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

[0001] Sequence Listing

[0002] This application contains a sequence listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy created on April 11, 2023 is named 51661-002WO2_Sequence_Listing_4_11_23 and is 31,499 bytes in size. Background Art

[0003] There is a need in the art for macromolecules that conditionally induce cellular effector functions (eg, biological or therapeutic activities) based on the presence of disease-signature ligands and methods of using these macromolecules. Summary of the invention

[0004] In one aspect, the present invention provides a macromolecule comprising a first binding domain connected to a second binding domain, wherein (a) the first binding domain specifically binds to a disease-characteristic ligand in a biological sample; and (b) the second binding domain specifically binds to an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of forming a multimer 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 multimer member to the disease-characteristic ligand.

[0005] In some embodiments, the polymer is a dimer, trimer or tetramer. In some embodiments, the polymer is a dimer. In some embodiments, the dimer is a homodimer.

[0006] On the other hand, the present invention provides a macromolecule pair, each macromolecule independently comprising a first binding domain connected to a second binding domain, wherein (a) the first binding domain of each macromolecule specifically binds to a disease-characteristic ligand in a biological sample; (b) the second binding domain of the first member of the macromolecule pair specifically binds to a first effector ligand in the biological sample; and (c) the second binding domain of the second member of the macromolecule pair specifically binds to a second effector ligand in the biological sample; wherein the second binding domain of the first member of the macromolecule pair and the second binding domain of the second member of the macromolecule pair induce cellular effector function after binding to the first effector ligand and the second effector ligand; wherein the macromolecule pair is capable of forming a heteropolymer 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 heteropolymer member to the disease-characteristic ligand.

[0007] On the other hand, the present invention provides a macromolecule pair, each macromolecule independently comprising a first binding domain connected to a second binding domain, 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-characteristic 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 a disease-characteristic ligand in a biological sample; and (c) the second binding domain specifically binds to an effector ligand in the biological sample and induces a cellular effector function after binding to the effector ligand; wherein the macromolecule is capable of forming a multimer 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 multimer member to the disease-characteristic ligand.

[0008] On the other hand, the present invention provides a macromolecule pair, each macromolecule independently comprising a first binding domain connected to a second binding domain, 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 characteristic 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 a disease characteristic ligand in a biological sample; (c) the second binding domain of the first member of the macromolecule pair specifically binds to a first effector ligand in the biological sample; and (d) the second binding domain of the second member of the macromolecule pair specifically binds to a second effector ligand in the biological sample; wherein the macromolecule is capable of forming a multimer 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 multimer member to the disease characteristic ligand.

[0009] In certain of the 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).

[0010] In some embodiments, the heteromultimer is a dimer.

[0011] In some embodiments of any of the above aspects, the disease characteristic ligand is a protein, a peptide or a small molecule. In some embodiments, the protein is a soluble protein or an insoluble protein.

[0012] In some embodiments, the disease characteristic ligand is a cytokine. In some embodiments, the cytokine is an interleukin, an interferon, a growth factor, a chemokine, or a TNF family member. 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 β (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 γ induced protein 10 (IP-10); or the TNF family member is TNF-α.

[0013] In some embodiments, the disease characteristic ligand is a cell surface receptor, a surface antigen, a membrane bound protein, an extracellular matrix component, or an integrin.

[0014] In some embodiments, the disease characteristic 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.

[0015] In some embodiments, the disease characteristic ligand is a nucleic acid.

[0016] In some embodiments, the disease-characteristic ligand is a carbohydrate, a lipid, a peptide, a nucleoside, or a combination of the foregoing.

[0017] In some embodiments, the disease-characteristic ligand is a hormone, an amino acid derivative, a steroid, or an eicosanoid.

[0018] In some embodiments, the disease characteristic ligand is a non-self antigen. In some embodiments, the disease characteristic ligand is a virus, bacteria, or a fragment or antigen thereof.

[0019] In some embodiments, the disease signature ligand is a multimer. In some embodiments, the disease signature ligand is a dimer, trimer or tetramer.

[0020] In some embodiments of any of the above aspects, the first binding domain, FBD1 or FBD2 comprises a polypeptide that specifically binds to the disease characteristic ligand. In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or a fragment thereof is 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).

[0021] 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 binder based on a fibronectin type III domain (FN3), a nanobody, a nanoCLAMP, an optimizer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR).

[0022] In some embodiments, the polypeptide is an endogenous binding domain. In some embodiments, the endogenous binding domain is a cell receptor domain, an enzyme domain, a variable lymphocyte receptor (VLR) domain, a receptor extracellular domain, a nuclear hormone receptor ligand binding domain, or a DNA binding domain.

[0023] In some embodiments, the first binding domain, FBD1 or FBD2 comprises an oligonucleotide that specifically binds to the disease characteristic ligand. In some embodiments, the oligonucleotide is a nucleic acid aptamer. In some embodiments, the nucleic acid aptamer is a DNA aptamer.

[0024] In some embodiments, the first binding domain comprises a chemical molecule that specifically binds to the disease characteristic ligand.

[0025] In some embodiments, the first binding domain, FBD1 or FBD2 has affinity for two or more disease characteristic portions.

[0026] In some embodiments of any of the above aspects, the effector ligand is a protein or a peptide.

[0027] In some embodiments, 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.

[0028] 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 a VEGFR, the RSK is a TGFBR2, the type 1 cytokine receptor is an IL-2R, the type 2 cytokine receptor is an IL10R, or the TNF superfamily receptor is a TNFR2 or 4-1BB.

[0029] In some embodiments, the second binding domain is an agonist of the effector ligand.

[0030] In some embodiments, the effector ligand must be homodimerized to exert cellular effector function. In some embodiments, the effector ligand is homodimerized in the presence of multimers of the macromolecule.

[0031] In some embodiments, the first effector ligand and the second effector ligand must associate to exert cellular effector function. In some embodiments, the first effector ligand and the second effector ligand associate in the presence of the macromolecular multimer. In some embodiments, the association is heterodimerization.

[0032] In some embodiments of any of the above aspects, the cellular effector function is a biological activity.

[0033] In some embodiments, the cellular effector function is a therapeutic activity.

[0034] In some embodiments, the cellular effector function is disease activity.

[0035] In some embodiments of any of the above aspects, the second binding domain comprises a polypeptide that specifically binds to the effector ligand. In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or a fragment thereof is a 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.

[0036] 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 alpha body, an anticalin, a lipocalin, an affinity polymer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monoclonal antibody, a FN3-based binder, a nanobody, a nanoCLAMP, an optimizer, a repebody, a pre-connexin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a VLR.

[0037] In some embodiments, the polypeptide is an endogenous binding domain. In some embodiments, the endogenous binding domain is a ligand of the effector ligand or a fragment thereof. In some embodiments, the endogenous binding domain is a viral binding protein or a fragment thereof.

[0038] In some embodiments, the second binding domain comprises an oligonucleotide that specifically binds to the effector ligand. In some embodiments, the oligonucleotide is a nucleic acid aptamer. In some embodiments, the nucleic acid aptamer is a DNA aptamer.

[0039] In some embodiments, the first binding domain, FBD1 or FBD2 comprises a chemical molecule that specifically binds to the disease characteristic ligand.

[0040] In some embodiments of aspects comprising a macromolecular pair, the second binding domain of the first member of the macromolecular pair is a first portion of a binding moiety and the second binding domain of the second member of the macromolecular pair is a second portion of a binding moiety.

[0041] In some embodiments of aspects comprising macromolecular pairs, the second binding domain of the first member of the macromolecular pair specifically binds to a first component of a heterodimeric receptor, and the second binding domain of the second member specifically binds to a second component of the heterodimeric receptor.

[0042] In some embodiments of aspects comprising a macromolecular pair, the second binding domain of the first member of the macromolecular pair is a first component of a dimer portion, and the second binding domain of the second member is a second component of a dimer portion.

[0043] In some embodiments of aspects comprising a macromolecular pair, the second binding domain of the first member of the macromolecular pair is a first fragment of a polypeptide chain, and the second binding domain of the second member is a second fragment of the polypeptide chain. In some embodiments, the polypeptide chain is a hormone, cytokine, or growth factor.

[0044] In some embodiments of aspects comprising macromolecular pairs, the second binding domain of the first member of the macromolecular pair and the second binding domain of the second member of the macromolecular pair have been engineered to have reduced affinity for each other.

[0045] In some embodiments of any of the above aspects, the macromolecule comprises a reporter portion. In some embodiments, the reporter portion is 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 a luciferase or a beta-lactamase.

[0046] In some embodiments of any of the above aspects, the macromolecule or the macromolecule pair further comprises one or more linker domains. In some embodiments, the one or more linker domains are peptide linkers. In some embodiments, the peptide linker comprises a GS linker. In some embodiments, the GS linker comprises a GS (G n S) mIn some embodiments, the GS linker comprises (G n S) m Connector.

[0047] In some embodiments of any of the above aspects, the macromolecule is a polypeptide.

[0048] In some embodiments of any of the above aspects, the biological sample is an extract, a fluid, a fraction, a cell, a tissue, or a subject.

[0049] In some embodiments of any of the above aspects, the macromolecule or one or both members of the macromolecule pair comprise a leader sequence. In some embodiments, the leader sequence comprises a secretion signal.

[0050] In some embodiments of any of the above aspects, one or both members of the macromolecule or macromolecule pair comprise a half-life extending moiety. In some embodiments, the half-life extending moiety is an Fc domain or a fragment thereof, an albumin domain or a fragment thereof, or polyethylene glycol (PEG) or a modified derivative thereof.

[0051] In another aspect, provided herein is a nucleic acid encoding any of the macromolecules provided herein.

[0052] In another aspect, provided herein is a nucleic acid pair encoding any of the macromolecule pairs provided herein.

[0053] In some embodiments, the nucleic acid is RNA or DNA.

[0054] In some embodiments, the nucleic acid is formulated with a delivery platform. In some embodiments, 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 includes (or is derived from) adenovirus, anellovirus, AAV or lentivirus.

[0055] In another aspect, provided herein is a nucleic acid encoding any of the macromolecules provided herein, wherein the nucleic acid is formulated with a carrier.

[0056] In another aspect, provided herein is a nucleic acid pair encoding any of the macromolecule pairs provided herein, wherein the nucleic acid pair is formulated with a carrier.

[0057] In some embodiments, the nucleic acid is RNA or DNA.

[0058] In some embodiments, the carrier is a lipid-based carrier. In some embodiments, the lipid-based carrier is LNP.

[0059] In another aspect, provided herein are vectors comprising any of the nucleic acids provided herein.

[0060] In another aspect, provided herein are vector pairs comprising any of the nucleic acid pairs provided herein.

[0061] In some embodiments, the vector or the vector pair is formulated with a carrier.

[0062] In another aspect, provided herein is a host cell comprising any nucleic acid or nucleic acid pair, or any vector or vector pair provided herein.

[0063] In another aspect, provided herein are multimers comprising any two or more macromolecules provided herein. In some embodiments, each macromolecule comprises the same amino acid sequence.

[0064] In another aspect, provided herein are multimers comprising at least one copy of each of any macromolecule pair provided herein.

[0065] In some embodiments of any of the above aspects, the macromolecular complex, macromolecule, nucleic acid or nucleic acid pair is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% pure or greater than 99% pure.

[0066] In some embodiments of any of the above aspects, the macromolecular complex, macromolecule, nucleic acid or nucleic acid pair is manufactured according to the Good Manufacturing Practice (GMP), Good Clinical Practice (GCP) and / or Good Laboratory Practice (GLP) standards of the US Food and Drug Administration (FDA).

[0067] In another aspect, provided herein is a method comprising providing access to a cell for any macromolecule or macromolecule pair, nucleic acid or nucleic acid pair, or multimer provided herein.

[0068] In another aspect, the present invention provides a method for regulating the state of a cell, the method comprising providing access to a cell for any macromolecule or macromolecule pair, nucleic acid or nucleic acid pair, or polymer provided herein, thereby regulating the state of the cell.

[0069] In another aspect, provided herein is a method for inducing a cellular effector function in a cell, the method comprising providing access to a cell for any macromolecule or macromolecule pair, nucleic acid or nucleic acid pair, or polymer provided herein, thereby inducing the cellular effector function in the cell.

[0070] In some embodiments, the cell is in a subject, and the macromolecule, nucleic acid, or multimer is administered in a therapeutically effective amount.

[0071] In some embodiments, the subject has or is suspected of having a disease or disorder characterized by abnormal levels of the disease-characteristic target, optionally wherein the subject was previously determined to have abnormal levels of the disease-characteristic target.

[0072] On the other hand, the present invention provides a method for determining the state of a cell, which method includes providing a pathway for any macromolecule or macromolecule pair comprising a reporter domain provided herein, a polymer thereof, or a nucleic acid or nucleic acid pair encoding the macromolecule or macromolecule pair to enter the cell, and detecting the presence of the reporter domain, thereby determining the state of the cell.

[0073] On the other hand, provided herein is a method for inducing cellular effector function in a cell, the method comprising contacting the cell with a macromolecule comprising a first binding domain connected to a second binding domain, wherein (a) the first binding domain specifically binds to a disease characteristic ligand in a biological sample containing the cell; and (b) the second binding domain specifically binds to an effector ligand in the biological sample and induces the cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of forming a multimer 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 multimer member to the disease characteristic ligand.

[0074] On the other hand, the present invention provides a method for inducing cellular effector function in a cell, the method comprising contacting the cell with a macromolecule pair, each macromolecule independently comprising a first binding domain connected to a second binding domain, wherein (a) the first binding domain of each macromolecule specifically binds to a disease-characteristic ligand in a biological sample containing the cell; (b) the second binding domain of the first member of the macromolecule pair specifically binds to the first effector ligand in the biological sample; and (c) the second binding domain of the second member of the macromolecule pair specifically binds to the second effector ligand in the biological sample; wherein the second binding domain of the first member of the macromolecule pair and the second binding domain of the second member of the macromolecule pair induce the cellular effector function after binding to the first effector ligand and the second effector ligand; wherein the macromolecule pair is capable of forming a heteropolymer 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 heteropolymer member to the disease-characteristic ligand.

[0075] On the other hand, the present invention provides a pair of macromolecules, each macromolecule comprising a first binding domain connected to a second binding domain, wherein (a) the first binding domain of the first member of the macromolecule pair specifically binds to a first portion of a disease-characteristic ligand in a biological sample; (b) the first binding domain of the second member of the macromolecule pair specifically binds to a second portion of the disease-characteristic ligand in the biological sample; and (c) the second binding domain of each macromolecule specifically binds to an effector ligand in the biological sample and induces cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of forming a heteropolymer 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 heteropolymer member to the disease-characteristic ligand.

[0076] On the other hand, the present invention provides a group consisting of three macromolecules, each macromolecule comprising a first binding domain connected to a second binding domain, wherein (a) the first binding domain of each macromolecule specifically binds to a disease-characteristic ligand in a biological sample; (b) the second binding domain of the first member of the macromolecule group specifically binds to a first effector ligand in the biological sample; (c) the second binding domain of the second member of the macromolecule group specifically binds to a second effector ligand in the biological sample; and (d) the second binding domain of the third member of the macromolecule group specifically binds to a third effector ligand in the biological sample; wherein the second binding domains of the first, second, and third members of the macromolecule pair induce cellular effector function after binding to the first, second, and third effector ligands; wherein the macromolecule group is capable of forming a heterotrimer 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 heterotrimeric member to the disease-characteristic ligand.

[0077] In some embodiments, the disease signature ligand is trimeric.

[0078] In some embodiments, binding of the second binding domain to the effector ligand is conditional upon binding of each multimeric member to the disease characteristic ligand.

[0079] In some embodiments, binding of the second binding domain to the effector ligand is not conditional upon binding of every multimeric member to the disease signature ligand.

[0080] On the other hand, the present invention provides a plurality of macromolecules, each macromolecule independently comprising a first binding domain connected to a second binding domain, wherein (a) the first binding domain of each macromolecule specifically binds to a disease characteristic ligand in a biological sample; and (b) the second binding domain of each macromolecule specifically binds to an effector ligand in the biological sample; wherein the plurality of macromolecules are capable of forming multimers 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 multimer member to the disease characteristic ligand.

[0081] In some embodiments, the multimer is a homodimer, homotrimer, or homotetramer.

[0082] In some embodiments, the multimer is a heterodimer, heterotrimer, or heterotetramer.

[0083] In some embodiments, at least one macromolecule comprises a disease signature ligand that is a protein, peptide, or small molecule.

[0084] In some embodiments, binding of the second binding domain to the effector ligand is conditional upon binding of each multimeric member to the disease characteristic ligand.

[0085] In some embodiments, binding of the second binding domain to the effector ligand is not conditional upon binding of every multimeric member to the disease signature ligand.

