Compositions and methods

Abstract

Provided herein are macromolecules that conditionally induce a cellular effector function (e.g., a biological or therapeutic activity) based on the presence of a disease signature ligand, compositions comprising the same, and methods of using the same.

Description

[0001] COMPOSITIONS AND METHODS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 495,509, filed on April 11 , 2023, and U.S. Provisional Patent Application No. 63 / 613,644, filed on December 21 , 2023, the entire contents of each of which are incorporated herein by reference in their entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 9, 2024, is named 51661 -005WO4_Sequence_Listing_4_9_24 and is 43,890 bytes in size.

[0006] BACKGROUND

[0007] There is a need in the art for macromolecules that conditionally induce a cellular effector function (e.g., a biological or therapeutic activity) based on the presence of a disease signature ligand and for methods of using the same.

[0008] SUMMARY OF THE INVENTION

[0009] In a first aspect, provided herein is a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand.

[0010] In a second aspect, provided herein is a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand, wherein the two macromolecules are identical.

[0011] In a third aspect, provided herein is a macromolecule complex comprising a pair of macromolecules, wherein each macromolecule independently comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample; (b) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1 ) that specifically binds a first effector ligand in the biological sample; and (c) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the pair of macromolecules binding the disease signature ligand.

[0012] In a fourth aspect, provided herein is a macromolecule complex comprising a pair of macromolecules, wherein each macromolecule independently comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the first member of the pair of macromolecules comprises a first binding domain 1 (FBD1 ) that specifically binds a first epitope of a disease signature ligand in a biological sample;

[0013] (b) the second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds a second epitope of the disease signature ligand in a biological sample; and (c) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand.

[0014] In some embodiments of the first through fourth aspects, the conjugation is covalent. In some embodiments, the covalent conjugation comprises a chemical linker or a polypeptide linker. In some embodiments, the covalent conjugation comprises disulfide linkages between the macromolecules.

[0015] In some embodiments of the first through fourth aspects, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair. In some embodiments, the pair of complementary moieties is biotin and avidin; barnase and barstar; a pair of complementary aptamers; or a pair of complementary polypeptides. In some embodiments, the pair of complementary polypeptides is a pair of engineered Fc fragments, e.g., a knob-into-hole pair.

[0016] In some embodiments of any of the above aspects, the conjugation sterically orients the two macromolecules to permit conditional concurrent binding of disease signal and effector ligands.

[0017] In some embodiments, the non-covalent conjugation is mediated by binding of the FBD to the disease signature ligand.

[0018] In some embodiments of the second through fourth aspects, the conjugation sterically orients the first member of the pair of macromolecules and the second member of the pair of macromolecules to permit conditional concurrent binding of disease signal and effector ligands.

[0019] In some embodiments, the non-covalent conjugation is mediated by binding of the FBD to the disease signature ligand.

[0020] In a fifth aspect, provided herein is a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are not conjugated to each other in the absence of the disease signature ligand; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand.

[0021] In a sixth aspect, provided herein is a macromolecule complex comprising a pair of macromolecules, wherein each macromolecule independently comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the first member of the pair of macromolecules comprises a first binding domain 1 (FBD1 ) that specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds a second epitope of the disease signature ligand in a biological sample; and (c) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are not conjugated to each other in the absence of the disease signature ligand; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand.

[0022] In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of the disease signature ligand, and the non-covalent conjugation is mediated by the disease signature ligand.

[0023] In a seventh aspect, provided herein is a macromolecule comprising two FBDs linked to two SBDs by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; and (b) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand.

[0024] In an eighth aspect, provided herein is a macromolecule comprising an FBD1 and an FBD2 linked to two SBDs by linker domains, wherein (a) the FBD1 specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the FBD2 specifically binds a second epitope of the disease signature ligand in the biological sample; and (c) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the FBD1 and the FBD2 binding the disease signature ligand.

[0025] In a ninth aspect, provided herein is a macromolecule comprising two FBDs linked to an SBD1 and an SBD2 by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; (b) the SBD1 specifically binds a first effector ligand in the biological sample; and (c) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand.

[0026] In a tenth aspect, provided herein is macromolecule comprising an FBD1 and an FBD2 linked to an SBD1 and an SBD2 by linker domains, wherein (a) the FBD1 specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the FBD2 specifically binds a second epitope of the disease signature ligand in the biological sample; (c) the SBD1 specifically binds a first effector ligand in the biological sample; and (d) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the FBD1 and the FBD2 binding the disease signature ligand.

[0027] In certain of the foregoing aspects, the first binding domain and second binding domain are linked covalently (e.g., by small molecule, peptide (e.g., as a single polypeptide chain comprising the first binding domain and second binding domain), or a combination thereof).

[0028] In some embodiments of any of the above aspects, the disease signature ligand comprises a protein, a peptide, or a small molecule. In some embodiments, the protein comprises a soluble protein or an insoluble protein. In some embodiments of any of the above aspects, the disease signature ligand is a protein, a peptide, or a small molecule. In some embodiments, the protein is a soluble protein or an insoluble protein.

[0029] In some embodiments of any of the above aspects, the disease signature ligand comprises a cytokine. In some embodiments of any of the above aspects, the disease signature ligand is a cytokine. In some embodiments, the cytokine is an interleukin, an interferon, a growth factor, a chemokine, a member of the TNF family, or VEGF. In some embodiments, the disease signature ligand is an interleukin. In some embodiments, the interleukin is IL-1 , IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15, IL-17, or IL-23; the interferon is IFN-gamma; the growth factor is transforming growth factor beta (TGF-beta), granulocyte colony stimulating factor (GCSF) granulocyte-macrophage colony-stimulating factor (GCSF), epidermal growth factor (EGF), or erythropoietin (EPO); the chemokine is monocyte chemoattractant protein-1 (MCP-1 ) or interferon gamma-induced protein 10 (IP-10); the member of the TNF family is TNF- alpha; or the VEGF is VEGF-A or VEGF-B.

[0030] In some embodiments of any of the above aspects, the disease signature ligand comprises a neurotransmitter. In some embodiments of any of the above aspects, the disease signature ligand is a neurotransmitter.

[0031] In some embodiments of any of the above aspects, the disease signature ligand comprises a cell surface receptor, a surface antigen, a membrane-bound protein, an extracellular matrix component, or an integrin. In some embodiments of any of the above aspects, the disease signature ligand is a cell surface receptor, a surface antigen, a membrane-bound protein, an extracellular matrix component, or an integrin.

[0032] In some embodiments of any of the above aspects, the disease signature ligand comprises a self antigen of an organism from which the biological sample is derived. In some embodiments of any of the above aspects, the disease signature ligand is a self antigen of an organism from which the biological sample is derived. In some embodiments, the self antigen is an anti-drug antibody (ADA), an autoantibody, or a tumor marker.

[0033] In some embodiments of any of the above aspects, the disease signature ligand comprises a nucleic acid. In some embodiments of any of the above aspects, the disease signature ligand is a nucleic acid.

[0034] In some embodiments of any of the above aspects, the disease signature ligand comprises a carbohydrate, a lipid, a peptide, a nucleoside, or a combination of the foregoing. In some embodiments of any of the above aspects, the disease signature ligand is a carbohydrate, a lipid, a peptide, a nucleoside, or a combination of the foregoing.

[0035] In some embodiments of any of the above aspects, the disease signature ligand comprises a hormone, an amino acid derivative, a steroid, or an eicosanoid. In some embodiments of any of the above aspects, the disease signature ligand is a hormone, an amino acid derivative, a steroid, or an eicosanoid.

[0036] In some embodiments of any of the above aspects, the disease signature ligand comprises a non-self antigen. In some embodiments of any of the above aspects, the disease signature ligand is a non-self antigen. In some embodiments, the disease signature ligand is a virus, a bacterium, or a fragment or antigen thereof.

[0037] In some embodiments of any of the above aspects, the disease signature ligand is multimeric. In some embodiments, the disease signature ligand is dimeric, trimeric, or tetrameric.

[0038] In some embodiments of any of the above aspects, the FBD, FBD1 , or FBD2 comprises a polypeptide that specifically binds the disease signature ligand.

[0039] In some embodiments, the polypeptide comprises an antibody or a fragment thereof. In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is an scFv, a monospecific tandem scFv (taFv), a bispecific taFv, a VHH, a VNAR, a Fab, a monospecific single-chain diabody, a bispecific single-chain diabody, or a dual-affinity re-targeting antibody (DART).

[0040] In some embodiments, the polypeptide comprises an antibody mimetic. In some embodiments, the polypeptide is an antibody mimetic. In some embodiments, the antibody mimetic is an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, a lipocalin, an avimer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monobody, a fibronectin type III domain (FN3)-based binder, a nanoantibody, a nanoCLAMP, an optimer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR).

[0041] In some embodiments, the polypeptide comprises an endogenous binding domain. In some embodiments, the endogenous binding domain comprises a cell receptor domain, an enzyme domain, a variable lymphocyte receptor (VLR) domain, a receptor ectodomain, a nuclear hormone receptor ligandbinding domain, or a DNA-binding domain. In some embodiments, the polypeptide is an endogenous binding domain. In some embodiments, the endogenous binding domain is a cell receptor domain, an enzyme domain, a variable lymphocyte receptor (VLR) domain, a receptor ectodomain, a nuclear hormone receptor ligand-binding domain, or a DNA-binding domain. In some embodiments of any of the above aspects, the FBD, FBD1 , or FBD2 comprises an oligonucleotide that specifically binds the disease signature ligand. In some embodiments, the oligonucleotide is a nucleic acid aptamer. In some embodiments, the nucleic acid aptamer is a DNA aptamer.

[0042] In some embodiments of any of the above aspects, the FBD, FBD1 , or FBD2 comprises a chemical molecule that specifically binds the disease signature ligand.

[0043] In some embodiments of any of the above aspects, the FBD, FBD1 , or FBD2 has affinity to two or more disease signature moieties.

[0044] In some embodiments of any of the above aspects, the effector ligand comprises a protein or a peptide. In some embodiments of any of the above aspects, the effector ligand is a protein or a peptide.

[0045] In some embodiments of any of the above aspects, the effector ligand comprises a cell-surface receptor or an intracellular receptor. In some embodiments, the cell-surface receptor comprises a catalytic receptor or the intracellular receptor is a nuclear hormone receptor. In some embodiments, the catalytic receptor comprises a receptor tyrosine kinase (RTK), a receptor serine / threonine kinase (RSK), a type 1 cytokine receptor, a type 2 cytokine receptor, or a tumor necrosis factor (TNF) superfamily receptor. In some embodiments, the RTK comprises VEGFR, the RSK is TGFBR2, the type 1 cytokine receptor is IL-2R, the type 2 cytokine receptor comprises IL10R, or the TNF superfamily receptor is TNFR2 or 4-1 BB. In some embodiments of any of the above aspects, the effector ligand is a cell-surface receptor or an intracellular receptor. In some embodiments, the cell-surface receptor is a catalytic receptor or the intracellular receptor is a nuclear hormone receptor. In some embodiments, the catalytic receptor is a receptor tyrosine kinase (RTK), a receptor serine / threonine kinase (RSK), a type 1 cytokine receptor, a type 2 cytokine receptor, or a tumor necrosis factor (TNF) superfamily receptor. In some embodiments, the RTK is VEGFR, the RSK is TGFBR2, the type 1 cytokine receptor is IL-2R, the type 2 cytokine receptor is IL1 OR, or the TNF superfamily receptor is TNFR2 or 4-1 BB.

[0046] In some embodiments of the first, second, fifth, seventh, and eighth aspects, the SBD comprises an agonist of the effector ligand. In some embodiments of the first, second, fifth, seventh, and eighth aspects, the SBD is an agonist of the effector ligand. In some embodiments, the effector ligand must be homodimerized to exert a cellular effector function. In some embodiments, the effector ligand is homodimerized in the presence of the macromolecule complex or macromolecule and the disease signature ligand.

[0047] In some embodiments of the third, fourth, ninth, and tenth aspects, the first effector ligand and the second effector ligand must be associated to exert a cellular effector function. In some embodiments, the first effector ligand and the second effector ligand are associated in the presence of the macromolecule complex or macromolecule and the disease signature ligand. In some embodiments, the association is heterodimerization.

[0048] In some embodiments of any of the above aspects, the cellular effector function comprises a biological activity. In some embodiments, the cellular effector function comprises a therapeutic activity. In some embodiments, the cellular effector function comprises a disease activity. In some embodiments of any of the above aspects, the cellular effector function is a biological activity. In some embodiments, the cellular effector function is a therapeutic activity. In some embodiments, the cellular effector function is a disease activity.

[0049] In some embodiments of any of the above aspects, the SBD, SBD1 , or SBD2 comprises a polypeptide that specifically binds the effector ligand.

[0050] In some embodiments, the polypeptide comprises an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof comprises an scFv, a monospecific taFv, a bispecific taFv, a VHH, a VNAR, a Fab, a monospecific single-chain diabody, a bispecific single-chain diabody, or a DART. In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is an scFv, a monospecific taFv, a bispecific taFv, a VHH, a VNAR, a Fab, a monospecific single-chain diabody, a bispecific single-chain diabody, or a DART.

[0051] In some embodiments, the polypeptide comprises an antibody mimetic. In some embodiments, the antibody mimetic comprises an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, a lipocalin, an avimer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monobody, a FN3-based binder, a nanoantibody, a nanoCLAMP, an optimer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a VLR. In some embodiments, the polypeptide is an antibody mimetic. In some embodiments, the antibody mimetic is an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, a lipocalin, an avimer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monobody, a FN3-based binder, a nanoantibody, a nanoCLAMP, an optimer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a VLR.

[0052] In some embodiments, the polypeptide comprises an endogenous binding domain. In some embodiments, the endogenous binding domain comprises a ligand of the effector ligand or a fragment thereof. In some embodiments, the endogenous binding domain comprises a viral binding protein or a fragment thereof. In some embodiments, the polypeptide is an endogenous binding domain. In some embodiments, the endogenous binding domain is a ligand of the effector ligand or a fragment thereof. In some embodiments, the endogenous binding domain is a viral binding protein or a fragment thereof.

[0053] In some embodiments of any of the above aspects, the SBD, SBD1 , or SBD2 comprises an oligonucleotide that specifically binds the effector ligand. In some embodiments, the oligonucleotide is a nucleic acid aptamer. In some embodiments, the nucleic acid aptamer is a DNA aptamer.

[0054] In some embodiments of any of the above aspects, the FBD comprises a chemical molecule that specifically binds the disease signature ligand.

[0055] In some embodiments of the third, fourth, ninth, and tenth aspects, the SBD1 comprises a first portion of a binding moiety and the SBD2 comprises a second portion of the binding moiety. In some embodiments of the third, fourth, ninth, and tenth aspects, the SBD1 is a first portion of a binding moiety and the SBD2 is a second portion of the binding moiety.

[0056] In some embodiments of the third, fourth, ninth, and tenth aspects, the SBD1 specifically binds to a first component of a heterodimeric receptor and the SBD2 specifically binds to a second component of the heterodimeric receptor.

[0057] In some embodiments of the third, fourth, ninth, and tenth aspects, the SBD1 comprises a first component of a dimeric moiety and the SBD2 comprises a second component of a dimeric moiety. In some embodiments of the third, fourth, ninth, and tenth aspects, the SBD1 is a first component of a dimeric moiety and the SBD2 is a second component of a dimeric moiety.

[0058] In some embodiments of the third, fourth, ninth, and tenth aspects, the SBD1 comprises a first fragment of a polypeptide chain and the SBD2 is a second fragment of the polypeptide chain. In some embodiments, the polypeptide chain comprises a hormone, a cytokine, or a growth factor. In some embodiments of the third, fourth, ninth, and tenth aspects, the SBD1 is a first fragment of a polypeptide chain and the SBD2 is a second fragment of the polypeptide chain. In some embodiments, the polypeptide chain is a hormone, a cytokine, or a growth factor.

[0059] In some embodiments of the third, fourth, and ninth, and tenth aspects, the SBD1 and the SBD2 have been engineered to have reduced affinity for one another.

[0060] In some embodiments of any of the above aspects, the macromolecule comprises a reporter moiety. In some embodiments, the reporter moiety comprises an affinity tag, a fluorescent marker, a radioactive marker, or a chromogenic marker. In some embodiments, the affinity tag is a FLAG affinity tag or the chromogenic marker is luciferase or beta-lactamase.

[0061] In some embodiments of any of the above aspects, the macromolecule complex or macromolecule comprises one or more linker domains. In some embodiments, the one or more linker domains are peptide linkers. In some embodiments, the peptide linkers comprise one or more GS linkers. In some embodiments, the GS linkers comprise one or more GS(GnS)mlinkers or one or more (GnS)m linkers. In some embodiments, the GS linkers comprise one or more (G4S)mlinkers.

[0062] In some embodiments of any of the above aspects, the macromolecule comprises a polypeptide.

[0063] In some embodiments of any of the above aspects, the macromolecule is a polypeptide.

[0064] In some embodiments of any of the above aspects, the biological sample comprises an extract, fluid, fraction, cell, tissue, or subject. In some embodiments of any of the above aspects, the biological sample is an extract, fluid, fraction, cell, tissue, or subject.

[0065] In some embodiments of any of the above aspects, the macromolecule or one or both members of pair of macromolecules comprises a leader sequence. In some embodiments, the leader sequence comprises a secretion signal.

[0066] In some embodiments of any of the above aspects, the macromolecule complex or one or both macromolecules of the macromolecule complex comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof, an albumin domain or a fragment thereof, or polyethylene glycol (PEG) or a modified derivative thereof.

[0067] In another aspect, provided herein is a nucleic acid encoding the macromolecule of any one of the first, fifth, seventh, eighth, ninth, and tenth aspects.

[0068] In another aspect, provided herein is a pair of nucleic acids encoding the pair of macromolecules of the second, third, fourth, or sixth aspect.

[0069] In some aspects, the nucleic acid is an RNA or a DNA.

[0070] In some aspects, the nucleic acid is formulated with a delivery platform.

[0071] In some aspects, the delivery platform is a lipid-based carrier or a vector delivery system. In some embodiments, the lipid-based carrier is a lipid nanoparticle (LNP). In some embodiments, the vector delivery system comprises or is derived from an adenovirus, an anellovirus, an AAV, or a lentivirus. In another aspect, provided herein is a nucleic acid encoding a macromolecule according to any one of the first, fifth, seventh, eighth, ninth, and tenth aspects, wherein the nucleic acid is formulated with a carrier.

[0072] In another aspect, provided herein is a pair of nucleic acids encoding a pair of macromolecules according to the second, third, fourth, or sixth aspect, wherein the pair of nucleic acids is formulated with a carrier.

[0073] In some embodiments, the nucleic acid is an RNA or a DNA.

[0074] In some embodiments, the carrier is a lipid-based carrier. In some embodiments, the lipid-based carrier is a LNP.

[0075] In another aspect, the disclosure provides a vector comprising the nucleic acid of any of the above aspects.

[0076] In another aspect, the disclosure provides a vector or pair of vectors comprising the pair of nucleic acids of any of the above aspects.

[0077] In some embodiments, the vector or pair of vectors is formulated with a carrier.

[0078] In another aspect, the disclosure provides a host cell comprising the nucleic acid, pair of nucleic acids, vector or pair of vectors of any one of the above aspects.

[0079] In some embodiments, the macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids is about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure or more than 99% pure.

[0080] In some embodiments, the macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids is manufactured according to the U.S. Food and Drug Administration (FDA)’s Good Manufacturing Practice (GMP), Good Clinical Practice (GCP), and / or Good Laboratory Practice (GLP) standards.

[0081] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a VEGF binding domain linked to a 4-1 BB binding domain, wherein the 4-1 BB binding domain induces a cellular effector function upon binding to 4- 1 BB; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding to VEGF. See, e.g., the drawings and examples herein.

[0082] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair. In some embodiments, the pair of complementary polypeptides is a pair of Fc fragments. In some embodiments, the pair of Fc fragments is a knob-into-hole pair. In some embodiments, the two macromolecules are identical.

[0083] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a VEGF binding domain linked to a 4-1 BB binding domain, wherein the 4-1 BB binding domain induces a cellular effector function upon binding to 4- 1 BB; wherein the two macromolecules are not conjugated to each other in the absence of VEGF; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding VEGF. In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of VEGF, and the non-covalent conjugation is mediated by VEGF. See, e.g., the drawings and examples herein.

[0084] In some embodiments, the macromolecule or one or both members of pair of macromolecules comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0085] In some embodiments of either of the above two aspects, the VEGF binding domain is an anti- VEGF scFv. In some embodiments, the VEGF binding domain is a VEGF receptor trap.

[0086] In some embodiments of either of the above two aspects, the 4-1 BB binding domain is an anti-4- 1 BB scFv.

[0087] In some embodiments, one of the macromolecules comprises SEQ ID NOs: 36 and 38.

[0088] In some embodiments, one of the macromolecules comprises SEQ ID NOs: 37 and 38.

[0089] In some embodiments, one of the macromolecules comprises SEQ ID NOs: 36 and 39.

[0090] In some embodiments, one of the macromolecules comprises SEQ ID NOs: 37 and 39.

[0091] In some embodiments, both macromolecules comprise SEQ ID NOs: 36 and 38.

[0092] In some embodiments, both macromolecules comprise SEQ ID NOs: 37 and 38.

[0093] In some embodiments, both macromolecules comprise SEQ ID NOs: 36 and 39.

[0094] In some embodiments, both macromolecules comprise SEQ ID NOs: 37 and 39.

[0095] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a TGFb binding domain linked to a TpoR binding domain, wherein the TpoR binding domain induces a cellular effector function upon binding to TpoR; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding to TGFb. See, e.g., the drawings and examples herein.

[0096] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair. In some embodiments, the pair of complementary polypeptides is a pair of Fc fragments. In some embodiments, the pair of Fc fragments is a knob-into-hole pair. In some embodiments, the two macromolecules are identical.

[0097] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a TGFb binding domain linked to a TpoR binding domain, wherein the TpoR binding domain induces a cellular effector function upon binding to TpoR; wherein the two macromolecules are not conjugated to each other in the absence of TGFb; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding TGFb. In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of TGFb, and the non-covalent conjugation is mediated by TGFb. See, e.g., the drawings and examples herein. In some embodiments, the macromolecule or one or both members of pair of macromolecules comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0098] In some embodiments of either of the above two aspects, the TGFb binding domain is an anti- TGFb scFv.

[0099] In some embodiments of either of the above two aspects, the TpoR binding domain is an anti- TpoR scFv.

[0100] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises an IL-8 binding domain linked to a TpoR binding domain, wherein the TpoR binding domain induces a cellular effector function upon binding to TpoR; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding to IL-8. See, e.g., the drawings and examples herein.

[0101] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair. In some embodiments, the pair of complementary polypeptides is a pair of Fc fragments. In some embodiments, the pair of Fc fragments is a knob-into-hole pair. In some embodiments, the two macromolecules are identical.

[0102] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises an IL-8 binding domain linked to a TpoR binding domain, wherein the TpoR binding domain induces a cellular effector function upon binding to TpoR; wherein the two macromolecules are not conjugated to each other in the absence of IL-8; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding IL-8. In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of IL-8, and the non-covalent conjugation is mediated by IL-8. See, e.g., the drawings and examples herein.

[0103] In some embodiments, the macromolecule or one or both members of pair of macromolecules comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0104] In some embodiments of either of the above two aspects, the IL-8 binding domain is an anti-IL8 scFv.

[0105] In some embodiments of either of the above two aspects, the TpoR binding domain is an anti- TpoR scFv.

[0106] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a TGFb binding domain linked to an IL2R binding domain, wherein the IL2R binding domain induces a cellular effector function upon binding to IL2R; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding to TGFb. See, e.g., the drawings and examples herein. In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair. In some embodiments, the pair of complementary polypeptides is a pair of Fc fragments. In some embodiments, the pair of Fc fragments is a knob-into-hole pair. In some embodiments, the two macromolecules are identical.

[0107] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a TGFb binding domain linked to an IL2R binding domain, wherein the IL2R binding domain induces a cellular effector function upon binding to IL2R; wherein the two macromolecules are not conjugated to each other in the absence of TGFb; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding TGFb. In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of TGFb, and the non-covalent conjugation is mediated by TGFb. See, e.g., the drawings and examples herein.

[0108] In some embodiments, the macromolecule or one or both members of pair of macromolecules comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0109] In some embodiments of either of the above two aspects, the TGFb binding domain is an anti- TGFb scFv.

[0110] In some embodiments of either of the above two aspects, the IL2R binding domain comprises the N-terminus of IL2. In some embodiments of either of the above two aspects, the IL2R binding domain comprises the C-terminus of IL2.

[0111] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises an IFNg binding domain linked to an IL10R binding domain, wherein the IL10R binding domain induces a cellular effector function upon binding to IL1 OR; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding to IFNg. See, e.g., the drawings and examples herein.

[0112] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair. In some embodiments, the pair of complementary polypeptides is a pair of Fc fragments. In some embodiments, the pair of Fc fragments is a knob-into-hole pair. In some embodiments, the two macromolecules are identical.

[0113] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises an IFNg binding domain linked to an IL10R binding domain, wherein the IL10R binding domain induces a cellular effector function upon binding to IL1 OR; wherein the two macromolecules are not conjugated to each other in the absence of IFNg; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding IFNg. In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of I FNg, and the non-covalent conjugation is mediated by IFNg. See, e.g., the drawings and examples herein.

[0114] In some embodiments, the macromolecule or one or both members of pair of macromolecules comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0115] In some embodiments of either of the above two aspects, the IFNg binding domain is an anti- IFNg scFv.

[0116] In some embodiments of either of the above two aspects, the IL10R binding domain comprises an anti-IL1 OR VHH.

[0117] In some embodiments of either of the above two aspects, the IL10R binding domain comprises IL10.

[0118] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises an IL6 binding domain linked to an IL10R binding domain, wherein the IL10R binding domain induces a cellular effector function upon binding to IL1 OR; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding to IL6. See, e.g., the drawings and examples herein.

[0119] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair. In some embodiments, the pair of complementary polypeptides is a pair of Fc fragments. In some embodiments, the pair of Fc fragments is a knob-into-hole pair. In some embodiments, the two macromolecules are identical.

[0120] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises an IL6 binding domain linked to an IL10R binding domain, wherein the IL10R binding domain induces a cellular effector function upon binding to IL1 OR; wherein the two macromolecules are not conjugated to each other in the absence of IL6; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding IL6. In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of IL6, and the non-covalent conjugation is mediated by IL6. See, e.g., the drawings and examples herein.

