non-target molecule.

[0263] The value of this dissociation constant can be determined by known methods. For example, Kd can be determined using a double-filter nitrocellulose filter binding assay such as that 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 blot, RIA, and flow cytometry analysis. The binding kinetics (e.g., binding affinity) of an antibody can also be evaluated by standard assays known in the art, such as Biacore™ system analysis.

[0264] Instead of Kd, EC 50 or IC 50 The relative affinity can also be determined using EC 50 IC indicates the concentration at which a polypeptide achieves 50% of its maximum binding amount to a given amount of binding partner.50 indicates the concentration at which a polypeptide inhibits 50% of the maximal binding of a given amount of competitor to a given amount of binding partner. 50 or IC 50 A lower EC2 indicates a higher affinity for the target. 50 and IC 50 All values ​​can be determined by well-known methods, for example, ELISA.

[0265] In some embodiments, the Kd of a therapeutic effector-target binder can be greater than about 1 pM, about 10 pM, about 100 pM, about 1 nM, about 10 nM, about 100 nM, about 500 nM, or about 1 uM (e.g., can be between 1 pM and 10 pM, 10 pM and 100 pM, 100 pM and 1 nM, 1 nM and 10 nM, 10 nM and 100 nM, 100 nM and 500 nM, or 500 nM and 1 uM). In some embodiments, the Kd of an address target binder can be less than about 1 uM, less than about 500 nM, less than about 100 nM, less than about 10 nM, less than about 1 nM, less than about 100 pM, less than about 10 pM, or less than about 1 pM (e.g., 1 uM to 500 nM, 500 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1 nM to 100 pM, 100 pM to 10 pM, or 10 pM to 1 pM). In some embodiments, the Kd of a 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.

[0266] In some embodiments, the EC of the therapeutic effector target binder 50 can be greater than about 1 pM, about 10 pM, about 100 pM, about 1 nM, about 10 nM, about 100 nM, about 500 nM, or about 1 uM (e.g., 1 pM to 10 pM, 10 pM to 100 pM, 100 pM to 1 nM, 1 nM to 10 nM, 10 nM to 100 nM, 100 nM to 500 nM, or 500 nM to 1 uM). 50may be less than about 1 uM, less than about 500 nM, less than about 100 nM, less than about 10 nM, less than about 1 nM, less than about 100 pM, less than about 10 pM, or less than about 1 pM (e.g., 1 uM to 500 nM, 500 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1 nM to 100 pM, 100 pM to 10 pM, or 10 pM to 1 pM). 50 Addresses the target binder's EC 50 In some cases, the concentration may be about 6 times, about 5 times, about 4 times, about 3 times, or about 2 times higher than the concentration.

[0267] In some embodiments, IC of a therapeutic effector-target binder 50 may be greater than about 1 pM, about 10 pM, about 100 pM, about 1 nM, about 10 nM, about 100 nM, about 500 nM, or about 1 uM (e.g., 1 pM to 10 pM, 10 pM to 100 pM, 100 pM to 1 nM, 1 nM to 10 nM, 10 nM to 100 nM, 100 nM to 500 nM, or 500 nM to 1 uM). In some embodiments, the IC50 of an address target binder may be less than about 1 uM, less than about 500 nM, less than about 100 nM, less than about 10 nM, less than about 1 nM, less than about 100 pM, less than about 10 pM, or less than about 1 pM (e.g., 1 uM to 500 nM, 500 nM to 100 nM, 100 nM to 10 nM, 10 nM to 1 nM, 1 nM to 100 pM, 100 pM to 10 pM, or 10 pM to 1 pM). 50 Addresses the IC of the target binder 50 It may be about 6 times, about 5 times, about 4 times, about 3 times, or about 2 times higher.

[0268] The cell or tissue densities of the effector and address targets bound by an ANDbody may be different. In embodiments, the density of the therapeutic effector target on a cell to which the effector target binding portion of an ANDbody binds is 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, or more than about 100,000-fold lower than the density of the address target on a cell to which the address target binding portion binds.

[0269] In some embodiments, the affinity of the first binding site for the therapeutic effector target to which it binds is about half (1 / 2) x Kd less than the affinity of the second binding site for the address target to which it binds, and the density of the therapeutic effector target on a cell to which the first binding site binds is about half (1 / 2) x Kd less than the density of the address target on a cell to which the second binding site binds.

[0270] In some embodiments, an ANDbody has both affinity and density parameters as previously described herein.

[0271] In some embodiments, the first and second binding sites of an ANDbody are directly linked to each other. Directly linked means that the first binding site coding sequence is adjacent to the second binding site coding sequence, and that no sequences from other sequences (such as linkers) are present. In some embodiments, the first and second binding sites of an ANDbody are not directly linked to each other.

[0272] In addition to small molecules, ANDbodies as disclosed herein can also be linked to additional moieties or moieties, such as extracellular components, intracellular components, soluble factors (e.g., enzymes, hormones, cytokines, growth factors, toxins, poisons, pollutants, etc.), or transmembrane proteins (e.g., cell surface receptors).

[0273] Exemplary effector target sequences and address target sequences for which ANDbodies of the present technology may have affinity are listed in Table 4 and the Sequence Listing. In some instances, the sequences include full-length protein sequences with or without signal peptide regions and / or Fc fusion sequences. In some embodiments, ANDbodies of the present technology include a binding domain that binds to a target protein or effector target protein. In several embodiments, the binding domain of an ANDbody of the present invention may bind to a protein sequence that includes a signal peptide. In other embodiments, the binding domain of an ANDbody of the present invention may bind to a protein lacking a signal protein. In some embodiments, the binding domain of an ANDbody of the present invention may bind to a full-length protein. In other embodiments, the binding domain of an ANDbody of the present invention may bind to a protein fusion, such as a full-length protein sequence or a peptide fragment thereof, with or without a signal peptide region, fused to another protein, such as an Fc sequence. In other embodiments, the binding domain of an ANDbody of the present invention may bind to a protein smaller than the full-length protein sequence, such as a peptide fragment of an address target or effector target.

[0274] [Table 4-1]

[0275] [Table 4-2]

[0276] [Table 4-3]

[0277] [Table 4-4]

[0278] [Table 4-5]

[0279] [Table 4-6]

[0280] [Table 4-7]

[0281] [Table 4-8]

[0282] [Table 4-9]

[0283] [Table 4-10]

[0284] [Table 4-11]

[0285] [Table 4-12]

[0286] [Table 4-13]

[0287] Production of ANDbody Compositions Production of ANDbody polypeptides Small polypeptide compositions (ANDbodies) of the invention can be produced by any suitable means. For example, all or part of an ANDbody can be expressed by a host cell comprising one or more nucleotides encoding the ANDbody. Thus, in some embodiments, the invention provides one or more nucleic acids encoding any one of the polymers provided herein or one or more subunits thereof. The nucleic acid can be, for example, a linear nucleic acid, a circular nucleic acid, and / or a modified nucleic acid. The invention further provides one or more vectors comprising one or more nucleic acids encoding any one of the polymers provided herein or one or more subunits thereof.

[0288] The present invention also provides one or more host cells (e.g., mammalian host cells) comprising one or more nucleic acids encoding any one of the polymers provided herein or one or more subunits thereof (e.g., comprising a vector comprising said one or more nucleic acids). The present invention further provides a method of producing any one of the polymers provided herein, the method comprising culturing one or more host cells in a medium. The method may further comprise recovering the polymer from the one or more host cells or the culture medium.

[0289] Such methods for producing therapeutic polypeptides are routine in the art. See generally, e.g., Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).

[0290] Methods for producing ANDbodies can include expression in mammalian cells, although recombinant proteins can also be produced using insect cells, yeast, bacteria, or other cells under the control of an appropriate promoter. Mammalian expression vectors can include nontranscribed elements such as an origin of replication, a suitable promoter and enhancer, and other 5' or 3' flanking nontranscribed sequences, as well as 5' or 3' nontranslated 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, such as the SV40 origin of replication, early promoter, enhancer, splice, and polyadenylation sites, can be used to provide other genetic elements required for expression of heterologous DNA sequences. Appropriate cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).

[0291] Various mammalian cell culture systems can be used to express and produce the ANDbodies described herein. Examples of mammalian expression systems include CHO cells, COS cells, HeLA cell lines, and BHK cell lines. Host cell culture processes for the production of protein therapeutics are described, for example, in Zhou and Kantardjieff (Eds.), Mammalian Cell Cultures for Biologics Manufacturing (Advances in Biochemical Engineering / Biotechnology), Springer (2014). Purification of protein therapeutics is described in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010). Formulation of protein therapeutics is described in Meyer (Ed.), Therapeutic Protein Drug Products: Practical Approaches to Formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).

[0292] Antibody production techniques are known. For example, for methods of producing recombinant antibodies, including antibody engineering, the 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, see: Zhiqiang (Editor), Therapeutic Monoclonal Antibodies: From Bench to Clinic. 1st Edition. Wiley 2009; Greenfield (Ed.) Antibodies: A Laboratory Manual. (Second Edition) Cold Spring Harbor Laboratory Press 2013; Ferrara et al. 2012. Using Phage and Yeast Display to Select Hundreds of Monoclonal Antibodies: Antigen 85, a Tuberculosis Biomarker. PLoS ONE 7(11):e49535.

[0293] ANDbody RNA production In some embodiments, ANDbody RNA can be produced, for example, for delivery to a subject. Therapeutic mRNAs are typically produced by in vitro transcription. Modifications such as the incorporation of modified bases, 5' cap analogs, and poly(A) tails can optimize activity and function. For example, mRNA translation and stability can be improved by cap and poly(A) tail modifications. For example, incorporating a cap analog, such as ARCA (anti-reverse cap analog), and a 100-200 bp poly(A) tail into in vitro-transcribed (IVT) mRNA improves expression and stability (Kaczmarek et al. Genome Medicine (2017) 9:60). Novel types of 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 destabilization by rare codons (Presnyak et al. Cell. 2015;160:1111-24.93; Thess et al. Mol Ther. 2015;23:1456-64). Protein product levels can be increased by modifying the 3' and 5' untranslated regions (UTRs), which contain sequences responsible for recruiting RNA-binding proteins (RBPs) and miRNAs (Kaczmarek). Furthermore, UTRs can be engineered to encode regulatory elements (e.g., K-turn motifs and miRNA-binding sites) to control RNA expression in a cell-specific manner (Wroblewska et al. 2015;33:839-41). RNA base modifications (e.g., pseudouridine-incorporated mRNA, e.g., N1-methyl-pseudouridine) contribute to masking mRNA immunostimulatory activity and increase mRNA translation by facilitating 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 producing them are known and are disclosed, for example, in the following documents: WO 2016011306; WO 2016014846; WO 2016022914; WO 2016077123; WO 2016164762; WO 2016201377; WO 2017049275; U.S. Patent Nos. 9937233; 8710200; 10022425; 9878056; 9572897; Jemielity et al. RNA. 2003; 9:1108-22.90; Mockey et al. Biochem Biophys Res Commun. 2006; 340:1062-8.91; Strenkowska et al. al.Nucleic Acids Res.2016;44:9578-90.92;Presnyak et al.Cell.2015;160:1111-24.93;Kaczmarek et al.Genome Medicine (2017) 9:60.

[0294] Production of affinity-modified ANDbodies Affinity-altered ANDbodies can be produced using methods known in the art; for example, ANDbodies can be designed to have a target binding site with reduced affinity for the effector target. See, for example, U.S. Patent No. 10,654,928. Generally, ANDbodies can be modified to alter the affinity of the effector target binding site for the effector target, or to alter the affinity of the address target binding site for the address target. Modifications can increase or decrease the affinity of the binding site for its binding partner.

[0295] Target and Address Assessment The expression of a therapeutic target can be assessed at either the RNA level or the protein level using methods known in the art. In some embodiments, the expression of a therapeutic target is assessed by measuring RNA expression, for example, using an RNA-seq dataset as a proxy for protein expression levels. RNA datasets include the Genotype-Tissue Expression (GTEx) dataset (e.g., https: / / www.genome.gov / Funded-Programs-Projects / Genotype-Tissue-Expression-Project) or the Human Protein Atlas (HPA) dataset (https: / / www.proteinatlas.org / ).

[0296] A non-limiting list of tissues in which expression of a therapeutic target can be assessed includes, for example: minor salivary glands, thyroid, lung, breast (mammary tissue), pancreas, adrenal glands, liver, kidney (renal cortex), kidney (renal medulla), adipose visceral tissue (omentum), small intestine - terminal ileum, fallopian tube, ovary, uterus, skin, non-sun-exposed skin (suprapubic skin); cervix - endocervix, cervix - cervicovaginal, vagina, sun-exposed skin (calf skin), Cells of anterior cingulate cortex (BA24), caudate nucleus (basal ganglia), putamen (basal ganglia), nucleus accumbens (basal ganglia), hypothalamus, amygdala, hippocampus, cerebellum / cerebellar hemispheres, substantia nigra, pituitary gland, spinal cord (cervical spinal cord), artery-aorta, heart-atrial appendage, coronary artery-heart, left ventricle, esophagus, esophageal-mucosa, esophageal-muscularis, gastroesophageal junction, spleen, stomach, transverse colon, sigmoid colon, testis, whole blood cells, cells (EBV-transformed lymphocytes), tibial artery, or tibial nerve tissue.

[0297] Address markers can be assessed using methods known in the art, for example, gene expression can be assessed at the mRNA level using Northern blots, cDNA or oligonucleotide microarrays, or sequencing (e.g., RNA-Seq), or at the protein expression level using protein microarrays, Western blots, flow cytometry, immunohistochemistry, etc. Modifications can be assessed, for example, using antibodies specific for particular modified forms of the protein, e.g., phosphorylation-specific antibodies, or mass spectrometry.

[0298] Using ANDbody The ANDbodies and pharmaceutical compositions thereof provided herein are suitable for administration to a subject in need thereof (the subject may be a human or a non-human animal), e.g., for human therapeutic or veterinary use. Accordingly, in some aspects, provided herein are methods comprising administering a therapeutically effective amount of any one of the macromolecules (i.e., ANDbodies) provided herein, a composition comprising same, or one or more nucleic acids encoding same, to a subject in need thereof.

[0299] Veterinary applications include use for the treatment of mammals, including commercially relevant mammals, e.g., pet and livestock animals, e.g., cows, pigs, horses, sheep, goats, cats, dogs, mice, and / or rats; and / or birds, e.g., commercially relevant birds, including parrots, poultry, chickens, ducks, geese, hens or roosters, and / or turkeys; zoo animals, e.g., felines; non-mammals, e.g., reptiles, fish, amphibians, etc.

[0300] The present invention further relates to a subject or subject cell comprising an ANDbody composition described herein. In some embodiments, the subject or subject cell is a plant, insect, bacterium, fungus, vertebrate, mammal (e.g., human), or other organism or cell.

[0301] In some embodiments, a subject or subject's cells are contacted (e.g., delivered or administered) with an ANDbody composition. In some embodiments, the subject is a mammal, such as a human. The amount of the ANDbody composition, expression product, or both in the subject can be measured at any time after administration.

[0302] In some aspects, provided herein are methods of enhancing activation of a signal transduction pathway in a tissue or cell, the methods comprising contacting the tissue or cell with a macromolecule provided herein, wherein neither the first binding moiety nor the second binding moiety substantially activates the signal transduction pathway in the absence of localization by the second binding moiety; and wherein contacting the tissue or cell with the macromolecule substantially increases activation of the signal transduction pathway in the tissue or cell by the first binding moiety compared to a reference macromolecule lacking the second binding moiety.

[0303] Pharmaceutical Composition Polypeptide Pharmaceutical Composition The ANDbody compositions (e.g., ANDbody polypeptide or RNA compositions) described herein can be administered to a subject in need thereof. The invention includes, for example, pharmaceutical compositions containing small molecules provided herein (i.e., ANDbodies) in combination with one or more pharmaceutically acceptable carriers or excipients. In some embodiments, the compositions are in unit dosage form suitable for administration (e.g., intravenous, oral, or subcutaneous administration) to a human subject.

[0304] 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.

[0305] RNA pharmaceutical composition Alternatively or additionally, a nucleic acid (e.g., RNA) encoding an ANDBody can be administered to a subject. Typically, therapeutic mRNA is produced by in vitro transcription. Modifications such as the incorporation of modified bases, 5' cap analogs, and poly(A) tails can optimize activity and function. For example, mRNA translation and stability can be improved by cap and poly(A) tail modifications. For example, incorporating a cap analog such as ARCA (anti-reverse cap analog) and a 100-200 bp poly(A) tail into in vitro-transcribed (IVT) mRNA improves expression and stability (Kaczmarek et al., Genome Medicine (2017) 9:60). Novel types of 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 destabilization by rare codons (Presnyak et al. 2015;160:1111-24.93; Thess et al., Mol Ther. 2015;23:1456-64). Modifying the 3' and 5' untranslated regions (UTRs), which contain sequences responsible for recruiting RNA-binding proteins (RBPs) and miRNAs, can increase the levels of protein product (Kaczmarek). Furthermore, UTRs can be modified to encode regulatory elements (e.g., K-turn motifs and miRNA-binding sites) to control RNA expression in a cell-specific manner (Wroblewska et al. Nat Biotechnol. 2015;33:839-41). RNA base modifications (e.g., pseudouridine incorporated into mRNA, e.g., N1-methyl-pseudouridine) contribute to masking mRNA immunostimulatory activity and increase mRNA translation by facilitating 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 producing them are known and are disclosed, for example, in the following documents: WO 2016011306; WO 2016014846; WO 2016022914; WO 2016077123; WO 2016164762; WO 2016201377; WO 2017049275; U.S. Patent Nos. 9937233; 8710200; 10022425; 9878056; 9572897; Jemielity et al. RNA. 2003; 9:1108-22.90; Mockey et al. Biochem Biophys Res Commun. 2006; 340:1062-8.91; Strenkowska et al. Nucleic Acids Res.2016;44:9578-90.92;Presnyak et al.Cell.2015;160:1111-24.93;Kaczmarek et al.Genome Medicine (2017) 9:60.

[0306] In some embodiments, the RNA is circular RNA. See, for example, International Publication No. WO2019118919, which describes the expression of therapeutic RNA, such as antibody RNA, from circular RNA. In some embodiments, the invention comprises a circular polyribonucleotide comprising: (a) an internal ribosome entry site (IRES); (b) an expression sequence encoding an ANDbody described herein and lacking a polyA sequence; and (c) a termination element. Circular RNA encoding an ANDbody described herein can be delivered naked (i.e., without formulation with a carrier) or with a carrier.