[0086] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

[0087] definition

[0088] As used herein, the term "macromolecule" refers to a macromolecule (e.g., a molecule greater than 1000 Daltons (1 kDa) in size) comprising one or more polypeptide, oligonucleotide, chemical, lipid and / or carbohydrate moieties. In some embodiments, the macromolecule is a recombinant protein (e.g., a fusion protein).

[0089] As used herein, the term "polymer" refers to a molecule consisting of at least two subunits (e.g., at least two subunits comprising one or more polypeptides, oligonucleotides, chemicals, lipids and / or carbohydrate moieties). Polymers include homopolymers and heteropolymers. "Homopolymers" are polymers consisting of two or more identical or substantially identical subunits (e.g., two or more macromolecules). Homopolymers 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 such differences in the amino acid sequence that these differences do not significantly affect the function of the subunits, for example, these differences do not significantly affect the affinity of the subunits to one or more ligands. "Heteropolymers" are polymers consisting of two or more non-identical subunits. Heteropolymers include heterodimers (comprising a first and a second subunit, e.g., comprising a non-identical pair of macromolecules), heterotrimers (comprising one copy of a first subunit and two copies of a second subunit), and homotetramers (comprising two copies or forms of each of the first and second subunits). Multimers further include higher order multimers, e.g., hexamers, heptamers, octamers, nonamers, and decamers.

[0090] As used herein, the term "binding domain" refers to any domain that has a specific affinity for a ligand. Binding domains include, but are not limited to, polypeptides (e.g., antibodies or fragments thereof (e.g., scFv, monospecific tandem scFv (taFv), bispecific taFv, VHH, VNAR, Fab, monospecific single-chain diabody, bispecific single-chain diabody, or dual affinity retargeting antibody (DART)), antibody mimetics (e.g., affibody, affilin, affimer, affitin, alpha body, anticalcalin, lipocalin, affinity polymer, DARPin, fynomer, gastrobody, knottin, Kunitz domain peptide, mono Antibodies, fibronectin type III domain (FN3)-based binders, nanobodies, nanoCLAMPs, optimizers, repebodies, pre-connectins, centyrins, obodies, peptide aptamers, synthetic peptides or variable lymphocyte receptors (VLRs)), endogenous binding domains (e.g., cell receptor domains, enzyme domains, variable lymphocyte receptor (VLR) domains, receptor extracellular domains, nuclear hormone receptor ligand binding domains, or DNA binding domains)), oligonucleotides (e.g., nucleic acid aptamers (e.g., DNA aptamers)) and chemical molecules and combinations thereof.

[0091] As used herein, the term "ligand" refers to any portion to which a binding domain as described herein can have affinity. Ligands include, without limitation, a chemical moiety, a portion of a molecule, a molecule (e.g., an allergen or 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 of phosphorylation, glycosylation, acylation, alkylation, 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.

[0092] The term "disease signature ligand" refers to a ligand associated with a disease state or disorder in a cell, tissue or subject (e.g., a mammal, such as a human). A disease signature ligand can be a protein, such as a soluble protein, an insoluble protein, a monomeric protein, and a multimeric protein. Disease signature ligands include, but are not limited to, cell surface receptors, cell surface antigens, membrane-bound proteins, extracellular matrix components, integrins, cytokines, neurotransmitters, anti-drug antibodies (ADA), autoantibodies, nucleic acids, carbohydrates, lipids, peptides, nucleosides, hormones, viruses, bacteria, fungi, or fragments or antigens thereof.

[0093] The term "effector ligand" refers to a ligand that can achieve a cellular effector function after being bound by a binding domain (e.g., a polymer of the present invention). 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).

[0094] As used herein, the term "associated with a disease, disorder, or condition" refers to 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 causal agent of the disease, disorder, or condition. For example, a virus can be a causal agent of a viral infection, a bacterium can be a causal agent of a bacterial infection, a fungus can be a causal agent of a fungal infection, or a parasite can be a causal agent of a parasitic infection, a cancer cell can be a causal agent of cancer, a toxin can be a causal agent of toxicity, or an allergen can be a causal 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 an increased severity of the disease, disorder, or condition.

[0095] As used herein, the term "carrier" means a compound, composition, agent or molecule that promotes the stability of a composition (e.g., a macromolecule or macromolecule pair as described herein), the transport or delivery of the composition into a subject, tissue or cell. Non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified phytoglycogen or glycogen-based materials), nanoparticles (e.g., nanoparticles encapsulating or covalently linked to cyclic or linear polyribonucleotides), liposomes, fusosomes, in vitro differentiated reticulocytes, exosomes, protein carriers (e.g., proteins covalently linked to polyribonucleotides), and cationic carriers (e.g., cationic lipopolymers or transfection agents).

[0096] As used herein, the terms "disease," "disorder," and "condition" all refer to a sub-health condition, such as a condition that is or would typically be diagnosed or treated by a medical professional.

[0097] As used herein, the term "polynucleotide" means a molecule comprising one or more nucleic acid subunits or nucleotides, and can be used interchangeably with "nucleic acid" or "oligonucleotide". A polynucleotide may comprise one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) or variants thereof. A nucleotide may comprise a nucleoside and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphate (PO3) groups. A nucleotide may comprise a nucleoside, a pentose (ribose or deoxyribose) and one or more phosphate groups. A ribonucleotide is a nucleotide in which the sugar is ribose. A polyribonucleotide or ribonucleic acid or RNA may refer to a macromolecule comprising a plurality of ribonucleotides polymerized via a phosphodiester bond. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose. A polynucleotide provided herein may comprise one or more modified nucleotides.

[0098] Polydeoxyribonucleotide or deoxyribonucleic acid or DNA means a macromolecule comprising a plurality of deoxyribonucleotides polymerized via a phosphodiester bond. Nucleotide can be a nucleoside monophosphate or a nucleoside polyphosphate. Nucleotide means a deoxyribonucleoside polyphosphate, such as deoxyribonucleoside triphosphate (dNTP), which can be selected from deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP) and deoxythymidine triphosphate (dTTP) dNTP, and can include a detectable label, such as a luminescent label or a marker (e.g., a fluorophore). One or more nucleotides can be modified nucleotides. Nucleotide can include any subunit that can be incorporated into a growing nucleic acid chain. This subunit can be A, C, G, T or U, or any other subunit that has specificity or is complementary to one or more complementary A, C, G, T or U or to purine (i.e., A or G or its variants) or pyrimidine (i.e., C, T or U or its variants). In some instances, polynucleotides are deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or derivatives or variants thereof. In some cases, polynucleotides are short interfering RNA (siRNA), microRNA (miRNA), plasmid DNA (pDNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (pre-mRNA), antisense RNA (asRNA), and encompass nucleotide sequences and any structural embodiments thereof, such as single-stranded, double-stranded, triple-stranded, spiral, hairpin, etc. In some cases, polynucleotide molecules are circular (e.g., circular RNA). Polynucleotides can have various lengths. Nucleic acid molecules 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), 2kb, 3kb, 4kb, 5kb, 10kb, 50kb or more. Polynucleotides can be isolated from cells or tissues. As embodied herein, polynucleotide sequences can include isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and synthetic DNA / RNA analogs.

[0099] As used herein, "polypeptide" means a polymer of amino acid residues (natural or non-natural, including D, L or a combination thereof) most commonly 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, homologues, orthologues, paralogues, fragments and other equivalents, variants and analogs of the aforementioned. Polypeptides can be single molecules, or can be multimolecular complexes, such as dimers, trimers or tetramers. They can also include single-chain or multi-chain polypeptides, such as antibodies or insulin, and can be associated or linked. The most common disulfide bonds are present in multi-chain polypeptides. The term polypeptide can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of corresponding naturally occurring amino acids.

[0100] As used herein, the term "sequence identity" is determined by comparing two peptides or two nucleotide sequences using a global or local comparison algorithm. When a sequence (when, for example, the best comparison is performed using default parameters by the program GAP or BESTFIT) has at least a certain minimum percentage of sequence identity, then they can be referred to as "substantially identical" or "basically similar". GAP uses the Needleman and Wunsch global comparison algorithm to compare it over the entire length of the two sequences, maximizing the number of matches and minimizing the number of gaps. Typically, using the GAP default parameters, gaps generate penalty points=50 (nucleotides) / 8 (proteins), and gap extension penalty points=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 [Proceedings of the National Academy of Sciences of the United States] 89,915-919). The scores of sequence alignment and sequence identity percentage can be determined using computer programs, such as GCG Wisconsin software package version 10.3 or EmbossWin version 2.10.0 (using program "needle") available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA. Alternatively or additionally, the identity percentage can be determined by searching the database using algorithms such as FASTA, BLAST, etc. Sequence identity refers to the sequence identity over the entire length of the sequence.

[0101] 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 and which targets the polypeptide sequence to the secretory pathway.

[0102] As used herein, the term "treat" or "treating" refers to the therapeutic treatment of a disease or disorder (e.g., an infectious disease, cancer, poisoning, or allergic reaction) of a subject. The effect of treatment may include reversing, alleviating, reducing the severity of, curing, inhibiting, reducing the progression of, stabilizing (i.e., not worsening) the state of, and / or preventing the spread of, a disease or disorder, compared to the state and / or condition of the disease or disorder in the absence of therapeutic treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 This is a schematic diagram showing the domain structure of a fusion protein that exerts an effector function on a second target (e.g., a biological effector ligand) under the condition of the presence of a first target (e.g., a disease signal ligand). From the N-terminus to the C-terminus, the fusion protein comprises a leader polypeptide; a single-chain variable fragment (scFv) containing 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 the first target; (G4S) n a linker; a VH domain and a VL domain having affinity for a second target upon multimerization with a second molecule having the same VH domain and VL domain, wherein the VH domain and the VL domain are connected by a G4S linker; and a FLAG affinity tag.

[0104] Figure 2 This is a schematic diagram showing the domain structure of a fusion protein pair that exerts an effector function on a second target (e.g., a biological effector ligand) under the condition of the presence of a first target (e.g., a disease signal ligand). From the N-terminus to the C-terminus, the fusion protein comprises a leader polypeptide; a FLAG affinity tag; 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 the first target; (G4S) n linker; and conditional effector domain A (in the first fusion protein) or conditional effector domain B (in the second fusion protein).

[0105] Figure 3This is a schematic diagram showing the domain structure of a fusion protein pair that exerts an effector function on a second target (e.g., a biological effector ligand) under the condition of the presence of a first target (e.g., a disease signal ligand). From the N-terminus to the C-terminus, the fusion protein comprises a leader polypeptide; 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 the first target; (G4S) n a linker; a VHH with affinity for the first part of the second target (in the first fusion protein ("anti-a")) or a VHH with affinity for the second part of the second target (in the second fusion protein ("anti-b")); and a FLAG affinity tag.

[0106] Figure 4 It is a schematic diagram showing the domain structure of a fusion protein pair that exerts an effector function on a second target (e.g., a biological effector ligand) under the condition of the presence of a first target (e.g., a disease signal 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 a scFv, wherein the VH domain and the VL domain of the scFv have affinity for the second target when assembled into a functional scFv, wherein the VH domain and the VL domain have low affinity for each other; a GS (G4S) 3 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 the first target; and a FLAG affinity tag.

[0107] Figure 5A This is a schematic diagram showing the domain structure of a fusion protein that conditionally regulates TPO receptor (TpoR) in the presence of TGF-β1 (TGFb). From the N-terminus to the C-terminus, the fusion protein comprises a mouse immunoglobulin kappa variable 3 (Ms IgKVIII) leader polypeptide; an anti-TGF-β1 scFv containing VH and VL connected by a (G4S)3 linker; (G4S) n linker; anti-TpoR scFv containing VH and VL connected by a (G4S)3 linker; and a FLAG affinity tag.

[0108] Figure 5B is a schematic diagram showing the binding of TGFb and TpoR Figure 5A Two copies of the fusion protein are shown. The membrane orientation of TpoR and downstream signaling partners are shown.

[0109] Figure 5C is a graph showing the Figure 5ASEAP activity (grey) in culture supernatants of TpoR secreted alkaline phosphatase (SEAP) reporter cells stimulated overnight with fusion proteins of β-actin and various concentrations of TGFb. TPO native cytokine is shown as a positive control for constitutive activity (black).

[0110] Fig. 6A Schematic diagram showing the domain structure of a fusion protein that conditionally regulates TpoR in the presence of interleukin-8 (IL-8). From N-terminus to C-terminus, the fusion protein comprises the Ms IgKVIII leader polypeptide; an anti-IL-8 scFv comprising VH and VL connected by a (G4S)3 linker; (G4S) n linker; anti-TpoRscFv containing VH and VL connected by a (G4S)3 linker; and a FLAG affinity tag.

[0111] Figure 6B is a schematic diagram showing the binding of IL-8 and TpoR Fig. 6A Two copies of the fusion protein are shown. The membrane orientation of TpoR and downstream signaling partners are shown.

[0112] Figure 6C is a graph showing the Fig. 6A SEAP activity in culture supernatants of TpoR SEAP reporter cells stimulated overnight with fusion proteins and varying concentrations of IL-8 (grey). TPO native cytokine is shown as a positive control for constitutive activity (black).

[0113] Fig. 7A This is a schematic diagram showing the domain structure of a complementary fusion protein pair (fusion proteins (a) and (b)) that regulates the activity of human interleukin-2 receptor (IL-2R) in the presence of TGFb. Fusion protein (a) comprises the Ms IgKVIII leader peptide from the N-terminus to the C-terminus; an anti-TGF-β1 scFv containing VH and VL connected by a (G4S)3 linker; (G4S) n linker; and conditional IL-2R effector domain A (IL-2 (N terminus)). Fusion protein (b) comprises MsIgKVIII leader peptide from N terminus to C terminus; anti-TGF-β1 scFv comprising VH and VL connected by a (G4S)3 linker; (G4S) n linker; and conditional IL-2R effector domain B (IL-2 (C-terminus)).

[0114] Figure 7B is a schematic diagram showing the binding of IL-2R receptor components IL2Rβ and IL2RγC in the absence (left) or presence (right) of TGFb, respectively. Fig. 7AFusion proteins of (a) and (b). The membrane orientation and downstream signaling partners of IL-2R are shown.

[0115] Figure 7C is a graph showing the Fig. 7A SEAP activity (grey squares) in the culture supernatant of IL-2SEAP reporter cells stimulated overnight with different concentrations of TGFβ and IL-2 cytokine (a) and (b). IL-2 cytokine is shown as a positive control for constitutive activity (black circles). TGFβ cytokine is shown as a negative control for conditional activity (grey triangles).

[0116] Fig. 8A The present invention is a schematic diagram showing the domain structure of a fusion protein pair that exerts an effector function on a second target (e.g., a biological effector ligand) under the condition of the presence of a first target (e.g., a disease signal ligand), wherein from the N-terminus to the C-terminus, the fusion protein comprises a leader polypeptide; 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 the first target; (G4S) n linker; a VHH with affinity for a first portion of a second target (in a first fusion protein ("anti-a")) or a VHH with affinity for a second portion of a second target (in a second fusion protein ("anti-b")); and a FLAG affinity tag (left); and a set of such schematic diagrams showing such fusion protein pairs in which the first target is IFN-γ (IFNg) and the second target is the IL-10 receptor (IL-10R) (right).

[0117] Figure 8B is a schematic diagram showing binding of IFNg and to the IL-10 receptor components IL-10Ra and IL-10Rb Fig. 8A Fusion protein pairs. The membrane orientation and downstream signaling partners of IL-10R are shown.

[0118] Figure 8C This figure shows the Fig. 8A SEAP activity in the culture supernatant of IL-10SEAP reporter cells stimulated overnight with fusion protein pairs and different concentrations of IFNg (grey squares). IL-10 cytokine is shown as a positive control for constitutive activity (black circles). IFNg cytokine is shown as a negative control for conditional activity (grey triangles).

[0119] Fig. 9AThis is a schematic diagram showing the domain structure of a complementary fusion protein pair that conditionally regulates IL-10R activity in the presence of IL-6. The first fusion protein comprises a mouse IgKVIII leader peptide from the N-terminus to the C-terminus; an anti-IL10Ra VHH antibody; a (G4S)2 linker; a first anti-IL-6 VHH antibody; (G4S) n linker; and a polyhistidine tag. The second fusion protein comprises a mouse IgKVIII leader peptide from the N-terminus to the C-terminus; an anti-IL10Rb VHH antibody; a (G4S)2 linker; a second anti-IL-6 VHH antibody; (G4S) n a linker; and a polyhistidine tag.

[0120] Fig. 9B is a schematic diagram showing binding of IL-6 and to the IL-10 receptor components IL-10Ra and IL-10Rb Fig. 9A Fusion protein pairs. The membrane orientation and downstream signaling partners of IL-10R are shown.

[0121] Fig. 9C is a graph showing the Fig. 9A SEAP activity in the culture supernatant of IL-10SEAP reporter cells stimulated overnight with fusion protein pairs and different concentrations of IL-6 (grey circles). IL-10 cytokine is shown as a positive control for constitutive activity (black squares). IL-6 cytokine is shown as a negative control for conditional activity (grey triangles).

