Compositions and methods

Abstract

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

Description

Cross Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 495,509, filed April 11, 2023, and U.S. Provisional Patent Application No. 63 / 613,644, filed December 21, 2023, the entire contents of each of these provisional patent applications are incorporated herein by reference in their entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and which is hereby incorporated by reference in its entirety. The XML copy, created on April 9, 2024, is named 51661-005WO4_Sequence_Listing_4_9_24 and is 43,890 bytes in size. BACKGROUND

[0003] There is a need in the art for macromolecules that conditionally induce cellular effector function (e.g., biological or therapeutic activity) based on the presence of a disease signature ligand, and methods of using these macromolecules. SUMMARY

[0004] In a first aspect, provided herein is a macromolecular complex comprising two macromolecules, wherein each macromolecule comprises a first binding domain (FBD) connected to a second binding domain (SBD) by a linker domain, wherein (a) the FBD specifically binds to a disease signature ligand in a biological sample; and (b) the SBD specifically binds to 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 the induction of the effector function by the macromolecular complex is conditional on each of the two macromolecules binding to the disease signature ligand.

[0005] In a second aspect, provided herein is a macromolecular complex comprising two macromolecules, wherein each macromolecule comprises a first binding domain (FBD) connected to a second binding domain (SBD) by a linker domain, wherein (a) the FBD specifically binds to a disease signature ligand in a biological sample; and (b) the SBD specifically binds to 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 the induction of the effector function by the macromolecular complex is conditional on each of the two macromolecules binding to the disease signature ligand, wherein the two macromolecules are identical.

[0006] In a third aspect, provided herein is a macromolecular 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) a 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) a 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 the induction of the effector function by the macromolecular complex is contingent on each of the pair of macromolecules binding the disease signature ligand.

[0007] In a fourth aspect, provided herein is a macromolecular 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) a 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;

[0008] (b) a 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 the 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 the induction of the effector function by the macromolecular complex is contingent on each of the two macromolecules binding the disease signature ligand.

[0009] 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 a disulfide bond between the macromolecules.

[0010] 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 complementary pair of moieties, each macromolecule comprising one member of the pair. In some embodiments, the complementary pair of moieties is biotin and avidin; a barnase and a barstar; a complementary pair of aptamers; or a complementary pair of polypeptides. In some embodiments, the complementary pair of polypeptides is a pair of engineered Fc fragments, such as a knob-into-hole pair.

[0011] In some embodiments of any of the above aspects, the conjugation spatially orients the two macromolecules to allow for conditional simultaneous binding of a disease signal and an effector ligand.

[0012] In some embodiments, the non-covalent conjugation is mediated by binding of the FBD to the disease signature ligand.

[0013] In some embodiments of the second through fourth aspects, the conjugation spatially orients the first member of the pair of macromolecules and the second member of the pair of macromolecules to allow for conditional simultaneous binding of a disease signal and an effector ligand.

[0014] In some embodiments, the non-covalent conjugation is mediated by binding of the FBD to the disease signature ligand.

[0015] In a fifth aspect, provided herein is a macromolecular 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 to a disease signature ligand in a biological sample; and (b) the SBD specifically binds to 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 the induction of the effector function by the macromolecular complex is conditional on binding of the disease signature ligand by each of the two macromolecules.

[0016] In a sixth aspect, provided herein is a macromolecular 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) a first member of the pair of macromolecules comprises a first binding domain 1 (FBD1) that specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) a second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of the disease signature ligand in the biological sample; and (c) the SBD specifically binds to 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 the induction of the effector function by the macromolecular complex is contingent on each of the two macromolecules binding to the disease signature ligand.

[0017] 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.

[0018] In a seventh aspect, provided herein is a macromolecule comprising two FBDs linked to two SBDs by a linker domain, wherein (a) the FBDs specifically bind to a disease signature ligand in a biological sample; and (b) the SBDs specifically bind to 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 conformation that allows the two SBDs to bind to the effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is contingent on each of the two FBDs binding to the disease signature ligand.

[0019] In an eighth aspect, provided herein is a macromolecule comprising a FBD1 and a FBD2 linked to two SBDs by a linker domain, wherein (a) the FBD1 specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) the FBD2 specifically binds to a second epitope of the disease signature ligand in the biological sample; and (c) the SBDs specifically bind to 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 conformation that allows the two SBDs to bind to the effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is contingent on each of the FBD1 and the FBD2 binding to the disease signature ligand.

[0020] In a ninth aspect, provided herein is a macromolecule comprising two FBDs connected to SBD1 and SBD2 via a linker domain, 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 conformation that allows the SBD1 and the SBD2 to bind to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is contingent on each of the two FBDs binding the disease signature ligand.

[0021] In a tenth aspect, provided herein is a macromolecule comprising FBD1 and FBD2 connected to SBD1 and SBD2 via a linker domain, 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 conformation that allows the SBD1 and the SBD2 to bind to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is contingent on each of the FBD1 and the FBD2 binding the disease signature ligand.

[0022] In certain foregoing aspects, the first binding domain and the second binding domain are covalently linked (e.g., by a small molecule, a peptide (e.g., as a single polypeptide chain comprising the first binding domain and the second binding domain), or a combination thereof).

[0023] 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.

[0024] 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 TNF family member, or a 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-γ; the growth factor is transforming growth factor beta (TGF-β), 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 TNF family member is TNF-α; or the VEGF is VEGF-A or VEGF-B.

[0025] 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.

[0026] 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.

[0027] In some embodiments of any of the above aspects, the disease signature ligand comprises an autoantigen of the organism from which the biological sample is derived. In some embodiments of any of the above aspects, the disease signature ligand is an autoantigen of the organism from which the biological sample is derived. In some embodiments, the autoantigen is an anti-drug antibody (ADA), an autoantibody, or a tumor marker.

[0028] 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.

[0029] 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.

[0030] In some embodiments of any of the above aspects, the disease characteristic ligand comprises a hormone, an amino acid derivative, a steroid, or an eicosanoid. In some embodiments of any of the above aspects, the disease characteristic ligand is a hormone, an amino acid derivative, a steroid, or an eicosanoid.

[0031] In some embodiments of any of the above aspects, the disease characteristic ligand comprises a non-self antigen. In some embodiments of any of the above aspects, the disease characteristic ligand is a non-self antigen. In some embodiments, the disease characteristic ligand is a virus, a bacterium, or a fragment or antigen thereof.

[0032] In some embodiments of any of the above aspects, the disease characteristic ligand is multimeric. In some embodiments, the disease characteristic ligand is dimeric, trimeric, or tetrameric.

[0033] In some embodiments of any of the above aspects, the FBD, the FBD1, or the FBD2 comprises a polypeptide that specifically binds the disease characteristic ligand.

[0034] In some embodiments, the polypeptide comprises or is an antibody or 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 retargeting antibody (DART).

[0035] In some embodiments, the polypeptide comprises or 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 nanobody, a nanoCLAMP, an optimer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR).

[0036] In some embodiments, the polypeptide comprises an endogenous binding domain. In some embodiments, the endogenous binding domain comprises a cellular 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, the polypeptide is an endogenous binding domain. In some embodiments, the endogenous binding domain is a cellular 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.

[0037] In some embodiments of any of the above aspects, the FBD, the FBD1, or the 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.

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

[0039] In some embodiments of any of the above aspects, the FBD, the FBD1, or the FBD2 has affinity for two or more disease signature moieties.

[0040] 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.

[0041] 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 4-1BB. 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 IL10R, or the TNF superfamily receptor is TNFR2 or 4-1BB.

[0042] 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 macromolecular complex or the macromolecule and the disease signature ligand.

[0043] In some embodiments of the third, fourth, ninth, and tenth aspects, the first effector ligand and the second effector ligand must associate to exert a cellular effector function. In some embodiments, the first effector ligand and the second effector ligand associate in the presence of the macromolecular complex or the macromolecule and the disease signature ligand. In some embodiments, the association is heterodimerization.

[0044] 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.

[0045] In some embodiments of any of the above aspects, the SBD, the SBD1, or the SBD2 comprises a polypeptide that specifically binds the effector ligand.

[0046] In some embodiments, the polypeptide comprises an antibody or 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 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.

[0047] 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 nanobody, a nanoCLAMP, an optimer, a repebody, a pre- connectin, 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 nanobody, a nanoCLAMP, an optimer, a repebody, a pre- connectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a VLR.

[0048] 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 fragment thereof. In some embodiments, the endogenous binding domain comprises a viral binding protein or 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 fragment thereof. In some embodiments, the endogenous binding domain is a viral binding protein or fragment thereof.

[0049] In some embodiments of any of the above aspects, the SBD, the SBD1, or the 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.

[0050] In some embodiments of any of the above aspects, the FBD comprises a chemical molecule that specifically binds the disease characteristic ligand.

[0051] 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.

[0052] 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.

[0053] 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 the 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 the dimeric moiety.

[0054] 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.

[0055] 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 each other.

[0056] 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.

[0057] In some embodiments of any of the above aspects, the macromolecular complex or the macromolecule comprises one or more linker domains. In some embodiments, the one or more linker domains is a peptide linker. In some embodiments, the peptide linker comprises one or more GS linkers. In some embodiments, the GS linker comprises one or more GS(G n S) m linker or one or more (G n S) m linker. In some embodiments, the GS linker comprises one or more (G4S) m linkers.

[0058] In some embodiments of any of the above aspects, the macromolecule comprises a polypeptide.

[0059] In some embodiments of any of the above aspects, the macromolecule is a polypeptide.

[0060] 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.

[0061] In some embodiments of any of the above aspects, one or both members of the macromolecule pair comprises a leader sequence. In some embodiments, the leader sequence comprises a secretion signal.

[0062] In some embodiments of any of the above aspects, the macromolecular complex or one or both macromolecules of the macromolecular complex comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or fragment thereof, an albumin domain or fragment thereof, or a polyethylene glycol (PEG) or modified derivative thereof.

[0063] In another aspect, provided herein is a nucleic acid encoding the macromolecule of any of the first, fifth, seventh, eighth, ninth, and tenth aspects.

[0064] In another aspect, provided herein is a pair of nucleic acids encoding the pair of macromolecules of the second, third, fourth, or sixth aspects.

[0065] In some aspects, the nucleic acid is RNA or DNA.

[0066] In some aspects, the nucleic acid is formulated with a delivery platform.

[0067] In some aspects, the delivery platform is a lipid-based carrier or vector delivery system. In some embodiments, the lipid-based carrier is a lipid nanoparticle (LNP). In some embodiments, the vector delivery system comprises or derives from an adenovirus, an anellovirus, an AAV, or a lentivirus.

[0068] 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, wherein the nucleic acid is formulated with a carrier.

[0069] In another aspect, provided herein is a pair of nucleic acids encoding the pair of macromolecules of the second, third, fourth, or sixth aspects, wherein the pair of nucleic acids is formulated with a carrier.

[0070] In some embodiments, the nucleic acid is RNA or DNA.

[0071] In some embodiments, the carrier is a lipid-based carrier. In some embodiments, the lipid-based carrier is an LNP.

[0072] In another aspect, the disclosure provides a vector comprising the nucleic acid of any of the above aspects.

[0073] In another aspect, the disclosure provides a vector or pair of vectors comprising the pair of nucleic acids of any of the above aspects.

[0074] In some embodiments, the vector or pair of vectors is formulated with a carrier.

[0075] In another aspect, the disclosure provides a host cell comprising the nucleic acid, pair of nucleic acids, vector, or pair of vectors of any of the above aspects.

[0076] In some embodiments, the macromolecular complex, the macromolecule, the nucleic acid, or the pair of nucleic acids is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure or more than 99% pure.

[0077] In some embodiments, the macromolecular complex, the macromolecule, the nucleic acid, or the pair of nucleic acids is manufactured according to U.S. Food and Drug Administration (FDA) Good Manufacturing Practice (GMP), Good Clinical Practice (GCP), and / or Good Laboratory Practice (GLP) standards.

[0078] In another aspect, the disclosure provides a macromolecular complex comprising two macromolecules, wherein each macromolecule comprises a VEGF binding domain linked to a 4-1BB binding domain, wherein the 4-1BB binding domain induces a cellular effector function upon binding to 4-1BB; wherein the two macromolecules are conjugated to each other; and wherein induction of the effector function by the macromolecular complex is contingent upon binding of each of the two macromolecules to VEGF. See, e.g., the Figures and Examples herein.

[0079] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a complementary pair of moieties, each macromolecule comprising one member of the pair. In some embodiments, the complementary pair of polypeptides is a Fc fragment pair. In some embodiments, the Fc fragment pair is a knob-in-hole pair. In some embodiments, the two macromolecules are identical.

[0080] In another aspect, the disclosure provides a macromolecular complex comprising two macromolecules, wherein each macromolecule comprises a VEGF binding domain linked to a 4-1BB binding domain, wherein the 4-1BB binding domain induces a cellular effector function upon binding to 4-1BB; wherein the two macromolecules are not conjugated to each other in the absence of VEGF; and wherein induction of the effector function by the macromolecular complex is contingent upon binding of each of the two macromolecules to VEGF. In some embodiments, the first member of the macromolecular pair and the second member of the macromolecular pair 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 Figures and Examples herein.

[0081] In some embodiments, one or both members of the macromolecule or the macromolecular pair comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or fragment thereof.

[0082] In some embodiments of either of the two aspects above, the VEGF binding domain is an anti-VEGF scFv. In some embodiments, the VEGF binding domain is a VEGF receptor trap.

[0083] In some embodiments of either of the two aspects above, the 4-1BB binding domain is an anti-4-1BB scFv.

[0084] In some embodiments, one of the macromolecules comprises SEQ ID NOs: 36 and 38.

[0085] In some embodiments, one of the macromolecules comprises SEQ ID NOs: 37 and 38.

[0086] In some embodiments, one of the macromolecules comprises SEQ ID NO: 36 and 39.

[0087] In some embodiments, one of the macromolecules comprises SEQ ID NO: 37 and 39.

[0088] In some embodiments, both macromolecules comprise SEQ ID NO: 36 and 38.

[0089] In some embodiments, both macromolecules comprise SEQ ID NO: 37 and 38.

[0090] In some embodiments, both macromolecules comprise SEQ ID NO: 36 and 39.

[0091] In some embodiments, both macromolecules comprise SEQ ID NO: 37 and 39.

[0092] In another aspect, the disclosure provides a macromolecular 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 one another; and wherein induction of the effector function by the macromolecular complex is contingent upon binding of each of the two macromolecules to TGFb. See, e.g., the Figures and Examples herein.

[0093] 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 moieties is a pair of Fc fragments. In some embodiments, the pair of Fc fragments is a pair of knuckle-and-cup structures. In some embodiments, the two macromolecules are identical.

[0094] In another aspect, the disclosure provides a macromolecular 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 one another in the absence of TGFb; and wherein induction of the effector function by the macromolecular complex is contingent upon binding of each of the two macromolecules to 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 one another in the presence of TGFb, and the non-covalent conjugation is mediated by TGFb. See, e.g., the Figures and Examples herein.

[0095] In some embodiments, the macromolecule or one or both members of the macromolecule pair comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or fragment thereof.

[0096] In some embodiments of either of the two aspects above, the TGFb binding domain is an anti-TGFb scFv.

[0097] In some embodiments of either of the two aspects above, the TpoR binding domain is an anti-TpoR scFv.

[0098] In another aspect, the disclosure provides a macromolecular 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 macromolecular complex is contingent upon each of the two macromolecules binding to IL-8. See, e.g., the Figures and Examples herein.

[0099] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a complementary pair of moieties, each macromolecule comprising one member of the pair. In some embodiments, the complementary pair of polypeptides is a Fc fragment pair. In some embodiments, the Fc fragment pair is a knob-in-hole pair. In some embodiments, the two macromolecules are identical.

[0100] In another aspect, the disclosure provides a macromolecular 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 macromolecular complex is contingent upon each of the two macromolecules binding to IL-8. In some embodiments, the first member of the macromolecule pair and the second member of the macromolecule pair 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 Figures and Examples herein.

[0101] In some embodiments, the macromolecule or one or both members of the macromolecule pair comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or fragment thereof.

[0102] In some embodiments of either of the two aspects above, the IL-8 binding domain is an anti-IL8 scFv.

[0103] In some embodiments of either of the two aspects above, the TpoR binding domain is an anti-TpoR scFv.

[0104] In another aspect, the disclosure provides a macromolecular 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 the induction of the effector function by the macromolecular complex is contingent on the binding of each of the two macromolecules to TGFb. See, e.g., the Figures and Examples herein.

[0105] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a complementary pair of moieties, each macromolecule comprising one member of the pair. In some embodiments, the complementary pair of polypeptides is a Fc fragment pair. In some embodiments, the Fc fragment pair is a knob-in-hole pair. In some embodiments, the two macromolecules are identical.

[0106] In another aspect, the disclosure provides a macromolecular 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 the induction of the effector function by the macromolecular complex is contingent on the binding of each of the two macromolecules to TGFb. In some embodiments, the first member of the macromolecular pair and the second member of the macromolecular pair 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 Figures and Examples herein.

[0107] In some embodiments, one or both members of the macromolecule or the macromolecular pair comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or fragment thereof.

[0108] In some embodiments of either of the two aspects above, the TGFb binding domain is an anti-TGFb scFv.

[0109] In some embodiments of either of the two aspects above, the IL2R binding domain comprises the N terminus of IL2. In some embodiments of either of the two aspects above, the IL2R binding domain comprises the C terminus of IL2.

[0110] In another aspect, the disclosure provides a macromolecular 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 IL10R; wherein the two macromolecules are conjugated to one another; and wherein the induction of the effector function by the macromolecular complex is contingent upon each of the two macromolecules binding to IFNg. See, e.g., the figures and examples herein.

[0111] 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 pair of knobs-into-holes. In some embodiments, the two macromolecules are identical.

[0112] In another aspect, the disclosure provides a macromolecular 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 IL10R; wherein the two macromolecules are not conjugated to one another in the absence of IFNg; and wherein the induction of the effector function by the macromolecular complex is contingent upon each of the two macromolecules binding to IFNg. In some embodiments, the first member of the macromolecular pair and the second member of the macromolecular pair are non-covalently conjugated to one another in the presence of IFNg, and the non-covalent conjugation is mediated by IFNg. See, e.g., the figures and examples herein.

[0113] In some embodiments, one or both members of the macromolecule or the macromolecular pair comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or fragment thereof.

[0114] In some embodiments of either of the above two aspects, the IFNg binding domain is an anti-IFNg scFv.

[0115] In some embodiments of either of the above two aspects, the IL10R binding domain comprises an anti-IL10R VHH.

[0116] In some embodiments of either of the above two aspects, the IL10R binding domain comprises IL10.

[0117] In another aspect, the disclosure provides a macromolecular 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 IL10R; wherein the two macromolecules are conjugated to one another; and wherein the induction of the effector function by the macromolecular complex is contingent upon each of the two macromolecules binding to IL6. See, e.g., the Figures and Examples herein.

[0118] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a complementary pair of moieties, each macromolecule comprising one member of the pair. In some embodiments, the complementary pair of polypeptides is a pair of Fc fragments. In some embodiments, the pair of Fc fragments is a pair of knobs-into-holes. In some embodiments, the two macromolecules are identical.

[0119] In another aspect, the disclosure provides a macromolecular 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 IL10R; wherein the two macromolecules are not conjugated to one another in the absence of IL6; and wherein the induction of the effector function by the macromolecular complex is contingent upon each of the two macromolecules binding IL6. In some embodiments, the first member of the macromolecular pair and the second member of the macromolecular pair are non-covalently conjugated to one another in the presence of IL6, and the non-covalent conjugation is mediated by IL6. See, e.g., the Figures and Examples herein.

[0120] In some embodiments, one or both members of the macromolecule or the macromolecular pair comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or fragment thereof.

[0121] In some embodiments of either of the above two aspects, the IL6 binding domain is an anti-IL6 VHH.

[0122] In some embodiments of either of the above two aspects, the IL10R binding domain comprises an anti-IL10R VHH.

[0123] In some embodiments of either of the above two aspects, the IL10R binding domain comprises IL10.

[0124] In another aspect, the disclosure provides a macromolecular 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 one another; and wherein the induction of the effector function by the macromolecular complex is contingent upon each of the two macromolecules binding to IL8. See, e.g., the figures and examples herein.

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

[0126] In another aspect, the disclosure provides a macromolecular 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 one another in the absence of IL8; and wherein the induction of the effector function by the macromolecular complex is contingent upon each of the two macromolecules binding to IL8. In some embodiments, the first member of the macromolecular pair and the second member of the macromolecular pair are non-covalently conjugated to one another in the presence of IL8, and the non-covalent conjugation is mediated by IL8. See, e.g., the figures and examples herein.

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

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

[0129] In some embodiments of either of the two aspects above, 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 4-1BB binding domain. See, e.g., the figures 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 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 4-1BB binding domain is an anti-4-1BB 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 present disclosure provides a macromolecule comprising a TGFb binding domain linked to a TpoR binding domain. See, e.g., the Figures and Examples herein.

[0139] In some embodiments, the macromolecule comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or fragment thereof.

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

[0141] In some embodiments, the TpoR binding domain is an anti-TpoR scFv.

[0142] In another aspect, the present disclosure provides a macromolecule comprising an IL-8 binding domain linked to a TpoR binding domain. See, e.g., the Figures and Examples herein.

[0143] In some embodiments, the macromolecule comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or fragment thereof.

[0144] In some embodiments, the IL-8 binding domain is an anti-IL8 scFv.

[0145] In some embodiments, the TpoR binding domain is an anti-TpoR scFv.

[0146] In another aspect, the present disclosure provides a macromolecule comprising a TGFb binding domain linked to an IL2R binding domain. See, e.g., the Figures and Examples herein.

[0147] In some embodiments, the macromolecule comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or fragment thereof.

[0148] In some embodiments, the TGFb binding domain is an anti-TGFb scFv.

[0149] In some embodiments, the IL2R binding domain comprises the N-terminus of IL2. In some embodiments of either of the two aspects above, the IL2R binding domain comprises the C-terminus of IL2.

[0150] In another aspect, the disclosure provides a macromolecule comprising an IFNg binding domain linked to an IL10R binding domain. See, e.g., the Figures and Examples herein.

[0151] In some embodiments, the macromolecule comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or fragment thereof.

[0152] In some embodiments, the IFNg binding domain is an anti-IFNg scFv.

[0153] In some embodiments, the IL10R binding domain comprises an anti-IL10R VHH.

[0154] In some embodiments of either of the two aspects above, the IL10R binding domain comprises IL10.

[0155] In another aspect, the disclosure provides a macromolecule comprising an IL6 binding domain linked to an IL10R binding domain. See, e.g., the Figures and Examples herein.

[0156] In some embodiments, the macromolecule comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or fragment thereof.

[0157] In some embodiments, the IL6 binding domain is an anti-IL6 VHH.

[0158] In some embodiments, the IL10R binding domain comprises an anti-IL10R VHH.

[0159] In some embodiments, the IL10R binding domain comprises IL10.

[0160] In another aspect, the disclosure provides a macromolecule comprising an IL8 binding domain linked to a CD3 binding domain. See, e.g., the Figures and Examples herein.

[0161] In some embodiments, the macromolecule comprises a half-life extension moiety. In some embodiments, the half-life extension moiety is an Fc domain or fragment thereof.

[0162] In some embodiments, the IL8 binding domain is an anti-IL8 scFv.

[0163] In some embodiments, the CD3 binding domain comprises an anti-CD3 VH.

[0164] In some aspects, the disclosure provides a composition, wherein the composition comprises a macromolecular complex, a macromolecule, a nucleic acid, or a pair of nucleic acids provided herein.

[0165] In some embodiments, the composition comprises a macromolecular complex described herein.

[0166] In some embodiments, the composition comprises a macromolecule described herein.

