Compositions and methods for targeted delivery of therapeutic agents

Abstract

Provided are polymeric compositions and related methods 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. Described are compositions and methods relating to polymers, e.g., ANDbodies™, that include an effector target binding domain specific for an effector target and an address binding domain specific for an address target.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety into this application. The ASCII copy, created on Jun. 6, 2022, is named 51666-002WO2_Sequence_Listing_6_6_22_ST25 and is 276,968 bytes in size. [Background technology]

[0002] 2. Background of the Invention Unwanted off-target effects are a challenge for otherwise desirable therapeutic targets that are present in healthy tissue as well as diseased tissue. Summary of the Invention [Problem to be solved by the invention]

[0003] Summary of the Invention The present disclosure describes, in part, polymer compositions and related methods 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. In general, the compositions described herein include polymers, e.g., ANDbody™, that include an effector target binding domain specific to an effector target and an address binding domain specific to an address target. The address target is generally sufficiently limited in a subject to target the polymer to a desired cell, tissue or organ. In some embodiments, the effector target binding domain does not affect the effector target in the absence of the address target binding domain. 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 induce an effect on signal transduction by the effector target in the target cell or tissue. The compositions described herein can be used, for example, to specifically deliver a therapeutic agent 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 of 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, where (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 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 further, (iii) the first binding site does not substantially affect signaling 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.

[0005] In some embodiments, at least 25% of the macromolecules detectable in a subject are detectable in target tissues or cells 1-7 days after administration of the macromolecule to the subject.

[0006] 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.

[0007] In some embodiments, the first binding site has a low affinity for the effector target.

[0008] In some embodiments, the first binding site has low binding activity for the effector target.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] In some embodiments, the address target is expressed regionally in the subject. In some embodiments, the address target is expressed locally in the subject. In some embodiments, the expression of the address target is restricted to one cell type in the subject.

[0013] In some embodiments, an address target is expressed by only one cell of a subject when in a particular cellular state.

[0014] In some embodiments, the address target is expressed by only one cell of a subject in a disease state.

[0015] In some embodiments, the first binding site or the second binding site comprises a polypeptide.

[0016] In some embodiments, the polypeptide is an antibody or an antigen-binding fragment thereof.

[0017] In some embodiments, the macromolecule is an antibody that comprises a first binding site specific for an effector target in a subject and a second binding site specific for an address target.

[0018] In some embodiments, the polypeptide is a ligand of an effector target or a ligand of an address target.

[0019] 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.

[0020] In some embodiments, the target tissue is skin and the second binding site is specific for desmoglein-1 (DSG-1).

[0021] In some embodiments, the target tissue is lung tissue and the second binding site is specific for RAGE.

[0022] In some embodiments, the target tissue is kidney tissue and the second binding site is specific for cadherin 16 (CDH16).

[0023] In some embodiments, the target tissue is intestinal tissue and the second binding site is specific for cadherin 17 (CDH17).

[0024] In another aspect, the disclosure provides a macromolecule comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target 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.

[0025] 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 (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, 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.

[0026] 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 (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, 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.

[0027] 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 (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 at 1 to 7 days after administration.

[0028] In another aspect, the disclosure provides a macromolecule comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target 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.

[0029] In another aspect, the disclosure provides a macromolecule comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target 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 which 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.

[0030] In another aspect, the disclosure provides a macromolecule comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target 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 macromolecule lacking the second binding site.

[0031] In some embodiments, the first binding site has a low affinity for the effector target.

[0032] In some embodiments, the first binding site has low binding activity for the effector target.

[0033] 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.

[0034] 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.

[0035] 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 EC of the second binding site to address 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.

[0036] 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.

[0037] 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.

[0038] In some embodiments, the effector target is a protein, lipid, or sugar.

[0039] In some embodiments, the effector target is a cell membrane-associated target.

[0040] In some embodiments, the effector target is a protein, hi some embodiments, the effector target is a secreted protein.

[0041] In some embodiments, the effector target is encoded by a gene selected from the group consisting of the genes listed in Table 1.

[0042] In some embodiments, the macromolecule agonizes the effector target.

[0043] In some embodiments, the macromolecule antagonizes an effector target.

[0044] In some embodiments, the address target is a protein, a lipid, or a sugar.

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

[0046] In some embodiments, expression of an effector target or an address target is expression of an RNA sequence encoding the effector target or the address target.

[0047] In some embodiments, the expression levels of effector targets or address targets are assessed using an RNA-seq dataset.

[0048] In some embodiments, the RNA-seq dataset is a Genotype-Tissue Expression (GTEx) dataset or a Human Protein Atlas (HPA) dataset.

[0049] In some embodiments, expression of an effector target or an address target is protein expression.

[0050] In some embodiments, the effector target is systemically expressed in the subject.

[0051] In some embodiments, the effector target is regionally expressed in the subject.

[0052] In some embodiments, the effector target is expressed locally in the subject.

[0053] In some embodiments, the address target is regionally expressed in the subject.

[0054] In some embodiments, the address target is expressed locally in the subject.

[0055] In some embodiments, expression of an address target is restricted to one cell type of a subject.

[0056] 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.

[0057] In some embodiments, the address target is expressed in the ECM and is not present in detectable amounts elsewhere in the subject.

[0058] In some embodiments, the address target is only expressed in cells of interest when in a particular cellular state.

[0059] In some embodiments, the address target is only expressed in the subject's cells when in a disease state.

[0060] In some embodiments, the address target is not expressed in tissues where binding of the second binding site to the effector target is deleterious to the subject.

[0061] In some embodiments, a binding site for an address target does not detectably bind to a binding site for a natural ligand of the address target.

[0062] In some embodiments, expression of effector targets or address targets 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, cervix vaginalis, 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.

[0063] In some embodiments, expression of the effector target or address target comprises expression in skin tissue, lung tissue, kidney tissue, or intestinal tissue, hi some embodiments, expression of the address target is substantially higher in skin tissue, lung tissue, kidney tissue, or intestinal tissue than in any other tissue.

[0064] In some embodiments, the effector target and / or address target are expressed on a structural tissue of the subject.

[0065] In some embodiments, the effector target and the address target are on the same cell.

[0066] In some embodiments, the effector target and the address target are on different cells.

[0067] In some embodiments, the effector target and the address target are on different cells of the same cell type.

[0068] In some embodiments, the effector target and the address target are on different cells of different cell types.

[0069] In some embodiments, the effector target and the address target are on different cells within the same tissue.

[0070] 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.

[0071] In some embodiments, the effector target and the address target are on different cells located within 100 nm of each other in the subject.

[0072] In some embodiments, either the effector target or the address target is present on the cell surface.

[0073] In some embodiments, the polymer is a DNA polynucleotide.

[0074] In some embodiments, the macromolecule comprises an RNA or an RNA-polypeptide conjugate.

[0075] In some embodiments, the macromolecule comprises a polypeptide. In some embodiments, the macromolecule is a polypeptide.

[0076] In some embodiments, the polypeptide is an antibody or an antigen-binding fragment thereof.

[0077] In some embodiments, the first binding site and the second binding site comprise a VH and / or a VL, respectively.

[0078] In some embodiments, the macromolecule is an antibody that comprises a first binding site specific for an effector target in a subject and a second binding site specific for an address target.

[0079] In some embodiments, the macromolecule is an asymmetric or symmetric antibody.

[0080] In some embodiments, the antibody or antigen-binding fragment thereof includes: 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, adducted 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 type inhibitor, or receptor decoy.

[0081] In some embodiments, the polypeptide is a ligand of an effector target or a ligand of an address target.

[0082] In some embodiments, the ligand is a naturally occurring ligand, a modified ligand, or a synthetic ligand.

[0083] In some embodiments, the effector target or address target is a receptor and the polypeptide is its ligand.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] In some embodiments, the address target has a Geni 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.

[0088] In some embodiments, the address targets have a Tau coefficient greater than about 0.67, about 0.75, about 0.8, about 0.85, about 0.90, or about 0.95.

[0089] In some embodiments, the effector target has a Gini coefficient of less than about 0.25, about 0.20, or about 0.15.

[0090] In some embodiments, the effector target has a tau coefficient of less than about 0.25, about 0.20, or about 0.15.

[0091] 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.

[0092] In some embodiments, the first binding site and the second binding site are directly bound to each other in the polymer.

[0093] In some embodiments, the first binding site and the second binding site in the polymer are linked by a stable domain.

[0094] In some embodiments, the effector target is Notch2 and the address target is RAGE.

[0095] In some embodiments, RAGE signaling is not affected by the second moiety binding to the RAGE address target.

[0096] In some embodiments, the effector target is Notch2 and the address target is uromodulin (UMOD).

[0097] In some embodiments, UMOD signaling is not affected by the second moiety binding to the UMOD address target.

[0098] In some embodiments, the effector target is Notch2 and the address target is meprin A subunit beta (MEP1B).

[0099] In some embodiments, MEP1B signaling is not affected by the second moiety binding to the MEP1B address target.

[0100] In some embodiments, the effector target is IL11Ra and the address target is RAGE, hi some embodiments, RAGE signaling is not affected by the second moiety that binds to the RAGE address target.

[0101] 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 that binds to the UMOD address target.

[0102] In some embodiments, the subject is a human.

[0103] In another aspect, the 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.

[0104] In some embodiments, the moiety is a molecule.

[0105] In some embodiments, the moiety is not a toxin.

[0106] In some embodiments, the moiety is a cell.

[0107] In some embodiments, the moiety is not a T cell or a NK cell.

[0108] In some embodiments, the target tissue is not a tumor.

[0109] In another aspect, the 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 to 86, wherein the target tissue comprises an address target and an effector target.

[0110] In another aspect, the 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 according to any one of claims 1 to 86, wherein the address target is not substantially expressed in the heart or lung.

[0111] 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.

[0112] 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.

[0113] In another aspect, the disclosure provides a macromolecule comprising a first binding site and a second binding site, where (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; and where 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.

[0114] 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 first binding site and the second binding site are directly linked to each other in the polymer.

[0115] In another aspect, the disclosure provides a macromolecule comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target 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 one another by a stable domain.

[0116] In another aspect, the disclosure provides a macromolecule comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target 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 the host.

[0117] In another aspect, the disclosure provides a pharmaceutical composition comprising the macromolecule of any one of the preceding embodiments.

[0118] In another aspect, the disclosure provides a pharmaceutical composition comprising a polymer and one or more pharma- ceutically acceptable excipients, wherein the polymer comprises a first binding site and a second binding site, (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.

[0119] In some embodiments, the pharmaceutical composition is an RNA pharmaceutical composition.

[0120] In some embodiments, the pharmaceutical composition further comprises a carrier.

[0121] In some embodiments, the carrier is a lipid nanoparticle.

[0122] In some embodiments, the carrier is a viral vector.

[0123] In some embodiments, the support is a membrane support.

[0124] In some embodiments, the membrane carrier is a cell.

[0125] In some embodiments, the membrane carrier is a vesicle.

[0126] In another aspect, the disclosure provides a method of 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, where (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).

[0127] In another aspect, the disclosure provides a method of modulating 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, where (a) the first binding site is specific for an effector target in the subject and (b) the second binding site is specific for RAGE.

[0128] In another aspect, the disclosure provides a method of modulating 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, where (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).

[0129] In another aspect, the disclosure provides a method of modulating 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, where (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).

[0130] In another aspect, the disclosure provides a method of 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, where (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 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 further, (iii) the first binding site does not substantially affect signaling 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.

[0131] In another aspect, the disclosure provides a method of concentrating a macromolecule in a target tissue or cell of a subject, the method comprising administering to the subject a macromolecule comprising a first binding site and a second binding site, wherein (a) the first binding site is specific for an effector target 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 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 further, (iii) the first binding site does not substantially affect signaling of the effector target in the absence of localization by the second binding site; allowing 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 at 1 to 7 days after administration of the macromolecule to the subject.

[0132] 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.

[0133] 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.

[0134] In some embodiments, the polymer is a polymer of any one of the preceding embodiments.

[0135] 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 become apparent from the following drawings and detailed description of certain embodiments, as well as from the appended claims. [Brief description of the drawings]