[0307] Combination therapy In some embodiments, an ANDbody or ANDbody composition provided herein is administered in combination with one or more additional therapeutic agents.

[0308] Carrier lipid nanoparticles Formulations for in vivo delivery of the compositions described herein (e.g., polypeptide or RNA ANDbody compositions) together with a carrier include lipid nanoparticle (LNP) formulations. See, e.g., U.S. Patent Nos. 9,764,036; 9,682,139; Kauffman et al. Nano Lett. 2015; 15:7300-6.37; Fenton et al. Adv Mater. 2016; 28:2939-43). LNPs, in some embodiments, comprise one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids); one or more conjugated lipids (e.g., PEG-conjugated lipids, i.e., lipids conjugated to a polymer, as described in Table 5 of WO2019217941, which are incorporated herein by reference in their entireties); one or more sterols (e.g., cholesterol); and, optionally, one or more targeting molecules (e.g., conjugated receptors, receptor ligands, antibodies); or combinations thereof.

[0309] Lipids that can be used in nanoparticle formation (e.g., lipid nanoparticles) include, for example, those listed in Table 4 of WO 2019217941, which is incorporated herein by reference, e.g., the lipid-containing nanoparticles can include one or more of the lipids listed in Table 4 of WO 2019217941. The lipid nanoparticles may also include additional elements such as polymers listed in Table 5 of WO 2019217941, which is incorporated herein by reference.

[0310] In some embodiments, the conjugated lipid, if present, is selected from the following: PEG-diacylglycerol (DAG) (e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEGS-DAG) (e.g., 4-O-(2',3'-di( The copolymers may include one or more of: N-(carbonylmethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, N-(carbonylmethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, N-(carbonylmethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those listed in Table 2 of WO2019051289 (incorporated by reference), and combinations thereof.

[0311] In some embodiments, sterols that can be incorporated into the lipid nanoparticles include one or more cholesterol or cholesterol derivatives, such as those described in International Publication No. 2009 / 127060 or U.S. Patent Application Publication No. 2010 / 0130588, which are incorporated by reference. Additional exemplary sterols include plant sterols, such as those described in Eygeris et al. (2020), dx.doi.org / 10.1021 / acs.nanolett.0c01386, which are incorporated by reference herein.

[0312] In some embodiments, lipid particles comprise an ionizable lipid, a non-cationic lipid, a complex lipid that inhibits particle aggregation, and a sterol. The amounts of these components can be varied independently to achieve desired properties. For example, in some embodiments, lipid nanoparticles comprise about 20 mol% to about 90 mol% of the total lipids (in other embodiments, 20-70% (mol), 30-60% (mol), or 40-50% (mol) of the total lipids present in the lipid nanoparticles; or about 50 mol% to about 90 mol%), about 5 mol% to about 30 mol% of the total lipids, about 0.5 mol% to about 20 mol% of the total lipids, and about 20 mol% to about 50 mol% of the total lipids. The ratio of total lipid to nucleic acid can be varied as desired. For example, the ratio of total lipid to nucleic acid (mass or weight) can range from about 10:1 to about 30:1.

[0313] In some embodiments, the lipid to nucleic acid ratio (mass / mass ratio; w / w ratio) can range from 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 impart a desired N / P ratio, e.g., an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10, or more. Generally, the total lipid content of a lipid nanoparticle formulation can range from about 5 mg / mL to about 30 mg / mL.

[0314] Some non-limiting examples of lipid compounds that can be used (e.g., in combination with other lipid components) to form lipid nanoparticles for the compositions described herein, e.g., delivery of nucleic acids (e.g., RNA) described herein, include the following: [ka]

[0315] In some embodiments, LNPs comprising formula (i) are used to deliver ANDbody RNA compositions described herein to the liver and / or hepatocytes. [ka]

[0316] In some embodiments, LNPs comprising formula (ii) are used to deliver ANDbody RNA compositions described herein to the liver and / or hepatocytes. [ka]

[0317] In some embodiments, LNPs comprising formula (iii) are used to deliver ANDbody RNA compositions described herein to the liver and / or hepatocytes. [ka]

[0318] In some embodiments, LNPs comprising formula (v) are used to deliver ANDbody RNA compositions described herein to the liver and / or hepatocytes. [ka]

[0319] In some embodiments, LNPs comprising formula (vi) are used to deliver ANDbody RNA compositions described herein to the liver and / or hepatocytes. [ka]

[0320] In some embodiments, LNPs comprising formula (viii) are used to deliver ANDbody RNA compositions described herein to the liver and / or hepatocytes. [ka]

[0321] In some embodiments, LNPs comprising formula (ix) are used to deliver ANDbody RNA compositions described herein to the liver and / or hepatocytes. [ka] During the ceremony, X1 is O, NR1, or a direct bond, X2 is C2-5 alkylene, X3 is C(=O) or a direct bond, R1 is H or Me, R3 is Ci-3 alkyl, and R2 is Ci-3 alkyl, or R2 together with the nitrogen atom to which it is attached and 1 to 3 carbon atoms of X2 form a 4-, 5-, or 6-membered ring, or X1 is NR1, and R1 and R2 together with the nitrogen atom to which they are attached form a 5-, or 6-membered ring, or R2 together with R3 and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y1 is C2-12 alkylene, and Y2 is one of the following: [ka] (either orientation), [ka] (either orientation), [ka] (either orientation) is selected from n is 0 to 3, R4 is Ci-15 alkyl, and Z1 is Ci-6 alkylene or a direct bond; Z 2 teeth, [ka] (either orientation) or absent, with the proviso that when Z1 is a direct bond, Z2 is absent; R5 is a C5-9 alkyl or a C6-10 alkoxy, R6 is a C5-9 alkyl or a C6-10 alkoxy, W is methylene or a direct bond, R7 is H or Me, or a salt thereof, provided that when R3 and R2 are C2 alkyl, X1 is O, X2 is a straight-chain C3 alkylene, X3 is C(=0), Y1 is a straight-chain Ce alkylene, and (Y2)n-R4 is [ka] and R4 is a straight chain C5 alkyl, Z1 is a C2 alkylene, Z2 is absent, W is methylene, R7 is H, and R5 and R6 are not Cx alkoxy.

[0322] In some embodiments, an LNP comprising formula (xii) is used to deliver an ANDbody RNA composition described herein to the liver and / or hepatocytes. [ka]

[0323] In some embodiments, an LNP comprising formula (xi) is used to deliver an ANDbody RNA composition described herein to the liver and / or hepatocytes. [ka] [ka]

[0324] In some embodiments, the LNP comprises a compound of formula (xiii) and a compound of formula (xiv). [ka]

[0325] In some embodiments, an LNP comprising formula (xv) is used to deliver an ANDbody RNA composition described herein to the liver and / or hepatocytes. [ka]

[0326] In some embodiments, LNPs comprising a formulation of formula (xvi) are used to deliver ANDbody RNA compositions described herein to lung endothelial cells. [ka] [ka]

[0327] In some embodiments, the lipid compounds used to form lipid nanoparticles for delivering the compounds described herein, e.g., nucleic acids (e.g., RNA) described herein, are made by one of the following reactions: [ka]

[0328] In some embodiments, a compound (e.g., a nucleic acid or a protein) described herein is provided in an LNP comprising an ionizable lipid. In some embodiments, the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); for example, as described in Example 1 of U.S. Pat. No. 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)propyloctadeca-9,12-dienoate (LP01), for example, as synthesized in Example 13 of WO 2015 / 095340, incorporated herein by reference in its entirety. In some embodiments, the ionizable lipid is di((Z)-non-2-en-1-yl)9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., as synthesized in Examples 7, 8, or 9 of U.S. Patent Application Publication No. 2012 / 0027803 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), e.g., as synthesized in Examples 14 and 16 of WO 2010 / 053572 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is the 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)propanoate, e.g., structure (I) of WO 2020 / 106946, which is incorporated herein by reference in its entirety.

[0329] In some embodiments, the ionizable lipid may be a cationic lipid, an ionizable cationic lipid, e.g., a cationic lipid that can exist in a positively or 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, for example, under physiological conditions. Exemplary cationic lipids contain one or more positively charged amine groups. In some embodiments, the lipid particles contain the cationic lipid in a formulation together with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structured lipids (e.g., sterols), PEG, cholesterol, and polymer-conjugated lipids. In some embodiments, the cationic lipid may be an ionizable cationic lipid. Exemplary cationic lipids disclosed herein may have an effective pKa greater than 6.0. In some embodiments, the lipid nanoparticle may contain a second cationic lipid having an effective pKa different from that of the first cationic lipid (e.g., greater than the first effective pKa). The lipid nanoparticles may comprise 40-60 mol % cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and a therapeutic agent encapsulated within or associated with the lipid nanoparticles, such as a nucleic acid (e.g., RNA) as described herein. In some embodiments, the nucleic acid is co-formulated with the cationic lipid. The nucleic acid may be adsorbed to the surface of an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the nucleic acid may be encapsulated in an LNP, e.g., an LNP comprised of a cationic lipid. In some embodiments, the lipid nanoparticles may comprise a targeting moiety, e.g., coated with a targeting agent. In some embodiments, the LNP formulation is biodegradable.In some embodiments, lipid nanoparticles comprising one or more lipids described herein, e.g., formula (i), (ii), (vii) and / or (ix), encapsulate at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or 100% of the RNA molecules.

[0330] Exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, but are not limited to, those listed in Table 1 of International Publication No. 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 U.S. Patent Application Publication No. 2016 / 0311759; I of U.S. Patent Application Publication No. 20150376115 or U.S. Patent Application Publication No. 2016 / 0376224; I, II, or III of U.S. Patent Application Publication No. 20160151284; I, IA, II, or IIA of U.S. Patent Application Publication No. 20170210967; Ic of U.S. Patent Application Publication No. 20150140070; U.S. Patent Application Publication No. 2013 / 0178541 No. 2013 / 0303587 or U.S. Patent Application Publication No. 2013 / 0123338, U.S. Patent Application Publication No. 2015 / 0141678, U.S. Patent Application Publication No. 2015 / 0239926, II, III, IV, or V; U.S. Patent Application Publication No. 2017 / 0119904, U.S. Patent Application Publication No. 2017 / 117528, I or II; U.S. Patent Application Publication No. 2012 / 0149894, U.S. Patent Application Publication No. 2015 / 0057373, U.S. Patent Application Publication No. 2013 / 11 No. 6126, A; U.S. Patent Application Publication No. 2013 / 0090372, A; U.S. Patent Application Publication No. 2013 / 0274523, A; U.S. Patent Application Publication No. 2013 / 0274504, A; U.S. Patent Application Publication No. 2013 / 0053572, A; WO 2013 / 016058, A; WO 2012 / 162210, A; U.S. Patent Application Publication No. 2008 / 042973, I; U.S. Patent Application Publication No. 2012 / 01287670, I, II, III, or IV; U.S. Patent Application Publication No. 2014 / 02 No. 2015 / 00257, I or II; U.S. Patent Application Publication No. 2015 / 0203446, I, II, or III; U.S. Patent Application Publication No. 2015 / 0005363, I or III; U.S. Patent Application Publication No. 2014 / 0308304, I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV; U.S. Patent Application Publication No. 2013 / 0338210; WO 2009 / 132131, I, II, III, or IV; U.S. Patent Application Publication No. 2012 / 01011478, A;U.S. Patent Application Publication No. 2012 / 0027796, I or XXXV; U.S. Patent Application Publication No. 2012 / 0058144, XIV or XVII; U.S. Patent Application Publication No. 2013 / 0323269; U.S. Patent Application Publication No. 2011 / 0117125, I; U.S. Patent Application Publication No. 2011 / 0256175, I, II, or III; U.S. Patent Application Publication No. 2012 / 0202871, I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII; U.S. Patent Application No. I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of U.S. Patent Application Publication No. 2011 / 0076335; I or II of U.S. Patent Application Publication No. 2006 / 008378; I of U.S. Patent Application Publication No. 2013 / 0123338; ​​I or XAYZ of U.S. Patent Application Publication No. 2015 / 0064242; XVI, XVII, or XVIII of U.S. Patent Application Publication No. 2013 / 0022649; U.S. Patent Application Publication No. 2013 / 0116307 No. I, II, or III; U.S. Patent Application Publication No. 2013 / 0116307, I, II, or III; U.S. Patent Application Publication No. 2010 / 0062967, I or II; U.S. Patent Application Publication No. 2013 / 0189351, I-X; U.S. Patent Application Publication No. 2014 / 0039032, I; U.S. Patent Application Publication No. 2018 / 0028664, V; U.S. Patent Application Publication No. 2016 / 0317458, I; U.S. Patent Application Publication No. 2013 / 0195920, I; U.S. Patent No. 10,222,222 No. 1,127, No. 5, 6, or 10; No. WO 2018 / 081480, No. III-3; No. WO 2020 / 081938, No. I-5 or I-8; No. U.S. Pat. No. 9,867,888, No. 18 or 25; No. U.S. Pat. App. Pub. No. 2019 / 0136231, No. A; No. WO 2020 / 219876, No. II; No. U.S. Pat. App. Pub. No. 2012 / 0027803, No. OF-02; No. U.S. Pat. App. Pub. No. 2019 / 0240349, No. 23; No. U.S. Pat. No. 10,086,013, No. 23; Miao cKK-E12 / A6 from et al (2020); C12-200 from WO 2010 / 053572; 7C1 from Dahlman et al (2017); 304-O13 or 503-O13 from Whitehead et al; TS-P4C2 from U.S. Pat. No. 9,708,628;International Publication No. 2020 / 106946 I;International Publication No. 2020 / 106946 I. ;

[0331] In some embodiments, the ionizable lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-L9-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), for example, as described in Example 9 of WO2019051289A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is the lipid ATX-002, for example, as described in Example 10 of WO2019051289A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is (13Z,16Z)-A,A-dimethyl-3-nonyldecosa-13,16-dien-1-amine (compound 32), e.g., as described in Example 11 of WO2019051289A9 (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 WO2019051289A9 (incorporated herein by reference in its entirety).

[0332] Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC). , palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine (e.g., 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine ethanolamine (e.g., 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 (D EPC), palmitoyloleylphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleylphosphatidylcholine, or a mixture thereof.It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10 to C24 carbon chains, such as lauroyl, myristoyl, paimitoyl, 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. Such lipids include, in some embodiments, plant lipids that have been found to improve liver transfection with mRNA (e.g., DGTS).

[0333] Other examples of non-cationic lipids suitable for use in lipid nanoparticles include, but are not limited to, non-phospholipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphiphilic acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, sphingomyelin, etc. Other non-cationic lipids are described in WO 2017 / 099823 or U.S. Patent Application Publication No. 2018 / 0028664, the contents of which are incorporated herein by reference in their entirety.

[0334] In some embodiments, the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of U.S. Patent Application Publication No. 2018 / 0028664, the entire contents of which are incorporated herein by reference. The non-cationic lipid may comprise, for example, 0-30% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In several embodiments, the molar ratio of ionizable lipid to neutral lipid ranges 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).

[0335] In some embodiments, the lipid nanoparticles do not include any phospholipids.

[0336] In some embodiments, lipid nanoparticles may further comprise components such as sterols to provide membrane integrity. One example of a sterol that can be used in lipid nanoparticles is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs such as 5a-cholestanol, 53-coprostanol, cholesteryl-(2,-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog, for example, cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in International Publication No. 2009 / 127060 and U.S. Patent Application Publication No. 2010 / 0130588, each of which is incorporated herein by reference in its entirety.

[0337] In some embodiments, membrane integrity-conferring components, such as sterols, may comprise 0-50% (mol) of the total lipid present in the lipid nanoparticle (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%). In some embodiments, such components comprise 20-50% (mol), 30-40% (mol) of the total lipid content of the lipid nanoparticle.

[0338] In some embodiments, the lipid nanoparticles may contain polyethylene glycol (PEG) or conjugated lipid molecules. These are generally 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 (e.g., 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 a (methoxypolyethylene glycol)-conjugated lipid.

[0339] Exemplary PEG-lipid conjugates include, but are not limited to, the following: PEG-diacylglycerol (DAG) (e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEGS-DAG) (e.g., 4-O-(2',3'-di(tetramethyl) ... PEG-lipid conjugates include, for example, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, PEG-dialkoxypropylcarbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEG-lipid conjugates are described, for example, in U.S. Pat. No. 5,885,613, U.S. Pat. No. 6,287,591,

[0340] U.S. Patent Application Publication Nos. 2003 / 0077829, 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2010 / 0130588, 2016 / 0376224, 2017 / 0119904, and 2017 / 099823 (the entire contents of which are incorporated herein by reference). In some embodiments, the PEG-lipid is a compound of formula III, III-aI, III-a-2, III-b-1, III-b-2, or V of U.S. Patent Application Publication No. 2018 / 0028664, the entire contents of which are incorporated herein by reference. In some embodiments, the PEG-lipid is of Formula II in U.S. Patent No. 20150376115 or U.S. Patent Application Publication No. 2016 / 0376224, the contents of both of which are incorporated herein by reference in their entireties. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. PEG-lipids include PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (l-[8'-(cholest-5-en-3[β]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[ω]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-ditetradecanoic acid), and PEG-DMG (3,4-ditetradecanoic acid). PEG-lipids may be one or more of: PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], ... [ka]

[0341] In some embodiments, lipids conjugated with molecules other than PEG can also be used in place of PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used instead of or in addition to PEG-lipids.

[0342] Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids, are described in the PCT and LIS patent applications listed in Table 2 of WO2019051289A9, the contents of all of which are incorporated herein by reference in their entireties.