[0121] In some embodiments, the macromolecule or one or both members of pair of macromolecules comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0122] In some embodiments of either of the above two aspects, the IL6 binding domain is an anti- IL6 VHH.

[0123] In some embodiments of either of the above two aspects, the IL10R binding domain comprises an anti-IL1 OR VHH.

[0124] In some embodiments of either of the above two aspects, the IL10R binding domain comprises IL10. In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises an IL8 binding domain linked to a CD3 binding domain, wherein the CD3 binding domain induces a cellular effector function upon binding to CD3; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding to IL8. See, e.g., the drawings and examples herein.

[0125] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair. In some embodiments, the pair of complementary polypeptides is a pair of Fc fragments. In some embodiments, the pair of Fc fragments is a knob-into-hole pair. In some embodiments, the two macromolecules are identical.

[0126] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises an IL8 binding domain linked to a CD3 binding domain, wherein the CD3 binding domain induces a cellular effector function upon binding to CD3; wherein the two macromolecules are not conjugated to each other in the absence of IL8; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding IL8. In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of IL8, and the non-covalent conjugation is mediated by IL8. See, e.g., the drawings and examples herein.

[0127] In some embodiments, the macromolecule or one or both members of pair of macromolecules comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0128] In some embodiments of either of the above two aspects, the IL8 binding domain is an anti-IL8 scFv.

[0129] In some embodiments of either of the above two aspects, the CD3 binding domain comprises an anti-CD3 VH.

[0130] In another aspect, the disclosure provides a macromolecule comprising a VEGF binding domain linked to a 4-1 BB binding domain. See, e.g., the drawings and examples herein.

[0131] In some embodiments, the macromolecule comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0132] In some embodiments, the VEGF binding domain is an anti-VEGF scFv. In some embodiments, the VEGF binding domain is a VEGF receptor trap.

[0133] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB scFv.

[0134] In some embodiments, the macromolecule comprises SEQ ID NOs: 36 and 38.

[0135] In some embodiments, the macromolecule comprises SEQ ID NOs: 37 and 38.

[0136] In some embodiments, the macromolecule comprises SEQ ID NOs: 36 and 39.

[0137] In some embodiments, the macromolecule comprises SEQ ID NOs: 37 and 39.

[0138] In another aspect, the disclosure provides a macromolecule comprising a TGFb binding domain linked to a TpoR binding domain. See, e.g., the drawings and examples herein. In some embodiments, the macromolecule comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0139] In some embodiments, the TGFb binding domain is an anti-TGFb scFv.

[0140] In some embodiments, the TpoR binding domain is an anti-TpoR scFv.

[0141] In another aspect, the disclosure provides a macromolecule comprising an IL-8 binding domain linked to a TpoR binding domain. See, e.g., the drawings and examples herein.

[0142] In some embodiments, the macromolecule comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0143] In some embodiments, the IL-8 binding domain is an anti-IL8 scFv.

[0144] In some embodiments, the TpoR binding domain is an anti- TpoR scFv.

[0145] In another aspect, the disclosure provides a macromolecule comprising a TGFb binding domain linked to an IL2R binding domain. See, e.g., the drawings and examples herein.

[0146] In some embodiments, the macromolecule comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0147] In some embodiments, the TGFb binding domain is an anti- TGFb scFv.

[0148] In some embodiments, the IL2R binding domain comprises the N-terminus of IL2. In some embodiments of either of the above two aspects, the IL2R binding domain comprises the C-terminus of IL2.

[0149] In another aspect, the disclosure provides a macromolecule comprising an IFNg binding domain linked to an IL10R binding domain. See, e.g., the drawings and examples herein.

[0150] In some embodiments, the macromolecule comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0151] In some embodiments, the IFNg binding domain is an anti- IFNg scFv.

[0152] In some embodiments, the IL10R binding domain comprises an anti-IL1 OR VHH.

[0153] In some embodiments of either of the above two aspects, the IL10R binding domain comprises IL10.

[0154] In another aspect, the disclosure provides a macromolecule comprising an IL6 binding domain linked to an IL10R binding domain. See, e.g., the drawings and examples herein.

[0155] In some embodiments, the macromolecule comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0156] In some embodiments, the IL6 binding domain is an anti- IL6 VHH.

[0157] In some embodiments, the IL10R binding domain comprises an anti-IL1 OR VHH.

[0158] In some embodiments, the IL10R binding domain comprises IL10.

[0159] In another aspect, the disclosure provides a macromolecule comprising an IL8 binding domain linked to a CD3 binding domain. See, e.g., the drawings and examples herein.

[0160] In some embodiments, the macromolecule comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or a fragment thereof.

[0161] In some embodiments, the IL8 binding domain is an anti-l L8 scFv.

[0162] In some embodiments, the CD3 binding domain comprises an anti-CD3 VH. In some aspects, the disclosure provides a composition, wherein the composition comprises a macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids provided herein.

[0163] In some embodiments, the composition comprises a macromolecule complex described herein.

[0164] In some embodiments, the composition comprises a macromolecule described herein.

[0165] In some aspects, the disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids provided herein and a pharmaceutically acceptable excipient.

[0166] In some embodiments, the pharmaceutical composition comprises a macromolecule complex described herein and a pharmaceutically acceptable excipient.

[0167] In some embodiments, the pharmaceutical composition comprises a macromolecule described herein and a pharmaceutically acceptable excipient.

[0168] In some aspects, the disclosure provides method for preparing a pharmaceutical composition, the method comprising: combining a macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids provided herein with a pharmaceutically acceptable excipient, thereby preparing the pharmaceutical composition.

[0169] In some embodiments, the pharmaceutical composition comprises a macromolecule complex described herein and a pharmaceutically acceptable excipient.

[0170] In some embodiments, the pharmaceutical composition comprises a macromolecule described herein and a pharmaceutically acceptable excipient.

[0171] In another aspect, the disclosure provides a met hod comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects access to a cell.

[0172] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for providing access to a cell.

[0173] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in providing access to a cell.

[0174] In another aspect, the disclosure provides a method of modulating the state of a cell, comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects access to the cell, thereby modulating the state of the cell.

[0175] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for modulating the state of a cell.

[0176] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in modulating the state of a cell.

[0177] In another aspect, the disclosure provides a method of inducing a cellular effector function in a cell, comprising providing the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects access to the cell, thereby inducing the cellular effector function in the cell.

[0178] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for inducing a cellular effector function in a cell.

[0179] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in inducing a cellular effector function in a cell.

[0180] In some embodiments, the cell is in a subject and the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition is administered in a therapeutically effective amount.

[0181] In some embodiments, the subject has, or is suspected of having, a disease or disorder characterized by abnormal levels of the disease signature target, optionally wherein the subject was previously determined to have abnormal levels of the disease signature target.

[0182] In another aspect, the disclosure provides a method of determining the state of a cell, comprising providing the macromolecule complex, or macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects access to the cell, and detecting the presence of the reporter domain, thereby determining the state of the cell.

[0183] In another aspect, the disclosure provides the use of the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for determining the state of a cell.

[0184] In another aspect, the disclosure provides the macromolecule complex, macromolecule, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition of any one of the above aspects for use in determining the state of a cell.

[0185] In another aspect, the disclosure provides a method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule complex comprising two macromolecules, each comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two copies of the macromolecule are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each copy of the macromolecule binding the disease signature ligand.

[0186] In another aspect, the disclosure provides the use of a macromolecule complex comprising two macromolecules, each comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two copies of the macromolecule are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each copy of the macromolecule binding the disease signature ligand in the manufacture of a medicament for inducing a cellular effector function in a cell.

[0187] In another aspect, the disclosure provides a macromolecule complex comprising two macromolecules, each comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two copies of the macromolecule are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each copy of the macromolecule binding the disease signature ligand for use in inducing a cellular effector function in a cell.

[0188] In another aspect, the disclosure provides a method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule complex comprising a pair of macromolecules, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample; (b) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1 ) that specifically binds a first effector ligand in the biological sample; and (c) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the pair of macromolecules binding the disease signature ligand.

[0189] In another aspect, the disclosure provides the use of a macromolecule complex comprising a pair of macromolecules, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample; (b) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1 ) that specifically binds a first effector ligand in the biological sample; and (c) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the pair of macromolecules binding the disease signature ligand in the manufacture of a medicament for inducing a cellular effector function in a cell.

[0190] In another aspect, the disclosure provides a macromolecule complex comprising a pair of macromolecules, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample; (b) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1 ) that specifically binds a first effector ligand in the biological sample; and (c) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the pair of macromolecules binding the disease signature ligand for use in inducing a cellular effector function in a cell.

[0191] In another aspect, the disclosure provides a method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule comprising two FBDs linked to two SBDs by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; and (b) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand.

[0192] In another aspect, the disclosure provides the use of a macromolecule comprising two FBDs linked to two SBDs by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; and (b) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand in the manufacture of a medicament for inducing a cellular effector function in a cell.

[0193] In another aspect, the disclosure provides a macromolecule comprising two FBDs linked to two SBDs by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; and (b) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand for use in inducing a cellular effector function in a cell.

[0194] In another aspect, the disclosure provides a method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule comprising two FBDs linked to an SBD1 and an SBD2 by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; (b) the SBD1 specifically binds a first effector ligand in the biological sample; and (c) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand.

[0195] In another aspect, the disclosure provides the use of a macromolecule comprising two FBDs linked to an SBD1 and an SBD2 by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; (b) the SBD1 specifically binds a first effector ligand in the biological sample; and (c) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand in the manufacture of a medicament for inducing a cellular effector function in a cell.

[0196] In another aspect, the disclosure provides a macromolecule comprising two FBDs linked to an SBD1 and an SBD2 by linker domains, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; (b) the SBD1 specifically binds a first effector ligand in the biological sample; and (c) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand for use in inducing a cellular effector function in a cell.

[0197] Other features and advantages of the invention will be apparent from the following Detailed Description and the Claims.

[0198] Definitions

[0199] As used herein, the term “macromolecule” refers to a large molecule (e.g., a molecule with a size greater than 1000 Daltons (1 kDa)) comprising one or more polypeptide, oligonucleotide, chemical, lipid, and / or carbohydrate moieties. In some embodiments, the macromolecule is a recombinant protein (e.g., a fusion protein).

[0200] As used herein, the term “multimer” refers to a molecule made up of at least two subunits (e.g., at least two subunits comprising one or more polypeptide, oligonucleotide, chemical, lipid, and / or carbohydrate moieties). Multimers include homomultimers and heteromultimers. A “homomultimer” is a multimer consisting of two or more identical or substantially identical subunits (e.g., two or more macromolecules). Homomultimers include homodimers (comprising two identical or substantially identical subunits), homotrimers (comprising three identical or substantially identical subunits), and homotetramers (comprising four identical or substantially identical subunits). “Substantially identical subunits” include subunits having differences in amino acid sequences that do not significantly affect the function of the subunit, e.g., that do not significantly affect the affinity of the subunit for one or more ligands. A “heteromultimer” is a multimer consisting of two or more non-identical subunits. Heteromultimers include heterodimers (comprising a first and a second subunit, e.g., comprising a pair of non-identical macromolecules), heterotrimers (comprising one copy of a first subunit and two copies of a second subunit), and homotetramers (comprising two copies or versions of each of a first and a second subunit). Multimers further include higher-order multimers, e.g., hexamers, heptamers, octamers, nonamers, and decamers.

[0201] As used herein, the term “binding domain” refers to any domain that has specific affinity for a ligand. Binding domains include, without limitation, polypeptides (e.g., an antibody or a fragment thereof (e.g., an scFv, a monospecific tandem scFv (taFv), a bispecific taFv, a VHH, a VNAR, a Fab, a monospecific single-chain diabody, a bispecific single-chain diabody, or a dual-affinity re-targeting antibody (DART)), an antibody mimetic (e.g., an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, a lipocalin, an avimer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monobody, a fibronectin type III domain (FN3)-based binder, a nanoantibody, a nanoCLAMP, an optimer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR)), an endogenous binding domain or a variant or derivative thereof (e.g., a cell receptor domain, an enzyme domain, a variable lymphocyte receptor (VLR) domain, a receptor ectodomain, a nuclear hormone receptor ligand-binding domain, a DNA-binding domain, or a receptor trap))), oligonucleotides (e.g., a nucleic acid aptamer (e.g., a DNA aptamer)), and chemical molecules, as well as combinations thereof.

[0202] As used herein, the term “ligand” refers to any moiety for which a binding domain as described herein may have affinity. Ligands include, without limitation, a chemical moiety, a portion of a molecule, a molecule (e.g., an allergen or a toxin), a macromolecule (e.g., a polypeptide, a nucleic acid, or carbohydrate), a post-translational modification state of a macromolecule (e.g., a macromolecule that is phosphorylated, glycosylated, acylated, alkylated, and the like), a higher-order macromolecular structure (e.g., a complex of two or more polypeptides), a cell (e.g., a cancer cell), a portion of a cell (e.g., a tumor antigen), a receptor on the surface of a cell, a pathogen (e.g., a virus or a portion or a virus; a bacterium or a portion of a bacterium; a fungus or a portion of a fungus; or a parasite or a portion of a parasite), or a tissue-type.

[0203] The term “disease signature ligand” refers to a ligand that is associated with a disease state or a disorder of a cell, tissue, or subject (e.g., mammal, e.g., human). Disease signature ligands may be proteins, e.g., soluble proteins, insoluble proteins, monomeric proteins, and multimeric proteins. Disease signature ligands include, without limitation, cell surface receptors, cell surface antigens, membranebound proteins, extracellular matrix components, integrins, cytokines, neurotransmitters, anti-drug antibodies (ADAs), autoantibodies, nucleic acids, carbohydrates, lipids, peptides, nucleosides, hormones, viruses, bacteria, fungi, or a fragment or antigen thereof.

[0204] The term “effector ligand” refers to a ligand that is capable of effecting a cellular effector function upon being bound by a binding domain, e.g., a multimer of the invention. Disease signature ligands include proteins and peptides, e.g., cell-surface receptors (e.g., catalytic receptors, e.g., a receptor tyrosine kinase (RTK), a receptor serine / threonine kinase (RSK), a type 1 cytokine receptor, a type 2 cytokine receptor, a tumor necrosis factor (TNF) superfamily receptor (e.g., TNFR2 or 4-1 BB), or a nuclear hormone receptor).

[0205] As used herein, the term “associated with” a disease, disorder, or condition refers to a relationship, either causative or correlative, between an entity and the occurrence or severity of a disease, disorder, or condition in a subject. For example, if a target is associated with a disease, disorder, or condition, the target may be the causative agent of the disease, disorder, or condition. For example, a virus may be the causative agent in a viral infection, bacteria may be the causative agent in a bacterial infection, a fungus may be the causative agent in a fungal infection, or a parasite may be the causative agent in a parasitic infection, a cancer cell may be the causative agent of a cancer, a toxin may be the causative agent of toxicity, or an allergen may the causative agent of an allergic reaction. The target associated with a disease, disorder, or condition may also or alternately be correlated with an increased likelihood of occurrence or an increased severity of a disease disorder, or condition.

[0206] As used herein, the term “carrier” means a compound, composition, reagent, or molecule that facilitates the stability, transport or delivery of a composition (e.g., a macromolecule or pair or macromolecules as described herein) into a subject, a tissue, or a cell. Non-limiting examples of carriers include carbohydrate carriers (e.g., an anhydride-modified phytoglycogen or glycogen-type material), nanoparticles (e.g., a nanoparticle that encapsulates or is covalently linked binds to the circular or linear polyribonucleotide), liposomes, fusosomes, ex vivo differentiated reticulocytes, exosomes, protein carriers (e.g., a protein covalently linked to the polyribonucleotide), and cationic carriers (e.g., a cationic lipopolymer or transfection reagent).

[0207] As used herein, the terms “disease,” “disorder,” and “condition” each refer to a state of sub- optimal health, for example, a state that is or would typically be diagnosed or treated by a medical professional.

[0208] The term “pharmaceutically acceptable excipient” as used herein means a pharmaceutically acceptable material, or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material useful for formulating a macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids for medicinal or therapeutic use.

[0209] The term “polynucleotide” as used herein means a molecule comprising one or more nucleic acid subunits, or nucleotides, and can be used interchangeably with “nucleic acid” or “oligonucleotide”. A polynucleotide can include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), or variants thereof. A nucleotide can include a nucleoside and at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (PO3) groups. A nucleotide can include a nucleobase, a five- carbon sugar (either ribose or deoxyribose), and one or more phosphate groups. Ribonucleotides are nucleotides in which the sugar is ribose. Polyribonucleotides or ribonucleic acids, or RNA, can refer to macromolecules that include multiple ribonucleotides that are polymerized via phosphodiester bonds. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose. The polynucleotides provided herein may include one or more modified nucleotides.

[0210] Polydeoxyribonucleotides or deoxyribonucleic acids, or DNA, means macromolecules that include multiple deoxyribonucleotides that are polymerized via phosphodiester bonds. A nucleotide can be a nucleoside monophosphate or a nucleoside polyphosphate. A nucleotide means a deoxyribonucleoside polyphosphate, such as, e.g., a deoxyribonucleoside triphosphate (dNTP), which can be selected from deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP) and deoxythymidine triphosphate (dTTP) dNTPs, and may include detectable tags, such as luminescent tags or markers (e.g., fluorophores). One or more of the nucleotides may be a modified nucleotide. A nucleotide can include any subunit that can be incorporated into a growing nucleic acid strand. Such subunit can be an A, C, G, T, or U, or any other subunit that is specific to one or more complementary A, C, G, T or U, or complementary to a purine (i.e. , A or G, or variant thereof) or a pyrimidine (i.e. , C, T or U, or variant thereof). In some examples, a polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or derivatives or variants thereof. In some cases, a polynucleotide is a short interfering RNA (siRNA), a microRNA (miRNA), a plasmid DNA (pDNA), a short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (pre-mRNA), antisense RNA (asRNA), to name a few, and encompasses both the nucleotide sequence and any structural embodiments thereof, such as singlestranded, double-stranded, triple-stranded, helical, hairpin, etc. In some cases, a polynucleotide molecule is circular (e.g., a circular RNA). A polynucleotide can have various lengths. A nucleic acid molecule can have a length of at least about 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3, kb, 4 kb, 5 kb, 10 kb, 50 kb, or more. A polynucleotide can be isolated from a cell or a tissue. As embodied herein, the polynucleotide sequences may include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and synthetic DNA / RNA analogs.

[0211] As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural, including D, L, or a combination thereof) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a single molecule or may be a multi- molecular complex such as a dimer, trimer, or tetramer. They can also comprise single chain or multichain polypeptides such as antibodies or insulin and can be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0212] As used herein, the term “sequence identity” is determined by alignment of two peptide or two nucleotide sequences using a global or local alignment algorithm. Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) 18 (proteins) and gap extension penalty = 3 (nucleotides) 12 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121 -3752 USA, or EmbossWin version 2.10.0 (using the program “needle”). Alternatively or additionally, percent identity may be determined by searching against databases, using algorithms such as FASTA, BLAST, etc. Sequence identity refers to the sequence identity over the entire length of the sequence.

[0213] A “signal sequence” or “leader sequence” refers to a polypeptide sequence, e.g., between 10 and 30 amino acids in length, that is present at the N-terminus of a polypeptide sequence of a nascent protein which targets the polypeptide sequence to the secretory pathway.

[0214] As used herein, the term “specifically binds” refers to a preferential interaction between a binding domain and its target or ligand (such as binding between an antibody and an antigen or epitope) that may be determinative of the presence of the target or ligand in the presence of a heterogeneous population of molecules including biological molecules. For example, a binding domain that specifically binds to a ligand (e.g., an antibody that specifically binds to an antigen or epitope) may be a binding domain that binds this ligand with greater affinity, avidity, more readily, and / or with greater duration than it binds to other ligands. In one aspect, the extent of binding of a binding domain to an unrelated molecule (nonligand) is less than about 10% of the binding of the binding domain to the ligand as measured using an appropriate assay. In certain aspects, a binding domain that specifically binds to a ligand has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM. Specific binding can include, but does not require exclusive binding. In one aspect, “specific binding” refers to binding wherein a binding domain binds to a particular ligand (e.g., a polypeptide or antigen or epitope on a particular polypeptide) without substantially binding to any other ligand (e.g., polypeptide or polypeptide antigen or epitope).

[0215] As used herein, the term “treat,” or “treating,” refers to a therapeutic treatment of a disease or disorder (e.g., an infectious disease, a cancer, a toxicity, or an allergic reaction) in a subject. The effect of treatment can include reversing, alleviating, reducing severity of, curing, inhibiting the progression of, reducing the likelihood of recurrence of the disease or one or more symptoms or manifestations of the disease or disorder, stabilizing (i.e. , not worsening) the state of the disease or disorder, and / or preventing the spread of the disease or disorder as compared to the state and / or the condition of the disease or disorder in the absence of the therapeutic treatment.

[0216] BRIEF DESCRIPTION OF THE DRAWINGS

[0217] Fig. 1 is a schematic diagram showing the domain structure of a fusion protein for exerting an effector function on a second target (e.g., a biological effector ligand) conditional on the presence of a first target (e.g., a disease signal ligand). From N- to C-terminus, the fusion protein includes a leader polypeptide; a single-chain variable fragment (scFv) comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) connected by a (G4S)3 linker, wherein the VH and VL domains have affinity for the first target; a (G4S)n linker; VH and VL domains having affinity for the second target after multimerization with a second molecule having the same VH and VL domains, wherein the VH and VL domains are connected by a G4S linker; and a FLAG affinity tag.

[0218] Fig. 2 is a schematic diagram showing the domain structure of a pair of fusion proteins for exerting an effector function on a second target (e.g., a biological effector ligand) conditional on the presence of a first target (e.g., a disease signal ligand). From N- to C-terminus, the fusion proteins include a leader polypeptide; a FLAG affinity tag; a scFv comprising a VH domain and a VL domain connected by a (648)3 linker, wherein the VH and VL domains have affinity for the first target; a (G4S)n linker; and a conditional effector domain A (in the first fusion protein) or a conditional effector domain B (in the second fusion protein).

[0219] Fig. 3 is a schematic diagram showing the domain structure of a pair of fusion proteins for exerting an effector function on a second target (e.g., a biological effector ligand) conditional on the presence of a first target (e.g., a disease signal ligand). From N- to C-terminus, the fusion protein includes a leader polypeptide; a scFv comprising a VH domain and a VL domain connected by a (G4S)3 linker, wherein the VH and VL domains have affinity for the first target; a (G4S)n linker; a VHH having affinity for a first moiety of the second target (in the first fusion protein (“anti-a”)) or a VHH having affinity for a second moiety of the second target (in the second fusion protein (“anti-b”)); and a FLAG affinity tag.

[0220] Fig. 4 is a schematic diagram showing the domain structure of a pair of fusion proteins for exerting an effector function on a second target (e.g., a biological effector ligand) conditional on the presence of a first target (e.g., a disease signal ligand). From N- to C-terminus, the fusion proteins include a leader polypeptide; a VH domain (in the first fusion protein) or a VL domain (in the second fusion protein) of an scFv, wherein the VH and VL domains of the scFv have affinity for the second target when assembled as a functional scFv, wherein the VH and VL domains have low affinity for one another; a GS(G4S)3linker; a scFv comprising a VH domain and a VL domain connected by a (648)3 linker, wherein the VH and VL domains have affinity for the first target; and a FLAG affinity tag.

[0221] Fig. 5A is a schematic diagram showing the domain structure of a fusion protein that modulates the TPO receptor (TpoR) conditional on the presence of TGF-beta1 (TGFb). From N- to C-terminus, the fusion protein includes a mouse immunoglobulin kappa variable 3 (Ms IgKVIll) leader polypeptide; an anti-TGF-beta1 scFv comprising a VH and a VL connected by a (648)3 linker; a (G4S)n linker; an anti- TpoR scFv comprising a VH and a VL connected by a (648)3 linker; and a FLAG affinity tag.

[0222] Fig. 5B is a schematic diagram showing two copies of the fusion protein of Fig. 5A bound to TGFb and TpoR. The membrane orientation and downstream signaling partners of TpoR are shown.

[0223] Fig. 5C is a graph showing secreted alkaline phosphatase (SEAP) activity (gray) in the culture supernatant of TpoR SEAP reporter cells that were stimulated overnight with a constant amount of the fusion protein of Fig. 5A and varying concentrations of TGFb. TPO native cytokine is shown as a positive control for constitutive activity (black).

[0224] Fig. 6A is a schematic diagram showing the domain structure of a fusion protein that modulates TpoR conditional on the presence of interleukin-8 (IL-8). From N- to C-terminus, the fusion protein includes a Ms IgKVIll leader polypeptide; an anti-IL-8 scFv comprising a VH and a VL connected by a (G4S)3 linker; a (G4S)n linker; an anti-TpoR scFv comprising a VH and a VL connected by a (G4S)3 linker; and a FLAG affinity tag.

[0225] Fig. 6B is a schematic diagram showing two copies of the fusion protein of Fig. 6A bound to IL-8 and TpoR. The membrane orientation and downstream signaling partners of TpoR are shown.

[0226] Fig. 6C is a graph showing SEAP activity (gray) in the culture supernatant of TpoR SEAP reporter cells that were stimulated overnight with a constant amount of the fusion protein of Fig. 6A and varying concentrations of IL-8. TPO native cytokine is shown as a positive control for constitutive activity (black).

[0227] Fig. 7A is a schematic diagram showing the domain structure of a pair of complementary fusion proteins (fusion proteins (a) and (b)) that modulate human interleukin-2 receptor (IL-2R) activity conditional on the presence of TGFb. Fusion protein (a) includes, from N- to C-terminus, a Ms IgKVIll leader peptide; an anti-TGF-beta1 scFv comprising a VH and a VL connected by a (G4S)3 linker; a (G4S)n linker; and a conditional IL-2R effector domain A (IL-2 (N-term)). Fusion protein (b) includes, from N- to C-terminus, a Ms IgKVIll leader peptide; an anti-TGF-beta1 scFv comprising a VH and a VL connected by a (G4S)3 linker; a (G4S)n linker; and a conditional IL-2R effector domain B (IL-2 (C-term)).

[0228] Fig. 7B is a schematic diagram showing fusion proteins (a) and (b) of Fig. 7A bound to the IL-2R receptor components IL2Rp and IL2RyC, respectively, in the absence (left) or presence (right) of TGFb. The membrane orientation and downstream signaling partners of IL-2R are shown.