[0136] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]FIG. 1 is a schematic diagram showing an exemplary ANDbody™ molecule and its use as a logic gate pharmaceutical. FIG. 1 shows the broad distribution of therapeutic targets, e.g., effector targets, in a human subject without address targeting (right side) and the localized and restricted distribution due to address targeting achieved by the address target binding domain (left side). FIG. 1 also provides a representative bipartite structure of an ANDbody having an address target binding domain linked to an effector target binding domain, which includes a functional moiety, e.g., a moiety that modulates, e.g., agonizes or antagonizes, a target effector in an address targeted cell or tissue. The address target binding domain directs the ANDbody to a desired location, such as a target cell or tissue, and enables the effector target binding domain to bind to a 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 trigger an effect on signal transduction 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. [Diagram 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. [Diagram 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, Fab2, IgG(L,H)-Fv, or BiTEs. [Figure 4] FIG. 4 shows the EC50 curves of an exemplary single effector targeting domain (dashed line), such as a monospecific biologic having a single binding domain to an effector target (e.g., scFv), compared to the EC50 (solid line) of an exemplary bispecific ANDbody biologic comprising an address target binding domain and an effector target binding domain (e.g., di-scFc), whereby the single effector targeting domain (usually ubiquitously expressed) is targeted / restricted to a local, address target specific tissue and / or cell, 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] 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 shown as the average of three mice. Values ​​are shown with the most intense signal set to 1 to adjust for differences in labeling efficiency. [Figure 5B] Figure 5B is a bar graph showing the levels of fluorescence intensity detected in the indicated tissues in mice treated with anti-DSG1 antibody PRO004 conjugated to IRDYE® 800CW or treated with vehicle control (untreated). Data are shown as the average of three mice. Values ​​are shown with the most intense signal set to 1 to adjust for differences in labeling efficiency. [Figure 6A] Figure 6A is a bar graph showing the levels of fluorescence intensity detected in the indicated tissues in mice treated with anti-RAGE antibody PRO001 conjugated to IRDYE® 800CW or vehicle control. Data are presented as the average of three mice. Values ​​are presented with the most intense signal set to 1 to adjust for differences in labeling efficiency. [Figure 6B]Figure 6B is a bar graph showing the levels of fluorescence intensity detected in the indicated tissues in mice treated with anti-RAGE antibody PRO002 conjugated to IRDYE® 800CW or vehicle control. Data are shown as the average of three mice. Values ​​are shown with the most intense signal set to 1 to adjust for differences in labeling efficiency. [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 Balb / C mice treated with 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] 8 is a bar graph showing the levels of fluorescence intensity detected in the indicated tissues in mice treated with anti-CDH16 antibody PRO056 conjugated to IRDYE® 800CW or vehicle control. Data are shown as the average of three mice. Values ​​are shown with the most intense signal set to 1 to adjust for differences in labeling efficiency. [Figure 9] 9 is a bar graph showing the levels of fluorescence intensity detected in the indicated tissues in mice treated with anti-CDH17 antibody PRO061 conjugated to IRDYE® 800CW or vehicle control. Data are shown as the average of three mice. Values ​​are shown with the most intense signal set to 1 to adjust for differences in labeling efficiency. [Figure 10] Figure 10 is a series of photomicrographs showing staining of Notch2 antagonist mAbs; PRO034, PRO035, PRO036 and 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 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 schematics showing the design of the PRO023, PRO025, PRO024, PRO027, and PRO026 IL-10 / DSG1 ANDbodies. [Figure 13A] FIG. 13A is a bar graph showing levels of tumor necrosis factor alpha (TNFα) in peripheral blood mononuclear cell (PBMC) cell cultures following pre-stimulation with hrIL-10 followed by treatment with lipopolysaccharide (LPS) for the indicated times. [Figure 13B] FIG. 13B is a bar graph showing levels of TNFα in PBMC cell cultures after pre-stimulation with anti-DSG1 monoclonal antibody (mAb) followed by treatment with LPS for the indicated times. [Figure 13C] FIG. 13C is a bar graph showing levels of TNFα in PBMC cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO024 followed by treatment with LPS for the indicated times. [Figure 13D] FIG. 13D is a bar graph showing levels of TNFα in PBMC cell cultures after pre-stimulation with IL-10 / DSG1 ANDbody PRO026 followed by treatment with LPS for the indicated times. [Figure 13E] FIG. 13E is a bar graph showing 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] FIG. 13F is a bar graph showing 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]FIG. 13G is a bar graph showing 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. [Figure 14A] FIG. 14A is a bar graph showing levels of TNFα in primary macrophage cell cultures after pre-stimulation with hrIL-10 followed by treatment with LPS for the times indicated. [Figure 14B] FIG. 14B is a bar graph showing 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] FIG. 14C is a bar graph showing 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] FIG. 14D is a bar graph showing 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. [Figure 14E] FIG. 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] FIG. 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] FIG. 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]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 the expression of secreted embryonic alkaline phosphatase (SEAP). OD630: optical density at 630 nm. Curve fitting was performed using GraphPad Prism 9 to fit a 4-parameter log(agonist) vs. response. [Figure 16A] FIG. 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 antibody or ANDbody via tail vein injection. Concentrations of circulating molecules were measured by ELISA. Mean concentrations and standard deviations are shown. N=3. [Figure 16B] FIG. 16B is a plot showing the concentration (ng of target protein per mg of total protein) over time of PRO003, PRO024, and PRO058 (functionally equivalent to PRO026) in skin tissue samples from BALB / c mice administered 3 mg / kg of the indicated antibody or ANDbody via tail vein injection. Skin samples were harvested at the indicated time points, homogenized, and protein 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. [Figure 18A]Figure 18A is a plot showing the levels 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 expression of SEAP. OD630: optical density at 630 nm. Curve fitting was performed using GraphPad Prism 9 to fit a 4 parameter log(agonist) vs response. [Figure 18B] FIG. 18B is a plot showing the levels 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 4 parameter log(agonist) vs. response. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0137] Detailed Description of the Invention Provided herein are ANDbody™ molecules that include a therapeutic effector target binding domain and an address target binding domain. 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 a target tissue or cell, localizing the effector target to the target cell or tissue, for example, forming an AND gate type logic gate. For example, in some embodiments, an ANDbody is a macromolecule that includes at least (a) a first binding site specific for a therapeutic effector target that is expressed, for example, ubiquitously expressed, in a mammalian subject, for example, on a cell surface; and (b) a second binding site specific for an address target. In some embodiments, expression of the address target is limited in vivo in the subject. In some embodiments, the binding of the first binding site to the therapeutic effector target is weaker than the binding of the second binding site to the address marker. The effector target and the address target may be on the same cell, or may be in different cells or compartments within the same tissue.

[0138] In some embodiments, at least 25% of the macromolecules (e.g., ANDbodies) detectable in a subject are detectable in target tissues or cells 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 macromolecules detectable in a subject are detectable in target tissues or cells 1-7 days after administration of the macromolecule to the subject.

[0139] Effector Target The ANDbodies™ of the present invention include effectors that modulate 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 to produce a biological response, such as a therapeutic effect, in the subject. However, in some embodiments, the effector target binding domains provided herein may not induce 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 present invention.

[0140] In some embodiments, the effector target binding domain may produce a small / weak biological effect when provided alone, but may provide a larger / 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, the effector target binding domain may produce an acceptable biological effect when provided alone, but may provide a larger / stronger biological effect when provided together with an address targeting domain for localizing the effector target binding domain to a targeted cell or tissue. In some embodiments, the 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 for localizing the effector target binding domain to a targeted cell or tissue. In some embodiments, the effector target binding domain, when provided alone, may produce biological effects, including undesirable off-target biological effects, but when provided together with an address targeting domain, may be targeted, concentrated, and concentrated to a desired address in a targeted cell or tissue to reduce or eliminate undesirable off-target biological effects. Thus, the effector target binding domain of the present technology, when provided together with an address target binding domain as described herein, provides a superior therapeutic agent that produces a more potent targeted biological effect with reduced side effects, including reduced unintended off-target biological effects.

[0141] Examples of such therapeutic signaling effects include, but are not limited to, the following: (i) blocking signaling pathways that promote or maintain a disease state; (ii) activation of a signaling pathway that ameliorates or prevents 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; (vii) Inducing tissue remodeling to reduce or prevent fibrosis.

[0142] 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.

[0143] 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 between 10 nM and 1 μM, e.g., an affinity of between 10 nM and 100 nM).

[0144] 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 are listed in Table 1, along with exemplary functions of the effector targets.

[0145] [Table 1]

[0146] 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 cell state of interest in the organism. An address target can be, for example, a cell surface protein, or a structure localized in the extracellular matrix. As used herein, "restricted" expression of an address target means that the address target has differential, e.g., less widespread, in vivo expression, as opposed to systematic expression. In certain embodiments, an address target is expressed in a single cell type, tissue, or cell state in a mammalian subject, such as a human subject.

[0147] In some embodiments, the address target binding domains currently provided do not substantially affect biological signaling upon binding to the address target, e.g., do not modulate signaling pathways or other biological responses in the target cell or tissue. For example, the address target binders may be inert or inactive in that they have no additional activity (other than binding), including lack of catalytic activity, after binding to the address target. For example, the address target binders bind to non-signaling sites or motifs of the address target. "Signal" is used herein to indicate a conformational, enzymatic, and / or electrical result that occurs as a result of target binding. Thus, as described herein, the address target binding domains do not signal upon binding of the address target. A domain that "substantially" does not affect biological signaling, as used herein, is a domain that modulates a signaling pathway or other biological response in the 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%).

[0148] Similarly, the effector target binding domain may not signal substantially or at all when not localized by the address target binding domain. In some embodiments, the effector target binding domain signals with higher efficacy (e.g., has higher binding activity) when localized by the address target binding domain compared to when not localized by the address target binding domain. When the effector target binding domain is localized to a targeted cell or tissue by the address target binding domain as part of the same macromolecule, the signal transduction of the effector target can be affected as described above.

[0149] In some embodiments, the address target is used for organ-specific addressing, tissue-specific addressing, or cell-specific addressing.

[0150] The specificity of the address target binding domain to a cell or tissue can be detected using methods known in the art. In one embodiment, the Gini coefficient (GC) score is used, which is a method for assessing the change in expression of a particular gene in a dataset. (See: O'Hagan et al., GeneGini: assessment via the Gini coefficient of reference "housekeeping" genes and diverse human transporter expression profiles. Cell systems 6, 230-244, https: / / doi.org / 10.1016 / j. cels.2018.01.003 (2018); Wright Muelas et al., The role and robustness of the Gini coefficient as an unbiased tool for the selection of Gini genes for normalising expression profiling data. Sci Rep 9, 17960 (2019). https: / / doi.org / 10.1038 / s41598-019-54288-7). Address target binders can be identified using cell 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, exhibit a Gini score of 0.15 to 0.19.

[0151] In one embodiment, a Tau score is used, which represents the expression change of a particular gene in a dataset. The calculation of Tau uses information on the expression of the gene in each tissue and its maximum expression across all tissues, while also taking into account the number of tissues in which expression is measured (see: Itai Yanai, et al., Genome-wide midrange transcription profiles reveal expression level relationships in human tissue specification, Bioinformatics, Volume 21, Issue 5, 1 March 2005, Pages 650-659; Kryuchkova-Mostacci N, Robinson-Rechavi M. A benchmark of gene expression tissue-specificity metrics. Brief Bioinform. 2017 Mar 1;18(2):205-214. doi: 10.1093 / bib / bbw008). In some embodiments, the address target markers exhibit a Tau score of more than 0.6, such as 0.74 to 1.00. Conversely, non-addressable markers that are more systematically expressed exhibit tau scores below 0.3, such as 0.15-0.19.

[0152] In some embodiments, the specificity of the address target binding domain for a particular cell or tissue, as indicated by the appropriate Gini score and / or Tau score, is determined using tissue-based analysis that does not include tissue with a natural biological separation barrier (i.e., blood-brain barrier). For example, in some embodiments, the Gini score and / or Tau score can be calculated without data from tissues such as, but not limited to, central nervous system, brain, eye, and / or testis tissue. In some embodiments, the address target as provided herein reveals a cell state. As used herein, "cell state" refers to a given physiological state of a cell. A cell state can be, for example, a disease state (vs. a non-disease or normal state of a cell or tissue); or an activation state (vs. 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., pre-cancerous or cancerous cell states). In some aspects, a cell state represents the fact that a cell of a particular type retains the characteristics of that particular cell type and does not acquire characteristics that would classify it 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 may 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 only 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), may exhibit differences in protein modifications such as phosphorylation, acetylation, or may exhibit differences in appearance.Thus, a cell state may be a cell state in which the cell expresses, produces, secretes one or more markers, exhibits a particular protein modification, has a particular appearance, and / or exhibits or does not exhibit one or more biological responses to a stimulus or environmental condition. Exemplary address targets of the present technology are listed below in Table 2A (HPA database analysis) and Table 2B (Gtex database analysis).

[0153] [Table 2]

[0154] [Table 3]

[0155] [Table 4]

[0156] [Table 5]

[0157] [Table 6]

[0158] [Table 7]

[0159] [Table 8]

[0160] [Table 9]

[0161] [Table 10]

[0162]

Table 11

[0163]

Table 12

[0164]

Table 13

[0165]

Table 14

[0166]

Table 15

[0167]

Table 16

[0168]

Table 17

[0169]

Table 18

[0170]

Table 19

[0171]

Table 20

[0172]

Table 21

[0173]

Table 22

[0174]

Table 23

[0175]

Table 24

[0176]

Table 25

[0177]

Table 26

[0178]

Table 27

[0179]

Table 28

[0180]

Table 29

[0181]

Table 30

[0182] [Table 31]

[0183] [Table 32]

[0184] [Table 33]

[0185] [Table 34]

[0186] [Table 35]

[0187] [Table 36]

[0188] [Table 37]

[0189] Table 2B contains address targets based on the Gtex database analysis.

[0190] [Table 38]

[0191] [Table 39]

[0192] [Table 40]

[0193]

Table 41

[0194]

Table 42

[0195]

Table 43

[0196]

Table 44

[0197]

Table 45

[0198]

Table 46

[0199]

Table 47

[0200]

Table 48

[0201]

Table 49

[0202]

Table 50

[0203]

Table 51

[0204]

Table 52

[0205]

Table 53

[0206]

Table 54

[0207]

Table 55

[0208]

Table 56

[0209]

Table 57

[0210]

Table 58

[0211]

Table 59

[0212]

Table 60

[0213]

Table 61

[0214] [Table 62]

[0215] [Table 63]

[0216] [Table 64]

[0217] [Table 65]

[0218] [Table 66]

[0219] [Table 67]

[0220] ANDbody structure In general, an ANDbody can be any macromolecule, such as a polypeptide or protein, that contains both an effector target binding site or binding domain and an address target binding site or binding domain. The binding sites can be on the same polypeptide chain or on different polypeptide chains that are linked to each other, for example, via disulfide bonds.

[0221] In some embodiments, the binding site of the ANDbody for the effector target and the binding site for the address target each comprise an antibody heavy and / or light chain domain. In some embodiments, an ANDbody comprises a first antibody variable domain that has binding specificity for an effector target and a second antibody variable domain that has binding specificity for an address target. In other embodiments, an ANDbody comprises a first antigen binding site of an antibody, which first antigen binding site has binding specificity for an effector target, and a second antigen binding site of an antibody, which second antigen binding site has binding specificity for an address target.

[0222] 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, four, e.g., an antibody molecule comprises a plurality of variable domain sequences, where 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).

[0223] In some embodiments, an ANDbody is an antibody molecule having an arm or domain that binds an effector target and an arm or domain that binds an address target, in some embodiments, an ANDbody is an antibody molecule that includes a light chain that binds either the effector target or the address target and a heavy chain that binds the other of the effector target and the address target.

[0224] 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, adducted 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 type inhibitor, or receptor decoy.

[0225] The affinity of the effector target binding site and the address target binding site of the ANDbody to their respective binding partners can be different. In some embodiments, the affinity of the first binding site to the therapeutic effector target to which the first binding site binds is weaker than the affinity of the second binding site to the address target. In some embodiments, the affinity of the first binding site to the therapeutic effector target to which the first binding site binds is 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold weaker than the affinity of the second binding site to the address target.

[0226] The terms "binding affinity" and "binding activity" refer to the tendency of a macromolecule, such as a polypeptide molecule, to bind or not bind to a target. For the purposes of the present invention, which combines two binding sites, the relative affinity of 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 affinity of two binding sites to be compared while eliminating any interference from other binding sites present on the macromolecule of the present invention.

[0227] 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.

[0228] 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 as 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.

[0229] 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, such as, for example, ELISA.

[0230] 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 targeted 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). In some embodiments, the Kd of a therapeutic effector targeted binder may be about 6-fold, about 5-fold, about 4-fold, about 3-fold, or about 2-fold higher than the Kd of an address targeted binder.

[0231] In some embodiments, the EC of a therapeutic effector target binder 50 can be about 1 pM, about 10 pM, about 100 pM, about 1 nM, about 10 nM, about 100 nM, about 500 nM, or greater than about 1 uM (e.g., can be 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.

[0232] In some embodiments, IC of 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., may 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 IC50 of the 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). In some embodiments, the IC50 of the therapeutic effector 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 100 pM, or less than about 10 pM. 50 is the IC of the address target binder 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.

[0233] The cell or tissue densities of the effector and address targets to which an ANDbody binds may be different. In some 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 of an ANDbody binds.