[0343] In some embodiments, PEG or conjugated lipids may comprise 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 be varied as needed. For example, lipid particles may comprise 30-70% ionizable lipids by molar or total weight of the composition, 0-60% cholesterol by molar or total weight of the composition, 0-30% non-cationic lipids by molar or total weight of the composition, and 1-10% conjugated lipids by molar or total weight of the composition. Preferably, the composition comprises 30-40% ionizable lipids by molar or total weight of the composition, 40-50% cholesterol by molar or total weight of the composition, and 10-20% non-cationic lipids by molar or total weight of the composition. In some other embodiments, the composition contains 50-75% ionizable lipid by molar or total weight, 20-40% cholesterol by molar or total weight, 5-10% non-cationic lipid by molar or total weight, and 1-10% complex lipid by molar or total weight. The composition may contain 60-70% ionizable lipid by molar or total weight, 25-35% cholesterol by molar or total weight, and 5-10% non-cationic lipid by molar or total weight. The composition may also contain up to 90% ionizable lipid by molar or total weight and 2-15% non-cationic lipid by molar or total weight.The formulation may also be a lipid nanoparticle formulation, for example, 8-30% ionizable lipid by molar or total weight of the composition, 5-30% non-cationic lipid by molar or total weight of the composition, and 0-20% cholesterol by molar or total weight of the composition; 4-25% ionizable lipid by molar or total weight of the composition, 4-25% non-cationic lipid by molar or total weight of the composition, 2-25% cholesterol by molar or total weight of the composition, 10-35% complex lipid by molar or total weight of the composition, and 5% cholesterol by molar or total weight of the composition. Alternatively, the lipid particle formulation may comprise 2-30% ionizable lipid by molar or total weight of the composition, 2-30% noncationic lipid by molar or total weight of the composition, 1-15% cholesterol by molar or total weight of the composition, 2-35% complex lipid by molar or total weight of the composition, and 1-20% cholesterol by molar or total weight of the composition; alternatively, up to 90% ionizable lipid by molar or total weight of the composition and 2-10% noncationic lipid by molar or total weight of the composition, or up to 100% cationic lipid by molar or total weight of the composition. In some embodiments, the lipid particle formulation comprises an ionizable lipid, phospholipid, cholesterol, and PEGylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation comprises an ionizable lipid, cholesterol, and PEGylated lipid in a molar ratio of 60:38.5:1.5.

[0344] In some embodiments, the lipid particles comprise an ionizable lipid, a non-cationic lipid (e.g., a phospholipid), a sterol (e.g., cholesterol), and a PEGylated lipid, wherein the molar ratio of lipids ranges from 20-70 mole percent with a target of 40-60 for the ionizable lipid, the molar percent of non-cationic lipid ranges from 0-30 with a target of 0-15, the molar percent of sterol ranges from 20-70 with a target of 30-50, and the molar percent of PEGylated lipid ranges from 1-6 with a target of 2-5.

[0345] In some embodiments, the lipid particles comprise ionizable lipid / non-cationic lipid / sterol / complex lipid in a molar ratio of 50:10:38.5:1.5.

[0346] In one aspect, the present disclosure provides a lipid nanoparticle formulation comprising a phospholipid, a lecithin, a phosphatidylcholine, and a phosphatidylethanolamine.

[0347] In some embodiments, one or more additional compounds may also be included. These compounds may be administered separately, or the additional compounds may be contained in the lipid nanoparticles of the present invention. In other words, the lipid nanoparticles may contain other compounds in addition to the nucleic acid or at least a second nucleic acid different from the first nucleic acid. The other additional compounds may be selected from the group consisting of organic or inorganic small or macromolecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, or any combination thereof.

[0348] In some embodiments, LNPs are targeted to specific tissues by adding an LNP targeting domain. For example, by displaying a biological ligand on the surface of LNPs, it can enhance the interaction with cells that display their cognate receptor, thereby promoting the association of cells with tissues that express the receptor and the delivery of cargo to the tissue. In some embodiments, the biological ligand can be a ligand that promotes delivery to the liver, for example, LNPs that display GalNAc achieve the delivery of nucleic acid cargo to hepatocytes that display asialoglycoprotein receptor (ASGPR). Akinc et al. Mol Ther 18(7):1357-1364 (2010) teaches that the conjugation of trivalent GalNAc ligands with PEG-lipids (GalNAc-PEG-DSG) can produce ASGPR-dependent LNPs for observable LNP cargo effects (see, for example, Akinc et al. 2010, supra, Figure 6).For LNP formulations displaying other ligands, e.g., LNP formulations incorporating folate, transferrin, or antibodies, see WO 2017223135, which is incorporated herein by reference in its entirety, as well as the references used therein, i.e., 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 US 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.

[0349] In some embodiments, LNPs are selected for tissue-specific activity by adding Selective ORgan Targeting (SORT) molecules to formulations containing traditional components such as ionizable cationic lipids, amphipathic phospholipids, cholesterol, and poly(ethylene glycol) (PEG) lipids. The teachings of Cheng et al. Nat Nanotechnol 15(4):313-320 (2020) demonstrate that the addition of supplemental "SORT" components precisely alters in vivo RNA delivery profiles and mediates tissue-specific (e.g., lung, liver, spleen) gene delivery and editing as a function of the percentage and biophysical properties of the SORT molecules.

[0350] In some embodiments, the LNPs comprise a biodegradable, ionizable lipid, such as (9Z,l2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,l2-dienoate (also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,l2Z)-octadeca-9,l2-dienoate)) or another ionizable lipid. See, e.g., the lipids of WO 2019 / 067992, WO 2017 / 173054, WO 2015 / 095340, and WO 2014 / 136086 and the references provided therein. In some embodiments, the terms cationic and ionizable in relation to LNP lipids are interchangeable, e.g., ionizable lipids are cationic depending on pH.

[0351] In some embodiments, the mean LNP diameter of an LNP formulation may be tens to hundreds of nanometers, as measured, for example, by dynamic light scattering (DLS). In some embodiments, the mean LNP diameter of an LNP formulation may be about 40 nm to about 150 nm, e.g., about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm. In some embodiments, the mean LNP diameter of the LNP formulation can be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In some embodiments, the mean LNP diameter of the LNP formulation can be about 70 nm to about 100 nm. In certain embodiments, the mean LNP diameter of the LNP formulation can be about 80 nm. In some embodiments, the mean LNP diameter of the LNP formulation can be about 100 nm. In some embodiments, the mean LNP diameter of the LNP formulation ranges from about 1 mm to about 500 mm, about 5 mm to about 200 mm, about 10 mm to about 100 mm, about 20 mm to about 80 mm, about 25 mm to about 60 mm, about 30 mm to about 55 mm, about 35 mm to about 50 mm, or about 38 mm to about 42 mm.

[0352] LNPs may, in some cases, be relatively uniform. The polydispersity index can be used to indicate the homogeneity of LNPs, e.g., the particle size distribution of lipid nanoparticles. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. LNPs may have a polydispersity index of about 0 to about 0.25, e.g., 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 LNPs may be about 0.10 to about 0.20.

[0353] The zeta potential of LNPs can be used to indicate the kinetic potential of the composition. In some embodiments, the zeta potential can represent the surface charge of the LNP. Generally, lipid nanoparticles with a relatively low positive or negative charge are desirable, as more highly charged species may have undesirable interactions with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the LNP can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0354] The efficiency of protein and / or nucleic acid encapsulation represents the amount of protein and / or nucleic acid encapsulated or otherwise bound to the LNPs after preparation relative to the initial amount provided. A high encapsulation efficiency (e.g., approaching 100%) is desirable. For example, encapsulation efficiency can be measured by comparing the amount of protein or nucleic acid in a solution containing lipid nanoparticles before and after disintegrating 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 solution. Fluorescence can be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in solution. For the lipid nanoparticles described herein, the encapsulation efficiency of proteins and / or nucleic acids may be at least 50%, e.g., 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 may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0355] The LNPs may optionally include one or more coatings. In some embodiments, the LNPs may be formulated into capsules, films, or tablets having a coating. The capsules, films, or tablets containing the compositions described herein can have any useful size, tensile strength, hardness, or density.

[0356] Further exemplary lipids, formulations, methods, and characterization of LNPs are taught by International Publication No. WO2020061457, which is incorporated by reference in its entirety.

[0357] In some embodiments, in vitro or ex vivo cell lipofection is performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In certain embodiments, LNPs are formulated with GenVoy_ILM ionizable lipid mix (Precision NanoSystems). In certain embodiments, LNPs are formulated with 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which is described in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), which is incorporated herein by reference in its entirety.

[0358] LNP formulations optimized for delivery of CRISPR-Cas systems, e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA, are described in WO 2019067992 and WO 2019067910, both of which are incorporated by reference.

[0359] Another specific LNP formulation useful for delivery of nucleic acids is described in U.S. Pat. No. 8,158,601 and U.S. Pat. No. 8,168,775, both of which are incorporated by reference, and includes the formulation used in patisiran, sold under the name ONPATTRO.

[0360] Exemplary doses of LNPs comprising the RNA compositions described herein can 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 AAV comprising nucleic acids encoding one or more components of the system can include MOIs of about 10, 10, 10, and 10 vg / kg.

[0361] In some embodiments, the invention includes lipid nanoparticles (LNPs) comprising an ANDbody polypeptide (or RNA encoding same), a nucleic acid molecule, or DNA encoding an ANDbody described herein. In embodiments, the LNPs comprise a cationic lipid. In some embodiments, the LNPs further comprise one or more neutral lipids, e.g., DSPC, DPPC, DMPC, DOPC, POPC, DOPE, SM, a steroid, e.g., cholesterol, and / or one or more polymer-conjugated lipids, e.g., a pegylated lipid, e.g., PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkyoxypropylcarbamate. In some embodiments, the cationic lipid of the LNP has the following structure: [ka]

[0362] For a general review of LNPs, see, e.g., Li et al. 2017, Nanomaterials 7, 122; doi:10.3390 / nano7060122.

[0363] Other Carriers viral vectors The compositions described herein (e.g., polypeptide or RNA ANDbody compositions) can be delivered by a viral vector (e.g., a viral vector that expresses RNA). The viral vector can be administered to a cell or a subject (e.g., a human subject or a non-human animal). The viral vector can be administered locally or systemically.

[0364] 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 viruses), coronaviruses; negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., paramyxoviruses (e.g., measles and Sendai)); positive-strand RNA viruses such as picornaviruses and alphaviruses; and adenoviruses, herpesviruses (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus, replication-deficient herpes viruses), and poxviruses (e.g., vaccinia, modified vaccinia Ankara). Examples of retroviruses include double-stranded DNA viruses such as variola (Ankara), avian pox (MVA), avian pox, and canary pox. Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, human papillomaviruses, human foamy viruses, and hepatitis viruses. Examples of retroviruses include avian leukosis sarcoma viruses, avian C viruses, mammalian C viruses, B viruses, D viruses, oncoretroviruses, HTLV-BLV complex, lentiviruses, alpharetroviruses, gammaretroviruses, and spumaviruses (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 ape 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, the teachings of which are incorporated herein by reference.

[0365] Anellovirus vectors can also be used to deliver the ANDbody compositions described herein. Anellovectors are known in the art and are described, for example, in International Publication Nos. 2020123773, 2020123816, 2018232017, and 2020123773. In certain embodiments, anellovector compositions contain a genomic element comprising a promoter operably linked to a nucleic acid sequence encoding an ANDbody described herein, wherein the genetic element is encapsulated by a proteinaceous outer layer comprising anellovirus ORF1, e.g., anellovirus capsid protein.

[0366] Cell- and Vesicle-Based Carriers The compositions described herein (e.g., polypeptide or RNA ANDbody compositions) can be administered to cells, vesicles, or other membrane-based carriers. In one embodiment, the compositions and systems described herein can be formulated into liposomes or other similar vesicles. Liposomes are spherical vesicular structures composed of a single or multilayer lipid bilayer membrane surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer membrane. Liposomes can be anionic, neutral, or cationic. Liposomes are biocompatible, non-toxic, capable of delivering both hydrophilic and lipophilic drug molecules, protecting their cargo from degradation by plasma enzymes, and transporting their load across biological membranes and the blood-brain barrier (BBB) ​​(see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679 for a review). Vesicles can be made from several kinds of lipids; however, phospholipids are most commonly used to produce liposomes as drug carriers.The method of preparing multilamellar vesicle lipids is known in the art (see U.S. Patent No. 6,693,086; the teaching of this patent on the preparation of multilamellar vesicle lipids is incorporated herein by reference).Vesicle formation can occur spontaneously when lipid membrane is mixed with aqueous solution, but can also be promoted by applying force in the form of shaking using a homogenizer, sonicator or extrusion device (for example, for general review, see Spuch and Navarro, Journal of Drug Delivery, vol.2011, Article ID 469679, 12 pages, 2011.doi:10.1155 / 2011 / 469679). Extruded lipids can be prepared by extrusion through size-reducing filters 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.

[0367] Exosomes can also be used as drug delivery vehicles for the compositions and systems described herein. For a review, see Ha et al. July 2016. Acta Pharmaceutica Sinica B. Volume 6, Issue 4, Pages 287-296; https: / / doi.org / 10.1016 / j.apsb.2016.02.001.

[0368] Additionally, ex vivo differentiated erythrocytes can also be used as carriers of agents (eg, inhibitors) described herein, such as antibodies or nucleic acids described herein. See, for example, WO 2015073587; WO 2017123646; WO 2017123644; WO 2018102740; WO 2016183482; WO 2015153102; WO 2018151829; WO 2018009838; Shi et al. 2014. Proc Natl Acad Sci USA. 111(28):10131-10136; U.S. Patent No. 9,644,180; Huang et al. Nature Communications 8:423; Shi et al. Proc Natl Acad Sci USA. 111(28):10131-10136.

[0369] For example, fusosome compositions such as those described in WO2018208728 may also be used as carriers for delivering the agents or preparations described herein.

[0370] Plant nanovesicles and plant messenger packs (PMPs), such as those described in WO 2011097480, WO 2013070324, WO 2017004526, or WO 2020041784, can also be used as carriers for delivering the compositions described herein.

[0371] Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications and sections thereof cited herein are incorporated by reference for the purpose or subject matter discussed herein. [Example]

[0372] Example The present invention is further illustrated in the following non-limiting examples.

[0373] [Table 14]

[0374] Example 1. Antibody binding mouse and human RAGE and NOTCH2 1.1 Generation of anti-RAGE antibodies by vaccination An exemplary target of this technology, an antibody against the human RAGE extracellular domain, was generated by immunization. The extracellular domain of human RAGE (NCBI protein accession Q15109, positions N24-A344) fused to the Fc region of human IgG1 (UniProt ID P01857, positions P100-K330) (huRAGE) was expressed in HEK293F cells. Briefly, the DNA sequence was codon-optimized for mammalian expression and appropriately sequenced within 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), as previously described (Rothschilds et al. 2019). Female BALB / c mice were immunized with 50 μg of huRAGE-Fc fusion protein via intravenous injection in CFA / IFA (Millipore Sigma, catalog numbers F5881-10ML and F5506-10ML) adjuvant. Hybridomas were then generated (Listek et al. 2020). Clones were first screened for IgG reactivity specific to the huRAGE-Fc fusion protein used for immunization in an ELISA format, followed by flow cytometry testing using cells stably (CHO) or transiently (HEK293F) transfected with full-length huRAGE. Anti-RAGE hybridoma clones were then evaluated based on mouse cross-reactivity. Flow cytometry testing was performed using cells stably (CHO) or transiently (HEK293F) transfected with full-length murine RAGE (mRAGE) to select clones that bind to mRAGE. Positive clones expressing cross-reactive anti-RAGE mAbs between human and mouse were then further purified by limiting dilution cloning. Hybridomas were grown in DMEM / 2% ultra-low IgG serum, and mAbs were purified by protein G chromatography using Millipore Sigma, P3296-1ML, according to the manufacturer's instructions.

[0375] 1.2 Selection of inactive anti-RAGE antibodies The address target binding moieties of the present technology are designed so as not to affect signal transduction upon binding to an address target, such as the exemplary RAGE address target. Thus, the anti-RAGE hybridoma clones produced as described above are further evaluated based on their inability to block RAGE ligand binding. Human RAGE ligands tested included: HMGB1 (full length, Creative BioMart catalog #HMGB1-29332TH), Advanced Glycation Endproduct (fused to bovine serum albumin, Millipore Sigma catalog #121800-10MG-M), S100A12 (full length, R&D Systems catalog #1052-ER-050), S100A1 (full length, R&D Systems catalog #9705-S1-100), S100A4 (R&D Systems catalog #137-S4-050), S100A10 (full length, Creative BioMart catalog #S100A10-157H), S100A11 (R&D Systems catalog #9015-S11-050), S100A13 (R&D R&D Systems catalog #4327-SA-050), S100B (R&D Systems catalog #1820-SB-050), amyloid beta peptide (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA from NM_000484.2, Millipore Sigma catalog #AG912-1MG), and Mac-1 (F17-N1105 from NP_001139280 and Q23-N700 from UniProt P05107, R&D Systems catalog #4047-AM-050). ELISA was used to quantify the ability of the ligand to bind in the presence of anti-RAGE antibodies. huRAGE-Fc was adsorbed to ELISA plates, blocked, and then the plates were incubated with hybridoma-derived anti-RAGE antibody clones at concentrations ranging from 10 fM to 10 μM (one condition for each concentration and each clone). After washing the plates, the ligands are each biotinylated according to the manufacturer's instructions (ThermoFisher catalog #21435) and then incubated on the plates at concentrations ranging from 10 fM to 10 uM.After washing, SA-conjugated HRP secondary antibody is added, followed by TMB substrate, and a colorimetric readout is quantified by absorbance. To isolate anti-RAGE clones that are inactive and do not affect the binding of one or more ligands, ligand binding is compared for a given ligand with and without the addition of anti-RAGE antibody.

[0376] The inability of anti-RAGE antibodies to inhibit the IFNα-induced gene signature was also assessed, and anti-RAGE hybridoma clones that did not alter cell signaling were selected based on the IFNα-induced gene signature assay. PBMCs from healthy human donors were stimulated with 50% serum from SLE patients for 4 hours. The assay was completed in the presence of either anti-RAGE antibodies or an unrelated (negative) isotype control human IgG1 antibody (Bio X Cell catalog #BE0297) at antibody concentrations ranging from 10 fM to 10 μM. Additionally, a huRAGE-Fc fusion molecule was used as a positive control. Total RNA was purified, and the expression of type I IFN-inducible genes, including DDX58, G1P2, MX1, OAS3, RSAD2, IFIT1, and IFI35, was measured by real-time qRT-PCR analysis as previously described (International Publication No. WO 2008 / 137552 A2, https: / / patentimages.storage.googleapis.com / 94 / 26 / c8 / 7b9f27f693c4b6 / WO2008137552A2.pdf). Gene expression inhibition values ​​were normalized to those of a negative control Ab.

[0377] 1.3 Generation of anti-Notch2 antibodies by vaccination An exemplary effector target of this technology, an antibody against the extracellular domain of human Notch2 (huNotch2) fused to the Fc region of human IgG1, is generated by immunization similar to that described above for RAGE. After immunization and hybridoma generation, clones are screened exactly as described above, but for binding to full-length human and mouse Notch2 (instead of RAGE). Positive clones expressing cross-reactive anti-Notch2 mAbs between human and mouse are then further purified by limiting dilution cloning. Hybridomas are grown in DMEM / 2% ultra-low IgG serum, and mAbs are purified by protein G chromatography.