[0229] Fig. 7C is a graph showing SEAP activity in the culture supernatant of IL-2 SEAP reporter cells that were stimulated overnight with a constant amount of fusion proteins (a) and (b) of Fig. 7A and varying concentrations of TGFb (gray squares). IL-2 cytokine is shown as a positive control for constitutive activity (black circles). TGFp cytokine is shown as a negative control for conditional activity (gray triangles).

[0230] Fig. 8A is a schematic diagram showing the domain structure of a pair of fusion proteins for exerting an effector function on a second target (e.g., a biological effector ligand) conditional on the presence of a first target (e.g., a disease signal ligand), in which from N- to C-terminus, the fusion protein includes a leader polypeptide; a scFv comprising a VH domain and a VL domain connected by a (G4S)3 linker, wherein the VH and VL domains have affinity for the first target; a (G4S)n linker; a VHH having affinity for a first moiety of the second target (in the first fusion protein (“anti-a”)) or a VHH having affinity for a second moiety of the second target (in the second fusion protein (“anti-b”)); and a FLAG affinity tag (left); and a set of schematic diagrams showing such a pair of fusion proteins in which the first target is IFN-gamma (IFNg) and the second target is the IL-10 receptor (IL-1 OR) (right).

[0231] Fig. 8B is a schematic diagram showing the domain structure of a pair of fusion proteins for exerting an effector function on a second target (e.g., a biological effector ligand) conditional on the presence of a first target (e.g., a disease signal ligand), in which the pair of fusion proteins self-assemble as heterodimers, in which from N- to C-terminus, the fusion protein includes a leader polypeptide; a VHH having affinity for a first moiety of the second target (in the first fusion protein; “anti-a”) or a VHH having affinity for a second moiety of the second target (in the second fusion protein; “anti-b”); a (G4S)2 linker; a scFv comprising a VH domain and a VL domain connected by a (G4S)3 linker, wherein the VH and VL domains have affinity for the first target; a (G4S)n linker; a human IgG 1 fragment crystallizable region (Fc region) with “knob” mutations (in the first fusion protein) or a human IgG 1 Fc region with “hole” mutations (in the second fusion protein) and a 6x histidine (His) affinity tag (left) and schematic diagrams showing such a pair of fusion proteins in which the second target is IL-1 OR (right).

[0232] Fig. 8C is a schematic diagram showing the domain structure of a fusion protein for exerting an effector function on a second target (e.g., a biological effector ligand) conditional on the presence of a first target (e.g., a disease signal ligand), in which from N- to C-terminus, the fusion protein includes a leader polypeptide; a VHH having affinity for a first moiety of the second target (“anti-a”); a (648)2 linker; a scFv comprising a VH domain and a VL domain connected by a (648)3 linker, wherein the VH and VL domains have affinity for the first target; a (64S)n linker; a second copy of the scFv comprising a VH domain and a VL domain connected by a (648)3 linker, wherein the VH and VL domains have affinity for the first target; a (64S)n linker; a VHH having affinity for a second moiety of the second target (in the second fusion protein (“anti-b”); and a polyhistidine affinity tag (top left); (b) a schematic diagram showing a fusion protein as in (a), wherein the order of the anti-a and anti-b VHH domains is reversed (bottom left); and schematic diagrams showing such fusion proteins in which the second target is IL-1 OR (right).

[0233] Fig. 8D is a schematic diagram showing the pair of fusion proteins of Fig. 8A bound to IFNg and to the IL-10 receptor components IL-1 ORa and IL-1 ORb. The membrane orientation and downstream signaling partners of IL-1 OR are shown.

[0234] Fig. 8E is a graph showing SEAP activity in the culture supernatant of IL-10 SEAP reporter cells that were stimulated overnight with 10 nM of the pair of fusion proteins of Fig. 8A and varying concentrations of IFNg (gray squares). IL-10 cytokine is shown as a positive control for constitutive activity (black circles). IFNg cytokine is shown as a negative control for conditional activity (gray triangles).

[0235] Fig. 8F is a pair of schematic diagrams showing variants of the pair of fusion proteins of Fig. 8B in which the first target is IFNg, the second target is IL-1 OR, and the linker between the Fc hinge and the IFNg-binding scFv consists of 5 amino acid residues (P482+P483; left) or 10 amino acid residues (P484+P485; right).

[0236] Fig. 8G is a graph showing SEAP activity in the culture supernatant of IL-10 SEAP reporter cells that were stimulated overnight with a constant amount of the fusion proteins of Fig. 8F and varying concentrations of IFNg.

[0237] Fig. 9A is a schematic diagram showing the domain structure of a pair of complementary fusion proteins that modulate IL-1 OR activity conditional on the presence of IL-6. The first fusion protein includes, from N- to C-terminus, a mouse IgKVIll leader peptide; an anti-IL10Ra VHH antibody; a (648)2 linker; a first anti-IL-6 VHH antibody; a (64S)n linker; and a polyhistidine tag. The second fusion protein includes, from N- to C-terminus, a mouse IgKVIll leader peptide; an anti-IL1 ORb VHH antibody; a (648)2 linker; a second anti-IL-6 VHH antibody; a (64S)n linker; and a polyhistidine tag.

[0238] Fig. 9B is a schematic diagram showing the pair of fusion proteins of Fig. 9A bound to IL-6 and to the IL-10 receptor components IL-1 ORa and IL-1 ORb. The membrane orientation and downstream signaling partners of IL-1 OR are shown.

[0239] Fig. 9C is a graph showing SEAP activity in the culture supernatant of IL-10 SEAP reporter cells that were stimulated overnight with a constant amount of the pair of fusion proteins of Fig. 9A and varying concentrations of IL-6 (gray circles). IL-10 cytokine is shown as a positive control for constitutive activity (black squares). IL-6 cytokine is shown as a negative control for conditional activity (gray triangles).

[0240] Fig. 10A is a schematic diagram showing the domain structure of a pair of complementary fusion proteins that modulate cluster of differentiation 3 (CD3) activity conditional on the presence of IL-8. The first fusion protein includes, from N- to C-terminus, a mouse IgKVIll leader peptide; an anti-CD3 VH domain of an scFv; a GS(G4S) linker; an anti-IL-8 scFv; and a FLAG affinity tag. The second fusion protein includes, from N- to C-terminus, a mouse IgKVIll leader peptide; an anti-CD3 VL domain of an scFv; a GS(G4S) linker; an anti-IL-8 scFv; and a FLAG affinity tag.

[0241] Fig. 10B is a schematic diagram showing the pair of fusion proteins of Fig. 10A bound to IL-8 and CD3. The membrane orientation and downstream signaling partners of CD3 are shown.

[0242] Fig. 10C is a bar graph showing luciferase activity (via NFAT-luc reporter assay) in the culture supernatant of NFAT-luc Jurkat reporter cells that were stimulated for 24 hours with a constant amount of the pair of fusion proteins of Fig. 10A in the presence or absence of 25nM IL-8. The full-length reference anti-CD3 scFv is shown as a positive control for constitutive activity.

[0243] Fig. 11 A is a schematic diagram showing the domain structure of a pair of fusion proteins that modulate CD3 activity conditional on the presence of IL-8, in which the pair of fusion proteins selfassemble as heterodimers, in which from N- to C-terminus, the first fusion protein includes a leader polypeptide; an anti-CD3 VH domain of an scFv; a GS(G4S) linker; an anti-IL-8 scFv; a (G4S)n linker; a Fc region with “knob” mutations; and a polyhistidine tag and the second fusion protein includes a leader polypeptide; an anti-CD3 VL domain of an scFv; a GS(G4S) linker; an anti-IL-8 scFv; a (G4S)n linker; a Fc region with “hole” mutations; and a polyhistidine tag.

[0244] Fig. 11 B is a schematic diagram the domain structure of a pair of fusion proteins that modulate CD3 activity conditional on the presence of IL-8, in which from N- to C-terminus, the fusion protein includes a leader polypeptide; a VH domain having affinity for a first moiety of CD3; a (G4S)2 linker; a scFv comprising a VH domain and a VL domain connected by a (G4S)3 linker, wherein the VH and VL domains have affinity for IL-8; a (G4S)n linker; a second copy of the scFv comprising a VH domain and a VL domain connected by a (G4S)3 linker, wherein the VH and VL domains have affinity for IL-8; a (G4S)n linker; a VL domain having affinity for a second moiety of CD3; and a polyhistidine affinity tag (top); and a schematic diagram showing a fusion protein as in (a), wherein the order of the anti-CD3 VH and VL domains is reversed (bottom).

[0245] Fig. 12A is a schematic diagram showing a modified version of the pair of fusion proteins shown in Figs. 8B and 8F in which the first target is I FNg , the second target is IL-1 OR, and the linker length between the Fc hinge and the I FNg-binding scFv has been modulated.

[0246] Fig. 12B is a schematic diagram showing a modified version of the pair of fusion proteins shown in Figs. 8B and 8F in which the first target is I FNg , the second target is IL-1 OR, and the linker length between the IFNg-binding scFv and the IL1 OR-binding VHH has been modulated.

[0247] Fig. 12C is a schematic diagram showing a modified version of the pair of fusion proteins shown in Figs. 8B and 8F in which the first target is I FNg , the second target is IL-1 OR, and the IL1 OR-binding VHH has been substituted with a scFv agonist against IL-1 OR. Fig. 12D is a schematic diagram showing a modified version of the pair of fusion proteins shown in Figs. 8B and 8F in which the first target is I FNg , the second target is IL-1 OR, and the IL1 OR-binding VHH has been substituted with a monomeric IL-10 cytokine.

[0248] Fig. 12E is a schematic diagram showing a modified version of the pair of fusion proteins shown in Figs. 8B and 8F in which the first target is I FNg , the second target is IL-1 OR, and the knob-into-hole Fc regions and the I FNg-binding scFv have been replaced with a knob-into-hole full-length IgG having an IFNg-binding Fab region.

[0249] Fig. 12F is a schematic diagram showing a modified version of the pair of fusion proteins shown in Figs. 8B and 8F in which the first target is I FNg , the second target is IL-1 OR, and the fusion protein is fused to a full-length IgG antibody at the N-terminus of the heavy chain.

[0250] Fig. 12G is a schematic diagram showing a modified version of the pair of fusion proteins shown in Figs. 8B and 8F in which the first target is I FNg , the second target is IL-1 OR, and the fusion protein is fused to a full-length IgG antibody at the N-terminus of the light chain.

[0251] Fig. 12H is a schematic diagram showing a modified version of the pair of fusion proteins shown in Figs. 8B and 8F in which the first target is I FNg , the second target is IL-1 OR, and the fusion protein is fused to a full-length IgG antibody at the C-terminus of the light chain.

[0252] Fig. 121 is a schematic diagram showing a modified version of the pair of fusion proteins shown in Figs. 8B and 8F in which the first target is I FNg , the second target is IL-1 OR, and the fusion protein is fused to a full-length IgG antibody at the C-terminus of the heavy chain Fc region.

[0253] Fig. 13A is a schematic diagram showing the domain structure of a fusion protein that modulates 4-1 BB activity conditional on the presence of VEGF (VEGF / 4-1 BB fusion protein constructs), in which from N- to C-terminus, the fusion protein includes a leader polypeptide; an anti-4-1 BB scFv; a (G4S)n linker; an anti-IL-8 scFv; a VEGF binder; and an Fc region.

[0254] Fig. 13B is a schematic diagram showing the fusion proteins of Fig. 13A in unbound format and bound to VEGF and to a 4-1 BB receptor on the surface of a T cell. Left: monovalent construct in which the fusion protein comprises a monomeric Fc region. Right: bivalent construct in which the fusion protein comprises an Fc region capable of dimerization.

[0255] Fig. 14A is a graph showing fold induction of luminescence in Jurkat T cells that express 4-1 BB and a NFkB-driven luciferase reporter gene that were treated with a VEGF / 4-1 BB fusion protein construct (“fusion protein construct”) comprising a urelumab-based effector domain, a VEGF receptor trap-based binding domain, and a monovalent Fc region alone or in the presence of human VEGF-A, mouse VEGF-A, or mouse VEGF-B. A urelumab reference monoclonal antibody (mAb) is shown as a control.

[0256] Fig. 14B is a graph showing fold induction of luminescence in Jurkat T cells that express 4-1 BB and a NFkB-driven luciferase reporter gene that were treated with a VEGF / 4-1 BB fusion protein construct comprising a urelumab-based effector domain, a VEGF receptor trap-based binding domain, and a divalent Fc region alone or in the presence of human VEGF-A or mouse VEGF-A. A urelumab reference monoclonal antibody (mAb) is shown as a control.

[0257] Fig. 15A is a graph showing fold induction of luminescence in Jurkat T cells that express 4-1 BB and a NFkB-driven luciferase reporter gene that were treated with a VEGF / 4-1 BB fusion protein construct comprising a utomilumab-based effector domain, a VEGF binding domain (anti-VEGF scFv), and a monovalent Fc region alone or in the presence of human VEGF-A or mouse VEGF-A. A utomilumab reference mAb is shown as a control.

[0258] Fig. 15B is a graph showing fold induction of luminescence in Jurkat T cells that express 4-1 BB and a NFkB-driven luciferase reporter gene that were treated with a VEGF / 4-1 BB fusion protein construct comprising a utomilumab-based effector domain, a VEGF binding domain (receptor trap; “VEGF-trap"), and a divalent Fc region alone or in the presence of human VEGF-A or mouse VEGF-A. A utomilumab reference mAb is shown as a control.

[0259] Fig. 16A is a schematic diagram showing the design of a lymphopenia-induced proliferation study designed to assess T cell expansion in vivo following intravenous (IV) administration of a VEGF / 4-1 BB fusion protein construct (“4-1 BB SB”) in the presence or absence of VEGF. CFSE: carboxyfluorescein succinimidyl ester.

[0260] Fig. 16B is a plot showing the percentage of T cells in each of the indicated treatment groups (see Table 4) that were undergoing fast lymphopenia-induced proliferation (LIP) at Day 7, as measured by dilution of CFSE dye.

[0261] Fig. 16C is a plot showing the percentage of T cells derived from the donor population that were effector T cells following treatment in each of the indicated treatment groups (see Table 4).

[0262] Fig. 16D is a plot showing the percentage of donor-derived T cells that were naive T cells in each of the indicated treatment groups (see Table 4).

[0263] Fig. 17A is a schematic diagram showing the design of a tumor efficacy study designed to assess the anti-tumor activity of the VEGF / 4-1 BB fusion protein construct anti-4-1 BBAx VEGF-Trap (Bi) (“VEGF-4-1 bb SB”) at doses of 10, 3, 1 , and 0.5 milligrams per kilogram of body weight (MPK) in mice having MC38 tumors.

[0264] Fig. 17B is a plot showing tumor size (in mm3) over time in mice having MC38 tumors that were treated with various doses of anti-4-1 BBAx VEGF-Trap (Bi), a urelumab comparator, or a hlgG4 isotype control.

[0265] Fig. 17C is a set of stacked bar graphs showing the proportion of mice that experienced a partial response (PR) (black bar; tumor size was smaller than average tumor size in the isotype control group) or a complete response (CR) (gray bar; mouse became tumor-free after treatment) at 17 days after treatment with anti-4-1 BBAx VEGF-Trap (Bi) (“SB”), a urelumab comparator, PD1 blockade (“PD1 ”), or PD1 blockade in combination with anti-4-1 BBAx VEGF-Trap (Bi) (low dose) at the indicated doses.

[0266] Fig. 18 is a plot showing tumor size (in mm3) over time in mice that had had MC38 tumors, became tumor-free following treatment with anti-4-1 BBAx VEGF-Trap (Bi) or a urelumab comparator, and were re-challenged with a lethal dose of MC38 tumor cells as compared to treatment-naive mice.

[0267] Fig. 19A is a schematic diagram showing mice that have been implanted with either a “cold” tumor line that does not secrete human IFNg or a “hot” tumor line that secretes human IFNg. Both tumor lines carry a pSTAT3-driven luciferase reporter system capable of reading out IL-1 OR signaling activity.

[0268] Fig. 19B is a schematic diagram showing the design of an assay for conditional IL-1 OR activity. NSG mice were inoculated with hot or cold tumor cells. When tumors reached 5 mm diameter, an IFNg / IL-1 OR fusion protein construct was injected. IL-1 OR activity was assessed by luciferase injection 24 hours after administration of the IFNg I IL-10 fusion protein construct. A constitutively active IL-10 protein was provided as a control.

[0269] Fig. 20A is a bar graph showing quantification of luciferase signal in mice bearing an IFNg- positive “hot” tumor or an IFNg-negative “cold” tumor that were treated with an IFNg I IL-10 fusion protein construct.

[0270] Fig. 20B is a bar graph showing quantification of luciferase signal in mice bearing an IFNg- positive “hot” tumor or an IFNg-negative “cold” tumor that were treated with a constitutively active bivalent IL-10 construct.

[0271] Fig. 21 A is a pair of graphs showing the results of dimeric anti-4-1 BBAx VEGF-trap (Bi) construct purification. The top graph shows that the protein product from the first peak was 96.5% monodisperse and eluted at a size consistent with dimeric (-130 kDa) stoichiometry. The bottom graph shows the migration of gel filtration standards run to generate elution time estimates for various protein sizes. POI: Peak of interest.

[0272] Fig. 21 B is an image showing dimeric stoichiometry of dimeric anti-4-1 BBAx VEGF-trap (Bi) construct confirmed by SDS-PAGE. Under non-reducing conditions (lane “NR”), the macromolecule complex runs as a dimer. Under reducing conditions (lane “R”), the macromolecule complex collapses to its constituent monomeric subunits, as expected. Estimated monomer size is ~65kDa, and dimer is -130 kDa. The molecular weight standard is in the left-hand lane.

[0273] DETAILED DESCRIPTION

[0274] Featured herein are macromolecules that conditionally induce a cellular effector function (e.g., a biological or therapeutic activity) based on the presence of a disease signature ligand, multimers thereof, compositions comprising the same, and methods of using the same.

[0275] I. Compositions

[0276] A. Macromolecules and macromolecule complexes i. Homomultimeric macromolecule complexes

[0277] In one aspect, provided herein is a macromolecule complex comprising two macromolecules, each comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand (e.g., the macromolecule complex does not induce effector function in the absence of the disease signature ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0278] Exemplary first binding domains and disease signature ligands are provided in Section IB herein. Exemplary second binding domains and effector ligands are provided in Section IC herein. In some embodiments, the conjugation between the two macromolecules is covalent. For example, in some embodiments, the two macromolecules are conjugated by a chemical linker or a polypeptide linker. In other embodiments, the conjugation between the two macromolecules is non- covalent. For example, in some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, wherein each macromolecule comprises (e.g., is covalently or non-covalently linked to) one member of the pair. The pair of complementary moieties may be, e.g., biotin and avidin; barnase and barstar; a pair of complementary aptamers; or a pair of complementary polypeptides, (e.g., a pair of Fc fragments (e.g., a pair of engineered Fc fragments). For example, in some embodiments, the pair of complementary moieties is a knob-into-hole Fc pair (e.g., one of the macromolecules comprises (e.g., is covalently or non-covalently conjugated to) an Fc region with “knob” mutations and the other macromolecule comprises (e.g., is covalently or non-covalently conjugated to) an Fc region with “hole” mutations. Knob-into-hole Fc pairs are described, e.g., in Xu et al., mAbs, 7(1 ): 231 -242, 2015. As a general principle, the conjugation (e.g., covalent or non-covalent conjugation) facilitates concurrent binding of the disease signal and effector ligands conditional on the presence of the disease signature ligands, e.g., the linker or pair of complementary moieties is configured to sterically orient the two copies of the macromolecule to permit conditional concurrent binding of disease signal and effector ligands. In some embodiments, the non-covalent conjugation is mediated by the disease signature ligand (e.g., the two macromolecules are conjugated only in the presence of the disease signature ligand).

[0279] In another aspect, provided herein is a macromolecule complex comprising two macromolecules, each comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are not conjugated to each other in the absence of the disease signature ligand; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand (e.g., the macromolecule complex does not induce effector function in the absence of the disease signature ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand). In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of the disease signature ligand, and wherein the non-covalent conjugation is mediated by the disease signature ligand.

[0280] In some embodiments, the two macromolecules are identical (e.g., are identical in amino acid sequence and / or nucleotide sequence). Alternatively, the two macromolecules may be non-identical in sequence, but comprise substantially the same first binding domain and second binding domain. For example, the two macromolecules may comprise binding domains that are identical in sequence (e.g., amino acid sequence and / or nucleotide sequence) or that differ in sequence but have substantially the same affinity for the disease signature ligand or the effector ligand.

[0281] In some embodiments of the invention, the second binding domain is an antibody or antibody fragment that does not bind the effector ligand when the two macromolecules are not bound to the disease signature ligand. For example, in some embodiments, the second binding domain comprises heavy chain variable domains (VH domains) and light chain variable domains (VL domains) having affinity for the second target, wherein the VH and VL domains are connected by a short linker (e.g., G4S linker) that does not allow intra-chain pairing of the VH and VL domains. In some embodiments, the second binding domain is a diabody.

[0282] In one exemplary embodiment, each of the two macromolecules is a fusion protein comprising, from N- to C-terminus, a leader polypeptide; a first binding domain comprising a single-chain variable fragment (scFv) comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) connected by a (648)3 linker, wherein the VH and VL domains have affinity for the disease signal ligand; a (G4S)n linker; a second binding domain comprising VH and VL domains having affinity for the effector ligand, wherein the VH and VL domains are connected by a G4S linker; and a FLAG affinity tag.

[0283] In another aspect of the invention, provided herein is a complex comprising a macromolecule complex as described herein in complex with one or both of the disease signature ligand and the effector ligand.

[0284] In some embodiments, the macromolecules are polypeptides. In other embodiments, the macromolecules comprise one or more non-polypeptide components, e.g., comprises one or more nucleic acid or chemical components, as further described below.

[0285] In some embodiments, the macromolecules further comprise one or more of a leader domain (e.g., a leader polypeptide), one or more linker domains, and one or more reporter domains, as further described in Section 1 (E) below.

[0286] Further provided herein are macromolecule complexes comprising three or more (e.g., three, four, five, or more than five) macromolecules, each comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the three or more macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the three or more macromolecules binding the disease signature ligand (e.g., the macromolecule complex does not induce effector function in the absence of the disease signature ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0287] Heteromultimeric macromolecule complexes

[0288] In another aspect, provided herein is a macromolecule complex providing a pair of macromolecules, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample; (b) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1 ) that specifically binds a first effector ligand in the biological sample; and (c) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule is conditional upon each member of the pair of macromolecules binding the disease signature ligand (e.g., the macromolecule complex does not induce effector function in the absence of the disease signature ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0289] Exemplary first binding domains and disease signature ligands are provided in Section IB herein. Exemplary second binding domains and effector ligands are provided in Section IC herein.

[0290] In some embodiments, the conjugation between the two macromolecules is covalent. For example, in some embodiments, the two macromolecules are conjugated by a chemical linker or a polypeptide linker. In other embodiments, the conjugation between the two macromolecules is non- covalent. For example, in some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, wherein each macromolecule comprises (e.g., is covalently or non-covalently linked to) one member of the pair. The pair of complementary moieties may be, e.g., biotin and avidin; barnase and barstar; a pair of complementary aptamers; or a pair of complementary polypeptides, (e.g., a pair of Fc fragments (e.g., a pair of engineered Fc fragments). For example, in some embodiments, the pair of complementary moieties is a knob-into-hole Fc pair (e.g., one of the macromolecules comprises (e.g., is covalently or non-covalently conjugated to) an Fc region with “knob” mutations and the other macromolecule comprises (e.g., is covalently or non-covalently conjugated to) an Fc region with “hole” mutations. Knob-into-hole Fc pairs are described, e.g., in Xu et al., mAbs, 7(1 ): 231 -242, 2015. As a general principle, the conjugation (e.g., covalent or non-covalent conjugation) facilitates concurrent binding of the disease signal and effector ligands conditional on the presence of the disease signature ligands, e.g., the linker or pair of complementary moieties is configured to sterically orient the two copies of the macromolecule to permit conditional concurrent binding of disease signal and effector ligands. In some embodiments, the non-covalent conjugation is mediated by the disease signature ligand (e.g., the two macromolecules are conjugated only in the presence of the disease signature ligand).

[0291] In another aspect, provided herein is a macromolecule complex comprising a pair of macromolecules, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the first member of the pair of macromolecules comprises a first binding domain 1 (FBD1 ) that specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds a second epitope of the disease signature ligand in a biological sample; and (c) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are not conjugated to each other in the absence of the disease signature ligand; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand (e.g., the macromolecule complex does not induce effector function in the absence of the disease signature ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand). In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of the disease signature ligand, and wherein the non-covalent conjugation is mediated by the disease signature ligand.

[0292] In another aspect of the invention, provided herein is a complex comprising a macromolecule complex as described herein in complex with one, two, or all three of the disease signature ligand, the first effector ligand, and the second effector ligand.

[0293] In some embodiments, each member of the pair of macromolecules is a polypeptide. In other embodiments, one or both members of the pair of macromolecules comprises one or more nonpolypeptide components, e.g., comprises one or more nucleic acid or chemical components, as further described below.

[0294] In another aspect, provided herein is a macromolecule complex comprising a pair of macromolecules, each comprising a FBD linked to a SBD, wherein (a) the FBD of a first member of the pair of macromolecules specifically binds a first moiety of a disease signature ligand in a biological sample; (b) the FBD of a second member of the pair of macromolecules specifically binds a second moiety of the disease signature ligand in the biological sample; and (c) the SBD of each macromolecule specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule is conditional upon each member of the macromolecule complex binding the disease signature ligand (e.g., the macromolecule complex does not induce effector function in the absence of the disease signature ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0295] In another aspect of the invention, provided herein is a macromolecule complex comprising a set of three macromolecules, each comprising a FBD linked to a SBD, wherein (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample; (b) the first member of the set of macromolecules comprises a SBD1 that specifically binds a first effector ligand in the biological sample; (c) the second member of the set of macromolecules comprises a SBD2 that specifically binds a second effector ligand in the biological sample; and (d) the third member of the set of macromolecules comprises a second binding domain 3 (SBD3) that specifically binds a third effector ligand in the biological sample; wherein the SBD1 , SBD2, and SBD3 induce a cellular effector function upon binding to the first, second, and third effector ligands; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each member of the set of three macromolecules binding the disease signature ligand. In some embodiments, the disease signature ligand is trimeric.