[0122] Fig. 10A Schematic diagram showing the domain structure of a complementary fusion protein pair that conditionally regulates differentiation cluster 3 (CD3) activity in the presence of IL-8. The first fusion protein comprises a mouse IgKVIII leader peptide from the N-terminus to the C-terminus; 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 a mouse IgKVIII leader peptide from the N-terminus to the C-terminus; an anti-CD3 VL domain of a scFv; a GS (G4S) linker; an anti-IL-8 scFv; and a FLAG affinity tag.

[0123] Fig. 10B is a schematic diagram showing the binding of IL-8 and CD3 Fig. 10A Fusion protein pairs. The membrane orientation and downstream signaling partners of CD3 are shown.

[0124] Fig. 10C is a bar graph showing the expression of a constant amount of Fig. 10AThe fusion protein of NFAT-luc has an effect on the luciferase activity in the culture supernatant of NFAT-luc Jurkat reporter cells stimulated for 24 hours (measured via the NFAT-luc reporter). The full-length parental anti-CD3 scFv is shown as a positive control for constitutive activity. DETAILED DESCRIPTION

[0125] Featured herein are macromolecules, multimers thereof, compositions comprising the same, and methods of use thereof that conditionally induce cellular effector functions (eg, biological or therapeutic activities) based on the presence of a disease-signature ligand.

[0126] I. Composition

[0127] A. Macromolecules

[0128] i. Macromolecules that form homodimers

[0129] In one aspect, the present invention provides a macromolecule comprising a first binding domain connected to a second binding domain, wherein (a) the first binding domain specifically binds to a disease characteristic ligand in a biological sample; and (b) the second binding domain specifically binds to an effector ligand in the biological sample, and induces a cellular effector function after binding to the effector ligand; wherein the macromolecule is capable of forming a multimer in the presence of the disease characteristic ligand (e.g., forming a multimer only in the presence of the disease characteristic ligand; forming a multimer substantially only in the presence of the disease characteristic ligand; or preferentially forming a multimer 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 multimer member to the disease characteristic ligand. In the presence of the disease characteristic ligand, the multimer can be formed by direct or indirect interactions between the component macromolecules (e.g., can be formed by direct association or indirect association between the macromolecules (e.g., via a disease characteristic ligand)). Exemplary first binding domains and disease characteristic ligands are provided in Section IB of the present invention. Exemplary second binding domains and effector ligands are provided in Section IC of the present invention.

[0130] In some embodiments, the multimer is a dimer (i.e., comprising two copies or forms of the macromolecule), a trimer (i.e., comprising three copies or forms of the macromolecule), or a tetramer (i.e., comprising four copies or forms of the macromolecule). The present invention also contemplates higher order multimers (e.g., hexamers, heptamers, octamers, nonamers, and decamers).

[0131] In some embodiments, the multimer is a homopolymer, that is, a multimer in which each of the two or more macromolecules is identical (e.g., identical in amino acid sequence and / or nucleotide sequence). For example, in some embodiments, the macromolecule is a polypeptide, and the two or more macromolecules are identical in amino acid sequence. Alternatively, the multimer can include two or more forms of macromolecules that are not identical in sequence, but include substantially the same first binding domain and second binding domain. For example, the two or more forms of macromolecules can be included in the same or different in sequence (e.g., amino acid sequence and / or nucleotide sequence) but have substantially the same affinity for the disease characteristic ligand or the effector ligand.

[0132] In some embodiments of the invention, the multimer is a dimer, for example, a homodimer or a heterodimer.

[0133] In some embodiments of the present invention, the second binding domain is an antibody or antibody fragment that does not bind to the effector ligand when the macromolecule is in monomeric form. 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) having affinity for the second target, wherein the VH domain and the VL domain are connected by a short linker (e.g., a G4S linker) (which does not allow intrachain pairing of the VH domain and the VL domain). In some embodiments, the second binding domain is a diabody.

[0134] In an exemplary embodiment, from N-terminus to C-terminus, the macromolecule is a fusion protein comprising 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 signal ligand; (G4S) n A linker; a second binding domain comprising a VH domain and a VL domain having affinity for the effector ligand, wherein the VH domain and the VL domain are connected by a G4S linker; and a FLAG affinity tag. In one embodiment, the disease signal ligand is TGF-β1, and the effector ligand is a TPO receptor.

[0135] In another aspect of the invention, provided herein is a multimer (eg, a dimer, trimer or tetramer) comprising two or more copies or forms of the macromolecule of the above aspects.

[0136] In another aspect of the present invention, provided herein is a complex comprising the multimer as described herein complexed with one or both of the disease characteristic ligand and the effector ligand.

[0137] In some embodiments, the macromolecule is a polypeptide. In other embodiments, the macromolecule comprises one or more non-polypeptide components, such as one or more nucleic acids or chemical components, as further described below.

[0138] In some embodiments, the macromolecule further comprises one or more of: a leader domain (e.g., a leader polypeptide), one or more linker domains, and one or more reporter domains, as further described in Section 1(E) below.

[0139] Nucleic acids, vectors and host cells

[0140] In another aspect of the present invention, a nucleic acid (e.g., RNA molecule or DNA molecule) encoding one or more of any of the above-mentioned macromolecules is provided herein. The nucleic acid can be formulated with a carrier and / or a delivery platform, such as a lipid-based carrier (e.g., lipid nanoparticle (LNP)) and / or a vector delivery system (e.g., adenovirus, adeno-associated virus (AAV), anellovirus or slow virus). Other examples of lipid-based carriers that can be used for the present invention are provided in Part I (I) of this article. For example, in some aspects, a nucleic acid (e.g., RNA molecule or DNA molecule, e.g., circular or linear: RNA molecule or DNA molecule) encoding one or more of any of the above-mentioned macromolecules is provided herein, wherein the nucleic acid is formulated with a carrier (e.g., lipid carrier, e.g., LNP). In some aspects, these nucleic acids include one or more modified nucleotides.

[0141] Further provided herein is a vector (e.g., a plasmid or viral vector) comprising or encoding any of the above nucleic acids. The vector can be formulated with a carrier (e.g., a carrier suitable for delivery to a target cell (e.g., a mammalian cell), such as a lipid-containing carrier, such as a preparation containing LNP).

[0142] Further provided herein is a host cell that has been modified to contain the above-mentioned nucleic acid or vector. Suitable host cells include bacterial cells and eukaryotic cells (e.g., mammalian cells). In certain embodiments, nucleic acid or vector as described herein are manufactured in a host cell and separated from the host cell.

[0143] ii. Formation of heterodimer macromolecules

[0144] On the other hand, the present invention provides a macromolecule pair, each macromolecule independently comprising a first binding domain connected to a second binding domain, wherein (a) the first binding domain of each macromolecule specifically binds to a disease-characteristic ligand in a biological sample; (b) the second binding domain of the first member of the macromolecule pair specifically binds to a first effector ligand in the biological sample; and (c) the second binding domain of the second member of the macromolecule pair specifically binds to a second effector ligand in the biological sample; wherein the second binding domain of the first member of the macromolecule pair and the second binding domain of the second member of the macromolecule pair induce cellular effector function after binding to the first effector ligand and the second effector ligand; wherein the macromolecule pair is capable of forming a heteromultimer in the presence of the disease-characteristic ligand (e.g., forming a heteromultimer only in the presence of the disease-characteristic ligand; forming a heteromultimer essentially only in the presence of the disease-characteristic ligand; or preferentially forming a heteromultimer 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 heteromultimer member to the disease-characteristic ligand. The multimer can be formed by direct or indirect interactions between the component macromolecules (e.g., can be formed by direct association or indirect association between the macromolecules (e.g., via a disease characteristic ligand)). Exemplary first binding domains and disease characteristic ligands are provided in Section IB of this article. Exemplary second binding domains and effector ligands are provided in Section IC of this article.

[0145] In some embodiments, the heteromultimer is a dimer (ie, comprises one copy of each member of the macromolecule).

[0146] In another aspect of the present invention, provided herein is a heteromultimer (eg, dimer) comprising at least one copy of each of the macromolecular pairs of the above aspects.

[0147] In another aspect of the present invention, provided herein is a complex comprising a heteromultimer as described herein complexed with one, two or all three of the disease characteristic ligand, the first effector ligand and the second effector ligand.

[0148] In some embodiments, each member of the macromolecular pair is a polypeptide. In other embodiments, one or both members of the macromolecular pair comprise one or more non-polypeptide components, such as one or more nucleic acids or chemical components, as further described below.

[0149] On the other hand, the present invention provides a pair of macromolecules, each macromolecule comprising a first binding domain connected to a second binding domain, wherein (a) the first binding domain of the first member of the macromolecule pair specifically binds to a first portion of a disease-characteristic ligand in a biological sample; (b) the first binding domain of the second member of the macromolecule pair specifically binds to a second portion of the disease-characteristic ligand in the biological sample; and (c) the second binding domain of each macromolecule specifically binds to an effector ligand in the biological sample and induces cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of forming a heteropolymer 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 heteropolymer member to the disease-characteristic ligand.

[0150] In another aspect of the present invention, a group consisting of three macromolecules is provided herein, each macromolecule comprising a first binding domain connected to a second binding domain, wherein (a) the first binding domain in each macromolecule specifically binds to a disease characteristic ligand in a biological sample; (b) the second binding domain of the first member of the macromolecule group specifically binds to a first effector ligand in the biological sample; (c) the second binding domain of the second member of the macromolecule group specifically binds to a second effector ligand in the biological sample; and (d) the second binding domain of the third member of the macromolecule group specifically binds to a third effector ligand in the biological sample; wherein the second binding domains of the first, second, and third members of the macromolecule pair induce cellular effector functions after binding to the first, second, and third effector ligands; wherein the macromolecule group is capable of forming heterotrimers 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 heterotrimer member to the disease characteristic ligand. In some embodiments, the disease characteristic ligand is a trimer.

[0151] In some embodiments, one or two members of the macromolecule pair or one, two, or all three of the group of three 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.

[0152] Nucleic acids, vectors and host cells

[0153] In another aspect of the present invention, a nucleic acid pair (e.g., RNA molecule pair or DNA molecule pair, e.g., circular or linear RNA molecule pair or DNA molecule pair) encoding any of the above-mentioned macromolecules is provided herein. The nucleic acid pair can be formulated together or separately with one or more carriers and / or delivery platforms (e.g., lipid-based carriers (e.g., lipid nanoparticles (LNP)) or vector delivery systems (e.g., adenovirus, AAV, anellovirus or slow virus)). Other examples of lipid-based carriers that can be used for the present invention are provided in Section I (I) herein. For example, in some aspects, a nucleic acid pair (e.g., RNA molecule pair or DNA molecule pair) encoding any of the above-mentioned macromolecules is provided herein, wherein the nucleic acid pair is formulated together with a carrier (e.g., a lipid carrier, such as LNP).

[0154] Further provided herein are vectors (e.g., plasmids or viral vectors) comprising or encoding any of the above nucleic acids. In some embodiments, each member of the nucleic acid pair is composed or encoded by a separate vector. The vector pair can be formulated together or separately with a carrier (e.g., a carrier suitable for delivery to a target cell (e.g., a mammalian cell).

[0155] Further provided herein is a host cell that has been modified to include the above-mentioned nucleic acid or vector. Suitable host cells include bacterial cells and eukaryotic cells (e.g., mammalian cells). In certain embodiments, a single host cell comprises two members of the nucleic acid pair or a vector pair comprising each member of the nucleic acid pair encoding the nucleic acid pair. In other embodiments, the first host cell comprises the first member of the nucleic acid pair (or a vector comprising or encoding the first member of the nucleic acid pair), and the second host cell comprises the second member of the nucleic acid pair (or a vector comprising or encoding the second member of the nucleic acid pair). In certain embodiments, nucleic acid or vector as described herein are manufactured in a host cell and separated from the host cell.

[0156] B. Disease-signature ligands and first binding domain

[0157] i. Disease signature ligands

[0158] The disease-characteristic ligand bound by the macromolecule or the macromolecule pair can be any moiety (eg, protein, peptide or small molecule) associated with a disease state or disorder in a cell, tissue or subject (eg, mammal, such as a human).

[0159] 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 may be present in a solution in the biological sample (e.g., may be present in the extracellular space), or may be embedded in a membrane present in the biological sample (e.g., may be embedded in a cell membrane).

[0160] 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.

[0161] In some embodiments, the disease signature ligand is a multimeric protein (e.g., a homomultimeric protein), such as 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 to one member of the dimerized disease signature ligand.

[0162] In other embodiments, the disease signature ligand is a monomeric protein.

[0163] In some embodiments, the disease characteristic 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-γ); a growth factor (e.g., transforming growth factor β (TGF-β), 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 γ-induced protein 10 (IP-10; also known as CXCL10); or a TNF family member (e.g., TNF-α). In some aspects, the cytokine is multimerized (e.g., dimerized, trimerized, or tetramerized). In some embodiments, the disease characteristic ligand is a neurotransmitter (e.g., serotonin, dopamine, or histamine).

[0164] In some embodiments, the disease characteristic ligand is an autoantigen of the organism from which the biological sample is derived (e.g., an autoantigen produced by a mammalian subject (e.g., a human subject)). In some embodiments, the autoantigen is an anti-drug antibody (ADA), such as an ADA targeting a therapeutic agent for treating a disease state or disorder of the cell, tissue, or subject. In some embodiments, the autoantigen is an autoantibody. In some embodiments, the autoantigen is a cancer antigen, such as a tumor marker.

[0165] In some embodiments, the disease characteristic ligand is a nucleic acid.

[0166] In some embodiments, the disease-characteristic ligand is a carbohydrate, a lipid, a peptide, a nucleoside, or a combination of the foregoing.

[0167] 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).

[0168] In some embodiments, the disease signature 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 signature ligand is a virus, bacteria, fungus, or fragment thereof, or an antigen (e.g., a virus, bacteria, or fungus, or fragment thereof, that causes a disease state or disorder in the cell, tissue, or subject).

[0169] ii. Disease characteristic ligand binding domain (first binding domain)

[0170] Each macromolecule provided herein comprises a first binding domain that specifically binds to the disease characteristic ligand (e.g., binds to the disease characteristic ligand as described in Section IB (i) above). In some embodiments, the first binding domain allows another binding domain to bind to the disease characteristic ligand (e.g., designed or selected so that at least two copies of the first binding domain can bind to the disease characteristic ligand and / or so that the first binding domain of each member of the macromolecule pair can bind to the disease characteristic ligand).

[0171] In some embodiments, the first binding domain comprises a polypeptide that specifically binds the disease characteristic ligand.

[0172] In certain embodiments, the polypeptide is an antibody or a fragment thereof. In certain embodiments, the antibody or a fragment thereof is 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).

[0173] 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 binder based on a fibronectin type III domain (FN3), a nanobody, a nanoCLAMP, an optimizer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR).

[0174] In some embodiments, the polypeptide is an endogenous binding domain of the organism from which the biological sample and / or the disease characteristic ligand is derived, such as 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 extracellular domain, a nuclear hormone receptor ligand binding domain, or a DNA binding domain. For example, in some embodiments, the first binding domain is a receptor comprising a cytokine (e.g., a polymerized cytokine, such as a dimer, trimer, or tetramer cytokine) or a polypeptide consisting thereof, or a receptor comprising an immunologically active polymer (e.g., an immunologically active dimer, trimer, or tetramer) or a polypeptide consisting thereof. In some embodiments, the disease characteristic ligand is IL-6, and the first binding domain is a polypeptide comprising an IL-6 receptor. In some embodiments, the disease characteristic ligand is TNF-α, and the first binding domain is a polypeptide comprising a TNF-α receptor.

[0175] In some embodiments, the first binding domain comprises an oligonucleotide that specifically binds to the disease characteristic ligand. In some embodiments, the oligonucleotide is a nucleic acid aptamer (eg, a DNA aptamer).

[0176] In some embodiments, the first binding domain comprises a chemical molecule that specifically binds to the disease characteristic ligand.

[0177] Additional binding domains that can be used in the present invention are described, for example, in Zhong and D'Antona, Antibodies, 10(2):13, 2021.

[0178] In some embodiments, the disease characteristic ligand binding domain has an affinity (K > 10 to > 100 pM, < 10 nM, > 10 nM, or > 100 nM) for the disease characteristic ligand.D value), or have a micromolar affinity for the disease-specific ligand (e.g., K D ≤1 μM). In some aspects, the disease characteristic ligand binding domain has a K of 1 nM or less. D Binds to a ligand characteristic of the disease.

[0179] In some embodiments of any macromolecule provided herein, the first binding domain has affinity for two or more disease characteristic parts. For example, the first binding domain can include at least two binding parts as described above, wherein the at least two binding parts specifically bind to at least two different disease characteristic parts.