[0378] 1.4 Wide IC 50 Selection of anti-Notch2 antibodies with a broad range of activity The effector target binding site of this technology, such as Notch2, is designed so that it does not affect signal transduction upon binding to the effector target unless it is localized to the target tissue by an address target binding site such as RAGE. Therefore, the binding affinity of the effector target (e.g., Notch2) binding site is analyzed. Specifically, the IC of a Notch2 antibody against the ligand human Jagged-2-Fc fusion protein (Creative BioMart, JAG2-382H) that binds to surface Notch2 is analyzed. 50 were assessed using flow cytometry to determine IC 50 Select antibodies with a concentration ranging from less than 1 nM to 5 uM. Jagged-2-Fc is labeled with Alexa Fluor 647 (AF647) according to the manufacturer's instructions (ThermoFisher, A20186) and as previously described (Tzeng et al. 2015).

[0379] HEK293F cells are transiently transfected with full-length huNotch2. The cells are incubated with increasing concentrations of anti-Notch2 antibodies ranging from 1 pM to 50 uM. Then (without washing the cells), the cells are incubated with AF647-labeled Jagged-2-Fc at constant concentrations ranging from 1 pM to 50 uM for 1 hour at 4°C. Each IC 50 A fixed concentration of AF647 Jagged-2-Fc is selected along with various anti-Notch2 antibodies in the assay. Binding of AF647 Jagged-2-Fc to increasing concentrations of anti-Notch2 antibodies is quantified on cells by flow cytometry using a ThermoFisher Attune NxT (B2R3Y3V6).

[0380] 1.5 Expression and purification of bispecific ANDbodies I C 50 DNA sequences from 10 RAGE antibodies and 10 Notch2 antibodies ranging in potency (<1 nM to 5 μM) were cloned into a human IgG1 framework with a single matching point mutation within the CH3 domain Fc region using In-Fusion HD Cloning (Takara Bio, catalog #638911) according to the "Controlled Fab-Arm Exchange" (cFAE) method (Labrijn et al. 2014). The antibodies were individually expressed from transient HEK293 cells and purified using Protein A affinity resin. The parent antibodies (combination of one RAGE antibody and one Notch2 antibody) were then converted into a bispecific RAGE / Notch2 ANDbody according to the cFAE method. Briefly, the parent antibodies were mixed under permissive redox conditions to allow recombination of half molecules. The reducing agent was then removed to allow reoxidation of the interchain disulfide bonds. Finally, the exchange efficiency was quantified using chromatography or mass spectrometry-based methods. Approximately 100 RAGExNotch2 mutant ANDbodies will be created.

[0381] 1.6 Affinity of ANDbody variants To identify ANDbody™ variants that meet the desired effector-target and address-target binding affinity criteria, SPR-based affinity measurements are performed on a BIAcore model 2000 or T100 (Biacore / GE Healthcare, Piscataway, NJ) at 25°C using HBS-EP+ buffer (Cytiva catalog #BR100669) containing 0.1 mg / ml BSA (Millipore Sigma catalog #A9418) as the running buffer. A Sensor Chip Protein A (Cytiva catalog #29127557) is used to capture mouse RAGE-Fc, human RAGE-Fc, mouse Notch2-Fc, or human Notch2-Fc. ANDbodies are injected in a 3-fold dilution series from 60 to 0.74 nM, and dissociation is monitored for all proteins for 10 minutes. Kinetic analysis is performed by simultaneously fitting the association and dissociation phases of the sensorgrams with a 1:1 Langmuir binding model in the manufacturer-supplied BIAevaluation software (Biacore). A double reference is applied in each analysis to remove background responses from the reference surface and buffer-only controls.

[0382] This assay allows quantitative evaluation of the affinity of all ANDbody variants for RAGE and Notch2. ANDbody variants with higher affinity for RAGE than for Notch2, as well as variants with no difference in affinity or higher affinity for Notch2, are used for subsequent in vitro and in vivo experiments.

[0383] 1.7 In vitro assay of Notch2 antagonism in cells with or without RAGE expression To characterize ANDbodies in vitro, HEK293F cells were transiently transfected with full-length huRAGE (R+) or full-length huNotch2 (N+), or co-transfected with both (RN+). Many mutant RAGExNotch2 ANDbodies were fluorescently labeled with Alexa Fluor 647 (AF647) according to the manufacturer's instructions (ThermoFisher, A20186) and as previously described (Tzeng et al. 2015). ANDbodies were then incubated with R+, N+, RN+, or combined R+ and N+ cells at ANDbody concentrations ranging from 10 fM to 10 μM. Parental anti-Notch2 monospecific antibodies (one from each mutant) were fluorescently labeled with FITC according to the manufacturer's instructions (ThermoFisher, 53027). In some conditions, the parental anti-Notch2 FITC-labeled antibody was incubated with cells pre-bound with AF647 RAGExNotch2 (the corresponding Notch2 variant) at concentrations between 10 fM and 10 uM to saturate the remaining binding sites of Notch2. The binding EC values ​​of both the AF647-labeled RAGExNotch2 ANDbody variant and the parental FITC-labeled anti-Notch2 antibody were measured. 50 was quantified using flow cytometry. When AF647 RAGExNotch2 was added followed by FITC Notch2 antibody, the FITC signal from those cells was subtracted from the FITC signal from FITC Notch2 alone on the cells (then normalized to the Notch2 alone signal) to quantify the % Notch2 bound by RAGExNotch2. These values ​​at different concentrations of RAGExNotch2 ANDbody were used to calculate the EC 50 A curve is generated. This assay can also be performed using AF647-labeled anti-Notch2 antibody instead of AF647-labeled ANDbody.

[0384] ECs observed in N+ and RN+ cells 50 The difference reveals that the presence of RAGE enhances Notch2 blockade and identifies cells expressing the RAGExNotch2 ANDbody.

[0385] 1.8 Biodistribution of ANDbodies and Parental Antibodies (In Vivo) To analyze the biodistribution of the ANDbody, female Balb / c and C57BL / 6 mice will be used to quantify the biodistribution of the RAGExNotch2 ANDbody and its respective parental antibodies (anti-Notch2 or anti-RAGE used in the cFAE of the ANDbody).

[0386] To quantify cellular biodistribution, proteins (ANDbodies and antibodies) were first individually labeled with AF647 according to the manufacturer's instructions (ThermoFisher, A20186) and previously described methods (Tzeng et al.). Each labeled antibody was then injected individually (tail vein) at doses of 10 μg, 100 μg, and 500 μg. An equal volume of saline (PBS) was also injected as a control.

[0387] For cell biodistribution, mice were euthanized using CO2 at 12 h, 1 day, 2 days, 3 days, 7 days, and 14 days postinjection. Tissues, including heart, lung, spleen, blood, kidney, liver, and intestine, were processed into single-cell suspensions according to previously described methods (Tzeng et al. 2015). Briefly, blood was collected by cardiac puncture into EDTA-treated tubes (BD catalog #365974). Other tissues were collected, weighed, mechanically dissociated between frosted glass slides, and filtered through a 70 μm mesh screen (Millipore Sigma, catalog #CLS431751-50EA) to form single-cell suspensions. Spleen cells, whole blood, and lungs were processed in ammonium chloride-potassium (ACK) lysis buffer (Thermofisher Scientific, catalog #A1049201). Hearts were digested with collagenase and processed into single-cell suspensions according to conventional methods (Covarrubias et al. 2019). Flow cytometry was performed on immune cells 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- SSC1o), dendritic cells (CD3e- CD11chi), NK cells (NK1.1+ CD3e-), and NKT cells (NK1.1+ CD3e+) as previously described (Tzeng et al. 2015). Lung cells, including epithelial (CD326+CD31-CD45-), endothelial (CD326-CD31+CD45-), and hematopoietic (CD326-CD31-CD45+) lineages, were analyzed as previously defined (Singer et al.). Antibodies were purchased from Biolegend, and flow cytometry was performed on a ThermoFisher Attune NxT (B2R3Y3V6) instrument. The presence of labeled ANDbody or other labeled antibodies on the cell surface was determined by the fluorescence of AF647 (this fluorophore was avoided in the flow panel).

[0388] To quantify tissue biodistribution, proteins (ANDbody and antibody) are first individually labeled with NHS-5 / 6-FAM (Thermofisher Scientific, catalog #46409) according to the manufacturer's instructions.

[0389] For tissue biodistribution, mice were euthanized using CO2 at 12 hours, 1 day, 2 days, 3 days, 7 days, and 14 days after injection, and tissues including lung, spleen, blood, kidney, liver, and intestine were collected, weighed, and imaged using an IVIS Spectrum imaging system (Caliper Life Sciences; excitation wavelength: 500 nm, emission wavelength: 540 nm). Images were analyzed using Living Image software.

[0390] 1.9 In vivo bioactivity to quantify gene expression changes To analyze ANDbody activity, we quantify bioactivity in vivo using female Balb / c and C57BL / 6 mice. To quantify bioactivity across tissues, we injected the RAGExNotch2 ANDbody or its parent antibody (anti-Notch2 or anti-RAGE used in the cFAE of the ANDbody) intravenously (tail vein) at doses of 10 μg, 100 μg, or 500 μg. As a control, we also injected the same volume of saline (PBS).

[0391] At 12 hours, 1 day, 2 days, 3 days, 7 days, and 14 days post-injection, tissues including lung, spleen, blood, kidney, liver, heart, and intestine were processed into single-cell suspensions according to a previously described method (Tzeng et al. 2015). Blood was collected by cardiac puncture into EDTA-treated tubes (BD catalog #365974), while other tissues were collected, weighed, mechanically dissociated between frosted glass slides, and filtered through a 70 μm mesh screen (Millipore Sigma, catalog #CLS431751-50EA) to form a single-cell suspension. Spleen cells, whole blood, and lungs were processed in ammonium chloride-potassium (ACK) lysis buffer (Thermofisher Scientific, catalog #A1049201).

[0392] 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 of RT-qPCR reaction mix containing 1X iqSYBR Green Supermix (Bio-Rad) and a total of 450 nM of forward and reverse primers. Reactions were performed on a BioRad CFX Real Time PCR Detection System using a two-step amplification protocol with the following thermal cycling parameters: 95°C for 3 minutes, followed by 40 cycles of 95°C for 10 seconds (melting) and 55°C for 30 seconds (annealing + extension). All reactions were performed in duplicate.

[0393] The genes involved in Notch2 signaling are mouse Hes1, Hey1, and HeyL, and the reference gene is SdhA. The primers used for amplification were the mouse Hes1 primer set (forward, 5'-CAACACGACCGGACAAAC-3' and reverse, 5'-AAGAATAAATGAAAGTCTAAGCCAA-3'), the mouse Hey1 primer set (forward, 5'-GCCGAAGTTG CCCGTTATCT-3' and reverse, 5'-CGCTGGATG CGTAGTTGTT-3'), the mouse HeyL primer set (forward, 5'-GAGCTGAC TTCCCACAACCA-3' and reverse, 5'-GAGAGG TGCCTTTGCGTAGA-3'), and the mouse SdhA primer set (forward, 5'-AGTGGGCT GTCTTCCTTAAC-3' and reverse, 5'-GGATTGCTTCT GTTTGCTTGG-3'), which have been previously described (Nandagopal et al. 2018). All primers were purchased from IDT DNA.

[0394] Hes1, Hey1, and HeyL gene expression is measured in ANDbody-treated, untreated, and anti-Notch2-treated mice, including in the lungs.

[0395] 1.10 In vivo bioactivity using body weight and histology Female Balb / c and C57BL / 6 mice were used to compare the pathology of organs, including the spleen, kidney, liver, heart, intestine, teeth, and lungs, by histology. Treatment with ANDbody, anti-Notch2 antibody alone, or saline (PBS) was compared. Starting at 8 weeks of age, mice were injected IV (tail vein) with 10 μg, 100 μg, or 500 μg of ANDbody or the corresponding Notch2 antibody (before cFAE) once or twice weekly. As a control, mice were injected with the same volume of saline (PBS) once or twice weekly. Mice were weighed twice weekly starting before the first treatment. Two, four, and six weeks after treatment, mice were euthanized, and organs were processed for histology.

[0396] Unless otherwise noted, organs are removed into cassettes and directly transferred to 10% neutral-buffered formalin (Sigma-Aldrich) for 12–24 h before being embedded in paraffin. Lungs are perfused with 10% neutral-buffered formalin before being placed in cassettes for immersion in neutral-buffered formalin. Intestines are thoroughly washed before being placed in cassettes for immersion in 10% neutral-buffered formalin. Paraffin sections (1–2 μm) are cut, dewaxed, and then subjected to histochemical staining. Sections are stained with hematoxylin / eosin (H&E; Merck, Darmstadt, Germany) and blindly scored according to immune infiltration and tissue morphology.

[0397] In addition to lung morphology, weight loss (or lack thereof) over the treatment period in mice treated with ANDbody is compared to weight loss (or lack thereof) in mice treated with anti-Notch2 and to weight loss (or lack thereof) in untreated mice.

[0398] Example 2. Antibodies that bind to mouse and human UMOD and NOTCH2 2.1 Generation of anti-UMOD antibodies by yeast surface display Yeast surface display (Chao et al. 2006) was used to generate antibodies against mouse UMOD (Creative BioMart, catalog #UMOD-17835M, untagged), an exemplary target addressed by this technology. This was performed using methods previously described (Angelini et al. 2015) and summarized below. Yeast display was initiated with a synthetic antibody library from the Sidhu lab, which is based on a natural framework, library 'G' (Van Deventer et al. 2015). The scFvs displayed on the yeast surface were selected for binding to mouse UMOD. Selection against human UMOD antigen (Creative BioMart, catalog #UMOD-001H, untagged) was then performed to ensure that binders were cross-reactive between the human and mouse forms. To increase the affinity of the scFv binders, affinity maturation was performed using error-prone PCR, as previously described (Angelini et al. 2015), and the resulting library was reselected for binding to both mouse and human UMOD. After engineering, many of the broadly cross-reactive scFvs are recloned into a human IgG1 antibody format.

[0399] 2.2 Selection of inactive anti-UMOD antibodies The address target binding sites of the present technology are designed so as not to affect signal transduction upon binding to an address target, such as an exemplary UMOD address target. Thus, anti-UMOD antibodies are further evaluated based on their inability to block UMOD ligand binding in assays such as those described above for RAGE antibodies or in in vivo assays. Inactive UMOD antibodies can be identified in assays in which the kidney structure is not affected or modified by the screened UMOD antibody.

[0400] 2.3 Generation of anti-Notch2 antibodies by vaccination Antibodies cross-reactive to mouse and human Notch2 are generated, cloned, and expressed into a human IgG1 framework according to conventional methods as described above.

[0401] 2.4 Wide range of ICs 50 Selection of active Notch2 antibodies Antibodies against Notch2 were used with broad IC 50 Select by range.

[0402] 2.5 Expression and Purification of ANDbodies as Bispecific Antibodies Wide range of ICs 50 The DNA sequences of 10 UMOD antibodies and 10 Notch2 antibodies with ribonucleotides (<1 nM to 5 uM) are made into 100 mutant ANDbodies as described above.

[0403] 2.6 Affinity of ANDbodies for UMOD and Notch2 The affinity of the ANDbodies for UMOD and Notch2 is assessed using BIAcore as described above. In this case, the human and mouse versions of his-tagged UMOD are immobilized on a Sensor Chip NTA (Cytiva catalog #BR100034). Human and mouse notch2-Fc are immobilized as described above.

[0404] 2.7 Biodistribution of ANDbodies and Parental Antibodies (In Vivo) Cell and tissue biodistribution studies are performed using the methods described above (as described above), except that the ANDbody used in this case is UMODxNotch2, and the parent antibodies correspond to anti-UMOD and anti-Notch2.

[0405] 2.8 In vivo bioactivity to quantify changes in gene expression The in vivo biological activity of the UMODxNotch2 ANDbody will be quantified using the gene expression methods described above.

[0406] 2.9 In vivo bioactivity using weight and histology The in vivo biological activity of the UMODxNotch2 ANDbody will be quantified using the weight and histology methods described above.

[0407] Example 3. Antibodies that bind to mouse and human MEP1B and Notch2 3.1 Production of anti-MEP1B antibodies by yeast surface display Yeast surface display (Chao et al. 2006) was used to generate antibodies against mouse MEP1B (Cusabio, CSB-MP730755MO), an exemplary target addressed by this technology. Yeast display was performed as described above (2.1) to obtain a cross-reactive mouse / human MEP1B binder (human MEP1B, Cusabio, CSB-MP618098HU). After generation, a number of scFvs cross-reactive with mouse and human MEP1B were cloned into human IgG1, transiently transfected into HEK293F cells, and purified using Protein A resin as described above.

[0408] 3.2 Vaccination to generate anti-Notch2 antibodies Antibodies cross-reactive to mouse and human Notch2 are generated, cloned, and expressed into a human IgG1 framework according to conventional methods previously described.

[0409] 3.3 Wide range of ICs 50 Selection of active Notch2 antibodies Antibodies against Notch2 were tested using broad IC as described above. 50 Select by range.

[0410] 3.4 Expression and Purification of ANDbodies as Bispecific Antibodies Wide range of ICs 50 The DNA sequences of 10 MEP1B antibodies and 10 Notch2 antibodies with ribonucleotides (<1 nM to 5 uM) are made into 100 mutant ANDbodies as described above.

[0411] 3.5 Affinity of ANDbodies for MEP1B and Notch2 The affinity of the ANDbodies for MEP1B and Notch2 is assessed using BIAcore as described above. In this case, His-tagged human and mouse versions of MEP1B are immobilized on Sensor Chip NTA (Cytiva catalog #BR100034). Human and mouse notch2-Fc are immobilized as described above.

[0412] 3.6 Biodistribution (in vivo) of ANDbodies and parent antibodies Cell and tissue biodistribution studies are performed using the methods described above, except that the ANDbody used in this case is MEP1BxNotch2, and the parent antibodies correspond to anti-MEP1B and anti-Notch2.

[0413] 3.7 In vivo bioactivity to quantify gene expression changes The in vivo biological activity of the MEP1BxNotch2 ANDbody will be quantified using the gene expression method described above.

[0414] 3.8 In vivo bioactivity using gravimetric and histological methods The in vivo bioactivity of the MEP1BxNotch2 ANDbody will be quantified using the weight and histology methods described above.

[0415] Example 4. ANDbodies that bind to mouse and human RAGE and IL11RA 4.1 Generation of anti-RAGE antibodies by vaccination Methods for vaccinating mice / ratios to generate cross-reactive anti-RAGE antibodies are described above.

[0416] 4.2 Selection of inactive anti-RAGE antibodies Methods for selecting inactive anti-RAGE antibodies are described above.