[0167] In some aspects, the disclosure provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a macromolecular complex, a macromolecule, a nucleic acid, or a pair of nucleic acids provided herein and a pharmaceutically acceptable excipient.

[0168] In some embodiments, the pharmaceutical composition comprises a macromolecular complex described herein and a pharmaceutically acceptable excipient.

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

[0170] In some aspects, the disclosure provides a method for making a pharmaceutical composition, the method comprising: combining a macromolecular complex, a macromolecule, a nucleic acid, or a pair of nucleic acids provided herein with a pharmaceutically acceptable excipient, thereby making the pharmaceutical composition.

[0171] In some embodiments, the pharmaceutical composition comprises a macromolecular complex described herein and a pharmaceutically acceptable excipient.

[0172] In some embodiments, the pharmaceutical composition comprises a macromolecule described herein and a pharmaceutically acceptable excipient.

[0173] In another aspect, the disclosure provides a method, the method comprising providing a macromolecular complex, a macromolecule, a nucleic acid, a pair of nucleic acids, a composition, or a pharmaceutical composition of any of the above aspects a pathway into a cell.

[0174] In another aspect, the disclosure provides use of a macromolecular complex, a macromolecule, a nucleic acid, a pair of nucleic acids, a composition, or a pharmaceutical composition of any of the above aspects in the manufacture of a medicament for providing a pathway into a cell.

[0175] In another aspect, the disclosure provides the macromolecular complex, the macromolecule, the nucleic acid, the nucleic acid pair, the composition, or the pharmaceutical composition of any of the above aspects for use in providing access to a cell.

[0176] In another aspect, the disclosure provides a method of modulating a state of a cell, the method comprising providing the macromolecular complex, the macromolecule, the nucleic acid, the nucleic acid pair, the composition, or the pharmaceutical composition of any of the above aspects access to the cell, thereby modulating the state of the cell.

[0177] In another aspect, the disclosure provides use of the macromolecular complex, the macromolecule, the nucleic acid, the nucleic acid pair, the composition, or the pharmaceutical composition of any of the above aspects in the manufacture of a medicament for modulating a state of a cell.

[0178] In another aspect, the disclosure provides the macromolecular complex, the macromolecule, the nucleic acid, the nucleic acid pair, the composition, or the pharmaceutical composition of any of the above aspects for use in modulating a state of a cell.

[0179] In another aspect, the disclosure provides a method of inducing a cellular effector function in a cell, the method comprising providing the macromolecular complex, the macromolecule, the nucleic acid, the nucleic acid pair, the composition, or the pharmaceutical composition of any of the above aspects access to the cell, thereby inducing the cellular effector function in the cell.

[0180] In another aspect, the disclosure provides use of the macromolecular complex, the macromolecule, the nucleic acid, the nucleic acid pair, the composition, or the pharmaceutical composition of any of the above aspects in the manufacture of a medicament for inducing a cellular effector function in a cell.

[0181] In another aspect, the disclosure provides the macromolecular complex, the macromolecule, the nucleic acid, the nucleic acid pair, the composition, or the pharmaceutical composition of any of the above aspects for use in inducing a cellular effector function in a cell.

[0182] In some embodiments, the cell is in a subject, and the macromolecular complex, the macromolecule, the nucleic acid, the nucleic acid pair, the composition, or the pharmaceutical composition is administered in a therapeutically effective amount.

[0183] In some embodiments, the subject has or is suspected of having a disease or disorder characterized by an abnormal level of the disease characteristic target, optionally wherein the subject was previously determined to have an abnormal level of the disease characteristic target.

[0184] In another aspect, the disclosure provides a method of determining a state of a cell, the method comprising providing a macromolecular complex, or a macromolecule, a nucleic acid, a pair of nucleic acids, a composition, or a pharmaceutical composition of any of the above aspects access to the cell, and detecting the presence of the reporter domain, thereby determining the state of the cell.

[0185] In another aspect, the disclosure provides use of a macromolecular complex, a macromolecule, a nucleic acid, a pair of nucleic acids, a composition, or a pharmaceutical composition of any of the above aspects in the manufacture of a medicament for determining a state of a cell.

[0186] In another aspect, the disclosure provides a macromolecular complex, a macromolecule, a nucleic acid, a pair of nucleic acids, a composition, or a pharmaceutical composition of any of the above aspects for use in determining a state of a cell.

[0187] 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 macromolecular complex comprising two macromolecules, each macromolecule 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 the induction of the effector function by the macromolecular complex is conditional on the binding of the disease signature ligand by each copy of the macromolecule.

[0188] In another aspect, the disclosure provides use of a macromolecular complex comprising two macromolecules in the manufacture of a medicament for inducing a cellular effector function in a cell, each macromolecule 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 the induction of the effector function by the macromolecular complex is conditional on the binding of the disease signature ligand by each copy of the macromolecule.

[0189] In another aspect, the disclosure provides a macromolecular complex comprising two macromolecules, each macromolecule 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 the induction of the effector function by the macromolecular complex is conditional on each copy of the macromolecule binding the disease signature ligand, for use in inducing a cellular effector function in a cell.

[0190] 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 macromolecular complex comprising a pair of macromolecules, each macromolecule 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) a 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) a 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 the induction of the effector function by the macromolecular complex is conditional on each of the pair of macromolecules binding the disease signature ligand.

[0191] In another aspect, the disclosure provides a macromolecular complex comprising a pair of macromolecules, each macromolecule 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) a 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) a 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 the induction of the effector function by the macromolecular complex is conditional on each of the pair of macromolecules binding the disease signature ligand, for use in the manufacture of a medicament for inducing a cellular effector function in a cell.

[0192] In another aspect, the disclosure provides a macromolecular complex comprising a pair of macromolecules, each macromolecule independently comprising a first binding domain (FBD) connected 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) a 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) a 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 macromolecular complex is conditional on each of the pair of macromolecules binding the disease signature ligand, the macromolecular complex for use in inducing a cellular effector function in a cell.

[0193] 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 connected to two SBDs by a linker domain, 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 conformation that allows the two SBDs to bind to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional on each of the two FBDs binding the disease signature ligand.

[0194] In another aspect, the disclosure provides the use of a macromolecule comprising two FBDs connected to two SBDs by a linker domain in the manufacture of a medicament for inducing a cellular effector function in a cell, 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 conformation that allows the two SBDs to bind to the effector ligand in the presence of the disease signature ligand, and wherein induction of the effector function by the macromolecule is conditional on each of the two FBDs binding the disease signature ligand.

[0195] In another aspect, the disclosure provides a macromolecule comprising two FBDs connected to two SBDs via a linker domain, 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 conformation that allows the two SBDs to bind to the effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is conditional on each of the two FBDs binding the disease signature ligand, the macromolecule for use in inducing a cellular effector function in a cell.

[0196] 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 connected to SBD1 and SBD2 via a linker domain, 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 conformation that allows the SBD1 and the SBD2 to bind to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is conditional on each of the two FBDs binding the disease signature ligand.

[0197] In another aspect, the disclosure provides the use of a macromolecule comprising two FBDs connected to SBD1 and SBD2 via a linker domain in the manufacture of a medicament for inducing a cellular effector function in a cell, 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 conformation that allows the SBD1 and the SBD2 to bind to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is conditional on each of the two FBDs binding the disease signature ligand.

[0198] In another aspect, the disclosure provides a macromolecule comprising two FBDs linked to SBD1 and SBD2 by a linker domain, 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 conformation that allows the SBD1 and the SBD2 to bind to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is conditional on the binding of the disease signature ligand by each of the two FBDs, the macromolecule for use in inducing a cellular effector function in a cell.

[0199] Other features and advantages of the present application will be apparent from the following detailed description, and from the claims.

[0200] Definitions

[0201] As used herein, the term "macromolecule" refers to a large molecule (e.g., a molecule having a size greater than 1000 daltons (1 kDa)) comprising one or more polypeptide, oligonucleotide, chemical, lipid, and / or carbohydrate moieties. In some embodiments, a macromolecule is a recombinant protein (e.g., a fusion protein).

[0202] As used herein, the term "multimer" refers to a molecule composed 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 composed 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 sequence 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 composed 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 heterotetramers (comprising two copies of each of a first and a second subunit or form). Multimers further include higher order multimers, e.g., hexamers, heptamers, octamers, nonamers, and decamers.

[0203] 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 fragment thereof (e.g., a 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 retargeting 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 kunz domain peptide, a monobody, a fibronectin type III domain (FN3)-based binder, a nanobody, a nanoCLAMP, an optimer, a repebody, a pre- connectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR)), an endogenous binding domain or variant or derivative thereof (e.g., a cellular 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), an oligonucleotide (e.g., an aptamer (e.g., a DNA aptamer)) and a chemical molecule and combinations thereof.

[0204] As used herein, the term "ligand" refers to any moiety that a binding domain as described herein can have affinity for. 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 a carbohydrate), a post- translational modification state of a macromolecule (e.g., a macromolecule that is phosphorylated, glycosylated, acylated, alkylated, etc.), 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 of 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.

[0205] The term "disease characteristic ligand" refers to a ligand that is associated with a disease state or disorder of a cell, tissue, or subject (e.g., a mammal, e.g., a human). A disease characteristic ligand can be a protein, e.g., a soluble protein, an insoluble protein, a monomeric protein, and a multimeric protein. Disease characteristic ligands include, without limitation, a cell surface receptor, a cell surface antigen, a membrane-bound protein, an extracellular matrix component, an integrin, a cytokine, a neurotransmitter, an anti-drug antibody (ADA), an autoantibody, a nucleic acid, a carbohydrate, a lipid, a peptide, a nucleoside, a hormone, a virus, a bacterium, a fungus, or a fragment or antigen thereof.

[0206] The term "effector ligand" refers to a ligand that is capable of effecting a cellular effector function upon binding by a binding domain (e.g., a multimer of the application). Disease characteristic ligands include proteins and peptides, such as cell surface receptors (e.g., catalytic receptors, such as receptor tyrosine kinases (RTKs), receptor serine / threonine kinases (RSKs), type 1 cytokine receptors, type 2 cytokine receptors, tumor necrosis factor (TNF) superfamily receptors (e.g., TNFR2 or 4-1BB), or nuclear hormone receptors).

[0207] As used herein, the term "associated with" a disease, disorder, or condition means a causal or correlational relationship 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 can be a causative agent of the disease, disorder, or condition. For example, a virus can be a causative agent of a viral infection, a bacterium can be a causative agent of a bacterial infection, a fungus can be a causative agent of a fungal infection, or a parasite can be a causative agent of a parasitic infection, a cancer cell can be a causative agent of a cancer, a toxin can be a causative agent of a toxicity, or an allergen can be a causative agent of an allergic reaction. A target associated with a disease, disorder, or condition can also or alternatively be associated with an increased likelihood of occurrence or increased severity of the disease, disorder, or condition.

[0208] As used herein, the term "carrier" means a compound, composition, reagent, or molecule that facilitates the stability of a composition (e.g., a macromolecule or pair of macromolecules as described herein), facilitates transport or delivery of the composition into a subject, tissue, or cell. Non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified plant glycogen or glycogen-type materials), nanoparticles (e.g., nanoparticles encapsulating or covalently linked to circular or linear polyribonucleotides), liposomes, fusosomes, ex vivo differentiated reticulocytes, exosomes, protein carriers (e.g., proteins covalently linked to polyribonucleotides), and cationic carriers (e.g., cationic lipopolymers or transfection reagents).

[0209] As used herein, the terms "disease," "disorder," and "condition" all refer to a state of health that is suboptimal, e.g., a state that is diagnosed or treated by a medical professional.

[0210] The term "pharmaceutically acceptable excipient" as used herein means a pharmaceutically-acceptable material or vehicle that can be used for formulating a macromolecular complex, macromolecule, nucleic acid, or pair of nucleic acids for medical or therapeutic use, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulation material.

[0211] 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 comprise one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. A nucleotide can comprise a nucleoside and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (PO3) groups. A nucleotide can comprise a nucleobase, a five-carbon sugar (ribose or deoxyribose), and one or more phosphate groups. A ribonucleotide is a nucleotide with a ribose sugar. A polyribonucleotide or ribonucleic acid or RNA can refer to a macromolecule comprising a plurality of ribonucleotides polymerized via phosphodiester bonds. A deoxyribonucleotide is a nucleotide with a deoxyribose sugar. The polynucleotides provided herein can comprise one or more modified nucleotides.

[0212] A polynucleotide or nucleic acid or DNA means a macromolecule comprising a plurality of deoxyribonucleotides polymerized via phosphodiester bonds. The nucleotides can be nucleoside monophosphates or nucleoside polyphosphates. Nucleotide means a deoxyribonucleoside polyphosphate, such as, for example, 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 can comprise a detectable tag, such as a luminescent tag or label (e.g., a fluorophore). One or more nucleotides can be modified nucleotides. The nucleotides can comprise any subunit that can be incorporated into a growing nucleic acid chain. Such subunits can be A, C, G, T, or U, or any other subunit that is specific for one or more complementary A, C, G, T, or U, or is complementary to a purine (i.e., A or G or variants thereof) or pyrimidine (i.e., C, T, or U or variants thereof). In some examples, the polynucleotide is a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), or a derivative or variant thereof. In some cases, the polynucleotide is, by way of example only, a short interfering RNA (siRNA), a microRNA (miRNA), a plasmid DNA (pDNA), a short hairpin RNA (shRNA), a small nuclear RNA (snRNA), a messenger RNA (mRNA), a pre-mRNA, an antisense RNA (asRNA), and encompasses both the nucleotide sequence and any structural embodiment thereof, such as single-stranded, double-stranded, triple-stranded, helical, hairpin, and the like. In some cases, the polynucleotide molecule is circular (e.g., a circular RNA). The polynucleotide can have a variety of lengths. The 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. The polynucleotide can be isolated from a cell or tissue. As embodied herein, a polynucleotide sequence can include an isolated and purified DNA / RNA molecule, a synthetic DNA / RNA molecule, and a synthetic DNA / RNA analog.

[0213] As used herein, "polypeptide" means a polymer of amino acid residues (natural or non-natural, including D, L, or combinations thereof) most often linked together by peptide bonds. As used herein, the term 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. Polypeptides can be single molecules, or can be multi-molecular complexes, such as dimers, trimers, or tetramers. They can also include single- or multi-chain polypeptides, such as antibodies or insulin, and can be associated or linked. The most common disulfide bonds occur in multi-chain polypeptides. The term polypeptide can also apply to an amino acid polymer in which one or more of the amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids.

[0214] As used herein, the term "sequence identity" is determined by aligning two peptides or two nucleotide sequences using a global or local alignment algorithm. Sequences can be referred to as "essentially identical" or "essentially similar" when they have at least some minimum percentage of sequence identity, when optimally aligned, e.g., by the program GAP or BESTFIT using default parameters. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Typically, the default parameters for GAP are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and a gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is Blosum62 (Henikoff and Henikoff, 1992, PNAS 89, 915-919). Scoring of sequence alignments and percent sequence identity can be determined using computer programs, such as the GCG Wisconsin Software 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 can be determined by searching a database using algorithms such as FASTA, BLAST, etc. Sequence identity refers to sequence identity over the entire length of the sequence.

[0215] A "signal sequence" or "leader sequence" refers to a polypeptide sequence, for example between 10 and 30 amino acids in length, which is present at the N-terminus of the polypeptide sequence of a nascent protein, which targets the polypeptide sequence to the secretory pathway.

[0216] As used herein, the term "specifically binds" refers to a preferential interaction between a binding domain and its target or ligand (e.g., binding between an antibody and an antigen or epitope), which can determine the presence of the target or ligand in the presence of a heterogeneous population of molecules comprising 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) can be a binding domain that binds to this ligand with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other ligands. In one aspect, the extent of binding of a binding domain to an unrelated molecule (non-ligand) 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) for the ligand of < 1 μΜ, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM. D Specific binding can include, but does not require, exclusive binding. In one aspect, "specifically binds" refers to binding of a binding domain to a particular ligand (e.g., a polypeptide or an antigen or epitope on a particular polypeptide), and not substantially to any other ligand (e.g., polypeptide or polypeptide antigen or epitope).

[0217] As used herein, the term "treat" or "treating" refers to therapeutic treatment of a disease or disorder (e.g., an infectious disease, a cancer, a poisoning, or an allergic reaction) in a subject. The effects of treatment can include reversing, alleviating, reducing, curing, inhibiting progression of, decreasing likelihood of recurrence of, stabilizing (i.e., not worsening), and / or preventing spread of one or more symptoms or manifestations of the disease or disorder, as compared to the state and / or condition of the disease or disorder in the absence of therapeutic treatment. BRIEF DESCRIPTION OF DRAWINGS

[0218] Figure 1is a schematic showing the domain structure of a fusion protein that exerts effector function on a second target (e.g., a biological effector ligand) contingent on the presence of a first target (e.g., a disease signaling ligand). From N- to C-terminus, the fusion protein comprises 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 VH and VL domain having affinity for the second target upon 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.

[0219] Figure 2 is a schematic showing the domain structure of a pair of fusion proteins that exert effector function on a second target (e.g., a biological effector ligand) contingent on the presence of a first target (e.g., a disease signaling ligand). From N- to C-terminus, the fusion protein comprises a leader polypeptide; a FLAG affinity tag; 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; and a conditional effector domain A (in the first fusion protein) or a conditional effector domain B (in the second fusion protein).

[0220] Figure 3 is a schematic showing the domain structure of a pair of fusion proteins that exert effector function on a second target (e.g., a biological effector ligand) contingent on the presence of a first target (e.g., a disease signaling ligand). From N- to C-terminus, the fusion protein comprises 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 portion of the second target (in the first fusion protein (“anti-a”)) or a VHH having affinity for a second portion of the second target (in the second fusion protein (“anti-b”)); and a FLAG affinity tag.

[0221] Figure 4This is a schematic diagram illustrating the domain structures of a fusion protein pair that function as an effector to a second target (e.g., a biological effector ligand) conditioned in the presence of a first target (e.g., a disease signaling ligand). From the N-terminus to the C-terminus, the fusion protein comprises a leader polypeptide; a VH domain (in the first fusion protein) or a VL domain (in the second fusion protein) of the scFv, wherein the VH and VL domains of the scFv have affinity for the second target when assembled into a functional scFv, wherein the VH and VL domains have low affinity for each other; a GS(G4S)3 linker; an scFv containing the VH and VL domains linked by the (G4S)3 linker, wherein the VH and VL domains have affinity for the first target; and a FLAG affinity tag.

[0222] Figure 5A This schematic diagram illustrates the domain structure of a fusion protein that conditioned on the TPO receptor (TpoR) in the presence of TGF-β1 (TGFb). From the N-terminus to the C-terminus, the fusion protein contains a mouse immunoglobulin κ variable 3 (Ms IgKVIII) leader polypeptide; it also contains anti-TGF-β1 scFv molecules linked to VH and VL via a (G4S)3 linker; (G4S) n Connector; containing anti-TpoR scFv for VH and VL connected via (G4S)3 connector; and FLAG affinity label.

[0223] Figure 5B This is a schematic diagram illustrating the binding of TGFb and TpoR. Figure 5A Two copies of the fusion protein. The membrane orientation and downstream signal transduction partner of TpoR are shown.

[0224] Figure 5C The graph shows the use of constant quantities. Figure 5A The fusion protein and different concentrations of TGFb stimulated overnight in the culture supernatant of TpoR secreted alkaline phosphatase (SEAP) reporter cells showed SEAP activity (gray). TPO native cytokines are shown as a positive control (black) representing constitutive activity.

[0225] Figure 6A This schematic diagram illustrates the domain structure of a fusion protein in which TpoR is conditioned to be regulated in the presence of interleukin-8 (IL-8). From the N-terminus to the C-terminus, the fusion protein contains a Ms IgKVIII leader polypeptide; anti-IL-8 scFvs for VH and VL linked via a (G4S)3 linker; (G4S) n Connector; containing anti-TpoR scFv for VH and VL connected via (G4S)3 connector; and FLAG affinity label.

[0226] Figure 6B is a schematic showing two copies of the fusion protein binding to IL-8 and TpoR Figure 6A is a schematic showing the membrane orientation of TpoR and downstream signaling partners.

[0227] Figure 6C is a graph showing SEAP activity in culture supernatant of TpoR SEAP reporter cells stimulated overnight with a constant amount of Figure 6A fusion protein and different concentrations of IL-8 (grey). TPO natural cytokine is shown as positive control for constitutive activity (black).

[0228] Figure 7A is a schematic showing the domain structure of a complementary fusion protein pair (fusion proteins (a) and (b)) regulating human interleukin-2 receptor (IL-2R) activity conditioned on the presence of TGFb. Fusion protein (a) comprises from N- to C-terminus a Ms IgK VI II I leader peptide; an anti-TGF-β1 scFv containing VH and VL connected by a (G4S)3 linker; a (G4S) n linker; and a conditional IL-2R effector domain A (IL-2 (N-terminal)). Fusion protein (b) comprises from N- to C-terminus a Ms IgK VI II I leader peptide; an anti-TGF-β1 scFv containing VH and VL connected by a (G4S)3 linker; a (G4S) n linker; and a conditional IL-2R effector domain B (IL-2 (C-terminal)).

[0229] Figure 7B is a schematic showing fusion proteins (a) and (b) binding to IL-2R receptor components IL2Rβ and IL2RγC, respectively, in the absence (left) or presence (right) of TGFb. The membrane orientation of IL-2R and downstream signaling partners are shown. Figure 7A

[0230] Figure 7C is a graph showing SEAP activity in culture supernatant of IL-2 SEAP reporter cells stimulated overnight with a constant amount of Figure 7A fusion proteins (a) and (b) and different concentrations of TGFb (grey squares). IL-2 cytokine is shown as positive control for constitutive activity (black circles). TGFβ cytokine is shown as negative control for conditional activity (grey triangles).

[0231] Figure 8A ​is a schematic showing the domain structure of a pair of fusion proteins exerting effector function on a second target (e.g., a biological effector ligand) contingent on the presence of a first target (e.g., a disease signaling ligand), wherein the fusion proteins comprise, from N- to C-terminus, a leader polypeptide; a scFv containing 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 portion of the second target (in the first fusion protein (“anti-a”)) or a VHH having affinity for a second portion of the second target (in the second fusion protein (“anti-b”)); and a FLAG affinity tag (left); and a set of such schematics showing such a pair of fusion proteins with the first target being IFN-gamma (IFNg) and the second target being IL-10 receptor (IL-10R) (right).

[0232] Figure 8B is a schematic showing the domain structure of a fusion protein exerting effector function on a second target (e.g., a biological effector ligand) contingent on the presence of a first target (e.g., a disease signaling ligand), wherein the fusion protein self-assembles into a heterodimer, wherein the fusion protein comprises, from N- to C-terminus, a leader polypeptide; a VHH having affinity for a first portion of the second target (“anti-a”) or a VHH having affinity for a second portion of the second target (“anti-b”); a (G4S)2 linker; a scFv containing 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 IgGl fragment crystallizable region (Fc region) with a “knob” mutation (in the first fusion protein) or a human IgGl Fc region with a “hole” mutation (in the second fusion protein) and a 6x histidine (His) affinity tag (left); and such a schematic showing such a pair of fusion proteins with the second target being IL-10R (right).

[0233] Figure 8C is a schematic showing the domain structure of a fusion protein exerting effector function on a second target (e.g., a biological effector ligand) contingent on the presence of a first target (e.g., a disease signaling ligand), wherein the fusion protein comprises, from N- to C-terminus, a leader polypeptide; a VHH having affinity for a first portion of the second target (“anti-a”); a (G4S)2 linker; a scFv containing 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) nlinker; second copy of scFv containing a VH domain and a VL domain connected by a (G4S)3 linker, wherein the VH domain and VL domain pair have affinity for a first target; (G4S) n linker; VHH to a second part of a second target (in the second fusion protein (“anti-b”)); and polyhistidine affinity tag (top left); (b) such a schematic showing the fusion proteins as in (a) with the order of the anti-a and anti-b VHH domains reversed (bottom left); and such a schematic showing the fusion protein pair with the second target being IL-10R (right).