[0296] In some embodiments, one or both members of the pair of macromolecules, or one, two, or all three of the set of three macromolecules, further comprise one or more of a leader domain (e.g., a leader polypeptide), one or more linker domains, and one or more reporter domains, as further described in Section 1 (E) below. iii. Macromolecules comprising two FBDs and two SBDs

[0297] In another aspect, provided herein is a macromolecule comprising two first binding domains (FBDs) linked to two second binding domains (SBDs), wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; and (b) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand (e.g., the macromolecule does not induce effector function in the absence of the disease signature ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0298] Exemplary first binding domains and disease signature ligands are provided in Section IB herein. Exemplary second binding domains and effector ligands are provided in Section IC herein.

[0299] In some embodiments, the two FBDs are identical (e.g., are identical in amino acid sequence and / or nucleotide sequence). Alternatively, the two FBDs may differ in sequence, but have substantially the same affinity for the disease signature ligand.

[0300] In some embodiments, the two SBDs are identical (e.g., are identical in amino acid sequence and / or nucleotide sequence). Alternatively, the two SBDs may differ in sequence, but have substantially the same affinity for the effector ligand.

[0301] In some embodiments of the invention, the SBDs are antibodies or antibody fragments that do not bind the effector ligand when the two FBDs of the macromolecule are not bound to the disease signature ligand. For example, in some embodiments, the SBD comprises heavy chain variable domains (VH domains) and light chain variable domains (VL domains) having affinity for the second target, wherein the VH and VL domains are connected by a short linker (e.g., G4S linker) that does not allow intra-chain pairing of the VH and VL domains. In some embodiments, the second binding domain is a diabody.

[0302] In another aspect of the invention, provided herein is a complex comprising a macromolecule as described herein in complex with one or both of the disease signature ligand and the effector ligand.

[0303] In some embodiments, the macromolecule is a polypeptide (e.g., comprises a single polypeptide chain). In other embodiments, the macromolecule comprises one or more non-polypeptide components, e.g., comprises one or more nucleic acid or chemical components, as further described below.

[0304] In some embodiments, the macromolecule further comprises one or more of a leader domain (e.g., a leader polypeptide), one or more linker domains, and one or more reporter domains, as further described in Section 1 (E) below. iv. Macromolecules comprising two FBDs, an SBD1, and an SBD2

[0305] In another aspect, provided herein is a macromolecule comprising two first binding domains (FBDs) linked to two non-identical second binding domains (SBDs), e.g., a first second binding domain (SBD1 ) and a second second binding domain (SBD2), wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; (b) the SBD1 specifically binds a first effector ligand in the biological sample; and (c) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand (e.g., the macromolecule does not induce effector function in the absence of the disease signature ligand; does not substantially induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0306] Exemplary first binding domains and disease signature ligands are provided in Section IB herein. Exemplary second binding domains and effector ligands are provided in Section IC herein.

[0307] In some embodiments of the invention, the SBDs (e.g., SBD1 and / or SBD2) are antibodies or antibody fragments that do not bind the effector ligand when the two FBDs of the macromolecule are not bound to the disease signature ligand. For example, in some embodiments, the SBDs (e.g., SBD1 and / or SBD2) comprise heavy chain variable domains (VH domains) and light chain variable domains (VL domains) having affinity for the second target, wherein the VH and VL domains are connected by a short linker (e.g., G4S linker) that does not allow intra-chain pairing of the VH and VL domains. In some embodiments, the SBDs (e.g., SBD1 and / or SBD2) are diabodies.

[0308] In another aspect of the invention, provided herein is a complex comprising a macromolecule as described herein in complex with one or both of the disease signature ligand and the effector ligand.

[0309] In some embodiments, the macromolecule is a polypeptide (e.g., comprises a single polypeptide chain). In other embodiments, the macromolecule comprises one or more non-polypeptide components, e.g., comprises one or more nucleic acid or chemical components, as further described below.

[0310] In some embodiments, the macromolecule further comprises one or more of a leader domain (e.g., a leader polypeptide), one or more linker domains, and one or more reporter domains, as further described in Section 1 (E) below. v. Nucleic acids, vectors, and host cells

[0311] In another aspect of the invention, provided herein are one or more nucleic acids (e.g., one or more RNA molecules or DNA molecules) encoding one or more of any of the macromolecules described above (e.g., a pair of nucleic acids (e.g., a pair of RNA molecules or a pair of DNA molecules) encoding any of the pairs of macromolecules described above). The one or more nucleic acids may be circular or linear. The one or more nucleic acids may be formulated with a carrier and / or a delivery platform, e.g., a lipid-based carrier (e.g., a lipid nanoparticle (LNP)) and / or a vector delivery system (e.g., an adenovirus, an adeno-associated virus (AAV), an anellovirus, or a lentivirus). Further examples of lipid-based carriers that may be used in the invention are provided in Section l(l) herein. For example, in some aspects, provided herein are one or more nucleic acids (e.g., one or more RNA molecules or DNA molecules, e.g., circular or linear RNA molecules or DNA molecules) encoding one or more of any of the macromolecules described above, wherein the one or more nucleic acids are formulated with a carrier, e.g., a lipid carrier, e.g., a LNP. In some aspects, the nucleic acids include one or more modified nucleotides. Further provided herein are vectors (e.g., plasmids or viral vectors) comprising or encoding any of the above-described nucleic acids. The vector may be formulated with a carrier, e.g., a carrier appropriate for delivery to a target cell (e.g., a mammalian cell), such as, for example, a lipid-containing carrier, such as an LNP-containing formulation.

[0312] Further provided herein are host cells that have been modified to comprise the above-described nucleic acids or vectors. Suitable host cells include bacterial and eukaryotic cells (e.g., mammalian cells). In some embodiments, a nucleic acid or vector as described herein is manufactured in and isolated from a host cell.

[0313] B. Disease signature ligands and first binding domains

[0314] / . Disease signature ligands

[0315] The disease signature ligand bound by the macromolecule, pair of macromolecules, or macromolecule complex may be any moiety (e.g., protein, peptide, or small molecule) associated with a disease state or a disorder of a cell, tissue, or subject (e.g., mammal, e.g., human).

[0316] In some embodiments, the disease signature ligand is a protein. In some embodiments, the protein is a soluble protein or an insoluble protein. For example, the disease signature ligand may be present in solution in the biological sample (e.g., may be present in the extracellular space) or may be embedded in a membrane present in the biological sample (e.g., may be embedded in a cell membrane).

[0317] In some embodiments, the disease signature ligand is a cell surface receptor (e.g., HER2), a cell surface antigen (e.g., prostate specific membrane antigen (PSMA)), a membrane-bound protein (e.g., ASCT2), an extracellular matrix component (e.g., fibronectin or collagen), or an integrin.

[0318] In some embodiments, the disease signature ligand is a multimeric protein (e.g., a homomultimeric protein), e.g., a dimeric, trimeric, or tetrameric protein (e.g., an immunologically active multimeric protein). For example, in some embodiments comprising a single species of macromolecule capable of forming a dimer in the presence of the disease signature ligand (e.g., embodiments as described in Section I A(i) herein), the disease signature ligand may be a dimer, such that each macromolecule binds one member of the dimerized disease signature ligand. For example, in some embodiments comprising two identical or substantially identical first binding domains (FBDs) (e.g., embodiments as described in Sections I A(i) and IA(iii) herein), the disease signature ligand may be a dimer, such that each macromolecule binds one member of the dimerized disease signature ligand.

[0319] In other embodiments, the disease signature ligand is a monomeric protein.

[0320] In some embodiments, the disease signature ligand is a cytokine. In some embodiments, the cytokine is an interleukin (e.g., IL-1 , IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15, IL-17, or IL-23); an interferon (e.g., IFN-gamma); a growth factor (e.g., transforming growth factor beta (TGF-beta), granulocyte colonystimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GCSF), epidermal growth factor (EGF), or erythropoietin (EPO)); a chemokine (e.g., monocyte chemoattractant protein-1 (MCP-1 ) or interferon gamma-induced protein 10 (IP-10; also called CXCL10); or a member of the TNF family (e.g., TNF-alpha). In some aspects, the cytokine is multimerized (e.g., dimerized, trimerized, or tetramerized). In some embodiments, the disease signature ligand is a neurotransmitter (e.g., serotonin, dopamine, or histamine). In some embodiments, the disease signature ligand is VEGF (e.g., VEGF-A or VEGF-B).

[0321] In some embodiments, the disease signature ligand is a self antigen of an organism from which the biological sample is derived (e.g., a self antigen produced by a mammalian subject, e.g., a human subject). In some embodiments, the self antigen is an anti-drug antibody (ADA), e.g., an ADA that targets a therapeutic agent used for treatment of the disease state or a disorder of the cell, tissue, or subject. In some embodiments, the self antigen is an autoantibody. In some embodiments, the self antigen is a cancer antigen, e.g., a tumor marker.

[0322] In some embodiments, the disease signature ligand is a nucleic acid.

[0323] In some embodiments, the disease signature ligand is a carbohydrate, a lipid, a peptide, a nucleoside, or a combination of the foregoing.

[0324] In some embodiments, the disease signature ligand is a hormone (e.g., a peptide / protein hormone (e.g., insulin, oxytocin, or a growth hormone)), an amino acid derivative (e.g., melatonin or thyroxine), a steroid (e.g., a glucocorticoid), or an eicosanoid (e.g., prostaglandin).

[0325] In some embodiments, the disease signature ligand is a non-self antigen, i.e., an antigen of an organism other than the one from which the biological sample is derived. In some embodiments, the disease signature ligand is a virus, a bacterium, a fungus, or a fragment or antigen thereof (e.g., a virus, bacterium, or fungus that causes a disease state or a disorder of the cell, tissue, or subject or a fragment thereof).

[0326] / / . Disease signature ligand binding domains (first binding domains)

[0327] Each of the macromolecules provided herein comprises at least one first binding domain (FBD) that specifically binds to the disease signature ligand (e.g., binds to a disease signature ligand as described in Section I B(i) , above). In some embodiments, the first binding domain permits binding of an additional binding domain to the disease signature ligand (e.g., is designed or selected such that at least two copies of the first binding domain can bind to the disease signature ligand and / or such that the first binding domain of each member of a pair of macromolecules can bind to the disease signature ligand).

[0328] In some embodiments, the first binding domain comprises a polypeptide that specifically binds the disease signature ligand.

[0329] In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is an scFv, a monospecific tandem scFv (taFv), a bispecific taFv, a VHH, a VNAR, a Fab, a monospecific single-chain diabody, a bispecific single-chain diabody, or a dual-affinity re-targeting antibody (DART).

[0330] In some embodiments, the polypeptide is an antibody mimetic. In some embodiments, the antibody mimetic is an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, a lipocalin, an avimer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monobody, a fibronectin type III domain (FN3)-based binder, a nanoantibody, a nanoCLAMP, an optimer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR).

[0331] In some embodiments, the polypeptide is an endogenous binding domain of an organism from which the biological sample and / or the disease signature ligand is derived, e.g., a binding domain that is naturally produced by the organism. In some embodiments, the endogenous binding domain is a cell receptor domain, an enzyme domain, a variable lymphocyte receptor (VLR) domain, a receptor ectodomain, a nuclear hormone receptor ligand-binding domain, or a DNA-binding domain. For example, in some embodiments, the first binding domain is a polypeptide that comprises or consists of a receptor for a cytokine (e.g., a multimerized cytokine, e.g., a dimeric, trimeric, or tetrameric cytokine) or is a polypeptide that comprises or consists of a receptor for an immunologically active multimer (e.g., an immunologically active dimer, trimer, or tetramer). In some embodiments, the disease signature ligand is IL-6 and the first binding domain is a polypeptide comprising the IL-6 receptor. In some embodiments, the disease signature ligand is TNF-alpha and the first binding domain is a polypeptide comprising the TNF-alpha receptor. In some embodiments, the disease signature ligand is VEGF and the first binding domain comprises a receptor trap derived from the VEGF receptor VEGFR1 or VEGFR2 (e.g. aflibercept).

[0332] In some embodiments, the first binding domain comprises an oligonucleotide that specifically binds the disease signature ligand. In some embodiments, the oligonucleotide is a nucleic acid aptamer (e.g., a DNA aptamer).

[0333] In some embodiments, the first binding domain comprises a chemical molecule that specifically binds the disease signature ligand.

[0334] Additional binding domains that may be used in the invention are described, e.g., in Zhong and D’Antona, Antibodies, 10(2): 13, 2021.

[0335] In some embodiments, the disease signature ligand-binding domain has an affinity (KD value) for the disease signature ligand of >10 to >100 pM, <10 nM, >10 nM, or >100 nM or has micromolar affinity for the disease signature ligand (e.g., KD of < 1 pM). In some aspects, the disease signature ligandbinding domain binds the disease signature ligand with a KD of 1 nM or lower.

[0336] In some embodiments of any of the macromolecules provided herein, the first binding domain has affinity to two or more disease signature moieties. For example, the first binding domain may comprise at least two binding moieties as described above, wherein the at least two binding moieties specifically bind to at least two different disease signature moieties.

[0337] In some embodiments comprising pairs of macromolecules (e.g., embodiments as described in Sections I A(i) and IA(ii) herein), the first and second members of the pair of macromolecules comprise first binding domains that are identical in sequence (e.g., amino acid sequence and / or nucleotide sequence) or that differ in sequence, but have substantially the same affinity for the disease signature ligand or the effector ligand. Alternatively, in other embodiments, the first and second members of the pair of macromolecules comprise different first binding domains. For example, in some embodiments, the first binding domain of a first member of the pair of macromolecules specifically binds a first epitope or moiety of a disease signature ligand in a biological sample, and the second binding domain of a second member of the pair of macromolecules specifically binds a second epitope or moiety of the disease signature ligand in the biological sample.

[0338] For example, in some embodiments, the disclosure provides a macromolecule complex comprising two macromolecules, each comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein: (a) the first member of the pair of macromolecules comprises a first binding domain 1 (FBD1 ) that specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds a second epitope of the disease signature ligand in a biological sample; and (c) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand.

[0339] In a further example, in some embodiments, the disclosure provides a macromolecule complex comprising a pair of macromolecules, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the first member of the pair of macromolecules comprises a first binding domain 1 (FBD1 ) that specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds a second epitope of the disease signature ligand in a biological sample; (c) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1 ) that specifically binds a first effector ligand in the biological sample; and (d) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the pair of macromolecules binding the disease signature ligand.

[0340] Similarly, in some embodiments comprising macromolecules comprising two FBDs (e.g., embodiments as described in Sections IA(iii) and IA(iv) herein), the two FBDs are identical in sequence (e.g., amino acid sequence and / or nucleotide sequence) or that differ in sequence, but have substantially the same affinity for the disease signature ligand or the effector ligand. Alternatively, in other embodiments, the macromolecule comprises two different FBDs. For example, in some embodiments, the macromolecule comprises a first FBD that specifically binds a first epitope or moiety of a disease signature ligand in a biological sample and a second FBD that specifically binds a second epitope or moiety of the disease signature ligand in the biological sample.

[0341] For example, in some embodiments, the disclosure provides a macromolecule comprising an FBD1 and an FBD2 linked to two SBDs, wherein (a) the FBD1 specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the FBD2 specifically binds a second epitope of the disease signature ligand in the biological sample; and (c) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the FBD1 and the FBD2 binding the disease signature ligand.

[0342] In a further example, in some embodiments, the disclosure provides a macromolecule comprising an FBD1 and an FBD2 linked to an SBD1 and an SBD2, wherein (a) the FBD1 specifically binds a first epitope of a disease signature ligand in a biological sample; (b) the FBD2 specifically binds a second epitope of the disease signature ligand in the biological sample; (c) the SBD1 specifically binds a first effector ligand in the biological sample; and (d) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs (FBD1 and FBD2) binding the disease signature ligand.

[0343] In embodiments comprising non-identical first binding domains (e.g., a first binding domain 1 (FBD1 ) and a first binding domain 2 (FBD2)), the first binding domains may bind to different epitopes of the disease signature ligand, e.g., partially overlapping epitopes or non-overlapping epitopes, e.g., such that the two first binding domains do not sterically hinder each other from binding their respective epitopes on the disease signature ligand; only partially sterically hinder each other from binding their respective epitopes on the disease signature ligand; or do not completely hinder each other from binding their respective epitopes on the disease signature ligand. Accordingly, in some aspects, the first binding domains are able to concurrently bind the disease signature ligand.

[0344] In some embodiments comprising non-identical first binding domains (e.g., an FBD1 and an FBD2), the two first binding domains have substantially similar affinities for their respective epitopes (e.g., affinities that differ by less than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%). In other embodiments, the FBD1 and FBD2 have substantially different affinities for their respective epitopes (e.g., affinities that differ by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, e.g., 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2500%, 5000%, 7500%, or 10000%).

[0345] In some embodiments in which the disease signature ligand is a polypeptide, the first binding domain binds to the disease signature ligand at a binding site that contains an Arg, Lys, Asp, His or Glu amino acid residue (or a combination thereof) (e.g., a binding site that is enriched for one or more of these residues).

[0346] In one embodiment, the disease signature ligand is TGFb.

[0347] In one embodiment, the disease signature ligand is VEGF.

[0348] In one embodiment, the disease signature ligand is IL-8.

[0349] In one embodiment, the disease signature ligand is IL-6.

[0350] In one embodiment, the disease signature ligand is IFNg.

[0351] In one embodiment, the disease signature ligand is IL-10 and the first binding domain is a polypeptide comprising the IL-10 receptor.

[0352] In one embodiment, the disease signature ligand is IL-12 and the first binding domain comprises the p40 subunit and / or the p35 subunit of IL-12.

[0353] In one embodiment, the disease signature ligand is VEGF and the first binding domain comprises (i) a VEGF-neutralizing antibody (e.g. bevacizumab) or (ii) a receptor trap derived from the VEGF receptor VEGFR1 or VEGFR2 (e.g. aflibercept).

[0354] C. Effector ligands and second binding domains / . Effector ligands

[0355] The effector ligand bound by the macromolecule, pair of macromolecules, or macromolecule complex may be any moiety (e.g., protein or peptide) that is present in the biological sample and is capable of effecting a cellular effector function upon being bound by a multimer of the invention.

[0356] In some embodiments, the effector ligand is a protein or a peptide.

[0357] In some embodiments, the effector ligand is a cell-surface receptor.

[0358] In some embodiments, the effector ligand is TpoR.

[0359] In some embodiments, the effector ligand is 4-1 BB.

[0360] In some embodiments, the effector ligand is IL2R.

[0361] In some embodiments, the effector ligand is IL10R.

[0362] In some embodiments, the effector ligand is CD3.

[0363] In some embodiments, the cell-surface receptor is a catalytic receptor, e.g., a receptor tyrosine kinase (RTK), a receptor serine / threonine kinase (RSK), a type 1 cytokine receptor, a type 2 cytokine receptor, or a tumor necrosis factor (TNF) superfamily receptor (e.g., TNFR2 or 4-1 BB). In some embodiments, the cell-surface receptor is 4-1 BB. In some embodiments, the cell-surface receptor is TpoR.

[0364] In some embodiments, the RTK is VEGFR, the RSK is TGFBR2, the type 1 cytokine receptor is IL2R, the type 2 cytokine receptor is IL10R, or the TNF superfamily receptor is TNFR2. In some embodiments, the receptor is CD3.

[0365] In some embodiments, the effector ligand is an intracellular receptor. In some embodiments, the intracellular receptor is a nuclear hormone receptor (e.g., a glucocorticoid receptor). In some embodiments in which the effector ligand is an intracellular receptor, the macromolecule is delivered as an RNA.

[0366] In some embodiments, the second binding domain (SBD) (e.g., SBD1 , SBD2, and / or SBD3) is an agonist of the effector ligand. In other embodiments, the second binding domain is an antagonist of the effector ligand.

[0367] In some embodiments, the effector ligand must be homodimerized to exert a cellular effector function. In some embodiments, the effector ligand is capable of homodimerization and exerts a cellular effector function with at least 2-fold, 5-fold, 10-fold, 10O-fold, or 10OO-fold greater strength in the homodimerized form as compared to a monomeric form. In some embodiments, the effector ligand is homodimerized in the presence of the macromolecule or macromolecule complex. In some embodiments, the effector ligand is activated by binding of the macromolecule or macromolecule complex in the absence of its endogenous ligand.

[0368] In some embodiments, the macromolecule or macromolecule complex exhibits conditional avidity, triggered avidity, and / or dimerization avidity. For example, in some aspects, a plurality of macromolecules or macromolecule complexes bind to a disease signature ligand (e.g., a tumor antigen or a pathogen surface marker), and the proximity results in increased avidity which then activates a potent downstream effect. In some embodiments comprising non-identical second binding domains (e.g., comprising a SBD1 and an SBD2) (e.g., embodiments as described in Sections IA(ii) and IA(iv) herein), the first effector ligand and the second effector ligand must be associated (e.g., in proximity and correctly oriented (e.g., heterodimerized)) to exert a cellular effector function. In some embodiments, the effector ligand is capable of heterodimerization and exerts a cellular effector function with at least 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold greater strength in the heterodimerized form as compared to a monomeric form. Accordingly, in some embodiments, the first effector ligand and the second effector ligand are associated (e.g., in proximity and correctly oriented (e.g., heterodimerized)) in the presence of the macromolecule or macromolecule complex.

[0369] In other embodiments comprising non-identical second binding domains (e.g., comprising a SBD1 and an SBD2) (e.g., embodiments as described in Sections IA(ii) and IA(iv) herein), the first effector ligand and the second effector ligand must be associated (e.g., in proximity and correctly oriented (e.g., heterodimerized)) and must further be associated with one or more additional moieties to exert a cellular effector function. For example, in some embodiments, the first effector ligand and the second effector ligand are members of a receptor complex comprising at least three members (e.g., a homotrimeric receptor complex, a heterotrimeric receptor complex, a homotetrameric receptor complex, or a heterotetrameric receptor complex).

[0370] In some embodiments of any of the macromolecules, pairs of macromolecules, and macromolecule complexes provided herein, the disease signature ligand is a soluble protein (e.g., a cytokine) and the effector ligand is a catalytic receptor (e.g., a catalytic receptor that exerts a cellular function upon multimerization (e.g., homomultimerization or heteromultimerization)).

[0371] Hi. Cellular effector functions

[0372] In some embodiments, the cellular effector function of the disease signature ligand is a biological activity.

[0373] In some embodiments, the cellular effector function of the disease signature ligand is a therapeutic activity.

[0374] In some embodiments, the cellular effector function of the disease signature ligand is a disease activity (e.g., an aberrant activity associated with a disease state), and the cellular effector function is repressed by binding of the multimerized macromolecule or pair of macromolecules to the effector ligand. iv. Effector ligand binding domains (second binding domains)

[0375] In some embodiments, the second binding domain (SBD) (e.g., SBD1 , SBD2, or SBD3) comprises a polypeptide that specifically binds the effector ligand. In some embodiments, the first binding domain permits binding of an additional binding domain to the disease signature ligand (e.g., is designed or selected such that at least two copies of the second binding domain can bind to the effector ligand).

[0376] In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is an scFv, a monospecific tandem scFv (taFv), a bispecific taFv, a VHH, a VNAR, a Fab, a monospecific single-chain diabody, a bispecific single-chain diabody, or a dual-affinity re-targeting antibody (DART). In some embodiments, the polypeptide is an antibody mimetic. In some embodiments, the antibody mimetic is an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, a lipocalin, an avimer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monobody, a fibronectin type III domain (FN3)-based binder, a nanoantibody, a nanoCLAMP, an optimer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR).

[0377] In some embodiments, the polypeptide is an endogenous binding domain. In some embodiments, the endogenous binding domain is a ligand of the effector ligand or a fragment thereof. In some embodiments, the endogenous binding domain is a viral binding protein or a fragment thereof.

[0378] In some embodiments, the second binding domain comprises an oligonucleotide that specifically binds the effector ligand. In some embodiments, the oligonucleotide is a nucleic acid aptamer (e.g., a DNA aptamer).

[0379] Additional binding domains that may be used in the invention are described, e.g., in Zhong and D’Antona, Antibodies, 10(2): 13, 2021.

[0380] In some embodiments, the effector ligand-binding domain has an affinity for the effector ligand that is similar to that of a native ligand of the effector ligand. In some embodiments, the effector ligandbinding domain has an affinity (KD value) for the effector ligand that is in the picomolar (pM) range or is <1 pM. In some embodiments, the effector ligand-binding domain has an affinity for the effector ligand that is 500 nM or lower. In some embodiments in which the effector ligand binding domain comprises two monomers, the affinity of each of the monomers for the effector ligand is 1 -2 orders of magnitude higher than the affinity of the effector ligand binding domain monomers for one another.

[0381] In some embodiments of any of the macromolecules provided herein, the second binding domain has affinity to two or more effector ligands. For example, the second binding domain may comprise at least two binding moieties as described above, wherein the at least two binding moieties specifically bind to at least two different effector moieties.

[0382] Pairs of second binding domains

[0383] In some embodiments comprising macromolecules or pairs of macromolecules comprising nonidentical second binding domains (e.g., comprising a SBD1 and an SBD2) (e.g., embodiments as described in Sections IA(ii) and IA(iv) herein), one of the two SBDs (e.g., the SBD1 ) specifically binds a first effector ligand in a biological sample, and the other SBD (e.g., SBD2) specifically binds a second effector ligand in the biological sample.

[0384] For example, in some embodiments, the SBD1 is a first portion of a binding moiety and the SBD2 is a second portion of the binding moiety.

[0385] In another example, one of the two SBDs (e.g., the SBD1 ) specifically binds to a first component of a heteromultimeric (e.g., heterodimeric) receptor and the other SBD (e.g., the SBD2) specifically binds to a second component of the heteromultimeric (e.g., heterodimeric) receptor.

[0386] In another example, one of the two SBDs (e.g., the SBD1 ) is a first component of a dimeric moiety and the other SBD (e.g., the SBD2) is a second component of a dimeric moiety. In another example, one of the two SBDs (e.g., the SBD1 ) is a first fragment of a polypeptide chain and the other SBD (e.g., the SBD2) is a second fragment of the polypeptide chain. In some embodiments, the polypeptide chain is a hormone, a cytokine, or a growth factor.