[0180] In some embodiments comprising a macromolecular pair (e.g., embodiments as described in Sections IA(i) and IA(ii) herein), the first member and the second member of the macromolecular pair comprise a first binding domain that is identical in sequence (e.g., amino acid sequence and / or nucleotide sequence) or that is different in sequence but has substantially the same affinity for the disease characteristic ligand or the effector ligand. Alternatively, in other embodiments, the first member and the second member of the macromolecular pair comprise different first binding domains. For example, in some embodiments, the first binding domain of the first member of the macromolecular pair specifically binds to a first epitope or portion of a disease characteristic ligand in a biological sample, and the second binding domain of the second member of the macromolecular pair specifically binds to a second epitope or portion of the disease characteristic ligand in the biological sample.

[0181] For example, in some embodiments, the present disclosure provides a macromolecule pair, each macromolecule independently comprising a first binding domain connected to a second binding domain, 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-characteristic 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-characteristic ligand in a biological sample; (c) the second binding domain of the first member of the macromolecule pair specifically binds to a first effector ligand in the biological sample; and (d) the second binding domain of the second member of the macromolecule pair specifically binds to a second effector ligand in the biological sample; wherein the second binding domain of the first member of the macromolecule pair and the second binding domain of the second member of the macromolecule pair induce cellular effector function after binding to the first effector ligand and the second effector ligand; wherein the macromolecule pair is capable of forming a heteromultimer 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 heteromultimer member to the disease-characteristic ligand.

[0182] In other examples, in some embodiments, the present disclosure provides a macromolecular pair, each macromolecule independently comprising a first binding domain connected to a second binding domain, wherein (a) the first member of the macromolecular pair comprises a first binding domain 1 (FBD1) that specifically binds to a first epitope of a disease characteristic ligand in a biological sample; (b) the second member of the macromolecular pair comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of a disease characteristic ligand in a biological sample; and (c) the second binding domain specifically binds to an effector ligand in the biological sample, and induces a cellular effector function after binding to the effector ligand; wherein the macromolecule is capable of forming a multimer 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 multimer member to the disease characteristic ligand. In some embodiments, each macromolecule in the macromolecular pair comprises a second binding domain that is identical in sequence (e.g., amino acid sequence and / or nucleotide sequence) or different in sequence but has substantially the same affinity for the effector ligand.

[0183] 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, such as such that the two first binding domains do not sterically hinder each other from binding to their respective epitopes on the disease signature ligand; only partially sterically hinder each other from binding to their respective epitopes on the disease signature ligand; or do not completely hinder each other from binding to their respective epitopes on the disease signature ligand. Thus, in some aspects, the first binding domains are capable of binding to the disease signature ligand simultaneously.

[0184] 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., the affinities 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 significantly different affinities for their respective epitopes (e.g., the affinities differ by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, such as 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2500%, 5000%, 7500% or 10000%).

[0185] In some embodiments where the disease signature ligand is a polypeptide, the first binding domain binds to the disease signature ligand at a binding site containing an Arg, Lys, Asp, His, or Glu amino acid residue (or a combination thereof) (e.g., a binding site enriched in one or more of these residues).

[0186] In one embodiment, the disease signature ligand is IL-10 and the first binding domain is a polypeptide comprising an IL-10 receptor.

[0187] In one embodiment, the disease signature ligand is IL-12, and the first binding domain comprises the p40 subunit and / or the p35 subunit of IL-12.

[0188] C. Effector ligand and second binding domain

[0189] i. Effector ligand

[0190] The effector ligand bound by the macromolecule or the macromolecule pair may be any moiety (eg, protein or peptide) present in the biological sample and capable of achieving a cellular effector function after being bound by the multimer of the present invention.

[0191] In some embodiments, the effector ligand is a protein or peptide.

[0192] In some embodiments, the effector ligand is a cell surface receptor.

[0193] 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).

[0194] 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.

[0195] In some embodiments, the effector ligand is an intracellular receptor. In some embodiments, the intracellular receptor is a nuclear hormone receptor (e.g., a glucocorticoid receptor). In some embodiments where the effector ligand is an intracellular receptor, the macromolecule is delivered as RNA.

[0196] ii. Mechanism of effector ligand activation

[0197] In some embodiments, the second binding domain is an agonist of the effector ligand. In other embodiments, the second binding domain is an antagonist of the effector ligand.

[0198] In some embodiments, the effector ligand must be homodimerized to exert a cellular effector function. In some embodiments, the effector ligand is capable of homodimerization and exerts a cellular effector function at least 2, 5, 10, 100, or 1000 times as strong in homodimerized form as compared to the monomeric form. In some embodiments, the effector ligand is homodimerized in the presence of a multimer of the macromolecule. In some embodiments, the effector ligand is activated by homodimerization in the absence of its endogenous ligand.

[0199] In some embodiments, the macromolecule or the macromolecule pair exhibits conditional affinity, triggering affinity and / or dimerization affinity. For example, in some aspects, multiple macromolecules or macromolecule pairs bind to disease characteristic ligands (e.g., tumor antigens or pathogen surface markers), and this proximity results in increased affinity, which then activates effective downstream effects.

[0200] In some embodiments comprising macromolecular pairs (e.g., embodiments as described in Section IA(ii) herein), the first effector ligand and the second effector ligand must associate (e.g., be in proximity and properly 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 at least 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold stronger in heterodimerized form than in monomeric form. Thus, in some embodiments, the first effector ligand and the second effector ligand associate (e.g., be in proximity and properly oriented (e.g., heterodimerize)) in the presence of a multimer of the macromolecule.

[0201] In other embodiments comprising macromolecular pairs (e.g., embodiments as described in Section IA(ii) herein), the first effector ligand and the second effector ligand must be associated (e.g., in proximity and properly oriented (e.g., heterodimerization)) 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).

[0202] In some embodiments of any of the macromolecules and macromolecule pairs provided herein, the disease characteristic 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 after multimerization (e.g., homomultimerization or heteromultimerization)).

[0203] iii. Cell effector function

[0204] In some embodiments, the cellular effector function of the disease signature ligand is a biological activity.

[0205] In some embodiments, the cellular effector function of the disease characteristic ligand is a therapeutic activity.

[0206] 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 binding of the multimerized macromolecule or macromolecule pair to the effector ligand.

[0207] iv. Effector ligand binding domain (second binding domain)

[0208] In some embodiments, the second binding domain comprises a polypeptide that specifically binds to the effector ligand. In some embodiments, the first binding domain allows an additional binding domain to bind to the disease characteristic ligand (e.g., designed or selected so that at least two copies of the second binding domain can bind to the effector ligand).

[0209] In certain embodiments, the polypeptide is an antibody or a fragment thereof. In certain embodiments, the antibody or a fragment thereof is 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).

[0210] 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 binder based on a fibronectin type III domain (FN3), a nanobody, a nanoCLAMP, an optimizer, a repebody, a pronectin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR).

[0211] In some embodiments, the polypeptide is an endogenous binding domain. In some embodiments, the endogenous binding domain is a ligand of the effector ligand or a fragment thereof. In some embodiments, the endogenous binding domain is a viral binding protein or a fragment thereof.

[0212] 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 (eg, a DNA aptamer).

[0213] Additional binding domains that can be used in the present invention are described, for example, in Zhong and D'Antona, Antibodies, 10(2):13, 2021.

[0214] 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) in the picomolar (pM) range or <1 μM for the effector ligand. D In some embodiments, the effector ligand binding domain has an affinity for the effector ligand of 500 nM or less. In some embodiments where the effector ligand binding domain comprises two monomers, the affinity of each monomer for the effector ligand is 1-2 orders of magnitude higher than the affinity of the effector ligand binding domain monomers for each other.

[0215] In some embodiments of any macromolecule provided herein, the second binding domain has 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.

[0216] The second binding domain pair

[0217] In some embodiments comprising a macromolecular pair (e.g., embodiments as described in Section IA(ii) herein), the first member and the second member of the macromolecular pair comprise a second binding domain that is identical in sequence (e.g., amino acid sequence and / or nucleotide sequence) or different in sequence but has substantially the same affinity for the disease characteristic ligand or the effector ligand. Alternatively, in other embodiments, the first member and the second member of the macromolecular pair comprise different second binding domains. For example, in some embodiments, the second binding domain of the first member of the macromolecular pair specifically binds to a first effector ligand in a biological sample, and the second binding domain of the second member of the macromolecular pair specifically binds to a second effector ligand in the biological sample.

[0218] For example, in some embodiments comprising a macromolecular pair, the second binding domain of the first member of the macromolecular pair is a first portion of a binding moiety, and the second binding domain of the second member of the macromolecular pair is a second portion of a binding moiety.

[0219] In an exemplary embodiment, the macromolecular pair is a polypeptide pair, which comprises a leader polypeptide from N-terminus to C-terminus; a VH domain (in the first fusion protein) or a VL domain (in the second fusion protein) of a scFv, the scFv having affinity for the effector ligand, wherein the VH domain and the VL domain have low affinity for each other; a GS (G4S) 3 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 a disease signal ligand; and a FLAG affinity tag. In some embodiments, the disease characteristic ligand is TGF-β1, and the effector ligand is cluster of differentiation 3 (CD3).

[0220] In another example, the second binding domain of the first member of the macromolecular pair specifically binds to a first component of a heteromultimeric (e.g., heterodimeric) receptor, and the second binding domain of the second member specifically binds to a second component of the heteromultimeric (e.g., heterodimeric) receptor.

[0221] In an exemplary embodiment, the macromolecular pair is a polypeptide pair, wherein (i) the first member of the polypeptide pair comprises a leader polypeptide from the N-terminus to the C-terminus; a scFv, the scFv comprising a VH domain and a VL domain connected by a (G4S)3 linker, wherein the VH domain and the VL domain have affinity for a disease characteristic ligand; (G4S) n A linker; a VHH having affinity for a first component of a heteromultimeric (e.g., heterodimeric) receptor; and a FLAG affinity tag, and (ii) the second member of the polypeptide pair comprises a leader polypeptide from the N-terminus to the C-terminus; an scFv comprising a VH domain and a VL domain connected by a (G4S)3 linker, wherein the VH domain and the VL domain have affinity for a disease characteristic ligand; (G4S) n A linker; a VHH having affinity for the second component of the heteromultimer (e.g., heterodimer) receptor; and a FLAG affinity tag. In one embodiment, the disease characteristic ligand is IFN-γ, and the VHH of the first polypeptide and the second polypeptide have affinity for IL10Ra and IL10Rb, respectively.

[0222] In another example, the second binding domain of the first member of the macromolecular pair is a first component of the dimer portion and the second binding domain of the second member is a second component of the dimer portion.

[0223] In another example, the second binding domain of the first member of the macromolecular pair is a first fragment of a polypeptide chain, and the second binding domain of the second member is a second fragment of the polypeptide chain. In some embodiments, the polypeptide chain is a hormone, cytokine, or growth factor.

[0224] In an exemplary embodiment, the macromolecular pair is a polypeptide pair, wherein (i) the first member of the polypeptide pair comprises a leader polypeptide from the N-terminus to the C-terminus; a FLAG affinity tag; a first binding domain, which consists of a scFv comprising a VH domain and a VL domain connected by a (G4S)3 linker, wherein the VH domain and the VL domain have affinity for a disease signal ligand; (G4S) n A linker; and a second binding domain comprising a first portion of a binding moiety, and (ii) the second member of the polypeptide pair comprises a leader polypeptide; a FLAG affinity tag; a first binding domain consisting of a scFv comprising a VH domain and a VL domain connected by a (G4S)3 linker, wherein the VH domain and the VL domain have affinity for a first target; (G4S) n a linker; and a second binding domain comprising a second portion of the binding moiety. In one embodiment, the disease signaling ligand is TGF-β1, the effector ligand is IL-2 receptor, and the binding moiety is IL-2.

[0225] In some embodiments of any of the above examples, the second binding domain of the first member of the macromolecular pair and the second binding domain of the second member of the macromolecular pair have been engineered to have a reduced affinity for each other. In some embodiments, the second binding domain of the first member of the macromolecular pair and the second binding domain of the second member of the macromolecular pair have an affinity (K) of >1 μM (e.g., >5-10 μM) but less than 1 mM (e.g., 10-200 μM) for each other. D ).

[0226] In some embodiments of any of the macromolecules or macromolecule pairs described herein, the second binding domain comprises a conditional effector domain.

[0227] D. First Binding Domain and Second Binding Domain Pair

[0228] In some embodiments, the disease characteristic ligand is TGFβ, and the effector ligand is TPO receptor. For example, in some embodiments, the first binding domain includes 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 includes 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 a light chain variable domain (VL)).

[0229] In some embodiments, the disease characteristic 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).

[0230] In some embodiments, the disease characteristic ligand is TGFβ, and the effector ligand is IL-2 receptor (IL-2R). For example, in some embodiments, the first binding domain includes 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 include an IL-2R binding domain (e.g., a fragment and / or a modified form of IL-2). In some embodiments, the present disclosure provides a macromolecule or macromolecule pair comprising two second binding domains containing complementary fragments of IL-2R.

[0231] In some embodiments, the disease characteristic ligand is IL-8, and the effector ligand is an IL-2 receptor (IL-2R). For example, in some embodiments, the first binding domain includes an IL-8 binding domain (e.g., an anti-IL-8 antibody or antibody fragment), and the one or more second binding domains include an IL-2R binding domain (e.g., a fragment and / or modified form of IL-2). In some embodiments, the disclosure provides a macromolecule or macromolecule pair comprising two second binding domains containing complementary fragments of IL-2R.

[0232] In some embodiments, the disease characteristic ligand is IFN-γ, and the effector ligand is IL-10 receptor. For example, in some embodiments, the first binding domain includes an IFN-γ binding domain (e.g., an anti-IFN-γ antibody or antibody fragment, such as an anti-IFN-γ scFv), and the one or more second binding domains include an IL-10 receptor binding domain (e.g., an antibody or antibody fragment targeting one or more components of the IL-10 receptor). In some embodiments, the present disclosure provides a macromolecule or macromolecule pair comprising two second binding domains targeting different components of the IL-10 receptor (e.g., IL-10Ra and IL-10Rb).

[0233] In some embodiments, the disease characteristic ligand is IL-6, and the effector ligand is an IL-10 receptor. For example, in some embodiments, the first binding domain includes 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 include an IL-10 receptor binding domain (e.g., an antibody or antibody fragment targeting one or more components of the IL-10 receptor). In some embodiments, the present disclosure provides a macromolecule or macromolecule pair comprising two second binding domains targeting different components of the IL-10 receptor (e.g., IL-10Ra and IL-10Rb) and / or two first binding domains targeting different epitopes of IL-6.

[0234] In some embodiments, the disease characteristic ligand is TGFβ, and the effector ligand is differentiation cluster 3 (CD3). For example, in some embodiments, the first binding domain includes 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 one or more second binding domains include a CD3 binding domain (e.g., an anti-CD3 antibody or antibody fragment, such as an anti-CD3 heavy chain variable domain (VH) and / or a light chain variable domain (VL)). In some embodiments, the present disclosure provides a macromolecule or macromolecule pair comprising two second binding domains of different components (e.g., containing anti-CD3 VH and VL, respectively) containing a CD3 targeting portion.

[0235] In some embodiments, the disease characteristic ligand is IL-8, and the effector ligand is CD3. For example, in some embodiments, the first binding domain includes an IL-8 binding domain (e.g., an anti-IL-8 antibody or antibody fragment, such as an anti-IL-8 single chain variable fragment (scFv)), and the one or more second binding domains include a CD3 binding domain (e.g., an anti-CD3 antibody or antibody fragment, such as an anti-CD3 heavy chain VH and / or VL). In some embodiments, the present disclosure provides a macromolecule or macromolecule pair comprising two second binding domains of different components (e.g., containing anti-CD3 VH and VL, respectively) containing a CD3 targeting portion.

[0236] 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.

[0237] In some embodiments, the disease characteristic ligand binding domain (first binding domain) comprises TNFα, MCP-1 or IL-12 or a fragment and / or a modified form thereof, and the effector ligand binding domain (second binding domain) comprises IL-10 or a fragment and / or a modified form thereof. In some aspects, the effector ligand is an IL-10 receptor.

[0238] E. Leader, Reporter and Adapter

[0239] Precursor

[0240] In some embodiments of any compositions and methods provided herein, the macromolecule comprises a leader peptide, such as a leader peptide targeting a macromolecule for secretion. The leader peptide can be cut from the macromolecule before forming a polymer. In certain embodiments, the leader peptide is a mouse immunoglobulin kappa variable 3 (IgKVIII) leader peptide (e.g., UniProt ID A0A140T8P0 position M1 to G20). Other exemplary leader sequences are provided in Table 1 (SEQ ID NO: 15-31).