[0234] Figure 8D is a pair of such schematics showing the fusion protein pair of Figure 8A bound to IFNg as well as to the IL-10 receptor components IL-10Ra and IL-10Rb.

[0235] Figure 8E is a graph showing SEAP activity in culture supernatant of IL-10 SEAP reporter cells stimulated overnight with a constant amount of Figure 8A the fusion protein pair and different concentrations of IFNg. The IL-10 cytokine shows constitutive activity as a positive control (black circles). The IFNg cytokine shows conditional activity as a negative control (grey triangles).

[0236] Figure 8F is a pair of such schematics showing Figure 8B the fusion protein pair with the first target being IFNg and the second target being IL-10R and the linker between the Fc hinge and the IFNg binding scFv consisting of 5 amino acid residues (P482+P483; left) or 10 amino acid residues (P484+P485; right).

[0237] Figure 8G is a graph showing SEAP activity in culture supernatant of IL-10 SEAP reporter cells stimulated overnight with a constant amount of Figure 8F the fusion protein and different concentrations of IFNg.

[0238] Figure 9A is a schematic showing the domain structure of a complementary fusion protein pair regulating IL-10R activity in the presence of IL-6. The first fusion protein comprises from N- to C-terminus a mouse IgKVIII leader peptide; an anti-IL10Ra VHH antibody; a (G4S)2 linker; a first anti-IL-6 VHH antibody; a (G4S) nlinker; and a polyhistidine tag. The second fusion protein comprises from N-terminus to C-terminus a mouse IgKVIII leader peptide; an anti-IL10Rb VHH antibody; a (G4S)2 linker; a second anti-IL-6 VHH antibody; a (G4S)2 linker; and a polyhistidine tag. n linker; and a polyhistidine tag.

[0239] Figure 9B is a schematic showing the fusion protein pair binding to IL-6 and to the IL-10 receptor components IL-10Ra and IL-10Rb Figure 9A is a schematic showing the membrane orientation of the IL-10R and downstream signaling partners.

[0240] Figure 9C is a graph showing SEAP activity in culture supernatant of IL-10 SEAP reporter cells stimulated overnight with a constant amount of the fusion protein pair Figure 9A and different concentrations of IL-6 (grey circles). The IL-10 cytokine shows as positive control for constitutive activity (black squares). The IL-6 cytokine shows as negative control for conditional activity (grey triangles).

[0241] Figure 10A is a schematic showing the domain structure of a complementary fusion protein pair regulating cluster of differentiation 3 (CD3) activity in the presence of IL-8. The first fusion protein comprises from N-terminus to C-terminus a mouse IgKVIII leader peptide; an anti-CD3 VH domain of a scFv; a GS(G4S) linker; an anti-IL-8 scFv; and a FLAG affinity tag. The second fusion protein comprises from N-terminus to C-terminus a mouse IgKVIII leader peptide; an anti-CD3 VL domain of a scFv; a GS(G4S) linker; an anti-IL-8 scFv; and a FLAG affinity tag.

[0242] Figure 10B is a schematic showing the fusion protein pair binding to IL-8 and CD3. The membrane orientation of CD3 and downstream signaling partners are shown. Figure 10A

[0243] Figure 10C is a bar graph showing luciferase activity (via NFAT-luc reporter assay) in culture supernatant of NFAT-luc Jurkat reporter cells stimulated for 24 hours with a constant amount of the fusion protein pair Figure 10A in the presence or absence of 25 nM IL-8. The full-length reference anti-CD3 scFv shows as positive control for constitutive activity.

[0244] Figure 11A ​is a schematic showing the domain structure of a fusion protein pair that conditions CD3 activity in the presence of IL-8, wherein the fusion protein pair self-assembles into a heterodimer, wherein from N- to C-terminus, the first fusion protein comprises a leader polypeptide; an anti-CD3 VH domain of a scFv; a GS(G4S) linker; an anti-IL-8 scFv; a (G4S) n linker; an Fc region with a "knob" mutation; and a polyhistidine tag, and the second fusion protein comprises a leader polypeptide; an anti-CD3 VL domain of a scFv; a GS(G4S) linker; an anti-IL-8 scFv; a (G4S) n linker; an Fc region with a "hole" mutation; and a polyhistidine tag.

[0245] Figure 11B is a schematic showing the domain structure of a fusion protein pair that conditions CD3 activity in the presence of IL-8, wherein from N- to C-terminus, the fusion protein comprises a leader polypeptide; a VH domain with affinity for a first portion of CD3; a (G4S)2 linker; a scFv containing a VH domain and a VL domain connected by a (G4S)3 linker, wherein the VH domain and the VL domain have affinity for IL-8; a (G4S) n linker; a second copy of a scFv containing a VH domain and a VL domain connected by a (G4S)3 linker, wherein the VH domain and the VL domain have affinity for IL-8; a (G4S) n linker; a VL domain with affinity for a second portion of CD3; and a polyhistidine affinity tag (top); and a schematic showing the fusion protein as in (a) with the order of the anti-CD3 VH and VL domains reversed (bottom).

[0246] Figure 12A is a schematic showing Figure 8B and Figure 8F a modified version of the fusion protein pair shown in (a) and (b), wherein the first target is IFNg, the second target is IL-10R, and the length of the linker between the Fc hinge and the IFNg-binding scFv has been adjusted.

[0247] Figure 12B is a schematic showing Figure 8B and Figure 8F a modified version of the fusion protein pair shown in (a) and (b), wherein the first target is IFNg, the second target is IL-10R, and the length of the linker between the IFNg-binding scFv and the IL10R-binding VHH has been adjusted.

[0248] Figure 12C is a schematic showing Figure 8Band Figure 8F A modified version of the pair of fusion proteins shown in

[0249] Figure 12D is a schematic showing a modified version of the pair of fusion proteins shown in Figure 8B and Figure 8F A modified version of the pair of fusion proteins shown in

[0250] Figure 12E is a schematic showing a modified version of the pair of fusion proteins shown in Figure 8B and Figure 8F A modified version of the pair of fusion proteins shown in

[0251] Figure 12F is a schematic showing a modified version of the pair of fusion proteins shown in Figure 8B and Figure 8F A modified version of the pair of fusion proteins shown in

[0252] Figure 12G is a schematic showing a modified version of the pair of fusion proteins shown in Figure 8B and Figure 8F A modified version of the pair of fusion proteins shown in

[0253] Figure 12H is a schematic showing a modified version of the pair of fusion proteins shown in Figure 8B and Figure 8F A modified version of the pair of fusion proteins shown in

[0254] Figure 12I is a schematic showing a modified version of the pair of fusion proteins shown in Figure 8B and Figure 8F A modified version of the pair of fusion proteins shown in

[0255] Figure 13A is a schematic diagram illustrating the domain structure of a fusion protein that modulates 4-1BB activity as a function of the presence of VEGF (VEGF / 4-1BB fusion protein construct), wherein from N- to C-terminus, the fusion protein comprises a leader polypeptide; an anti-4-1BB scFv; (G4S) n a linker; an anti-IL-8 scFv; a VEGF binding agent; and an Fc region.

[0256] Figure 13B is a schematic diagram illustrating the domain structure of a fusion protein that modulates 4-1BB activity as a function of the presence of VEGF (VEGF / 4-1BB fusion protein construct), wherein from N- to C-terminus, the fusion protein comprises a leader polypeptide; an anti-4-1BB scFv; (G4S) Figure 13A in unbound form and bound to VEGF and to the 4-1BB receptor on the surface of a T cell. Left: monovalent construct of the fusion protein comprising a monomeric Fc region. Right: bivalent construct of the fusion protein comprising an Fc region capable of dimerization.

[0257] Figure 14A is a graph illustrating the fold induction of luminescence in Jurkat T cells expressing 4-1BB and an NFkB-driven luciferase reporter treated with a VEGF / 4-1BB fusion protein construct comprising an effector domain based on urelumab, a binding domain based on a VEGF receptor trap, and a monovalent Fc region, either alone or in the presence of human VEGF-A, mouse VEGF-A, or mouse VEGF-B. Urelumab reference monoclonal antibody (mAb) is shown as a control.

[0258] Figure 14B is a graph illustrating the fold induction of luminescence in Jurkat T cells expressing 4-1BB and an NFkB-driven luciferase reporter treated with a VEGF / 4-1BB fusion protein construct comprising an effector domain based on urelumab, a binding domain based on a VEGF receptor trap, and a bivalent Fc region, either alone or in the presence of human VEGF-A or mouse VEGF-A. Urelumab reference monoclonal antibody (mAb) is shown as a control.

[0259] Figure 15A is a graph illustrating the fold induction of luminescence in Jurkat T cells expressing 4-1BB and an NFkB-driven luciferase reporter treated with a VEGF / 4-1BB fusion protein construct comprising an effector domain based on utomilumab, a VEGF binding domain (anti-VEGF scFv), and a monovalent Fc region, either alone or in the presence of human VEGF-A or mouse VEGF-A. Utomilumab reference mAb is shown as a control.

[0260] Figure 15Bis a graph showing luminescence induction fold in Jurkat T cells expressing 4-1BB and an NFkB-driven luciferase reporter treated with VEGF / 4-1BB fusion protein constructs containing an utomilumab-based effector domain, a VEGF-binding domain (receptor trap; "VEGF-trap"), and a bivalent Fc region alone or in the presence of human VEGF-A or mouse VEGF-A. The utomilumab reference mAb is shown as a control.

[0261] Figure 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 VEGF / 4-1BB fusion protein constructs ("4-1BB SB") in the presence or absence of VEGF. CFSE: carboxyfluorescein succinimidyl ester.

[0262] Figure 16B is a graph showing the percentage of T cells in each indicated treatment group (see Table 4) undergoing rapid lymphopenia-induced proliferation (LIP) on day 7, as measured by dilution of CFSE dye.

[0263] Figure 16C is a graph showing the percentage of T cells derived from the donor population in each indicated treatment group (see Table 4) that were effector T cells following treatment.

[0264] Figure 16D is a graph showing the percentage of donor-derived T cells that were naive T cells in each indicated treatment group (see Table 4).

[0265] Figure 17A is a schematic diagram showing the design of a tumor efficacy study designed to assess the anti-tumor activity of VEGF / 4-1BB fusion protein constructs anti-4-1BB A x VEGF-trap (bivalent) ("VEGF-4-1bb SB") in mice bearing MC38 tumors.

[0266] Figure 17B is a graph showing tumor size (in mm 3 ) in mice bearing MC38 tumors over time, treated with different doses of anti-4-1BB A x VEGF-trap (bivalent), urelumab comparator, or hlgG4 isotype control.

[0267] Figure 17Cis a set of such stacked bar graphs showing the fraction of mice that experienced a partial response (PR) (black bars; tumor size less than the average tumor size in the isotype control group) or a complete response (CR) (gray bars; mice became tumor-free after treatment) 17 days after treatment with the indicated dose of anti-4-1BB A xVEGF-trap (bivalent) ("SB"), urelumab comparator, PD1 blocker ("PD1"), or PD1 blocker in combination with anti-4-1BB A xFraction of mice that experienced a partial response (PR) (black bars; tumor size less than the average tumor size in the isotype control group) or a complete response (CR) (gray bars; mice became tumor-free after treatment) 17 days after treatment with the indicated dose of VEGF-trap (bivalent) (low dose).

[0268] Figure 18 is a graph showing tumor size (in mm 3 ) over time in mice with MC38 tumors that became tumor-free after treatment with anti-4-1BB A xVEGF-trap (bivalent) or urelumab comparator and re-challenged with a lethal dose of MC38 tumor cells.

[0269] Figure 19A is a schematic 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 that is able to read out IL-10R signaling activity.

[0270] Figure 19B is a schematic showing the design of the assay for conditional IL-10R activity. NSG mice are inoculated with hot or cold tumor cells. When tumors reach 5 mm in diameter, IFNg / IL-10R fusion protein constructs are injected. IL-10R activity is assessed by luciferase injection 24 hours after administration of the IFNg / IL-10 fusion protein construct. Constitutively active IL-10 protein is provided as a control.

[0271] Figure 20A is a bar graph showing quantification of luciferase signal in mice carrying IFNg positive "hot" tumors or IFNg negative "cold" tumors treated with IFNg / IL-10 fusion protein constructs.

[0272] Figure 20B is a bar graph showing quantification of luciferase signal in mice carrying IFNg positive "hot" tumors or IFNg negative "cold" tumors treated with constitutively active bivalent IL-10 constructs.

[0273] Figure 21A is a pair of graphs showing the fraction of mice that experienced a partial response (PR) (black bars; tumor size less than the average tumor size in the isotype control group) or a complete response (CR) (gray bars; mice became tumor-free after treatment) 17 days after treatment with the indicated dose of anti-4-1BB AResults of xVEGF-trap (bivalent) construct purification. The top panel shows that the protein product from the first peak is 96.5% monodisperse and eluted at a size consistent with a stoichiometry of two dimers (~130 kDa). The bottom panel shows the migration of a gel filtration standard run to produce elution time estimates for various protein sizes. POI: peak of interest.

[0274] Figure 21B is such an image showing the dimeric anti-4-1BB A Stoichiometry of dimers of xVEGF-trap (bivalent) construct. Under non-reducing conditions (lane "NR"), the macromolecular complex runs as a dimer. Under reducing conditions (lane "R"), the macromolecular complex resolves into its constituent monomeric subunits, as expected. The estimated monomer size is about 65 kDa, and the dimer is about 130 kDa. Molecular weight standards are in the left lanes. DETAILED DESCRIPTION

[0275] Characterized herein are macromolecules, multimers thereof, compositions comprising the same, and methods of using the same that conditionally induce a cellular effector function (e.g., a biological or therapeutic activity) based on the presence of a disease signature ligand.

[0276] I. Compositions

[0277] A. Macromolecules and macromolecular complexes

[0278] i. Homomultimeric macromolecular complexes

[0279] In one aspect, provided herein is a macromolecular complex comprising two macromolecules, each macromolecule 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 one another; and wherein the induction of the effector function by the macromolecular complex is conditional on the binding of the disease signature ligand by each of the two macromolecules (e.g., the macromolecular complex does not induce the effector function in the absence of the disease signature ligand; does not substantially induce the effector function in the absence of the disease signature ligand, or preferentially induces the effector function in the presence of the disease signature ligand).

[0280] 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.

[0281] 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 complementary moiety pair, wherein each macromolecule comprises (e.g., is covalently or non-covalently linked to) one member of the pair. The complementary moiety pair can be, for example, biotin and avidin; Bacillus amyloliquefaciens ribonuclease and Bacillus amyloliquefaciens ribonuclease inhibitor; a complementary aptamer pair; or a complementary polypeptide pair. (e.g., an Fc fragment pair (e.g., an engineered Fc fragment pair)). For example, in some embodiments, the complementary moiety pair is a knob-in-hole Fc pair (e.g., one of the macromolecules comprises (e.g., is covalently or non-covalently conjugated to) an Fc region having a “knob” mutation, and the other macromolecule comprises (e.g., is covalently or non-covalently conjugated to) an Fc region having a “hole” mutation. Knob-in-hole Fc pairs are described, for example, in Xu et al., mAbs, 7(1): 231-242, 2015. As a general principle, the conjugation (e.g., covalent or non-covalent conjugation) conditions the simultaneous binding of the disease signal and effector ligands with the presence of the disease signature ligand, e.g., the linker or the complementary moiety pair is configured to spatially orient the two copies of the macromolecule to allow for the conditional simultaneous binding of the disease signal and effector ligands. In some embodiments, the non-covalent conjugation is mediated by the disease signature ligand (e.g., the two macromolecules only conjugate in the presence of the disease signature ligand).

[0282] In another aspect, provided herein is a macromolecular complex comprising two macromolecules, each macromolecule 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 do not conjugate to each other in the absence of the disease signature ligand; and wherein the induction of the effector function by the macromolecular complex is conditional on each of the two macromolecules binding the disease signature ligand (e.g., the macromolecular complex does not induce the effector function in the absence of the disease signature ligand; does not substantially induce the effector function in the absence of the disease signature ligand, or preferentially induces the effector function in the presence of the disease marker ligand). In some embodiments, the first member of the macromolecule pair and the second member of the macromolecule pair non-covalently conjugate to each other in the presence of the disease signature ligand, and wherein the non-covalent conjugation is mediated by the disease signature ligand.

[0283] In some embodiments, the two macromolecules are identical (e.g., identical in terms of amino acid sequence and / or nucleotide sequence). Alternatively, the two macromolecules can not be identical in sequence, but comprise substantially identical first and second binding domains. For example, the two macromolecules can comprise binding domains that are identical in sequence (e.g., amino acid sequence and / or nucleotide sequence) or different in sequence but have substantially identical affinity for the disease signature ligand or the effector ligand.

[0284] In some embodiments of the application, 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 a heavy chain variable domain (VH domain) and a light chain variable domain (VL domain) that have affinity for a second target, wherein the VH domain and the VL domain are connected by a short linker (e.g., a G4S linker) that does not allow for intrachain pairing of the VH domain and the VL domain. In some embodiments, the second binding domain is a diabody.

[0285] In one exemplary embodiment, each of the two macromolecules is a fusion protein comprising, from N-terminus 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 (G4S)3 linker, wherein the VH domain and the VL domain have affinity for a disease signature ligand; a (G4S) n linker; a second binding domain comprising a VH domain and a VL domain that have affinity for the effector ligand, wherein the VH domain and the VL domain are connected by a G4S linker; and a FLAG affinity tag.

[0286] In another aspect of the application, provided herein is a complex comprising a macromolecular complex as described herein complexed with one or both of the disease signature ligand and the effector ligand.

[0287] In some embodiments, the macromolecules are polypeptides. In other embodiments, the macromolecules comprise one or more non-polypeptide components, e.g., comprising one or more nucleic acids or chemical components, as further described below.

[0288] In some embodiments, the macromolecules further comprise one or more of the following: 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.

[0289] Further provided herein are macromolecular complexes comprising three or more (e.g., three, four, five, or more than five) macromolecules, each macromolecule 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 one another; and wherein the induction of the effector function by the macromolecular complex is conditional on each of the three or more macromolecules binding the disease signature ligand (e.g., the macromolecular complex does not induce the effector function in the absence of the disease signature ligand; does not substantially induce the effector function in the absence of the disease signature ligand, or preferentially induces the effector function in the presence of the disease signature ligand).

[0290] ii. Heteromultimeric macromolecular complexes

[0291] In another aspect, provided herein is a macromolecular complex providing a pair of macromolecules, each macromolecule 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) a 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) a 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 one another; and wherein the induction of the effector function by the pair of macromolecules is conditional on each member of the pair of macromolecules binding the disease signature ligand (e.g., the macromolecular complex does not induce the effector function in the absence of the disease signature ligand; does not substantially induce the effector function in the absence of the disease signature ligand, or preferentially induces the effector function in the presence of the disease signature ligand).

[0292] 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.

[0293] 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, where each macromolecule comprises (e.g., is covalently or non-covalently linked to) one member of the pair. The pair of complementary moieties can be, for example, biotin and avidin; Bacillus amyloliquefaciens ribonuclease and Bacillus amyloliquefaciens ribonuclease inhibitor; a pair of complementary aptamers; or a pair of complementary polypeptides. (e.g., a pair of Fc fragments (e.g., engineered Fc fragments). For example, in some embodiments, the pair of complementary moieties is a knob-in-hole Fc pair (e.g., one of the macromolecules comprises (e.g., is covalently or non-covalently conjugated to) an Fc region having a “knob” mutation, and the other macromolecule comprises (e.g., is covalently or non-covalently conjugated to) an Fc region having a “hole” mutation. Knob-in-hole Fc pairs are described, for example, in Xu et al., mAbs, 7(1): 231-242, 2015. As a general principle, the conjugation (e.g., covalent or non-covalent conjugation) conditions the simultaneous binding of the disease signal and effector ligands with the presence of the disease signature ligand, e.g., the linker or the pair of complementary moieties is configured to spatially orient the two copies of the macromolecule to allow for the conditional simultaneous binding of the disease signal and effector ligands. In some embodiments, the non-covalent conjugation is mediated by the disease signature ligand (e.g., the two macromolecules only conjugate in the presence of the disease signature ligand).

[0294] In another aspect, provided herein is a macromolecular complex comprising a pair of macromolecules, each macromolecule independently comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) a first member of the pair of macromolecules comprises a first binding domain 1 (FBD1) that specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) a second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of the disease signature ligand in the biological sample; and (c) the SBD specifically binds to 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 the induction of the effector function by the macromolecular complex is contingent on each of the two macromolecules binding to the disease signature ligand (e.g., the macromolecular complex does not induce the effector function in the absence of the disease signature ligand; does not substantially induce the effector function in the absence of the disease signature ligand, or preferentially induces the 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.

[0295] In another aspect of the application, provided herein is a complex comprising a macromolecular complex as described herein complexed with one, two, or all three of the disease signature ligand, the first effector ligand, and the second effector ligand.

[0296] 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 non-polypeptide components, e.g., comprising one or more nucleic acids or chemical components, as further described below.

[0297] In another aspect, provided herein is a macromolecular complex comprising a pair of macromolecules, each macromolecule comprising an FBD linked to a SBD, wherein (a) the FBD of a first member of the pair of macromolecules specifically binds a first portion 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 portion 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 the induction of the effector function by the pair of macromolecules is contingent upon the binding of each member of the pair of macromolecules to the disease signature ligand (e.g., the macromolecular complex does not induce the effector function in the absence of the disease signature ligand; does not substantially induce the effector function in the absence of the disease signature ligand, or preferentially induces the effector function in the presence of the disease signature ligand).

[0298] In another aspect of the application, provided herein is a macromolecular complex comprising a set of three macromolecules, each macromolecule comprising an FBD linked to a SBD, wherein (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample; (b) a first member of the set of macromolecules comprises a SBD1 that specifically binds a first effector ligand in the biological sample; (c) a second member of the set of macromolecules comprises a SBD2 that specifically binds a second effector ligand in the biological sample; and (d) a 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, the SBD2, and the SBD3 induce a cellular effector function upon binding to the first, the second, and the third 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 the induction of the effector function by the set of three macromolecules is contingent upon the binding of each member of the set of three macromolecules to the disease signature ligand. In some embodiments, the disease signature ligand is trimeric.

[0299] In some embodiments, one or both members of the pair of macromolecules or one, two, or all three members of the set of three macromolecules further comprises one or more of the following: 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.

[0300] iii. Macromolecule comprising two FBDs and two SBDs

[0301] 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 conformation that allows the two SBDs to bind to the effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is contingent on the binding of the disease signature ligand by each of the two FBDs (e.g., the macromolecule does not induce the effector function in the absence of the disease signature ligand; does not substantially induce the effector function in the absence of the disease signature ligand, or preferentially induces the effector function in the presence of the disease signature ligand).

[0302] 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.

[0303] In some embodiments, the two FBDs are identical (e.g., identical in terms of amino acid sequence and / or nucleotide sequence). Alternatively, the two FBDs can differ in sequence but have substantially identical affinity for the disease signature ligand.

[0304] In some embodiments, the two SBDs are identical (e.g., identical in terms of amino acid sequence and / or nucleotide sequence). Alternatively, the two SBDs can differ in sequence but have substantially identical affinity for the effector ligand.

[0305] In some embodiments of the application, 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 a heavy chain variable domain (VH domain) and a light chain variable domain (VL domain) having affinity for a second target, wherein the VH domain and the VL domain are linked by a short linker (e.g., a G4S linker) that does not allow for intrachain pairing of the VH domain and the VL domain. In some embodiments, the second binding domain is a diabody.

[0306] In another aspect of the application, provided herein is a complex comprising a macromolecule as described herein complexed with one or both of the disease signature ligand and the effector ligand.