[0417] 4.3 Production of anti-IL11Ra antibodies by yeast surface display Using yeast surface display as described above, we generated antibodies of various affinities that cross-react with mouse and human IL11Ra, exemplary effector targets of this technology. The DNA sequences encoding the extracellular domains of mouse IL11Ra (positions 24-372 of UniProt ID Q64385) and human IL11Ra (positions 24-370 of UniProt ID Q14626) were codon-optimized for mammalian expression and appropriately sequenced with a C-terminal His tag in the pcDNA3.4-TOPO expression vector (ThermoFisher Scientific). The proteins were transiently transfected into HEK293F cells and then purified using TALON® Metal Affinity Resin according to the manufacturer's instructions (Clontech), as per conventional methods (Rothschilds et al. 2019). These soluble recombinant mouse and human IL11Ra were used as antigens for yeast surface display.

[0418] ScFvs cross-reactive against mouse and human IL11Ra are cloned into human IgG1, transiently transfected into HEK293F cells, and then purified using Protein A resin as described above.

[0419] 4.4 Wide range of ICs 50 Selection of active anti-IL11Ra antibodies As previously described, IC of IL11Ra antibody against the ligand human IL11 (R&D Systems, catalog #218-IL-025 / CF) that binds to surface IL11Ra 50 were assessed using flow cytometry and showed IC values ​​ranging from less than 1 nM to 5 uM. 50 In this example, full-length human IL11Ra is transiently transfected onto the surface of HEK293F cells. The method of the previous example is used, substituting IL11Ra antibody for Notch2 antibody and IL11 for Jagged-2-Fc.

[0420] 4.5 Expression and Purification of ANDbodies as Bispecific Antibodies Wide range of ICs50 DNA sequences from 10 RAGE antibodies and 10 IL11Ra antibodies with potency (from less than 1 nM to 5 uM) are made into approximately 100 mutant ANDbodies as described above.

[0421] 4.6 Affinity of ANDbody for RAGE and IL11Ra The affinity of the ANDbodies for RAGE and IL11Ra is assessed using BIAcore as described above, where His-tagged human and mouse versions of IL11Ra are immobilized on Sensor Chip NTA (Cytiva catalog #BR100034) and human and mouse versions of RAGE-Fc are captured on Sensor Chip Protein A (Cytiva catalog #29127557).

[0422] 4.7 In vitro assay of IL11Ra antagonism on cells with or without RAGE expression The assay is performed as described above, except that full-length human IL11Ra is used instead of Notch2 and anti-IL11Ra antibody is used instead of anti-Notch antibody. The corresponding RAGExIL11Ra ANDbody is also used.

[0423] 4.8 Biodistribution (In Vivo) of ANDbodies and Parental Antibodies Cell and tissue biodistribution tests are performed as described above, except that the ANDbody used in this case is RAGExIL11Ra, and the parent antibodies correspond to anti-RAGE and anti-IL11Ra.

[0424] 4.9 In vivo biological activity of RAGExIL11Ra ANDbody In response to mouse treatment with murine IL11, increased collagen content is observed in both the ventricle and kidney (Schafer et al. 2017). Therefore, we measured collagen content to quantify the amount of IL11Ra bioactivity following ANDbody treatment.

[0425] As previously described (Schafer et al. 2017), 10-week-old male C57BL / 6 mice were injected subcutaneously with 2 μg of murine IL11 or an equal volume of saline daily for 21 days. Murine IL11 was produced recombinantly by synthesizing codon-optimized DNA using the C-terminal His-tagged murine IL11 sequence (UniProt ID P47873), followed by transient transfection of HEK293F cells and purification of the His-tagged IL11 using TALON resin. Starting 3 days before the first IL11 injection and twice weekly thereafter, IL11- and saline-treated mice received IP treatment injections consisting of 250 μg of ANDbody RAGExIL11Ra, parental anti-IL11Ra alone, or an equal volume of saline (PBS).

[0426] At the end of 21 days of IL11 treatment, mice will be euthanized and the amount of total collagen in the lungs, spleen, blood, kidneys, liver, heart, and intestine will be quantified as previously described (Schafer et al. 2017) based on colorimetric detection of hydroxyproline using the Quickzyme Total Collagen Assay Kit (Quickzyme Biosciences).

[0427] Example 5. Antibodies that bind to mouse and human UMOD and IL11RA 5.1 Generation of anti-UMOD antibodies by yeast surface display Anti-UMOD (e.g., address target) antibodies are selected as above and cloned into human IgG1.

[0428] 5.2 Selection of inactive anti-UMOD antibodies Anti-UMOD antibodies are further evaluated based on their inability to block UMOD ligand binding in assays such as those described above.

[0429] 5.3 Generation of anti-IL11Ra antibodies by yeast surface display The same IL11Ra antibody generated above is used in yeast surface display as described above. ScFvs cross-reactive with mouse and human IL11Ra are cloned into human IgG1, transiently transfected into HEK293F cells, and purified using Protein A resin as described above.

[0430] 5.4 Wide range of ICs 50 Selection of active anti-IL11Ra antibodies Using the above method, various IC 50 Select an IL11Ra antibody.

[0431] 5.5 Expression and Purification of ANDbodies as Bispecific Antibodies Wide range of ICs 50 DNA sequences from 10 UMOD antibodies and 10 IL11Ra antibodies with potency (from less than 1 nM to 5 uM) are made into approximately 100 mutant ANDbodies as described above.

[0432] 5.6 Affinity of ANDbody for UMOD and IL11Ra The affinity of ANDbodies for UMOD and IL11Ra is assessed using BIAcore as described above. In this case, His-tagged human and mouse versions of UMOD are immobilized on Sensor Chip NTA (Cytiva catalog #BR100034). Some ANDbody variants are expected to have higher affinity than IL11Ra, but all variants will be tested in additional assays.

[0433] 5.7 Biodistribution of ANDbodies and Parental Antibodies (In Vivo) Cell and tissue biodistribution studies are performed as described above, except that the ANDbody used in this case is UMODxIL11Ra, and the parent antibodies correspond to anti-UMOD and anti-IL11Ra.

[0434] 5.8 In vivo biological activity of UMODxIL11Ra ANDbody The in vivo biological activity of the UMODxIL11Ra ANDbody will be quantified using the same method as above.

[0435] Example 6. Antibodies that bind to mouse and human MEP1B and IL11Ra 6.1 Generation of anti-MEP1B antibodies by yeast surface display Anti-MEP1B (e.g., address target) antibodies are selected as described above and cloned into human IgG1.

[0436] 6.2 Production of anti-IL11Ra antibodies by yeast surface display The same IL11Ra (e.g., effector target) antibodies generated by yeast surface display above are used here as well. ScFvs cross-reactive with mouse and human IL11Ra are cloned into human IgG2, transiently transfected into HEK293F cells, and purified using Protein A resin as described above.

[0437] 6.3 Wide range of ICs 50 Selection of active anti-IL11Ra antibodies Using the above method, various IC 50 Select an IL11Ra antibody.

[0438] 6.4 Expression and Purification of ANDbodies as Fusion Proteins to Human IgG2 The 10 highest affinity MEP1B scFvs and 10 IL11Ra antibodies with a wide range of affinities (from less than 1 nM to 5 uM) were generated as ANDbodies with a human IgG2 Fc region. To accomplish this, scFv sequences from MEP1B mutants were cloned into each IgG2 IL11Ra antibody mutant. The MEP1B scFvs were separated from either the N-terminus or C-terminus of the IL11Ra antibody light or heavy chain by a flexible linker (3xGGGGS) (each ANDbody has two MEP1B scFvs). By cloning the MEP1B scFvs (always separated by a linker) before the N-terminus and after the C-terminus of the heavy or light chain, respectively, mutants with a total of four MEP1B scFvs per ANDbody were generated. Location of scFvs on IL11Ra antibodies: N-terminus of both heavy and light chains; C-terminus of both heavy and light chains; N-terminus of heavy chain and C-terminus of light chain; other variants with four or more MEP1B scFvs per IL11Ra antibody on the MEP1BxIL11Ra ANDbody by mixing and matching C-terminus of heavy chain and N-terminus of light chain; and other variants with scFvs in three or four different positions (resulting in a total of six or eight scFvs per ANDbody, respectively).

[0439] 6.5 Affinity of ANDbody mutants for MEP1B and IL11Ra The affinity of the ANDbodies for MEP1B and IL11Ra is assessed using BIAcore as described above, where the human and mouse versions of His-tagged MEP1B and His-tagged IL11Ra are immobilized on Sensor Chip NTA (Cytiva catalog #BR100034).

[0440] 6.6 Biodistribution of ANDbodies and Parental Antibodies (In Vivo) The cell and tissue biodistribution test was performed using the above method, except that the ANDbody used in this case was MEP1BxIL11Ra, and the parent antibodies corresponded to anti-MEP1B and anti-IL11Ra.

[0441] 6.7 In vivo biological activity of MEP1BxIL11Ra ANDbody The in vivo biological activity of the MEP1BxIL11Ra ANDbody will be quantified using the same method as above.

[0442] Example 7. Exemplary Address-Limited Binders to Skin This example demonstrates the restricted expression of anti-DSG1 antibodies (address binders) in the skin.

[0443] 7.1 Expression and purification of anti-DSG1 monoclonal antibodies The sequences encoding the variable heavy chain regions (HC: SEQ ID NO: 24 and SEQ ID NO: 26, shown in Table 5) of two anti-desmoglein-1 (anti-DSG1) antibodies, designated 3-09*5 and 3-07 / 1e (Yamagami et al., J Immunol: 5615-5621, 2009), were fused to a human IgG1 (huIgG1) backbone with effector-null mutations L234A, L235A, and P329G (LALA-PG) and cloned into the PCDNA3.4™ vector (ThermoFisher Scientific). The variable light chain regions (SEQ ID NO: 25 and SEQ ID NO: 27) were fused to the constant kappa light chain (for 3-09*5) (SEQ ID NO: 22) or the constant lambda light chain (for 3-07 / 1e) (SEQ ID NO: 23) and then cloned into PCDNA3.4™.

[0444] To express and purify the antibodies, EXPI293F™ cells (ThermoFisher Scientific) were transfected with a 1:1 ratio of heavy and light chain DNA using the EXPIFECTAMINE™ 293 Transfection Kit (ThermoFisher Scientific) according to the manufacturer's recommendations. Transiently expressed antibodies were purified from the conditioned medium by filtering the transfected cells 5 days after transfection. The conditioned medium was incubated with Protein A agarose beads for 1 hour. After washing the bound beads with phosphate-buffered saline (PBS) pH 7.4, 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 designated PRO003 (3-09*5 HC) (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).

[0445] The purified mAbs were analyzed by analytical size exclusion chromatography (SEC) for monodispersity and by SDS-PAGE for purity.

[0446] [Table 5-1]

[0447] [Table 5-2]

[0448] 7.2 Anti-DSG1 antibodies PRO003 and PRO004 bind to mouse DSG1 expressed on cells Mouse DSG1 (NCBI accession number NP_034209.2), which carries a c-Myc epitope tag at the C-terminus of the protein, was transiently expressed in RAW 264.7 cells using LIPOFECTAMINE™ 3000 (ThermoFisher Scientific) according to the manufacturer's protocol. After fixation and permeabilization, cells were stained with an anti-c-Myc antibody (Life Technologies A-21281) and analyzed by flow cytometry to confirm DSG1 expression. PRO003 and PRO004 specifically bound to mouse DSG1-transfected cells, demonstrating the expected binding specificity and suitability for mouse studies.

[0449] 7.3 Anti-DSG1 antibodies injected into mice selectively accumulate in the skin To demonstrate that conjugation to a skin address can result in accumulation of antibodies in the skin, the anti-DSG1 antibodies PRO003 and PRO004 were chemically conjugated to a near-infrared (IR) dye: IRDYE® 800CW, according to the manufacturer's instructions (LI-COR® 928-38044).

[0450] Mice were administered labeled antibodies at a dose of 3 mg / kg via tail vein injection. Each antibody was administered to two groups of three mice, and the mice were euthanized 3 and 7 days after administration. After euthanasia, nine organs (heart, lung, pancreas, kidney, small intestine, large intestine, skin, liver, and stomach) were collected, and the near-infrared fluorescence of each tissue was measured using an IVIS® imager (PERKINELMER®). For skin imaging, a patch of skin was shaved and cut into approximately 1 cm sections for imaging. 2Samples from each mouse were arranged in a standard format and the total fluorescence intensity was measured. Fluorescence intensity from each organ was quantified by measuring the total signal and subtracting the local background, and the fluorescence intensity for each tissue was averaged. High background signals were observed in the livers of all treated mice, so the livers were excluded from analysis. While not wishing to be bound by theory, it is believed that the liver may take up fluorescent dye independently of antibody targeting. Similarly, background signals were observed in the stomachs of all groups, including mice not treated with any antibody. Because fluorescence was also observed from the food given to the mice, the stomachs were excluded from analysis.

[0451] Figures 5A and 5B show the tissue-wide fluorescent signal of PRO003 (Figure 5A) and PRO004 (Figure 5B). The distribution of both antibodies is strongly biased to the skin. These data demonstrate that the DSG1 antibody can be used as an address for selective skin targeting of ANDbodies.

[0452] Example 8. Exemplary Address-Restricted Binders to the Lung This example demonstrates the restricted expression of anti-RAGE antibodies (address binders) in the lung.

[0453] 8.1 Expression and Purification of Anti-RAGE Monoclonal Antibodies Sequences encoding the variable heavy chain regions (SEQ ID NO: 32 and SEQ ID NO: 34 shown in Table 6) of two anti-RAGE mAbs, designated h11E6.8 and XT-M4 (Creative Biolabs), were fused to a huIgG1 backbone with effector-null mutations L234A, L235A, and P329G (LALA-PG) and cloned into the PCDNA3.4™ vector (ThermoFisher Scientific). Sequences encoding the variable light chain regions (SEQ ID NO: 33 and SEQ ID NO: 35) were fused to a constant kappa light chain and cloned into PCDNA3.4™.

[0454] For expression and purification, heavy and light chain DNA was transfected into EXPI293F™ cells (ThermoFisher Scientific) at a 1:1 ratio using the EXPIFECTAMINE™ 293 Transfection Kit (ThermoFisher Scientific) according to the manufacturer's recommendations. Transiently expressed antibodies were purified from the conditioned medium by filtering the transfected cells 5 days after transfection. The conditioned medium was incubated with Protein A agarose beads for 1 hour. After washing the bound beads with phosphate-buffered saline (PBS) pH 7.4, 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 designated 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).

[0455] The purified mAbs were analyzed for monodispersity by analytical size-exclusion chromatography and for purity by SDS-PAGE. PRO001 and PRO002 were highly monodisperse and resolved by SDS-PAGE at the expected molecular weights. Binding studies confirmed binding to the RAGE antigen (not shown).

[0456] [Table 6]

[0457] To examine the binding of anti-RAGE antibodies by ELISA, recombinant His-tagged mouse RAGE protein (Abcam ab276858) was coated overnight onto NUNC-IMMUNO™ MAXISORP™ ELISA plates at a concentration of 1 μg / mL. The next day, the coated antigen was removed, and wells were blocked with 1% IgG-free bovine serum albumin (BSA) and then incubated with 11 anti-RAGE antibodies (PRO001 and PRO002) in a 4-fold serial dilution starting at 20 nM. Bound antibodies were detected with a peroxidase-conjugated anti-human IgG antibody along with tetramethylbenzidine (TMB) and acid quenching reagent. Both PRO001 and PRO002 bound to the mouse RAGE antigen with similar affinities, with apparent affinities of around 90 pM by ELISA, suggesting that both antibodies are strong binders.

[0458] 8.2 Anti-RAGE antibodies PRO001 and PRO002 bind to mouse RAGE expressed in cells PRO001 and PRO002 were tested for binding to mouse RAGE in cell culture. To confirm the binding activity and specificity of these two antibodies, mouse RAGE (NCBI accession NP_031451.2) carrying a c-Myc epitope tag at the C-terminus of the protein was transiently expressed in EXPI293™ cells (ThermoFisher Scientific) using EXPIFECTAMINE™ (ThermoFisher Scientific) according to the manufacturer's protocol. After fixing and permeabilizing the cells, RAGE expression was confirmed by staining with an anti-c-Myc antibody (Life Technologies A-21281) and analyzing by flow cytometry. Both antibodies specifically bound to mouse RAGE expressed on the cells, confirming their expected binding specificity and suitability for mouse studies.

[0459] 8.3 Anti-RAGE antibodies injected into mice selectively accumulate in the lungs To demonstrate that binding to the lung address induces accumulation of the antibodies in the lung, the anti-RAGE antibodies PRO001 and PRO002 were chemically conjugated to a near-infrared dye as described in Example 7.

[0460] Each labeled antibody was administered to mice via tail vein injection and imaged as described in Example 7. Figures 6A and 6B show the fluorescence signals measured from whole tissues of mice treated with the two antibodies, normalized so that the brightest signal was equal to 1. A group of three untreated mice was included as a negative control for autofluorescence. Compared to the other antibodies tested, the distribution of both antibodies was strongly biased toward the lung. These data demonstrate that the two RAGE-binding antibodies selectively accumulate in the lung, suggesting that either can be used as an address for selective pulmonary targeting of ANDbodies.

[0461] 8.4 Anti-RAGE antibodies specifically accumulate on alveolar epithelial cells Single-cell expression analysis demonstrated that RAGE is specifically expressed in type 1 alveolar epithelial cells and less expressed in type 2 alveolar epithelial cells. To test the hypothesis that antibodies address specific cell types, three Balb / C mice were injected via the tail vein with 3 mg / kg PRO002. Three untreated mice served as negative controls. Three days after administration, the mice were euthanized, and lungs and other tissues were collected. All tissues were fixed in formalin. Sections from each tissue were analyzed by immunohistochemistry (IHC) using an anti-human secondary antibody conjugated to horseradish peroxidase. Figure 7 shows representative staining from treated and untreated mice. Strong staining was observed in the alveolar tissue of mice treated with PRO002, but not in the adjacent airways or negative control conditions. These results demonstrate that binders specific to specific cell types can be used to direct antibody distribution to those cells within a larger tissue.

[0462] Example 9. Exemplary Address-Restricted Binders to the Kidney This example demonstrates the restricted expression of anti-CDH16 antibodies (address binders) in the kidney.