[0234] In some embodiments, the affinity of the first binding site for the therapeutic effector target to which it binds is about one-half (1 / 2) x Kd lower 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 one-half (1 / 2) x Kd lower than the density of the address target on a cell to which the second binding site binds.

[0235] In some embodiments, an ANDbody has both affinity and density parameters as previously described herein.

[0236] In some embodiments, the first and second binding sites of the 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, without any sequences from other sequences (such as linkers). In some embodiments, the first and second binding sites of the ANDbody are not directly linked to each other.

[0237] ANDbodies as disclosed herein can be linked to additional sites 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).

[0238] Exemplary effector target sequences and address target sequences for which the ANDbodies of the present technology may have affinity are listed in Table 3 and the Sequence Listing. In some instances, the sequences include full-length protein sequences with or without a signal peptide region and / or Fc fusion sequences. In some embodiments, the ANDbodies of the present technology include a binding domain that binds to a target protein or an effector target protein. In some embodiments, the binding domain of the ANDbodies of the present invention may bind to a protein sequence that includes a signal peptide. In other embodiments, the binding domain of the ANDbodies of the present invention may bind to a protein that does not include a signal protein. In some embodiments, the binding domain of the ANDbodies of the present invention may bind to a full-length protein. In other embodiments, the binding domain of the ANDbodies 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 the ANDbodies of the present invention may bind to a protein that is smaller than one that includes a full-length protein sequence, such as a peptide fragment of an address target or an effector target.

[0239] [Table 68]

[0240] [Table 69]

[0241] [Table 70]

[0242] [Table 71]

[0243] [Table 72]

[0244] [Table 73]

[0245] [Table 74]

[0246] [Table 75]

[0247] [Table 76]

[0248] [Table 77]

[0249] [Table 78]

[0250] [Table 79]

[0251] [Table 80]

[0252] Production of ANDbody Compositions Production of ANDbody Polypeptides The ANDbody polypeptides of the invention can be produced by any suitable means. For example, all or part of the ANDbody can be expressed by a host cell that contains nucleotides encoding the ANDbody. Such methods of making 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).

[0253] Methods for producing ANDbodies may include expression in mammalian cells, although recombinant proteins may also be produced using insect cells, yeast, bacteria, or other cells under the control of an appropriate promoter. Mammalian expression vectors may include non-transcribed elements such as an origin of replication, a suitable promoter and enhancer, and other 5' or 3' flanking non-transcribed sequences, as well as 5' or 3' non-translated sequences such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and termination sequences. DNA sequences from the SV40 viral genome, such as the SV40 origin of replication, early promoter, enhancer, splice, and polyadenylation sites, may be used to provide other genetic elements required for expression of heterologous DNA sequences. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).

[0254] A variety of 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).

[0255] Antibody production techniques are known. For example, methods for 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.

[0256] Production of ANDbody RNA In some embodiments, the ANDbody RNA can be produced, for example, for delivery to a subject. Generally, therapeutic mRNA is produced by in vitro transcription. Modifications such as incorporation of modified bases, 5'cap analogs, and polyA tails can optimize activity and function. For example, mRNA translation and stability can be achieved by cap and polyA tail modifications. For example, incorporation of cap analogs such as ARCA (anti-reverse cap analog) and a 100-200 bp poly(A) tail into in vitro transcribed (IVT) mRNA improves expression and stability (Kaczmarek et al. Genome Medicine (2017) 9:60). Novel 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 al. Mol Ther. 2015;23: 1456-64). The levels of protein products can be increased by modifying the 3' and 5' untranslated regions (UTRs), which contain sequences responsible for the recruitment of RNA-binding proteins (RBPs) and miRNAs (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. 2015;33:839-41). RNA base modifications (e.g., pseudouridine-incorporated mRNAs, such as 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. Pat. Nos. 9937233, 8710200, 10022425, 9878056, and 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.

[0257] Production of affinity-modified ANDbodies Affinity-modified ANDbodies can be made 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. Pat. No. 10,654,928. In general, ANDbodies can be modified to modify the affinity of the effector target binding site for the effector target, or to modify the affinity of the address target binding site for the address target. Modifications can increase or decrease the affinity of the binding site for the binding partner.

[0258] Target and Address Assessment The expression of therapeutic targets can be evaluated at either the RNA or protein level using methods known in the art. In some embodiments, the expression of therapeutic targets is evaluated by measuring RNA expression, for example, using an RNA sequence 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 / ).

[0259] 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 gland, 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 (crural skin), Cells 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-auricle, coronary artery-heart, left ventricle, esophagus, esophagus-mucosa, esophagus-muscularis, gastroesophageal junction, spleen, stomach, transverse colon, sigmoid colon, testis, whole blood cell, cell-(EBV-transformed lymphocyte, tibial artery, or tibial nerve tissue.

[0260] 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, such as phosphorylation-specific antibodies, or mass spectrometry.

[0261] Use of 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 use or veterinary use.

[0262] Veterinary applications include uses 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.

[0263] The present invention further relates to a subject or a 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.

[0264] In some embodiments, a subject or subject 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.

[0265] Pharmaceutical Compositions Polypeptide pharmaceutical compositions The ANDbody compositions (e.g., ANDbody polypeptide or RNA compositions) described herein can be administered to a subject in need thereof. The invention includes pharmaceutical compositions containing the ANDbody compositions in combination with one or more pharma- ceutically acceptable excipients.

[0266] 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.

[0267] RNA pharmaceutical composition Nucleic acids (e.g., RNA) encoding the ANDBody can alternatively or additionally be administered to a subject. Generally, therapeutic mRNAs are produced by in vitro transcription. Modifications such as incorporation of modified bases, 5'cap analogs, and polyA tails can optimize activity and function. For example, translation and stability of mRNAs can be achieved by cap and polyA tail modifications. For example, incorporation of cap analogs such as ARCA (anti-reverse cap analog) and 100-200 bp poly(A) tails into in vitro transcribed (IVT) mRNAs improves expression and stability (Kaczmarek et al., Genome Medicine (2017) 9:60). New types of cap analogs such as 1,2-dithiodiphosphate modified caps can further improve the efficiency of translation (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 al., Mol Ther. 2015;23: 1456-64). Modification of the 3' and 5' untranslated regions (UTRs), which contain sequences responsible for the recruitment of RNA-binding proteins (RBPs) and miRNAs, can increase the levels of protein products (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 their production 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. Pat. Nos. 9937233, 8710200, 10022425, 9878056, and 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.

[0268] In some embodiments, the RNA is a 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. The circular RNA encoding the ANDbody described herein can be delivered naked (i.e., not formulated with a carrier) or with a carrier.

[0269] Carrier Lipid Nanoparticles Formulations for in vivo delivery of the compositions described herein (e.g., polypeptide or RNA ANDbody compositions) with carriers include lipid nanoparticle (LNP) formulations.See, for example, 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 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.

[0270] Lipids that can be used in the nanoparticle formers (e.g., lipid nanoparticles) include, for example, those described in Table 4 of WO2019217941, which is incorporated herein by reference, e.g., the lipid-containing nanoparticles can include one or more of the lipids set forth in Table 4 of WO2019217941. The lipid nanoparticles may also include additional elements such as polymers, as described in Table 5 of WO2019217941, which is incorporated herein by reference.

[0271] In some embodiments, the conjugated lipids, if present, are 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., 4-0-(2',3'-diaminophenyl)-2,3-diaminophenyl)-1,2-dimethylphenyl-2,4-diaminophenyl-1,2-diaminophenyl-2,3-diaminophenyl-1,2-diaminophenyl-2,4 ... (tetradecanoyloxy)propyl-l-0-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, 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.

[0272] In some embodiments, sterols that can be incorporated into the lipid nanoparticles include one or more of 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.

[0273] In some embodiments, the lipid particles include ionized lipids, non-cationic lipids, complex lipids that inhibit particle aggregation, and sterols. The amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, the lipid nanoparticles include ionized lipids in an amount of 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; about 50 mol% to about 90 mol%), non-cationic lipids in an amount of about 5 mol% to about 30 mol% of the total lipids, complex lipids in an amount of about 0.5 mol% to about 20 mol% of the total lipids, and sterols in an amount of about 20 mol% to about 50 mol% of the total lipids. The ratio of total lipids to nucleic acids can be varied as desired. For example, the ratio of total lipids to nucleic acids (mass or weight) can range from about 10:1 to about 30:1.

[0274] In some embodiments, the ratio of lipid to nucleic acid (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 amount of lipid and nucleic acid can be adjusted to provide a desired N / P ratio, for example, an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or more. Generally, the total lipid content of the lipid nanoparticle formulation can range from about 5 mg / mL to about 30 mg / mL.

[0275] 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]

[0276] In some embodiments, LNPs comprising formula (i) are used to deliver the ANDbody RNA compositions described herein to the liver and / or liver cells. [ka]

[0277] In some embodiments, LNPs comprising formula (ii) are used to deliver the ANDbody RNA compositions described herein to the liver and / or liver cells. [ka]

[0278] In some embodiments, LNPs comprising formula (iii) are used to deliver the ANDbody RNA compositions described herein to the liver and / or liver cells. [ka]

[0279] In some embodiments, LNPs comprising formula (v) are used to deliver the ANDbody RNA compositions described herein to the liver and / or liver cells. [ka]

[0280] In some embodiments, LNPs comprising formula (vi) are used to deliver the ANDbody RNA compositions described herein to the liver and / or liver cells. [ka]

[0281] In some embodiments, LNPs comprising formula (viii) are used to deliver the ANDbody RNA compositions described herein to the liver and / or liver cells. [ka]

[0282] In some embodiments, LNPs comprising formula (ix) are used to deliver the ANDbody RNA compositions described herein to the liver and / or liver cells. [ka] During the ceremony, X1 is O, NR1 or a direct bond, X2 is a C2-5 alkylene, X3 is C(=O) or a direct bond, R1 is H or Me, R3 is a Ci-3 alkyl, R2 is a Ci-3 alkyl, or R2 together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X2 form a 4-, 5-, or 6-membered ring, or X1 is NR1, R1 and R2 together with the nitrogen atom to which they are attached form a 5-, or 6-membered ring, or R2 together with R3 and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y1 is a 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.

[0283] In some embodiments, LNPs comprising formula (xii) are used to deliver the ANDbody RNA compositions described herein to the liver and / or liver cells. [ka]

[0284] In some embodiments, LNPs comprising formula (xi) are used to deliver the ANDbody RNA compositions described herein to the liver and / or liver cells. [ka]

[0285] In some embodiments, the LNP comprises a compound of formula (xiii) and a compound of formula (xiv). [ka]

[0286] In some embodiments, LNPs comprising formula (xv) are used to deliver the ANDbody RNA compositions described herein to the liver and / or liver cells. [ka]

[0287] In some embodiments, LNPs comprising a formulation of formula (xvi) are used to deliver the ANDbody RNA compositions described herein to pulmonary endothelial cells. [ka]

[0288] In some embodiments, the lipid compounds used to form lipid nanoparticles for delivering the compounds described herein, e.g., the nucleic acids (e.g., RNA) described herein, are made by one of the following reactions: [ka]

[0289] In some embodiments, the compounds (e.g., nucleic acids or proteins) described herein are provided in LNPs that include 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, which is incorporated 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, which is incorporated 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, which is 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, which is 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 by reference in its entirety.

[0290] In some embodiments, the ionizable lipid may be a cationic lipid, an ionizable cationic lipid, for example, a cationic lipid that may exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that may be easily protonated. In some embodiments, the cationic lipid is a lipid that may be positively charged, for example, under physiological conditions. Exemplary cationic lipids include one or more amine groups that are positively charged. In some embodiments, the lipid particles include cationic lipids in the 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 of greater than 6.0. In some embodiments, the lipid nanoparticle may include a second cationic lipid that has a different effective pKa (e.g., greater than the first effective pKa) than the first cationic lipid. 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 nanoparticle, e.g., 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 the LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the nucleic acid may be encapsulated in the 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.

[0291] 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. WO2019051289, which is incorporated herein by reference. Additional exemplary lipids include, but are not limited to, one or more of the following formulas: X of US2016 / 0311759; I of US20150376115 or US2016 / 0376224; I, II or III of US20160151284; I, IA, II or IIA of US20170210967; Ic of US20150140070; US2013 / 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, U.S. Patent Application Publication No. 2017 / 0119904, U.S. Patent Application Publication No. 2017 / 117528, 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, U.S. Pat. Appl. Pub. No. 2013 / 0090372, U.S. Pat. Appl. Pub. No. 2013 / 0274523, U.S. Pat. Appl. Pub. No. 2013 / 0274504, U.S. Pat. Appl. Pub. No. 2013 / 0053572, WO 2013 / 016058, WO 2012 / 162210, U.S. Pat. Appl. Pub. No. I, II, III, or IV of U.S. Pat. Appl. Pub. 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, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of U.S. Patent Application Publication No. 2014 / 0308304, U.S. Patent Application Publication No. 2013 / 0338210, I, II, III, or IV of WO 2009 / 132131, A of U.S. Patent Application Publication No. 2012 / 01011478,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, I; U.S. Patent Application Publication No. 2011 / 0117125, I, II, or III; U.S. Patent Application Publication No. 2011 / 0256175, I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII; U.S. Patent Application Publication No. 2012 / 0202871, I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII; 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, No. I, II, or III; U.S. Patent Application Publication No. 2010 / 0062967, No. I or II; U.S. Patent Application Publication No. 2013 / 0189351, Nos. I-X; U.S. Patent Application Publication No. 2014 / 0039032, No. I; U.S. Patent Application Publication No. 2018 / 0028664, No. V; U.S. Patent Application Publication No. 2016 / 0317458, No. I; U.S. Patent Application Publication No. 2013 / 0195920, No. I; U.S. Patent Application Publication No. U.S. Pat. No. 1,127, No. 5, 6, or 10; No. III-3 of WO 2018 / 081480; No. I-5 or No. I-8 of WO 2020 / 081938; No. 18 or 25 of U.S. Patent Application Publication No. 9,867,888; No. A of U.S. Patent Application Publication No. 2019 / 0136231; No. II of WO 2020 / 219876; No. 1 of U.S. Patent Application Publication No. 2012 / 0027803; No. OF-02 of U.S. Patent Application Publication No. 2019 / 0240349; No. 23 of U.S. Patent Application Publication No. 10,086,013; 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. WO 2020 / 106946 I; International Publication No. WO 2020 / 106946 I;

[0292] In some embodiments, the ionizable lipid is MC3(6Z,9Z,28Z,3lZ)-heptatriaconta-6,9,28,3l-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 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), for example, 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, for example, as described in Example 12 of WO2019051289A9 (incorporated herein by reference in its entirety).

[0293] 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 (DM ... phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), di-glyceryl 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 mixtures 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-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).