[0307] In some embodiments, the macromolecule is a polypeptide (e.g., comprising a single polypeptide chain). In other embodiments, the macromolecule comprises one or more non-polypeptide components, e.g., comprising one or more nucleic acids or chemical components, as further described below.

[0308] In some embodiments, the macromolecule further comprises one or more of the following: 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.

[0309] iv. Macromolecule comprising two FBDs, SBD1, and SBD2

[0310] 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 conformation that allows the SBD1 and the SBD2 to bind to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is contingent on the binding of the disease signature ligand by each of the two FBDs (e.g., the macromolecule does not induce the effector function in the absence of the disease signature ligand; does not substantially induce the effector function in the absence of the disease signature ligand, or preferentially induces the effector function in the presence of the disease signature ligand).

[0311] 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.

[0312] In some embodiments of the application, 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 do not bind the disease signature ligand. For example, in some embodiments, the SBDs (e.g., SBD1 and / or SBD2) comprise a heavy chain variable domain (VH domain) and a light chain variable domain (VL domain) with affinity for a second target, wherein the VH domain and the VL domain are connected by a short linker (e.g., a G4S linker) that does not allow for intrachain pairing of the VH domain and the VL domain. In some embodiments, the SBDs (e.g., SBD1 and / or SBD2) are diabodies.

[0313] In another aspect of the application, provided herein is a complex comprising a macromolecule as described herein complexed with one or both of the disease signature ligand and the effector ligand.

[0314] In some embodiments, the macromolecule is a polypeptide (e.g., comprising a single polypeptide chain). In other embodiments, the macromolecule comprises one or more non-polypeptide components, e.g., comprising one or more nucleic acids or chemical components, as further described below.

[0315] In some embodiments, the macromolecule further comprises one or more of the following: a lead domain (e.g., a lead polypeptide), one or more linker domains, and one or more reporter domains, as further described in Section 1(E) below.

[0316] v. Nucleic Acids, Vectors, and Host Cells

[0317] In another aspect of the application, provided herein is one or more nucleic acids (e.g., one or more RNA molecules or DNA molecules) encoding one or more of any of the above-described macromolecules (e.g., a pair of nucleic acids (e.g., a pair of RNA molecules or DNA molecules) encoding a pair of any of the above-described macromolecules). The one or more nucleic acids can be circular or linear. The one or more nucleic acids can 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), a circovirus, or a lentivirus). Additional examples of lipid-based carriers that can be used in the present application are provided in Section I(I) herein. For example, in some aspects, provided herein is 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 above-described macromolecules, wherein the one or more nucleic acids are formulated with a carrier (e.g., a lipid carrier, e.g., an LNP). In some aspects, the nucleic acids comprise one or more modified nucleotides.

[0318] Further provided herein are vectors (e.g., plasmid or viral vectors) comprising or encoding any of the above-described nucleic acids. The vectors can be formulated with a carrier (e.g., a carrier suitable for delivery to a target cell (e.g., a mammalian cell), such as, e.g., a lipid-containing carrier, such as an LNP-containing formulation).

[0319] Further provided herein are host cells that have been modified to comprise the above-described nucleic acids or vectors. Suitable host cells include bacterial cells and eukaryotic cells (e.g., mammalian cells). In some embodiments, the nucleic acids or vectors as described herein are manufactured in a host cell and isolated from the host cell.

[0320] B. Disease characteristic ligand and first binding domain

[0321] i. Disease characteristic ligand

[0322] The disease characteristic ligand bound by the macromolecule, the pair of macromolecules, or the macromolecular complex can be any moiety (e.g., protein, peptide, or small molecule) associated with a disease state or disorder of a cell, tissue, or subject (e.g., mammal, such as a human).

[0323] In some embodiments, the disease characteristic ligand is a protein. In some embodiments, the protein is a soluble protein or an insoluble protein. For example, the disease characteristic ligand can be present in solution in the biological sample (e.g., can be present in the extracellular space), or can be embedded in a membrane present in the biological sample (e.g., can be embedded in a cell membrane).

[0324] In some embodiments, the disease characteristic 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.

[0325] In some embodiments, the disease signature ligand is a multimeric protein (e.g., a homo-multimeric 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 IA(i) herein), the disease signature ligand can 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 IA(i) and IA(iii) herein), the disease signature ligand can be a dimer, such that each macromolecule binds one member of the dimerized disease signature ligand.

[0326] In other embodiments, the disease signature ligand is a monomeric protein.

[0327] 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-g); a growth factor (e.g., transforming growth factor beta (TGF-b), granulocyte colony-stimulating 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 inducible protein 10 (IP-10; also known as CXCL10); or a TNF family member (e.g., TNF-a). 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).

[0328] In some embodiments, the disease signature ligand is a VEGF (e.g., VEGF-A or VEGF-B).

[0329] In some embodiments, the disease signature ligand is a self-antigen of the 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 targeting a therapeutic agent used to treat a disease state or 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.

[0330] In some embodiments, the disease signature ligand is a nucleic acid.

[0331] In some embodiments, the disease characteristic ligand is a carbohydrate, a lipid, a peptide, a nucleoside, or a combination of the foregoing.

[0332] In some embodiments, the disease characteristic ligand is a hormone (e.g., a peptide / protein hormone (e.g., insulin, oxytocin, or growth hormone)), an amino acid derivative (e.g., melatonin or thyroxine), a steroid (e.g., a glucocorticoid), or an eicosanoid (e.g., a prostaglandin).

[0333] In some embodiments, the disease characteristic ligand is a non-self antigen, i.e., an antigen of an organism other than the organism from which the biological sample is derived. In some embodiments, the disease characteristic ligand is a virus, a bacterium, a fungus, or a fragment or antigen thereof (e.g., a virus, bacterium, or fungus, or a fragment thereof, that causes a disease state or disorder of the cell, tissue, or subject).

[0334] ii. Disease characteristic ligand binding domain (first binding domain)

[0335] Each macromolecule provided herein comprises at least one first binding domain (FBD) that specifically binds to the disease characteristic ligand (e.g., binds to a disease characteristic ligand as described in Section IB(i) above). In some embodiments, the first binding domain allows for additional binding domains to bind to the disease characteristic ligand (e.g., is designed or selected such that at least two copies of the first binding domain can bind to the disease characteristic ligand and / or such that the first binding domain of each member of a macromolecule pair can bind to the disease characteristic ligand).

[0336] In some embodiments, the first binding domain comprises a polypeptide that specifically binds to the disease characteristic ligand.

[0337] In some embodiments, the polypeptide is an antibody or 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 retargeting antibody (DART).

[0338] In some embodiments, the polypeptide is an antibody mimetic. In some embodiments, the antibody mimetic is an affibody, affilin, affimer, affitin, alphabody, anticalin, lipocalin, avimer, DARPin, fynomer, gastrobody, knottin, kunitz domain peptide, monobody, fibronectin type III domain (FN3)-based binder, nanobody, nanoCLAMP, optimer, repebody, pre- connectin, centyrin, obody, peptide aptamer, synthetic peptide, or variable lymphocyte receptor (VLR).

[0339] In some embodiments, the polypeptide is an endogenous binding domain of the organism from which the biological sample and / or the disease signature ligand is derived, e.g., a binding domain 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 comprising or consisting of a receptor for a cytokine (e.g., a multimerized cytokine, e.g., a dimeric, trimeric, or tetrameric cytokine), or a polypeptide comprising or consisting 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 an IL-6 receptor. In some embodiments, the disease signature ligand is TNF-a, and the first binding domain is a polypeptide comprising a TNF-a receptor. In some embodiments, the disease signature ligand is VEGF, and the first binding domain comprises a receptor trap derived from VEGF receptor VEGFR1 or VEGFR2 (e.g., aflibercept).

[0340] 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).

[0341] In some embodiments, the first binding domain comprises a chemical molecule that specifically binds the disease signature ligand.

[0342] Additional binding domains that can be used in the present application are described, e.g., in Zhong and D’Antona, Antibodies, 10(2): 13, 2021.

[0343] In some embodiments, the disease signature ligand binding domain has an affinity (K D value) for the disease signature ligand of > 10 to > 100 pM, < 10 nM, > 10 nM, or > 100 nM, or has a micromolar affinity (e.g., K D ≤ 1 μM) for the disease signature ligand. In some aspects, the disease signature ligand binding domain binds the disease signature ligand with a K D of 1 nM or less.

[0344] In some embodiments of any of the macromolecules provided herein, the first binding domain has affinity for two or more disease signature moieties. For example, the first binding domain can 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.

[0345] In some embodiments comprising a macromolecule pair (e.g., embodiments as described in Sections IA(i) and IA(ii) herein), the first member and the second member of the macromolecule pair comprise a first binding domain that is identical in sequence (e.g., amino acid sequence and / or nucleotide sequence) or differs in sequence but has substantially the same affinity for the disease signature ligand or the effector ligand. Alternatively, in other embodiments, the first member and the second member of the macromolecule pair comprise different first binding domains. For example, in some embodiments, the first binding domain of the first member of the macromolecule pair specifically binds to a first epitope or moiety of a disease signature ligand in a biological sample, and the second binding domain of the second member of the macromolecule pair specifically binds to a second epitope or moiety of the disease signature ligand in the biological sample.

[0346] For example, in some embodiments, the disclosure provides a macromolecular complex comprising two macromolecules, each macromolecule comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein: (a) the first member of the macromolecule pair comprises a first binding domain 1 (FBD1) that specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) the second member of the macromolecule pair comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of the disease signature ligand in the biological sample; and (c) the SBD specifically binds to 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 the induction of the effector function by the macromolecular complex is contingent on each of the two macromolecules binding to the disease signature ligand.

[0347] In further examples, in some embodiments, the present disclosure provides a macromolecular complex comprising a pair of macromolecules, each macromolecule independently comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) a first member of the pair of macromolecules comprises a first binding domain 1 (FBD1) that specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) a second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of the disease signature ligand in the biological sample; (c) the first member of the pair of macromolecules comprises a second binding domain 1 (SBD1) that specifically binds to 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 to 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 the induction of the effector function by the macromolecular complex is contingent on each of the FBD1 and the FBD2 binding to the disease signature ligand.

[0348] Similarly, in some embodiments comprising a macromolecule 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 are different 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 to a first epitope or portion of a disease signature ligand in a biological sample and a second FBD that specifically binds to a second epitope or portion of the disease signature ligand in the biological sample.

[0349] For example, in some embodiments, the present disclosure provides a macromolecule comprising a FBD1 and a FBD2 linked to two SBDs, wherein (a) the FBD1 specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) the FBD2 specifically binds to a second epitope of the disease signature ligand in the biological sample; and (c) the SBDs specifically bind to 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 conformation that allows the two SBDs to bind to the effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is contingent on each of the FBD1 and the FBD2 binding to the disease signature ligand.

[0350] In further examples, in some embodiments, the present disclosure provides a macromolecule comprising FBD1 and FBD2 linked to SBD1 and 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 conformation that allows the SBD1 and the SBD2 to bind to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is contingent on each of the two FBDs (FBD1 and FBD2) binding the disease signature ligand.

[0351] In embodiments comprising non-identical first binding domains (e.g., first binding domain 1 (FBD1) and first binding domain 2 (FBD2)), the first binding domains can bind to different epitopes (e.g., partially overlapping epitopes or non-overlapping epitopes) of the disease signature ligand, 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. Thus, in some aspects, the first binding domains are capable of simultaneously binding the disease signature ligand.

[0352] In some embodiments comprising non-identical first binding domains (e.g., FBD1 and FBD2), the two first binding domains have substantially similar affinities for their respective epitopes (e.g., 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 the FBD2 have substantially different affinities for their respective epitopes (e.g., 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%).

[0353] In some embodiments in which the disease characteristic ligand is a polypeptide, the first binding domain binds to the disease characteristic ligand at a binding site containing Arg, Lys, Asp, His, or Glu amino acid residues (or combinations thereof) (e.g., a binding site enriched for one or more of these residues).

[0354] In one embodiment, the disease characteristic ligand is TGFb.

[0355] In one embodiment, the disease characteristic ligand is VEGF.

[0356] In one embodiment, the disease characteristic ligand is IL-8.

[0357] In one embodiment, the disease characteristic ligand is IL-6.

[0358] In one embodiment, the disease characteristic ligand is IFNg.

[0359] In one embodiment, the disease characteristic ligand is IL-10, and the first binding domain is a polypeptide comprising an IL-10 receptor.

[0360] In one embodiment, the disease characteristic ligand is IL-12, and the first binding domain comprises a p40 subunit and / or a p35 subunit of IL-12.

[0361] In one embodiment, the disease characteristic ligand is VEGF, and the first binding domain comprises (i) a VEGF neutralizing antibody (e.g., bevacizumab) or (ii) a receptor trap derived from a VEGF receptor VEGFR1 or VEGFR2 (e.g., aflibercept).

[0362] C. Effector Ligand and Second Binding Domain

[0363] i. Effector Ligand

[0364] The effector ligand bound by the macromolecule, the pair of macromolecules, or the macromolecular complex can be any moiety (e.g., protein or peptide) present in the biological sample and capable of effecting a cellular effector function upon binding by the multimer of the application.

[0365] In some embodiments, the effector ligand is a protein or peptide.

[0366] In some embodiments, the effector ligand is a cell surface receptor.

[0367] In some embodiments, the effector ligand is TpoR.

[0368] In some embodiments, the effector ligand is 4-1BB.

[0369] In some embodiments, the effector ligand is IL2R.

[0370] In some embodiments, the effector ligand is IL10R.

[0371] In some embodiments, the effector ligand is CD3.

[0372] In some embodiments, the cell surface receptor is a catalytic receptor, such as 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 4-1BB). In some embodiments, the cell surface receptor is 4-1BB. In some embodiments, the cell surface receptor is TpoR.

[0373] 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.

[0374] In some embodiments, the effector ligand is an intracellular receptor. In some embodiments, the intracellular receptor is a nuclear hormone receptor (e.g., glucocorticoid receptor). In some embodiments where the effector ligand is an intracellular receptor, the macromolecule is delivered as RNA.

[0375] ii. Mechanism of effector ligand activation

[0376] 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.

[0377] 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 at least 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold stronger cellular effector function in the homodimerized form compared to the monomeric form. In some embodiments, the effector ligand is homodimerized in the presence of the macromolecule or macromolecular complex. In some embodiments, the effector ligand is activated by binding to the macromolecule or macromolecular complex in the absence of its endogenous ligand.

[0378] In some embodiments, the macromolecule or the macromolecular complex exhibits conditional avidity, triggered avidity, and / or dimerization avidity. For example, in some aspects, multiple macromolecules or macromolecular complexes bind to a disease signature ligand (e.g., a tumor antigen or a pathogen surface marker), and this proximity results in an increase in avidity, which then activates potent downstream effects.

[0379] In some embodiments that include a second binding domain that is not identical to the first binding domain (e.g., that include SBD1 and SBD2) (e.g., embodiments as described in Sections IA(ii) and IA(iv) herein), the first effector ligand and the second effector ligand must associate (e.g., come into proximity and be correctly oriented (e.g., heterodimerize)) to exert a cellular effector function. In some embodiments, the effector ligand is capable of heterodimerization, and exerts a cellular effector function that is at least 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold stronger in the heterodimerized form compared to the monomeric form. Thus, in some embodiments, the first effector ligand and the second effector ligand associate (e.g., come into proximity and be correctly oriented (e.g., heterodimerize)) in the presence of the macromolecule or the macromolecular complex.

[0380] In other embodiments that include a second binding domain that is not identical to the first binding domain (e.g., that include SBD1 and SBD2) (e.g., embodiments as described in Sections IA(ii) and IA(iv) herein), the first effector ligand and the second effector ligand must associate (e.g., come into proximity and be correctly oriented (e.g., heterodimerize)) and must further associate 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).

[0381] In some embodiments of any of the macromolecules, macromolecule pairs, and macromolecular 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)).

[0382] iii. Cellular Effector Function

[0383] In some embodiments, the cellular effector function of the disease signature ligand is a biological activity.

[0384] In some embodiments, the cellular effector function of the disease signature ligand is a therapeutic activity.

[0385] In some embodiments, the cellular effector function of the disease signature ligand is disease activity (e.g., abnormal activity associated with a disease state), and the cellular effector function is inhibited by multimerization of the macromolecule or binding of the macromolecule pair to the effector ligand.

[0386] iv. Effector Ligand Binding Domain (Second Binding Domain)

[0387] 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 allows additional binding domains to bind to the disease signature ligand (e.g., designed or selected such that at least two copies of the second binding domain can bind to the effector ligand).

[0388] In some embodiments, the polypeptide is an antibody or 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 retargeting antibody (DART).

[0389] In some embodiments, the polypeptide is an antibody mimetic. In some embodiments, the antibody mimetic is an affimer, an affilin, an affimer, an affitin, an alphabody, an anticaline, 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 nanobody, a nanoCLAMP, an optimer, a repebody, a prelamin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR).

[0390] 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 fragment thereof. In some embodiments, the endogenous binding domain is a viral binding protein or fragment thereof.

[0391] 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).

[0392] Additional binding domains that can be used in the present application are described, for example, in Zhong and D’Antona, Antibodies, 10(2): 13, 2021.

[0393] In some embodiments, the effector ligand binding domain has an affinity for the effector ligand that is similar to the affinity of the natural ligand of the effector ligand. In some embodiments, the effector ligand binding domain has an affinity (K D In some embodiments, the effector ligand binding domain has an affinity for the effector ligand that is similar to the affinity of the natural ligand of the effector ligand. In some embodiments, the effector ligand binding domain has an affinity (K

[0394] In some embodiments of any of the macromolecules provided herein, the second binding domain has an affinity for two or more effector ligands. For example, the second binding domain can 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.

[0395] The second binding domain has an affinity for

[0396] In some embodiments comprising a macromolecule or pair of macromolecules comprising non-identical second binding domains (e.g., comprising SBD1 and 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 to a first effector ligand in a biological sample, and the other SBD (e.g., SBD2) specifically binds to a second effector ligand in the biological sample.

[0397] 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.

[0398] 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.

[0399] 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 the dimeric moiety.

[0400] 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. In some embodiments, the effector ligand binding domain has an affinity for the effector ligand that is similar to the affinity of the natural ligand of the effector ligand. In some embodiments, the effector ligand binding domain has an affinity (K D In some embodiments, the effector ligand binding domain has an affinity for the effector ligand that is similar to the affinity of the natural ligand of the effector ligand. In some embodiments, the effector ligand binding domain has an affinity (K

[0394] In some embodiments of any of the macromolecules provided herein, the second binding domain has an affinity for two or more effector ligands. For example, the second binding domain can 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.

[0395] The second binding domain has an affinity for

[0396] In some embodiments comprising a macromolecule or pair of macromolecules comprising non-identical second binding domains (e.g., comprising SBD1 and 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 to a first effector ligand in a biological sample, and the other SBD (e.g., SBD2) specifically binds to a second effector ligand in the biological sample.

[0397] 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.

[0398] 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.

[0399] 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 the dimeric moiety.

[0400] 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.

[0401] In some embodiments of any of the above examples, the non-identical second binding domains (e.g., SBD1 and SBD2) have been engineered to have reduced affinity for one another. In some embodiments, the non-identical second binding domains (e.g., SBD1 and SBD2) have an affinity (Kd) for one another of > 1 µM (e.g., > 5-10 µM) but less than 1 mM (e.g., 10-200 µM). D .

[0402] In some embodiments of any of the macromolecules or macromolecular complexes described herein, the second binding domain comprises a conditionally effective domain.

[0403] D. First and second binding domain pairs

[0404] In some embodiments, the disease signature ligand is TGFβ and the effector ligand is a TPO receptor. For example, in some embodiments, the first binding domain comprises a TGFβ binding domain (e.g., an anti-TGF-β1 antibody or antibody fragment, such as an anti-TGF-β1 single chain variable fragment (scFv)) and the second binding domain comprises a TPO receptor (TpoR) binding domain (e.g., an anti-TpoR antibody or antibody fragment, such as an anti-TpoR heavy chain variable domain (VH) and light chain variable domain (VL)).

[0405] In some embodiments, the disease signature ligand is IL-8 and the effector ligand is a 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, such as an anti-IL-8 scFv) and the second binding domain comprises a TpoR binding domain (e.g., an anti-TpoR antibody or antibody fragment, such as an anti-TpoR VH and VL).

[0406] In some embodiments, the disease signature ligand is TGFβ and the effector ligand is an IL-2 receptor (IL-2R). For example, in some embodiments, the first binding domain comprises a TGFβ binding domain (e.g., an anti-TGF-β1 antibody or antibody fragment, such as an anti-TGF-β1 scFv) and the one or more second binding domains comprise an IL-2R binding domain (e.g., a fragment and / or modified form of IL-2). In some embodiments, the disclosure provides a macromolecule, macromolecular complex, or macromolecular pair comprising two second binding domains comprising complementary fragments of an IL-2R.

[0407] 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 form of IL-2). In some embodiments, the disclosure provides a macromolecule, macromolecular complex, or macromolecular pair comprising two second binding domains comprising a complementary fragment of the IL-2R.

[0408] In some embodiments, the disease signature ligand is IFN-g, and the effector ligand is the IL-10 receptor. For example, in some embodiments, the first binding domain comprises an IFN-g binding domain (e.g., an anti-IFN-g antibody or antibody fragment, such as an anti-IFN-g scFv), and the one or more second binding domains comprise an IL-10 receptor binding domain (e.g., an antibody or antibody fragment that targets one or more components of the IL-10 receptor). In some embodiments, the disclosure provides a macromolecule, macromolecular complex, or macromolecular pair comprising two second binding domains that target different components of the IL-10 receptor (e.g., IL-10Ra and IL-10Rb).

[0409] 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, such as 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 that targets one or more components of the IL-10 receptor). In some embodiments, the disclosure provides a macromolecule, macromolecular complex, or macromolecular pair comprising two second binding domains that target different components of the IL-10 receptor (e.g., IL-10Ra and IL-10Rb) and / or two first binding domains that target different epitopes of IL-6.

[0410] In some embodiments, the disease signature ligand is TGFβ, and the effector ligand is cluster of differentiation 3 (CD3). For example, in some embodiments, the first binding domain comprises a TGFβ binding domain (e.g., an anti-TGF-β1 antibody or antibody fragment, e.g., an anti-TGF-β1 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 present disclosure provides a macromolecule, macromolecular complex, or macromolecular pair comprising two second binding domains comprising different components containing CD3 targeting moieties (e.g., containing anti-CD3 VH and VL, respectively).

[0411] 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 present disclosure provides a macromolecule, macromolecular complex, or macromolecular pair comprising two second binding domains comprising different components containing CD3 targeting moieties (e.g., containing anti-CD3 VH and VL, respectively).

[0412] 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 form thereof. In some aspects, the effector ligand is an IL-10 receptor.

[0413] In some embodiments, the disease signature ligand binding domain (first binding domain) comprises TNFα, MCP-1, or IL-12 or a fragment and / or modified form thereof, and the effector ligand binding domain (second binding domain) comprises IL-10 or a fragment and / or modified form thereof. In some aspects, the effector ligand is an IL-10 receptor.

[0414] In some embodiments, the disease signature ligand is VEGF, and the effector ligand is 4-1BB. 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 a VEGF receptor VEGFR1 or VEGFR2 (e.g., aflibercept), and the second binding domain comprises a 4-1BB binding domain (e.g., an anti-4-1BB antibody or antibody fragment, e.g., an anti-4-1BB scFv). In some embodiments, the 4-1BB binding domain is an scFv reformatted from a full-length agonistic mAb (e.g., urelumab or utomilumab) (e.g., as set forth in SEQ ID NO: 36 and SEQ ID NO: 37, respectively).