[0463] 9.1 Expression and Purification of Anti-CDH16 Monoclonal Antibodies The sequence encoding the variable heavy chain region (SEQ ID NO: 40; shown in Table 6) of anti-cadherin 16 (anti-CDH16) mAb Ab270263 (Abcam) was fused to a huIgG1 backbone with effector-null mutations L234A, L235A, P329G (LALA-PG) and cloned into the PCDNA3.4™ vector (ThermoFisher Scientific). The sequence encoding the variable light chain region (SEQ ID NO: 41) was fused to a constant kappa light chain and cloned into PCDNA3.4™.

[0464] For expression and purification, heavy and light chain DNAs were transfected into EXPI293F™ cells (ThermoFisher Scientific) at a 1:1 ratio using the EXPIFECTAMINE™ Transfection Kit (ThermoFisher Scientific) according to the manufacturer's recommendations. Transiently expressed antibodies were purified from the conditioned medium by filtering the transfected cells 5 days after transfection. The conditioned medium was incubated with protein A agarose beads for 1 hour. After washing the bound beads with phosphate-buffered saline (PBS) pH 7.4, 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 designated PRO056 (heavy chain sequence: SEQ ID NO: 42; light chain sequence: SEQ ID NO: 43).

[0465] The purified mAb was analyzed for monodispersity by analytical size exclusion chromatography (SEC) and for purity by SDS-PAGE. PRO056 was expressed as highly monodisperse and resolved by SDS-PAGE at the expected molecular weight.

[0466] [Table 7]

[0467] To examine anti-CDH16 antibody binding by ELISA, recombinant His-tagged mouse CDH16 protein, expressed and purified in-house, was coated onto NUNC-IMMUNO™ MAXISORP™ ELISA plates overnight at a concentration of 1 μg / mL. The next day, the coated antigen was removed, and wells were blocked with 1% IgG-free bovine BSA and then incubated with 11 anti-CDH16 antibodies (PRO056) in 3-fold serial dilutions starting at 533 nM. Bound antibodies were detected with a peroxidase-conjugated anti-human IgG antibody along with TMB and acid stop reagent. PRO056 bound to the mouse CDH16 antigen with an affinity of 200 pM by ELISA.

[0468] 9.2 Anti-CDH16 antibodies selectively accumulate in the kidney The anti-CDH16 antibody, PRO056, was chemically conjugated to the near-infrared fluorescent dye IRDYE® 800CW as described in Example 7. The labeled antibody was administered to mice via tail vein injection at a dose of 3 mg / kg. Two groups of three mice were used, and they were euthanized 3 and 7 days after administration. After euthanasia, organs were collected, and the near-infrared fluorescence of each tissue was measured using a model IVIS® imager (PERKINELMER®) as described above. The fluorescence intensity from each organ was quantified by measuring the total signal and subtracting the local background, and the fluorescence intensity across each tissue was averaged. Figure 8 shows the fluorescence signal measured from whole tissues of a mouse treated with PRO056. Each signal was normalized so that the brightest signal was equal to 1. One group of three untreated mice was included as a negative control for autofluorescence.

[0469] The distribution is strongly biased toward the kidney compared to the antibodies provided herein that target addresses in the skin, lung, or kidney. These data demonstrate that antibodies that bind to CDH16 selectively accumulate in the kidney, indicating that they can be used as an addressing domain for ANDbodies to selectively target the kidney.

[0470] Example 10. Exemplary Address-Restricted Binders to the Intestine This example demonstrates the restricted expression of anti-CDH17 antibodies (address binders) in the intestine.

[0471] 10.1 Expression and Purification of Anti-CDH17 Monoclonal Antibodies The sequence encoding the variable heavy chain region (SEQ ID NO: 44; shown in Table 8) of anti-cadherin 17 (anti-CDH17) mAb MAB8524 (R&D Systems) was fused to a huIgG1 backbone with effector-null mutations L234A, L235A, P329G (LALA-PG) and cloned into the PCDNA3.4™ vector (ThermoFisher Scientific). The sequence encoding the variable light chain region (SEQ ID NO: 45) was fused to a constant kappa light chain and cloned into PCDNA3.4™.

[0472] For expression and purification, heavy and light chain DNAs were transfected into EXPI293F™ cells (ThermoFisher Scientific) at a 1:1 ratio using the EXPIFECTAMINE™ Transfection Kit (ThermoFisher Scientific) according to the manufacturer's recommendations. Transiently expressed antibodies were purified from the conditioned medium by filtering the transfected cells 5 days after transfection. The conditioned medium was incubated with protein A agarose beads for 1 hour. After washing the bound beads with phosphate-buffered saline (PBS) pH 7.4, 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 designated PRO061 (heavy chain sequence: SEQ ID NO: 46; light chain sequence: SEQ ID NO: 47).

[0473] The purified mAb was analyzed by analytical size exclusion chromatography (SEC) for monodispersity and by SDS-PAGE for purity. PRO061 was expressed as highly monodisperse and resolved by SDS-PAGE at the expected molecular weight.

[0474] [Table 8]

[0475] To confirm binding activity and specificity, mouse CDH17 (NCBI accession NP_062727.1) carrying a c-Myc epitope tag at the C-terminus of the protein was transiently expressed in RAW 264.7 cells using LIPOFECTAMINE™ 3000 (ThermoFisher Scientific) according to the manufacturer's protocol. CDH17 expression was confirmed by fixing and permeabilizing the cells, staining with an anti-c-Myc antibody (Life Technologies A-21281), and analyzing by flow cytometry. PRO061 specifically bound to cells transfected with mouse CDH17 and exhibited the expected binding specificity, confirming its suitability for mouse studies.

[0476] 10.2 Anti-CDH17 antibodies injected into mice selectively accumulate in the intestine As described in Example 7, PRO061 was chemically conjugated to the near-infrared fluorescent dye IRDYE® 800CW. The labeled antibody was administered to mice via tail vein injection at a dose of 3 mg / kg. Two groups of three mice were used, and they were euthanized 3 and 7 days after administration. After euthanasia, organs were collected, and the near-infrared fluorescence of each tissue was measured using a model IVIS® imager (PERKINELMER®) as described above. The fluorescence intensity from each organ was quantified by measuring the total signal and subtracting the local background, and the fluorescence intensity across each tissue was averaged. Figure 9 shows the fluorescence signals measured from whole tissues of a mouse treated with PRO061. Each signal was normalized so that the brightest signal was equal to 1. One group of three untreated mice was included as a negative control for autofluorescence. The distribution was strongly biased towards the intestine, indicating that antibodies that bind to CDH17 selectively accumulate in the intestine and that ANDbodies can use these as address targeting domains to selectively target the intestine.

[0477] Example 11. Organizational Limitation of Predicted Addresses To assay whether predicted organ-specific or selective expression addresses are indeed most abundant in a given organ, and to determine which monoclonal antibody clones (mAbs) most selectively bind to the desired organ, immunohistochemistry (IHC) of fresh frozen (FF) healthy mouse tissue microarray (TMA) sections mounted on glass slides was used.

[0478] FF TMA-coated slides were prepared by first assembling freshly frozen tissue microarray blocks. To allow for the formation of TMA blocks, individual organs harvested from freshly sacrificed C57BL / 6 mice were embedded in optimal cutting (OCT) medium in separate cryomolds and frozen. A cylindrical core of tissue was then removed from each block and placed within the block to create the final FF TMA. Using a cryostat, layers of the TMA were cut and placed onto positively charged microscope slides and stored at -80°C until staining.

[0479] Addresses were verified by direct binding of either polyclonal antibodies or mAbs raised against the address of interest to slides coated with FF TMA. These address-specific antibodies were detected with horseradish peroxidase (HRP)-conjugated antibodies specific for the IgG of the host in which the primary address-specific antibody was raised. The location and intensity of binding were determined by the addition of the HRP substrate 3,3'-diaminobenzidine (DAB), which produces a brown color at the binding site of the primary antibody, proportional to the amount of antibody deposited. Nuclei were counterstained with hematoxylin, resulting in a blue color. For each address, various different mAb clones were assayed for tissue specificity, and the pattern of tissue binding was assessed by performing IHC on the same FF TMA described above.

[0480] Table 9 summarizes the observed binding of the tested antibodies. All tested antibodies reacted primarily with the expected target tissues, with varying degrees of weak reactivity to other tissues. Without wishing to be bound by theory, much of the extra-tissue reactivity may represent nonspecific binding by the antibody. For example, each of the four antibodies tested for binding to RAGE showed binding to the lung, while three antibodies showed low levels of binding to other tissues.

[0481] [Table 9]

[0482] Example 12. Production and Use of ANDbodies In this example, we demonstrate the production of an exemplary ANDbody that blocks Notch2 and binds to RAGE as an addresser.

[0483] 12.1 Expression and Purification of Anti-Notch2 Monoclonal Antibodies The sequences encoding the variable heavy chain regions (SEQ ID NOS: 48, 50, 52, and 54; shown in Table 10) of four anti-Notch2 mAbs (Wu et al., Nature, 464:1052-1057, 2010) were each individually fused to a huIgG1 backbone with effector-null mutations L234A, L235A, and P329G (LALA-PG) and cloned into the PCDNA3.4™ vector (ThermoFisher Scientific). The sequences encoding the corresponding variable light chain regions (SEQ ID NOS: 49, 51, 53, and 55) were fused to a constant kappa light chain and cloned into PCDNA3.4™.

[0484] For expression and purification, heavy and light chain DNAs were transfected at a 1:1 ratio into EXPI293F™ cells (ThermoFisher Scientific) using the EXPIFECTAMINE™ 293 Transfection Kit (ThermoFisher Scientific) according to the manufacturer's recommendations. Transiently expressed antibodies were purified from the conditioned medium by filtering the transfected cells 5 days after transfection. The conditioned medium was incubated with protein A agarose beads for 1 hour. After washing the bound beads with phosphate-buffered saline (PBS) pH 7.4, 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 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).

[0485] [Table 10-1]

[0486] [Table 10-2]

[0487] 12.2 Binding and Affinity Studies of Anti-Notch2 Antibodies To test anti-Notch2 antibody binding by ELISA, recombinant His-tagged human and mouse Notch2 NRR domains expressed and purified in-house were coated onto NUNC-IMMUNO™ MAXISORP™ ELISA plates overnight at a concentration of 1 μg / mL. The next day, the coated antigen was removed, and wells were blocked with 1% IgG-free bovine BSA. Then, plates were incubated with 11 anti-Notch2 antibodies (PRO034, PRO035, PRO036, and PRO037) in a 3-fold serial dilution starting at 200 nM for PRO035 and PRO037, and 666 nM for PRO034 and PRO036. Bound antibodies were detected with a peroxidase-conjugated anti-human IgG antibody along with TMB and acid quenching reagent. Each antibody bound to the human and mouse NRR domains of Notch2 with affinities ranging from 47 pM to 140 nM.

[0488] To examine the binding affinity of anti-Notch2 antibodies by biolayer interferometry (BLI), PRO034, PRO035, PRO036, and PRO037 were immobilized on an anti-human IgG Fc biosensor and immersed in recombinant His-tagged mouse and human Notch2 NRR proteins at various concentrations (1000 nM to 31 nM). The association rate with the antigen was measured. The dissociation rate was then measured by immersing the biosensor in buffer. Binding affinity was calculated as the ratio of the dissociation rate to the association rate. The intrinsic affinities determined by BLI were all in the nM range, suggesting that binding activity improves affinity in an ELISA format.

[0489] 12.3 Design and Production of Notch2 / RAGE ANDbodies An ANDbody containing the first fragment antigen-binding (Fab) arms of the above-mentioned anti-Notch2 antibodies (PRO034, PRO035, and PRO036) and the second Fab arm of the anti-RAGE antibody PRO002 (Example 8) was generated using a controlled Fab arm exchange (cFAE) reaction (Labrijn et al., Proc Natl Acad Sci USA, 110(13):5145-5150, 2013). Site-directed mutagenesis was performed to introduce an F405L amino acid substitution mutation into the Fc fragment of the anti-RAGE antibody (PRO002) and a K409R amino acid substitution mutation into the Fc fragment of each of the anti-Notch2 antibodies (PRO034, PRO035, and PRO036). These antibodies were expressed and purified as previously described for the parent antibodies. Next, the individual monoclonal antibodies were mixed in equimolar ratios, resulting in a controlled reduction-reoxidation reaction, which promoted the recombination of bispecific antibodies induced by matching point mutations (F405L-K409R). The formation of ANDbodies was analyzed by analytical chromatography and SDS-PAGE. The resulting ANDbodies were designated PRO051, PRO052, and PRO053 (containing PRO034, PRO035, and PRO036, respectively).

[0490] SDS-PAGE and analytical size-exclusion chromatography showed that the major product formed after the cFAE reaction had a typical IgG1 molecular weight (150 kDa), suggesting complete reoxidation. Analytical hydrophobic interaction chromatography demonstrated the formation of a new product, the desired heterodimeric antibody.

[0491] 12.4 Notch2 / RAGE ANDbody demonstrates simultaneous binding to Notch2 and RAGE by BLI To examine simultaneous dual antigen binding of the Notch2 / RAGE ANDbody by BLI, PRO051, PRO052, and PRO053, along with a monovalent parent antibody control, were immobilized on an anti-human IgG Fc biosensor and immersed in recombinant His-tagged mouse RAGE protein at 150 nM. Dual antigen binding was then measured by performing a second association step in a well containing recombinant mouse Notch2 NRR at 150 nM. Dissociation rates were then measured by immersing the biosensor in buffer.

[0492] The sensorgrams revealed that ANDbodies PRO051, PRO052, and PRO053 were able to simultaneously bind both RAGE and Notch2 antigens, whereas the monovalent parent antibody bound only to either RAGE or Notch2 NRR, supporting the conclusion that the ANDbodies were of correct composition and functional in simultaneously binding both antigens.

[0493] 12.5 Notch2 / RAGE ANDbody demonstrates selective binding to human lung tissue by immunohistochemistry Immunohistochemistry (IHC) on slide-mounted fresh frozen healthy mouse tissue microarray (FF TMA) sections was used to evaluate tissue binding by ANDbodies containing the above Notch2 inhibitory antibodies. TMAs were constructed and stained as described in Example 11. Figure 10 shows staining of mouse TMAs with three anti-Notch2 antibodies: PRO034, PRO035, and PRO036, as well as Notch2 / RAGE ANDbodies: PRO051, PRO052, and PRO053. In both cases, the RAGE-targeting ANDbodies exhibited significantly enhanced lung tissue binding. These data demonstrate that the tissue specificity of the addressing arm can be conferred to ANDbodies by combining a receptor-targeting binder with an antibody-format address binder.

[0494] 12.6 Notch2 / RAGE ANDbodies selectively distribute to the lung compared with corresponding non-targeted anti-Notch2 antibodies To evaluate the in vivo behavior of ANDbodies targeting Notch2 and RAGE, mice were treated with PRO051, PRO052, and PRO053 antibodies at 3 mg / kg intravenously. Each group contained three mice. Tissues were collected from each mouse 3, 7, 14, and 21 days after administration. Lung accumulation of each antibody was measured by homogenizing a fixed amount of lung tissue, normalizing each sample to a fixed amount of extracted protein, and then detecting human antibodies using a sandwich ELISA.

[0495] Figure 11 shows the lung accumulation of PRO052 compared to an antibody that binds to RAGE and a control target, respiratory syncytial virus (RSV) glycoprotein F (RAGE XT-M4 / motavizumab), and an antibody that binds to Notch and RSV glycoprotein F (Notch2-2 / motavizumab). Antibodies targeting 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 to RAGE and RSV glycoprotein F, indicating that the overall specificity of PRO052 is intermediate between the two arms independently. These results indicate that the addressing arm of the ANDbody can significantly redirect the binding specificity of the target-binding arm.

[0496] Example 13. Production and Use of ANDbodies This example describes the production of an exemplary ANDbody that (i) contains a ligand effector that targets the IL-10 pathway and (ii) binds to DSG1 as an address.

[0497] 13.1 Description, Design, and Production of IL-10 / Anti-DSG1 ANDbodies To validate the IL-10 / anti-DSG1 ANDbody format, three parameters were investigated: IL-10 valency (one or two IL-10 moieties), anti-DSG1 arm valency (one or two Fab arms), and two versions of IL-10 (dimeric or monomeric IL-10). Formats representing different combinations of IL-10 molecules, IL-10 valency, and antibody valency were evaluated. Wild-type (WT) IL-10 (accession number P22301), a monomeric modified IL-10 sequence (Josephson et al., J Biol Chem:13552-7, 2000), and a dimeric modified IL-10 sequence (Minsshawi et al., Front Immunol., 11:1794, 2020) were fused to the PRO003 sequence (Example 7) at the C-terminus of the heavy chain to obtain a bivalent format. For the monovalent format, monomeric and dimeric IL-10 were fused to the N-terminus of the Fc and co-expressed with PRO003. The monovalent format was asymmetric, and mutations in the Fc domain (A chain: S364K / K409S; B chain: K370S / F405K (WO 2017 / 106462 A1)) were used to force asymmetric pairing.

[0498] To express and purify antibodies, EXPI293F™ cells (ThermoFisher Scientific) were transfected with a 1:1 ratio of heavy and light chain DNA or a 1:1:1 ratio of heavy chain:light chain:IL-10-Fc (described below) using the EXPIFECTAMINE™ 293 Transfection Kit (ThermoFisher Scientific) according to the manufacturer's recommendations. Transiently expressed antibodies were purified from the conditioned medium by filtering the transfected cells 5 days after transfection. The conditioned medium was incubated with Protein A agarose beads for 1 hour. After washing the bound beads with phosphate-buffered saline (PBS) pH 7.4, 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.

[0499] The resulting mAbs were designated PRO023, PRO024, PRO025, PRO026 and PRO027 (Figure 12).

[0500] PRO023 comprises (a) a heavy chain sequence (SEQ ID NO: 67) that comprises the heavy chain sequence of PRO003 (SEQ ID NO: 28) and the wild-type human IL-10 sequence (SEQ ID NO: 64), and (b) a light chain sequence of PRO003 (SEQ ID NO: 29).

[0501] PRO024 comprises (a) a heavy chain sequence (SEQ ID NO: 68) which comprises the heavy chain sequence of PRO003 (SEQ ID NO: 28) and a monomeric human IL-10 sequence (SEQ ID NO: 64), and (b) a light chain sequence of PRO003 (SEQ ID NO: 29).

[0502] PRO025 comprises (a) a heavy chain sequence (SEQ ID NO: 69) comprising the heavy chain sequence of PRO003 (SEQ ID NO: 28) and a dimeric human IL-10 sequence (SEQ ID NO: 66), and (b) a light chain sequence of PRO003 (SEQ ID NO: 29).