[0294] 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.

[0295] In some embodiments, the non-cationic lipid is oleic acid or a compound of formula I, II, or IV of US Patent Application Publication No. 2018 / 0028664, the entirety of which is 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 some embodiments, the molar ratio of ionized 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).

[0296] In some embodiments, the lipid nanoparticles do not contain any phospholipids.

[0297] In some embodiments, the 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-choiestanol, 53-coprostanol, choiesteryl-(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. WO 2009 / 127060 and US Patent Publication No. 2010 / 0130588, each of which is incorporated herein by reference in its entirety.

[0298] In some embodiments, components that confer membrane integrity, 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 are 20-50% (mol), 30-40% (mol) of the total lipid content of the lipid nanoparticle.

[0299] In some embodiments, the lipid nanoparticles may contain polyethylene glycol (PEG) or conjugated lipid molecules. These are generally used to inhibit lipid nanoparticle aggregation 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 (methoxypolyethylene glycol)-conjugated lipid.

[0300] 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., 4-0-(2',3'-di(tetramethylphenyl)-2,3-diphenylamine ( ...-phospholipid, PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-phospholipid, PEG-phospholipid, PEG-phospholipid, PEG-phospholipid, PEG-phospholipid, PEG-phospholipid, PEG-phospholipid, PEG-phospholipid, PEG-phospholipid, PEG- Decanoyloxy)propyl-l-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-l,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,

[0301] US Patent Publication Nos. 2003 / 0077829, 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2010 / 0130588, 2016 / 0376224, 2017 / 0119904, and 2017 / 099823, the contents of all of which are incorporated herein by reference in their entirety. 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 US Patent Publication No. 2018 / 0028664, the contents of which are incorporated herein by reference in their entirety. 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-ditetradecylglycerol), and PEG-DMG (3,4-ditetradecylglycerol). Coxylbenzyl-[ω]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises a structure selected from the following: [ka]

[0302] 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-polymeric lipid (GPL) conjugates can be used in place of or in addition to PEG-lipids.

[0303] 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.

[0304] In some embodiments, the PEG or complex lipid may comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the content of the PEG or complex lipid is 0.5-10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle. The molar ratio of the ionized lipid, the non-cationic lipid, the sterol, and the PEG / complex lipid may be varied as required. For example, the lipid particle may comprise 30-70% ionized lipid by mole or total weight of the composition, 0-60% cholesterol by mole or total weight of the composition, 0-30% non-cationic lipid by mole or total weight of the composition, and 1-10% complex lipid by mole or total weight of the composition. Preferably, the composition comprises 30-40% ionized lipid by mole or total weight of the composition, 40-50% cholesterol by mole or total weight of the composition, and 10-20% non-cationic lipid by mole or total weight of the composition. In some other embodiments, the composition comprises 50-75% ionizable lipid by molar or total weight of the composition, 20-40% cholesterol by molar or total weight of the composition, and 5-10% non-cationic lipid by molar or total weight of the composition, and 1-10% complex lipid by molar or total weight of the composition. The composition may comprise 60-70% ionizable lipid by molar or total weight of the composition, 25-35% cholesterol by molar or total weight of the composition, and 5-10% non-cationic lipid by molar or total weight of the composition. The composition may also comprise up to 90% ionizable lipid by molar or total weight of the composition and 2-15% non-cationic lipid by molar or total weight of the composition.The formulation may also be a lipid nanoparticle formulation, e.g., 8-30% ionized 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% ionized 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. or 2-30% ionized lipid by molar or total weight of the composition, 2-30% non-cationic 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; or up to 90% ionized lipid by molar or total weight of the composition, and 2-10% non-cationic 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 ionized 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 ionized lipid, cholesterol, and PEGylated lipid in a molar ratio of 60:38.5:1.5.

[0305] 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, where the molar ratio of lipids ranges from 20-70 mole percent for the ionizable lipid with a target of 40-60, the molar percent of the non-cationic lipid ranges from 0-30 with a target of 0-15, the molar percent of the sterol ranges from 20-70 with a target of 30-50, and the molar percent of the PEGylated lipid ranges from 1-6 with a target of 2-5.

[0306] 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.

[0307] In one aspect, the disclosure provides a lipid nanoparticle formulation comprising a phospholipid, a lecithin, a phosphatidylcholine, and a phosphatidylethanolamine.

[0308] In some embodiments, one or more additional compounds may also be included. These compounds may be administered separately, or the additional compounds may be included 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. Without being limited thereto, the other additional compounds may be selected from the group consisting of organic or inorganic small or large molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and their derivatives, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, or any combination thereof.

[0309] In some embodiments, LNPs are directed to specific tissues by the addition of LNP targeting domains. For example, biological ligands can be displayed on the surface of LNPs to enhance interaction with cells that display cognate receptors, thereby promoting cell association with tissues expressing the receptors as well as cargo delivery to the tissues. In some embodiments, the biological ligands can be ligands that promote delivery to the liver, for example, LNPs that display GalNAc achieve delivery of nucleic acid cargo to hepatocytes that display asialoglycoprotein receptors (ASGPR). Akinc et al. Mol Ther 18(7):1357-1364(2010) teaches that conjugation of trivalent GalNAc ligands with PEG-lipids (GalNAc-PEG-DSG) results in 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 WO2017223135, which is incorporated 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.

[0310] 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.

[0311] In some embodiments, the LNPs comprise a biodegradable, ionizable lipid, hi some embodiments, the LNPs comprise (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, in the context of LNP lipids, the terms cationic and ionizable are interchangeable, e.g., ionizable lipids are cationic depending on the pH.

[0312] In some embodiments, the average LNP diameter of an LNP formulation may be tens of nm to hundreds of nm, e.g., as measured by dynamic light scattering (DLS). In some embodiments, the average 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 average LNP diameter of the LNP formulation may be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation may be about 70 nm to about 100 nm. In certain embodiments, the average LNP diameter of the LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of the LNP formulation may be about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation ranges from about 1 mm to about 500 mm, about 5 mm to about 200 mm, about 10 mm to about 100 mm, about 20 mm to about 80 mm, about 25 mm to about 60 mm, about 30 mm to about 55 mm, about 35 mm to about 50 mm, or about 38 mm to about 42 mm.

[0313] The LNPs may be relatively uniform in some cases. The polydispersity index can be used to indicate the homogeneity of the LNPs, e.g., the size distribution of the lipid nanoparticles. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow size distribution. The 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 the LNPs may be about 0.10 to about 0.20.

[0314] 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 LNPs. In general, lipid nanoparticles with relatively low positive or negative charges are desirable, since 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.

[0315] The efficiency of protein and / or nucleic acid encapsulation represents the amount of protein and / or nucleic acid encapsulated or otherwise bound to the LNP after preparation relative to the initial amount provided. It is desirable for the encapsulation efficiency to be high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing lipid nanoparticles before and after the lipid nanoparticles are degraded 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 the protein and / or nucleic acid 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%.

[0316] 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 comprising the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0317] Further exemplary lipids, formulations, methods, and characterization of LNPs are taught by International Publication No. WO2020061457, which is incorporated by reference in its entirety.

[0318] 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 are described in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), which is incorporated herein by reference in its entirety.

[0319] Optimized LNP formulations for delivery of CRISPR-Cas systems, e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA, are described in WO2019067992 and WO2019067910, both of which are incorporated by reference.

[0320] Other specific LNP formulations useful for delivery of nucleic acids are 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 include the formulation used in patisiran, sold under the name ONPATTRO.

[0321] 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.

[0322] 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 several 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 dialkyloxypropylcarbamate. In some embodiments, the cationic lipid of the LNP has the following structure: [ka]

[0323] For a general review of LNPs, see, e.g., Li et al. 2017, Nanomaterials 7, 122; doi:10.3390 / nano7060122.

[0324] 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 expressing an 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.

[0325] Examples of viral vectors include retroviruses (e.g., Retroviridae family viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses; negative-stranded RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., paramyxoviruses (e.g., measles and Sendai); positive-stranded 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 viruses include double-stranded DNA viruses such as Variola ankara (MVA), avian pox and canary pox. Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, human papilloma viruses, 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, spumaviruses (Coffin, JM, 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.

[0326] 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. WO 2020123773, WO 2020123816, WO 2018232017, and WO 2020123773. In certain embodiments, the anellovector composition comprises 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.

[0327] Cell- and Vesicle-Based Carriers The compositions described herein (e.g., polypeptide or RNA ANDbody compositions) can be administered to cells, vesicles, or cells in other membrane-based carriers. In one embodiment, the compositions and systems described herein can be formulated into liposomes or other similar vesicles. Liposomes are spherical vesicular structures composed of a single or multi-layer 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, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load through biological membranes and the blood-brain barrier (BBB) ​​(see, e.g., for a review, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155 / 2011 / 469679). Vesicles can be made from several kinds of lipids; however, phospholipids are most commonly used to produce liposomes as drug carriers. Methods for preparing multilamellar vesicle lipids are known in the art (see U.S. Pat. No. 6,693,086; the teachings of this patent on preparing multilamellar vesicle lipids are 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 homogenizer, sonicator, or extrusion device (for example, for a 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.

[0328] 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.

[0329] 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.

[0330] For example, fusosome compositions such as those described in WO2018208728 may also be used as carriers for delivery of the Agents or preparations described herein.

[0331] Plant nanovesicles and plant messenger pak (PMP), 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.

[0332] Without further elaboration, it is believed that a person skilled in the art can utilize the present invention to its fullest extent based on the above description. Therefore, the following specific embodiments are to be construed as merely illustrative, and not limiting of the remainder of the disclosure in any way. All publications and sections thereof cited herein are incorporated by reference for the purposes or subject matter discussed herein. EXAMPLES

[0333] Working Example The present invention is further illustrated in the following non-limiting examples.

[0334] [Table 81]

[0335] Example 1. Antibody binding mouse and human RAGE and NOTCH2 1.1 Generation of anti-RAGE antibodies by vaccination Antibodies against the human RAGE extracellular domain, an exemplary address target of the technology, are generated by immunization. The extracellular domain of human RAGE (NCBI protein accession Q15109 position N24-A344) (huRAGE) fused to the Fc region of human IgG1 (UniProt ID P01857 position P100-K330) is expressed in HEK293F cells. Briefly, the DNA sequence is codon-optimized for mammalian expression and appropriately sequenced within the pcDNA3.4-TOPO expression vector (ThermoFisher Scientific). The protein is transiently transfected into HEK293 cells and purified using rProtein A Sepharose Fast Flow resin according to the manufacturer's instructions (GE Healthcare) as in conventional methods (Rothschilds et al. 2019). Female BALB / c mice are immunized with 50ug of huRAGE-Fc fusion protein by ip injection with CFA / IFA (Millipore Sigma, Cat. No. F5881-10ML and F5506-10ML) adjuvant. Hybridomas are then generated (Listek et al. 2020). Clones are first screened in an ELISA format for IgG reactivity specific to the huRAGE-Fc fusion protein used for immunization, followed by flow cytometry testing with cells stably (CHO) or transiently (HEK293F) transfected with full-length huRAGE. Anti-RAGE hybridoma clones are then evaluated based on mouse cross-reactivity. Flow cytometry testing is performed with cells stably (CHO) or transiently (HEK293F) transfected with full-length mouse RAGE (mRAGE) to select clones that bind to mRAGE. Positive clones expressing human-mouse cross-reactive anti-RAGE mAbs 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 using (Millipore Sigma, P3296-1ML) according to the manufacturer's instructions.

[0336] 1.2 Selection of inactive anti-RAGE 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 the exemplary RAGE address target, and therefore 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 Systems catalog #1016-S13-050), S100A14 (R&D Systems catalog #1015-S14-050), S100A15 (R&D Systems catalog #1016-S13-050), S100A16 (R&D Systems catalog #1016-S13-050), S100A17 (R&D Systems catalog #1016-S13-050), S100A18 (R&D Systems catalog #1016-S13-050), S100A19 (R&D Systems catalog #1016-S13-050), S100A20 (R&D Systems catalog #1016-S13-050), S100A21 (R&D Systems catalog #1016-S13-050), S100A22 (R&D Systems catalog #1016-S13-050), S100A23 (R&D Systems catalog #1016-S13-050), S100A2 R&D Systems catalog #4327-SA-050), S100B (R&D Systems catalog #1820-SB-050), amyloid beta peptide (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA in NM_000484.2, Millipore Sigma catalog # AG912-1MG) and Mac-1 (F17-N1105 in NP_001139280 and Q23-N700 in UniProt P05107, R&D Systems catalog #4047-AM-050). ELISA is used to quantify the ability of the ligands to bind in the presence of anti-RAGE antibodies. huRAGE-Fc is adsorbed to ELISA plates and then, after blocking, the plates are incubated with anti-RAGE antibody clones from hybridomas at concentrations ranging from 10 fM to 10 uM (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 the 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.

[0337] The inability of anti-RAGE antibodies to inhibit the IFNα-inducible gene signature is also evaluated, and anti-RAGE hybridoma clones that do not alter cell signaling based on the IFNα-inducible gene signature assay are selected. PBMCs from healthy human donors are stimulated with 50% serum from SLE patients for 4 hours. The assay is completed at antibody concentrations ranging from 10 fM to 10 uM in the presence of either anti-RAGE antibodies or a non-relevant (negative) isotype control human IgG1 antibody (Bio X Cell catalog #BE0297). Additionally, huRAGE-Fc fusion molecule is used as a positive control. Total RNA is purified and the expression of type I IFN-inducible genes, including DDX58, G1P2, MXl, OAS3, RSAD2, IFITl, and IFI35, is 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). The inhibition values ​​of gene expression were normalized to the negative control Ab.

[0338] 1.3 Generation of anti-Notch2 antibodies by vaccination Antibodies against the extracellular domain of human Notch2 (huNotch2) fused to the Fc region of human IgG1, an exemplary effector target of the present technology, are generated by immunization similar to RAGE described above. After immunization and hybridoma generation, clones are screened exactly as 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.

[0339] 1.4 Wide IC 50 Selection of anti-Notch2 antibodies with a range of activities The effector target binding site of the present 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 Notch2 antibody against the ligand human Jagged-2-Fc fusion protein (Creative BioMart, JAG2-382H) that binds to surface Notch2 is analyzed. 50 The IC was assessed using flow cytometry. 50 Select antibodies with a concentration ranging from <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).

[0340] 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. Afterwards (without washing the cells), the cells are incubated with constant concentrations of AF647-labeled Jagged-2-Fc ranging from 1 pM to 50 uM for 1 h at 4°C. Each IC 50 One fixed concentration of AF647 Jagged-2-Fc is selected along with various anti-Notch2 antibodies in the assay. Binding of AF647 Jagged-2-Fc with increasing concentrations of anti-Notch2 antibodies is quantified on cells by flow cytometry using a ThermoFisher Attune NxT (B2R3Y3V6).