[0241] Table 1. Leader sequences

[0242]

[0243]

[0244] Report

[0245] 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.

[0246] Exemplary reporter moieties include, but are not limited to, affinity tags (e.g., FLAG affinity tags), fluorescent markers, and chromogenic markers (e.g., luciferase or β-lactamase). For example, in some embodiments, the reporter moiety is a near-infrared probe (e.g., indocyanine green (ICG) or methylene blue (MB)) or a near-infrared fluorescent protein or a fragment thereof. In other embodiments, the reporter moiety includes a fragment of the bait protein and is detected by adding an exogenous dye that detects the bait protein.

[0247] In some embodiments including a macromolecular pair, the first member and the second member of the macromolecular pair comprise complementary reporter moieties, such as reporter moieties that are detectable (e.g., produce a fluorescent signal) when the first member and the second member of the macromolecular pair form a multimer. For example, the first member and the second member of the macromolecular 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).

[0248] Connectors

[0249] In some embodiments of any of the compositions and methods provided herein, the macromolecule comprises one or more linker domains, such as a linker domain that connects a first binding domain to a second binding domain; a linker domain that connects one or more subdomains within the first binding domain or the second binding domain; and / or a linker domain that connects the first binding domain or the second binding domain to a leader peptide or a reporter portion.

[0250] In some embodiments, the one or more linker domains are peptide linkers. In some embodiments, the peptide linker is a GS linker. In some embodiments, the peptide linker is a glycine-serine (GS) linker, e.g., having the form GS (GS). n S) m GS connector or having the form (G n S) m GS linker, e.g., wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., n=1-5 or 5-10, e.g., n=4) and m=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (e.g., m=1-5, 5-10, or 10-15, e.g., m=5).

[0251] F. Half-life extension

[0252] Any macromolecule (e.g., polypeptide) provided herein can be modified to alter (e.g., extend) its half-life (e.g., alter (e.g., extend) its half-life (e.g., half-life in circulation (e.g., in serum)) and / or to elicit desired effector functions. 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).

[0253] In some aspects, the polypeptide provided herein is modified to include an Fc region, which extends the half-life of the polypeptide relative to the form of the polypeptide not including the Fc region. In some embodiments, the Fc region is an IgG isotype Fc region, such as 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 includes 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 can induce one or more Fc effector functions. One or more mutations that enhance the recycling based on the neonatal Fc receptor (FcRn) can be used to modify the Fc region to extend the half-life. Other Fc variants that can be used for the present invention include previously described changes in Fcγ receptor binding or Fc neonatal receptor binding and recycling through mutations Fc variants and Fc variants comprising glycosylation modifications. Variant Fc regions that can be used in the present invention include those provided in the following references: 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.

[0254] In some aspects, the polypeptides provided herein are modified to include an Fc region that alters Fcγ receptor binding and / or effector function or Fc neonatal receptor binding and / or recycling. In some aspects, the polypeptides provided herein are modified to include an Fc region that includes one or more glycosylation modifications.

[0255] In some aspects, the macromolecules (e.g., polypeptides) provided herein are modified to include human serum albumin (HSA) or a binding agent thereof, which HSA or a binding agent thereof prolongs the half-life (e.g., circulating half-life) of the polypeptide relative to a form of the polypeptide that does not include HSA or a binding agent thereof. For example, in some embodiments, the polypeptide is directly fused to HSA. In other embodiments, the polypeptide is fused to an HSA binding agent (e.g., a short peptide sequence, a VHH, or any other antibody or natural scaffold targeting HSA).

[0256] In some aspects, the modification (e.g., a heterologous moiety) increases the half-life of the macromolecule (e.g., a 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%).

[0257] G. Manufacture and Purity

[0258] In some embodiments of any of the compositions and methods provided herein, the macromolecule, macromolecule pair, nucleic acid, nucleic acid pair, polymer, or composition comprising the same is at least 95% pure (e.g., at least 95% free of any impurities or undesirable substances). In some embodiments, the macromolecule, macromolecule pair, macromolecular complex, nucleic acid, nucleic acid pair, polymer, or composition comprising the same is more than 95% pure, for example, at least 96%, 97%, 98% or 99% pure or 100% pure.

[0259] In some embodiments of any of the compositions and methods provided herein, the macromolecule, macromolecule pair, nucleic acid, nucleic acid pair, polymer, or composition comprising the same is manufactured according to one or more International Organization for Standardization (ISO) standards.

[0260] In some embodiments, the macromolecule, macromolecule pair, macromolecule complex, nucleic acid, nucleic acid pair, polymer, or composition comprising the same is manufactured according to the Good Manufacturing Practice (GMP), Good Clinical Practice (GCP) and / or Good Non-clinical Research Practice (GLP) standards of the U.S. Food and Drug Administration (FDA).

[0261] H. Biological samples

[0262] 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, such as a human subject).

[0263] In some embodiments, the biological sample is from a vertebrate (e.g., a mammal, a bird, a fish, a reptile, or an 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 an embodiment, the non-human mammal is a non-human primate (e.g., a monkey, an ape), an ungulate (e.g., a cow, a buffalo, a sheep, a goat, a pig, a camel, a llama, an alpaca, a deer, a horse, a donkey), a carnivore (e.g., a dog, a cat), a rodent (e.g., a rat, a mouse), or a lagomorph (e.g., a rabbit). In some embodiments, the biological sample is from a bird, such as a member of the avian taxon Galliformes (e.g., a chicken, a turkey, a pheasant, a quail), an Anseriformes (e.g., a duck, a goose), a Paleognathus (e.g., an ostrich, an emu), a Columbiformes (e.g., a pigeon, a pigeon), or a Psittaciformes (e.g., a parrot). In some embodiments, the biological sample is from an invertebrate, such as an arthropod (eg, an insect, arachnid, crustacean), a nematode, an annelid, a worm, or a mollusk.

[0264] I. Lipid Nanoparticles

[0265] The compositions (e.g., macromolecules, macromolecule pairs, polypeptides, nucleic acids, and compositions comprising them), methods, and delivery systems provided by the present disclosure can adopt any suitable carrier or delivery form as described herein, including lipid nanoparticles (LNPs) in certain embodiments. In certain embodiments, lipid nanoparticles include one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic or zwitterionic lipids); one or more conjugated lipids (PEG conjugated lipids or lipids conjugated to polymers as described in Table 5 of WO 2019217941; incorporated herein by reference in its entirety); one or more sterols (e.g., cholesterol).

[0266] 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, the lipid-containing nanoparticles may comprise one or more lipids in Table 4 of WO 2019217941. The lipid nanoparticles may comprise additional elements, such as polymers, such as those described in Table 5 of WO 2019217941, which is incorporated by reference.

[0267] In some embodiments, the conjugated lipid (when present) may include one or more of the following: PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), polyethylene glycolated phosphatidylethanolamine (PEG-PE), PEG succinic diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those described in Table 2 of WO 2019051289 (incorporated by reference), and combinations of the foregoing.

[0268] In some embodiments, the sterol that can be incorporated into the lipid nanoparticle includes one or more of cholesterol or cholesterol derivatives, such as those in WO 2009 / 127060 or US 2010 / 0130588 incorporated by reference. Additional exemplary sterols include plant sterols, including those described in Eygeris et al. (2020), dx.doi.org / 10.1021 / acs.nanolett.0c01386, incorporated herein by reference.

[0269] In some embodiments, lipid particles include ionizable lipids, non-cationic lipids, conjugated lipids and sterols that inhibit particle aggregation. The amount of these components can be changed independently to obtain desired characteristics. For example, in some embodiments, lipid nanoparticles include ionizable lipids, and their amount is about 20mol% to about 90mol% of total lipids (in other embodiments, it can be 20%-70% (mol), 30%-60% (mol) or 40%-50% (mol) of total lipids present in lipid nanoparticles; about 50mol% to about 90mol%); non-cationic lipids, their amount is about 5mol% to about 30mol% of total lipids; conjugated lipids, their amount is about 0.5mol% to about 20mol% of total lipids; and sterols, their amount is about 20mol% to about 50mol% of total lipids. The ratio of total lipids to nucleic acids can be changed as needed. For example, the ratio of total lipids to nucleic acids (mass or weight) can be about 10: 1 to about 30: 1.

[0270] In some embodiments, the ratio of lipid to nucleic acid (mass / mass ratio; w / w ratio) can be in the range of about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amount of lipid and nucleic acid can be adjusted to provide a desired N / P ratio, such as 3, 4, 5, 6, 7, 8, 9, 10 or higher N / P ratios. Typically, the total lipid content of the lipid nanoparticle formulation can be in the range of about 5 mg / ml to about 30 mg / mL.

[0271] Some non-limiting examples of lipid compounds that can be used (e.g., in combination with other lipid components) to form lipid nanoparticles for delivering the compositions described herein, e.g., nucleic acids described herein (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)) include:

[0272]

[0273] In some embodiments, LNPs comprising formula (i) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0274]

[0275] In some embodiments, LNPs comprising formula (ii) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0276]

[0277] In some embodiments, LNPs comprising formula (iii) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0278]

[0279] In some embodiments, LNPs comprising formula (v) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0280]

[0281] In some embodiments, LNPs comprising formula (vi) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0282]

[0283] In some embodiments, LNPs comprising formula (viii) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0284]

[0285] In some embodiments, LNPs comprising formula (ix) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0286]

[0287] in

[0288] X 1 Yes O, NR 1 Or direct key, X 2 is C2-5 alkylene, X 3 is C(=O) or a direct bond, R 1 is H or Me, R 3 is a C1-3 alkyl group, R 2 is C1-3 alkyl, or R 2 The nitrogen atom to which it is attached and X 2 1-3 carbon atoms of X together form a 4-membered, 5-membered or 6-membered ring, or X 1 YesNR 1 , R 1 and R 2 Together with the nitrogen atom to which they are attached, they form a 5- or 6-membered ring, or R 2 With R 3 Together with the nitrogen atom to which they are attached, they form a 5-, 6- or 7-membered ring, Y 1 is C2-12 alkylene, Y 2 Selected from

[0289]

[0290] n is 0 to 3, R 4 is a C1-15 alkyl group, Z 1 is a C1-6 alkylene group or a direct bond,

[0291]

[0292] (in either orientation) or not present, provided that if Z 1 is a direct key, then Z 2 does not exist;

[0293] R 5 is C5-9 alkyl or C6-10 alkoxy, R6 is C5-9 alkyl or C6-10 alkoxy, 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 a C2 alkyl group, X 1 It's O, X 2 is a straight chain C3 alkylene group, X 3 is C(=0), Y 1 is a straight chain Ce alkylene group, (Y 2 )nR 4 yes

[0294]

[0295] , R 4 is a straight chain C5 alkyl group, Z 1 is C2 alkylene, Z 2 does not exist, W is a methylene group, and R 7 is H, then R 5 and R 6 Not Cx alkoxy.

[0296] In some embodiments, LNPs comprising formula (xii) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0297]

[0298] In some embodiments, LNPs comprising formula (xi) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0299] in

[0300] In some embodiments, the LNP comprises a compound of formula (xiii) and a compound of formula (xiv).

[0301]

[0302] In some embodiments, LNPs comprising formula (xv) are used to deliver polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0303]

[0304] In some embodiments, LNPs comprising the formulation of formula (xvi) are used to deliver the polyribonucleotide (eg, cyclic polyribonucleotide, linear polyribonucleotide) compositions described herein to cells.

[0305]

[0306] in

[0307]

[0308] In some embodiments, lipid compounds used to form lipid nanoparticles for delivering compositions described herein, e.g., nucleic acids described herein (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)) are prepared by one of the following reactions:

[0309]

[0310] In certain embodiments, the LNP comprising formula (xxi) is used for delivering polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions as described herein to cells. In certain embodiments, the LNP of formula (xxi) is the LNP described in WO 2021113777 (e.g., the lipid of formula (1), such as the lipid of Table 1 of WO 2021113777).

[0311]

[0312] in

[0313] Each n is independently an integer from 2 to 15; L1 and L3 are each independently -OC(O)-* or -C(O)O-*, wherein "*" represents the point of attachment to R1 or R3;

[0314] R1 and R3 are each independently a linear or branched C9-C10-C12-methyl-1-nitropropene optionally substituted with one or more substituents selected from the group consisting of 20 Alkyl or C9-C 20alkenyl: oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkoxycarbonyl, aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonealkyl; and

[0315] R2 is selected from the group consisting of:

[0316]

[0317] In certain embodiments, the LNP comprising formula (xxii) is used for delivering polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions as described herein to cells. In certain embodiments, the LNP of formula (xxii) is the LNP described in WO 2021113777 (e.g., lipids of formula (2), such as lipids of Table 2 of WO 2021113777).

[0318]

[0319] in

[0320] Each n is independently an integer from 1 to 15;

[0321] R1 and R2 are each independently selected from the group consisting of:

[0322]

[0323] R3 is selected from the group consisting of:

[0324]

[0325] In certain embodiments, the LNP comprising formula (xxiii) is used for delivering polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions as described herein to cells. In certain embodiments, the LNP of formula (xxiii) is the LNP described in WO 2021113777 (e.g., lipids of formula (3), such as lipids of Table 3 of WO 2021113777).

[0326]

[0327] in

[0328] X is selected from -O-, -S- or -OC(O)-*, wherein * represents the point of attachment to R1;

[0329] R1 is selected from the group consisting of:

[0330]

[0331] And R2 is selected from the group consisting of:

[0332]

[0333] In some embodiments, compositions described herein (e.g., nucleic acids (e.g., cyclic polyribonucleotides, linear polyribonucleotides) or proteins) are provided in LNPs comprising ionizable lipids. In some embodiments, the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); For example, as described in Example 1 of US 9,867,888 (incorporated herein in its entirety by reference). In some embodiments, the ionizable lipid is 9Z, 12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadecane-9,12-dienoate (LP01), for example, as synthesized in Example 13 of WO 2015 / 095340 (incorporated herein in its entirety by reference). In some embodiments, the ionizable lipid is di((Z)-non-2-en-1-yl)9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), for example, as synthesized in Example 7, Example 8, or Example 9 of US2012 / 0027803 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), for example, as synthesized in Example 14 and Example 16 of WO 2010 / 053572 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is an imidazolyl cholesteryl ester (ICE) lipid (3S, 10R, 13R, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-(1H-imidazol-4-yl)propanoate, such as structure (I) from WO 2020 / 106946 (incorporated herein by reference in its entirety).

[0334] In some embodiments, the ionizable lipid can be a cationic lipid, an ionizable cationic lipid, such as a cationic lipid that can exist in a positively charged form or a neutral form according to pH, or an amine-containing lipid that can be easily protonated. In some embodiments, the cationic lipid is, for example, a lipid that can be positively charged under physiological conditions. Exemplary cationic lipids include one or more positively charged amine groups. In some embodiments, the lipid particle includes a cationic lipid prepared with one or more of a neutral lipid, an ionizable amine-containing lipid, a biodegradable acetylenic lipid, a steroid, a phospholipid including a polyunsaturated lipid, a structural lipid (e.g., a sterol), PEG, 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 exceeding 6.0. In an embodiment, the lipid nanoparticle can include a second cationic lipid with an effective pKa different from the first cationic lipid (e.g., greater than the first effective pKa). The lipid nanoparticles can comprise between 40mol% and 60mol% of cationic lipids, neutral lipids, steroids, polymer-conjugated lipids and therapeutic agents encapsulated in or associated with the lipid nanoparticles, such as nucleic acids described herein (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)). In some embodiments, nucleic acids are co-formulated with cationic lipids. Nucleic acids can be adsorbed onto the surface of LNPs (e.g., LNPs comprising cationic lipids). In some embodiments, nucleic acids can be encapsulated in LNPs (e.g., LNPs comprising cationic lipids). In some embodiments, lipid nanoparticles can comprise targeting moieties, such as targeting moieties coated with targeting agents. In embodiments, LNP formulations are biodegradable. In some embodiments, lipid nanoparticles comprising one or more lipids described herein (e.g., formula (i), (ii), (vii), and / or (ix)) encapsulate at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or 100% of the RNA molecules.