[0503] PRO026 comprises (a) the heavy chain sequence of PRO003 (SEQ ID NO: 70), further comprising mutations in the Fc domain to effect asymmetric pairing; (b) the light chain sequence of PRO003 (SEQ ID NO: 29); and (c) an IL-10-Fc fusion protein (SEQ ID NO: 72) comprising an Fc region (SEQ ID NO: 71) comprising mutations to effect asymmetric pairing and a monomeric human IL-10 sequence (SEQ ID NO: 64).

[0504] PRO027 comprises (a) the heavy chain sequence of PRO003 (SEQ ID NO: 70), further comprising mutations in the Fc domain to effect asymmetric pairing; (b) the light chain sequence of PRO003 (SEQ ID NO: 29); and (c) an IL-10-Fc fusion protein (SEQ ID NO: 73) comprising an Fc region (SEQ ID NO: 71) comprising mutations to effect asymmetric pairing and a dimeric human IL-10 sequence (SEQ ID NO: 66).

[0505] The purified ANDbodies were analyzed for monodispersity by analytical size-exclusion chromatography and for purity by SDS-PAGE. PRO024 and PRO026 had the highest yield and monodispersity after a single purification step. PRO023 and PRO027 had moderate yields and approximately 70% monodispersity. PRO025 had a low yield and approximately 89% monodispersity.

[0506] [Table 11-1]

[0507] [Table 11-2]

[0508] [Table 11-3]

[0509] 13.2 IL-10 / DSG1 ANDbody binds to both IL-10 receptors To examine the binding of IL-10 / anti-DSG1 ANDbodies to IL-10 receptor alpha (IL-10Ra) by ELISA, recombinant His-tagged human IL-10Ra (Creative Biomart) was coated onto NUNC-IMMUNO™ MAXISORP™ ELISA plates overnight at a concentration of 1 μg / mL. The next day, the coated antigen was removed, and wells were blocked with 1% IgG-free BSA before incubation with 11 anti-IL-10 / anti-DSG1 ANDbodies (as described above) in 3-fold serial dilutions starting at 30 μg / mL. Bound antibodies were detected with a peroxidase-conjugated anti-human IgG antibody along with TMB and acid stop reagent.

[0510] Of the molecules tested, only those containing an IL-10 moiety (PRO023, PRO024, PRO025, PRO026, PRO027, and the positive control IL-10 Fc) (Creative Biomart IL10-326H) bound to IL-10Ra, demonstrating that binding was driven by IL-10, and not the negative control anti-DSG1 antibody (PRO003), and that the IL-10 moiety was functional in binding to its receptor.

[0511] 13.3 The IL-10 / DSG1 ANDbody activates the IL-10 signaling pathway To demonstrate that IL-10 retains 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 examined. HEK-BLUE™ IL-10 cells (InvivoGen) were used to assess 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). Upon activation of the IL-10 signal in these cells, the cells express and secrete SEAP into the cell culture medium. The extent of IL-10 signaling was measured by adding QUANTI-BLUE™ solution colorimetric reagent (InvivoGen) to the cell culture medium and reading the absorbance at 630 nm.

[0512] In this experiment, PRO023, PRO024, PRO025, PRO026, and PRO027 were each titrated from 1 pM to >1 nM with overnight incubation in cell culture medium. Figures 18A and 18B show representative activity data for each IL-10 / DSG1 ANDbody, along with positive and negative controls, as described in Table 12. Table 12 shows the EC50 values ​​for the HEK-BLUE™ cell response to three control molecules: (1) recombinant human IL-10 (BioLegend #573204) (rhIL-10), (2) recombinant human IL-10 fused to a human Fc domain (hIL-10 Fc fusion), and (3) the parental anti-DSG1 antibody (PRO003), as well as to each of the above IL-10 / DSG1 ANDbodies. These data confirmed that all five IL-10 / DSG1 ANDbodies retain the signaling activity of human IL-10. These data demonstrate that the relative biological potency of ANDbodies can be modulated by altering the format of the molecule, the structure of the biologically active moiety, and the valency of the active moiety.

[0513] [Table 12]

[0514] 13.4 IL-10 / DSG1 ANDbody suppresses inflammatory responses in primary mouse macrophages To demonstrate that the IL-10 / DSG1 ANDbody can suppress inflammatory immune responses, we evaluated the effects of the IL-10 / DSG1 ANDbody on murine peripheral blood mononuclear cells (PBMCs) and macrophages treated with lipopolysaccharide (LPS) as an inflammatory stimulus. For these experiments, PBMCs were isolated from the blood, and macrophages were isolated from the spleens of Balb / C mice by negative enrichment with magnetic beads (Miltenyi Biotech #130-110-434). Macrophage activation was assessed by measuring the levels of TNFα cytokine present in the culture medium after 3 and 5–6 hours of stimulation with LPS.

[0515] Figures 13A-13G show tumor necrosis factor alpha (TNFα) levels in PBMC cell cultures after pre-stimulation with the indicated IL-10 / DSG1 ANDbodies or control molecules followed by treatment with LPS for the indicated times. Figures 14A-14G show TNFα levels in primary macrophage cell cultures after pre-stimulation with the indicated IL-10 / DSG1 ANDbodies or control molecules followed by treatment with LPS for the indicated times. Because panels represent experimental runs over multiple days, data cannot be compared between panels. These data demonstrate that all five IL-10 / DSG1 ANDbodies can suppress inflammatory stimuli in primary macrophages.

[0516] 13.5 Address Target Binding Enhances Activity / Potency of ANDbody Effector Function Combining addressing (e.g., using an address targeting domain) with a biologically active molecule can enhance biological activity in a variety of ways. One exemplary enhancement is increasing the potency of the effector moiety against specific cells in which the address target is also present.

[0517] To verify whether the presence of an address targeting domain can enhance the signaling ability of the effector targeting domain, human DSG1 was expressed in HEK-BLUE™ IL-10 cells using stable lentiviral expression (the stable expressing cells are designated HEKBLUE™ IL-10 / DSG1). The DSG1 gene (NP_034209.2) was cloned into an appropriate lentiviral plasmid backbone, packaged into viral particles using VIRAPOWER™ Lentiviral Packing Mix (ThermoFisher Scientific), and transduced according to the manufacturer's instructions. DSG1 expression was confirmed by qPCR.

[0518] We evaluated the potency of an ANDbody in which one Fab fragment of an anti-DSG1 mAb (PRO058, functionally equivalent to PRO026) was substituted with recombinant human IL-10, i.e., monomeric IL-10, and a corresponding control containing motavizumab as a negative control in the antibody sequence. Activity against HEK-BLUE™ IL-10 cells and HEK-BLUE™ IL-10 cells stably expressing DSG1 was assayed. Figure 15 shows representative signaling responses of each molecule in parental HEK-BLUE™ IL-10 cells and HEK-BLUE™ IL-10 / DSG1 cells. The two cell lines responded similarly to recombinant IL-10, confirming that DSG1 expression does not significantly affect their sensitivity to IL-10. The DSG1 / IL-10 ANDbody exhibited approximately 15-fold increased potency when DSG1 was expressed in target cells. This effect was confirmed to be mediated by binding to DSG1, as the potency of the corresponding IL-10 / motavizumab protein was not affected at all, demonstrating that the ANDbody designs provided herein are capable of address-mediated enhanced biological potency and that unwanted off-target effects can be further reduced by using weakly targeted binder moieties in the ANDbody.

[0519] 13.6 IL-10 / DSG1 ANDbodies retain the pharmacokinetic and tissue distribution properties of the parent anti-DSG1 antibody Some ANDbodies are intended to confer tissue or cell targeting of a parent mAb or other targeting molecule to a biologically active site that would otherwise have undesirable pharmacokinetics or tissue distribution.

[0520] The IL-10 / DSG1 ANDbody is intended to target IL-10 activity to the skin. In Example 7, this anti-DSG1 antibody was shown to selectively distribute to mouse skin. In contrast, IL-10 has been reported to clear from the human circulation with a half-life of approximately 2 hours (Radwanski et al., Pharm Res. 1998 Dec;15(12):1895-901). Therefore, we evaluated whether the IL-10 / DSG1 ANDbody retained the skin-targeting ability of the parent antibody.

[0521] BALB / c mice were administered 3 mg / kg of PRO003, PRO024, or PRO058 via tail vein injection. PRO058 is functionally equivalent to PRO026 and contains substitutions in the Fc domain to improve purification of the recombinant protein. PRO058 contains (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) containing the Fc region (SEQ ID NO:74) and the monomeric human IL-10 sequence (SEQ ID NO:64).

[0522] Serum samples were collected at 1 hour to 48 hours. Tissue samples were collected 1, 2, 4, and 7 days after administration. The amount of anti-DSG1 or ANDbody in each serum or tissue sample was measured by ELISA. Figures 16A and 16B show that the IL-10 / anti-DSG1 ANDbodies PRO024 and PRO058 have PK profiles in skin and serum similar to those of the parent anti-DSG1 antibody PRO003. These data indicate that antibody-cytokine fusions containing recombinant IL-10 can retain the pharmacokinetic properties of the parent antibody.

[0523] Example 14. TNFα-blocking molecules conjugated to DSG1 targeting moieties This example describes the production of an exemplary ANDbody that blocks TNFα and binds to DSG1 as an addresser.

[0524] 14.1 Expression and Purification of Anti-TNFα Monoclonal Antibodies Anti-TNFα antibodies were generated with the VH and VL sequences of a commercially available antibody fused to a huIgG1 backbone containing the effector-null mutations L234A, L235A, and P329G (LALA-PG), and their binding and affinity to TNFα were characterized as previously described. The resulting mAbs were designated PRO076 and PRO078.

[0525] 14.2 Design, Expression, and Purification of Anti-TNFα-DSG1 ANDbodies ANDbodies were designed by combining PRO004 (Example 7) with a previously reported dominant-negative TNFα (Steed et al., Science. 2003 Sep 26;301(5641)) or the clinically effective anti-TNFα antibodies listed below, with the aim of locally downregulating TNFα in the extracellular environment of inflamed skin. In designing anti-DSG1 antibodies to block TNFα, various formats and valencies were thoroughly explored, including cytokine / antibody and TNF receptor 2 (TNFR2) / antibody fusions.

[0526] To express and purify the antibodies, EXPI293F™ cells (ThermoFisher Scientific) were transfected with a 1:1 ratio of heavy and light chain DNA using the EXPIFECTAMINE™ 293 Transfection Kit (ThermoFisher Scientific) according to the manufacturer's recommendations. Transiently expressed antibodies were purified from the conditioned medium by filtering the transfected cells 5 days after transfection. The conditioned medium was incubated with Protein A agarose beads for 1 hour. After washing the bound beads with phosphate-buffered saline (PBS) pH 7.4, 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 designated PRO070, PRO074, PRO075, and PRO077 (Figure 17).

[0527] The purified ANDbodies were analyzed for monodispersity by analytical size-exclusion chromatography and for purity by SDS-PAGE. Additional standard purification steps were performed to remove aggregates. PRO077 had the highest final yield, followed by PRO074 and PRO075, with PRO070 having the lowest final yield. The purified ANDbodies were highly pure and of the correct composition, as confirmed by SDS-PAGE.

[0528] 14.3 Anti-TNFα-DSG1 ANDbody Binding and Affinity Studies ELISA binding assays showed that all constructs were active in binding to human and mouse TNFα with varying affinities. PRO074 and PRO075 showed similar affinities for both human and mouse TNFα, within 2 / 3-fold of the parental antibody. PRO077 showed 5-fold and 12-fold reduced binding affinity for human and mouse TNFα, respectively, compared to the parental antibody. This reduced affinity is likely due to the change in format from Fab to single-chain variable fragment (scFv).

[0529] 14.4 Anti-TNFα-DSG1 ANDbody in In Vitro Activity Assays The ability of each of the anti-TNFα / anti-DSG1 ANDbodies to inhibit TNFα signaling was assessed using HEK-BLUE™ TNFα cells (InvivoGen). These cells were engineered to express secreted embryonic alkaline phosphatase (SEAP) in response to TNFα signaling. TNFα was measured according to the manufacturer's instructions. To assess inhibitory activity, the concentration of TNFα was adjusted to 225 pM (the approximate EC of recombinant human TNFα in this assay). 80 ) and pre-incubated with TNFα blocking molecules at concentrations ranging from 10 nM to approximately 10 pM. Table 13 shows the IC of each anti-TNFα-DSG1 ANDbody, along with the corresponding parent antibody as a positive control. 50These data demonstrate that the ANDbodies retain TNFα blocking activity comparable to the parent anti-TNFα antibodies from which they are derived.

[0530] [Table 13]

[0531] VII. Other Embodiments Some embodiments of the technology described herein can be defined according to any of the following numbered embodiments:

[0532] 1. A polymer comprising a first binding site and a second binding site, (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for an address target expressed in a target tissue or cell of interest; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signal transduction upon binding to the address target; and (iii) a macromolecule, wherein the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site, and wherein the macromolecule is linked to a small molecule.

[0533] 2. A polymer comprising a first binding site and a second binding site, (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for an address target expressed in a target tissue or cell of interest; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signal transduction upon binding to the address target; and (iii) the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; wherein localization of the macromolecule to non-target tissues or cells is substantially reduced compared to the localization of a control macromolecule lacking the second binding moiety, wherein the macromolecule is linked to a small molecule.

[0534] 3. A polymer comprising a first binding site and a second binding site, (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for an address target expressed in a target tissue or cell of interest; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signal transduction upon binding to the address target; and (iii) the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; wherein localization of the macromolecule to non-target tissues or cells is substantially increased compared to the localization of a control macromolecule lacking the second binding moiety, wherein the macromolecule is linked to a small molecule.

[0535] 4. A macromolecule comprising a first binding site and a second binding site, (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for an address target expressed in a target tissue or cell of interest; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signal transduction upon binding to the address target; and (iii) the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; wherein at least 25% of the macromolecule administered to a subject is detected in the target tissue or cells 1 to 7 days after administration, wherein the macromolecule is linked to a small molecule.

[0536] 5. A polymer comprising a first binding site and a second binding site, (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for an address target expressed in a target tissue or cell of interest; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signal transduction upon binding to the address target; and (iii) the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; 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.

[0537] 6. A macromolecule comprising a first binding site and a second binding site, (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for an address target expressed in a target tissue or cell of interest; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signal transduction upon binding to the address target; and (iii) the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; 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, and wherein the macromolecule is linked to a small molecule.

[0538] 7. A macromolecule comprising a first binding site and a second binding site, (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for an address target expressed in a target tissue or cell of interest; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signal transduction upon binding to the address target; and (iii) the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; A macromolecule wherein the efficacy of the first binding moiety in the target tissue or cell is substantially increased compared to a control macromolecule lacking the second binding moiety, wherein the macromolecule is linked to a small molecule.

[0539] 8. The macromolecule of any one of embodiments 1 to 7, wherein the first binding site has a low affinity for the effector target.

[0540] 9. The macromolecule of any one of embodiments 1 to 7, wherein the first binding site has low binding activity for the effector target.

[0541] 10. The macromolecule of any one of embodiments 1 to 4 and 6 to 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.

[0542] 11. The macromolecule of any one of embodiments 1 to 10, wherein the binding activity of the first binding site for the effector target is lower than the binding activity of the second binding site for the address target.

[0543] 12. (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) EC of the first binding site for the effector target 50 EC of the second binding site to address the target 50 Higher; (c) IC of the first binding site for the effector target 50 addresses the IC of the second binding site to the target 50 12. The polymer of any one of embodiments 1 to 11, wherein the molecular weight is higher than the molecular weight of the polymer.

[0544] 13. The macromolecule of any one of embodiments 1 to 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.

[0545] 14. The macromolecule of any one of embodiments 1 to 13, wherein the affinity of the second binding site to the address target has a Kd of greater than about 1 nM, greater than about 2 nM, or greater than about 50 nM.

[0546] 15. The macromolecule according to any one of embodiments 1 to 14, wherein the effector target is a protein, lipid, or sugar.

[0547] 16. The macromolecule according to any one of embodiments 1 to 15, wherein the effector target is a cell membrane-associated target.

[0548] 17. The macromolecule according to any one of embodiments 15 or 16, wherein the effector target is a protein.

[0549] 18. The macromolecule of embodiment 17, wherein the effector target is a secreted protein.

[0550] 19. The macromolecule of any one of embodiments 17 or 18, wherein the effector target is encoded by a gene selected from the group consisting of the genes listed in Table 1.

[0551] 20. The macromolecule of any one of embodiments 1 to 19, wherein the macromolecule agonizes an effector target.

[0552] 21. The macromolecule according to any one of embodiments 1 to 19, wherein the macromolecule antagonizes an effector target.

[0553] 22. The macromolecule according to any one of embodiments 1 to 21, wherein the address target is a protein, lipid, or sugar.

[0554] 23. The macromolecule according to embodiment 22, wherein the address target is a protein.

[0555] 24. The macromolecule according to any one of embodiments 17 to 23, wherein expression of the effector target or address target is expression of an RNA sequence encoding the effector target or address target.

[0556] 25. The macromolecule according to embodiment 24, wherein the expression level of the effector target or address target is assessed using an RNA-seq dataset.

[0557] 26. The macromolecule of embodiment 2, wherein the RNA-seq dataset is a Genotype-Tissue Expression (GTEx) dataset or a Human Protein Atlas (HPA) dataset.

[0558] 27. The macromolecule according to embodiment 23, wherein expression of the effector target or address target is protein expression.

[0559] 28. The macromolecule according to any one of embodiments 1 to 27, wherein the effector target is systemically expressed in the subject.

[0560] 29. The macromolecule according to any one of embodiments 1 to 27, wherein the effector target is regionally expressed in the subject.

[0561] 30. The macromolecule according to any one of embodiments 1 to 27, wherein the effector target is expressed locally in the subject.

[0562] 31. The macromolecule of any one of embodiments 1 to 30, wherein the address target is regionally expressed in the subject.

[0563] 32. The macromolecule according to any one of embodiments 1 to 30, wherein the address target is expressed locally in the subject.

[0564] 33. The macromolecule according to any one of embodiments 1 to 30, wherein expression of the addressed target is restricted to one cell type of interest.

[0565] 34. The macromolecule according to any one of embodiments 1 to 33, wherein the address target is a soluble protein or an extracellular matrix (ECM)-associated protein that is not present in detectable amounts on the cell surface.

[0566] 35. The macromolecule of embodiment 34, wherein the address target is expressed in the ECM and is not present in detectable amounts elsewhere in the subject.

[0567] 36. The macromolecule of any one of embodiments 1 to 35, wherein the address target is expressed only in cells of interest when in a specific cellular state.