[0387] In some embodiments of any of the above examples, the non-identical second binding domains (e.g., SBD1 and an SBD2) have been engineered to have reduced affinity for one another. In some embodiments, the non-identical second binding domains (e.g., SBD1 and an SBD2) have an affinity (KD) for one another of >1 pM, e.g., >5-10 pM, but less than 1 mM (e.g., 10-200 pM).

[0388] In some embodiments of any of the macromolecules or macromolecule complexes described herein, the second binding domain comprises a conditional effector domain.

[0389] D. Pairs of first and second binding domains

[0390] In some embodiments, the disease signature ligand is TGFbeta and the effector ligand is the TPO receptor. For example, in some embodiments, the first binding domain comprises a TGFbeta- binding domain (e.g., an anti-TGF-beta1 antibody or antibody fragment, e.g., an anti-TGF-beta1 singlechain variable fragment (scFv)) and the second binding domain comprises a TPO receptor (TpoR) binding domain (e.g., an anti-TpoR antibody or antibody fragment, e.g., an anti-TpoR heavy chain variable domain (VH) and light chain variable domain (VL)).

[0391] In some embodiments, the disease signature ligand is IL-8 and the effector ligand is the TPO receptor. For example, in some embodiments, the first binding domain comprises an IL-8-binding domain (e.g., an anti-IL-8 antibody or antibody fragment, e.g., an anti-IL-8 scFv) and the second binding domain comprises a TpoR binding domain (e.g., an anti-TpoR antibody or antibody fragment, e.g., an anti-TpoR VH and VL).

[0392] In some embodiments, the disease signature ligand is TGFbeta and the effector ligand is the IL-2 receptor (IL-2R). For example, in some embodiments, the first binding domain comprises a TGFbeta- binding domain (e.g., an anti-TGF-beta1 antibody or antibody fragment, e.g., an anti-TGF-beta1 scFv) and the one or more second binding domains comprise an IL-2R binding domain (e.g., a fragment and / or modified version of IL-2). In some embodiments, the disclosure provides a macromolecule, macromolecule complex, or pair of macromolecules comprising two second binding domains comprising complementary fragments of IL-2R.

[0393] In some embodiments, the disease signature ligand is IL-8 and the effector ligand is the IL-2 receptor (IL-2R). For example, in some embodiments, the first binding domain comprises an IL-8-binding domain (e.g., an anti-IL-8 antibody or antibody fragment) and the one or more second binding domains comprise an IL-2R binding domain (e.g., a fragment and / or modified version of IL-2). In some embodiments, the disclosure provides a macromolecule, macromolecule complex, or pair of macromolecules comprising two second binding domains comprising complementary fragments of IL-2R.

[0394] In some embodiments, the disease signature ligand is IFN-gamma and the effector ligand is the IL-10 receptor. For example, in some embodiments, the first binding domain comprises an IFN-gamma- binding domain (e.g., an anti-IFN-gamma antibody or antibody fragment, e.g., an anti-IFN-gamma scFv) and the one or more second binding domains comprise an IL-10-receptor binding domain (e.g., an antibody or antibody fragment targeting one or more components of the IL-10 receptor). In some embodiments, the disclosure provides a macromolecule, macromolecule complex, or pair of macromolecules comprising two second binding domains targeting different components of the IL-10 receptor, e.g., IL-10Ra and IL-1 ORb.

[0395] In some embodiments, the disease signature ligand is IL-6 and the effector ligand is the IL-10 receptor. For example, in some embodiments, the first binding domain comprises an IL-6-binding domain (e.g., an anti-IL-6 antibody or antibody fragment, e.g., an anti-IL-6 VHH antibody) and the one or more second binding domains comprise an IL-10-receptor binding domain (e.g., an antibody or antibody fragment targeting one or more components of the IL-10 receptor). In some embodiments, the disclosure provides a macromolecule, macromolecule complex, or pair of macromolecules comprising two second binding domains targeting different components of the IL-10 receptor, e.g., IL-1 ORa and IL-1 ORb and / or two first binding domains targeting different epitopes of IL-6.

[0396] In some embodiments, the disease signature ligand is TGFbeta and the effector ligand is cluster of differentiation 3 (CD3). For example, in some embodiments, the first binding domain comprises a TGFbeta-binding domain (e.g., an anti-TGF-beta1 antibody or antibody fragment, e.g., an anti-TGF-beta1 single-chain variable fragment (scFv)) and the one or more second binding domains comprise a CD3- binding domain (e.g., an anti-CD3 antibody or antibody fragment, e.g., an anti-CD3 heavy chain variable domain (VH) and / or light chain variable domain (VL)). In some embodiments, the disclosure provides a macromolecule, macromolecule complex, or pair of macromolecules comprising two second binding domains comprising different components of a CD3-targeting moiety, e.g., respectively comprising an anti-CD3 VH and VL.

[0397] In some embodiments, the disease signature ligand is IL-8 and the effector ligand is CD3. For example, in some embodiments, the first binding domain comprises an IL-8-binding domain (e.g., an anti- IL-8 antibody or antibody fragment, e.g., an anti-IL-8 single-chain variable fragment (scFv)) and the one or more second binding domains comprise a CD3- binding domain (e.g., an anti-CD3 antibody or antibody fragment, e.g., an anti-CD3 heavy chain VH and / or VL). In some embodiments, the disclosure provides a macromolecule, macromolecule complex, or pair of macromolecules comprising two second binding domains comprising different components of a CD3-targeting moiety, e.g., respectively comprising an anti-CD3 VH and VL.

[0398] In some embodiments, the disease signature ligand-binding domain (first binding domain) is part of an inflammatory cytokine system and the effector ligand-binding domain (second binding domain) comprises IL-10 or a fragment and / or modified version thereof. In some aspects, the effector ligand is the IL-10 receptor.

[0399] In some embodiments, the disease signature ligand-binding domain (first binding domain) comprises TNFalpha, MCP-1 , or IL-12 or a fragment and / or modified version thereof and the effector ligand-binding domain (second binding domain) comprises IL-10 or a fragment and / or modified version thereof. In some aspects, the effector ligand is the IL-10 receptor.

[0400] In some embodiments, the disease signature ligand is VEGF and the effector ligand is 4-1 BB. For example, in some embodiments, the first binding domain comprises a VEGF binding domain (e.g., (i) a VEGF-neutralizing antibody (e.g. bevacizumab) or (ii) a receptor trap derived from the VEGF receptor VEGFR1 or VEGFR2 (e.g. aflibercept)) and the second binding domain comprises a 4-1 BB binding domain (e.g., an anti-4-1 BB antibody or antibody fragment, e.g., an anti-4-1 BB scFv). In some embodiments, the 4-1 BB binding domain is a scFv reformatted from a full-length agonistic mAb, such as urelumab or utomilumab (e.g., as shown in SEQ ID NO: 36 and SEQ ID NO: 37, respectively).

[0401] In one aspect, provided herein is a macromolecule complex comprising two macromolecules, each comprising a VEGF binding domain linked to a 4-1 BB binding domain, wherein the 4-1 BB binding domain induces a cellular effector function upon binding to 4-1 BB; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding to VEGF. In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair. In some embodiments, the pair of complementary polypeptides is a pair of Fc fragments. In some embodiments, the pair of Fc fragments is a knob-into-hole pair. In some embodiments, the two macromolecules are identical.

[0402] In another aspect, provided herein is a macromolecule complex comprising two macromolecules, each comprising a VEGF binding domain linked to a 4-1 BB binding domain, wherein the 4-1 BB binding domain induces a cellular effector function upon binding to 4-1 BB; wherein the two macromolecules are not conjugated to each other in the absence of the disease signature ligand; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding VEGF. In some embodiments, the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of the disease signature ligand, wherein the non-covalent conjugation is mediated by the disease signature ligand. In some embodiments, the macromolecule or one or both members of pair of macromolecules comprises a half-life extension moiety (e.g., an Fc domain or a fragment thereof).

[0403] In some embodiments of either of the above aspects, the VEGF binding domain is an anti-VEGF scFv (e.g., as provided in SEQ ID NO: 39). In some embodiments, the VEGF binding domain is a VEGF receptor trap (e.g., as provided in SEQ ID NO: 38). In some embodiments, the VEGF binding domain is (i) a VEGF-neutralizing antibody (e.g. bevacizumab) or (ii) a receptor trap derived from the VEGF receptor VEGFR1 or VEGFR2 (e.g. aflibercept).

[0404] In some embodiments of either of the above aspects, the 4-1 BB binding domain is an anti-4-1 BB scFv. In some embodiments, the 4-1 BB binding domain is a scFv reformatted from a full-length agonistic mAb, such as urelumab or utomilumab (e.g., as shown in SEQ ID NO: 36 and SEQ ID NO: 37, respectively).

[0405] Exemplary reference polypeptides (e.g., antibodies and antigen-binding fragments thereof) can be employed in the invention, including variants of a reference sequence. For example, a “variant of a TPO receptor (TpoR)-binding polypeptide,” a “variant of an IFN-gamma-binding polypeptide,” a “variant of an IL-1 Ob-binding polypeptide,” a “variant of a CD3-binding polypeptide,” a “variant of an IL-8-binding polypeptide,” a “variant of a TGFb-binding polypeptide,” a “variant of an IL6-binding polypeptide,” a “variant of a 4-1 BB-binding polypeptide,” (e.g., a “variant” in reference to a polypeptide provided by the invention), and the like, comprises an amino acid sequence with, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or more amino acid substitutions relative to the reference sequence (e.g., one of SEQ ID NOs: 1 -9, 12-14, and 32-43). In some embodiments, the variant retains a function of the disclosed polypeptide, e.g., in an assay provided herein, e.g., in an example provided herein.

[0406] In certain embodiments, a variant of a polypeptide provided by the invention includes up to 1 , 2, 3, 4, 5, 6, or 7 amino acid substitutions, e.g., 1 -3 amino acid substitutions. A variety of amino acid substitutions for variants of a polypeptide provided by the invention are possible, including substitution with non-canonical amino acids. In some embodiments, the variant of a polypeptide provided by the invention retains a function of the disclosed polypeptide, e.g., in an assay provided herein, e.g., in an example provided herein.

[0407] In some embodiments, a variant of a polypeptide comprises conservative substitutions or highly conservative substitutions, relative to the reference sequence. “Conservative substitutions” relative to a reference sequence means a given amino acid substitution has a value of 0 or greater in BLOSUM62.

[0408] “Highly conservative substitutions” relative to a reference sequence means a given amino acid substitution has a value of 1 or greater (e.g., in some embodiments, 2, or more) in BLOSUM62.

[0409] In certain embodiments, a variant of a polypeptide provided by the invention comprises the paratope of a polypeptide provided by the invention (e.g., an antibody or fragment thereof provided by the invention), where only highly conservative substitutions are made in the residues of the paratope (e.g., up to 1 , 2, 3, 4, or 5 substitutions in the paratope), while in certain embodiments, no substitutions are made in the paratope, and any substitutions are outside of the paratope and, in various embodiments, these non-paratope residues may be either highly conservative substitutions, conservative substitutions, or nonconservative substitutions, or a combination thereof. For example, in certain embodiments, any substitutions of non-paratope residues that are in the CDRs of a variable region are either conservative substitutions or, in some embodiments, highly conservative substitutions — e.g., in certain embodiments, the CDRs of a variant of a polypeptide may have up to 1 , 2, 3, 4, or 5 substitutions per CDR in residues other than those that make up the paratope, wherein the substitutions are conservative substitutions or, in more particular embodiments, highly conservative substitutions. In more particular embodiments, nonconservative substitutions can be made outside of the CDRs, although the skilled artisan will appreciate that substitutions outside of the CDRs may, in some embodiments, be conservative or highly conservative. In certain embodiments, a variant polypeptide provided by the invention, relative to a reference immunoglobulin light chain variable region or immunoglobulin heavy chain variable region is at least 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99%, or more, identical, with due regard for the various substitution criteria described above (e.g., substitutions relative to the paratope, CDRs, non-paratope residues, and non-CDRs residues).

[0410] E. Leader, reporter and linker moieties Leaders

[0411] In some embodiments of any of the compositions and methods provided herein, the macromolecule comprises a leader peptide, e.g., a leader peptide that targets the macromolecule for secretion. The leader peptide may be cleaved from the macromolecule prior to formation of the multimer. In some embodiments, the leader peptide is a mouse immunoglobulin kappa variable 3 (IgKVIll) leader peptide (e.g., UniProt ID A0A140T8P0 positions M1 to G20). Further exemplary leader sequences are provided in Table 1 (SEQ ID NOs: 15-31 ). Table 1. Leader sequences

[0412] Reporters In some embodiments of any of the compositions and methods provided herein, the macromolecule comprises a reporter moiety. For example, in embodiments comprising a pair of macromolecules, one or both members of the pair of macromolecules may comprise the reporter moiety, or the members of the pair of macromolecules may each comprise different reporter moieties.

[0413] Exemplary reporter moieties include, without limitation, affinity tags (e.g., FLAG affinity tags), fluorescent markers, and chromogenic markers. For example, in some embodiments, the reporter moiety is a near-infrared probe (e.g., indocyanine green (ICG) or methylene blue (MB)) or a near-infrared fluorescent protein or a fragment thereof. In other embodiments, the reporter moiety comprises a fragment of a bait protein and is detected by adding an exogenous dye that detects the bait protein.

[0414] In some embodiments comprising a pair of macromolecules, the first and second members of the pair of macromolecules comprise complementary reporter moieties, e.g., reporter moieties that are detectable (e.g., produce a fluorescent signal) when the first and second members of the pair of macromolecules form a multimer. For example, the first and second members of the pair of macromolecules may comprise members of a fluorescence resonance energy transfer (FRET) pair (e.g., a near-infrared FRET pair), e.g., a peptide-based or protein-based FRET pair.

[0415] Linkers

[0416] In some embodiments of any of the compositions and methods provided herein, the macromolecule comprises one or more linker domains, e.g., linker domains that connect the first binding domain to the second binding domain; connect one or more sub-domains within the first binding domain or the second binding domain; and / or connect the first binding domain or the second binding domain to a leader peptide or a reporter moiety.

[0417] In some embodiments, the one or more linker domains are peptide linkers. In some embodiments, the peptide linkers are GS linkers. In some embodiments, the peptide linkers are glycineserine (GS) linkers, e.g., GS linkers having the format GS(GnS)mor GS linkers having the format (GnS)m, e.g., wherein n = 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., n = 1 -5 or 5-10, e.g., n = 4) and m = 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 (e.g., m = 1 -5, 5-10, or 10-15, e.g., m = 5). In some embodiments, the peptide linkers are GS linkers having the format (G4S)n, e.g., wherein n = 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., n = 1 -5 or 5-10).

[0418] F. Half-life extension moieties

[0419] Any of the macromolecules (e.g., polypeptides) provided herein may be modified to alter (e.g., extend) their half-life (e.g., to alter (e.g., extend) their half-life (e.g., half, life in circulation (e.g., in serum)) and / or to elicit a desired effector function. For example, in some embodiments, any of the macromolecules provided herein may include a moiety (e.g., a heterologous moiety) that extends the halflife of the macromolecule. Exemplary half-life extension moieties include polypeptides (e.g., a fragment crystallizable region (Fc region) or a fragment or variant thereof or an albumin domain or a fragment or variant thereof) and non-polypeptide moieties (e.g., polyethylene glycol (PEG) or a modified derivative thereof).

[0420] In some aspects, a polypeptide provided herein is modified to include a Fc region that extends the half-life of the polypeptide relative to a version of the polypeptide not comprising the Fc region. In some embodiments, the Fc region is an IgG isotype Fc region, e.g., an IgG 1 , lgG2, or lgG4 subtype Fc region (e.g., such an Fc region from a human, a mouse, or a non-human primate (NHP)). In some embodiments, the Fc region comprises one or more Fc effector function-silencing mutations (e.g., LALA or LALAPG mutations (mutations in lgG1 at positions L234, L235, G236, N297, or P329)); in other aspects, the Fc region is capable of eliciting one or more Fc effector functions. The Fc region may be modified to extend half-life using one or more mutations that enhance neonatal Fc receptor (FcRn)-based recycling. Further Fc variants that may be used in the invention include mutated Fc variants previously described to alter Fc gamma receptor binding or Fc neonatal receptor binding and recycling and Fc variants comprising glycosylation modifications. Variant Fc regions that may be used in the invention include those provided in Saunders, Frontiers in Immunology, 10: Article 1296, 2019; Delidakis et al., Annual Review of Biomedical Engineering, 24: 249-274, 2022; and Wilkinson et al., PLoS ONE, 16(12): e0260954, 2021 . In some aspects, a polypeptide provided herein is modified to include an Fc region that alters Fc gamma receptor binding and / or effector function or Fc neonatal receptor binding and / or recycling. In some aspects, a polypeptide provided herein is modified to include an Fc region that comprises one or more glycosylation modifications.

[0421] In some aspects, a macromolecule (e.g., polypeptide) provided herein is modified to include a human serum albumin (HSA) or binder thereof that extends the half-life (e.g., half-life in circulation) of the polypeptide relative to a version of the polypeptide not comprising the HSA or binder thereof. For example, in some embodiments, the polypeptide is directly fused to HSA. In other embodiments, the polypeptide is fused to a HSA binder, e.g., a short peptide sequence, a VHH, or any other antibody or natural scaffold that targets HSA.

[0422] In some aspects, the modification (e.g., heterologous moiety) increases the half-life of the macromolecule (e.g., polypeptide) by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100% (e.g., 1 -5%, 5-10%, 10-15%, 15-20%, 20-25%, 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%) relative to a control macromolecule, e.g., a version of the macromolecule not comprising the modification.

[0423] G. Manufacturing and purity

[0424] In some embodiments of any of the compositions and methods provided herein, the macromolecule, pair of macromolecules, macromolecule complex, nucleic acid, pair of nucleic acids, multimer, or composition comprising the same is at least 95% pure (e.g., at least 95% free of any impurity or undesired substance). In some embodiments, the macromolecule, pair of macromolecules, macromolecule complex, nucleic acid, pair of nucleic acids, multimer, or composition comprising the same is more than 95% pure, e.g., is at least 96%, 97%, 98%, or 99% pure or is 100% pure.

[0425] In some embodiments of any of the compositions and methods provided herein, the macromolecule, pair of macromolecules, macromolecule complex, nucleic acid, pair of nucleic acids, multimer, or composition comprising the same are manufactured in accordance with one or more International Organization for Standardization (ISO) standards.

[0426] In some embodiments, the macromolecule, pair of macromolecules, macromolecule complex, nucleic acid, pair of nucleic acids, multimer, or composition comprising the same is manufactured according to the U.S. Food and Drug Administration (FDA)’s Good Manufacturing Practice (GMP), Good Clinical Practice (GCP), and / or Good Laboratory Practice (GLP) standards.

[0427] H. Biological samples

[0428] In some embodiments of any of the compositions and methods provided herein, the biological sample is an extract, fluid, or fraction (e.g., an extract, fluid, or fraction derived from a subject and comprising a cell); a cell; a tissue; or a subject (e.g., a mammalian subject, e.g., a human subject).

[0429] In some embodiments of any of the compositions and methods provided herein, the biological sample is a human subject.

[0430] In some embodiments, the biological sample is from a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In some embodiments, the biological sample is from a human (e.g., the subject is a human). In other embodiments, the biological sample is from a non-human animal (e.g., the subject is a non-human mammal). In embodiments, the non-human mammal is a non-human primate (e.g., monkeys, apes), an ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), a carnivore (e.g., dog, cat), a rodent (e.g., rat, mouse), or a lagomorph (e.g., rabbit). In some embodiments, the biological sample is from a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In some embodiments, the biological sample is from is an invertebrate such as an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid, a helminth, or a mollusk.

[0431] I. Lipid nanoparticles

[0432] The compositions (e.g., macromolecules, pairs of macromolecules, macromolecule complexes, polypeptides, nucleic acids, and compositions comprising the same), methods, and delivery systems provided by the present disclosure may employ any suitable carrier or delivery modality described herein, including, in certain embodiments, lipid nanoparticles (LNPs). Lipid nanoparticles, in some embodiments, include one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids); one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of WO2019217941 ; incorporated herein by reference in its entirety); one or more sterols (e.g., cholesterol).

[0433] Lipids that can be used in nanoparticle formations (e.g., lipid nanoparticles) include, for example those described in Table 4 of WO2019217941 , which is incorporated by reference — e.g., a lipid- containing nanoparticle can include one or more of the lipids in Table 4 of WO2019217941 . Lipid nanoparticles can include additional elements, such as polymers, such as the polymers described in Table 5 of WO2019217941 , incorporated by reference.

[0434] In some embodiments, conjugated lipids, when present, can include one or more of PEG- diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'- di(tetradecanoyioxy)propyi-l-0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypoly ethylene glycol 2000)- 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt, and those described in Table 2 of WO2019051289 (incorporated by reference), and combinations of the foregoing.

[0435] In some embodiments, sterols that can be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in W02009 / 127060 or US2010 / 0130588, which are incorporated by reference. Additional exemplary sterols include phytosterols, including those described in Eygeris et al. (2020), dx.doi.org / 10.1021 / acs.nanolett.0c01386, incorporated herein by reference.

[0436] In some embodiments, the lipid particle includes an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a sterol. The amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, the lipid nanoparticle includes an ionizable lipid is in an amount from about 20 mol % to about 90 mol % of the total lipids (in other embodiments it may be 20-70% (mol), 30-60% (mol) or 40-50% (mol); about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle), a non-cationic lipid in an amount from about 5 mol % to about 30 mol % of the total lipids, a conjugated lipid in an amount from about 0.5 mol % to about 20 mol % of the total lipids, and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipids. The ratio of total lipid to nucleic acid can be varied as desired. For example, the total lipid to nucleic acid (mass or weight) ratio can be from about 10: 1 to about 30: 1 .

[0437] In some embodiments, the lipid to nucleic acid ratio (mass / mass ratio; w / w ratio) can be in the range of from about 1 :1 to about 25:1 , from about 10:1 to about 14:1 , from about 3:1 to about 15:1 , from about 4:1 to about 10:1 , from about 5:1 to about 9:1 , or about 6:1 to about 9:1 . The amounts of lipids and nucleic acid can be adjusted to provide a desired N / P ratio, for example, N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Generally, the lipid nanoparticle formulation’s overall lipid content can range from about 5 mg / ml to about 30 mg / mL.

[0438] Some non-limiting example of lipid compounds that may be used (e.g., in combination with other lipid components) to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA (e.g., circular polyribonucleotide, linear polyribonucleotide)) described herein includes,

[0439] In some embodiments an LNP including Formula (i) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0440] In some embodiments an LNP including Formula (ii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0441] In some embodiments an LNP including Formula (iii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0442]

[0443] In some embodiments an LNP including Formula (v) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0444] In some embodiments an LNP including Formula (vi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0445] In some embodiments an LNP including Formula (viii) is used to deliver a polyribonucleotide

[0446] (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0447] In some embodiments an LNP including Formula (ix) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells. wherein

[0448] X1is O, NR1, or a direct bond, X2is C2-5 alkylene, X3is C(=O) or a direct bond, R1is H or Me, R3is C1 -3 alkyl, R2is C1 -3 alkyl, or R2taken together with the nitrogen atom to which it is attached and 1 -3 carbon atoms of X2form a 4-, 5-, or 6-membered ring, or X1is NR1, R1and R2taken together with the nitrogen atoms to which they are attached form a 5- or 6-membered ring, or R2taken together with R3and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y1is C2-12 alkylene, Y2is selected from

[0449] (in either orientation), (in either orientation), (in either orientation), n is 0 to 3, R4is C1 -15 alkyl, Z1is C1 -6 alkylene or a direct bond,

[0450] (in either orientation) or absent, provided that if Z1is a direct bond, Z2is absent;

[0451] R5is C5-9 alkyl or C6-10 alkoxy, R6is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and R7is H or Me, or a salt thereof, provided that if R3and R2are C2 alkyls, X1is O, X2is linear C3 alkylene, X3is C(=0), Y1is linear Ce alkylene, (Y2)n-R4is

[0452] , R4is linear C5 alkyl, Z1is C2 alkylene, Z2is absent, W is methylene, and R7is H, then R5and R6are not Cx alkoxy.

[0453] In some embodiments an LNP including Formula (xii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0454] In some embodiments an LNP including Formula (xi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells. In some embodiments an LNP includes a compound of Formula (xiii) and a compound of Formula

[0455] (xiv).

[0456] In some embodiments an LNP including Formula (xv) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0457] In some embodiments an LNP including a formulation of Formula (xvi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells.

[0458] In some embodiments, a lipid compound used to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA (e.g., circular polyribonucleotide, linear polyribonucleotide)) described herein is made by one of the following reactions: 5

[0459] In some embodiments an LNP including Formula (xxi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells. In some embodiments the LNP of Formula (xxi) is an LNP described by WO2021 1 13777 (e.g., a lipid of Formula (1 ) such as a lipid of Table 1 of WO2021 1 13777). wherein each n is independently an integer from 2-15; Li and L3 are each independently -OC(O)-* or - C(O)O-*, wherein indicates the attachment point to R1 or R3;

[0460] R1 and R3 are each independently a linear or branched C9-C20 alkyl or C9-C20 alkenyl, optionally substituted by one or more substituents selected from a group consisting of oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkyl sulfonyl, and alkyl sulfonealkyl; and

[0461] R2 is selected from a group consisting of:

[0462]

[0463] In some embodiments an LNP including Formula (xxii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells. In some embodiments the LNP of Formula (xxii) is an LNP described by WO2021 1 13777 (e.g., a lipid of Formula (2) such as a lipid of Table 2 of WO2021 1 13777). wherein each n is independently an integer from 1 -15;

[0464] Ri and R2 are each independently selected from a group consisting of:

[0465]

[0466] Rs is selected from a group consisting of:

[0467]

[0468] In some embodiments an LNP including Formula (xxiii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to cells. In some embodiments the LNP of Formula (xxiii) is an LNP described by WO2021113777 (e.g., a lipid of Formula (3) such as a lipid of Table 3 of WO2021113777). (xxiii) wherein

[0469] X is selected from -O-, -S-, or -OC(O)-*, wherein * indicates the attachment point to Ri;

[0470] Ri is selected from a group consisting of: and R2 is selected from a group consisting of:

[0471]

[0472] In some embodiments, a composition described herein (e.g., a nucleic acid (e.g., a circular polyribonucleotide, a linear polyribonucleotide) or a protein) is provided in an LNP that includes 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); e.g., as described in Example 1 of US9,867,888 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is 9Z,12Z)-3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12- dienoate (LP01 ), e.g., as synthesized in Example 13 of WO2015 / 095340 (incorporated by reference herein 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 Example 7, 8, or 9 of US2012 / 0027803 (incorporated by reference herein 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 WO2010 / 053572 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is 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-IH- cyclopenta[a]phenanthren-3-yl 3-(1 H-imidazol-4-yl)propanoate, e.g., Structure (I) from W02020 / 106946 (incorporated by reference herein in its entirety).