[0415] In one aspect, provided herein is a macromolecular complex comprising two macromolecules, each macromolecule comprising a VEGF binding domain linked to a 4-1BB binding domain, wherein the 4-1BB binding domain induces a cellular effector function upon binding to 4-1BB; wherein the two macromolecules are conjugated to one another; and wherein induction of the effector function by the macromolecular complex is contingent 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 moieties is a pair of Fc fragments. In some embodiments, the pair of Fc fragments is a pair of knobby-stomy structures. In some embodiments, the two macromolecules are identical.

[0416] In another aspect, provided herein is a macromolecular complex comprising two macromolecules, each macromolecule comprising a VEGF binding domain linked to a 4-1BB binding domain, wherein the 4-1BB binding domain induces a cellular effector function upon binding to 4-1BB; wherein the two macromolecules are not conjugated to one another in the absence of the disease signature ligand; and wherein induction of the effector function by the macromolecular complex is contingent upon each of the two macromolecules binding to 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 one another in the presence of the disease signature ligand, wherein the non-covalent conjugation is mediated by the disease signature ligand. In some embodiments, one or both members of the pair of macromolecules or the macromolecule comprises a half-life extending moiety (e.g., an Fc domain or fragment thereof).

[0417] In some embodiments of any 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 VEGF receptor VEGFR1 or VEGFR2 (e.g., aflibercept).

[0418] In some embodiments of any of the above aspects, the 4-1BB binding domain is an anti-4-1BB scFv. In some embodiments, the 4-1BB binding domain is an scFv reformatted from a full-length agonistic mAb (e.g., urelumab or utomilumab) (e.g., as set forth in SEQ ID NO: 36 and SEQ ID NO: 37, respectively).

[0419] Exemplary reference polypeptides (e.g., antibodies and antigen-binding fragments thereof) can be used in the application, including variants of the reference sequences. For example, “variants of TPO receptor (TpoR) binding polypeptides,” “variants of IFN-g binding polypeptides,” “variants of IL-10b binding polypeptides,” “variants of CD3 binding polypeptides,” “variants of IL-8 binding polypeptides,” “variants of TGFb binding polypeptides,” “variants of IL6 binding polypeptides,” “variants of 4-1BB binding polypeptides” (e.g., “variants” of the polypeptides provided in the application) and the like comprise an amino acid sequence having, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions relative to a reference sequence (e.g., one of SEQ ID NOs: 1-9, 12-14, and 32-43). In some embodiments, for example, in the examples provided herein, for example, in the assays provided herein, the variants retain the function of the disclosed polypeptides.

[0420] In certain embodiments, variants of the polypeptides provided in the application comprise up to 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, for example, 1-3 amino acid substitutions. Multiple amino acid substitutions of variants of the polypeptides provided in the application are contemplated, including substitutions with non-canonical amino acids. In some embodiments, for example, in the examples provided herein, for example, in the assays provided herein, the variants of the polypeptides provided in the application retain the function of the disclosed polypeptides.

[0421] In some embodiments, variants of the polypeptides comprise conservative substitutions or highly conserved substitutions relative to a reference sequence. By “conservative substitution” relative to a reference sequence is meant a given amino acid substitution has a value of 0 or greater in BLOSUM62.

[0422] A "highly conserved substitution" with respect to a reference sequence means that a given amino acid substitution has a value of 1 or greater (e.g., in some embodiments, 2 or greater) in BLOSUM62.

[0423] In certain embodiments, a variant of a polypeptide provided herein comprises a paratope of a polypeptide provided herein (e.g., an antibody or fragment thereof provided herein), wherein only highly conserved substitutions are made in the residues of the paratope (e.g., up to 1, 2, 3, 4, or 5 substitutions in the paratope), and 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 can be highly conserved substitutions, conserved substitutions, or non-conserved substitutions, or combinations thereof. For example, in certain embodiments, any substitutions of non-paratope residues in the CDRs of the variable region are conserved substitutions, or in some embodiments, are highly conserved substitutions, e.g., in certain embodiments, a CDR of a variant of a polypeptide can have up to 1, 2, 3, 4, or 5 substitutions in residues other than those that make up the paratope, wherein the substitutions are conserved substitutions, or in more specific embodiments, are highly conserved substitutions. In more specific embodiments, non-conserved substitutions can be made outside of the CDRs, although the skilled artisan will appreciate that in some embodiments, substitutions outside of the CDRs can be conserved or highly conserved. In certain embodiments, a variant polypeptide provided herein 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 to a reference immunoglobulin light chain variable region or immunoglobulin heavy chain variable region, with appropriate consideration of the various substitution criteria described above (e.g., substitutions with respect to paratope, CDR, non-paratope residues, and non-CDR residues).

[0424] E. Leader, reporter, and linker moieties

[0425] Leader

[0426] 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 can be cleaved from the macromolecule prior to formation of the multimer. In some embodiments, the leader peptide is a mouse immunoglobulin kappa variable 3 (IgKVIII) leader peptide (e.g., UniProt ID A0A140T8P0 positions M1 to G20). Other exemplary leader sequences are provided in Table 1 (SEQ ID NOs: 15-31).

[0427] Table 1. Leader sequences

[0428]

[0429] Reporter

[0430] In some embodiments of any of the compositions and methods provided herein, the macromolecule comprises a reporter moiety. For example, in embodiments comprising a macromolecule pair, one or both members of the macromolecule pair can comprise a reporter moiety, or the members of the macromolecule pair can each comprise a different reporter moiety.

[0431] Exemplary reporter moieties include, without limitation, affinity tags (e.g., FLAG affinity tag), fluorescent labels, and chromogenic labels. 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 fragment thereof. In other embodiments, the reporter moiety comprises a fragment of a decoy protein, and is detected by the addition of an exogenous dye that detects the decoy protein.

[0432] In some embodiments comprising a macromolecule pair, the first member and the second member of the macromolecule pair comprise complementary reporter moieties, e.g., reporter moieties that are detectable (e.g., produce a fluorescent signal) when the first member and the second member of the macromolecule pair form a multimer. For example, the first member and the second member of the macromolecule pair can 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).

[0433] Linker

[0434] In some embodiments of any of the compositions and methods provided herein, the macromolecule comprises one or more linker domains, e.g., a linker domain connecting a first binding domain to a second binding domain; a linker domain connecting one or more subdomains within a first binding domain or a second binding domain; and / or a linker domain connecting a first binding domain or a second binding domain to a leader peptide or a reporter moiety.

[0435] In some embodiments, the one or more linker domains is a peptide linker. In some embodiments, the peptide linker is a GS linker. In some embodiments, the peptide linker is a glycine-serine (GS) linker, e.g., a GS linker having the form GS(GS)2, GS(GS)3, GS(GS)4, GS(GS)5, GS(GS)6, GS(GS)7, GS(GS)8, GS(GS)9, GS(GS)10, GS(GS)11, GS(GS)12, GS(GS)13, GS(GS)14, GS(GS)15, GS(GS)16, GS(GS)17, GS(GS)18, GS(GS)19, GS(GS)20, GS(GS)21, GS(GS)22, GS(GS)23, GS(GS)24, GS(GS)25, GS(GS)26, GS(GS)27, GS(GS)28, GS(GS)29, GS(GS)30, GS(GS)31, GS(GS)32, GS(GS)33, GS(GS)34, GS(GS)35, GS(GS)36, GS(GS)37, GS(GS)38, GS(GS)39, GS(GS)40, GS(GS)41, GS(GS)42, GS(GS)43, GS(GS)44, GS(GS)45, GS(GS)46, GS(GS)47, GS(GS)48, GS(GS)49, GS(GS)50, or a GS linker having the form GS(GS)n, where n is an integer from 1 to 50. n S) m In some embodiments, the one or more linker domains is a peptide linker. In some embodiments, the peptide linker is a GS linker. In some embodiments, the peptide linker is a glycine-serine (GS) linker, e.g., a GS linker having the form GS(GS)2, GS(GS)3, GS(GS)4, GS(GS)5, GS(GS)6, GS(GS)7, GS(GS)8, GS(GS)9, GS(GS)10, GS(GS)11, GS(GS)12, GS(GS)13, GS(GS)14, GS(GS)15, GS(GS)16, GS(GS)17, GS(GS)18, GS(GS)19, GS(GS)20, GS(GS)21, GS(GS)22, GS(GS)23, GS(GS)24, GS(GS)25, GS(GS)26, GS(GS)27, GS(GS)28, GS(GS)29, GS(GS)30, GS(GS)31, GS(GS)32, GS(GS)33, GS(GS)34, GS(GS)35, GS(GS)36, GS(GS)37, GS(GS)38, GS(GS)39, GS(GS)40, GS(GS)41, GS(GS)42, GS(GS)43, GS(GS)44, GS(GS)45, GS(GS)46, GS(GS)47, GS(GS)48, GS(GS)49, GS(GS)50, or a GS linker having the form GS(GS)n, where n is an integer from 1 to 50. n S) mGS linker of the form (G4S)n-m, e.g., where 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, 11, 12, 13, 14, or 15 (e.g., m = 1-5, 5-10, or 10-15, e.g., m = 5). In some embodiments, the peptide linker is a GS linker of the form (G4S)n-m, e.g., where n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., n = 1-5 or 5-10). n GS linker of the form (G4S)n-m, e.g., where n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., n = 1-5 or 5-10).

[0436] F. Half-life Extending Moieties

[0437] Any macromolecule (e.g., polypeptide) provided herein can be modified to alter (e.g., extend) its half-life (e.g., to alter (e.g., extend) its half-life (e.g., circulating (e.g., in serum)) and / or to elicit a desired effector function. For example, in some embodiments, any macromolecule provided herein can comprise a moiety (e.g., a heterologous moiety) that extends the half-life of the macromolecule. Exemplary half-life extending moieties include polypeptides (e.g., a crystallizable fragment 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).

[0438] In some aspects, a polypeptide provided herein is modified to comprise an Fc region that extends the half-life of the polypeptide relative to a version of the polypeptide that does not comprise an Fc region. In some embodiments, the Fc region is an IgG isotype Fc region, e.g., an IgG1, IgG2, or IgG4 subtype Fc region (e.g., such an Fc region from a human, mouse, or non-human primate (NHP)). In some embodiments, the Fc region comprises one or more Fc effector function silent mutations (e.g., LALA or LALAPG mutations (mutations at positions L234, L235, G236, N297, or P329 in IgG1)); in other aspects, the Fc region is capable of eliciting one or more Fc effector functions. The Fc region can be modified to extend half-life using one or more mutations that enhance recycling based on the neonatal Fc receptor (FcRn). Other Fc variants that can be used in the application include mutated Fc variants that alter Fcy receptor binding or Fc neonatal receptor binding and recycling, and Fc variants comprising glycosylation modifications, as previously described. Variant Fc regions that can be used in the application 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.

[0439] In some aspects, a polypeptide provided herein is modified to comprise an Fc region that alters Fcy receptor binding and / or effector function or Fc neonatal receptor binding and / or recycling. In some aspects, a polypeptide provided herein is modified to comprise an Fc region that comprises one or more glycosylation modifications.

[0440] In some aspects, a macromolecule (e.g., polypeptide) provided herein is modified to comprise human serum albumin (HSA) or a binder thereof that extends the half-life (e.g., circulating half-life) of the polypeptide relative to a version of the polypeptide that does not comprise HSA or a 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).

[0441] 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 form of the macromolecule that does not comprise the modification).

[0442] G. Manufacture and Purity

[0443] In some embodiments of any of the compositions and methods provided herein, the macromolecule, macromolecule pair, macromolecular complex, nucleic acid, nucleic acid pair, multimer, or composition comprising the same is at least 95% pure (e.g., at least 95% free of any impurities or undesired substances). In some embodiments, the macromolecule, macromolecule pair, macromolecular complex, nucleic acid, nucleic acid pair, 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.

[0444] In some embodiments of any of the compositions and methods provided herein, the macromolecule, macromolecule pair, macromolecular complex, nucleic acid, nucleic acid pair, multimer, or composition comprising the same is manufactured according to one or more International Organization for Standardization (ISO) standards.

[0445] In some embodiments, the macromolecule, macromolecule pair, macromolecular complex, nucleic acid, nucleic acid pair, multimer, or composition comprising the same is manufactured according to United States Food and Drug Administration (FDA) good manufacturing practice (GMP), good clinical practice (GCP), and / or good laboratory practice (GLP) standards.

[0446] H. Biological Sample

[0447] 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 cells); a cell; a tissue; or a subject (e.g., a mammalian subject, e.g., a human subject).

[0448] In some embodiments of any of the compositions and methods provided herein, the biological sample is a human subject.

[0449] In some embodiments, the biological sample is from a vertebrate (e.g., a 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, non-human mammals are non-human primates (e.g., monkeys, apes), ungulates (e.g., cattle, buffalo, sheep, goats, pigs, camels, llamas, alpacas, deer, horses, donkeys), carnivores (e.g., dogs, cats), rodents (e.g., rats, mice), or rabbits (e.g., rabbits). In some embodiments, the biological sample is from a bird, such as a member of the order Galliformes (e.g., chickens, turkeys, pheasants, quails), Anseriformes (e.g., ducks, geese), Paleognathea (e.g., ostriches, emus), Columbiformes (e.g., pigeons, wild pigeons), or Psittaciformes (e.g., parrots). In some embodiments, the biological sample is derived from invertebrates such as arthropods (e.g., insects, arachnids, crustaceans), nematodes, annelids, worms, or mollusks.

[0450] I. Lipid nanoparticles

[0451] The compositions (e.g., macromolecules, macromolecule pairs, macromolecular complexes, peptides, nucleic acids, and compositions comprising them), methods, and delivery systems provided in this disclosure may employ any suitable carrier or delivery form described herein, and in some embodiments include lipid nanoparticles (LNPs). In some embodiments, the lipid nanoparticles comprise 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 as described in Table 5 of WO2019217941; which is incorporated herein by reference in its entirety); and one or more sterols (e.g., cholesterol).

[0452] Lipids that can be used for nanoparticle formation (e.g., lipid nanoparticles) include, for example, those described in Table 4 of WO 2019217941, which is incorporated herein by reference—for example, lipid-containing nanoparticles may contain one or more lipids as described in Table 4 of WO 2019217941. Lipid nanoparticles may contain additional elements, such as polymers, as described in Table 5 of WO 2019217941, which is incorporated herein by reference.

[0453] In some embodiments, the conjugated lipid (when present) can include one or more of: PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxylpropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinic diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxylpropyl carbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those described in Table 2 of WO 2019051289 (incorporated by reference) and combinations of the foregoing.

[0454] In some embodiments, the sterol that can be incorporated into the lipid nanoparticle includes one or more of cholesterol or a cholesterol derivative, such as those in WO 2009 / 127060 or US 2010 / 0130588, 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 by reference herein.

[0455] In some embodiments, the lipid particle comprises an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits particle aggregation, and a sterol. The amounts of these components can be varied independently to achieve desired properties. For example, in some embodiments, the lipid nanoparticle comprises an ionizable lipid in an amount of about 20 mol% to about 90 mol% of the total lipid (in other embodiments, it can be 20-70% (mol), 30-60% (mol), or 40-50% (mol) of the total lipid present in the lipid nanoparticle; about 50 mol% to about 90 mol%); a non-cationic lipid in an amount of about 5 mol% to about 30 mol% of the total lipid; a conjugated lipid in an amount of about 0.5 mol% to about 20 mol% of the total lipid; and a sterol in an amount of about 20 mol% to about 50 mol% of the total lipid. The ratio of total lipid to nucleic acid can be varied as desired. For example, the ratio of total lipid to nucleic acid (mass or weight) can be about 10 : 1 to about 30 : 1.

[0456] In some embodiments, the ratio of lipid to nucleic acid (mass / mass ratio; w / w ratio) can range from about 1 : 1 to about 25: 1, about 10 : 1 to about 14 : 1, about 3 : 1 to about 15 : 1, about 4 : 1 to about 10 : 1, about 5 : 1 to about 9 : 1, or about 6 : 1 to about 9 : 1. The amount of lipid and nucleic acid can be adjusted to provide a desired N / P ratio, e.g., an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10, or higher. Generally, the total lipid content of the lipid nanoparticle formulation can range from about 5 mg / ml to about 30 mg / mL.

[0457] Some non-limiting examples of lipid compounds that can be used (e.g., in combination with other lipid components) to form lipid nanoparticles for delivering compositions described herein, e.g., nucleic acids (e.g., RNAs (e.g., circular polyribonucleotides, linear polyribonucleotides)) described herein include:

[0458] (i)

[0459] In some embodiments, LNPs comprising Formula (i) are used to deliver polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0460] (ii)

[0461] In some embodiments, LNPs comprising Formula (ii) are used to deliver polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0462] (iii)

[0463] In some embodiments, LNPs comprising Formula (iii) are used to deliver polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0464] (iv)

[0465] (v)

[0466] In some embodiments, LNPs comprising Formula (v) are used to deliver polyribonucleotide (e.g., circular polyribonucleotide, linear polyribonucleotide) compositions described herein to a cell.

[0467] (vi)

[0468] In some embodiments, an LNP comprising Formula (vi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to a cell.

[0469] (vii)

[0470] (viii)

[0471] In some embodiments, an LNP comprising Formula (viii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to a cell.

[0472] (ix)

[0473] In some embodiments, an LNP comprising Formula (ix) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to a cell.

[0474] (x)

[0475] wherein

[0476] X 1 is O, NR 1 or a direct bond, X 2 is C2-5 alkylene, X 3 is C(=O) or a direct bond, R 1 is H or Me, R 3 is C1-3 alkyl, R 2 is C1-3 alkyl, or R 2 together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X 2 form a 4-, 5-, or 6-membered ring, or X 1 is NR 1 , R 1 and R 2 together with the nitrogen atom to which they are attached form a 5- or 6-membered ring, or R 2 together with R 3 and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y 1 is C2-12 alkylene, Y 2 is selected from

[0477]

[0478] (in either orientation), (in either orientation), (in either orientation),

[0479] n is 0 to 3, R 4 is C1-15alkyl, Z 1 is C1-6alkylene or a direct bond,

[0480]

[0481] is absent, provided that if Z 1 is a direct bond, then Z 2 is absent;

[0482] R 5 is C5-9alkyl or C6-10alkoxy, R 6 is C5-9alkyl or C6-10alkoxy, W is methylene or a direct bond, and R 7 is H or Me, or a salt thereof, provided that if R 3 and R 2 is C2alkyl, X 1 is O, X 2 is linear C3alkylene, X 3 is C(=0), Y 1 is linear Cealkylene, (Y 2 )n-R 4 is

[0483]

[0484] , R 4 is linear C5alkyl, Z 1 is C2alkylene, Z 2 is absent, W is methylene, and R 7 is H, then R 5 and R 6 are not Cxalkoxy.

[0485] In some embodiments, an LNP comprising Formula (xii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to a cell.

[0486] (xi)

[0487] In some embodiments, an LNP comprising Formula (xi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to a cell.

[0488] wherein R = (xii)

[0489] (xiii)

[0490] (xiv)

[0491] In some embodiments, the LNP comprises a compound of Formula (xiii) and a compound of Formula (xiv).

[0492] (xv)

[0493] In some embodiments, an LNP comprising Formula (xv) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to a cell.

[0494] (xvi)

[0495] In some embodiments, an LNP comprising a formulation of Formula (xvi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to a cell.

[0496] (xvii)

[0497] wherein X= (xviii)(a)

[0498] (xviii)(b)

[0499] (xix)

[0500] In some embodiments, a lipid compound used to form a lipid nanoparticle for delivering a composition described herein, e.g., a nucleic acid (e.g., RNA (e.g., a circular polyribonucleotide, a linear polyribonucleotide)) described herein, is prepared by one of the following reactions:

[0501] + (xx)(a)

[0502] + (xx)(b).

[0503] In some embodiments, an LNP comprising Formula (xxi) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to a cell. In some embodiments, the LNP of Formula (xxi) is an LNP described in WO 2021113777 (e.g., a lipid of Formula (1), such as a lipid of Table 1 of WO 2021113777).

[0504] (xxi)

[0505] wherein

[0506] each n is independently an integer from 2-15; L1and L3are each independently -OC(O)- or -C(O)O- wherein “ ” denotes the point of attachment to R1or R3;

[0507] R1and R3are each independently a linear or branched C9-C 20 alkyl or C9-C 20 alkenyl: oxo, halo, hydroxyl, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (amino carbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkoxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; and

[0508] R2is selected from the group consisting of:

[0509]

[0510] In some embodiments, an LNP comprising Formula (xxii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to a cell. In some embodiments, the LNP of Formula (xxii) is an LNP described in WO 2021113777 (e.g., a lipid of Formula (2), such as a lipid of Table 2 of WO 2021113777).

[0511] (xxii)

[0512] wherein

[0513] each n is independently an integer from 1-15;

[0514] R1and R2are each independently selected from the group consisting of:

[0515]

[0516] R3is selected from the group consisting of:

[0517] .

[0518] In some embodiments, the LNP comprising Formula (xxiii) is used to deliver a polyribonucleotide (e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition described herein to a cell. In some embodiments, the LNP of Formula (xxiii) is an LNP described in WO 2021113777 (e.g., a lipid of Formula (3), such as a lipid of Table 3 of WO 2021113777).

[0519] (xxiii)

[0520] wherein

[0521] X is selected from -O-, -S-, or -OC(O)- wherein denotes the point of attachment to R1;

[0522] R1is selected from the group consisting of:

[0523]

[0524] and R2is selected from the group consisting of:

[0525]

[0526] In some embodiments, the compositions described herein (e.g., nucleic acids (e.g., circular polyribonucleotides, linear polyribonucleotides) or proteins) are provided in LNPs comprising an ionizable lipid. In some embodiments, the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)octanoate (SM-102); e.g., as described in Example 1 of US 9,867,888 (incorporated by reference in its entirety herein). 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 (LPOl), e.g., as synthesized in Example 13 of WO 2015 / 095340 (incorporated by reference in its entirety herein). In some embodiments, the ionizable lipid is di((Z)-non-2-en-l-yl) 9-((4-dimethylamino)butanoyl)oxy)heptadecandioate (L319), e.g., as synthesized in Example 7, Example 8, or Example 9 of US 2012 / 0027803 (incorporated by reference in its entirety herein). In some embodiments, the ionizable lipid is l,l’-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), e.g., as synthesized in Example 14 and Example 16 of WO 2010 / 053572 (incorporated by reference in its entirety herein). 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-lH- cyclopenta[a]phenanthren-3-yl 3-(lH-imidazol-4-yl)propanoate, e.g., from WO 2020 / 106946 (incorporated by reference in its entirety herein) Structure (I).

[0527] In some embodiments, the ionizable lipid can be a cationic lipid, an ionizable cationic lipid, for example, a cationic lipid that can exist in a positively charged form or a neutral form depending on the pH, or an amine-containing lipid that can readily be protonated. In some embodiments, the cationic lipid is a lipid that is capable of carrying a positive charge, for example, under physiological conditions. Exemplary cationic lipids include one or more amine groups that carry a positive charge. In some embodiments, the lipid particle includes a cationic lipid formulated with one or more of a neutral lipid, an ionizable amine-containing lipid, a biodegradable alkyne lipid, a steroid, a phospholipid including a polyunsaturated lipid, a structural lipid (e.g., a sterol), a PEG, a cholesterol, and a polymer-conjugated lipid. In some embodiments, the cationic lipid can be an ionizable cationic lipid. Exemplary cationic lipids as disclosed herein can have an effective pKa of more than 6.0. In embodiments, the lipid nanoparticle can include a second cationic lipid having an effective pKa that is different from (e.g., greater than) the first effective pKa. The lipid nanoparticle can include between 40 mol% and 60 mol% of a cationic lipid, a neutral lipid, a steroid, a polymer-conjugated lipid, and a therapeutic agent, for example, a nucleic acid (e.g., an RNA (e.g., a circular polyribonucleotide, a linear polyribonucleotide)) as described herein, encapsulated within or associated with the lipid nanoparticle. In some embodiments, the nucleic acid is co-formulated with the cationic lipid. The nucleic acid can be adsorbed to the surface of the LNP (e.g., an LNP including a cationic lipid). In some embodiments, the nucleic acid can be encapsulated in the LNP (e.g., an LNP including a cationic lipid). In some embodiments, the lipid nanoparticle can include a targeting moiety, for example, a targeting moiety coated with a targeting agent. In embodiments, the LNP formulation is biodegradable. In some embodiments, the lipid nanoparticle including one or more lipids 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 the RNA molecules.