[0568] 37. The macromolecule according to any one of embodiments 1 to 36, wherein the address target is expressed only in the cells of the subject when in a disease state.

[0569] 38. The macromolecule of any one of embodiments 1 to 37, wherein the address target is not expressed in tissues in which binding of the second binding moiety to the effector target is deleterious to the subject.

[0570] 39. The macromolecule according to any one of embodiments 1 to 38, wherein the binding site for the address target does not detectably bind to the binding site of the address target's natural ligand.

[0571] 40. Expression of the effector target or address target is in the following areas: minor salivary gland, thyroid, lung, breast, mammary gland tissue, pancreas, adrenal gland, liver, kidney, renal cortex, renal medulla, adipose visceral tissue, omentum, small intestine, terminal ileum, fallopian tube, ovary, uterus, skin, non-sun-exposed skin, suprapubic skin, cervix, endocervix, cervicovaginal region, vagina, sun-exposed skin, calf skin, anterior cingulate cortex, Brodmann area 24 (BA24), cerebrum 40. The macromolecule of any one of embodiments 1-39, comprising expression in one or more of basal ganglia, caudate nucleus, putamen, nucleus accumbens, hypothalamus, amygdala, hippocampus, cerebellum, cerebellar hemispheres, substantia nigra, pituitary gland, spinal cord, cervical spinal cord, artery, aorta, heart, atrial appendage, coronary artery, left ventricle, esophagus, esophageal mucosa, esophageal muscularis, gastroesophageal junction, spleen, stomach, colon, transverse colon, sigmoid colon, testis, whole blood cells, EBV-transformed lymphocytes, tibial artery, or tibial nerve tissue.

[0572] 41. The macromolecule according to embodiment 40, wherein expression of the effector target or address target comprises expression in skin tissue, lung tissue, kidney tissue, or intestinal tissue.

[0573] 42. The macromolecule according to embodiment 41, wherein expression of the addressed target is substantially higher in skin tissue, lung tissue, kidney tissue, or intestinal tissue than in any other tissue.

[0574] 43. The macromolecule according to any one of embodiments 1 to 42, wherein the effector target and / or the address target is expressed on a structural tissue of the subject.

[0575] 44. The macromolecule according to any one of embodiments 1 to 43, wherein the effector target and the address target are on the same cell.

[0576] 45. The macromolecule according to any one of embodiments 1 to 43, wherein the effector target and the address target are on different cells.

[0577] 46. ​​The macromolecule according to embodiment 45, wherein the effector target and the address target are on different cells of the same cell type.

[0578] 47. The macromolecule according to embodiment 45, wherein the effector target and the address target are on different cells of different cell types.

[0579] 48. The macromolecule according to embodiment 45, wherein the effector target and the address target are on different cells within the same tissue.

[0580] 49. (a) The effector target is on a circulating cell and the address target is on a tissue-restricted cell; or (b) The macromolecule of any one of embodiments 45, 47, and 48, wherein the effector target is on a tissue-restricted cell and the address target is on a circulating cell.

[0581] 50. The macromolecule of any one of embodiments 45-49, wherein the effector target and the address target are on different cells located within 100 nm of each other in the subject.

[0582] 51. The macromolecule according to any one of embodiments 45 to 49, wherein either the effector target or the address target is present on the cell surface.

[0583] 52. The polymer according to any one of embodiments 1 to 51, wherein the polymer is a DNA polynucleotide.

[0584] 53. The polymer according to any one of embodiments 1 to 51, wherein the polymer comprises an RNA or an RNA-polypeptide conjugate.

[0585] 54. The polymer of any one of embodiments 1 to 51 and 53, wherein the polymer comprises a polypeptide.

[0586] 55. The polymer according to any one of embodiments 1 to 51, wherein the polymer is a polypeptide.

[0587] 56. The macromolecule of embodiment 54 or 55, wherein the polypeptide is an antibody or an antigen-binding fragment thereof.

[0588] 57. The macromolecule of embodiment 56, wherein the first binding site and the second binding site comprise a VH and / or a VL, respectively.

[0589] 58. The macromolecule of embodiment 57, wherein the macromolecule is an antibody comprising a first binding site specific for an effector target in a subject and a second binding site specific for an address target.

[0590] 59. The macromolecule of embodiment 57 or 58, wherein the macromolecule is an asymmetric antibody or a symmetric antibody.

[0591] 60. The antibody or antigen-binding fragment thereof is selected from the group consisting of scFv, BsIgG, BsAb fragment, BiTE, dual-affinity re-targeting protein (DART), tandem diabody (TandAb), diabody, Fab2, bivalent scFv (di-scFv), chemically conjugated F(ab')2, Ig ​​molecule with two, three or four different antigen-binding sites, DVI-IgG four-in-one (DVI-IgG four-in-one), and the like. four-in-one), ImmTac, HSAbody, IgG-IgG, Cov-X-Body, scFv1-PEG-scFv2, conjugated IgG, DVD-IgG, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, monobody, 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 triple body, BiKE, CrossMAb, dsDb, scDb, tandem dAb / VHH, triple dAb 60. The macromolecule of any one of embodiments 56 to 59, comprising a VHH, a tetravalent dAb / VHH, a Fab-scFv, a Fab-Fv, or a DART-Fc, an adectin, a Kunitz-type inhibitor, or a receptor decoy.

[0592] 61. The macromolecule according to embodiment 54, wherein the polypeptide is a ligand of an effector target or a ligand of an address target.

[0593] 62. The macromolecule according to embodiment 61, wherein the ligand is a natural ligand, a modified ligand, or a synthetic ligand.

[0594] 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.

[0595] 64. The macromolecule according to any one of embodiments 61 to 63, wherein the first binding site comprises an antibody or an antigen-binding fragment thereof, and the second binding site comprises a ligand of the address target.

[0596] 65. The macromolecule according to any one of embodiments 61 to 63, wherein 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.

[0597] 66. The polymer of any one of embodiments 1 to 51 and 54 to 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.

[0598] 67. The polymer of any one of embodiments 1 to 66, wherein the address target has a Gini coefficient greater than about 0.4, about 0.5, about 0.57, about 0.65, about 0.7, about 0.85, about 0.90, or about 0.95.

[0599] 68. The polymer of any one of embodiments 1 to 67, wherein the address target has a Tau coefficient greater than about 0.67, about 0.75, about 0.8, about 0.85, about 0.90, or about 0.95.

[0600] 69. The macromolecule according to any one of embodiments 1 to 68, wherein the effector target has a Gini coefficient lower than about 0.25, about 0.20, or about 0.15.

[0601] 70. The macromolecule according to any one of embodiments 1 to 69, wherein the effector target has a tau coefficient lower than about 0.25, about 0.20, or about 0.15.

[0602] 71. The macromolecule of any one of embodiments 1-70, further comprising a third binding site.

[0603] 72. The macromolecule of embodiment 71, wherein the third binding site is the same as the first binding site.

[0604] 73. The macromolecule of embodiment 71, wherein the third binding site is the same as the second binding site.

[0605] 74. The polymer of any one of embodiments 1-73, wherein the first binding site and the second binding site are directly bonded to each other in the polymer.

[0606] 75. The polymer according to any one of embodiments 1 to 73, wherein the first binding site and the second binding site in the polymer are connected by a stable domain.

[0607] 76. The macromolecule according to any one of embodiments 1 to 75, wherein the effector target is Notch2 and the address target is RAGE.

[0608] 77. The macromolecule of embodiment 76, wherein RAGE signaling is not affected by the second moiety that binds to the RAGE address target.

[0609] 78. The macromolecule according to any one of embodiments 1 to 75, wherein the effector target is Notch2 and the address target is uromodulin (UMOD).

[0610] 79. The macromolecule of embodiment 78, wherein UMOD signaling is not affected by the second moiety that binds to the UMOD address target.

[0611] 80. The macromolecule according to any one of embodiments 1-75, wherein the effector target is Notch2 and the address target is meprin A subunit beta (MEP1B).

[0612] 81. The macromolecule of embodiment 80, wherein MEP1B signaling is not affected by the second moiety that binds to the MEP1B address target.

[0613] 82. The macromolecule according to any one of embodiments 1 to 75, wherein the effector target is IL11Ra and the address target is RAGE.

[0614] 83. The macromolecule of embodiment 82, wherein RAGE signaling is not affected by the second moiety that binds to the RAGE address target.

[0615] 84. The macromolecule of any one of embodiments 1 to 75, wherein the effector target is IL11Ra and the address target is UMOD.

[0616] 85. The macromolecule of embodiment 84, wherein UMOD signaling is not affected by the second moiety that binds to the UMOD address target.

[0617] 86. The macromolecule according to any one of embodiments 1 to 85, wherein the subject is a human.

[0618] 87. A method for delivering a moiety to a target tissue or cell of a subject, comprising administering to the subject a macromolecule described in any one of embodiments 1 to 86, wherein the target tissue comprises an address target.

[0619] 88. The macromolecule of embodiment 87, wherein the moiety is a molecule.

[0620] 89. The macromolecule of embodiment 87 or 88, wherein the moiety is not a toxin.

[0621] 90. The macromolecule of embodiment 87, wherein the moiety is a cell.

[0622] 91. The macromolecule of embodiment 90, wherein the moiety is not a T cell or an NK cell.

[0623] 92. The macromolecule according to any one of embodiments 87 to 91, wherein the target tissue is not a tumor.

[0624] 93. A method for modulating an effector target in a target tissue, comprising administering to the tissue a macromolecule described in any one of embodiments 1 to 86, wherein the target tissue comprises an address target and an effector target.

[0625] 94. A method for biasing a binder away from binding to an effector target when the effector target is found in the heart or lung, comprising administering a macromolecule described in any one of embodiments 1 to 86, wherein the address target is not substantially expressed in the heart or lung.

[0626] 95. A method for modulating a target tissue in a subject, comprising administering to the subject a macromolecule described in any one of embodiments 1 to 86, wherein the target tissue comprises an address target and an effector target.

[0627] 96. A method of treating a subject having a disease or condition associated with an effector target, comprising administering to the subject a macromolecule described in any one of embodiments 1 to 86, wherein a first binding moiety of the macromolecule binds to the effector target.

[0628] 97. A macromolecule comprising a first binding site and a second binding site, (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for an address target expressed in a target tissue or cell of interest; wherein the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of the effector target in the target tissue or cell; wherein the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; and The second binding site does not bind to the binding site of the natural ligand of the address target, where the macromolecule is linked to a small molecule.

[0629] 98. A polymer comprising a first binding site and a second binding site, (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for an address target expressed in a target tissue or cell of interest; wherein the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of the effector target in the target tissue or cell; wherein the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; and A macromolecule, wherein the first binding site and the second binding site are directly linked to each other in said macromolecule, wherein the macromolecule is linked to a small molecule.

[0630] 99. A polymer comprising a first binding site and a second binding site, (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for an address target expressed in a target tissue or cell of interest; wherein the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of the effector target in the target tissue or cell; wherein the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; and A macromolecule, wherein the first binding site and the second binding site are linked to each other by a stable domain, wherein the macromolecule is linked to a small molecule.

[0631] 100. A polymer comprising a first binding site and a second binding site, (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for an address target expressed in a target tissue or cell of interest; wherein the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of the effector target in the target tissue or cell; wherein the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; and The effector target and / or address target is a macromolecule expressed on a structural tissue of the host, where the macromolecule is linked to a small molecule.

[0632] 101. A pharmaceutical composition comprising the polymer according to any one of embodiments 1 to 86.

[0633] 102. A pharmaceutical composition comprising a polymer and one or more pharmaceutically acceptable excipients, The macromolecule comprises a first binding site and a second binding site, wherein: (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for an address target expressed in a target tissue or cell of interest; wherein the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of the effector target in the target tissue or cell; wherein the first binding site does not substantially affect signal transduction of the effector target in the absence of localization by the second binding site, and wherein the macromolecule is linked to a small molecule.

[0634] 103. The pharmaceutical composition according to embodiment 101 or 102, wherein the pharmaceutical composition is an RNA pharmaceutical composition.

[0635] 104. The pharmaceutical composition of any one of embodiments 101-103, further comprising a carrier.

[0636] 105. The pharmaceutical composition according to embodiment 104, wherein the carrier is a lipid nanoparticle.

[0637] 106. The pharmaceutical composition according to embodiment 104, wherein the carrier is a viral vector.

[0638] 107. The pharmaceutical composition according to embodiment 104, wherein the carrier is a membrane carrier.

[0639] 108. The pharmaceutical composition according to embodiment 107, wherein the membrane carrier is a cell.

[0640] 109. The pharmaceutical composition according to embodiment 107, wherein the membrane carrier is a vesicle.

[0641] 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: (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for desmoglein-1 (DSG-1), wherein the macromolecule is linked to a small molecule.

[0642] 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: (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for RAGE, wherein the macromolecule is linked to the small molecule.

[0643] 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: (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for cadherin 16 (CDH16), wherein the macromolecule is linked to the small molecule.

[0644] 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: (a) the first binding site is specific for an effector target of interest; (b) the second binding site is specific for cadherin 17 (CDH17), wherein the macromolecule is linked to the small molecule.

[0645] 114. A method for localizing a macromolecule to a target tissue or cell in 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 of interest; (b) the second binding site is specific for an address target expressed in a target tissue or cell of interest; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signal transduction upon binding to the address target; and (iii) the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; A method for allowing a macromolecule to localize to a target tissue or cell of a subject, wherein the macromolecule is linked to a small molecule.

[0646] 115. A method for localizing a macromolecule to a target tissue or cell in 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 of interest; (b) the second binding site is specific for an address target expressed in a target tissue or cell of interest; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of the effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signal transduction upon binding to the address target; and (iii) the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; A method for allowing a macromolecule to concentrate in a target tissue or cell of a subject, wherein at least 25% of the macromolecule detectable in the subject is detected in the target tissue or cell 1 to 7 days after administration of the macromolecule to the subject, wherein the macromolecule is linked to a small molecule.

[0647] 116. The method of embodiment 114 or 115, wherein the efficacy of the first binding moiety in the target tissue or cell is substantially increased compared to a control macromolecule lacking the second binding moiety.

[0648] 117. The method of embodiment 114 or 115, wherein signaling of the effector target by the macromolecule in non-target tissues or cells of the subject is substantially reduced compared to a control macromolecule lacking the second binding moiety.

[0649] 118. The method according to any one of embodiments 110 to 117, wherein the polymer is a polymer according to any one of embodiments 1 to 86.

[0650] 119. The polymer, method, or pharmaceutical composition of any one of claims 1-118, wherein the polymer and small molecule are linked by a linker.

[0651] 120. The polymer, method, or pharmaceutical composition of claim 119, wherein the linker is a cleavable linker.

[0652] 121. The polymer, method, or pharmaceutical composition of claim 119, wherein the linker is a non-cleavable linker.

[0653] 122. The macromolecule, method, or pharmaceutical composition of any one of claims 1-121, wherein the first binding site, the second binding site, or both, is a polypeptide.

[0654] 123. The polymer, method, or pharmaceutical composition of any one of claims 1-122, wherein a single small molecule is linked to the polymer.

[0655] 124. The polymer, method, or pharmaceutical composition of any one of claims 1-122, wherein multiple small molecules are linked to the polymer.

[0656] 125. The macromolecule, method, or pharmaceutical composition of claim 124, wherein each of the small molecules is the same.

[0657] 126. The macromolecule, method, or pharmaceutical composition of claim 124, wherein at least two of the small molecules are different from each other.

[0658] 127. The macromolecule, method, or pharmaceutical composition of any one of embodiments 1-126, wherein the target cell is an immune cell and the second binding site is specific for CD20.

[0659] 128. The macromolecule, method, or pharmaceutical composition of any one of embodiments 1-126, wherein the target cell is an immune cell and the second binding site is specific for CD33.

[0660] 129. The macromolecule, method, or pharmaceutical composition of any one of embodiments 1-128, wherein the small molecule is fluocinolone.

[0661] 130. The macromolecule, method, or pharmaceutical composition of any one of embodiments 1-128, wherein the small molecule is ibrutinib.

[0662] 131. The macromolecule, method, or pharmaceutical composition of any one of embodiments 1-128, wherein the small molecule is tofacitinib.

[0663] 132. The polymer, method, or pharmaceutical composition of any one of embodiments 1-128, wherein the polymer linked to a small molecule exhibits increased internalization compared to a control polymer not linked to a small molecule.

[0664] 133. The polymer, method, or pharmaceutical composition according to any one of embodiments 1-128, wherein the polymer does not substantially induce cell death.

[0665] 134. The macromolecule, method, or pharmaceutical composition of any one of embodiments 1-128, wherein the macromolecule modulates immune cell function, for example, of B cells or myeloid cells.

[0666] Although the invention has been described herein in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention.

Claims

1. 1. A method for localizing a macromolecule to a target tissue or cell in a subject, the method comprising administering to the subject a macromolecule comprising a first binding moiety and a second binding moiety; (a) the first binding site is specific for an effector target of the subject; (b) the second binding site is specific for an address target expressed in a target tissue or cell of the subject; (i) the second binding site localizes the first binding site to the address target such that the first binding site affects signaling of an effector target in the target tissue or cell; (ii) the second binding site does not substantially affect signal transduction upon binding to the address target; (iii) the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; A method that allows the macromolecule to localize to a target tissue or cell of the subject, wherein the macromolecule is linked to a small molecule.

2. The method of claim 1 , wherein the polymer 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. 2. The method of claim 1, wherein at least 25% of the macromolecule detectable in the subject is detected in the target tissue or cell at 1 to 7 days after administration of the macromolecule to the subject.

6. 10. The method of claim 1, wherein the efficacy of the first binding moiety in the target tissue or cell is substantially increased compared to a control macromolecule lacking the second binding moiety.

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 low binding activity for the effector target.

9. 2. 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. 2. The method of claim 1, wherein the binding activity of the first binding site for the effector target is lower than the binding activity of the second binding site for the address target.

11. 10. The method of claim 1, wherein effector target signaling by the macromolecule in a non-target tissue or cell of the subject is substantially reduced compared to a control macromolecule lacking the second binding moiety.

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 expression of the address target is restricted to a cell type of the subject.

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

16. 10. The method of claim 1, wherein the address target is expressed only by cells of the subject in a disease state.

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

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

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

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

21. (a) the first binding site comprises an antibody or antigen-binding fragment thereof and the second binding site comprises a ligand of the address target; or 21. The method of claim 20, wherein (b) the first binding site comprises a ligand of the effector target and the second binding site comprises an antibody or antigen-binding fragment thereof.

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

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

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

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

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

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

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