[0473] In some embodiments, an ionizable lipid may be a cationic lipid, an ionizable cationic lipid, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine- containing lipid that can be readily protonated. In some embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. In some embodiments, the lipid particle includes a cationic lipid in formulation with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, and polymer conjugated lipids. In some embodiments, the cationic lipid may be an ionizable cationic lipid. An exemplary cationic lipid as disclosed herein may have an effective pKa over 6.0. In embodiments, a lipid nanoparticle may include a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa), than the first cationic lipid. A lipid nanoparticle may include between 40 and 60 mol percent of a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, and a therapeutic agent, e.g., a nucleic acid (e.g., RNA (e.g., a circular polyribonucleotide, a linear polyribonucleotide)) described herein, encapsulated within or associated with the lipid nanoparticle. In some embodiments, the nucleic acid is co-formulated with the cationic lipid. The nucleic acid may be adsorbed to the surface of an LNP, e.g., an LNP including a cationic lipid. In some embodiments, the nucleic acid may be encapsulated in an LNP, e.g., an LNP including a cationic lipid. In some embodiments, the lipid nanoparticle may include a targeting moiety, e.g., coated with a targeting agent. In embodiments, the LNP formulation is biodegradable. In some embodiments, a lipid nanoparticle including one or more lipid described herein, e.g., Formula (i), (ii), (ii), (vii) and / or (ix) encapsulates at least 1 %, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or 100% of an RNA molecule.

[0474] Exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, without limitation, those listed in Table 1 of WO2019051289, incorporated herein by reference. Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US2016 / 031 1759; I of US201503761 15 or in US2016 / 0376224; I, II or III of US20160151284; I, IA, II, or 11 A of US20170210967; l-c of US20150140070; A of US2013 / 0178541 ; I of US2013 / 0303587 or US2013 / 0123338; I of US2015 / 0141678; II, III, IV, or V of US2015 / 0239926; I of US2017 / 01 19904; I or II of WO2017 / 1 17528; A of US2012 / 0149894; A of US2015 / 0057373; A of WO2013 / 1 16126; A of US2013 / 0090372; A of US2013 / 0274523; A of US2013 / 0274504; A of US2013 / 0053572; A of W02013 / 016058; A of W02012 / 162210; I of US2008 / 042973; I, II, III, or IV of US2012 / 01287670; I or II of US2014 / 0200257; I, II, or III of US2015 / 0203446; I or III of US2015 / 0005363; I, IA, IB, IC, ID, II, HA, IIB, IIC, HD, or lll-XXIV of US2014 / 0308304; of US2013 / 0338210; I, II, III, or IV of W02009 / 132131 ; A of US2012 / 0101 1478; I or XXXV of US2012 / 0027796; XIV or XVII of US2012 / 0058144; of US2013 / 0323269; I of US201 1 / 01 17125; I, II, or III of US201 1 / 0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US2012 / 0202871 ; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US201 1 / 0076335; I or II of US2006 / 008378; I of US2013 / 0123338; I or X-A-Y-Z of US2015 / 0064242; XVI, XVII, or XVIII of US2013 / 0022649; I, II, or III of US2013 / 01 16307; I, II, or III of US2013 / 01 16307; I or II of US2010 / 0062967; l-X of US2013 / 0189351 ; I of US2014 / 0039032; V of US2018 / 0028664; I of US2016 / 0317458; I of US2013 / 0195920; 5, 6, or 10 of US10,221 , 127; HI-3 of WO2018 / 081480; I-5 or I-8 of W02020 / 081938; 18 or 25 of US9,867,888; A of US2019 / 0136231 ; II of WO2020 / 219876; 1 of US2012 / 0027803; OF-02 of US2019 / 0240349; 23 of US10,086,013; cKK-E12 / A6 of Miao et al (2020); C12-200 of WO2010 / 053572; 7C1 of Dahlman et al (2017); 304-013 or 503-013 of Whitehead et al ; TS- P4C2 of US9,708,628; I of W02020 / 106946; I of W02020 / 106946; and (1 ), (2), (3), or (4) of WO2021 / 1 13777. Exemplary lipids further include a lipid of any one of Tables 1 -16 of WO2021 / 1 13777.

[0475] In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,3 IZ)-heptatriaconta- 6,9,28,3 I- tetraen-l9-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is the lipid ATX-002, e.g., as described in Example 10 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is (l3Z,l6Z)-A,A-dimethyl-3- nonyldocosa-13, 16-dien-l-amine (Compound 32), e.g., as described in Example 1 1 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO2019051289A9 (incorporated by reference herein in its entirety).

[0476] 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), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 - carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl- ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethylphosphatidylethanolamine (such as 16-O-dimethyl PE), 18-l-trans PE, l-stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoylphosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, 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, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Additional exemplary lipids, in certain embodiments, include, without limitation, those described in Kim et al. (2020) dx.doi.org / 10.1021 / acs.nanolett.0c01386, incorporated herein by reference. Such lipids include, in some embodiments, plant lipids found to improve liver transfection with mRNA (e.g., DGTS).

[0477] Other examples of non-cationic lipids suitable for use in the lipid nanoparticles include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationic lipids are described in WO2017 / 099823 or US patent publication US2018 / 0028664, the contents of which is incorporated herein by reference in their entirety.

[0478] In some embodiments, the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US2018 / 0028664, incorporated herein by reference in its entirety. The non-cationic lipid can include, for example, 0-30% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the noncationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In embodiments, the molar ratio of ionizable lipid to the 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 ).

[0479] In some embodiments, the lipid nanoparticles do not include any phospholipids.

[0480] In some aspects, the lipid nanoparticle can further include a component, such as a sterol, to provide membrane integrity. One exemplary sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 53-coprostanol, cholesteryl-(2’-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p- cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue, e.g., cholesteryl-(4 '-hydroxy)-buty1 ether. Exemplary cholesterol derivatives are described in PCT publication W02009 / 127060 and US patent publication US2010 / 0130588, each of which is incorporated herein by reference in its entirety.

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

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

[0483] Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(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 a mixture thereof. Additional exemplary PEG-lipid conjugates are described, for example, in US5, 885,613, US6,287,59I, US2003 / 0077829, US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2010 / 0130588, US2016 / 0376224, US2017 / 0119904, and US / 099823, the contents of all of which are incorporated herein by reference in their entirety. In some embodiments, a PEG-lipid is a compound of Formula III, lll-a-l, lll-a-2, lll-b-1 , lll-b-2, or V of US2018 / 0028664, the content of which is incorporated herein by reference in its entirety. In some embodiments, a PEG-lipid is of Formula II of US20150376115 or US2016 / 0376224, the content of both of which is incorporated herein by reference in its entirety. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG- disterylglycerol, PEG- dilaurylglycamide, PEG-dimyristylglycamide, PEG- dipalmitoylglycamide, PEG-disterylglycamide, PEG- cholesterol (l-[8'-(Cholest-5-en-3[beta]- oxy)carboxamido-3',6'-dioxaoctanyl] carbamoyl-[omega]-methyl- poly(ethylene glycol), PEG- DMB (3,4-Ditetradecoxylbenzyl- [omega]-methyl-poly(ethylene glycol) ether), and 1 ,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid includes PEG-DMG, 1 ,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid includes a structure selected from:

[0484] In some embodiments, lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.

[0485] 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 entirety.

[0486] In some embodiments, the PEG or the conjugated lipid can include 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG or the conjugated lipid content is 0.5- 10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle. Molar ratios of the ionizable lipid, non- cationic-lipid, sterol, and PEG / conjugated lipid can be varied as needed. For example, the lipid particle can include 30-70% ionizable lipid by mole or by total weight of the composition, 0-60% cholesterol by mole or by total weight of the composition, 0-30% non-cationic-lipid by mole or by total weight of the composition and 1 -10% conjugated lipid by mole or by total weight of the composition. Preferably, the composition includes 30-40% ionizable lipid by mole or by total weight of the composition, 40-50% cholesterol by mole or by total weight of the composition, and 10- 20% non-cationic-lipid by mole or by total weight of the composition. In some other embodiments, the composition is 50-75% ionizable lipid by mole or by total weight of the composition, 20-40% cholesterol by mole or by total weight of the composition, and 5 to 10% non-cationic-lipid, by mole or by total weight of the composition and 1 -10% conjugated lipid by mole or by total weight of the composition. The composition may contain 60-70% ionizable lipid by mole or by total weight of the composition, 25-35% cholesterol by mole or by total weight of the composition, and 5-10% non-cationic-lipid by mole or by total weight of the composition. The composition may also contain up to 90% ionizable lipid by mole or by total weight of the composition and 2 to 15% non-cationic lipid by mole or by total weight of the composition. The formulation may also be a lipid nanoparticle formulation, for example including 8-30% ionizable lipid by mole or by total weight of the composition, 5-30% non-cationic lipid by mole or by total weight of the composition, and 0-20% cholesterol by mole or by total weight of the composition; 4-25% ionizable lipid by mole or by total weight of the composition, 4-25% non-cationic lipid by mole or by total weight of the composition, 2 to 25% cholesterol by mole or by total weight of the composition, 10 to 35% conjugate lipid by mole or by total weight of the composition, and 5% cholesterol by mole or by total weight of the composition; or 2-30% ionizable lipid by mole or by total weight of the composition, 2-30% non-cationic lipid by mole or by total weight of the composition, 1 to 15% cholesterol by mole or by total weight of the composition, 2 to 35% conjugate lipid by mole or by total weight of the composition, and 1 -20% cholesterol by mole or by total weight of the composition; or even up to 90% ionizable lipid by mole or by total weight of the composition and 2-10% non-cationic lipids by mole or by total weight of the composition, or even 100% cationic lipid by mole or by total weight of the composition. In some embodiments, the lipid particle formulation includes ionizable lipid, phospholipid, cholesterol and a PEG-ylated lipid in a molar ratio of 50: 10:38.5: 1 .5. In some other embodiments, the lipid particle formulation includes ionizable lipid, cholesterol and a PEG-ylated lipid in a molar ratio of 60:38.5: 1 .5.

[0487] In some embodiments, the lipid particle includes ionizable lipid, non-cationic lipid (e.g., phospholipid), a sterol (e.g., cholesterol) and a PEG-ylated lipid, where the molar ratio of lipids ranges from 20 to 70 mole percent for the ionizable lipid, with a target of 40-60, the mole percent of non-cationic lipid ranges from 0 to 30, with a target of 0 to 15, the mole percent of sterol ranges from 20 to 70, with a target of 30 to 50, and the mole percent of PEG-ylated lipid ranges from 1 to 6, with a target of 2 to 5. In some embodiments, the lipid particle includes ionizable lipid I non-cationic- lipid / sterol I conjugated lipid at a molar ratio of 50:10:38.5: 1 .5.

[0488] In an aspect, the disclosure provides a lipid nanoparticle formulation including phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine.

[0489] In some embodiments, one or more additional compounds can also be included. Those compounds can be administered separately, or the additional compounds can be included in the lipid nanoparticles of the invention. In other words, the lipid nanoparticles can contain other compounds in addition to the nucleic acid or at least a second nucleic acid, different than the first. Without limitations, other additional compounds can be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials, or any combinations thereof.

[0490] In some embodiments, the LNPs include biodegradable, ionizable lipids. In some embodiments, the LNPs include (9Z,l2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,l2-dienoate, also called 3- ((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,l2Z)-octadeca- 9,12-dienoate) or another ionizable lipid. See, e.g., lipids of WO2019 / 067992, WO / 2017 / 173054, WO2015 / 095340, and WO2014 / 136086, as well as references provided therein. In some embodiments, the term cationic and ionizable in the context of LNP lipids is interchangeable, e.g., wherein ionizable lipids are cationic depending on the pH.

[0491] In some embodiments, the average LNP diameter of the LNP formulation may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 70 nm to about 100 nm. In a particular embodiment, the average LNP diameter of the LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of the LNP formulation may be about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation ranges from about I mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, or from about 38 mm to about 42 mm.

[0492] A LNP may, in some instances, be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A LNP may have a polydispersity index from about 0 to about 0.25, such as 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21 , 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a LNP may be from about 0.10 to about 0.20.

[0493] The zeta potential of a LNP may be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of an LNP. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0494] The efficiency of encapsulation of a protein and / or nucleic acid, describes the amount of protein and / or nucleic acid that is encapsulated or otherwise associated with a LNP after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence may be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of a protein and / or nucleic acid may be at least 50%, for example 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%.

[0495] A LNP may optionally include one or more coatings. In some embodiments, a LNP may be formulated in a capsule, film, or table having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness, or density.

[0496] Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by W02020 / 061457 and WO2021 / 113777, each of which is incorporated herein by reference in its entirety. Further exemplary lipids, formulations, methods, and characterization of LNPs are taught by Hou et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater (2021 ). doi.org / 10.1038 / s41578-021 -00358-0, which is incorporated herein by reference in its entirety (see, for example, exemplary lipids and lipid derivatives of Figure 2 of Hou et al.).

[0497] In some embodiments, in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In certain embodiments, LNPs are formulated using the GenVoyJLM ionizable lipid mix (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl 51 (34):8529-8533 (2012), incorporated herein by reference in its entirety.

[0498] LNP formulations optimized for the delivery of CRISPR-Cas systems, e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA, are described in WO2019067992 and WO2019067910, both incorporated by reference, and are useful for delivery of circular polyribonucleotides and linear polyribonucleotides described herein.

[0499] Additional specific LNP formulations useful for delivery of nucleic acids (e.g., circular polyribonucleotides, linear polyribonucleotides) are described in US8158601 and US8168775, both incorporated by reference, which include formulations used in patisiran, sold under the name ONPATTRO. Exemplary dosing of polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) LNP may include about 0.1 , 0.25, 0.3, 0.5, 1 , 2, 3, 4, 5, 6, 8, 10, or 100 mg / kg (RNA). Exemplary dosing of AAV including a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) may include an MOI of about 1011, 1012, 1013, and 1014vg / kg.

[0500] II. Methods of Use

[0501] In some aspects, provided herein are methods (e.g., methods of inducing cellular effector functions, modulating the state of a cell, and / or treating disease) comprising providing any of the macromolecules, pairs of macromolecules, or macromolecule complexes; nucleic acids or pairs of nucleic acids; or multimers described herein access to a cell.

[0502] In some aspects, provided herein is use of any of the macromolecules, pairs of macromolecules, or macromolecule complexes; nucleic acids or pairs of nucleic acids; or multimers described herein in the manufacture of a medicament for accessing a cell (e.g., a medicament for use in a method of inducing cellular effector functions, modulating the state of a cell, and / or treating disease).

[0503] In some aspects, provided herein are any of the macromolecules, pair of macromolecules, or macromolecule complexes; nucleic acids or pairs of nucleic acids; or multimers described herein for use in accessing a cell (e.g., a medicament for use in a method of inducing cellular effector functions, modulating the state of a cell, and / or treating disease).

[0504] A. Methods of inducing cellular effector functions

[0505] In one aspect, provided herein is a method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule complex (e.g., a macromolecule complex as described in Section I A(i)) comprising two macromolecules, each comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the FBD specifically binds a disease signature ligand in a biological sample; and (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two copies of the macromolecule are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each copy of the macromolecule binding the disease signature ligand.

[0506] In another aspect, provided herein is a method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule complex (e.g., a macromolecule complex as described in Section IA(ii)) comprising a pair of macromolecules, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample; (b) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1 ) that specifically binds a first effector ligand in the biological sample; and (c) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the pair of macromolecules binding the disease signature ligand.

[0507] In another aspect, provided herein is a method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule (e.g., a macromolecule complex as described in Section IA(iii)) comprising two FBDs linked to two SBDs, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; and (b) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand.

[0508] In another aspect, provided herein is a method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule (e.g., a macromolecule complex as described in Section IA(iv)) comprising two FBDs linked to an SBD1 and an SBD2, wherein (a) the FBDs specifically bind a disease signature ligand in a biological sample; (b) the SBD1 specifically binds a first effector ligand in the biological sample; and (c) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand.

[0509] In some aspects, provided herein is use any of the macromolecules, pairs of macromolecules, or macromolecule complexes; nucleic acids or pairs of nucleic acids; or multimers described herein in the manufacture of a medicament for inducing a cellular effector function in a cell.

[0510] In some aspects, provided herein are any of the macromolecules, pairs of macromolecules, or macromolecule complexes; nucleic acids or pairs of nucleic acids; or multimers described herein for use in inducing a cellular effector function in a cell.

[0511] In some embodiments of any of the above aspects, the cell is in a subject and the macromolecule, macromolecule complex, or nucleic acid is administered in a therapeutically effective amount.

[0512] In some embodiments, the subject has, or is suspected of having, a disease or disorder characterized by abnormal levels of the disease signature target, optionally wherein the subject was previously determined to have abnormal levels of the disease signature target.

[0513] In some embodiments, the cell is a T cell. In some embodiments, the effector ligand is 4-1 BB and the cell is a T cell. In some embodiments, the cellular effector function is T cell proliferation. In some embodiments, the cellular effector function is anti-tumor activity. B. Methods of modulating the state of a cell

[0514] In another aspect, provided herein is a method of modulating the state of a cell, comprising providing any of the macromolecules or macromolecule complexes or nucleic acids or pairs of nucleic acids described herein access to the cell (e.g., wherein the disease signature ligand(s) and effector ligand(s) bound by the macromolecule or macromolecule complex are present on or in proximity to the cell), thereby modulating the state of the cell.

[0515] In some aspects, provided herein is use any of the macromolecules, pairs of macromolecules, or macromolecule complexes; nucleic acids or pairs of nucleic acids; or multimers described herein in the manufacture of a medicament for modulating the state of a cell.

[0516] In some aspects, provided herein are any of the macromolecules, pairs of macromolecules, or macromolecule complexes; nucleic acids or pairs of nucleic acids; or multimers described herein for use in modulating the state of a cell.

[0517] In some embodiments of any of the above aspects, the cell is in a subject and the macromolecule, macromolecule complex, or nucleic acid is administered in a therapeutically effective amount.

[0518] In some embodiments, the subject has, or is suspected of having, a disease or disorder characterized by abnormal levels of the disease signature target, optionally wherein the subject was previously determined to have abnormal levels of the disease signature target.

[0519] C. Methods of determining the state of a cell

[0520] In another aspect, provided herein is a method of determining the state of a cell, comprising providing any of the macromolecules or macromolecule complexes comprising a reporter domain or nucleic acids or pairs of nucleic acids encoding the same described herein access to the cell (e.g., wherein the disease signature ligand(s) and effector ligand(s) bound by the macromolecule or macromolecule complex are present on or in proximity to the cell), and detecting the presence of the reporter domain, thereby determining the state of the cell.

[0521] In some aspects, provided herein is use any of the macromolecules, pairs of macromolecules, or macromolecule complexes; nucleic acids or pairs of nucleic acids; or multimers described herein in the manufacture of a medicament for determining the state of a cell.

[0522] In some aspects, provided herein are any of the macromolecules, pairs of macromolecules, or macromolecule complexes; nucleic acids or pairs of nucleic acids; or multimers described herein for use in determining the state of a cell.

[0523] In some embodiments of any of the above aspects, the cell is in a subject and the macromolecule, macromolecule complex, or nucleic acid is administered in a therapeutically effective amount.

[0524] In some embodiments, the subject has a disease or disorder characterized by abnormal levels of the disease signature target. III. Dimeric macromolecule assembly

[0525] A. Compositions

[0526] In one aspect, provided herein is a composition comprising a macromolecule complex (e.g., a macromolecule complex described herein), wherein the macromolecule complex comprises a dimer, and wherein at least about 90% of the dimer is monodisperse.

[0527] In some embodiments, at least about 92% of the dimer is monodisperse. In some embodiments, at least about 93% of the dimer is monodisperse. In some embodiments, at least about 94% of the dimer is monodisperse. In some embodiments, at least about 95% of the dimer is monodisperse. In some embodiments, at least about 96% of the dimer is monodisperse. In some embodiments, at least about 97% of the dimer is monodisperse. In some embodiments, at least about 98% of the dimer is monodisperse. In some embodiments, at least about 99% of the dimer is monodisperse.

[0528] In another aspect, provided herein is a composition comprising a macromolecule complex (e.g., a macromolecule complex described herein), wherein the macromolecule complex comprises two macromolecules that are capable of dimerization, and wherein the composition is at least about 90% monodisperse at a size consistent with the dimerized macromolecule complex.

[0529] In some embodiments, the composition is at least about 92% monodisperse, at least about 93% monodisperse, at least about 94% monodisperse, at least about 95% monodisperse, at least about 96% monodisperse, at least about 97% monodisperse, at least about 98% monodisperse, or at least about 99% monodisperse (e.g., at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% monodisperse) at a size consistent with the dimerized macromolecule complex.

[0530] The composition may be, e.g., a composition produced from one or more cell lines that produce one or more components of the macromolecule complex (e.g., a cell line that produces a single macromolecule that forms a homodimer, a cell line that expresses two macromolecules that form a heterodimer, or two cell lines that each express one macromolecule of a pair that forms a heterodimer). For example, the cell line may be transiently or stably transfected with a plasmid encoding a secreted form of the macromolecule or macromolecules. The composition may be purified before monodispersity is assessed. The purification may include, e.g., clarifying culture supernatant of the cell line(s) (e.g., via a two-step purification scheme using Protein A affinity chromatography followed by polishing using cation exchange chromatography).

[0531] In some embodiments, the percent monodispersity is determined by size exclusion chromatography (SEC), e.g., as described in the examples provided herein. In some embodiments, the percent monodispersity is determined by analytical SEC. In some embodiments, the percent monodispersity is determined by analytical SEC using a SUPERDEX® 200 column.

[0532] In some embodiments, the size of the macromolecule complex is consistent with the size of a dimer, e.g., as described in the examples provided herein.

[0533] In some embodiments, the macromolecule complex comprises two macromolecules, each comprising a VEGF binding domain linked to a 4-1 BB binding domain.

[0534] In some embodiments, the VEGF binding domain is an anti-VEGF scFv.

[0535] In some embodiments, the VEGF binding domain is a VEGF receptor trap.

[0536] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB scFv. In some embodiments, the macromolecule complex comprises a 4-1 BB X VEGF-TRAP.

[0537] In some embodiments, the macromolecule complex comprises a 4-1 BB X VEGF-scFV.

[0538] In some embodiments, the macromolecule complex comprises two macromolecules, each comprising a TGFb binding domain linked to a TpoR binding domain.

[0539] In some embodiments, the TGFb binding domain is an anti-TGFb scFv.

[0540] In some embodiments, the TpoR binding domain is an anti-TpoR scFv.

[0541] In some embodiments, the macromolecule complex comprises two macromolecules, each comprising an IL-8 binding domain linked to a TpoR binding domain.

[0542] In some embodiments, the IL-8 binding domain is an anti-l L8 scFv.

[0543] In some embodiments, the TpoR binding domain is an anti-TpoR scFv.

[0544] In some embodiments, the macromolecule complex comprises two macromolecules, each comprising a TGFb binding domain linked to an IL2R binding domain.

[0545] In some embodiments, the TGFb binding domain is an anti- TGFb scFv.

[0546] In some embodiments, the IL2R binding domain comprises the N-terminus of IL2. In some embodiments, the IL2R binding domain comprises the C-terminus of IL2.

[0547] In some embodiments, the macromolecule complex comprises two macromolecules, each comprising an IFNg binding domain linked to an IL10R binding domain.

[0548] In some embodiments, the IFNg binding domain is an anti- IFNg scFv.

[0549] In some embodiments, the IL10R binding domain comprises an anti-IL1 OR VHH.

[0550] In some embodiments, the IL10R binding domain comprises IL10.

[0551] In some embodiments, the macromolecule complex comprises two macromolecules, each comprising an IL6 binding domain linked to an IL1 OR binding domain.

[0552] In some embodiments, the IL6 binding domain is an anti- IL6 VHH.

[0553] In some embodiments, the IL10R binding domain comprises an anti-IL1 OR VHH.

[0554] In some embodiments, the IL10R binding domain comprises IL10.

[0555] In some embodiments, the macromolecule complex comprises two macromolecules, each comprising an IL8 binding domain linked to a CD3 binding domain.

[0556] In some embodiments, the IL8 binding domain is an anti-IL8 scFv.

[0557] In some embodiments, the CD3 binding domain comprises an anti-CD3 VH.

[0558] B. Methods

[0559] In one aspect, the present disclosure provides a method for preparing a pharmaceutical composition, the method comprising:

[0560] (a) preparing or obtaining a composition comprising a macromolecule complex (e.g., a macromolecule complex described herein) (e.g., a sample thereof);

[0561] (b) measuring or having measured the percent of the macromolecule in the composition (or the sample thereof) that is monodispersed; and

[0562] (c) formulating or having formulated the composition as a pharmaceutical composition if the percent of the macromolecule in the composition (or the sample thereof) that is monodispersed in the sample is equal to or greater than a pre-determined threshold. In another aspect, the present disclosure provides a method for preparing a pharmaceutical composition, the method comprising:

[0563] (a) preparing or obtaining a composition comprising a macromolecule complex (e.g., a macromolecule complex described herein) (e.g., a sample thereof);

[0564] (b) measuring or having measured the percent monodispersity of the macromolecule in the composition (or the sample thereof); and

[0565] (c) formulating or having formulated the composition as a pharmaceutical composition if the percent monodispersity of the macromolecule in the composition (or the sample thereof) is equal to or greater than a pre-determined threshold.

[0566] In some embodiments of the above aspects, formulating or having formulated the composition as a pharmaceutical composition comprises combining the composition with one or more pharmaceutically acceptable excipients.

[0567] In some embodiments, the macromolecule is a dimer.

[0568] In some embodiments, the pre-determined threshold is at least about 90% of the dimer is monodisperse. In some embodiments, the pre-determined threshold is at least about 92% of the dimer is monodisperse. In some embodiments, the pre-determined threshold is at least about 93% of the dimer is monodisperse. In some embodiments, the pre-determined threshold is at least about 94% of the dimer is monodisperse. In some embodiments, the pre-determined threshold is at least about 95% of the dimer is monodisperse. In some embodiments, the pre-determined threshold is at least about 96% of the dimer is monodisperse. In some embodiments, the pre-determined threshold is at least about 97% of the dimer is monodisperse. In some embodiments, the pre-determined threshold is at least about 98% of the dimer is monodisperse. In some embodiments, the pre-determined threshold is at least about 99% of the dimer is monodisperse.