[0335] Exemplary ionizable lipids that can be used in the lipid nanoparticle formulation include, without limitation, those listed in Table 1 of WO 2019051289, which is incorporated herein by reference. Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US2016 / 0311759; I of US 20150376115 or US2016 / 0376224; I, II, or III of US20160151284; I, IA, II, or IIA of US20170210967; Ic of US20150140070; A of US2013 / 0178541; I of US2013 / 0303587 or US2013 / 0123338; ​​I of US2015 / 0141678; II, III, IV, or V of US 2015 / 0239926; I of US2017 / 0119904; I or II of 2017 / 117528; A of US2012 / 0149894; A of US2015 / 0057373; WO A of US2013 / 116126; A of US2013 / 0090372; A of US2013 / 0274523; A of US2013 / 0274504; A of US2013 / 0053572; A of WO2013 / 016058; A of WO2012 / 162210; I of US2008 / 042973; I, II, III or IV of US2012 / 01287670; I or II of US2014 / 0200257; I, II or III of US2015 / 0203446; I or III of US2015 / 0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID or III-XXIV of 2014 / 0308304; I, II, III or IV of WO2009 / 132131; A of US2012 / 01011478; I or XXXV of US2012 / 0027796; XIV or XVII of US2012 / 0058144; I of US2013 / 0323269; I of US2011 / 0117125; I, II or III of US2011 / 0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US2012 / 0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV or XVI of US2011 / 0076335; I or II of US2006 / 008378; I of US2013 / 0123338; ​​I or XAYZ of US2015 / 0064242;XVI, XVII or XVIII of US2013 / 0022649; I, II or III of US2013 / 0116307; I, II or III of US2013 / 0116307; I or II of US2010 / 0062967; IX of US2013 / 0189351; I of US2014 / 0039032; V of US2018 / 0028664; I of US 2016 / 0317458; I of US2013 / 0195920; 5, 6 or 10 of US10,221,127; III-3 of WO2018 / 081480; I-5 or I-8 of WO 2020 / 081938; US 9,867,888 18 or 25; US2019 / 0136231 A; WO 2020 / 219876 II; US2012 / 0027803 1; US2019 / 0240349 OF-02; US10,086,013 23; Miao et al. (2020) cKK-E12 / A6; WO 2010 / 053572 C12-200; Dahlman et al. (2017) 7C1; Whitehead et al. 304-O13 or 503-O13; US 9,708,628 TS-P4C2; WO 2020 / 106946 I; WO 2020 / 106946 I; and WO (1), (2), (3) or (4) of WO 2021 / 113777. Exemplary lipids further include lipids of any one of Tables 1-16 of WO 2021 / 113777. ;

[0336] In some embodiments, the ionizable lipid is MC3 (6Z, 9Z, 28Z, 31Z) - heptathriacontane -6, 9, 28, 31-tetraen-19-yl-4- (dimethylamino) butyrate (DLin-MC3-DMA or MC3), for example as described in Example 9 of WO 2019051289A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is lipid ATX-002, for example as described in Example 10 of WO 2019051289A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is (13Z, 16Z) -A, A- dimethyl-3-nonyl docos-13, 16-dien-1-amine (compound 32), for example as described in Example 11 of WO 2019051289A9 (incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is Compound 6 or Compound 22, for example as described in Example 12 of WO 2019051289A9 (incorporated herein by reference in its entirety).

[0337] 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-1-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-monomethyl PE). -dimethyl PE), l8-l-trans PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleoylphosphatidylglycerol (POPG), di-re-oleoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dihexadecyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine or a mixture thereof. It should be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids with C10-C24 carbon chains, such as lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl. In certain embodiments, other exemplary lipids include but are not limited to those described in Kim et al. (2020) dx.doi.org / 10.1021 / acs.nanolett.0c01386 incorporated herein by reference. In certain embodiments, such lipids include plant lipids (e.g., DGTS) found to improve liver transfection with mRNA.

[0338] Other examples of non-cationic lipids suitable for use in lipid nanoparticles include, but are not limited to, non-phospholipids, such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, ricinoleic acid glyceride, hexadecyl stearate, isopropyl myristate, amphoteric acrylic acid polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, polyethoxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, etc. Other non-cationic lipids are described in WO2017 / 099823 or U.S. Patent Publication US2018 / 0028664, the contents of which are incorporated herein by reference in their entirety.

[0339] In some embodiments, the non-cationic lipid is oleic acid or a compound of formula I, II or IV of US 2018 / 0028664, which is incorporated by reference in its entirety. The non-cationic lipid may account for 0-30% (mol) of the total lipid present in, for example, a 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 an embodiment, 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).

[0340] In some embodiments, the lipid nanoparticles do not comprise any phospholipids.

[0341] In some aspects, the lipid nanoparticles may further comprise components such as sterols to provide membrane integrity. An exemplary sterol that can be used in lipid nanoparticles is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs such as 5a-cholestanol, 53-coprostanol, cholesteryl-(2 , -hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether and 6-ketocholestanol; non-polar analogs such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog, such as cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT Publication WO2009 / 127060 and U.S. Patent Publication US2010 / 0130588, each of which is incorporated herein by reference in its entirety.

[0342] In some embodiments, the components that provide membrane integrity (such as sterols) can account for 0%-50% (mol) of the total lipids present in the lipid nanoparticles (e.g., 0-10%, 10%-20%, 20%-30%, 30%-40% or 40%-50%). In some embodiments, such components are 20%-50% (mol), 30%-40% (mol) of the total lipid content of the lipid nanoparticles.

[0343] In certain embodiments, lipid nanoparticles can include polyethylene glycol (PEG) or conjugate lipid molecules. Typically, these are used to suppress the aggregation of lipid nanoparticles and / or provide spatial stabilization. Exemplary conjugated lipids include but are not limited to PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic polymer lipid (CPL) conjugates and mixtures thereof. In certain embodiments, conjugated lipid molecules are PEG-lipid conjugates, such as (methoxy polyethylene glycol) conjugated lipids.

[0344] Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinic diacylglycerol (PEGS-DAG) (e.g., 4-0-(2',3'-di(tetradecanoyloxy)propyl-1-0-(w-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEG dialkoxypropyl carbamate, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEG-lipid conjugates are described, for example, in US Pat. Nos. 5,885,613, 5,876,614, 6,615, 6,634, 6,677, 6,683, 6,691, 6,734, 6,776, 6,693, 6,694, 6,734, 6,695, 6,696, 6,697, 6,698, 6,699, 6,699, 6,691, 6,69 ... 6,287,591, US2003 / 0077829, US2003 / 0077829, US 2005 / 0175682, US2008 / 0020058, US2011 / 0117125, US2010 / 0130588, US2016 / 0376224, US2017 / 0119904 and US / 099823, all of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-lipid is a compound of formula III, III-aI, III-a-2, III-b-1, III-b-2 or V of US2018 / 0028664, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the PEG-lipid has US20150376115 or US 2016 / 0376224, 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-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of the following: PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-distearylglycerol, PEG-dilaurylglyceramide, PEG -Dimyristylglyceramide, PEG-dipalmitoylglyceramide, PEG-distearylglyceramide, PEG-cholesterol (l-[8'-(cholest-5-en-3[β]-oxy)formamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-ditetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].In some embodiments, the PEG-lipid comprises PEG-DMG, 1,2-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:.

[0345]

[0346] In some embodiments, lipids conjugated to molecules other than PEG can also be used to replace PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates) and cationic polymer lipid (GPL) conjugates can be used to replace PEG-lipids or together with PEG-lipids.

[0347] Exemplary conjugated lipids (i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids) are described in the PCT and LIS patent applications listed in Table 2 of WO 2019051289A9, the contents of all of which are incorporated herein by reference in their entirety.

[0348] In some embodiments, PEG or conjugated lipids can account for 0%-20% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the content of PEG or conjugated lipids is 0.5%-10% or 2%-5% (mol) of the total lipids present in the lipid nanoparticles. The molar ratio of ionizable lipids, non-cationic lipids, sterols and PEG / conjugated lipids can be changed as needed. For example, lipid particles can include ionizable lipids of 30%-70% by mole or total weight of the composition, cholesterol of 0-60% by mole or total weight of the composition, non-cationic lipids of 0-30% by mole or total weight of the composition, and conjugated lipids of 1%-10% by mole or total weight of the composition. Preferably, the composition includes ionizable lipids of 30%-40% by mole or total weight of the composition, cholesterol of 40%-50% by mole or total weight of the composition, and non-cationic lipids of 10%-20% by mole or total weight of the composition. In some other embodiments, the composition is 50%-75% ionizable lipids, 20%-40% cholesterol, and 5% to 10% non-cationic lipids by mole or total weight of the composition, and 1%-10% conjugated lipids by mole or total weight of the composition. The composition can contain 60%-70% ionizable lipids, 25%-35% cholesterol, and 5%-10% non-cationic lipids by mole or total weight of the composition. The composition can also contain up to 90% ionizable lipids and 2% to 15% non-cationic lipids 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% to 25% cholesterol by mole or total weight of the composition, 10% to 35% conjugated lipid by mole or total weight of the composition, and 0% to 20% cholesterol by mole or total weight of the composition. 5% cholesterol; or 2%-30% ionizable lipids by mole or total weight of the composition, 2%-30% non-cationic lipids by mole or total weight of the composition, 1% to 15% cholesterol by mole or total weight of the composition, 2% to 35% conjugated lipids 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 lipids by mole or total weight of the composition and 2%-10% non-cationic lipids by mole or total weight of the composition or even 100% cationic lipids by mole or total weight of the composition.In some embodiments, the lipid particle formulation comprises an ionizable lipid, a phospholipid, 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, cholesterol, and a PEGylated lipid in a molar ratio of 60:38.5:1.5.

[0349] In some embodiments, the lipid particles comprise an ionizable lipid, a non-cationic lipid (e.g., a phospholipid), a sterol (e.g., cholesterol), and a PEGylated lipid, wherein for the ionizable lipid, the molar ratio of the lipid is in the range of 20 to 70 mole percent, with a target of 40-60, the mole percent of the non-cationic lipid is in the range of 0 to 30, with a target of 0 to 15, the mole percent of the sterol is in the range of 20 to 70, with a target of 30 to 50, and the mole percent of the PEGylated lipid is in the range of 1 to 6, with a target of 2 to 5.

[0350] In some embodiments, the lipid particle comprises a molar ratio of ionizable lipid / non-cationic lipid / sterol / conjugated lipid of 50:10:38.5:1.5.

[0351] In one aspect, the present disclosure provides lipid nanoparticle formulations comprising phospholipids, phosphatidylcholine, phosphatidylcholine, and phosphatidylethanolamine.

[0352] In certain embodiments, one or more additional compounds may also be included. Those compounds may be administered alone, or additional compounds may be included in the lipid nanoparticles of the present invention. In other words, except nucleic acid or at least the second nucleic acid, the lipid nanoparticles may contain other compounds that are different from the first nucleic acid. In a non-limiting manner, other additional compounds may be selected from the group consisting of: small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptide mimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biomaterials, or any combination thereof.

[0353] In some embodiments, LNP comprises biodegradable, ionizable lipid. In some embodiments, LNP comprises (9Z, 12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadecane-9,12-dienoate, also referred to as 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadecane-9,12-dienoate) or another ionizable lipid. See, e.g., WO 2019 / 067992, WO / 2017 / 173054, WO 2015 / 095340 and WO2014 / 136086 and the lipids of the references provided therein. In some embodiments, the terms cationic and ionizable are interchangeable in the context of LNP lipids, for example, where the ionizable lipid is cationic depending on pH.

[0354] 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 from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of LNP formulations can be about 50nm to about 100nm, about 50nm to about 90nm, about 50nm to about 80nm, about 50nm to about 70nm, about 50nm to about 60nm, about 60nm to about 100nm, about 60nm to about 90nm, about 60nm to about 80nm, about 60nm to about 70nm, about 70nm to about 100nm, about 70nm to about 90nm, about 70nm to about 80nm, about 80nm to about 100nm, about 80nm to about 90nm or about 90nm to about 100nm. In some embodiments, the average LNP diameter of LNP formulations can be about 70nm to about 100nm. In specific embodiments, the average LNP diameter of LNP formulations can be about 80nm. In some embodiments, the average LNP diameter of LNP formulations can be about 100nm. In some embodiments, the LNP formulation has an average LNP diameter ranging 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.

[0355] In some cases, LNP can be relatively homogeneous.Polydispersity index can be used to indicate the homogeneity of LNP, for example the size distribution of lipid nanoparticles.Small (for example, less than 0.3) polydispersity index usually indicates narrow size distribution.The polydispersity index of 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 certain embodiments, the polydispersity index of LNP can be about 0.10 to about 0.20.

[0356] The zeta potential of LNP can be used to indicate the electrokinetic potential of the composition. In certain embodiments, the zeta potential can describe the surface charge of LNP. Lipid nanoparticles with relatively low charge (positive or negative charge) are generally desirable because higher charged materials may not interact with cells, tissues and other elements in the body undesirably. 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.

[0357] The encapsulation efficiency of protein and / or nucleic acid describes the amount of protein and / or nucleic acid that is encapsulated or otherwise associated with LNP after preparation relative to the initial amount provided. Encapsulation efficiency is ideally higher (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing lipid nanoparticles before and after the lipid nanoparticles are broken 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 solution. Fluorescence can be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in solution. For lipid nanoparticles as described herein, the encapsulation efficiency of protein and / or nucleic acid can be at least 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In some embodiments, the encapsulation efficiency can be at least 90%. In some embodiments, the encapsulation efficiency can be at least 95%.

[0358] The LNP may optionally include one or more layers of coating. In some embodiments, the LNP may be formulated in a capsule, film or tablet having a coating. The capsule, film or tablet comprising the composition described herein may have any available size, tensile strength, hardness or density.

[0359] 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. Other exemplary lipids, formulations, methods, and characterizations of LNPs are taught by Hou et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater [Nature Review Materials] (2021).doi.org / 10.1038 / s41578-021-00358-0, which is incorporated herein by reference in its entirety (see, e.g., Hou et al. Figure 2 Exemplary lipids and lipid derivatives).

[0360] In some embodiments, in vitro or ex vivo cell liposome transfection is performed using Lipofectamine MessengerMax (ThermoFisher) or TransIT-mRNA transfection reagent (Mirus Bio). In certain embodiments, LNPs are prepared using GenVoy_ILM ionizable lipid mixtures (Precision NanoSystems). In certain embodiments, LNPs are prepared using 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the preparation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl [German Applied Chemistry] 51 (34): 8529-8533 (2012), which is incorporated herein by reference in its entirety.

[0361] LNP formulations optimized for delivery of CRISPR-Cas systems (e.g., Cas9-gRNA RNPs, gRNA, Cas9mRNA) are described in WO 2019067992 and WO 2019067910, both of which are incorporated by reference, and can be used to deliver the cyclic polyribonucleotides and linear polyribonucleotides described herein.

[0362] Additional specific LNP formulations useful for delivery of nucleic acids (e.g., cyclic polyribonucleotides, linear polyribonucleotides) are described in US 8,158,601 and US 8,168,775, both of which are incorporated by reference, including the formulation sold under the name ONPATTRO used in patisiran.

[0363] Exemplary administration of polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) LNPs may include about 0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, or 100 mg / kg (RNA). Exemplary administration of AAVs containing polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) may include about 10 11 , 10 12 , 10 13 and 10 14 MOI of vg / kg.

[0364] II. How to use

[0365] In some aspects, provided herein are methods (e.g., methods of inducing cellular effector function, regulating the state of a cell, and / or treating a disease) comprising providing access to a cell for any macromolecule or macromolecule pair; nucleic acid or nucleic acid pair; or polymer described herein.

[0366] A. Methods of Inducing Cell Effector Functions

[0367] In one aspect, the present invention provides a method for inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule comprising a first binding domain connected to a second binding domain (e.g., a macromolecule as described in Section IA(i)), wherein (a) the first binding domain specifically binds to a disease characteristic ligand in a biological sample containing the cell (e.g., a disease characteristic ligand located on or near the cell); and (b) the second binding domain specifically binds to an effector ligand in the biological sample (e.g., an effector ligand located on or near the cell) and induces the cellular effector function upon binding to the effector ligand; wherein the macromolecule is capable of forming a multimer 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 multimer member to the disease characteristic ligand.

[0368] In another aspect, provided herein is a method for inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule pair, each macromolecule independently comprising a first binding domain linked to a second binding domain (e.g., a macromolecule pair as described in Section IA(i) herein), wherein (a) the first binding domain of each macromolecule specifically binds to a disease characteristic ligand in a biological sample containing the cell (e.g., a disease characteristic ligand located on or near the cell); and (b) the second binding domain of the first member of the macromolecule pair specifically binds to the first effector ligand in the biological sample (e.g., a disease characteristic ligand located on or near the cell). and (c) the second binding domain of the second member of the macromolecule pair specifically binds to the second effector ligand in the biological sample (e.g., a second effector ligand located on or near the cell); wherein the second binding domain of the first member of the macromolecule pair and the second binding domain of the second member of the macromolecule pair induce the cell effector function after binding to the first effector ligand and the second effector ligand; wherein the macromolecule pair is capable of forming a heteromultimer 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 heteromultimer member to the disease characteristic ligand.

[0369] On the other hand, provided herein is a method for inducing cellular effector function in a cell, the method comprising providing a macromolecule or macromolecule pair described herein; a nucleic acid or a nucleic acid pair; or a multimer (e.g., a homopolymer or a heteropolymer) with access to the cell (e.g., wherein the one or more disease characteristic ligands bound by the macromolecule or macromolecule pair and the one or more effector ligands are present on or near the cell), thereby inducing cellular effector function in the cell.