[0341] 1.5 Expression and purification of ANDbody bispecifics I C 50 DNA sequences from 10 RAGE and 10 Notch2 antibodies ranging in potency (from <1 nM to 5 uM) are cloned into a human IgG1 framework with a single matching point mutation in the CH3 domain Fc region using In-Fusion HD Cloning (Takara Bio, Cat #638911) according to the "Controlled Fab-Arm Exchange" (cFAE) method (Labrijn et al. 2014). After expressing the antibodies individually from a transient HEK293 expression and purifying each antibody using Protein A affinity resin, the parent antibodies (combination of one RAGE and one Notch2 antibody) are made into a bispecific RAGE / Notch2 ANDbody according to the cFAE method. Briefly, the parent antibodies are mixed under permissive redox conditions to allow recombination of the half molecules. The reducing agent is then removed to allow reoxidation of the interchain disulfide bonds. Finally, the exchange efficiency is quantified using chromatography or mass spectrometry based methods. Approximately 100 RAGExNotch2 mutant ANDbodies will be generated.

[0342] 1.6 Affinity of ANDbody mutants 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) with 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 10 min for all proteins. Kinetic analysis is performed by simultaneously fitting the association and dissociation phases of the sensorgrams with a 1:1 Langmuir binding model in the BIAevaluation software (Biacore) supplied by the manufacturer. A double reference is applied in each analysis to remove background responses from the reference surface and buffer-only controls.

[0343] 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 with higher affinity for Notch2, are used for subsequent in vitro and in vivo experiments.

[0344] 1.7 In vitro assay of Notch2 antagonism in cells with or without RAGE expression To characterize ANDbodies in vitro, HEK293F cells are transiently transfected with full-length huRAGE (R+) or full-length huNotch2 (N+) or co-transfected with both (RN+). Many mutant RAGExNotch2 ANDbodies are fluorescently labeled with alexa fluor 647 (AF647) according to the manufacturer's instructions (ThermoFisher, A20186) and as previously described (Tzeng et al. 2015). ANDbodies are then incubated with R+, N+, RN+, or combined R+ and N+ cells at ANDbody concentrations ranging from 10 fM to 10 uM. Parental anti-Notch2 monospecific antibodies (one from each mutant) are 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 (corresponding Notch2 variant) at concentrations between 10 fM and 10 uM to saturate the remaining binding sites of Notch2. The binding EC of both the AF647-labeled RAGExNotch2 ANDbody variant and the parental FITC-labeled anti-Notch2 antibody was 50 was quantified using flow cytometry. When FITC Notch2 antibody was added after AF647 RAGExNotch2, the FITC signal from those cells was subtracted from the FITC signal from FITC Notch2 alone on the cells (and 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.

[0345] ECs observed in N+ and RN+ cells 50 The differences reveal that the presence of RAGE enhances Notch2 blockade and identify cells expressing the RAGExNotch2 ANDbody.

[0346] 1.8 Biodistribution of ANDbodies and Parent 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 each parental antibody (anti-Notch2 or anti-RAGE used in the cFAE of the ANDbody).

[0347] To quantify cellular biodistribution, proteins (ANDbody and antibody) are first individually labeled with AF647 according to the manufacturer's instructions (ThermoFisher, A20186) and previously described methods (Tzeng et al.). Each labeled antibody is then individually injected (tail vein) at doses of 10ug, 100ug, and 500ug. The same amount of saline (PBS) is also injected as a control.

[0348] For cell biodistribution, mice are euthanized using CO2 at 12 hours, 1 day, 2 days, 3 days, 7 days, and 14 days after injection, and tissues such as heart, lung, spleen, blood, kidney, liver, and intestine are processed into single cell suspensions as previously described (Tzeng et al. 2015). Briefly, blood is collected by cardiac puncture into EDTA-treated tubes (BD catalog #365974), and other tissues are collected, weighed, mechanically dissociated between frosted glass slides, and filtered through a 70 μm mesh screen (Millipore Sigma, catalog #CLS431751-50EA) into single cell suspensions. Spleen cells, whole blood, and lungs are processed with 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 for 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- SSClo), 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, are also analyzed as previously defined (Singer et al.). Antibodies are purchased from Biolegend and flow cytometry is performed on a ThermoFisher Attune NxT (B2R3Y3V6). The presence of labeled ANDbody or other labeled antibodies on the cell surface is determined by the fluorescence of AF647 (this fluorophore was avoided in the flow panel).

[0349] 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.

[0350] For tissue biodistribution, mice are euthanized with 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 are harvested, weighed, and imaged with an IVIS Spectrum imaging system (Caliper Life Sciences; excitation wavelength: 500 nm, emission wavelength: 540 nm). Images are analyzed using Living Image software.

[0351] 1.9 In vivo bioactivity to quantify gene expression changes To analyze ANDbody activity, female Balb / c and C57BL / 6 mice are used to quantify bioactivity in vivo. To quantify bioactivity across tissues, RAGExNotch2 ANDbodies or their respective parental antibodies (anti-Notch2 or anti-RAGE used in the cFAE of the ANDbodies) are injected intravenously (tail vein) at doses of 10ug, 100ug, and 500ug. As a control, the same amount of saline (PBS) is also injected.

[0352] At 12 hours, 1 day, 2 days, 3 days, 7 days, and 14 days after injection, tissues including lungs, spleen, blood, kidneys, liver, heart, and intestines are processed into single cell suspensions as previously described (Tzeng et al. 2015). Blood is collected by cardiac puncture into EDTA-treated tubes (BD catalog #365974), and other tissues are collected, weighed, mechanically dissociated between frosted glass slides, and filtered through a 70 μm mesh screen (Millipore Sigma, catalog #CLS431751-50EA) into single cell suspensions. Spleen cells, whole blood, and lungs are processed in ammonium chloride-potassium (ACK) lysis buffer (Thermofisher Scientific, catalog #A1049201).

[0353] qRT-PCR is performed using a method previously described (Nandagopal et al. 2018). RNA is prepared using the RNeasy kit (QIAGEN). cDNA is prepared from 500 ng of RNA using the iScript cDNA synthesis kit (Bio-Rad). 0.5 μL of cDNA is used per 10 μL of RT-qPCR reaction mix containing 1X iqSYBR Green Supermix (Bio-Rad) and 450 nM total forward and reverse primers. Reactions are 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 min, followed by 40 cycles of 95°C for 10 s (melting) and 55°C for 30 s (annealing + extension). All reactions are performed in duplicate.

[0354] The genes involved in Notch2 signaling are mouse Hes1, Hey1, and HeyL, and the reference gene is SdhA. The primers used for amplification were mouse Hes1 primer set (forward, 5'-CAACACGACCGGACAAAC-3' and reverse, 5'-AAGAATAAATGAAAGTCTAAGCCAA-3'), mouse Hey1 primer set (forward, 5'-GCCGAAGTTG CCCGTTATCT-3' and reverse, 5'-CGCTGGATG CGTAGTTGTT-3'), mouse HeyL primer set (forward, 5'-GAGCTGAC TTCCCACAACCA-3' and reverse, 5'-GAGAGG TGCCTTTGCGTAGA-3'), and mouse SdhA primer set (forward, 5'-AGTGGGCT GTCTTCCTTAAC-3' and reverse, 5'-GGATTGCTTCT GTTTGCTTGG-3'), which were previously described (Nandagopal et al. 2018). All primers were purchased from IDT DNA.

[0355] Hes1, Hey1, and HeyL gene expression is measured in ANDbody-treated, untreated, and anti-Notch2-treated mice, including in the lungs.

[0356] 1.10 In vivo bioactivity using body weight and histology Female Balb / c and C57BL / 6 mice are used and histology is performed on organs including spleen, kidney, liver, heart, intestine, teeth, and lungs to compare pathology following ANDbody treatment, anti-Notch2 antibody treatment alone, or saline (PBS) treatment. Starting at 8 weeks of age, mice are injected IV (tail vein) once or twice weekly with 10ug, 100ug, and 500ug of ANDbody or corresponding Notch2 antibody (pre-cFAE). As a control, the same amount of saline (PBS) is also injected once or twice weekly. Mice are weighed twice weekly starting before the first treatment. Mice are euthanized 2, 4, and 6 weeks after treatment and organs are processed for histology.

[0357] Unless otherwise stated, organs are removed into cassettes and directly introduced into 10% neutral buffered formalin (Sigma-Aldrich) for 12–24 h before embedding in paraffin. Lungs are perfused with 10% neutral buffered formalin before being placed into cassettes for immersion in neutral buffered formalin. Intestines are thoroughly washed before being placed into 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.

[0358] In addition to lung morphology, the weight loss (or lack thereof) over the treatment period in mice treated with ANDbody is compared to the weight loss (or lack thereof) in mice treated with anti-Notch2, and to the weight loss (or lack thereof) in untreated mice.

[0359] 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) is used to generate antibodies against mouse UMOD (Creative BioMart, Cat#UMOD-17835M, untagged), an exemplary address target of the technology. This is performed using methods previously described (Angelini et al. 2015) and summarized below. Yeast display starts with a synthetic antibody library from the Sidhu lab, which is based on a natural framework, library 'G' (Van Deventer et al. 2015). scFvs displayed on the yeast surface are selected for binding to mouse UMOD. Selection can then be performed against human UMOD antigen (Creative BioMart, Cat#UMOD-001H, untagged) to ensure that binders are cross-reactive between the human and mouse forms. To increase the affinity of the scFv binders, affinity maturation is performed using error-prone PCR as previously described (Angelini et al. 2015), and the resulting library is reselected for binding to both mouse and human UMOD. After engineering, many of the poly-cross-reactive scFvs are recloned into a human IgG1 antibody format.

[0360] 2.2 Selection of inactive anti-UMOD antibodies The address target binding site of the present technology is designed so that it does not affect signal transduction when it binds to an address target, such as an exemplary UMOD address target. Therefore, the anti-UMOD antibody is further evaluated based on its inability to block UMOD ligand binding in an assay such as that described above for the RAGE antibody or in an in vivo assay. Inactive UMOD antibodies can be identified in an assay in which the kidney structure is not affected or modified by the screened UMOD antibody.

[0361] 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 previously described.

[0362] 2.4 Wide range of ICs 50 Selection of active Notch2 antibodies Antibodies against Notch2 were used with broad IC 50 Select by range.

[0363] 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 potency (<1 nM to 5 uM) are made into 100 mutant ANDbodies as described above.

[0364] 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 (see 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.

[0365] 2.7 Biodistribution of ANDbodies and Parent Antibodies (In Vivo) Cell and tissue biodistribution studies are performed using the methods described above (as above), except that the ANDbody used in this case is UMODxNotch2, and the parent antibodies correspond to anti-UMOD and anti-Notch2.

[0366] 2.8 In vivo bioactivity to quantify changes in gene expression The in vivo biological activity of the UMODxNotch2 ANDbody is quantified using the gene expression methods described above.

[0367] 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 previously described.

[0368] Example 3. Antibodies that bind to mouse and human MEP1B and Notch2 3.1 Generation of anti-MEP1B antibodies by yeast surface display Yeast surface display (Chao et al. 2006) is used to generate antibodies against mouse MEP1B (Cusabio, CSB-MP730755MO), an exemplary address target of the technology. Yeast display is performed as in (0) above to obtain cross-reactive mouse / human MEP1B binders (human MEP1B, Cusabio, CSB-MP618098HU). After generation, a number of scFvs cross-reactive with mouse and human MEP1B are cloned into human IgG1, transiently transfected into HEK293F cells, and purified using Protein A resin as described above.

[0369] 3.2 Vaccination to generate anti-Notch2 antibodies Antibodies cross-reactive against mouse and human Notch2 are generated, cloned and expressed into a human IgG1 framework according to conventional methods previously described.

[0370] 3.3 Wide range of ICs 50 Selection of active Notch2 antibodies Antibodies against Notch2 were used in broad IC 50 Select by range.

[0371] 3.4 Expression and purification of ANDbodies as bispecific antibodies Wide range of ICs 50 The DNA sequences of 10 MEP1B and 10 Notch2 antibodies with binding sites (<1 nM to 5 uM) are made into 100 mutant ANDbodies as described above.

[0372] 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, where His-tagged human and mouse versions of MEP1B are immobilized on a Sensor Chip NTA (Cytiva catalog #BR100034). Human and mouse notch2-Fc are immobilized as described above.

[0373] 3.6 Biodistribution of ANDbodies and Parent Antibodies (In Vivo) 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.

[0374] 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 methods described above.

[0375] 3.8 In vivo bioactivity using gravimetric and histological techniques The in vivo bioactivity of the MEP1BxNotch2 ANDbody will be quantified using the weight and histology methods previously described.

[0376] 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.

[0377] 4.2 Selection of inactive anti-RAGE antibodies Methods for selecting inactive anti-RAGE antibodies are described above.

[0378] 4.3 Production of anti-IL11Ra antibodies by yeast surface display Yeast surface display is used similarly as above to generate antibodies of various affinities that cross-react with mouse and human IL11Ra, the exemplary effector targets of the present technology. 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) are codon-optimized for mammalian expression and appropriately sequenced with a C-terminal His tag in the pcDNA3.4-TOPO expression vector (ThermoFisher Scientific). Proteins are transiently transfected into HEK293F cells and then purified using TALON® Metal Affinity Resin according to the manufacturer's instructions (Clontech) as in conventional methods (Rothschilds et al. 2019). These soluble recombinant mouse and human IL11Ra are used as antigens for yeast surface display.

[0379] ScFvs cross-reactive against mouse and human IL11Ra are cloned into human IgG1, transiently transfected into HEK293F cells and purified using Protein A resin as described above.

[0380] 4.4 Wide range of ICs 50 Selection of active anti-IL11Ra antibodies As previously described, IC of IL11Ra antibody against ligand human IL11 (R&D Systems, catalog #218-IL-025 / CF) that binds to surface IL11Ra 50 were assessed using flow cytometry and had IC values ​​ranging from <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.

[0381] 4.5 Expression and Purification of ANDbodies as Bispecific Antibodies Wide range of ICs50 DNA sequences from 10 RAGE antibodies and 10 IL11Ra antibodies having potency (from less than 1 nM to 5 uM) are made into approximately 100 mutant ANDbodies as described above.

[0382] 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).

[0383] 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 substituted for Notch2 and anti-IL11Ra antibody is substituted for anti-Notch antibody. The corresponding RAGExIL11Ra ANDbody is also used.

[0384] 4.8 Biodistribution of ANDbodies and Parent Antibodies (In Vivo) Cell and tissue biodistribution studies are carried out as described above, except that the ANDbody used in this case is RAGExIL11Ra, and the parent antibodies correspond to anti-RAGE and anti-IL11Ra.