[0569] In some embodiments, the pre-determined threshold is at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% monodispersity (e.g., at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% monodispersity) at a size consistent with the dimerized macromolecule complex.

[0570] In some embodiments, the percent monodispersity is determined by size exclusion chromatography (SEC), e.g., as described in the examples provided herein. In some embodiments, the percent monodispersity is determined by analytical SEC. In some embodiments, the percent monodispersity is determined by analytical SEC using a SUPERDEX® 200 column.

[0571] In some embodiments, the size of the macromolecule complex is consistent with the size of a dimer, e.g., as described in the examples provided herein.

[0572] In some embodiments, the macromolecule complex comprises two macromolecules, each comprising a VEGF binding domain linked to a 4-1 BB binding domain.

[0573] In some embodiments, the VEGF binding domain is an anti-VEGF scFv.

[0574] In some embodiments, the VEGF binding domain is a VEGF receptor trap.

[0575] In some embodiments, the 4-1 BB binding domain is an anti-4-1 BB scFv.

[0576] In some embodiments, the macromolecule complex comprises a 4-1 BB X VEGF-TRAP.

[0577] In some embodiments, the macromolecule complex comprises a 4-1 BB X VEGF-scFV. In some embodiments, the macromolecule complex comprises two macromolecules, each comprising a TGFb binding domain linked to a TpoR binding domain.

[0578] In some embodiments, the TGFb binding domain is an anti-TGFb scFv.

[0579] In some embodiments, the TpoR binding domain is an anti-TpoR scFv.

[0580] In some embodiments, the macromolecule complex comprises two macromolecules, each comprising an IL-8 binding domain linked to a TpoR binding domain.

[0581] In some embodiments, the IL-8 binding domain is an anti-l L8 scFv.

[0582] In some embodiments, the TpoR binding domain is an anti-TpoR scFv.

[0583] In some embodiments, the macromolecule complex comprises two macromolecules, each comprising a TGFb binding domain linked to an IL2R binding domain.

[0584] In some embodiments, the TGFb binding domain is an anti- TGFb scFv.

[0585] In some embodiments, the IL2R binding domain comprises the N-terminus of IL2. In some embodiments, the IL2R binding domain comprises the C-terminus of IL2.

[0586] In some embodiments, the macromolecule complex comprises two macromolecules, each comprising an IFNg binding domain linked to an IL10R binding domain.

[0587] In some embodiments, the IFNg binding domain is an anti- IFNg scFv.

[0588] In some embodiments, the IL10R binding domain comprises an anti-IL1 OR VHH.

[0589] In some embodiments, the IL10R binding domain comprises IL10.

[0590] In some embodiments, the macromolecule complex comprises two macromolecules, each comprising an IL6 binding domain linked to an IL1 OR binding domain.

[0591] In some embodiments, the IL6 binding domain is an anti- IL6 VHH.

[0592] In some embodiments, the IL10R binding domain comprises an anti-IL1 OR VHH.

[0593] In some embodiments, the IL10R binding domain comprises IL10.

[0594] In some embodiments, the macromolecule complex comprises two macromolecules, each comprising an IL8 binding domain linked to a CD3 binding domain.

[0595] In some embodiments, the IL8 binding domain is an anti-IL8 scFv.

[0596] In some embodiments, the CD3 binding domain comprises an anti-CD3 VH.

[0597] IV. OTHER EMBODIMENTS

[0598] Some embodiments of the technology described herein can be defined according to any of the following numbered embodiments:

[0599] 1 . A macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein:

[0600] (a) the FBD specifically binds a disease signature ligand in a biological sample; and

[0601] (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand. 2. A macromolecule complex comprising a pair of macromolecules, wherein each macromolecule independently comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein:

[0602] (a) the first member of the pair of macromolecules comprises a first binding domain 1 (FBD1 ) that specifically binds a first epitope of a disease signature ligand in a biological sample;

[0603] (b) the second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds a second epitope of the disease signature ligand in a biological sample; and

[0604] (c) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand.

[0605] 3. The macromolecule complex of embodiment 1 or 2, wherein the conjugation is covalent.

[0606] 4. The macromolecule complex of embodiment 3, wherein the covalent conjugation comprises a chemical linker or a polypeptide linker.

[0607] 5. The macromolecule complex of embodiment 1 or 2, wherein the conjugation is non-covalent.

[0608] 6. The macromolecule complex of embodiment 5, wherein the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair.

[0609] 7. The macromolecule complex of embodiment 6, wherein the pair of complementary moieties is biotin and avidin; barnase and barstar; a pair of complementary aptamers; or a pair of complementary polypeptides.

[0610] 8. The macromolecule complex of embodiment 7, wherein the pair of complementary polypeptides is a pair of engineered Fc fragments.

[0611] 9. The macromolecule complex of embodiment 8, wherein the pair of engineered Fc fragments is a knob-into-hole pair.

[0612] 10. The macromolecule complex of any one of embodiments 4 and 6-9, wherein the conjugation sterically orients the two macromolecules to permit conditional concurrent binding of disease signal and effector ligands.

[0613] 11 . The macromolecule complex of embodiment 5, wherein the non-covalent conjugation is mediated by the disease signature ligand.

[0614] 12. The macromolecule complex of any one of embodiments 1 , 3-5, 10, and 11 , wherein the two macromolecules are identical. 13. A macromolecule complex comprising a pair of macromolecules, wherein each macromolecule independently comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein:

[0615] (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample;

[0616] (b) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1 ) that specifically binds a first effector ligand in the biological sample; and

[0617] (c) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the pair of macromolecules binding the disease signature ligand.

[0618] 14. A macromolecule complex comprising a pair of macromolecules, wherein each macromolecule independently comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein:

[0619] (a) the first member of the pair of macromolecules comprises a first binding domain 1 (FBD1 ) that specifically binds a first epitope of a disease signature ligand in a biological sample;

[0620] (b) the second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds a second epitope of the disease signature ligand in a biological sample;

[0621] (c) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1 ) that specifically binds a first effector ligand in the biological sample; and

[0622] (d) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the pair of macromolecules binding the disease signature ligand.

[0623] 15. The macromolecule complex of embodiment 13 or 14, wherein the conjugation is covalent.

[0624] 16. The macromolecule complex of embodiment 15, wherein the covalent conjugation comprises a chemical linker or a polypeptide linker.

[0625] 17. The macromolecule complex of embodiment 13 or 14, wherein the conjugation is non-covalent. 18. The macromolecule complex of embodiment 17, wherein the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair.

[0626] 19. The macromolecule complex of embodiment 18, wherein the pair of complementary moieties is biotin and avidin; barnase and barstar; a pair of complementary aptamers; or a pair of complementary polypeptides.

[0627] 20. The macromolecule complex of embodiment 19, wherein the pair of complementary polypeptides is a pair of engineered Fc fragments.

[0628] 21 . The macromolecule complex of embodiment 20, wherein the pair of engineered Fc fragments is a knob-into-hole pair.

[0629] 22. The macromolecule complex of any one of embodiments 13, 14, and 18-21 , wherein the conjugation sterically orients the first member of the pair of macromolecules and the second member of the pair of macromolecules to permit conditional concurrent binding of disease signal and effector ligands.

[0630] 23. The macromolecule complex of embodiment 18, wherein the non-covalent conjugation is mediated by the disease signature ligand.

[0631] 24. A macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein:

[0632] (a) the FBD specifically binds a disease signature ligand in a biological sample; and

[0633] (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are not conjugated to each other in the absence of the disease signature ligand; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand.

[0634] 25. A macromolecule complex comprising a pair of macromolecules, wherein each macromolecule independently comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein:

[0635] (a) the first member of the pair of macromolecules comprises a first binding domain 1 (FBD1 ) that specifically binds a first epitope of a disease signature ligand in a biological sample;

[0636] (b) the second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds a second epitope of the disease signature ligand in a biological sample; and

[0637] (c) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are not conjugated to each other in the absence of the disease signature ligand; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand.

[0638] 26. The macromolecule complex of embodiment 24 or 25, wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of the disease signature ligand, and wherein the non-covalent conjugation is mediated by the disease signature ligand.

[0639] 27. A macromolecule comprising two FBDs linked to two SBDs by one or more linker domains, wherein:

[0640] (a) the FBDs specifically bind a disease signature ligand in a biological sample; and

[0641] (b) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand.

[0642] 28. A macromolecule comprising an FBD1 and an FBD2 linked to two SBDs by one or more linker domains, wherein:

[0643] (a) the FBD1 specifically binds a first epitope of a disease signature ligand in a biological sample;

[0644] (b) the FBD2 specifically binds a second epitope of the disease signature ligand in the biological sample; and

[0645] (c) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the FBD1 and the FBD2 binding the disease signature ligand.

[0646] 29. A macromolecule comprising two FBDs linked to an SBD1 and an SBD2 by one or more linker domains, wherein:

[0647] (a) the FBDs specifically bind a disease signature ligand in a biological sample;

[0648] (b) the SBD1 specifically binds a first effector ligand in the biological sample; and

[0649] (c) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand.

[0650] 30. A macromolecule comprising an FBD1 and an FBD2 linked to an SBD1 and an SBD2 by one or more linker domains, wherein:

[0651] (a) the FBD1 specifically binds a first epitope of a disease signature ligand in a biological sample;

[0652] (b) the FBD2 specifically binds a second epitope of the disease signature ligand in the biological sample;

[0653] (c) the SBD1 specifically binds a first effector ligand in the biological sample; and

[0654] (d) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the FBD1 and the FBD2 binding the disease signature ligand.

[0655] 31 . The macromolecule complex or macromolecule of any one of embodiments 1 -30, wherein the disease signature ligand is a protein, a peptide, or a small molecule.

[0656] 32. The macromolecule complex or macromolecule of embodiment 31 , wherein the protein is a soluble protein or an insoluble protein.

[0657] 33. The macromolecule complex or macromolecule of any one of embodiments 1 -30, wherein the disease signature ligand is a cytokine.

[0658] 34. The macromolecule complex or macromolecule of embodiment 33, wherein the cytokine is an interleukin, an interferon, a growth factor, a chemokine, or a member of the TNF family.

[0659] 35. The macromolecule complex or macromolecule of embodiment 34, wherein the interleukin is IL-1 , IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15, IL-17, or IL-23; the interferon is IFN-gamma; the growth factor is transforming growth factor beta (TGF-beta), granulocyte colony stimulating factor (GCSF) granulocytemacrophage colony-stimulating factor (GCSF), epidermal growth factor (EGF), erythropoietin (EPO), or VEGF; the chemokine is monocyte chemoattractant protein-1 (MCP-1 ) or interferon gamma-induced protein 10 (IP-10); or the member of the TNF family is TNF-alpha.

[0660] 36. The macromolecule complex or macromolecule of any one of embodiments 1 -30, wherein the disease signature ligand is a neurotransmitter. 37. The macromolecule complex or macromolecule of any one of embodiments 1 -30, wherein the disease signature ligand is a cell surface receptor, a surface antigen, a membrane-bound protein, an extracellular matrix component, or an integrin.

[0661] 38. The macromolecule complex or macromolecule of any one of embodiments 1 -30, wherein the disease signature ligand is a self antigen of an organism from which the biological sample is derived.

[0662] 39. The macromolecule complex or macromolecule of embodiment 38, wherein the self antigen is an anti-drug antibody (ADA), an autoantibody, or a tumor marker.

[0663] 40. The macromolecule complex or macromolecule of any one of embodiments 1 -30, wherein the disease signature ligand is a nucleic acid.

[0664] 41 . The macromolecule complex or macromolecule of any one of embodiments 1 -30, wherein the disease signature ligand is a carbohydrate, a lipid, a peptide, a nucleoside, or a combination of the foregoing.

[0665] 42. The macromolecule complex or macromolecule of any one of embodiments 1 -30, wherein the disease signature ligand is a hormone, an amino acid derivative, a steroid, or an eicosanoid.

[0666] 43. The macromolecule complex or macromolecule of any one of embodiments 1 -30, wherein the disease signature ligand is a non-self antigen.

[0667] 44. The macromolecule complex or macromolecule of embodiment 43, wherein the disease signature ligand is a virus, a bacterium, or a fragment or antigen thereof.

[0668] 45. The macromolecule complex or macromolecule of any one of embodiments 1 -44, wherein the disease signature ligand is multimeric.

[0669] 46. The macromolecule complex or macromolecule of embodiment 45, wherein the disease signature ligand is dimeric, trimeric, or tetrameric.

[0670] 47. The macromolecule complex or macromolecule of any one of embodiments 1 -46, wherein the FBD, FBD1 , or FBD2 comprises a polypeptide that specifically binds the disease signature ligand.

[0671] 48. The macromolecule complex or macromolecule of embodiment 47, wherein the polypeptide is an antibody or a fragment thereof.

[0672] 49. The macromolecule complex or macromolecule of embodiment 48, wherein the antibody or fragment thereof is an scFv, a monospecific tandem scFv (taFv), a bispecific taFv, a VHH, a VNAR, a Fab, a monospecific single-chain diabody, a bispecific single-chain diabody, or a dual-affinity re-targeting antibody (DART).

[0673] 50. The macromolecule complex or macromolecule of embodiment 47, wherein the polypeptide is an antibody mimetic.

[0674] 51 . The macromolecule complex or macromolecule of embodiment 50, wherein the antibody mimetic is an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, a lipocalin, an avimer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monobody, a fibronectin type III domain (FN3)-based binder, a nanoantibody, a nanoCLAMP, an optimer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR).

[0675] 52. The macromolecule complex or macromolecule of embodiment 47, wherein the polypeptide is an endogenous binding domain.

[0676] 53. The macromolecule complex or macromolecule of embodiment 52, wherein the endogenous binding domain is a cell receptor domain, an enzyme domain, a variable lymphocyte receptor (VLR) domain, a receptor ectodomain, a nuclear hormone receptor ligand-binding domain, or a DNA-binding domain.

[0677] 54. The macromolecule complex or macromolecule of any one of embodiments 1 -46, wherein the FBD, FBD1 , or FBD2 comprises an oligonucleotide that specifically binds the disease signature ligand.

[0678] 55. The macromolecule complex or macromolecule of embodiment 54, wherein the oligonucleotide is a nucleic acid aptamer.

[0679] 56. The macromolecule complex or macromolecule of embodiment 55, wherein the nucleic acid aptamer is a DNA aptamer.

[0680] 57. The macromolecule complex or macromolecule of any one of embodiments 1 -46, wherein the FBD, FBD1 , or FBD2 comprises a chemical molecule that specifically binds the disease signature ligand.

[0681] 58. The macromolecule complex or macromolecule of any one of embodiments 1 -57, wherein the FBD, FBD1 , or FBD2 has affinity to two or more disease signature moieties.

[0682] 59. The macromolecule complex or macromolecule of any one of embodiments 1 -58, wherein the effector ligand is a protein or a peptide.

[0683] 60. The macromolecule complex or macromolecule of any one of embodiments 1 -59, wherein the effector ligand is a cell-surface receptor or an intracellular receptor. 61 . The macromolecule complex or macromolecule of embodiment 60, wherein the cell-surface receptor is a catalytic receptor or the intracellular receptor is a nuclear hormone receptor.

[0684] 62. The macromolecule complex or macromolecule of embodiment 61 , wherein the catalytic receptor is a receptor tyrosine kinase (RTK), a receptor serine / threonine kinase (RSK), a type 1 cytokine receptor, a type 2 cytokine receptor, or a tumor necrosis factor (TNF) superfamily receptor.

[0685] 63. The macromolecule complex or macromolecule of embodiment 62, wherein the RTK is VEGFR, the RSK is TGFBR2, the type 1 cytokine receptor is IL-2R, the type 2 cytokine receptor is IL1 OR, or the TNF superfamily receptor is TNFR2 or 4-1 BB.

[0686] 64. The macromolecule complex or macromolecule of any one of embodiments 1 -12, 24, 26-28, and 31 -63, wherein the SBD is an agonist of the effector ligand.

[0687] 65. The macromolecule complex or macromolecule of embodiment 64, wherein the effector ligand must be homodimerized to exert a cellular effector function.

[0688] 66. The macromolecule complex or macromolecule of embodiment 65, wherein the effector ligand is homodimerized in the presence of the macromolecule complex or macromolecule and the disease signature ligand.

[0689] 67. The macromolecule complex or macromolecule of any one of embodiments 13-23 and 29-63, wherein the first effector ligand and the second effector ligand must be associated to exert a cellular effector function.

[0690] 68. The macromolecule complex or macromolecule of embodiment 67, wherein the first effector ligand and the second effector ligand are associated in the presence of the macromolecule complex or macromolecule and the disease signature ligand.

[0691] 69. The macromolecule complex or macromolecule of embodiment 67 or 68, wherein the association is heterodimerization.

[0692] 70. The macromolecule complex or macromolecule of any one of embodiments 1 -69, wherein the cellular effector function is a biological activity.

[0693] 71 . The macromolecule complex or macromolecule of any one of embodiments 1 -70, wherein the cellular effector function is a therapeutic activity.

[0694] 72. The macromolecule complex or macromolecule of any one of embodiments 1 -70, wherein the cellular effector function is a disease activity.

[0695] 73. The macromolecule complex or macromolecule of any one of embodiments 1 -72, wherein the SBD, SBD1 , or SBD2 comprises a polypeptide that specifically binds the effector ligand. 74. The macromolecule complex or macromolecule of embodiment 73, wherein the polypeptide is an antibody or a fragment thereof.

[0696] 75. The macromolecule complex or macromolecule of embodiment 74, wherein the antibody or fragment thereof is an scFv, a monospecific taFv, a bispecific taFv, a VHH, a VNAR, a Fab, a monospecific single-chain diabody, a bispecific single-chain diabody, or a DART.

[0697] 76. The macromolecule complex or macromolecule of embodiment 73, wherein the polypeptide is an antibody mimetic.

[0698] 77. The macromolecule complex or macromolecule of embodiment 76, wherein the antibody mimetic is an affibody, an affilin, an affimer, an affitin, an alphabody, an anticalin, a lipocalin, an avimer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monobody, a FN3-based binder, a nanoantibody, a nanoCLAMP, an optimer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a VLR.

[0699] 78. The macromolecule complex or macromolecule of embodiment 73, wherein the polypeptide is an endogenous binding domain.

[0700] 79. The macromolecule complex or macromolecule of embodiment 78, wherein the endogenous binding domain is a ligand of the effector ligand or a fragment thereof.

[0701] 80. The macromolecule complex or macromolecule of embodiment 78, wherein the endogenous binding domain is a viral binding protein or a fragment thereof.

[0702] 81 . The macromolecule complex or macromolecule of any one of embodiments 1 -72, wherein the SBD, SBD1 , or SBD2 comprises an oligonucleotide that specifically binds the effector ligand.

[0703] 82. The macromolecule complex or macromolecule of embodiment 81 , wherein the oligonucleotide is a nucleic acid aptamer.

[0704] 83. The macromolecule complex or macromolecule of embodiment 82, wherein the nucleic acid aptamer is a DNA aptamer.

[0705] 84. The macromolecule complex or macromolecule of any one of embodiments 1 -72, wherein the FBD, FBD1 , or FBD2 comprises a chemical molecule that specifically binds the disease signature ligand.

[0706] 85. The macromolecule complex of any one of embodiments 13-23, 29-63, and 67-84, wherein the SBD1 is a first portion of a binding moiety and the SBD2 is a second portion of the binding moiety. 86. The macromolecule complex of any one of embodiments 13-23, 29-63, and 67-84, wherein the SBD1 specifically binds to a first component of a heterodimeric receptor and the SBD2 specifically binds to a second component of the heterodimeric receptor.

[0707] 87. The macromolecule complex of any one of embodiments 13-23, 29-63, and 67-84, wherein the SBD1 is a first component of a dimeric moiety and the SBD2 is a second component of a dimeric moiety.

[0708] 88. The macromolecule complex of any one of embodiments 13-23, 29-63, and 67-84, wherein the SBD1 is a first fragment of a polypeptide chain and the SBD2 is a second fragment of the polypeptide chain.

[0709] 89. The macromolecule complex of embodiment 88, wherein the polypeptide chain is a hormone, a cytokine, or a growth factor.

[0710] 90. The macromolecule complex of any one of embodiments 13-23, 29-63, and 67-89, wherein the SBD1 and the SBD2 have been engineered to have reduced affinity for one another.

[0711] 91 . The macromolecule complex or macromolecule of any one of embodiments 1 -90, wherein the macromolecule comprises a reporter moiety.

[0712] 92. The macromolecule complex or macromolecule of embodiment 91 , wherein the reporter moiety is an affinity tag, a fluorescent marker, a radioactive marker, or a chromogenic marker.

[0713] 93. The macromolecule complex or macromolecule of embodiment 92, wherein the affinity tag is a FLAG affinity tag or the chromogenic marker is luciferase or beta-lactamase.

[0714] 94. The macromolecule complex or macromolecule of any one of embodiments 1 -93, further comprising one or more linker domains.

[0715] 95. The macromolecule complex or macromolecule of embodiment 94, wherein the one or more linker domains are peptide linkers.

[0716] 96. The macromolecule complex or macromolecule of embodiment 95, wherein the peptide linkers comprise one or more GS linkers.

[0717] 97. The macromolecule complex or macromolecule of embodiment 96, wherein the GS linkers comprise one or more GS(GnS)mlinkers.

[0718] 98. The macromolecule complex or macromolecule of embodiment 96, wherein the GS linkers comprise one or more (GnS)m linkers. 99. The macromolecule complex or macromolecule of any one of embodiments 1 -53, 58-80, and 85- 98, wherein the macromolecule is a polypeptide.

[0719] 100. The macromolecule complex or macromolecule of any one of embodiments 1 -99, wherein the biological sample is an extract, fluid, fraction, cell, tissue, or subject.

[0720] 101 . The macromolecule complex or macromolecule of any one of embodiments 1 -100, wherein the macromolecule or one or both members of pair of macromolecules comprises a leader sequence.

[0721] 102. The macromolecule complex or macromolecule of embodiment 101 , wherein the leader sequence comprises a secretion signal.

[0722] 103. The macromolecule complex or macromolecule of any one of embodiments 1 -102, wherein the macromolecule or one or both members of pair of macromolecules comprises a half-life extension moiety.

[0723] 104. The macromolecule complex or macromolecule of embodiment 103, wherein the half-life extension moiety is an Fc domain or a fragment thereof, an albumin domain or a fragment thereof, or polyethylene glycol (PEG) or a modified derivative thereof.

[0724] 105. A nucleic acid encoding the macromolecule of any one of embodiments 1 , 3-12, and 24-104.

[0725] 106. A pair of nucleic acids encoding the pair of macromolecules of any one of embodiments 2 and 13-23.

[0726] 107. The nucleic acid or pair of nucleic acids of embodiment 105 or 106, wherein the nucleic acid is an RNA or a DNA.

[0727] 108. The nucleic acid or pair of nucleic acids of any one of embodiments 105-107, wherein the nucleic acid is formulated with a delivery platform.

[0728] 109. The nucleic acid or pair of nucleic acids of embodiment 108, wherein the delivery platform is a lipid-based carrier or a vector delivery system.

[0729] 110. The nucleic acid or pair of nucleic acids of embodiment 109, wherein the lipid-based carrier is a lipid nanoparticle (LNP).

[0730] 111. The nucleic acid or pair of nucleic acids of embodiment 109, wherein the vector delivery system comprises or is derived from an adenovirus, an anellovirus, an AAV, or a lentivirus.

[0731] 112. A nucleic acid encoding the macromolecule of any one of embodiments 1 , 3-12, and 24-104, wherein the nucleic acid is formulated with a carrier. 1 13. A pair of nucleic acids encoding the pair of macromolecules of any one of embodiments 2 and 13-23, wherein the pair of nucleic acids is formulated with a carrier.

[0732] 1 14. The nucleic acid or pair of nucleic acids of embodiment 1 12 or 1 13, wherein the nucleic acid is an RNA or a DNA.

[0733] 1 15. The nucleic acid or pair of nucleic acids of embodiment 1 12 or 1 13, wherein the carrier is a lipid- based carrier.

[0734] 1 16. The nucleic acid or pair of nucleic acids of embodiment 1 15, wherein the lipid-based carrier is a LNP.

[0735] 1 17. A vector comprising the nucleic acid of embodiment 105 or 107.

[0736] 1 18. A vector or pair of vectors comprising the pair of nucleic acids of embodiment 106 or 107.

[0737] 1 19. The vector or pair of vectors of embodiment 1 17 or 1 18, wherein the vector or pair of vectors is formulated with a carrier.

[0738] 120. A host cell comprising the nucleic acid or pair of nucleic acids of any one of embodiments 105- 1 16 or the vector or pair of vectors of any one of embodiments 1 17-1 19.

[0739] 121 . The macromolecule complex or macromolecule of any one of embodiments 1 -104 or the nucleic acid or pair of nucleic acids of any one of embodiments 105-1 16, wherein the macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids is more than 95% pure.

[0740] 122. The macromolecule complex or macromolecule of any one of embodiments 1 -104 or the nucleic acid or pair of nucleic acids of any one of embodiments 105-1 16, wherein the macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids is manufactured according to the U.S. Food and Drug Administration (FDA)’s Good Manufacturing Practice (GMP), Good Clinical Practice (GCP), and / or Good Laboratory Practice (GLP) standards.

[0741] 123. A method comprising providing the macromolecule complex or macromolecule of any one of embodiments 1 -104 or the nucleic acid or pair of nucleic acids of any one of embodiments 105-1 16 access to a cell.

[0742] 124. A method of modulating the state of a cell, comprising providing the macromolecule complex or macromolecule of any one of embodiments 1 -104 or the nucleic acid or pair of nucleic acids of any one of embodiments 105-1 16 access to the cell, thereby modulating the state of the cell.

[0743] 125. A method of inducing a cellular effector function in a cell, comprising providing the macromolecule complex or macromolecule of any one of embodiments 1 -104 or the nucleic acid or pair of nucleic acids of any one of embodiments 105-116 access to the cell, thereby inducing the cellular effector function in the cell.

[0744] 126. The method of embodiment 124 or 125, wherein the cell is in a subject and the macromolecule complex, macromolecule, nucleic acid, or pair of nucleic acids is administered in a therapeutically effective amount.