[0528] Exemplary ionizable lipids that can be used in a lipid nanoparticle formulation include, without limitation, those listed in Table 1 of WO 2019051289, incorporated by reference herein. Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US 2016 / 0311759; I in US 20150376115 or US 2016 / 0376224; I, II, or III of US 20160151284; I, IA, II, or IIA of US 20170210967; I-c of US 20150140070; A of US 2013 / 0178541; I of US 2013 / 0303587 or US 2013 / 0123338; I of US 2015 / 0141678; II, III, IV, or V of US 2015 / 0239926; I of US 2017 / 0119904; I or II of WO 2017 / 117528; A of US 2012 / 0149894; A of US 2015 / 0057373; A of WO 2013 / 116126; A of US 2013 / 0090372; A of US 2013 / 0274523; A of US 2013 / 0274504; A of US 2013 / 0053572; A of WO 2013 / 016058; A of WO 2012 / 162210; I of US 2008 / 042973; I, II, III, or IV of US 2012 / 01287670; I or II of US 2014 / 0200257; I, II, or III of US 2015 / 0203446; I or III of US 2015 / 0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of US 2014 / 0308304; of US 2013 / 0338210; I, II, III, or IV of WO 2009 / 132131; A of US 2012 / 01011478; I or XXXV of US 2012 / 0027796; XIV or XVII of US 2012 / 0058144; of US 2013 / 0323269; I of US 2011 / 0117125; I, II, or III of US 2011 / 0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US 2012 / 0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US 2011 / 0076335; I or II of US 2006 / 008378; I of US 2013 / 0123338;I or X-A-Y-Z of US 2015 / 0064242; XVI, XVII, or XVIII of US 2013 / 0022649; I, II, or III of US 2013 / 0116307; I, II, or III of US 2013 / 0116307; I or II of US 2010 / 0062967; I-X of US 2013 / 0189351; I of US 2014 / 0039032; V of US 2018 / 0028664; I of US 2016 / 0317458; I of US 2013 / 0195920; 5, 6, or 10 of US 10,221,127; III-3 of WO 2018 / 081480; I-5 or I-8 of WO 2020 / 081938; 18 or 25 of US 9,867,888; A of US 2019 / 0136231; II of WO 2020 / 219876; 1 of US 2012 / 0027803; OF-02 of US 2019 / 0240349; 23 of US 10,086,013; cKK-E12 / A6 of Miao et al. (2020); C12-200 of WO 2010 / 053572; 7C1 of Dahlman et al. (2017); 304-O13 or 503-O13 of Whitehead et al.; TS-P4C2 of US 9,708,628; I of WO 2020 / 106946; I of WO 2020 / 106946; and (1), (2), (3), or (4) of WO 2021 / 113777. Exemplary lipids further include the lipids of any one of Tables 1-16 of WO 2021 / 113777.

[0529] In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-l9-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), for example as described in Example 9 of WO 2019051289 A9 (incorporated by reference in its entirety herein). In some embodiments, the ionizable lipid is the lipid ATX-002, for example as described in Example 10 of WO 2019051289 A9 (incorporated by reference in its entirety herein). In some embodiments, the ionizable lipid is (l3Z,l6Z)-A,A-dimethyl-3-nonyldocosa-l3,l6-dien-l-amine (Compound 32), for example as described in Example 11 of WO 2019051289 A9 (incorporated by reference in its entirety herein). In some embodiments, the ionizable lipid is Compound 6 or Compound 22, for example as described in Example 12 of WO 2019051289 A9 (incorporated by reference in its entirety herein).

[0530] Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)- cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (e.g., 16-O-monomethyl PE), dimethyl- phosphatidylethanolamine (e.g., 16-O-dimethyl PE), l8-l-trans PE, l-stearoyl-2-oleoyl- phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), diarachidonoylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, lyso-phosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It should be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. In certain embodiments, additional exemplary lipids include, without limitation, those described in Kim et al. (2020) dx.doi.org / 10.1021 / acs.nanolett.0c01386, incorporated herein by reference. In some embodiments, such lipids include plant lipids found to improve liver transfection with mRNA (e.g., DGTS).

[0531] Other examples of non-cationic lipids suitable for use in the lipid nanoparticles include, without limitation, non-phospholipids, such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glyceryl ricinoleate, hexadecyl sterate, isopropyl myristate, amphipropyl acrylic polymer, triethanolamine-lauryle sulfate, alkyl-aryl sulfates, polyethoxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, sphingomyelin, and the like. Other non-cationic lipids are described in WO 2017 / 099823 or U.S. Patent Publication US 2018 / 0028664, the contents of which are incorporated by reference in their entirety.

[0532] In some embodiments, the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US 2018 / 0028664, incorporated by reference in its entirety. The non-cationic lipid can comprise, for example, 0-30% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In embodiments, the molar ratio of ionizable lipid to neutral lipid is about 2: 1 to about 8: 1 (e.g., about 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, or 8: 1).

[0533] In some embodiments, the lipid nanoparticle does not comprise any phospholipids.

[0534] In some aspects, the lipid nanoparticle can further comprise 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 analogs such as 5a-cholestanol, 53- coprostanol, cholesteryl-(2 , -hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; nonpolar analogs such as 5a-cholestan, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog, for example, cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT Publication WO 2009 / 127060 and U.S. Patent Publication US 2010 / 0130588, each of which is incorporated by reference in its entirety.

[0535] In some embodiments, the component that provides membrane integrity (such as a sterol) can comprise 0-50% (mol) of the total lipid present in the lipid nanoparticle (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%). In some embodiments, such a component is 20-50% (mol), 30-40% (mol) of the total lipid content of the lipid nanoparticle.

[0536] In some embodiments, the lipid nanoparticle can comprise a polyethylene glycol (PEG) or conjugated lipid molecule. Generally, these serve to inhibit aggregation of the lipid nanoparticle 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 polymeric lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, e.g., a (methoxypolyethylene glycol) conjugated lipid.

[0537] Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diaclyglycerol (DAG) (such as l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinic acid diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-ditetradecanoyloxy)propyl-l-0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkyloxypropyl carbamates, N-(carbonyl-methoxypolyethylene glycol 2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEG-lipid conjugates are described, for example, in US 5,885,613, US 6,287,591, US 2003 / 0077829, US 2003 / 0077829, US 2005 / 0175682, US 2008 / 0020058, US 2011 / 0117125, US 2010 / 0130588, US 2016 / 0376224, US 2017 / 0119904, and US / 099823, the contents of all of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-lipid is a compound of Formula III, III-a-l, III-a-2, III-b-l, III-b-2, or V of US 2018 / 0028664, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-lipid has Formula II of US 20150376115 or US 2016 / 0376224, the contents of both of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmitoyloxypropyl, or PEG-distearoyloxypropyl. The PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-distearoylglycerol, PEG-dilaurylglycerolamide, PEG-dimyristylglycerolamide, PEG-dipalmitoylglycerolamide, PEG-distearoylglycerolamide, PEG-cholesterol (l-[8'-(cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxyoctyl]carbamoyl-[omega]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-bis tetradecyloxybenzyl-[omega]-methyl-poly(ethylene glycol) ether), and l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].In some embodiments, the PEG-lipid comprises PEG-DMG, l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises a structure selected from the group consisting of:

[0538]

[0539] In some embodiments, lipids conjugated to molecules other than PEG can also be used in place of or together with PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic polymeric-lipids (GPL) conjugates can be used in place of or together with PEG-lipids.

[0540] Exemplary conjugated lipids (i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymeric-lipids) are described in PCT and LIS patent applications listed in Table 2 of WO 2019051289 A9, the contents of all of which are incorporated herein by reference in their entirety.

[0541] In some embodiments, the PEG or conjugated lipid can comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the PEG or conjugated lipid is present in an amount of 0.5-10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle. The molar ratios of ionizable lipid, non-cationic lipid, sterol, and PEG / conjugated lipid can vary as desired. For example, the lipid particle can comprise 30-70% ionizable lipid by mole or total weight of the composition, 0-60% cholesterol by mole or total weight of the composition, 0-30% non-cationic lipid by mole or total weight of the composition, and 1-10% conjugated lipid by mole or total weight of the composition. Preferably, the composition comprises 30-40% ionizable lipid by mole or total weight of the composition, 40-50% cholesterol by mole or total weight of the composition, and 10-20% non-cationic lipid by mole or total weight of the composition. In some other embodiments, the composition is 50-75% ionizable lipid by mole or total weight of the composition, 20-40% cholesterol by mole or total weight of the composition, and 5-10% non-cationic lipid by mole or total weight of the composition, and 1-10% conjugated lipid by mole or total weight of the composition. The composition can contain 60-70% ionizable lipid by mole or total weight of the composition, 25-35% cholesterol by mole or total weight of the composition, and 5-10% non-cationic lipid by mole or total weight of the composition. The composition can also contain up to 90% ionizable lipid by mole or total weight of the composition and 2-15% non-cationic lipid by mole or total weight of the composition. The formulation can also be a lipid nanoparticle formulation, for example, comprising 8-30% ionizable lipid by mole or total weight of the composition, 5-30% non-cationic lipid by mole or total weight of the composition, and 0-20% cholesterol by mole or total weight of the composition; 4-25% ionizable lipid by mole or total weight of the composition, 4-25% non-cationic lipid by mole or total weight of the composition, 2-25% cholesterol by mole or total weight of the composition, 10-35% conjugated lipid by mole or total weight of the composition, and 5% cholesterol by mole or total weight of the composition; or 2-30% ionizable lipid by mole or total weight of the composition, 2-30% non-cationic lipid by mole or total weight of the composition, 1-15% cholesterol by mole or total weight of the composition, 2-35% conjugated lipid by mole or total weight of the composition, and 1-20% cholesterol by mole or total weight of the composition; or even up to 90% ionizable lipid by mole or total weight of the composition and 2-10% non-cationic lipid by mole or total weight of the composition or even 100% cationic lipid by mole or total weight of the composition.In some embodiments, the lipid particle formulation comprises an ionizable lipid, a phospholipid, a cholesterol, and a PEGylated lipid in a molar ratio of 50 : 10 : 38.5 : 1.5. In some other embodiments, the lipid particle formulation comprises an ionizable lipid, a cholesterol, and a PEGylated lipid in a molar ratio of 60 : 38.5 : 1.5.

[0542] In some embodiments, the lipid particle comprises an ionizable lipid, a non-cationic lipid (e.g., a phospholipid), a sterol (e.g., cholesterol), and a PEGylated lipid, wherein the molar ratio of the lipids is in the range of 20 to 70 mole percent for the ionizable lipid, targeting 40-60, 0 to 30 mole percent for the non-cationic lipid, targeting 0 to 15, 20 to 70 mole percent for the sterol, targeting 30 to 50, and 1 to 6 mole percent for the PEGylated lipid, targeting 2 to 5.

[0543] In some embodiments, the lipid particle comprises an ionizable lipid, a non-cationic lipid, a sterol, and a conjugated lipid in a molar ratio of 50 : 10 : 38.5 : 1.5.

[0544] In one aspect, the disclosure provides a lipid nanoparticle formulation comprising a phospholipid, a phosphatidylcholine, a phosphatidylethanolamine, and a phosphatidylserine.

[0545] 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 present application. In other words, in addition to the nucleic acid, or at least a second nucleic acid, the lipid nanoparticles can contain other compounds different from the first nucleic acid. Without limitation, the 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, their peptidic analogs and derivatives, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, or any combination thereof.

[0546] In some embodiments, the LNP comprises a biodegradable, ionizable lipid. In some embodiments, the LNP comprises (9Z, 12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9, 12-dienoate, also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadeca-9, 12-dienoate), or another ionizable lipid. See, e.g., WO 2019 / 067992, WO / 2017 / 173054, WO 2015 / 095340, and WO 2014 / 136086, and the lipids provided therein with references. In some embodiments, the terms cationic and ionizable are interchangeable in the context of LNP lipids, e.g., where the ionizable lipid is cationic depending on the pH.

[0547] In some embodiments, the average LNP diameter of the LNP formulation can be between tens of nm and hundreds of nm, for example, as measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation can be about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the LNP formulation can be about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, or about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation can be about 70 nm to about 100 nm. In particular embodiments, the average LNP diameter of the LNP formulation can be about 80 nm. In some embodiments, the average LNP diameter of the LNP formulation can be about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation ranges from about 1 mm to about 500 mm, about 5 mm to about 200 mm, about 10 mm to about 100 mm, about 20 mm to about 80 mm, about 25 mm to about 60 mm, about 30 mm to about 55 mm, about 35 mm to about 50 mm, or about 38 mm to about 42 mm.

[0548] In some cases, the LNP can be relatively homogenous. A polydispersity index can be used to indicate the homogeneity of the LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The polydispersity index of the LNP can be about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the LNP can be about 0.10 to about 0.20.

[0549] The zeta potential of the LNP can be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential can describe the surface charge of the LNP. Lipid nanoparticles with a relatively low charge (positive or negative) are generally desirable, as higher charged species can undesirably interact with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the LNP can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0550] The encapsulation efficiency of a protein and / or nucleic acid describes the amount of protein and / or nucleic acid that is encapsulated or otherwise associated with the LNP after preparation relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the lipid nanoparticles before and after breaking up the lipid nanoparticles with one or more organic solvents or detergents. Anion exchange resin can be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence can 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 can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In some embodiments, the encapsulation efficiency can be at least 90%. In some embodiments, the encapsulation efficiency can be at least 95%.

[0551] The LNP can optionally comprise one or more layers of coating. In some embodiments, the LNP can be formulated in a capsule, film, or tablet having a coating. The capsule, film, or tablet comprising the compositions described herein can have any useful size, tensile strength, hardness, or density.

[0552] WO 2020 / 061457 and WO 2021 / 113777, each of which is incorporated herein by reference in its entirety, teach additional exemplary lipids, formulations, methods, and characterizations of LNPs. Additional exemplary lipids, formulations, methods, and characterizations 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, e.g., Hou et al. Table 1, which lists exemplary lipids and lipid derivatives). Figure 2 Exemplary lipids and lipid derivatives of Hou et al.).

[0553] In some embodiments, in vitro or ex vivo cell lipofection is performed using Lipofectamine MessengerMax (ThermoFisher) or TransIT-mRNA transfection reagent (Mirus Bio). In certain embodiments, LNP is formulated using GenVoy ILM ionizable lipid mixture (Precision NanoSystems). In certain embodiments, LNP is formulated using 2,2-dilinoleyl-4-dimethylaminooctyl-[1,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which is taught in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), which is incorporated by reference in its entirety.

[0554] LNP formulations optimized for delivery of CRISPR-Cas systems (e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA) are described in WO 2019067992 and WO 2019067910, both of which are incorporated by reference, and can be used to deliver circular and linear polyribonucleotides described herein.

[0555] Additional specific LNP formulations that can be used to deliver nucleic acids (e.g., circular and linear polyribonucleotides) are described in US 8158601 and US 8168775, both of which are incorporated by reference, including the formulation sold under the name ONPATTRO used in patisiran.

[0556] Exemplary dosing of polyribonucleotides (e.g., circular and linear polyribonucleotides) LNP can include about 0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, or 100 mg / kg (RNA). Exemplary dosing of AAV comprising polyribonucleotides (e.g., circular and linear polyribonucleotides) can include about 10 11 , 10 12 , 10 13 , and 10 14 vg / kg MOI.

[0557] II. Methods of use

[0558] In some aspects, provided herein are methods (e.g., methods of inducing a cellular effector function, methods of modulating a state of a cell, and / or methods of treating a disease) comprising a pathway into a cell for any of the following described herein: a macromolecule, a pair of macromolecules, or a macromolecular complex; a nucleic acid or a pair of nucleic acids; or a multimer.

[0559] In some aspects, provided herein are uses of any of the following described herein in the manufacture of a medicament for use in entering a cell (e.g., a medicament for use in a method of inducing a cellular effector function, modulating a state of a cell, and / or treating a disease): a macromolecule, a pair of macromolecules, or a macromolecular complex; a nucleic acid or a pair of nucleic acids; or a multimer.

[0560] In some aspects, provided herein are any of the following described herein: a macromolecule, a pair of macromolecules, or a macromolecular complex; a nucleic acid or a pair of nucleic acids; or a multimer for use in entering a cell (e.g., a medicament for use in a method of inducing a cellular effector function, modulating a state of a cell, and / or treating a disease).

[0561] A. Methods of inducing a cellular effector function

[0562] 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 macromolecular complex comprising two macromolecules (e.g., a macromolecular complex as described in Section IA(i)), each macromolecule 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 the induction of the effector function by the macromolecular complex is conditional on the binding of each copy of the macromolecule to the disease signature ligand.

[0563] 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 macromolecular complex comprising a pair of macromolecules (e.g., a macromolecular complex as described in Section IA(ii)), each macromolecule 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) a 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) a 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 one another; and wherein the induction of the effector function by the macromolecular complex is contingent upon each of the pair of macromolecules binding the disease signature ligand.

[0564] 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 comprising two FBDs linked to two SBDs (e.g., a macromolecular complex as described in Section IA(iii)), 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 conformation that allows the two SBDs to bind to the effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is contingent upon each of the two FBDs binding the disease signature ligand.

[0565] 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 comprising two FBDs linked to a SBD1 and a SBD2 (e.g., a macromolecular complex as described in Section IA(iv)), 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 conformation that allows the SBD1 and the SBD2 to bind to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is contingent on the binding of the disease signature ligand by each of the two FBDs.

[0566] In some aspects, provided herein is use of any of the following described herein: a macromolecule, a pair of macromolecules, or a macromolecular complex; a nucleic acid, or a pair of nucleic acids; or a multimer, in the manufacture of a medicament for inducing a cellular effector function in a cell.

[0567] In some aspects, provided herein is any of the following described herein: a macromolecule, a pair of macromolecules, or a macromolecular complex; a nucleic acid, or a pair of nucleic acids; or a multimer, for use in inducing a cellular effector function in a cell.

[0568] In some embodiments of any of the above aspects, the cell is in a subject, and the macromolecule, the macromolecular complex, or the nucleic acid is administered in a therapeutically effective amount.

[0569] In some embodiments, the subject has or is suspected of having a disease or disorder characterized by an abnormal level of the disease signature target, optionally wherein the subject was previously determined to have an abnormal level of the disease signature target.

[0570] In some embodiments, the cell is a T cell. In some embodiments, the effector ligand is 4-1BB, 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.

[0571] B. Methods of modulating the state of a cell

[0572] In another aspect, provided herein is a method of modulating a state of a cell, the method comprising providing access to the cell (e.g., wherein the one or more disease signature ligand and the one or more effector ligand bound by the macromolecule or the macromolecular complex are present on or near the cell) to any of the macromolecules or macromolecular complexes or nucleic acids or nucleic acid pairs described herein, thereby modulating the state of the cell.

[0573] In some aspects, provided herein is use of any of the following described herein: a macromolecule, a macromolecule pair, or a macromolecular complex; a nucleic acid or nucleic acid pair; or a multimer, in the manufacture of a medicament for modulating a state of a cell.

[0574] In some aspects, provided herein is any of the following described herein: a macromolecule, a macromolecule pair, or a macromolecular complex; a nucleic acid or nucleic acid pair; or a multimer, for use in modulating a state of a cell.

[0575] In some embodiments of any of the above aspects, the cell is in a subject, and the macromolecule, the macromolecular complex, or the nucleic acid is administered in a therapeutically effective amount.

[0576] In some embodiments, the subject has or is suspected of having a disease or disorder characterized by an abnormal level of the disease signature target, optionally wherein the subject was previously determined to have an abnormal level of the disease signature target.

[0577] C. Methods of determining a state of a cell

[0578] In another aspect, provided herein is a method of determining a state of a cell, the method comprising providing access to the cell (e.g., wherein the one or more disease signature ligand and the one or more effector ligand bound by the macromolecule or the macromolecular complex are present on or near the cell) to any of the macromolecules or macromolecular complexes comprising a reporter domain or nucleic acids or nucleic acid pairs encoding these macromolecules or macromolecular complexes described herein, and detecting the presence of the reporter domain, thereby determining the state of the cell.

[0579] In some aspects, provided herein is use of any of the following described herein: a macromolecule, a macromolecule pair, or a macromolecular complex; a nucleic acid or nucleic acid pair; or a multimer, in the manufacture of a medicament for determining a state of a cell.

[0580] In some aspects, provided herein is any of the following described herein: a macromolecule, a macromolecule pair, or a macromolecular complex; a nucleic acid or nucleic acid pair; or a multimer, for use in determining a state of a cell.

[0581] In some embodiments of any of the above aspects, the cell is in a subject, and the macromolecule, the macromolecular complex, or the nucleic acid is administered in a therapeutically effective amount.

[0582] In some embodiments, the subject has a disease or disorder characterized by an abnormal level of the disease characteristic target.

[0583] III. Dimeric macromolecular assembly

[0584] A. Compositions

[0585] In one aspect, provided herein is a composition comprising a macromolecular complex (e.g., a macromolecular complex described herein), wherein the macromolecular complex comprises a dimer, and wherein at least about 90% of the dimers are monodisperse.

[0586] In some embodiments, at least about 92% of the dimers are monodisperse. In some embodiments, at least about 93% of the dimers are monodisperse. In some embodiments, at least about 94% of the dimers are monodisperse. In some embodiments, at least about 95% of the dimers are monodisperse. In some embodiments, at least about 96% of the dimers are monodisperse. In some embodiments, at least about 97% of the dimers are monodisperse. In some embodiments, at least about 98% of the dimers are monodisperse. In some embodiments, at least about 99% of the dimers are monodisperse.

[0587] In another aspect, provided herein is a composition comprising a macromolecular complex (e.g., a macromolecular complex described herein), wherein the macromolecular complex comprises two macromolecules capable of dimerization, and wherein the composition is at least about 90% monodisperse at a size consistent with a dimerized macromolecular complex.

[0588] 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 a dimerized macromolecular complex.

[0589] The composition can be, for example, a composition produced from one or more cell lines that produce one or more components of the macromolecular 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(s) can be transiently or stably transfected with a plasmid encoding a secreted form of the one or more macromolecules. The composition can be purified prior to assessing monodispersity. Purification can include, for example, clarifying the culture supernatant of the one or more cell lines (e.g., a two-step purification protocol via use of protein A affinity chromatography followed by fine purification using cation exchange chromatography).

[0590] 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.

[0591] In some embodiments, the macromolecular complex has a size consistent with a size of a dimer, e.g., as described in the examples provided herein.

[0592] In some embodiments, the macromolecular complex comprises two macromolecules, each macromolecule comprising a VEGF binding domain linked to a 4-1BB binding domain.

[0593] In some embodiments, the VEGF binding domain is an anti-VEGF scFv.

[0594] In some embodiments, the VEGF binding domain is a VEGF receptor trap.

[0595] In some embodiments, the 4-1BB binding domain is an anti-4-1BB scFv.

[0596] In some embodiments, the macromolecular complex comprises a 4-1BB X VEGF-trap.

[0597] In some embodiments, the macromolecular complex comprises a 4-1BB X VEGF-scFV.

[0598] In some embodiments, the macromolecular complex comprises two macromolecules, each macromolecule comprising a TGFb binding domain linked to a TpoR binding domain.

[0599] In some embodiments, the TGFb binding domain is an anti-TGFb scFv.

[0600] In some embodiments, the TpoR binding domain is an anti-TpoR scFv.

[0601] In some embodiments, the macromolecular complex comprises two macromolecules, each macromolecule comprising an IL-8 binding domain linked to a TpoR binding domain.