[0370] In some embodiments of any of the above aspects, the cell is in a subject, and the macromolecule, nucleic acid, or multimer is administered in a therapeutically effective amount.

[0371] In some embodiments, the subject has or is suspected of having a disease or disorder characterized by abnormal levels of the disease-characteristic target, optionally wherein the subject was previously determined to have abnormal levels of the disease-characteristic target.

[0372] B. Methods of regulating the state of cells

[0373] On the other hand, the present invention provides a method for regulating the state of a cell, which method includes providing a macromolecule or a macromolecule pair; a nucleic acid or a nucleic acid pair; or a multimer (e.g., a homopolymer or a heteropolymer) described herein with a pathway into the cell (e.g., wherein the one or more disease characteristic ligands and the one or more effector ligands bound by the macromolecule or macromolecule pair are present on or near the cell), thereby regulating the state of the cell.

[0374] In some embodiments of any of the above aspects, the cell is in a subject, and the macromolecule, nucleic acid, or multimer is administered in a therapeutically effective amount.

[0375] In some embodiments, the subject has or is suspected of having a disease or disorder characterized by abnormal levels of the disease-characteristic target, optionally wherein the subject was previously determined to have abnormal levels of the disease-characteristic target.

[0376] C. Methods for determining the state of a cell

[0377] On the other hand, the present invention provides a method for determining the state of a cell, which method includes providing access to the cell for any of the macromolecules or macromolecule pairs comprising a reporter domain described herein; or nucleic acids or nucleic acid pairs encoding these macromolecules or macromolecule pairs; or multimers (e.g., homomultimers or heteromultimers) of macromolecules comprising a reporter domain (e.g., wherein the one or more disease characteristic ligands and the one or more effector ligands bound by the macromolecule or macromolecule pair are present on or near the cell), and detecting the presence of the reporter domain, thereby determining the state of the cell.

[0378] In some embodiments of any of the above aspects, the cell is in a subject, and the macromolecule, nucleic acid, or multimer is administered in a therapeutically effective amount.

[0379] In some embodiments, the subject has a disease or disorder characterized by abnormal levels of the disease-characteristic target.

[0380] Examples

[0381] The following are examples of methods of the present invention. It is understood that various other embodiments may be practiced, given the general description provided above.

[0382] Content list (example):

[0383] Example 1. A protein complex that conditionally regulates TPO receptor activity in response to TGF-β1 Example 2. Protein complex that conditionally activates IL-2R activity in response to TGF-β1 or IL-8 Example 3. Protein complex that conditionally activates IL-10R activity in response to IFN-γ or IL-6 Example 4. Fusion protein that activates T lymphocytes conditionally with TGF-β1 or IL-8

[0384] Example 1. Protein complex that conditionally regulates TPO receptor activity by TGF-β1 or IL-8

[0385] This example describes the expression, purification, and characterization of (i) a protein complex that conditionally regulates the thrombopoietin (TPO) receptor in the presence of transforming growth factor β1 (TGF-β1) and (ii) a protein complex that conditionally regulates the TPO receptor in the presence of interleukin-8 (IL-8). The general format of the fusion protein is shown in Figure 1 middle.

[0386] A. Fusion protein

[0387] TGF-β1-TPO fusion protein

[0388] This example provides a protein complex composed of two identical fusion proteins that conditionally regulate the TPO receptor in the presence of TGF-β1 (TGFb) (referred to herein as "TGFb-TPO fusion protein"). Each fusion protein comprises (from N-terminus to C-terminus): mouse immunoglobulin kappa variable 3 (IgKVIII) leader peptide (UniProt IDA0A140T8P0 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, wherein n=2, 3 or 5; and an anti-TPO receptor heavy chain variable domain (VH) and light chain variable domain (VL) (SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 10 and SEQ ID NO: 11, respectively; Table 2), these domains are connected by a G4S linker ( Figure 5A ). The proposed mechanism of action of the construct is shown in Figure 5B Middle: TGFb recruits both copies of the fusion protein via binding to its TGFb targeting domain, thereby positioning the TpoR effector domain into close proximity to bind to TpoR and induce TpoR activity.

[0389] Optionally, each fusion protein further comprises a FLAG affinity tag after the anti-TPO receptor VH and VL.

[0390] IL-8-TPO fusion protein

[0391] This example also provides a protein complex composed of two identical fusion proteins, which conditionally regulates the TPO receptor in the presence of IL-8 (referred to herein as "IL-8-TPO fusion protein"). Each fusion protein comprises (from N-terminus to C-terminus): mouse immunoglobulin kappa variable 3 (IgKVIII) leader peptide (UniProt ID A0A140T8P0 position M1 to G20); anti-IL-8 scFv (PDB 6WZM, SEQ ID NO: 12; Table 2); (G4S) n linker, wherein n=2, 3 or 5; and an anti-TPO receptor heavy chain variable domain (VH) and light chain variable domain (VL) (SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 10 and SEQ ID NO: 11, respectively; Table 2), these domains are connected by a G4S linker ( Fig. 6A ). The proposed mechanism of action of the construct is shown in Figure 6BMiddle: IL-8 recruits both copies of the fusion protein via binding to its IL-8 targeting domain, thereby positioning the TpoR effector domain into close proximity to dimerize TpoR and induce TpoR activity.

[0392] Optionally, each fusion protein further comprises a FLAG affinity tag after the anti-TPO receptor VH and VL.

[0393] Expression and purification

[0394] Fusion protein is expressed via transient transfection human embryonic kidney 293 (HEK293) cells under the control of cytomegalovirus (CMV) promoter. Anti-FLAG capture antibody is used to analyze secreted protein by western blotting or ELISA to assess expression and quality. If necessary, FLAG capture step is used, and then size exclusion chromatography (SEC) fine purification step is carried out to purify fusion protein. By western blotting, it is expected that expression produces the strong band of expected molecular weight, with minimum product related variants.

[0395] B. In vitro assays for evaluating the effects of fusion proteins on the TPO signaling pathway in the presence or absence of soluble TPO or TGF-β1

[0396] Unless otherwise indicated, HEK-BLUE TM TPO cells (InvivoGen catalog code: hkb-tpo) were used to evaluate the activity of the fusion protein. TM TPO cells were transiently transfected with a fusion protein expression construct (as described in Example 1A) to secrete the fusion protein as an autosecretory in the assay. Alternatively, supernatant from HEK293 cells transiently expressing the fusion protein was applied as a test sample to HEK-BLUE TM TPO cells. Confirmatory assays were performed using FLAG purified fusion protein material. JAK2 / STAT5 pathway activation via secreted alkaline phosphatase (SEAP) was compared between positive controls (administration of purified recombinant human TPO; transient transfection to express and secrete human TPO as autocrine), negative controls (administration of recombinant human interleukin-2 (IL-2); mock transfection plasmid) and test samples (fusion protein variants). HEK-BLUE expressing the fusion protein was transfected with 1% paraformaldehyde (PNA) to obtain a STAT5 / JAK2 / STAT5 pathway. TMTPO cells were incubated overnight in the presence or absence of TPO and in the presence or absence of TGF-β1 (for TGFb-TPO fusion protein) or IL-8 (for IL-8-TPO fusion protein). Functional fusion protein samples were further evaluated by dose titration of fusion protein samples in the presence of TGF-β1 or IL-8 (to evaluate EC50) or in the presence of TGF-β1 and TPO or IL-8 (to evaluate IC50). It is expected that the fusion protein activates TPO receptor signaling in the presence of TGF-β1 (TGFb-TPO fusion protein) or IL-8 (IL-8-TPO fusion protein).

[0397] Figure 5C The dose-response relationship between TGFb concentration and the activity of TGFb-TPO fusion protein (comprising the anti-TPO receptor VH and VL sequences of SEQ ID NO: 10 and SEQ ID NO: 11, 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 SEAP activity in the culture supernatant was measured.

[0398] Figure 6C The dose-response relationship between IL-8 concentration and the activity of IL-8-TPO fusion protein (comprising the anti-TPO receptor VH and VL sequences of SEQ ID NO: 10 and SEQ ID NO: 11, respectively) is shown. TpoR SEAP reporter cells were stimulated overnight with a constant amount of TGFb-TPO fusion protein and varying concentrations of IL-8, and SEAP activity in the culture supernatant was measured.

[0399] C. Characterization of TPO receptor agonism achieved by fusion proteins compared to native agonists

[0400] After the overnight incubation step of the assay described in Example 1B, cell samples for analysis and comparison of gene expression between cells treated with fusion proteins or soluble ligands were collected. RNA was extracted and purified according to the Qiagen RNeasy kit (Cat. No. 74104), with a threshold RNA quality number >7. RNA libraries were prepared using the Roche KAPA HyperPrep kit for gene expression profiling and sequenced on an Illumina NextSeq to generate paired-end reads of 40 nucleotides. The quality of the sequenced reads was checked using standard procedures, and alignment, counting, and analysis were performed. See, e.g., Kukurba and Montgomery, RNA sequencing and analysis. Cold Spring Harbor Protocols, 2015 (11), doi: 10.1101 / pdb.top084970.

[0401] Table 2. Sequences

[0402]

[0403]

[0404]

[0405] Example 2. Protein complex that conditionally activates IL-2R activity with TGF-β1 or IL-6

[0406] This example describes the expression, purification, and characterization of a protein complex that conditionally agonizes human interleukin-2 receptor (IL-2R) activity with (i) TGF-β1 and (ii) interleukin-8 (IL-8; also known as CXCL8). The general format of the fusion protein pair is shown in Figure 2 middle.

[0407] A. Fusion protein

[0408] TGF-β1-IL-2R fusion protein

[0409] This example provides a protein complex composed of two complementary fusion proteins (fusion proteins (a) and (b)) that conditionally regulates the IL-2 receptor (IL-2R) in the presence of TGF-β1. Each fusion protein comprises (from N-terminus to C-terminus): mouse IgKVIII leader peptide (UniProt ID A0A140T8P0 position M1 to G20); anti-TGF-β1 scFv (PDB4KV5; SEQ ID NO: 13 and 14; Table 2); (G4S)n linker, wherein n=10, 15 or 25; and (a) conditional IL-2R effector domain A (SEQ ID NO:3; Table 2) or (b) conditional IL-2R effector domain B (SEQ ID NO:4; Table 2) ( Fig. 7A ). The proposed mechanism of action of the complementary fusion protein is shown in Figure 7B Middle: TGFb recruits each of the fusion proteins via binding to its TGFb targeting domain, thereby positioning the IL-2R effector domain into close proximity to dimerize IL-2R and induce IL-2R activity.

[0410] Optionally, the fusion proteins each further comprise a FLAG affinity tag between the leader peptide and the anti-TGF-β1 scFv.

[0411] IL-8-IL-2R fusion protein

[0412] This example also provides a protein complex composed of two complementary fusion proteins that conditionally regulate the IL-2 receptor in the presence of IL-8, wherein the two complementary fusion proteins each comprise an anti-IL-8 scFv (PDB6WZM, SEQ ID NO: 12; Table 2).

[0413] Expression and purification

[0414] Fusion proteins were expressed and purified as described in Example 1A by 1) transfecting with equal amounts of each complementation construct (fusion proteins (a) and (b)) or 2) independently transfecting, purifying and premixing equal amounts of fusion proteins (a) and (b).

[0415] B. In vitro assay of IL-2R agonism on cells in the presence or absence of TGF-β1

[0416] Unless otherwise indicated, the manufacturer's protocol was used and HEK-BLUE TM CD122 / CD132 cells (Invitrogen catalog code: hkb-il2bg) were used to evaluate the activity of the fusion protein. IL-2 signaling pathway activation was compared between positive controls (administration of purified recombinant human IL-2; transient transfection to express and secrete human IL-2 as autocrine), negative controls (administration of recombinant human TPO; mock transfection plasmid) and test samples (fusion proteins (a) and (b) and their variants) via SEAP production. HEK-BLUE expressing the fusion protein TMCD122 / CD132 cells were incubated overnight in the presence or absence of IL-2 and in the presence or absence of TGF-β1. Functional fusion protein samples were further evaluated by titrating the fusion protein samples in the presence of TGF-β1 (to assess EC50). It is expected that the fusion proteins (a) and (b) administered in combination activate IL-2 signaling in the presence of TGF-β1.

[0417] Figure 7C The dose-response relationship between fusion proteins (a) and (b) and TGFb concentration is shown. IL-2 reporter cells were stimulated overnight with a constant amount of fusion proteins (a) and (b) and different concentrations of TGFb, and then SEAP activity in the culture supernatant was measured.

[0418] C. Characterization of TGFR agonism achieved by fusion proteins compared to native agonists

[0419] Transcriptome comparison between fusion protein and soluble ligand was performed as described in Example 1C.

[0420] Example 3. Protein complex that conditionally activates IL-10R activity with IFN-γ or IL-6

[0421] This example describes the expression, purification, and characterization of a protein complex that conditionally activates the human interleukin 10 receptor (IL-10 receptor) in the presence of interferon-γ (IFN-γ) or interleukin-6 (IL-6).

[0422] A. Fusion protein

[0423] IFN-γ-IL-10 fusion protein

[0424] This example provides a protein complex consisting of two complementary fusion proteins (fusion proteins (c) and (d)) that conditionally regulate the IL-10 receptor in the presence of IFN-γ. Each fusion protein comprises (from N-terminus to C-terminus): mouse IgKVIII leader peptide (UniProt ID A0A140T8P0 position M1 to G20); anti-IFN-γ scFv (PDB1T3F; SEQ ID NO: 5; Table 2); (G4S) n linker, wherein n=1, 2 or 3; and anti-IL10Ra VHH antibody (SEQ ID NO: 6; Table 2) (fusion protein (c)) or anti-IL10Rb VHH antibody (SEQ ID NO: 7; Table 2) (fusion protein (d)) ( Fig. 8A ). The general format of complementary fusion proteins is shown in Figure 3Optionally, each fusion protein further comprises a FLAG affinity tag after the anti-IL10Ra VHH antibody (fusion protein (c)) or the anti-IL10Rb VHH antibody (fusion protein (d)). The mechanism of action of the proposed construct is shown in Figure 8B Middle: IFN-γ recruits each of the fusion proteins via binding to its IFN-γ targeting domain, thereby positioning the IL-10R binding effector domains into close proximity to dimerize IL-10R and induce IL-10R activity.

[0425] Fusion proteins were expressed and purified as described in Example 1A by 1) transfecting with equal amounts of each complementation construct (fusion proteins (c) and (d)) or 2) independently transfecting, purifying and premixing equal amounts of fusion proteins (c) and (d).

[0426] IL-6-IL-10 fusion protein

[0427] This example also provides a protein complex composed of two complementary fusion proteins that conditionally regulate the IL-10 receptor in the presence of IL-6. Each fusion protein comprises (from N-terminus to C-terminus): mouse IgKVIII leader peptide (UniProt ID A0A140T8P0 position M1 to G20); anti-IL10Ra VHH antibody (first fusion protein) or anti-IL10Rb VHH antibody (second fusion protein); (G4S)2 linker; first anti-IL-6 VHH antibody (SEQ ID NO:32; first fusion protein) or second anti-IL-6 VHH antibody (SEQ ID NO:33; second fusion protein), wherein the first and second anti-IL-6 VHH antibodies bind to different epitopes on IL-6; (G4S) n linker; and a polyhistidine tag ( Fig. 9A ). Alternatively, the binding domains can be exchanged such that (i) the first fusion protein comprises the first anti-IL-6 VHH antibody and the second anti-IL-6 VHH antibody (SEQ ID NO: 33), and (ii) the second fusion protein comprises the second anti-IL-6 VHH antibody and the first anti-IL-6 VHH antibody (SEQ ID NO: 32). The proposed mechanism of action of the construct is shown in Fig. 9B Middle: IL-6 recruits each of the fusion proteins via binding to its IL-6 targeting domain, thereby positioning the IL-10R binding effector domains into close proximity to dimerize IL-10R and induce IL-10R activity.

[0428] B. In vitro assay of IL-10 agonism on cells in the presence or absence of IFN-γ or IL-6

[0429] Unless otherwise indicated, the manufacturer's protocol was used and HEK-BLUE TM IL-10 cells (Invitrogen catalog code: hkb-il10) were used to evaluate the activity of the fusion protein. JAK1 / STAT3 pathway activation was compared between positive controls (administration of purified recombinant human IL-10; transient transfection to express and secrete human IL-10 as autocrine), negative controls (administration of recombinant human TPO; mock transfection plasmids) and test samples (fusion proteins or fusion protein pairs and variants thereof as described herein) via SEAP production. HEK-BLUE expressing the fusion protein TM IL-10 cells are incubated overnight in the presence or absence of IL-10 or in the presence or absence of IFN-γ (for IFN-γ-IL-10 fusion protein) or IL-6 (for IL-6-IL-10 fusion protein). Functional fusion protein samples are further evaluated by performing dose titration of fusion protein samples in the presence of IFN-γ or IL-6 (to evaluate EC50) or in the presence of IFN-γ or IL-6 and IL-10 (to evaluate IC50). It is expected that the fusion protein or fusion protein pairs (combined administration) activate IL-10 receptor signaling with the presence of IFN-γ (for IFN-γ-IL-10 fusion protein) or IL-6 (for IL-6-IL-10 fusion protein).