[0385] 4.9 In vivo biological activity of RAGExIL11Ra ANDbody In response to treatment of mice with murine IL11, an increase in collagen content is seen in both the ventricles and kidneys (Schafer et al. 2017). Therefore, collagen content will be measured to quantify the amount of IL11Ra bioactivity following ANDbody treatment.

[0386] As previously described (Schafer et al. 2017), 10-week-old male C57BL / 6 mice are injected subcutaneously with 2ug of murine IL11 or an equal volume of saline daily for 21 days. Murine IL11 is produced recombinantly by synthesizing codon-optimized DNA with the sequence of C-terminal His-tagged murine IL11 (UniProt ID P47873) followed by HEK293F transient transfection and His-tagged IL11 purification using TALON resin, as described above. Starting 3 days before the first IL11 injection and twice weekly thereafter, IL11 and saline treated mice receive treatment injections IP consisting of 250ug of ANDbody RAGExIL11Ra, parental anti-IL11Ra alone, or an equal volume of saline (PBS).

[0387] At the end of 21 days of IL11 treatment, mice are euthanized and the amount of total collagen in the lungs, spleen, blood, kidneys, liver, heart, and intestine is quantified as previously described (Schafer et al. 2017) based on colorimetric detection of hydroxyproline using the Quickzyme Total Collagen Assay Kit (Quickzyme Biosciences).

[0388] 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 (eg, address target) antibodies are selected as above and cloned into human IgG1.

[0389] 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.

[0390] 5.3 Generation of anti-IL11Ra antibodies by yeast surface display The same IL11Ra antibody generated above in yeast surface display is used as described above. ScFvs cross-reactive against mouse and human IL11Ra are cloned into human IgG1 and transiently transfected into HEK293F cells and then purified using Protein A resin as described above.

[0391] 5.4 Wide range of ICs 50 Selection of active anti-IL11Ra antibodies Using the above method, various IC 50 Select an IL11Ra antibody.

[0392] 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.

[0393] 5.6 Affinity of ANDbody for UMOD and IL11Ra The affinity of the 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). It is expected that some ANDbody variants will have higher affinity than IL11Ra, but all variants will be tested in additional assays.

[0394] 5.7 Biodistribution of ANDbodies and Parent 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.

[0395] 5.8 In vivo biological activity of UMODxIL11Ra ANDbody The in vivo biological activity of the UMODxIL11Ra ANDbody will be quantified using the same methods as described above.

[0396] 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 (eg, address target) antibodies are selected as above and cloned into human IgG1.

[0397] 6.2 Generation 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 against mouse and human IL11Ra are cloned into human IgG2, transiently transfected into HEK293F cells, and purified using Protein A resin as above.

[0398] 6.3 Wide range of ICs 50 Selection of active anti-IL11Ra antibodies Using the above method, various IC 50 Select an IL11Ra antibody.

[0399] 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) are generated as ANDbodies with human IgG2 Fc regions. To do this, scFv sequences from MEP1B variants are cloned into IgG2 IL11Ra antibody variants, respectively. MEP1B scFvs are separated by a flexible linker (3xGGGGS) from either the N-terminus or C-terminus of the light or heavy chain of the IL11Ra antibody (each ANDbody has two MEP1B scFvs). By cloning MEP1B scFvs (always separated by a linker) before the N-terminus and after the C-terminus of the heavy or light chain, respectively, variants with a total of four MEP1B scFvs per ANDbody are generated. Location of scFvs on IL11Ra antibody: 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; as well as other variants with scFvs in three or four different positions (resulting in a total of six or eight scFvs per ANDbody, respectively).

[0400] 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 human and mouse versions of His-tagged MEP1B and His-tagged IL11Ra are immobilized on Sensor Chip NTA (Cytiva catalog #BR100034).

[0401] 6.6 Biodistribution of ANDbodies and Parent Antibodies (In Vivo) The above method is used to perform cell and tissue biodistribution studies, except that the ANDbody used in this case is MEP1BxIL11Ra, and the parent antibodies correspond to anti-MEP1B and anti-IL11Ra.

[0402] 6.7 In vivo biological activity of MEP1BxIL11Ra ANDbody The in vivo biological activity of the MEP1BxIL11Ra ANDbody will be quantified using the same methods as described above.

[0403] Example 7. Exemplary Addressable Binders for Skin This example shows the restricted expression of anti-DSG1 antibodies (address binders) in the skin.

[0404] 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 4) of two anti-desmoglein-1 (anti-DSG1) antibodies named 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™.

[0405] To express and purify the antibodies, heavy and light chain DNAs were transfected in 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 conditioned medium by filtering the transfected cells 5 days after transfection. 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, bound antibodies were eluted with 0.1 M glycine pH 2.5 and neutralized with 1 / 10 volume of Tris pH 8.5. The neutralized eluate was buffer exchanged into PBS. The resulting mAbs were named 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).

[0406] The purified mAbs were analyzed for monodispersity by analytical size-exclusion chromatography (SEC) and for purity by SDS-PAGE.

[0407] [Table 82]

[0408] [Table 83]

[0409] 7.2 Anti-DSG1 antibodies PRO003 and PRO004 bind to mouse DSG1 expressed on cells Mouse DSG1 (NCBI accession number NP_034209.2) with 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 fixing and permeabilizing the cells, they were stained with an anti-c-Myc antibody (Life Technologies A-21281) and analyzed by flow cytometry to confirm the expression of DSG1. PRO003 and PRO004 specifically bound to cells transfected with mouse DSG1, and were confirmed to have the expected binding specificity and be suitable for studies in mice.

[0410] 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 the antibody 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).

[0411] Mice were administered the labeled antibodies at a dose of 3 mg / kg each via tail vein injection. Two groups of three mice were administered each antibody, 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 harvested, and the near-infrared fluorescence of each tissue was measured with an IVIS® imager (PERKINELMER®). For imaging of the skin, a patch of skin was shaved and cut to approximately 1 cm for imaging. 2were collected. Samples from each mouse were arranged in a standard format and 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 signal was observed in the liver of all treated mice, so the liver was excluded from the analysis. Without wishing to be bound by theory, it is believed that the liver may take up the fluorescent dye independent of antibody targeting. Similarly, background signal was observed in the stomach of all groups, including mice not treated with any antibody. Fluorescence was also observed from the food fed to the mice, so the stomach was excluded from the analysis.

[0412] Figures 5A and 5B show the tissue-wide fluorescent signals of PRO003 (Figure 5A) and PRO004 (Figure 5B). The distribution of both antibodies is strongly biased to the skin. These data indicate that the DSG1 antibody can be used as an address for selective skin targeting of ANDbodies.

[0413] Example 8. Exemplary Address-Restricted Binders to the Lung This example demonstrates the restricted expression of anti-RAGE antibodies (address binders) in the lung.

[0414] 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 5) of two anti-RAGE mAbs, named 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™.

[0415] For expression and purification, a 1:1 ratio of heavy and light chain DNA was transfected 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 conditioned medium by filtering the transfected cells 5 days after transfection. 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, bound antibodies were eluted with 0.1 M glycine pH 2.5 and neutralized with 1 / 10 volume of Tris pH 8.5. The neutralized eluate was buffer exchanged into PBS. The resulting mAbs were named PRO001 (h11E6.8) (heavy chain sequence: SEQ ID NO: 36; light chain sequence: SEQ ID NO: 37) and PRO002 (XT-M4) (heavy chain sequence: SEQ ID NO: 38; light chain sequence: SEQ ID NO: 39).

[0416] 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).

[0417] [Table 84]

[0418] To examine the binding of anti-RAGE antibodies by ELISA, recombinant His-tagged mouse RAGE protein (ab276858 from Abcam) was coated overnight at 1 μg / mL concentration onto NUNC-IMMUNO™ MAXISORP™ ELISA plates. The next day, the coated antigen was removed and wells were blocked with 1% IgG-free bovine serum albumin (BSA) and then incubated with eleven anti-RAGE antibodies (PRO001 and PRO002) in a four-fold serial dilution starting at 20 nM. Bound antibodies were detected with peroxidase-labeled anti-human IgG antibody along with tetramethylbenzidine (TMB) and acid stop reagent. Both PRO001 and PRO002 bound to mouse RAGE antigen with similar affinities by ELISA with apparent affinities around 90 pM, suggesting that both antibodies are strong binders.

[0419] 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 the two antibodies, mouse RAGE (NCBI accession NP_031451.2) with 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 bound specifically to mouse RAGE expressed on cells, confirming that they have the expected binding specificity and are suitable for studies in mice.

[0420] 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.

[0421] Each labeled antibody was administered to mice by 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, respectively. One group of three untreated mice is included as a negative control for autofluorescence. Compared to the other antibodies tested, the distribution of both antibodies is strongly biased towards the lung. These data show that the two antibodies that bind RAGE selectively accumulate in the lung, indicating that either can be used as an address for selective pulmonary targeting of ANDbody.

[0422] 8.4 Anti-RAGE antibodies specifically accumulate on alveolar epithelial cells Single cell expression analysis showed that RAGE was 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 tail vein injected with 3 mg / kg PRO002. Three untreated mice served as negative controls. Three days after administration, the mice were euthanized, lungs and other tissues were collected, and all tissues were fixed in formalin. Sections of each tissue were analyzed by immunohistochemistry (IHC) using anti-human secondary antibodies conjugated to horseradish peroxidase. Figure 7 shows representative staining of treated and untreated mice. Strong staining was observed in the alveolar tissue of mice treated with PRO002, but not in the adjacent airways or in the negative control condition. This result indicates that binders to address specific cell types can be used to direct the distribution of antibodies to those cells within a larger tissue.

[0423] Example 9. Exemplary Address-Restricted Binders to the Kidney This example demonstrates the restricted expression of anti-CDH16 antibodies (address binders) in the kidney.

[0424] 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 the constant kappa light chain and cloned into PCDNA3.4™.

[0425] For expression and purification, heavy and light chain DNAs were transfected into EXPI293F™ cells (ThermoFisher Scientific) in a 1:1 ratio using the EXPIFECTAMINE™ Transfection Kit (ThermoFisher Scientific) according to the manufacturer's recommendations. Transiently expressed antibodies were purified from conditioned medium by filtering the transfected cells 5 days after transfection. 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, bound antibodies were eluted with 0.1 M glycine pH 2.5 and neutralized with 1 / 10 volume of Tris pH 8.5. The neutralized eluate was buffer exchanged into PBS. The resulting mAb was named PRO056 (heavy chain sequence: SEQ ID NO: 42; light chain sequence: SEQ ID NO: 43).

[0426] The purified mAb was analyzed by analytical size-exclusion chromatography (SEC) for monodispersity and by SDS-PAGE for purity. PRO056 was expressed as highly monodisperse and resolved by SDS-PAGE at the expected molecular weight.

[0427] [Table 85]

[0428] To examine the binding of anti-CDH16 antibodies by ELISA, recombinant His-tagged mouse CDH16 protein expressed and purified in-house was coated overnight on 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 BSA, followed by incubation with eleven anti-CDH16 antibodies (PRO056) in three-fold serial dilutions at a starting concentration of 533 nM. Bound antibodies were detected with peroxidase-labeled anti-human IgG antibodies along with TMB and acid stop reagent. PRO056 bound to mouse CDH16 antigen with an affinity of 200 pM by ELISA.

[0429] 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 were euthanized 3 and 7 days after administration. After euthanasia, organs were collected and the near-infrared fluorescence of each tissue was measured with 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 between each tissue was averaged. Figure 8 shows the fluorescence signal measured from whole tissues of a mouse treated with PRO056, each normalized so that the brightest signal is equal to 1. One group of three untreated mice was included as a negative control for autofluorescence.

[0430] The distribution is strongly biased towards the kidney, compared to the antibodies provided herein that target addresses in the skin, lung or kidney. These data show 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.

[0431] Example 10. Exemplary Address-Restricted Binders to the Intestine This example shows the restricted expression of anti-CDH17 antibodies (address binders) in the intestine.

[0432] 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 7) 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 the constant kappa light chain and cloned into PCDNA3.4™.

[0433] For expression and purification, heavy and light chain DNAs were transfected into EXPI293F™ cells (ThermoFisher Scientific) in a 1:1 ratio using the EXPIFECTAMINE™ Transfection Kit (ThermoFisher Scientific) according to the manufacturer's recommendations. Transiently expressed antibodies were purified from conditioned medium by filtering the transfected cells 5 days after transfection. 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, bound antibodies were eluted with 0.1 M glycine pH 2.5 and neutralized with 1 / 10 volume of Tris pH 8.5. The neutralized eluate was buffer exchanged into PBS. The resulting mAb was named PRO061 (heavy chain sequence: SEQ ID NO: 46; light chain sequence: SEQ ID NO: 47).

[0434] 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.

[0435] [Table 86]

[0436] To confirm binding activity and specificity, mouse CDH17 (NCBI accession NP_062727.1) with a c-Myc epitope tag at the protein C-terminus 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 anti-c-Myc antibody (Life Technologies A-21281), and analyzing by flow cytometry. PRO061 specifically bound to cells transfected with mouse CDH17 and had the expected binding specificity, confirming its suitability for mouse studies.

[0437] 10.2 Anti-CDH17 antibodies injected into mice selectively accumulate in the intestine PRO061 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 with 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 between each tissue was averaged. Figure 9 shows the fluorescence signal measured from whole tissues of mice treated with PRO061. Each is normalized so that the brightest signal is equal to 1. One group of untreated mice of three mice is included as a negative control for autofluorescence. The distribution was strongly biased towards the intestine, indicating that antibodies that bind CDH17 preferentially accumulate in the intestine and that ANDbodies can use these as address targeting domains to selectively target the intestine.

[0438] Example 11. Organization Restriction of Predicted Addresses Immunohistochemistry (IHC) of fresh frozen (FF) healthy mouse tissue microarray (TMA) sections mounted on glass slides was used to assay whether predicted organ-specific or selective expression addresses were in fact most abundant in a given organ and to determine which monoclonal antibody clones (mAbs) most selectively bound to the desired organ.

[0439] FF TMA-coated slides were prepared by first assembling fresh 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 into 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.

[0440] 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 antibodies were raised. The location and intensity of binding was determined by addition of the HRP substrate 3,3'diaminobenzidine (DAB), which produces a brown color at the binding site of the primary antibody in proportion to the amount of antibody deposited. Nuclei were counterstained with hematoxylin, yielding a blue color. For each address, a variety of different mAb clones were assayed for tissue specificity, and the pattern of tissue binding was assessed by performing IHC on the same FF TMAs described above.

[0441] Table 8 summarizes the observed binding of the antibodies tested. All antibodies tested reacted primarily with the expected target tissues, with more or less weak reactivity to other tissues. Without wishing to be bound by theory, it is possible that much of the extra-tissue reactivity represents non-specific 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.