[0602] In some embodiments, the IL-8 binding domain is an anti-IL8 scFv.

[0603] In some embodiments, the TpoR binding domain is an anti-TpoR scFv.

[0604] In some embodiments, the macromolecular complex comprises two macromolecules, each macromolecule comprising a TGFb binding domain linked to an IL2R binding domain.

[0605] In some embodiments, the TGFb binding domain is an anti-TGFb scFv.

[0606] 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.

[0607] In some embodiments, the macromolecular complex comprises two macromolecules, each macromolecule comprising an IFNg binding domain linked to an IL10R binding domain.

[0608] In some embodiments, the IFNg binding domain is an anti-IFNg scFv.

[0609] In some embodiments, the IL10R binding domain comprises an anti-IL10R VHH.

[0610] In some embodiments, the IL10R binding domain comprises IL10.

[0611] In some embodiments, the macromolecular complex comprises two macromolecules, each macromolecule comprising an IL6 binding domain linked to an IL10R binding domain.

[0612] In some embodiments, the IL6 binding domain is an anti-IL6 VHH.

[0613] In some embodiments, the IL10R binding domain comprises an anti-IL10R VHH.

[0614] In some embodiments, the IL10R binding domain comprises IL10.

[0615] In some embodiments, the macromolecular complex comprises two macromolecules, each macromolecule comprising an IL8 binding domain linked to a CD3 binding domain.

[0616] In some embodiments, the IL8 binding domain is an anti-IL8 scFv.

[0617] In some embodiments, the CD3 binding domain comprises an anti-CD3 VH.

[0618] B. Methods

[0619] In one aspect, the disclosure provides a method for preparing a pharmaceutical composition, the method comprising:

[0620] (a) preparing or obtaining a composition (e.g., a sample thereof) comprising a macromolecular complex (e.g., a macromolecular complex described herein);

[0621] (b) measuring or having measured the percentage of monodisperse macromolecules in the composition (or sample thereof); and

[0622] (c) formulating or having formulated the composition as a pharmaceutical composition if the percentage of monodisperse macromolecules in the composition (or sample thereof) in the sample is equal to or greater than a predetermined threshold.

[0623] In another aspect, the disclosure provides a method for preparing a pharmaceutical composition, the method comprising:

[0624] (a) preparing or obtaining a composition (e.g., a sample thereof) comprising a macromolecular complex (e.g., a macromolecular complex described herein);

[0625] (b) measuring or having measured the percentage of monodisperse macromolecules in the composition (or sample thereof); and

[0626] (c) formulating or having formulated the composition as a pharmaceutical composition if the percentage of monodisperse macromolecules in the composition (or sample thereof) in the sample is equal to or greater than a predetermined threshold.

[0627] 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.

[0628] In some embodiments, the macromolecule is a dimer.

[0629] In some embodiments, the predetermined threshold is at least about 90% of the dimers are monodisperse. In some embodiments, the predetermined threshold is at least about 92% of the dimers are monodisperse. In some embodiments, the predetermined threshold is at least about 93% of the dimers are monodisperse. In some embodiments, the predetermined threshold is at least about 94% of the dimers are monodisperse. In some embodiments, the predetermined threshold is at least about 95% of the dimers are monodisperse. In some embodiments, the predetermined threshold is at least about 96% of the dimers are monodisperse. In some embodiments, the predetermined threshold is at least about 97% of the dimers are monodisperse. In some embodiments, the predetermined threshold is at least about 98% of the dimers are monodisperse. In some embodiments, the predetermined threshold is at least about 99% of the dimers are monodisperse.

[0630] In some embodiments, the predetermined 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 at the size consistent with the dimerized macromolecular complex (e.g., at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% monodispersity).

[0631] In some embodiments, the percentage monodispersity is determined by size exclusion chromatography (SEC), e.g., as described in the examples provided herein. In some embodiments, the percentage monodispersity is determined by analytical SEC. In some embodiments, the percentage monodispersity is determined by analytical SEC using a Superdex® 200 column.

[0632] In some embodiments, the size of the macromolecular complex is consistent with the size of the dimer, e.g., as described in the examples provided herein.

[0633] In some embodiments, the macromolecular complex comprises two macromolecules, each macromolecule comprising a VEGF binding domain linked to a 4-1BB binding domain.

[0634] In some embodiments, the VEGF binding domain is an anti-VEGF scFv.

[0635] In some embodiments, the VEGF binding domain is a VEGF receptor trap.

[0636] In some embodiments, the 4-1BB binding domain is an anti-4-1BB scFv.

[0637] In some embodiments, the macromolecular complex comprises a 4-1BB X VEGF-trap.

[0638] In some embodiments, the macromolecular complex comprises 4-1BB X VEGF-scFV.

[0639] In some embodiments, the macromolecular complex comprises two macromolecules, each macromolecule comprising a TGFb binding domain linked to a TpoR binding domain.

[0640] In some embodiments, the TGFb binding domain is an anti-TGFb scFv.

[0641] In some embodiments, the TpoR binding domain is an anti-TpoR scFv.

[0642] In some embodiments, the macromolecular complex comprises two macromolecules, each macromolecule comprising an IL-8 binding domain linked to a TpoR binding domain.

[0643] In some embodiments, the IL-8 binding domain is an anti-IL8 scFv.

[0644] In some embodiments, the TpoR binding domain is an anti-TpoR scFv.

[0645] In some embodiments, the macromolecular complex comprises two macromolecules, each macromolecule comprising a TGFb binding domain linked to an IL2R binding domain.

[0646] In some embodiments, the TGFb binding domain is an anti-TGFb scFv.

[0647] 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.

[0648] In some embodiments, the macromolecular complex comprises two macromolecules, each macromolecule comprising an IFNg binding domain linked to an IL10R binding domain.

[0649] In some embodiments, the IFNg binding domain is an anti-IFNg scFv.

[0650] In some embodiments, the IL10R binding domain comprises an anti-IL10R VHH.

[0651] In some embodiments, the IL10R binding domain comprises IL10.

[0652] In some embodiments, the macromolecular complex comprises two macromolecules, each macromolecule comprising an IL6 binding domain linked to an IL10R binding domain.

[0653] In some embodiments, the IL6 binding domain is an anti-IL6 VHH.

[0654] In some embodiments, the IL10R binding domain comprises an anti-IL10R VHH.

[0655] In some embodiments, the IL10R binding domain comprises IL10.

[0656] In some embodiments, the macromolecular complex comprises two macromolecules, each macromolecule comprising an IL8 binding domain linked to a CD3 binding domain.

[0657] In some embodiments, the IL8 binding domain is an anti-IL8 scFv.

[0658] In some embodiments, the CD3 binding domain comprises an anti-CD3 VH.

[0659] IV. Other Embodiments

[0660] Some embodiments of the technology described herein can be defined according to any of the following numbered embodiments:

[0661] 1. A macromolecular 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:

[0662] (a) the FBD specifically binds a disease signature ligand in a biological sample; and

[0663] (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand;

[0664] wherein the two macromolecules are conjugated to each other;

[0665] and wherein the induction of the effector function by the macromolecular complex is conditional on each of the two macromolecules binding the disease signature ligand.

[0666] 2. A macromolecular 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:

[0667] (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;

[0668] (b) the second member of the pair of macromolecules comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of the disease signature ligand in the biological sample; and

[0669] (c) the SBD specifically binds to the effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand;

[0670] wherein the two macromolecules are conjugated to each other;

[0671] and wherein the induction of the effector function by the macromolecular complex is conditional on each of the two macromolecules binding the disease signature ligand.

[0672] 3. The macromolecular complex of either of embodiments 1 or 2, wherein the conjugation is covalent.

[0673] 4. The macromolecular complex of embodiment 3, wherein the covalent conjugation comprises a chemical linker or a polypeptide linker.

[0674] 5. The macromolecular complex of either of embodiments 1 or 2, wherein the conjugation is non-covalent.

[0675] 6. The macromolecular complex of embodiment 5, wherein the non-covalent conjugation is mediated by a complementary moiety pair, each macromolecule comprising one member of the pair.

[0676] 7. The macromolecular complex of embodiment 6, wherein the complementary moiety pair is biotin and avidin; Bacillus amyloliquefaciens ribonuclease and Bacillus amyloliquefaciens ribonuclease inhibitor; a complementary aptamer pair; or a complementary polypeptide pair.

[0677] 8. The macromolecular complex of embodiment 7, wherein the complementary polypeptide pair is an engineered Fc fragment pair.

[0678] 9. The macromolecular complex of embodiment 8, wherein the engineered Fc fragment pair is a knob-in-hole pair.

[0679] 10. The macromolecular complex of any one of embodiments 4 and 6-9, wherein the conjugation sterically orients the two macromolecules to allow for conditional simultaneous binding of disease signal and effector ligands.

[0680] 11. The macromolecular complex of embodiment 5, wherein the non-covalent conjugation is mediated by the disease signature ligand.

[0681] 12. The macromolecular complex of any one of embodiments 1, 3-5, 10 and 11, wherein the two macromolecules are identical.

[0682] 13. A macromolecular complex comprising a pair of macromolecules, wherein each macromolecule independently comprises a first binding domain (FBD) connected to a second binding domain (SBD) by a linker domain, wherein:

[0683] (a) the FBD of each macromolecule specifically binds a disease signature ligand in a biological sample;

[0684] (b) a 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

[0685] (c) a 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;

[0686] wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand;

[0687] wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to each other;

[0688] and wherein the induction of the effector function by the macromolecular complex is conditional on each of the pair of macromolecules binding the disease signature ligand.

[0689] 14. A macromolecular complex comprising a pair of macromolecules, wherein each macromolecule independently comprises a first binding domain (FBD) connected to a second binding domain (SBD) by a linker domain, wherein:

[0690] (a) a 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;

[0691] (b) a 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;

[0692] (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

[0693] (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;

[0694] wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand;

[0695] wherein the first member of the pair of macromolecules and the second member of the pair of macromolecules are conjugated to one another;

[0696] and wherein induction of the effector function by the pair of macromolecules is conditional upon each of the pair of macromolecules binding the disease signature ligand.

[0697] 15. The pair of macromolecules of either of embodiments 13 or 14, wherein the conjugation is covalent.

[0698] 16. The pair of macromolecules of embodiment 15, wherein the covalent conjugation comprises a chemical linker or a polypeptide linker.

[0699] 17. The pair of macromolecules of either of embodiments 13 or 14, wherein the conjugation is non-covalent.

[0700] 18. The pair of macromolecules of embodiment 17, wherein the non-covalent conjugation is mediated by a complementary pair of moieties, each macromolecule comprising one member of the pair.

[0701] 19. The pair of macromolecules of embodiment 18, wherein the complementary pair of moieties is biotin and avidin; Bacillus amyloliquefaciens ribonuclease and Bacillus amyloliquefaciens ribonuclease inhibitor; a complementary pair of aptamers; or a complementary pair of polypeptides.

[0702] 20. The pair of macromolecules of embodiment 19, wherein the complementary pair of polypeptides is a pair of engineered Fc fragments.

[0703] 21. The pair of macromolecules of embodiment 20, wherein the pair of engineered Fc fragments is a pair of knuckle-and-socket structures.

[0704] 22. The pair of macromolecules 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 simultaneous binding of disease signal and effector ligands.

[0705] 23. The pair of macromolecules of embodiment 18, wherein the non-covalent conjugation is mediated by the disease signature ligand.

[0706] 24. A pair of macromolecules, comprising two macromolecules, wherein each macromolecule comprises a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein:

[0707] (a) the FBD specifically binds a disease signature ligand in a biological sample; and

[0708] (b) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand.

[0709] wherein the two macromolecules are not conjugated to each other in the absence of the disease characteristic ligand;

[0710] and wherein the induction of the effector function by the macromolecular complex is conditional on each of the two macromolecules binding the disease characteristic ligand.

[0711] 25. A macromolecular 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:

[0712] (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 characteristic ligand in a biological sample;

[0713] (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 characteristic ligand in the biological sample; and

[0714] (c) the SBD specifically binds an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand;

[0715] wherein the two macromolecules are not conjugated to each other in the absence of the disease characteristic ligand;

[0716] and wherein the induction of the effector function by the macromolecular complex is conditional on each of the two macromolecules binding the disease characteristic ligand.

[0717] 26. The macromolecular complex of either of embodiments 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 characteristic ligand, and wherein the non-covalent conjugation is mediated by the disease characteristic ligand.

[0718] 27. A macromolecule comprising two FBDs linked to two SBDs by one or more linker domains, wherein:

[0719] (a) the FBDs specifically bind a disease characteristic ligand in a biological sample; and

[0720] (b) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand;

[0721] wherein the macromolecule is capable of adopting a conformation that allows the two SBDs to bind to the effector ligand in the presence of the disease characteristic ligand, and wherein induction of the effector function by the macromolecule is conditional on each of the two FBDs binding to the disease characteristic ligand.

[0722] 28. A macromolecule comprising FBD1 and FBD2 linked to two SBDs by one or more linker domains, wherein:

[0723] (a) the FBD1 specifically binds a first epitope of a disease characteristic ligand in a biological sample;

[0724] (b) the FBD2 specifically binds a second epitope of the disease characteristic ligand in the biological sample; and

[0725] (c) the SBDs specifically bind an effector ligand in the biological sample and induce a cellular effector function upon binding to the effector ligand;

[0726] wherein the macromolecule is capable of adopting a conformation that allows the two SBDs to bind to the effector ligand in the presence of the disease characteristic ligand, and wherein induction of the effector function by the macromolecule is conditional on each of the FBD1 and FBD2 binding to the disease characteristic ligand.

[0727] 29. A macromolecule comprising two FBDs linked to SBD1 and SBD2 by one or more linker domains, wherein:

[0728] (a) the FBDs specifically bind a disease characteristic ligand in a biological sample;

[0729] (b) the SBD1 specifically binds a first effector ligand in the biological sample; and

[0730] (c) the SBD2 specifically binds a second effector ligand in the biological sample;

[0731] wherein the SBD1 and SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand;

[0732] wherein the macromolecule is capable of adopting a conformation that allows the SBD1 and SBD2 to bind to the first effector ligand and the second effector ligand in the presence of the disease characteristic ligand, and wherein induction of the effector function by the macromolecule is conditional on each of the two FBDs binding to the disease characteristic ligand.

[0733] 30. A macromolecule comprising FBD1 and FBD2 linked to SBD1 and SBD2 by one or more linker domains, wherein:

[0734] (a) the FBD1 specifically binds a first epitope of a disease characteristic ligand in a biological sample;

[0735] (b) the FBD2 specifically binds a second epitope of the disease characteristic ligand in the biological sample;

[0736] (c) the SBD1 specifically binds a first effector ligand in the biological sample; and

[0737] (d) the SBD2 specifically binds a second effector ligand in the biological sample;

[0738] wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand;

[0739] wherein the macromolecule is capable of adopting a conformation that allows the SBD1 and the SBD2 to bind to the first effector ligand and the second effector ligand in the presence of the disease characteristic ligand, and wherein the induction of the effector function by the macromolecule is conditional on the binding of each of the FBD1 and the FBD2 to the disease characteristic ligand.

[0740] 31. The macromolecular complex or macromolecule of any one of embodiments 1-30, wherein the disease characteristic ligand is a protein, a peptide, or a small molecule.

[0741] 32. The macromolecular complex or macromolecule of embodiment 31, wherein the protein is a soluble protein or an insoluble protein.

[0742] 33. The macromolecular complex or macromolecule of any one of embodiments 1-30, wherein the disease characteristic ligand is a cytokine.

[0743] 34. The macromolecular complex or macromolecule of embodiment 33, wherein the cytokine is an interleukin, an interferon, a growth factor, a chemokine, or a TNF family member.

[0744] 35. The macromolecular 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-γ; the growth factor is transforming growth factor beta (TGF-β), granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage 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 TNF family member is TNF-α.

[0745] 36. A macromolecular complex or macromolecule as described in any one of Examples 1-30, wherein the disease-characteristic ligand is a neurotransmitter.

[0746] 37. A macromolecular complex or macromolecule as described in any one of Examples 1-30, wherein the disease-characteristic ligand is a cell surface receptor, surface antigen, membrane-bound protein, extracellular matrix component, or integrin.

[0747] 38. A macromolecular complex or macromolecule as described in any one of Examples 1-30, wherein the disease-characteristic ligand is an autoantigen of the organism from which the biological sample is derived.

[0748] 39. A macromolecular complex or macromolecule as described in Example 38, wherein the self-antigen is an anti-drug antibody (ADA), an autoantibody, or a tumor marker.

[0749] 40. A macromolecular complex or macromolecule as described in any one of Examples 1-30, wherein the disease-characteristic ligand is a nucleic acid.

[0750] 41. A macromolecular complex or macromolecule as described in any one of Examples 1-30, wherein the disease-characteristic ligand is a carbohydrate, lipid, peptide, nucleoside, or a combination thereof.

[0751] 42. A macromolecular complex or macromolecule as described in any one of Examples 1-30, wherein the disease-characteristic ligand is a hormone, an amino acid derivative, a steroid, or an eicosanoic acid.

[0752] 43. A macromolecular complex or macromolecule as described in any one of Examples 1-30, wherein the disease-characteristic ligand is a non-self antigen.

[0753] 44. A macromolecular complex or macromolecule as described in Example 43, wherein the disease-characteristic ligand is a virus, bacteria, or a fragment or antigen thereof.

[0754] 45. A macromolecular complex or macromolecule as described in any one of Examples 1-44, wherein the disease-characteristic ligand is a polymer.

[0755] 46. ​​A macromolecular complex or macromolecule as described in Example 45, wherein the disease-characteristic ligand is a dimer, trimer, or tetramer.

[0756] 47. A macromolecular complex or macromolecule as described in any one of Examples 1-46, wherein the FBD, the FBD1 or the FBD2 comprises a polypeptide that specifically binds to the disease-characteristic ligand.

[0757] 48. A macromolecular complex or macromolecule as described in Example 47, wherein the polypeptide is an antibody or a fragment thereof.

[0758] 49. The macromolecular 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 retargeting antibody (DART).

[0759] 50. The macromolecular complex or macromolecule of embodiment 47, wherein the polypeptide is an antibody mimetic.

[0760] 51. The macromolecular complex or macromolecule of embodiment 50, wherein the antibody mimetic is an affimer, an affilin, an affimer, an affitin, an alphabody, an anticaline, 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 nanobody, a nanoCLAMP, an optimer, a repebody, a pre- connectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR).

[0761] 52. The macromolecular complex or macromolecule of embodiment 47, wherein the polypeptide is an endogenous binding domain.

[0762] 53. The macromolecular 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.

[0763] 54. The macromolecular complex or macromolecule of any one of embodiments 1-46, wherein the FBD, the FBD1, or the FBD2 comprises an oligonucleotide that specifically binds the disease signature ligand.

[0764] 55. The macromolecular complex or macromolecule of embodiment 54, wherein the oligonucleotide is a nucleic acid aptamer.

[0765] 56. The macromolecular complex or macromolecule of embodiment 55, wherein the nucleic acid aptamer is a DNA aptamer.

[0766] 57. The macromolecular complex or macromolecule of any one of embodiments 1-46, wherein the FBD, the FBD1, or the FBD2 comprises a chemical molecule that specifically binds the disease signature ligand.

[0767] 58. The macromolecular complex or macromolecule of any one of embodiments 1-57, wherein the FBD, the FBD1, or the FBD2 has affinity for two or more disease signature moieties.

[0768] 59. The macromolecular complex or macromolecule of any one of embodiments 1-58, wherein the effector ligand is a protein or a peptide.

[0769] 60. The macromolecular complex or macromolecule of any one of embodiments 1-59, wherein the effector ligand is a cell surface receptor or an intracellular receptor.

[0770] 61. The macromolecular complex or macromolecule of embodiment 60, wherein the cell surface receptor is a catalytic receptor or the intracellular receptor is a nuclear hormone receptor.

[0771] 62. The macromolecular 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.

[0772] 63. The macromolecular 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 IL10R, or the TNF superfamily receptor is TNFR2 or 4-1BB.

[0773] 64. The macromolecular 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.

[0774] 65. The macromolecular complex or macromolecule of embodiment 64, wherein the effector ligand must be homodimerized to exert a cellular effector function.

[0775] 66. The macromolecular complex or macromolecule of embodiment 65, wherein the effector ligand is homodimerized in the presence of the macromolecular complex or the macromolecule and the disease signature ligand.

[0776] 67. The macromolecular complex or macromolecule of any one of embodiments 13-23 and 29-63, wherein the first effector ligand and the second effector ligand must associate to exert a cellular effector function.

[0777] 68. The macromolecular complex or macromolecule of embodiment 67, wherein the first effector ligand and the second effector ligand associate in the presence of the macromolecular complex or the macromolecule and the disease signature ligand.

[0778] 69. The macromolecular complex or macromolecule of either of embodiments 67 or 68, wherein the association is heterodimerization.

[0779] 70. The macromolecular complex or macromolecule of any one of embodiments 1-69, wherein the cellular effector function is a biological activity.

[0780] 71. The macromolecular complex or macromolecule of any one of embodiments 1-70, wherein the cellular effector function is a therapeutic activity.

[0781] 72. The macromolecular complex or macromolecule of any one of embodiments 1-70, wherein the cellular effector function is a disease activity.

[0782] 73. The macromolecular complex or macromolecule of any one of embodiments 1-72, wherein the SBD, the SBD1, or the SBD2 comprises a polypeptide that specifically binds the effector ligand.

[0783] 74. The macromolecular complex or macromolecule of embodiment 73, wherein the polypeptide is an antibody or fragment thereof.

[0784] 75. The macromolecular 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.

[0785] 76. The macromolecular complex or macromolecule of embodiment 73, wherein the polypeptide is an antibody mimetic.

[0786] 77. The macromolecular 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 nanobody, a nanoCLAMP, an optimer, a repebody, a pre- connectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a VLR.

[0787] 78. The macromolecular complex or macromolecule of embodiment 73, wherein the polypeptide is an endogenous binding domain.

[0788] 79. The macromolecular complex or macromolecule of embodiment 78, wherein the endogenous binding domain is a ligand or fragment thereof of the effector ligand.

[0789] 80. The macromolecular complex or macromolecule of embodiment 78, wherein the endogenous binding domain is a virus binding protein or fragment thereof.

[0790] 81. The macromolecular complex or macromolecule of any one of embodiments 1-72, wherein the SBD, the SBD1, or the SBD2 comprises an oligonucleotide that specifically binds the effector ligand.

[0791] 82. The macromolecular complex or macromolecule of embodiment 81, wherein the oligonucleotide is a nucleic acid aptamer.

[0792] 83. The macromolecular complex or macromolecule of embodiment 82, wherein the nucleic acid aptamer is a DNA aptamer.

[0793] 84. The macromolecular complex or macromolecule of any one of embodiments 1-72, wherein the FBD, the FBD1, or the FBD2 comprises a chemical molecule that specifically binds the disease signature ligand.

[0794] 85. The macromolecular 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.

[0795] 86. The macromolecular 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.

[0796] 87. The macromolecular 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 the dimeric moiety.

[0797] 88. The macromolecular 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.

[0798] 89. The macromolecular complex of embodiment 88, wherein the polypeptide chain is a hormone, a cytokine, or a growth factor.

[0799] 90. The macromolecular 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 each other.

[0800] 91. The macromolecular complex or macromolecule of any one of embodiments 1-90, wherein the macromolecule comprises a reporter moiety.

[0801] 92. The macromolecular complex or macromolecule of embodiment 91, wherein the reporter moiety is an affinity tag, a fluorescent label, a radiolabel, or a chromogenic label.

[0802] 93. The macromolecular complex or macromolecule of embodiment 92, wherein the affinity tag is a FLAG affinity tag, or the chromogenic label is luciferase or beta-lactamase.

[0803] 94. The macromolecular complex or macromolecule of any one of embodiments 1-93, further comprising one or more linker domains.