[0745] 127. The method of embodiment 126, wherein the subject has, or is suspected of having, a disease or disorder characterized by abnormal levels of the disease signature target, optionally wherein the subject was previously determined to have abnormal levels of the disease signature target.

[0746] 128. A method of determining the state of a cell, comprising providing the macromolecule complex or macromolecule of any one of embodiments 91 -93 or a nucleic acid or pair of nucleic acids encoding the same access to the cell, and detecting the presence of the reporter domain, thereby determining the state of the cell.

[0747] 129. A method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein:

[0748] (a) the FBD specifically binds a disease signature ligand in a biological sample; and

[0749] (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two copies of the macromolecule are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each copy of the macromolecule binding the disease signature ligand.

[0750] 130. A method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule complex comprising a pair of macromolecules, wherein each macromolecule independently comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein:

[0751] (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample;

[0752] (b) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1 ) that specifically binds a first effector ligand in the biological sample; and

[0753] (c) the second member of the pair of macromolecules comprises a second binding domain 2 (SBD2) that specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the pair of macromolecules binding the disease signature ligand.

[0754] 131 . A method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule comprising two FBDs linked to two SBDs by one or more linker domains, wherein:

[0755] (a) the FBDs specifically bind a disease signature ligand in a biological sample; and

[0756] (b) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand.

[0757] 132. A method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule comprising two FBDs linked to an SBD1 and an SBD2 by one or more linker domains, wherein:

[0758] (a) the FBDs specifically bind a disease signature ligand in a biological sample;

[0759] (b) the SBD1 specifically binds a first effector ligand in the biological sample; and

[0760] (c) the SBD2 specifically binds a second effector ligand in the biological sample; wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; wherein the macromolecule is capable of adopting a configuration that allows binding of the SBD1 and the SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional upon each of the two FBDs binding the disease signature ligand.

[0761] 133. A macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a VEGF binding domain linked to a 4-1 BB binding domain by a linker domain, wherein the 4- 1 BB binding domain induces a cellular effector function upon binding to 4-1 BB; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding to VEGF.

[0762] 134. The macromolecule complex of embodiment 133, wherein the conjugation is non-covalent.

[0763] 135. The macromolecule complex of embodiment 134, wherein the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair. 136. The macromolecule complex of embodiment 135, wherein the pair of complementary polypeptides is a pair of Fc fragments.

[0764] 137. The macromolecule complex of embodiment 136, wherein the pair of Fc fragments is a knob- into-hole pair.

[0765] 138. The macromolecule complex of any one of embodiments 133-136, wherein the two macromolecules are identical.

[0766] 139. A macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a VEGF binding domain linked to a 4-1 BB binding domain by a linker domain, wherein the 4- 1 BB binding domain induces a cellular effector function upon binding to 4-1 BB; wherein the two macromolecules are not conjugated to each other in the absence of the disease signature ligand; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding VEGF.

[0767] 140. The macromolecule complex of embodiment 139, wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of the disease signature ligand, and wherein the non-covalent conjugation is mediated by the disease signature ligand.

[0768] 141 . The macromolecule complex of embodiment 139 or 140, wherein the macromolecule or one or both members of pair of macromolecules comprises a half-life extension moiety.

[0769] 142. The macromolecule complex of embodiment 103, wherein the half-life extension moiety is an Fc domain or a fragment thereof.

[0770] 143. The macromolecule complex of any one of embodiments 133-142, wherein the VEGF binding domain is an anti-VEGF scFv.

[0771] 144. The macromolecule complex of any one of embodiments 133-142, wherein the VEGF binding domain is a VEGF receptor trap.

[0772] 145. The macromolecule complex of any one of embodiments 133-144, wherein the 4-1 BB binding domain is an anti-4-1 BB scFv.

[0773] 146. A macromolecule complex comprising two macromolecules, wherein each macromolecule comprises an IFNg binding domain linked to an IL-1 OR binding domain by a linker domain, wherein the IL- 10R binding domain induces a cellular effector function upon binding to IL-1 OR; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding to IFNg.

[0774] 147. The macromolecule complex of embodiment 146, wherein the conjugation is non-covalent.

[0775] 148. The macromolecule complex of embodiment 147, wherein the non-covalent conjugation is mediated by a pair of complementary moieties, each macromolecule comprising one member of the pair.

[0776] 149. The macromolecule complex of embodiment 148, wherein the pair of complementary polypeptides is a pair of Fc fragments.

[0777] 150. The macromolecule complex of embodiment 149, wherein the pair of Fc fragments is a knob- into-hole pair.

[0778] 151 . The macromolecule complex of any one of embodiments 146-150, wherein the two macromolecules are identical.

[0779] 152. A macromolecule complex comprising two macromolecules, wherein each macromolecule comprises an IFNg binding domain linked to an IL-1 OR binding domain by a linker domain, wherein the IL- 10R binding domain induces a cellular effector function upon binding to IL-1 OR; wherein the two macromolecules are not conjugated to each other in the absence of the disease signature ligand; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding IFNg.

[0780] 153. The macromolecule complex of embodiment 152, wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are non-covalently conjugated to each other in the presence of the disease signature ligand, and wherein the non-covalent conjugation is mediated by the disease signature ligand.

[0781] 154. The macromolecule complex of embodiment 152 or 153, wherein the macromolecule or one or both members of pair of macromolecules comprises a half-life extension moiety.

[0782] 155. The macromolecule complex of embodiment 154, wherein the half-life extension moiety is an Fc domain or a fragment thereof.

[0783] 156. The macromolecule complex of any one of embodiments 146-155, wherein the IFNg binding domain is an anti- IFNg scFv.

[0784] 157. The macromolecule complex of any one of embodiments 146-156, wherein the IL-1 OR binding domain is IL-10. EXAMPLES

[0785] The following are examples of the methods of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

[0786] Table of Contents (Examples):

[0787] Example 1. Protein complex modulating TPO receptor activity conditional on TGF-beta1 or IL-8

[0788] This Example describes the expression, purification, and characterization of (i) a protein complex that modulates the thrombopoietin (TPO) receptor conditional on the presence of transforming growth factor beta 1 (TGF-beta1 ) and (ii) a protein complex that modulates the TPO receptor conditional on the presence of interleukin-8 (IL-8). A genericized version of the fusion protein is shown in Fig. 1 .

[0789] A. Fusion proteins

[0790] TGF-beta1 - TPO fusion protein

[0791] This Example provides a protein complex composed of two identical fusion proteins that modulate the TPO receptor conditional on the presence of TGF-beta1 (TGFb) (referred to herein as the “TGFb - TPO fusion protein”). Each fusion protein includes (from N- to C-terminus): a mouse immunoglobulin kappa variable 3 (IgKVIll) leader peptide (UniProt ID A0A140T8P0 positions M1 to G20); an anti-TGF-beta1 single-chain variable fragment (scFv) (PDB 4KV5; SEQ ID NOs: 13 and 14; Table 2); a (G4S)n linker, where n = 2, 3, or 5; and an anti-TPO receptor heavy chain variable domain (VH) and light chain variable domain (VL) (SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 32 and SEQ ID NO: 33, respectively; Table 2) joined by a G4S linker (Fig. 5A). The proposed mechanism of action of the construct is shown in Fig. 5B: TGFb recruits two copies of the fusion protein via binding their TGFb- targeting domains, thus positioning the TpoR effector domains in close proximity to bind to and induce TpoR activity.

[0792] Optionally, each fusion protein further includes a FLAG affinity tag following the anti-TPO receptor VH and VL.

[0793] IL-8 - TPO fusion protein

[0794] This Example also provides a protein complex composed of two identical fusion proteins that modulate the TPO receptor conditional on the presence of IL-8 (referred to herein as the “IL-8 - TPO fusion protein”). Each fusion protein includes (from N- to C-terminus): a mouse immunoglobulin kappa variable 3 (IgKVIll) leader peptide (UniProt ID A0A140T8P0 positions M1 to G20); an anti-IL-8 scFv (PDB 6WZM, SEQ ID NO: 12; Table 2); a (G4S)n linker, where n = 2, 3, or 5; and an anti-TPO receptor heavy chain variable domain (VH) and light chain variable domain (VL) (SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 32 and SEQ ID NO: 33, respectively; Table 2) joined by a G4S linker (Fig. 6A). The proposed mechanism of action of the construct is shown in Fig. 6B: IL-8 recruits two copies of the fusion protein via binding their IL-8-targeting domains, thus positioning the TpoR effector domains in close proximity to dimerize and induce TpoR activity.

[0795] Optionally, each fusion protein further includes a FLAG affinity tag following the anti-TPO receptor VH and VL.

[0796] Expression and purification

[0797] The fusion proteins are expressed via transient transfection of human embryonic kidney 293 (HEK293) cells under control of the cytomegalovirus (CMV) promoter. Secreted protein is analyzed by Western blot or ELISA using an anti-FLAG capture antibody to assess expression and quality. Fusion proteins are purified if necessary using a FLAG capture step, followed by a size exclusion chromatography (SEC) polishing step. Expression is expected to yield a strong band at the expected molecular weight by western blot, with minimal product-related variants.

[0798] B. In vitro assays to assess effect of fusion protein on TPO signaling pathway in presence or absence of soluble TPO or TGF-beta1

[0799] HEK-BLUE™ TPO cells (InvivoGen catalog code: hkb-tpo) are used to evaluate activity of the fusion protein, using the manufacturer’s prescribed protocol unless otherwise indicated. Briefly, HEK- BLUE™ TPO cells are transiently transfected with the fusion protein expression construct (as described in Example 1 A) to secrete the fusion protein as autocrine in the assay. Alternatively, supernatant from HEK293 cells transiently expressing the fusion proteins is applied to the HEK-BLUE™ TPO cells as the test sample. Confirmatory assays are performed using FLAG-purified fusion protein material. JAK2 / STAT5 pathway activation via secreted alkaline phosphatase (SEAP) production is compared among positive controls (administration of purified recombinant human TPO; transient transfection to express and secrete human TPO as an autocrine), negative controls (administration of recombinant human interleukin-2 (IL-2); mock transfection plasmid), and test samples (fusion protein variants). HEK- BLUE™ TPO cells expressing the fusion proteins are incubated overnight in the presence or absence of TPO, as well as in the presence or absence of TGF-beta1 (for the TGFb - TPO fusion protein) or IL-8 (for the IL-8 - TPO fusion protein). Functional fusion protein samples are further evaluated by performing dose titrations of the fusion protein samples in the presence of TGF-beta1 or IL-8 to assess EC50, or in the presence of TGF-beta1 and TPO or IL-8 to assess IC50. The fusion proteins are expected to activate TPO receptor signaling conditional on the presence of TGF-beta1 (TGFb - TPO fusion protein) or IL-8 (IL-8 - TPO fusion protein).

[0800] Fig. 5C shows a dose-response relationship between TGFb concentration and activity of the TGFb - TPO fusion protein (comprising the anti-TPO receptor VH and VL sequences of SEQ ID NO: 32 and SEQ ID NO: 33, respectively). TpoR SEAP reporter cells were stimulated overnight with a constant amount of the TGFb - TPO fusion protein and varying concentrations of TGFb, followed by measurement of SEAP activity in the culture supernatant.

[0801] Fig. 6C shows a dose-response relationship between IL-8 concentration and activity of the IL-8 - TPO fusion protein (comprising the anti-TPO receptor VH and VL sequences of SEQ ID NO: 32 and SEQ ID NO: 33, respectively). TpoR SEAP reporter cells were stimulated overnight with a constant amount of the IL8- TPO fusion protein and varying concentrations of IL-8, followed by measurement of SEAP activity in the culture supernatant.

[0802] C. Characterization of TPO receptor agonism by fusion protein versus native agonist

[0803] Cell samples for profiling and comparing gene expression between cells treated with the fusion proteins or soluble ligands are collected following the overnight incubation step of the assay described in Example 1 B. RNA is extracted and purified according to the Qiagen RNeasy kit (Cat. No. 74104) with a threshold RNA Quality Number > 7. RNA libraries for gene expression profiling are prepared using the Roche KAPA HyperPrep kit and sequenced on an Illumina NextSeq to generate 40-nucleotide paired-end reads. Sequenced reads are checked for quality, aligned, counted, and analyzed using standard procedures. See, for example, Kukurba and Montgomery, RNA sequencing and analysis. Cold Spring Harbor Protocols, 2015(11 ), doi: 10.1101 / pdb.top084970.

[0804] Table 2. Sequences

[0805] Example 2. Protein complex agonizing IL-2R activity conditional on TGF-beta1 or IL-8

[0806] This Example describes the expression, purification, and characterization of (i) a protein complex that agonizes human interleukin-2 receptor (IL-2R) activity conditional on TGF-beta1 and (ii) a protein complex that agonizes human IL-2R activity conditional on interleukin-8 (IL-8; also known as CXCL8). A genericized version of the pair of fusion proteins is shown in Fig. 2.

[0807] A. Fusion proteins TGF-beta1 - IL-2R fusion protein pair This Example provides a protein complex composed of two complementary fusion proteins

[0808] (fusion proteins (a) and (b)) that modulates the IL-2 receptor (IL-2R) conditional on the presence of TGF- betal . Each fusion protein includes (from N- to C-terminus): a mouse IgKVIll leader peptide (UniProt ID A0A140T8P0 positions M1 to G20); an anti-TGF-beta1 scFv (PDB 4KV5; SEQ ID NOs: 13 and 14; Table 2); a (G4S)n linker, where n = 10, 15, or 25; and (a) conditional IL-2R effector domain A (SEQ ID NO: 3; Table 2) or (b) conditional IL-2R effector domain B (SEQ ID NO: 4; Table 2) (Fig. 7A). The proposed mechanism of action of the complementary fusion proteins is shown in Fig. 7B: TGFb recruits each of the fusion proteins via binding their TGFb-targeting domains, thus positioning the IL-2R effector domains in close proximity to dimerize and induce IL-2R activity.

[0809] Optionally, the fusion proteins each also include a FLAG affinity tag between the leader peptide and the anti-TGF-beta1 scFv.

[0810] IL-8 - IL-2R fusion protein pair

[0811] This Example also provides a protein complex composed of two complementary fusion proteins that modulate the IL-2 receptor conditional on the presence of IL-8, wherein the two complementary fusion proteins each comprise an anti-IL-8 scFv (PDB 6WZM, SEQ ID NO: 12; Table 2).

[0812] Expression and purification

[0813] The fusion proteins are expressed and purified as described in Example 1 A by 1 ) transfecting with equal quantities of each complementary construct (fusion proteins (a) and (b)) or 2) independently transfecting, purifying, and pre-mixing equal quantities of fusion proteins (a) and (b).

[0814] B. In vitro assays for IL-2R agonism on cells in presence or absence of TGF-beta1 HEK-BLUE™ CD122 / CD132 cells (InvivoGen catalog code: hkb-il2bg) are used to evaluate fusion protein activity, using the manufacturer’s prescribed protocol unless otherwise indicated and as described in Example 1 B. IL-2 signaling pathway activation via SEAP production is compared among positive controls (administration of purified recombinant human IL-2; transient transfection to express and secrete human IL-2 as an autocrine), negative controls (administration of recombinant human TPO; mock transfection plasmid), and test samples (fusion proteins (a) and (b) and variants thereof). HEK-BLUE™ CD122 / CD132 cells expressing fusion proteins are incubated overnight in the presence or absence of IL- 2, as well as in the presence or absence of TGF-beta1 . Functional fusion protein samples are further evaluated by performing dose titrations of the fusion protein samples in the presence of TGF-beta1 to assess EC50. Fusion proteins (a) and (b), administered in combination, are expected to activate IL-2 signaling conditional on the presence of TGF-beta1 .

[0815] Fig. 7C shows a dose-response relationship between fusion proteins (a) and (b) and TGFb concentration. IL-2 reporter cells were stimulated overnight with a constant amount of fusion proteins (a) and (b) and varying concentrations of TGFb, followed by measurement of SEAP activity in the culture supernatant.

[0816] C. Characterization of TGFR agonism by fusion protein versus native agonist

[0817] Transcriptomic comparison among fusion proteins and soluble ligands is performed as described in Example 1 C. Example 3. Protein complex agonizing IL-1 OR activity conditional on IFN-gamma or IL-6

[0818] This Example describes the expression, purification, and characterization of proteins and protein complexes that agonize the human interleukin 10 receptor (IL-10 receptor) conditional on the presence of interferon-gamma (IFN-gamma) or interleukin-6 (IL-6).

[0819] A. Fusion proteins

[0820] IFN-gamma - IL- 10 fusion protein pair

[0821] This Example provides a protein complex composed of two complementary fusion proteins (fusion proteins (c) and (d)) that modulates the IL-10 receptor conditional on the presence of IFN-gamma. Each fusion protein includes (from N- to C-terminus): a mouse IgKVIll leader peptide (UniProt ID A0A140T8P0 positions M1 to G20); an anti-IFN-gamma scFv (PDB 1T3F; SEQ ID NO: 5; Table 2); a (G4S)n linker, where n = 1 , 2, or 3; and an anti-l L1 ORa VHH antibody (SEQ ID NO: 6; Table 2) (fusion protein (c)) or an anti-IL1 ORb VHH antibody (SEQ ID NO: 7; Table 2) (fusion protein (d)) (Fig. 8A). A genericized version of the complementary fusion proteins is shown in Fig. 3. Optionally, each fusion protein further includes a FLAG affinity tag following the anti-IL10Ra VHH antibody (fusion protein (c)) or anti-IL1 ORb VHH antibody (fusion protein (d)). The proposed mechanism of action of the construct is shown in Fig. 8D: IFN-gamma recruits each of the fusion proteins via binding their IFN-gamma-targeting domains, thus positioning the IL-1 OR-binding effector domains in close proximity to dimerize and induce IL-1 OR activity.

[0822] The fusion proteins are expressed and purified as described in Example 1 A by 1 ) transfecting with equal quantities of each complementary construct (fusion proteins (c) and (d)) or 2) independently transfecting, purifying, and pre-mixing equal quantities of fusion proteins (c) and (d).

[0823] Self-assembling IFN-gamma - IL- 10 fusion protein pair

[0824] This Example also provides a protein complex composed of two complementary fusion proteins (fusion proteins (g) and (h)) that modulates the IL-10 receptor conditional on the presence of IFN-gamma, in which the two complementary fusion proteins self-assemble as heterodimers. Each fusion protein includes (from N- to C-terminus): a mouse IgKVIll leader peptide (UniProt ID A0A140T8P0 positions M1 to G20); an anti-IL10Ra VHH antibody (SEQ ID NO: 6; Table 2) (fusion protein (g)) or an anti-IL1 ORb VHH antibody (SEQ ID NO: 7; Table 2) (fusion protein (h)); a (G4S)n linker in which n = 2; an anti-IFN- gamma scFv (SEQ ID NO: 5; Table 2); a (G4S)n linker in which n = 1 , 2, 3, or 4; and a human IgG 1 Fc with knob mutations (SEQ ID NO: 10; Table 2) or hole mutations (SEQ ID NO: 11 ; Table 2) (Fig. 8B). Fig. 8F shows variants of the pair of fusion proteins in which the linker between the Fc hinge and the IFNg- binding scFv consists of 5 amino acid residues (construct P482+P483) or 10 amino acid residues (P484+P485).

[0825] Optionally, each fusion protein further includes a polyhistidine (His) affinity tag.

[0826] The fusion proteins are expressed by transfecting with equal quantities of each complementary construct (fusion proteins (g) and (h)). The chains form spontaneous heterodimers via their knob-into- hole Fc regions to create a single molecule carrying one copy of an anti-IL10Ra domain and one copy of an IL1 ORb domain, and can be purified either via protein A or Ni-NTA resin. Variations of IFN-gamma - IL- 10 fusion protein pair

[0827] Variations of the above-described protein complexes are shown in Figs. 12A-12I. Fig. 12A shows a variation in which the length of the linker between the Fc hinge and the I FNg-binding scFv has been modulated (increased or decreased). Fig. 12B shows a variation in which the length of the linker between the IFNg-binding scFv and the IL1 OR-binding VHH has been modulated (increased or decreased) (Fig. 12B). Figs. 12B and 12C show variations in which the IL1 OR-binding VHH has been substituted with a scFv agonist against IL-1 OR and a monomeric IL-10 cytokine, respectively. Fig. 12E shows a variation in which the knob-into-hole Fc regions and the IFNg-binding scFv have been replaced with a knob-into-hole full-length IgG having its Fab region encoding for IFNg-binding. Figs. 12F-12I show variants in which each fusion protein (excluding the knob-into-hole Fc regions) is fused to a full-length IgG antibody at the N-terminus of the heavy chain; the N-terminus of the light chain; the C-terminus of the light chain; or the C-terminus of the heavy chain Fc region.

[0828] Single-polypeptide IFN-gamma - IL- 10 fusion protein

[0829] This example also provides a protein that modulates the IL-10 receptor conditional on the presence of IFN-gamma, in which the protein is composed of a single polypeptide chain encoded by the following (from N- to C-terminus): a mouse IgKVIll leader peptide (UniProt ID A0A140T8P0 positions M1 to G20); an anti-l L1 ORa VHH antibody (SEQ ID NO: 6; Table 2); an anti-IFN-gamma scFv (SEQ ID NO: 5; Table 2); a (G4S)n linker in which n = 2, 3, 4, 5, or 6; a second copy of the anti-IFN-gamma scFv (SEQ ID NO: 5; Table 2); and an anti-IL1 ORb VHH antibody (SEQ ID NO: 7; Table 2) (Fig. 8C). Alternatively, the locations of the anti-l L1 ORa VHH antibody (SEQ ID NO: 6; Table 2) and the anti-l L1 ORb VHH antibody (SEQ ID NO: 7; Table 2) may be reversed (Fig. 8C). Optionally, the protein further includes a histidine (His) affinity tag.

[0830] The fusion protein is expressed as described in Example 1 A and can be purified using Ni-NTA resin.

[0831] IL-6 - IL- 10 fusion protein pair

[0832] This Example also provides a protein complex composed of two complementary fusion proteins that modulate the IL-10 receptor conditional on the presence of IL-6. Each fusion protein includes (from N- to C-terminus): a mouse IgKVIll leader peptide (UniProt ID A0A140T8P0 positions M1 to G20); an anti-IL10Ra VHH antibody (first fusion protein) or an anti-IL1 ORb VHH antibody (second fusion protein); a (G4S)2 linker; a first anti-IL-6 VHH antibody (SEQ ID NO: 34; first fusion protein) or a second anti-IL-6 VHH antibody (SEQ ID NO: 35; second fusion protein), wherein the first and second anti-IL-6 VHH antibodies bind distinct epitopes on IL-6; a (G4S)n linker; and a polyhistidine tag (Fig. 9A). Alternatively, the binding domains may be swapped such that (i) the first fusion protein comprises the first anti-IL-6 VHH antibody and the second anti-IL-6 VHH antibody (SEQ ID NO: 35) and (ii) the second fusion protein comprises the second anti-IL-6 VHH antibody and the first anti-IL-6 VHH antibody (SEQ ID NO: 34). The proposed mechanism of action of the construct is shown in Fig. 9B: IL-6 recruits each of the fusion proteins via binding their IL-6-targeting domains, thus positioning the IL-1 OR-binding effector domains in close proximity to dimerize and induce IL-1 OR acti...

Claims

WHAT IS CLAIMED IS:

1. A macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein:(a) the FBD specifically binds a disease signature ligand in a biological sample; and(b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand.

2. A macromolecule complex comprising a pair of macromolecules, wherein each macromolecule independently comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein:(a) the first member of the pair of macromolecules comprises a first binding domain 1 (FBD1 ) that specifically binds a first epitope of a disease signature ligand in a biological sample;(b) the second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds a second epitope of the disease signature ligand in a biological sample; and(c) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding the disease signature ligand.

3. The macromolecule complex of claim 1 or 2, wherein the conjugation is covalent.

4. The macromolecule complex of claim 1 or 2, wherein the conjugation is non-covalent.

5. The macromolecule complex or macromolecule of any one of claims 1 -4, wherein the disease signature ligand is a protein, a peptide, or a small molecule.

6. The macromolecule complex or macromolecule of any one of claims 1 -5, wherein the FBD, FBD1 , or FBD2 comprises a polypeptide that specifically binds the disease signature ligand.

7. The macromolecule complex or macromolecule of claim 6, wherein the polypeptide is an antibody or a fragment thereof.

8. The macromolecule complex or macromolecule of any one of claims 1 -7, wherein the effector ligand is a protein or a peptide.

9. The macromolecule complex or macromolecule of any one of claims 1 -8, further comprising one or more additional linker domains.

10. The macromolecule complex or macromolecule of any one of claims 1 -9, wherein the one or more linker domains are peptide linkers.11 . The macromolecule complex or macromolecule of any one of claims 1 -10, wherein the macromolecule is a polypeptide.

12. A nucleic acid encoding the macromolecule of any one of claims 1 -1 1 .

13. A pair of nucleic acids encoding the pair of macromolecules of claim 2.

14. The nucleic acid or pair of nucleic acids of claim 12 or 13, wherein the nucleic acid is an RNA or a DNA.

15. A nucleic acid encoding the macromolecule of any one of claims 1 -1 1 , wherein the nucleic acid is formulated with a carrier.

16. A vector comprising the nucleic acid of claim 12, 14 or 15.

17. A vector or pair of vectors comprising the pair of nucleic acids of claim 13 or 14.

18. A host cell comprising the nucleic acid or pair of nucleic acids of any one of claims 12-15 or the vector or pair of vectors of any one of claims 16 or 17.

19. A method comprising providing the macromolecule complex or macromolecule of any one of claims 1 -1 1 or the nucleic acid or pair of nucleic acids of any one of claims 12-15 access to a cell.

20. A macromolecule complex comprising two macromolecules, wherein each macromolecule comprises a VEGF binding domain linked to a 4-1 BB binding domain by a linker domain, wherein the 4- 1 BB binding domain induces a cellular effector function upon binding to 4-1 BB; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding to VEGF.21 . A macromolecule complex comprising two macromolecules, wherein each macromolecule comprises an IFNg binding domain linked to an IL-1 OR binding domain by a linker domain, wherein the IL- 10R binding domain induces a cellular effector function upon binding to IL-1 OR; wherein the two macromolecules are conjugated to each other;and wherein induction of the effector function by the macromolecule complex is conditional upon each of the two macromolecules binding to IFNg.

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