[0442] [Table 87]

[0443] Example 12. Production and Use of ANDbodies In this example, we demonstrate the production of an exemplary ANDbody that blocks Notch2 and binds RAGE as an addresser.

[0444] 12.1 Expression and purification of anti-Notch2 monoclonal antibodies Sequences encoding the variable heavy chain regions (SEQ ID NOs: 48, 50, 52, and 54; shown in Table 9) 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, P329G (LALA-PG) and cloned into the PCDNA3.4™ vector (ThermoFisher Scientific). Sequences encoding the corresponding variable light chain regions (SEQ ID NOs: 49, 51, 53, 55) were fused to a constant kappa light chain and cloned into PCDNA3.4™.

[0445] For expression and purification, heavy and light chain DNAs were transfected in 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 conditioned medium by filtering the transfected cells 5 days after transfection. 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, bound antibodies were eluted with 0.1 M glycine pH 2.5 and neutralized with 1 / 10 volume of Tris pH 8.5. The neutralized eluate was buffer exchanged into PBS. The resulting mAbs were named PRO034 (heavy chain sequence: SEQ ID NO:56; light chain sequence: SEQ ID NO:57), PRO035 (heavy chain sequence: SEQ ID NO:58; light chain sequence: SEQ ID NO:59), PRO036 (heavy chain sequence: SEQ ID NO:60; light chain sequence: SEQ ID NO:61), and PRO037 (heavy chain sequence: SEQ ID NO:62; light chain sequence: SEQ ID NO:63).

[0446] [Table 88]

[0447] [Table 89]

[0448] 12.2 Binding and affinity studies of anti-Notch2 antibodies To examine the binding of anti-Notch2 antibodies by ELISA, recombinant His-tagged human and mouse Notch2 NRR domains expressed and purified in-house were coated overnight on 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 BSA and then incubated with eleven anti-Notch2 antibodies (PRO034, PRO035, PRO036, PRO037) in three-fold serial dilutions at starting concentrations of 200 nM for PRO035 and PRO037, and 666 nM for PRO034 and PRO036. Bound antibodies were detected with peroxidase-labeled anti-human IgG antibody along with TMB and acid stop reagent. Each of the antibodies bound to the human and mouse NRR domains of Notch2 with affinities ranging from 47 pM to 140 nM.

[0449] To investigate the binding affinity of anti-Notch2 antibodies by biolayer interferometry (BLI), each of PRO034, PRO035, PRO036 and PRO037 was immobilized on an anti-human IgG Fc biosensor and immersed in recombinant His-tagged mouse and human Notch2 NRR proteins at various concentrations from 1000 nM to 31 nM, and the association rate with the antigen was measured. The dissociation rate was then measured by immersing the biosensor in buffer. The 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 avidity improves affinity in the ELISA format.

[0450] 12.3 Design and Production of Notch2 / RAGE ANDbody An ANDbody comprising the first fragment antigen-binding (Fab) arm of the above anti-Notch2 antibodies (PRO034, PRO035, and PRO036) and the second Fab arm of the anti-RAGE antibody PRO002 (Example 8) 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. Then, 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 named PRO051, PRO052, and PRO053 (including PRO034, PRO035, and PRO036, respectively).

[0451] By SDS-PAGE and analytical size-exclusion chromatography, the major product formed after the cFAE reaction had a typical molecular weight for IgG1 (150 kDa), suggesting complete reoxidation. Analytical hydrophobic interaction chromatography indicated the formation of a new product, the desired heterodimeric antibody.

[0452] 12.4 Notch2 / RAGE ANDbody is shown to bind to Notch2 and RAGE simultaneously by BLI To study simultaneous dual antigen binding of Notch2 / RAGE ANDbody by BLI, PRO051, PRO052 and PRO053, together 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, followed by a second association step in a well containing recombinant mouse Notch2 NRR at 150 nM to measure dual antigen binding. Dissociation rates were then measured by immersing the biosensor in buffer.

[0453] The sensorgrams revealed that ANDbodies: PRO051, PRO052 and PRO053 were able to bind both RAGE and Notch2 antigens simultaneously, whereas the monovalent parental antibodies only bound to either RAGE or Notch2 NRR, supporting the conclusion that the ANDbodies were of correct composition and functional in binding both antigens simultaneously.

[0454] 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 Notch2 inhibitory antibodies described above. TMAs were constructed and stained as described in Example 11. Figure 10 shows mouse TMA staining with three anti-Notch2 antibodies: PRO034, PRO035, and PRO036, as well as staining with Notch2 / RAGE ANDbodies: PRO051, PRO052, and PRO053. In both cases, the ANDbodies targeting RAGE show a marked enhancement of binding to lung tissue. These data indicate 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.

[0455] 12.6 Notch2 / RAGE ANDbodies are preferentially distributed to the lung compared to corresponding non-targeted anti-Notch2 antibodies To evaluate how the ANDbodies targeting Notch2 and RAGE behave in vivo, mice were treated with PRO051, PRO052, and PRO053 antibodies at 3 mg / kg IV. All groups contained 3 mice. Tissues were harvested from each mouse 3, 7, 14, and 21 days after treatment. Accumulation of each antibody in the lung was measured by homogenizing a fixed amount of lung tissue and detecting human antibodies by sandwich ELISA after normalizing each sample to a fixed amount of extracted protein.

[0456] FIG. 11 shows the lung accumulation of PRO052 compared to an antibody that binds RAGE and a control target, respiratory syncytial virus (RSV) glycoprotein F (RAGE XT-M4 / Motavizumab), and an antibody that binds 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 the bispecific antibody that binds 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 may significantly redirect the binding specificity of the target binding arm.

[0457] 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.

[0458] 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 moieties of IL-10), 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), monomeric modified IL-10 sequence (Josephson et al., J Biol Chem: 13552-7, 2000), and 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 at the N-terminus of Fc and co-expressed with PRO003. The monovalent format is asymmetric and uses mutations in the Fc domain (A chain: S364K / K409S; B chain: K370S / F405K (WO 2017 / 106462 A1)) to enforce asymmetric pairing.

[0459] To express and purify antibodies, a 1:1 ratio of heavy and light chain DNA or a 1:1:1 ratio of heavy:light:IL-10-Fc (described below) was transfected 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 conditioned media by filtering the transfected cells 5 days after transfection. Conditioned media was incubated with Protein A agarose beads for 1 hour. After washing the bound beads with phosphate buffered saline (PBS) pH 7.4, bound antibodies were eluted with 0.1 M glycine pH 2.5 and neutralized with 1 / 10 volume of Tris pH 8.5. The neutralized eluate was buffer exchanged into PBS.

[0460] The resulting mAbs were named PRO023, PRO024, PRO025, PRO026 and PRO027 (Figure 12).

[0461] PRO023 comprises (a) a heavy chain sequence (SEQ ID NO:67) which 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) the light chain sequence of PRO003 (SEQ ID NO:29).

[0462] PRO024 contains (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) the light chain sequence of PRO003 (SEQ ID NO:29).

[0463] PRO025 contains (a) a heavy chain sequence (SEQ ID NO:69) which comprises 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).

[0464] 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).

[0465] 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).

[0466] The purified ANDbodies were analyzed for monodispersity by analytical size exclusion chromatography and purity by SDS-PAGE. PRO024 and PRO026 had the highest yields and monodispersity after a single purification step. PRO023 and PRO027 had moderate yields and were approximately 70% monodisperse. PRO025 had a low yield and was approximately 89% monodisperse.

[0467] [Table 90]

[0468] [Table 91]

[0469] [Table 92]

[0470] 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 overnight at a concentration of 1 μg / mL onto NUNC-IMMUNO™ MAXISORP™ ELISA plates. 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 at a starting concentration of 30 μg / mL. Bound antibodies were detected with a peroxidase-labeled anti-human IgG antibody with TMB and acid stop reagent.

[0471] 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 is driven by IL-10, and not the negative control anti-DSG1 antibody (PRO003), and that the IL-10 moiety is functional in binding to its receptor.

[0472] 13.3 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 evaluate the signaling activity and relative potency of each molecule. These cells express all components of the IL-10 signaling pathway, including the IL-10-inducible gene encoding secreted embryonic alkaline phosphatase (SEAP). Upon activation of the IL-10 signal in these cells, the cells express and secrete SEAP into the cell culture medium. The extent of the IL-10 signal is measured by reading the absorbance at 630 nm after adding QUANTI-BLUE™ solution colorimetric reagent (invivoGen) to the cell culture medium.

[0473] 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 11. Table 11 shows the EC50 of 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) 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 retained the signaling activity of human IL-10. These data demonstrate that the relative biological potencies 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.

[0474] [Table 93]

[0475] 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 effect of the IL-10 / DSG1 ANDbody on mouse peripheral blood mononuclear cells (PBMCs) and macrophages treated with lipopolysaccharide (LPS) as an inflammatory stimulus. In these experiments, PBMCs were isolated from blood and macrophages from the spleens of Balb / C mice by negative enrichment with magnetic beads (Miltenyi Biotech #130-110-434). Macrophage activation was assayed by measuring the levels of TNFα cytokine present in the culture medium after 3 and 5-6 h of stimulation with LPS.

[0476] Figures 13A-13G show levels of tumor necrosis factor alpha (TNFα) 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 levels of TNFα 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. Panels represent experimental runs over multiple days, so data are not comparable between panels. These data show that all five IL-10 / DSG1 ANDbodies can suppress inflammatory stimuli in primary macrophages.

[0477] 13.5 Binding of Address Targets Enhances Activity / Potency of ANDbody Effector Functions Combining addressing (e.g., using an address targeting domain) with biologically active molecules can enhance biological activity in a variety of ways. One exemplary enhancement is to increase the potency of the effector moiety against a particular cell in which the address target is also present.

[0478] To verify whether the presence of an address targeting domain can enhance the signaling ability of the effector targeting domain, human DSG1 was expressed on HEK-BLUE™ IL-10 cells using lentiviral stable expression (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. Expression of DSG1 was confirmed by qPCR.

[0479] The potency of the ANDbody, which replaces one Fab of an anti-DSG1 mAb (PRO058, functionally equivalent to PRO026) with recombinant human IL-10, i.e., monomeric IL-10, and the corresponding control, which includes Motavizumab as a negative control in the antibody sequence, was evaluated. Activity against HEK-BLUE™ IL-10 cells and HEK-BLUE™ IL-10 cells stably expressing DSG1, was assayed. Figure 15 shows representative signal responses of each molecule in parental HEK-BLUE™ IL-10 cells and HEK-BLUE™ IL-10 / DSG1 cells. The two cell lines showed similar responses to recombinant IL-10, confirming that DSG1 expression does not significantly affect their sensitivity to IL-10. The DSG1 / IL-10 ANDbody showed approximately 15-fold increased potency when DSG1 was expressed in the target cells. This effect was confirmed to be mediated by binding to DSG1, as no effect was observed on the potency of the corresponding IL-10 / motavizumab protein, 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.

[0480] 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.

[0481] The IL-10 / DSG1 ANDbody is intended to target IL-10 activity to the skin. In Example 7, it was shown that this anti-DSG1 antibody selectively distributes to mouse skin. In contrast, IL-10 is reported to clear from human circulation with a half-life of approximately 2 hours (Radwanski et al, Pharm Res. 1998 Dec;15(12):1895-901. Therefore, it was evaluated whether the IL-10 / DSG1 ANDbody retained the skin targeting ability of the parent antibody.

[0482] BALB / c mice were administered 3 mg / kg of PRO003, PRO024, and PRO058 via tail vein injection. PRO058 is functionally equivalent to PRO026, with 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) that contains an Fc region (SEQ ID NO:74) and a monomeric human IL-10 sequence (SEQ ID NO:64).

[0483] Serum samples were collected at 1-48 hours. Tissue samples were collected 1, 2, 4, and 7 days after dosing. The amount of anti-DSG1 or ANDbody in each serum or tissue sample was measured by ELISA. Figures 16A and 16B show that IL-10 / anti-DSG1 ANDbodies: PRO024 and PRO058 have similar PK properties in skin and serum as the parental anti-DSG1 antibody PRO003. These data indicate that antibody-cytokine fusions containing recombinant IL-10 can retain the pharmacokinetic properties of the parental antibody.

[0484] 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.

[0485] 14.1 Expression and purification of anti-TNFα monoclonal antibodies Anti-TNFα antibodies with the VH and VL sequences of the commercially available antibody fused to a huIgG1 backbone containing the effector null mutations; L234A, L235A, P329G (LALA-PG) were generated and characterized for binding and affinity to TNFα as previously described. The resulting mAbs were designated PRO076 and PRO078.

[0486] 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 with 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 explored, including cytokine / antibody and TNF receptor 2 (TNFR2) / antibody fusions.

[0487] To express and purify the antibodies, heavy and light chain DNAs were transfected in 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 conditioned medium by filtering the transfected cells 5 days after transfection. 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, bound antibodies were eluted with 0.1 M glycine pH 2.5 and neutralized with 1 / 10 volume of Tris pH 8.5. The neutralized eluate was buffer exchanged into PBS. The resulting mAbs were named PRO070, PRO074, PRO075, and PRO077 (Figure 17).

[0488] 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 of high purity and correct composition as confirmed by SDS-PAGE.

[0489] 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 various affinities. PRO074 and PRO075 showed similar affinities for both human and mouse TNFα, within 2 / 3-fold difference from the parent antibody. PRO077 showed 5-fold and 12-fold reduced binding affinity for human and mouse TNFα, respectively, compared to the parent antibody. This reduced affinity is likely due to the format change from Fab to single chain variable fragment (scFv).

[0490] 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 have been engineered to express secreted embryonic alkaline phosphatase (SEAP) in response to signaling for TNFα signaling. TNFα was measured according to the manufacturer's instructions. To assess inhibitory activity, the concentration of TNFα was increased to 225 pm (the approximate EC of recombinant human TNFα in this assay). 80 ) and preincubated with TNFα blocking molecules at concentrations ranging from 10 nM to approximately 10 pM. Table 12 shows the IC of each of the anti-TNFα-DSG1 AND bodies, 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 were derived.

[0491] [Table 94]

[0492] VII. Other Embodiments Some embodiments of the technology described herein can be defined according to any of the following numbering embodiments.

[0493] 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 the subject; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of an effector target in a 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.

[0494] 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 the subject; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of an effector target in a 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, in the absence of localization by the second binding site, does not substantially affect signaling of the effector target; wherein localization of the macromolecule to non-target tissues or cells is substantially reduced relative to the localization of a control macromolecule lacking the second binding moiety.

[0495] 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 the subject; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of an effector target in a 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, in the absence of localization by the second binding site, does not substantially affect signaling of the effector target; wherein localization of the macromolecule to non-target tissues or cells is substantially increased relative to the localization of a control macromolecule lacking the second binding moiety.