[0804] 95. The macromolecular complex or macromolecule of embodiment 94, wherein the one or more linker domains are peptide linkers.

[0805] 96. The macromolecular complex or macromolecule of embodiment 95, wherein the peptide linkers comprise one or more GS linkers.

[0806] 97. The macromolecular complex or macromolecule of embodiment 96, wherein the GS linkers comprise one or more GS(G n S) m linkers.

[0807] 98. The macromolecular complex or macromolecule of embodiment 96, wherein the GS linkers comprise one or more (G n S) m linkers.

[0808] 99. The macromolecular complex or macromolecule of any one of embodiments 1-53, 58-80, and 85-98, wherein the macromolecule is a polypeptide.

[0809] 100. The macromolecular complex or macromolecule of any one of embodiments 1-99, wherein the biological sample is an extract, a fluid, a fraction, a cell, a tissue, or a subject.

[0810] 101. The macromolecular complex or macromolecule of any one of embodiments 1-100, wherein one or both members of the macromolecule or the pair of macromolecules comprises a leader sequence.

[0811] 102. The macromolecular complex or macromolecule of embodiment 101, wherein the leader sequence comprises a secretion signal.

[0812] 103. The macromolecular complex or macromolecule of any one of embodiments 1-102, wherein one or both members of the macromolecule or the pair of macromolecules comprises a half-life extending moiety.

[0813] 104. The macromolecular complex or macromolecule of embodiment 103, wherein the half-life extending moiety is an Fc domain or fragment thereof, an albumin domain or fragment thereof, or a polyethylene glycol (PEG) or modified derivative thereof.

[0814] 105. A nucleic acid encoding the macromolecule of any one of embodiments 1, 3-12, and 24-104.

[0815] 106. A nucleic acid pair encoding the macromolecule pair of any one of embodiments 2 and 13-23.

[0816] 107. The nucleic acid or nucleic acid pair of embodiments 105 or 106, wherein the nucleic acid is RNA or DNA.

[0817] 108. The nucleic acid or nucleic acid pair of any one of embodiments 105-107, wherein the nucleic acid is formulated with a delivery platform.

[0818] 109. The nucleic acid or nucleic acid pair of embodiment 108, wherein the delivery platform is a lipid-based carrier or vector delivery system.

[0819] 110. The nucleic acid or nucleic acid pair of embodiment 109, wherein the lipid-based carrier is a lipid nanoparticle (LNP).

[0820] 111. The nucleic acid or nucleic acid pair of embodiment 109, wherein the vector delivery system comprises or derives from an adenovirus, an anellovirus, an AAV, or a lentivirus.

[0821] 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.

[0822] 113. A nucleic acid pair encoding the macromolecule pair of any one of embodiments 2 and 13-23, wherein the nucleic acid pair is formulated with a carrier.

[0823] 114. The nucleic acid or nucleic acid pair of embodiments 112 or 113, wherein the nucleic acid is RNA or DNA.

[0824] 115. The nucleic acid or nucleic acid pair of embodiments 112 or 113, wherein the carrier is a lipid-based carrier.

[0825] 116. The nucleic acid or nucleic acid pair of embodiment 115, wherein the lipid-based carrier is an LNP.

[0826] 117. A vector comprising the nucleic acid of embodiments 105 or 107.

[0827] 118. A vector or pair of vectors comprising the nucleic acid pair of embodiment 106 or 107.

[0828] 119. The vector or pair of vectors of embodiment 117 or 118, wherein the vector or the pair of vectors is formulated with a carrier.

[0829] 120. A host cell comprising the nucleic acid or nucleic acid pair of any one of embodiments 105-116 or the vector or pair of vectors of any one of embodiments 117-119.

[0830] 121. The macromolecular complex or macromolecule of any one of embodiments 1-104 or the nucleic acid or nucleic acid pair of any one of embodiments 105-116, wherein the macromolecular complex, the macromolecule, the nucleic acid, or the nucleic acid pair is more than 95% pure.

[0831] 122. The macromolecular complex or macromolecule of any one of embodiments 1-104 or the nucleic acid or nucleic acid pair of any one of embodiments 105-116, wherein the macromolecular complex, the macromolecule, the nucleic acid, or the nucleic acid pair is manufactured according to United States Food and Drug Administration (FDA) Good Manufacturing Practice (GMP), Good Clinical Practice (GCP), and / or Good Laboratory Practice (GLP) standards.

[0832] 123. A method comprising providing access to a cell for the macromolecular complex or macromolecule of any one of embodiments 1-104 or the nucleic acid or nucleic acid pair of any one of embodiments 105-116.

[0833] 124. A method of modulating a state of a cell, the method comprising providing access to the cell for the macromolecular complex or macromolecule of any one of embodiments 1-104 or the nucleic acid or nucleic acid pair of any one of embodiments 105-116, thereby modulating the state of the cell.

[0834] 125. A method of inducing a cellular effector function in a cell, the method comprising providing access to the cell for the macromolecular complex or macromolecule of any one of embodiments 1-104 or the nucleic acid or nucleic acid pair of any one of embodiments 105-116, thereby inducing the cellular effector function in the cell.

[0835] 126. The method of embodiment 124 or 125, wherein the cell is in a subject and the macromolecular complex, the macromolecule, the nucleic acid, or the nucleic acid pair is administered in a therapeutically effective amount.

[0836] 127. The method of embodiment 126, wherein the subject has or is suspected of having a disease or disorder characterized by an abnormal level of the disease-characteristic target, optionally wherein the subject was previously determined to have an abnormal level of the disease-characteristic target.

[0837] 128. A method of determining the state of a cell, the method comprising providing access to the cell for a macromolecular complex or macromolecule of any one of embodiments 91-93 or a nucleic acid or pair of nucleic acids encoding the macromolecular complex or macromolecule, and detecting the presence of the reporter domain, thereby determining the state of the cell.

[0838] 129. A method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecular 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:

[0839] (a) the FBD specifically binds a disease-characteristic ligand in a biological sample; and

[0840] (b) the SBD specifically binds an effector ligand in the biological sample, and induces a cellular effector function upon binding to the effector ligand;

[0841] wherein the two copies of the macromolecule are conjugated to one another;

[0842] and wherein the induction of the effector function by the macromolecular complex is conditional on the binding of the disease-characteristic ligand by each copy of the macromolecule.

[0843] 130. A method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecular 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:

[0844] (a) the FBD of each macromolecule specifically binds a disease-characteristic ligand in a biological sample;

[0845] (b) a 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

[0846] (c) a 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;

[0847] wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand;

[0848] wherein the first member of the macromolecular pair and the second member of the macromolecular pair are conjugated to each other;

[0849] and wherein the induction of the effector function by the macromolecular complex is conditional on the binding of the disease signature ligand by each of the macromolecular pair.

[0850] 131. A method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule comprising two FBDs connected to two SBDs by one or more linker domains, wherein:

[0851] (a) the FBDs specifically bind a disease signature ligand in a biological sample; and

[0852] (b) the SBDs specifically bind an effector ligand in the biological sample, and induce a cellular effector function upon binding to the effector ligand;

[0853] wherein the macromolecule is capable of adopting a conformation that allows the two SBDs to bind to the effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is conditional on the binding of the disease signature ligand by each of the two FBDs.

[0854] 132. A method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule comprising two FBDs connected to SBD1 and SBD2 by one or more linker domains, wherein:

[0855] (a) the FBDs specifically bind a disease signature ligand in a biological sample;

[0856] (b) the SBD1 specifically binds a first effector ligand in the biological sample; and

[0857] (c) the SBD2 specifically binds a second effector ligand in the biological sample;

[0858] wherein the SBD1 and the SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand;

[0859] wherein the macromolecule is capable of adopting a conformation that allows the SBD1 and the SBD2 to bind to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and wherein the induction of the effector function by the macromolecule is conditional on the binding of the disease signature ligand by each of the two FBDs.

[0860] 133. A macromolecular complex comprising two macromolecules, wherein each macromolecule comprises a VEGF binding domain linked to a 4-1BB binding domain by a linker domain, wherein the 4-1BB binding domain induces a cellular effector function upon binding to 4-1BB;

[0861] wherein the two macromolecules are conjugated to one another;

[0862] and wherein the induction of the effector function by the macromolecular complex is contingent upon each of the two macromolecules binding to VEGF.

[0863] 134. The macromolecular complex of embodiment 133, wherein the conjugation is non-covalent.

[0864] 135. The macromolecular complex of embodiment 134, wherein the non-covalent conjugation is mediated by a complementary pair of moieties, each macromolecule comprising one member of the pair.

[0865] 136. The macromolecular complex of embodiment 135, wherein the complementary pair of polypeptides is a Fc fragment pair.

[0866] 137. The macromolecular complex of embodiment 136, wherein the Fc fragment pair is a knob-in-hole pair.

[0867] 138. The macromolecular complex of any one of embodiments 133-136, wherein the two macromolecules are identical.

[0868] 139. A macromolecular complex comprising two macromolecules, wherein each macromolecule comprises a VEGF binding domain linked to a 4-1BB binding domain by a linker domain, wherein the 4-1BB binding domain induces a cellular effector function upon binding to 4-1BB;

[0869] wherein the two macromolecules are not conjugated to one another in the absence of the disease characteristic ligand;

[0870] and wherein the induction of the effector function by the macromolecular complex is contingent upon each of the two macromolecules binding to VEGF.

[0871] 140. The macromolecular complex of embodiment 139, wherein the first member of the macromolecular pair and the second member of the macromolecular pair are non-covalently conjugated to one another in the presence of the disease characteristic ligand, and wherein the non-covalent conjugation is mediated by the disease characteristic ligand.

[0872] 141. The macromolecular complex of embodiment 139 or 140, wherein the macromolecule or one or both members of the macromolecular pair comprises a half-life extending moiety.

[0873] 142. The macromolecular complex of embodiment 103, wherein the half-life extending moiety is an Fc domain or fragment thereof.

[0874] 143. The macromolecular complex of any one of embodiments 133-142, wherein the VEGF binding domain is an anti-VEGF scFv.

[0875] 144. The macromolecular complex of any one of embodiments 133-142, wherein the VEGF binding domain is a VEGF receptor trap.

[0876] 145. The macromolecular complex of any one of embodiments 133-144, wherein the 4-1BB binding domain is an anti-4-1BB scFv.

[0877] 146. A macromolecular complex comprising two macromolecules, wherein each macromolecule comprises an IFNg binding domain linked to an IL-10R binding domain by a linker domain, wherein the IL-10R binding domain induces a cellular effector function upon binding to an IL-10R;

[0878] wherein the two macromolecules are conjugated to one another;

[0879] and wherein the induction of the effector function by the macromolecular complex is contingent on each of the two macromolecules binding to IFNg.

[0880] 147. The macromolecular complex of embodiment 146, wherein the conjugation is non-covalent.

[0881] 148. The macromolecular complex of embodiment 147, wherein the non-covalent conjugation is mediated by a complementary pair of moieties, each macromolecule comprising one member of the pair.

[0882] 149. The macromolecular complex of embodiment 148, wherein the complementary pair of polypeptides is a Fc fragment pair.

[0883] 150. The macromolecular complex of embodiment 149, wherein the Fc fragment pair is a knob-in-hole pair.

[0884] 151. The macromolecular complex of any one of embodiments 146-150, wherein the two macromolecules are identical.

[0885] 152. A macromolecular complex comprising two macromolecules, wherein each macromolecule comprises an IFNg binding domain linked to an IL-10R binding domain by a linker domain, wherein the IL-10R binding domain induces a cellular effector function upon binding to an IL-10R;

[0886] wherein the two macromolecules do not conjugate to each other in the absence of the disease signature ligand;

[0887] and wherein the induction of the effector function by the macromolecular complex is conditional on the binding of IFNg by each of the two macromolecules.

[0888] 153. The macromolecular complex of embodiment 152, wherein the first member of the macromolecular pair and the second member of the macromolecular pair 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.

[0889] 154. The macromolecular complex of embodiment 152 or 153, wherein the macromolecule or one or both members of the macromolecular pair comprises a half-life extending moiety.

[0890] 155. The macromolecular complex of embodiment 154, wherein the half-life extending moiety is an Fc domain or fragment thereof.

[0891] 156. The macromolecular complex of any one of embodiments 146-155, wherein the IFNg binding domain is an anti-IFNg scFv.

[0892] 157. The macromolecular complex of any one of embodiments 146-156, wherein the IL-10R binding domain is IL-10. Examples

[0893] The following are examples of methods of the present application. It is to be understood that various other embodiments can be practiced in light of the general description provided above.

[0894] List of Contents (Examples):

[0895]

[0896] Example 1. Protein complexes that conditionally regulate TPO receptor activity with TGF-β1 or IL-8

[0897] This example describes the expression, purification, and characterization of (i) a protein complex that conditionally regulates the thrombopoietin (TPO) receptor with the presence of transforming growth factor beta 1 (TGF-β1) and (ii) a protein complex that conditionally regulates the TPO receptor with the presence of interleukin-8 (IL-8). The general format of the fusion proteins is shown in Figure 1 .

[0898] A. Fusion Proteins

[0899] TGF-β1-TPO Fusion Proteins

[0900] This example provides a protein complex composed of two identical fusion proteins that conditionally regulate TPO receptor in the presence of TGF-β1 (referred to herein as “TGFb-TPO fusion proteins”). Each fusion protein comprises (from N- to C-terminus): mouse immunoglobulin kappa variable 3 (IgKVIII) leader peptide (UniProt ID A0A140T8P0 positions M1 to G20); anti-TGF-β1 single-chain variable fragment (scFv) (PDB 4KV5; SEQ ID NO: 13 and 14; Table 2); (G4S) n linker, where n = 2, 3, or 5; and 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), which are connected by a G4S linker Figure 5A The mechanism of action of the proposed construct is illustrated in Figure 5B : TGFb recruits two copies of the fusion protein via binding to its TGFb targeting domain, positioning the TpoR effector domain in close proximity to bind to TpoR and induce TpoR activity.

[0901] Optionally, each fusion protein further comprises a FLAG affinity tag after the anti-TPO receptor VH and VL.

[0902] IL-8-TPO fusion proteins

[0903] This example also provides a protein complex composed of two identical fusion proteins that conditionally regulate TPO receptor in the presence of IL-8 (referred to herein as “IL-8-TPO fusion proteins”). Each fusion protein comprises (from N- to C-terminus): mouse immunoglobulin kappa variable 3 (IgKVIII) leader peptide (UniProt ID A0A140T8P0 positions M1 to G20); anti-IL-8 scFv (PDB 6WZM, SEQ ID NO: 12; Table 2); (G4S) n linker, where n = 2, 3, or 5; and 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), which are connected by a G4S linker Figure 6A The mechanism of action of the proposed construct is illustrated in Figure 6BIn brief, IL-8 recruits two copies of the fusion protein via binding to its IL-8 targeting domain, positioning the TpoR effector domain in close proximity to dimerize TpoR and induce TpoR activity.

[0904] Optionally, each fusion protein further comprises a FLAG affinity tag after the anti-TPO receptor VH and VL.

[0905] Expression and purification

[0906] Fusion proteins were expressed under the control of the cytomegalovirus (CMV) promoter via transient transfection of human embryonic kidney 293 (HEK293) cells. Secreted protein was analyzed by Western blot or ELISA using anti-FLAG capture antibodies to assess expression and quality. If needed, fusion proteins were purified using a FLAG capture step followed by a size exclusion chromatography (SEC) fine purification step. By Western blot, expression was expected to produce a strong band of the expected molecular weight with minimal product-related variants.

[0907] B. In vitro assay to assess the effect of fusion proteins on the TPO signaling pathway in the presence or absence of soluble TPO or TGF-β1

[0908] HEK-BLUE cells (InvivoGen catalog code: hkb-blue) were used to evaluate the activity of fusion proteins, unless otherwise indicated. In brief, HEK-BLUE cells were seeded in 96-well plates at 50,000 cells / well in 100 μΐ of RPMI-1640 medium supplemented with 10% FBS and incubated overnight at 37°C, 5% CO2. The next day, 100 μΐ of assay medium (RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin / streptomycin, 2.5 ng / ml recombinant human IL-2, and 50 ng / ml recombinant human IL-4) was added to each well. Test samples were prepared by diluting the fusion protein in assay medium. The diluted test sample was added to the cells, and the plate was incubated at 37°C, 5% CO2 for 24 hours. TM TPO cells (InvivoGen catalog code: hkb-tpo) were used to evaluate the activity of fusion proteins. In brief, HEK-BLUE TPO cells were seeded in 96-well plates at 50,000 cells / well in 100 μΐ of RPMI-1640 medium supplemented with 10% FBS and incubated overnight at 37°C, 5% CO2. The next day, 100 μΐ of assay medium (RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin / streptomycin, 2.5 ng / ml recombinant human IL-2, and 50 ng / ml recombinant human IL-4) was added to each well. Test samples were prepared by diluting the fusion protein in assay medium. The diluted test sample was added to the cells, and the plate was incubated at 37°C, 5% CO2 for 24 hours. TM TPO cells were transiently transfected with fusion protein expression constructs (as described in Example 1A) to secrete the fusion protein as an autocrine in the assay. Alternatively, supernatant from HEK293 cells transiently expressing the fusion protein was applied to HEK-BLUE TPO cells as the test sample. TM TPO cells. FLAG-purified fusion protein material was used to perform confirmation assays. JAK2 / STAT5 pathway activation via secreted alkaline phosphatase (SEAP) production was compared between a positive control (purified recombinant human TPO was applied; HEK293 cells transiently transfected to express and secrete human TPO as an autocrine), a negative control (recombinant human interleukin-2 (IL-2) was applied; mock transfected plasmid), and test samples (fusion protein variants). HEK-BLUE TPO cells expressing fusion proteins were seeded in 96-well plates at 50,000 cells / well in 100 μΐ of RPMI-1640 medium supplemented with 10% FBS and incubated overnight at 37°C, 5% CO2. The next day, 100 μΐ of assay medium (RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin / streptomycin, 2.5 ng / ml recombinant human IL-2, and 50 ng / ml recombinant human IL-4) was added to each well. Test samples were prepared by diluting the fusion protein in assay medium. The diluted test sample was added to the cells, and the plate was incubated at 37°C, 5% CO2 for 24 hours. TMTPO cells were incubated overnight in the presence or absence of TPO and in the presence or absence of TGF-βΙ (for TGFb-TPO fusion proteins) or IL-8 (for IL-8-TPO fusion proteins). Functional fusion protein samples were further evaluated by dose titration of fusion protein samples in the presence of TGF-βΙ or IL-8 (to assess EC50) or in the presence of TGF-βΙ and TPO or IL-8 (to assess IC50). Fusion proteins were expected to activate TPO receptor signaling contingent on the presence of TGF-βΙ (TGFb-TPO fusion proteins) or IL-8 (IL-8-TPO fusion proteins).

[0909] Figure 5C A dose-response relationship between TGFb concentration and activity of TGFb-TPO fusion proteins (comprising anti-TPO receptor VH and VL sequences of SEQ ID NO: 32 and SEQ ID NO: 33, respectively) is shown. TpoR SEAP reporter cells were stimulated overnight with a constant amount of TGFb-TPO fusion protein and different concentrations of TGFb, and then SEAP activity was measured in culture supernatant.

[0910] Figure 6C A dose-response relationship between IL-8 concentration and activity of IL-8-TPO fusion proteins (comprising anti-TPO receptor VH and VL sequences of SEQ ID NO: 32 and SEQ ID NO: 33, respectively) is shown. TpoR SEAP reporter cells were stimulated overnight with a constant amount of IL8-TPO fusion protein and different concentrations of IL-8, and then SEAP activity was measured in culture supernatant.

[0911] C. Characterization of TPO receptor agonism achieved by fusion proteins versus native agonists

[0912] Following the overnight incubation step of the assay described in Example IB, cell samples were collected for analysis and comparison of gene expression between cells treated with fusion proteins or soluble ligands. RNA was extracted and purified according to the Qiagen RNeasy kit (Cat# 74104) with threshold RNA quality number > 7. RNA libraries were prepared for gene expression profiling using the Roche KAPA HyperPrep kit, and sequenced on an...

Claims

1. A macromolecular complex comprising two macromolecules, wherein each macromolecule comprises a first binding domain (FBD) connected to a second binding domain (SBD) via a linker domain, wherein: (a) The FBD specifically binds to disease-signature ligands in biological samples; and (b) The SBD specifically binds to the effector ligand in the biological sample and induces cellular effector function upon binding to the effector ligand; These two macromolecules are interlinked; Furthermore, the induction of the effector function by the macromolecular complex is conditional upon the binding of each of the two macromolecules to the disease-characteristic ligand.

2. A macromolecular complex comprising macromolecular pairs, wherein each macromolecule independently comprises a first binding domain (FBD) connected to a second binding domain (SBD) via a linker domain, wherein: (a) The first member of the macromolecule pair contains a first binding domain 1 (FBD1) of a first epitope that specifically binds to a disease-characteristic ligand in a biological sample. (b) The second member of the macromolecule pair contains a first binding domain 2 (FBD2) of a second epitope that specifically binds to a disease-characteristic ligand in a biological sample; and (c) The SBD specifically binds to the effector ligand in the biological sample and induces cellular effector function after binding to the effector ligand; These two macromolecules are interlinked; Furthermore, the induction of the effector function by the macromolecular complex is conditional upon the binding of each of the two macromolecules to the disease-characteristic ligand.

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

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

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

6. The macromolecular complex or macromolecule of any one of claims 1-5, wherein the FBD, the FBD1 or the FBD2 comprises a polypeptide that specifically binds to the disease-characteristic ligand.

7. The macromolecular complex or macromolecule of claim 6, wherein the polypeptide is an antibody or a fragment thereof.

8. The macromolecular complex or macromolecule as described in any one of claims 1-7, wherein the effector ligand is a protein or peptide.

9. The macromolecular complex or macromolecule according to any one of claims 1-8, wherein the macromolecular complex or macromolecule further comprises one or more additional connector domains.

10. The macromolecular complex or macromolecule according to any one of claims 1-9, wherein the one or more linker domains are peptide linkers.

11. The macromolecular complex or macromolecule according to any one of claims 1-10, wherein the macromolecule is a polypeptide.

12. A nucleic acid that encodes a macromolecule as claimed in any one of claims 1-11.

13. A nucleic acid pair encoding a macromolecule pair as claimed in claim 2.

14. The nucleic acid or nucleic acid pair as described in claim 12 or 13, wherein the nucleic acid is RNA or DNA.

15. A nucleic acid encoding a macromolecule as claimed in any one of claims 1-11, wherein the nucleic acid is formulated together with a carrier.

16. A vector comprising the nucleic acid as described in claim 12, 14 or 15.

17. A vector or vector pair comprising the nucleic acid pair as described in claim 13 or 14.

18. A host cell comprising a nucleic acid or nucleic acid pair as described in any one of claims 12-15 or a vector or vector pair as described in any one of claims 16 or 17.

19. A method comprising providing a pathway for entry into cells for a macromolecular complex or macromolecule as described in any one of claims 1-11 or a nucleic acid or nucleic acid pair as described in any one of claims 12-15.

20. A macromolecular complex comprising two macromolecules, wherein each macromolecule comprises a VEGF-binding domain connected to a 4-1BB-binding domain via a linker domain, wherein the 4-1BB-binding domain induces cellular effector function upon binding to 4-1BB. These two macromolecules are interlinked; Furthermore, the induction of the effector function by the macromolecular complex is conditional upon the binding of each of the two macromolecules to VEGF.

21. A macromolecular complex comprising two macromolecules, wherein each macromolecule comprises an IFNg binding domain connected to an IL-10R binding domain via a linker domain, wherein the IL-10R binding domain induces cellular effector function upon binding to IL-10R. These two macromolecules are interlinked; Furthermore, the induction of the effector function by this macromolecular complex is conditional upon the binding of each of the two macromolecules to IFNg.

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