[0496] 4. 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 the subject; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of an effector target in a 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, in the absence of localization by the second binding site, does not substantially affect signaling of the effector target; wherein at least 25% of the polymer administered to a subject is detectable in target tissues or cells 1 to 7 days after administration.

[0497] 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 the subject; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of an effector target in a 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, in the absence of localization by the second binding site, does not substantially affect signaling of the effector target; wherein the affinity of the first binding site for the effector target is lower than the affinity of the second binding site for the address target, the macromolecule.

[0498] 6. 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 the subject; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of an effector target in a 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, in the absence of localization by the second binding site, does not substantially affect signaling of the effector target; 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,

[0499] 7. 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 the subject; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of an effector target in a 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, in the absence of localization by the second binding site, does not substantially affect signaling of the effector target; 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.

[0500] 8. The macromolecule according to any one of embodiments 1 to 7, wherein the first binding site has a low affinity for the effector target.

[0501] 9. The macromolecule according to any one of embodiments 1 to 7, wherein the first binding site has low binding activity for the effector target.

[0502] 10. The macromolecule according to 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.

[0503] 11. The macromolecule according to 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.

[0504] 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 target 50 Higher; (c) IC of the first binding site for the effector target 50 IC of the second binding site to the address target 50 The polymer according to any one of embodiments 1 to 11,

[0505] 13. The polymer 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 lower than the affinity of the second binding site for the address target.

[0506] 14. The macromolecule according to any one of embodiments 1 to 13, wherein 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.

[0507] 15. The macromolecule according to any one of the preceding embodiments, wherein the effector target is a protein, lipid, or sugar.

[0508] 16. The macromolecule according to any one of the preceding embodiments, wherein the effector target is a cell membrane-associated target.

[0509] 17. The macromolecule according to any one of embodiments 15 or 16, wherein the effector target is a protein.

[0510] 18. The macromolecule according to embodiment 17, wherein the effector target is a secreted protein.

[0511] 19. The macromolecule according to 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.

[0512] 20. The macromolecule according to any one of the preceding embodiments, wherein the macromolecule agonizes an effector target.

[0513] 21. The macromolecule according to any one of the preceding embodiments, wherein the macromolecule antagonizes an effector target.

[0514] 22. The macromolecule according to any one of the preceding embodiments, wherein the address target is a protein, lipid, or sugar.

[0515] 23. The macromolecule according to embodiment 22, wherein the address target is a protein.

[0516] 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.

[0517] 25. The macromolecule according to embodiment 24, wherein the expression level of the effector target or the address target is assessed using an RNA-seq dataset.

[0518] 26. The macromolecule of embodiment 2, wherein the RNA sequence dataset is a Genotype-Tissue Expression (GTEx) dataset or a Human Protein Atlas (HPA) dataset.

[0519] 27. The macromolecule according to embodiment 23, wherein expression of the effector target or address target is protein expression.

[0520] 28. The macromolecule according to any one of embodiments 1 to 27, wherein the effector target is systemically expressed in the subject.

[0521] 29. The macromolecule according to any one of embodiments 1 to 27, wherein the effector target is regionally expressed in the subject.

[0522] 30. The macromolecule according to any one of embodiments 1 to 27, wherein the effector target is expressed locally in the subject.

[0523] 31. The macromolecule according to any one of embodiments 1 to 30, wherein the address target is regionally expressed in the subject.

[0524] 32. The macromolecule according to any one of embodiments 1 to 30, wherein the address target is expressed locally in the subject.

[0525] 33. The macromolecule according to any one of embodiments 1 to 30, wherein expression of the address target is restricted to one cell type of the subject.

[0526] 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, which is not present in detectable amounts on the cell surface.

[0527] 35. The macromolecule described in embodiment 34, wherein the address target is expressed in the ECM and is not present in detectable amounts elsewhere in the subject.

[0528] 36. A macromolecule according to any one of embodiments 1 to 35, wherein the address target is expressed only in cells of interest when in a particular cellular state.

[0529] 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.

[0530] 38. The macromolecule according to any one of embodiments 1 to 37, wherein the address target is not expressed in tissues in which binding of the second binding site to the effector target is deleterious to the subject.

[0531] 39. The macromolecule according to any one of the preceding embodiments, wherein the binding site for the address target does not detectably bind to the binding site of the address target's natural ligand.

[0532] 40. Expression of the effector target or address target is in: minor salivary gland, 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, cervix-vaginal 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 the following tissues: 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.

[0533] 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.

[0534] 42. The macromolecule according to embodiment 41, wherein expression of the address target is substantially higher in skin tissue, lung tissue, kidney tissue, or intestinal tissue than in any other tissue.

[0535] 43. The macromolecule according to any one of the preceding embodiments, wherein the effector target and / or the address target are expressed on a structural tissue of the subject.

[0536] 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.

[0537] 45. The macromolecule according to any one of embodiments 1 to 43, wherein the effector target and the address target are on different cells.

[0538] 46. ​​The macromolecule according to embodiment 45, wherein the effector target and the address target are on different cells of the same cell type.

[0539] 47. The macromolecule according to embodiment 45, wherein the effector target and the address target are on different cells of different cell types.

[0540] 48. The macromolecule according to embodiment 45, wherein the effector target and the address target are on different cells within the same tissue.

[0541] 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.

[0542] 50. The macromolecule of any one of embodiments 45 to 49, wherein the effector target and the address target are on different cells located within 100 nm of each other in a subject.

[0543] 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.

[0544] 52. The polymer according to any one of the preceding embodiments, wherein the polymer is a DNA polynucleotide.

[0545] 53. The polymer according to any one of the preceding embodiments, wherein the polymer comprises an RNA or an RNA-polypeptide conjugate.

[0546] 54. The polymer according to any one of embodiments 1 to 51 and 53, wherein the polymer comprises a polypeptide.

[0547] 55. The polymer according to any one of the preceding embodiments, wherein the polymer is a polypeptide.

[0548] 56. The macromolecule of embodiment 54 or 55, wherein the polypeptide is an antibody or an antigen-binding fragment thereof.

[0549] 57. The macromolecule according to embodiment 56, wherein the first binding site and the second binding site comprise a VH and / or a VL, respectively.

[0550] 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.

[0551] 59. The macromolecule according to embodiment 57 or 58, wherein the macromolecule is an asymmetric or symmetric antibody.

[0552] 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 linked F(ab')2, Ig ​​molecule with two, 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, adducted 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 polymer 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.

[0553] 61. The macromolecule according to embodiment 54, wherein the polypeptide is a ligand of an effector target or a ligand of an address target.

[0554] 62. The macromolecule according to embodiment 61, wherein the ligand is a natural ligand, a modified ligand, or a synthetic ligand.

[0555] 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.

[0556] 64. The polymer 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 that addresses the target.

[0557] 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.

[0558] 66. The polymer described in 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.

[0559] 67. The polymer according to any one of the preceding embodiments, wherein the address target has a Geni coefficient of 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.

[0560] 68. The polymer according to any one of the preceding embodiments, wherein the address target has a Tau coefficient of greater than about 0.67, about 0.75, about 0.8, about 0.85, about 0.90, or about 0.95.

[0561] 69. The macromolecule according to any one of the preceding embodiments, wherein the effector target has a Gini coefficient of less than about 0.25, about 0.20, or about 0.15.

[0562] 70. The macromolecule according to any one of embodiments 1 to 69, wherein the effector target has a tau coefficient of less than about 0.25, about 0.20, or about 0.15.

[0563] 71. The macromolecule of any one of the preceding embodiments, further comprising a third binding site.

[0564] 72. The macromolecule according to embodiment 71, wherein the third binding site is the same as the first binding site.

[0565] 73. The macromolecule of embodiment 71, wherein the third binding site is the same as the second binding site.

[0566] 74. The polymer according to any one of embodiments 1 to 73, wherein the first binding site and the second binding site are directly bound to each other in the polymer.

[0567] 75. The polymer according to any one of the preceding embodiments, wherein the first binding site and the second binding site in the polymer are linked by a stable domain.

[0568] 76. The macromolecule according to any one of embodiments 1 to 75, wherein the effector target is Notch2 and the address target is RAGE.

[0569] 77. The macromolecule of embodiment 76, wherein RAGE signaling is not affected by the second moiety that binds to the RAGE address target.

[0570] 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).

[0571] 79. The macromolecule of embodiment 78, wherein UMOD signaling is not affected by the second site that binds to the UMOD address target.

[0572] 80. The macromolecule according to any one of embodiments 1 to 75, wherein the effector target is Notch2 and the address target is meprin A subunit beta (MEP1B).

[0573] 81. The macromolecule of embodiment 80, wherein MEP1B signaling is not affected by the second moiety that binds to the MEP1B address target.

[0574] 82. The macromolecule according to any one of embodiments 1 to 75, wherein the effector target is IL11Ra and the address target is RAGE.

[0575] 83. The macromolecule of embodiment 82, wherein RAGE signaling is not affected by the second moiety that binds to the RAGE address target.

[0576] 84. The macromolecule according to any one of embodiments 1 to 75, wherein the effector target is IL11Ra and the address target is UMOD.

[0577] 85. The macromolecule of embodiment 84, wherein UMOD signaling is not affected by the second site that binds to the UMOD address target.

[0578] 86. The polymer according to any one of the preceding embodiments, wherein the subject is a human.

[0579] 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.

[0580] 88. The polymer according to embodiment 87, wherein the moiety is a molecule.

[0581] 89. The macromolecule according to embodiment 87 or 88, wherein the moiety is not a toxin.

[0582] 90. The macromolecule according to embodiment 87, wherein the moiety is a cell.

[0583] 91. The macromolecule according to embodiment 90, wherein the moiety is not a T cell or a NK cell.

[0584] 92. The macromolecule according to any one of embodiments 87 to 91, wherein the target tissue is not a tumor.

[0585] 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.

[0586] 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.

[0587] 95. A method for regulating 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.

[0588] 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 site of the macromolecule binds to the effector target.

[0589] 97. 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 moiety 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 an address target such that the first binding site affects signaling of an effector target in a 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 is a macromolecule that does not bind to the binding site of a natural ligand of the address target.

[0590] 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 moiety 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 an address target such that the first binding site affects signaling of an effector target in a 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 A macromolecule, wherein the first binding site and the second binding site are directly bound to each other in said macromolecule.

[0591] 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 moiety 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 an address target such that the first binding site affects signaling of an effector target in a 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 A macromolecule, wherein the first binding site and the second binding site are linked to each other by a stable domain.

[0592] 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 moiety 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 an address target such that the first binding site affects signaling of an effector target in a 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 Effector targets and / or address targets are macromolecules that are expressed on structural tissues of the host.

[0593] 101. A pharmaceutical composition comprising a polymer according to any one of embodiments 1 to 86.

[0594] 102. A pharmaceutical composition comprising a polymer and one or more pharma- ceutically 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 moiety 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 an address target such that the first binding site affects signaling of an effector target in a 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.

[0595] 103. The pharmaceutical composition according to embodiment 101 or 102, wherein the pharmaceutical composition is an RNA pharmaceutical composition.

[0596] 104. A pharmaceutical composition according to any one of embodiments 101 to 103, further comprising a carrier.

[0597] 105. The pharmaceutical composition according to embodiment 104, wherein the carrier is a lipid nanoparticle.

[0598] 106. The pharmaceutical composition according to embodiment 104, wherein the carrier is a viral vector.

[0599] 107. The pharmaceutical composition according to embodiment 104, wherein the carrier is a membrane carrier.

[0600] 108. The pharmaceutical composition according to embodiment 107, wherein the membrane carrier is a cell.

[0601] 109. The pharmaceutical composition according to embodiment 107, wherein the membrane carrier is a vesicle.

[0602] 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),

[0603] 111. A method for modulating the activity of an effector target in the lungs 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 method, wherein the second binding site is specific for RAGE.

[0604] 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).

[0605] 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).

[0606] 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 the subject; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of an effector target in a 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, in the absence of localization by the second binding site, does not substantially affect signaling of the effector target; A method that allows a macromolecule to localize to a target tissue or cell of a subject.

[0607] 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 the subject; (i) the second binding site localizes the first binding site to an address target such that the first binding site affects signaling of an effector target in a 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, in the absence of localization by the second binding site, does not substantially affect signaling of the effector target; A method that allows 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 at 1 to 7 days after administration of the macromolecule to the subject.

[0608] 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.

[0609] 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 site.

[0610] 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.

[0611] 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

A composition for use in a method of localizing a polymer to a target tissue or cell of interest, the composition comprising a polymer comprising a first binding site and a second binding site, the method comprising administering the composition to the subject, wherein (a) the first binding site is specific for an effector target of the subject, wherein (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, wherein (ii) the second binding site does not substantially affect signal transduction upon binding to the address target, wherein (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, a composition that enables the polymer to localize to the target tissue or cell of the subject. **Claim 2** The composition according to claim 1, wherein at least 25% of the polymer detectable in the subject is detected in the target tissue or cell at a time point of 1 to 7 days after administration of the polymer to the subject. **Claim 3** The efficacy of the first binding site in the target tissue or cell is substantially increased compared to a control polymer without the second binding site, and optionally, the first binding site has a low affinity for the effector target or a low binding activity for the effector target. The composition according to claim 1. **Claim 4** The composition according to claim 1, wherein (a) 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, or (b) 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. **Claim 5** The composition according to claim 1, wherein signal transduction of the effector target by the polymer in non-target tissues or cells of the subject is substantially reduced compared to a control polymer without the second binding site. **Claim 6** The composition according to claim 1, wherein the address target is expressed regionally in the subject. **Claim 7** The composition according to claim 1, wherein the address target is expressed locally in the subject.

8. The composition according to claim 1, wherein the expression of the address target is limited to the cell type of the subject.

9. The composition according to claim 1, wherein the address target is expressed only by the cells of the subject when in a specific cell state.

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

11. The composition according to claim 1, wherein the first binding site or the second binding site comprises a polypeptide, and optionally, the polypeptide is an antibody or an antigen-binding fragment thereof.

12. The composition according to claim 11, wherein the polymer 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.

13. The composition according to claim 11, wherein the polypeptide is a ligand of the effector target or a ligand of the address target.

14. (a) the first binding site comprises an antibody or an antigen-binding fragment thereof, and the second binding site comprises a ligand of the address target, or (b) the first binding site comprises a ligand of the effector target, and the second binding site comprises an antibody or an antigen-binding fragment thereof, the composition according to claim 13.

15. a) the target tissue is skin, and the second binding site is specific for desmoglein-1 (DSG-1), or b) the target tissue is lung tissue, and the second binding site is specific for RAGE, or c) the target tissue is kidney tissue, and the second binding site is specific for cadherin 16 (CDH16), or d) the target tissue is intestinal tissue, and the second binding site is specific for cadherin 17 (CDH17), the composition according to claim 1.