[0430] Figure 8C The dose-response relationship between fusion proteins (c) and (d) and IFN-γ concentration is shown. IL-10SEAP reporter cells were stimulated with 10 nM fusion proteins (c) and (d) and different concentrations of IFNg overnight, and then SEAP activity in the culture supernatant was measured.

[0431] Fig. 9C Shown is the dose-response relationship between IL-6 concentration and the activity of two complementary fusion proteins that modulate the IL-10 receptor in the presence of IL-6. IL-10SEAP reporter cells were stimulated overnight with a constant amount of the fusion protein pair and varying concentrations of IL-6, and SEAP activity was measured in the culture supernatant.

[0432] C. Characterization of IL-10 agonism achieved by fusion protein compared to native agonist

[0433] Transcriptome comparison between fusion protein and soluble ligand was performed as described in Example 1C.

[0434] Example 4. Fusion protein that conditionally activates T lymphocytes using TGF-β1 or IL-8

[0435] This example describes the expression, purification, and characterization of a protein complex that activates T cells via cluster of differentiation 3 (CD3) in the presence of TGF-β1.

[0436] A. Expression and purification of fusion proteins

[0437] TGF-β1-CD3 fusion protein

[0438] This example provides a protein complex composed of two complementary fusion proteins (fusion proteins (e) and (f)), which activates T cells via CD3 in the presence of TGF-β1. Each fusion protein contains (from N-terminus to C-terminus): mouse IgKVIII leader peptide (UniProt ID A0A140T8P0 position M1 to G20); anti-CD3 VH domain of scFv, which is engineered to have low (KD>1μM) affinity for its matching variable domain (SEQ ID NO.8; Table 2) (fusion protein (e)), or anti-CD3 VL domain of scFv, which is engineered to have low (KD>1μM) affinity for its matching variable domain (SEQ ID NO.9; Table 2); GS (G4S) linker; and anti-TGF-β1 scFv (PDB 4KV5; SEQ ID NO:13 and 14; Table 2). The general format of the construct is shown in Figure 4 Optionally, each fusion protein further comprises a FLAG affinity tag after the anti-TGF-β1 scFv.

[0439] Fusion proteins were expressed and purified as described in Example 1A by 1) transfecting with equal amounts of each complementation construct (fusion proteins (e) and (f)) or 2) independently transfecting, purifying and premixing equal amounts of fusion proteins (e) and (f).

[0440] IL-8-CD3 fusion protein

[0441] This example provides a protein complex composed of two complementary fusion proteins that activate T cells via CD3 in the presence of IL-8. Each fusion protein comprises (from N-terminus to C-terminus): a mouse IgKVIII leader peptide (UniProt ID A0A140T8P0 position M1 to G20); an anti-CD3 VH domain of a scFv engineered to have a low (KD>1μM) affinity for its matching variable domain (SEQ ID NO.8; Table 2) (first fusion protein), or an anti-CD3 VL domain of a scFv engineered to have a low (KD>1μM) affinity for its matching variable domain (SEQ ID NO.9; Table 2) (second fusion protein); a GS (G4S) linker; and an anti-IL-8 scFv (SEQ ID NO: 12; Table 2). The general form of the construct is shown in Figure 4 The construct of this example is shown in Fig. 10A Optionally, each fusion protein further comprises a FLAG affinity tag after the anti-TGF-β1 scFv. The mechanism of action of the proposed construct is shown in Fig. 10B Middle: IL-8 recruits each of the fusion proteins via binding to its IL-8 targeting domain, thereby positioning the CD3 binding effector domains into close proximity to dimerize CD3 and induce CD3 activity.

[0442] Fusion proteins were expressed and purified as described in Example 1A by 1) transfecting with equal amounts of each complementation construct (fusion proteins (e) and (f)) or 2) independently transfecting, purifying and premixing equal amounts of fusion proteins (e) and (f).

[0443] B. In vitro assay of T cell activation in the presence or absence of TGF-β1 or IL-8

[0444] Unless otherwise indicated, the manufacturer's protocol was used and JURKAT-LUCIA TM The activity of the fusion protein was evaluated by using nuclear factor of activated T cells (NFAT) cells (Invitrogen catalog code: jktl-nfat). NFAT pathway activation was compared between positive control (administered anti-CD3 and anti-CD28 scFv), negative control (administered recombinant human TPO; mock transfection plasmid) and test samples (fusion proteins or fusion protein pairs and variants thereof as described herein) via luciferase production. JURKAT-LUCIA expressing complementary fusion protein pairs were added to the cells. TMNFAT cells are incubated overnight with anti-CD28 antibodies and in the presence or absence of anti-CD3 scFv or in the presence or absence of TGF-β1 (for TGF-β1-CD3 fusion protein) or IL-8 (for IL-8-CD3 fusion protein). By in the presence of anti-CD28 scFv+TGF-β1 (or IL-8) (to evaluate EC50) or in the presence of anti-CD28 scFv+TGF-β1 (or IL-8)+anti-CD3 scFv (to evaluate IC50) to further evaluate functional fusion protein pairs. It is expected that fusion protein or fusion protein pairs (combined administration) are activated T cells with the presence of TGF-β1 (for TGF-β1-CD3 fusion protein) or IL-8 (for IL-8-CD3 fusion protein) as conditions.

[0445] Fig. 10C The dose-response relationship between the fusion protein (e) and (f) and IL-8 concentration is shown.NFAT-lucJurkat reporter cells were stimulated with a constant amount of fusion protein in the presence or absence of 25 nM IL-8 for 24 hours, and then luciferase activity in the culture supernatant was measured.

[0446] C. Characterization of T cell activation achieved by fusion proteins compared to native agonists

[0447] Transcriptome comparison between fusion protein and soluble ligand was performed as in Example 1C.

[0448] Although the foregoing invention has been described in detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

[0449] Other embodiments are within the claims.

Claims

1. A macromolecule comprising a first binding domain connected to a second binding domain, wherein: (a) the first binding domain specifically binds to a disease characteristic ligand in a biological sample; and (b) the second binding domain specifically binds to an effector ligand in the biological sample and induces a cellular effector function after binding to the effector ligand; The macromolecule is capable of forming a multimer in the presence of the disease characteristic ligand, and the induction of the effector function by the macromolecule is conditional on the binding of each multimer member to the disease characteristic ligand.

2. The macromolecule of claim 1, wherein the multimer is a dimer, trimer or tetramer.

3. The macromolecule of claim 2, wherein the multimer is a dimer.

4. The macromolecule of claim 3, wherein the dimer is a homodimer.

5. A macromolecule pair, each macromolecule independently comprising a first binding domain linked to a second binding domain, wherein: (a) the first binding domain of each macromolecule specifically binds to a disease characteristic ligand in a biological sample; (b) the second binding domain of the first member of the macromolecular pair specifically binds to the first effector ligand in the biological sample; and (c) the second binding domain of the second member of the macromolecular pair specifically binds to a second effector ligand in the biological sample; wherein the second binding domain of the first member of the macromolecular pair and the second binding domain of the second member of the macromolecular pair induce cellular effector function upon binding to the first effector ligand and the second effector ligand; Wherein the macromolecule pair is capable of forming a heteromultimer 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 heteromultimer member to the disease characteristic ligand.

6. A macromolecule pair, each macromolecule independently comprising a first binding domain linked to a second binding domain, wherein: (a) the first member of the macromolecular pair comprises a first binding domain 1 (FBD1) that specifically binds to a first epitope of a disease characteristic ligand in a biological sample; (b) the second member of the macromolecular pair comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of a disease characteristic ligand in a biological sample; and (c) the second binding domain specifically binds to an effector ligand in the biological sample and induces a cellular effector function after binding to the effector ligand; The macromolecule is capable of forming a multimer in the presence of the disease characteristic ligand, and the induction of the effector function by the macromolecule is conditional on the binding of each multimer member to the disease characteristic ligand.

7. A macromolecule pair, each macromolecule independently comprising a first binding domain linked to a second binding domain, wherein: (a) the first member of the macromolecular pair comprises a first binding domain 1 (FBD1) that specifically binds to a first epitope of a disease characteristic ligand in a biological sample; (b) the second member of the macromolecular pair comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of a disease characteristic ligand in a biological sample; (c) the second binding domain of the first member of the macromolecular pair specifically binds to the first effector ligand in the biological sample; and (d) the second binding domain of the second member of the macromolecular pair specifically binds to a second effector ligand in the biological sample; The macromolecule is capable of forming a multimer in the presence of the disease characteristic ligand, and the induction of the effector function by the macromolecule is conditional on the binding of each multimer member to the disease characteristic ligand.

8. The macromolecule of claim 5 or 6, wherein the heteromultimer is a dimer.

9. The macromolecule or macromolecule pair according to any one of claims 1 to 8, wherein the disease characteristic ligand is a protein, a peptide or a small molecule.

10. The macromolecule or macromolecule pair as claimed in claim 9, wherein the protein is a soluble protein or an insoluble protein.

11. The macromolecule or macromolecule pair as claimed in any one of claims 1 to 8, wherein the disease characteristic ligand is a cytokine.

12. The macromolecule or macromolecule pair of claim 11, wherein the cytokine is an interleukin, an interferon, a growth factor, a chemokine, or a TNF family member.

13. The macromolecule or macromolecule pair of claim 12, 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 β (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 γ-induced protein 10 (IP-10); or the TNF family member is TNF-α.

14. The macromolecule or macromolecule pair as claimed in any one of claims 1 to 8, wherein the disease characteristic ligand is a neurotransmitter.

15. The macromolecule or macromolecule pair according to any one of claims 1 to 8, wherein the disease characteristic ligand is a cell surface receptor, a surface antigen, a membrane-bound protein, an extracellular matrix component or an integrin.

16. The macromolecule or macromolecule pair according to any one of claims 1 to 8, wherein the disease characteristic ligand is an autoantigen of the organism from which the biological sample is derived.

17. The macromolecule or macromolecule pair of claim 16, wherein the autoantigen is an anti-drug antibody (ADA), an autoantibody or a tumor marker.

18. The macromolecule or macromolecule pair as claimed in any one of claims 1 to 8, wherein the disease characteristic ligand is a nucleic acid.

19. The macromolecule or macromolecule pair according to any one of claims 1 to 8, wherein the disease characteristic ligand is a carbohydrate, a lipid, a peptide, a nucleoside or a combination thereof.

20. The macromolecule or macromolecule pair of any one of claims 1-8, wherein the disease-characteristic ligand is a hormone, an amino acid derivative, a steroid or an eicosanoid.

21. The macromolecule or macromolecule pair of any one of claims 1-8, wherein the disease characteristic ligand is a non-self antigen.

22. The macromolecule or macromolecule pair as claimed in claim 21, wherein the disease characteristic ligand is a virus, a bacterium or a fragment or antigen thereof.

23. The macromolecule or macromolecule pair as described in any one of claims 1-22, wherein the disease characteristic ligand is multimeric.

24. The macromolecule or macromolecule pair as claimed in claim 23, wherein the disease characteristic ligand is a dimer, trimer or tetramer.

25. The macromolecule or macromolecule pair of any one of claims 1-24, wherein the first binding domain, FBD1 or FBD2 comprises a polypeptide that specifically binds to the disease characteristic ligand.

26. The macromolecule or macromolecule pair of claim 25, wherein the polypeptide is an antibody or a fragment thereof.

27. The macromolecule or macromolecule pair of claim 26, wherein the antibody or fragment thereof is 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).

28. The macromolecule or macromolecule pair of claim 25, wherein the polypeptide is an antibody mimetic.

29. The macromolecule or macromolecule pair of claim 28, wherein the antibody mimetic is an affibody, an affilin, an affimer, an affitin, an alpha body, an anticalin, a lipocalin, an affinity polymer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monoclonal antibody, a fibronectin type III domain (FN3) based binder, a nanobody, a nanoCLAMP, an optimizer, a repebody, a pre-connexin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a variable lymphocyte receptor (VLR).

30. The macromolecule or macromolecule pair of claim 25, wherein the polypeptide is an endogenous binding domain.

31. The macromolecule or macromolecule pair of claim 30, wherein the endogenous binding domain is a cell receptor domain, an enzyme domain, a variable lymphocyte receptor (VLR) domain, a receptor extracellular domain, a nuclear hormone receptor ligand binding domain, or a DNA binding domain.

32. The macromolecule or macromolecule pair of any one of claims 1-24, wherein the first binding domain, FBD1 or FBD2 comprises an oligonucleotide that specifically binds to the disease characteristic ligand.

33. The macromolecule or macromolecule pair of claim 32, wherein the oligonucleotide is a nucleic acid aptamer.

34. The macromolecule or macromolecule pair as claimed in claim 33, wherein the nucleic acid aptamer is a DNA aptamer.

35. The macromolecule or macromolecule pair as described in any one of claims 1-24, wherein the first binding domain, FBD1 or FBD2 comprises a chemical molecule that specifically binds to the disease characteristic ligand.

36. The macromolecule or macromolecule pair of any one of claims 1-35, wherein the first binding domain, FBD1 or FBD2 has affinity for two or more disease characteristic portions.

37. The macromolecule or macromolecule pair of any one of claims 1-36, wherein the effector ligand is a protein or a peptide.

38. The macromolecule or macromolecule pair of any one of claims 1-37, wherein the effector ligand is a cell surface receptor or an intracellular receptor.

39. The macromolecule or macromolecule pair of claim 38, wherein the cell surface receptor is a catalytic receptor, or the intracellular receptor is a nuclear hormone receptor.

40. The macromolecule or macromolecule pair of claim 39, 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.

41. The macromolecule or macromolecule pair of claim 40, 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.

42. The macromolecule of any one of claims 1-4, 6, and 8-41, wherein the second binding domain is an agonist of the effector ligand.

43. The macromolecule of claim 42, wherein the effector ligand must be homodimerized to exert cellular effector function.

44. The macromolecule of claim 43, wherein the effector ligand is homodimerized in the presence of multimers of the macromolecule.

45. The macromolecule pair of any one of claims 5 and 7-39, wherein the first effector ligand and the second effector ligand must be associated to exert cellular effector function.

46. ​​The macromolecule pair of claim 45, wherein the first effector ligand and the second effector ligand associate in the presence of a multimer of the macromolecule.

47. The macromolecular pair of claim 45 or 46, wherein the association is heterodimerization.

48. The macromolecule or macromolecule pair of any one of claims 1-47, wherein the cellular effector function is a biological activity.

49. The macromolecule or macromolecule pair of any one of claims 1-48, wherein the cellular effector function is a therapeutic activity.

50. The macromolecule or macromolecule pair of any one of claims 1-48, wherein the cellular effector function is disease activity.

51. The macromolecule or macromolecule pair of any one of claims 1-50, wherein the second binding domain comprises a polypeptide that specifically binds to the effector ligand.

52. The macromolecule or macromolecule pair of claim 51, wherein the polypeptide is an antibody or a fragment thereof.

53. The macromolecule or macromolecule pair of claim 52, wherein the antibody or fragment thereof is a 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.

54. The macromolecule or macromolecule pair of claim 51, wherein the polypeptide is an antibody mimetic.

55. The macromolecule or macromolecule pair of claim 54, wherein the antibody mimetic is an affibody, an affilin, an affimer, an affitin, an alpha body, an anticalin, a lipocalin, an affinity polymer, a DARPin, a fynomer, a gastrobody, a knottin, a Kunitz domain peptide, a monoclonal antibody, a FN3-based binder, a nanobody, a nanoCLAMP, an optimizer, a repebody, a preconnexin, a centyrin, an obody, a peptide aptamer, a synthetic peptide, or a VLR.

56. The macromolecule or macromolecule pair of claim 51, wherein the polypeptide is an endogenous binding domain.

57. The macromolecule or macromolecule pair of claim 56, wherein the endogenous binding domain is a ligand of the effector ligand or a fragment thereof.

58. The macromolecule or macromolecule pair of claim 56, wherein the endogenous binding domain is a viral binding protein or a fragment thereof.

59. The macromolecule or macromolecule pair of any one of claims 1-50, wherein the second binding domain comprises an oligonucleotide that specifically binds to the effector ligand.

60. The macromolecule or macromolecule pair of claim 59, wherein the oligonucleotide is a nucleic acid aptamer.

61. The macromolecule or macromolecule pair of claim 60, wherein the nucleic acid aptamer is a DNA aptamer.

62. The macromolecule or macromolecule pair as described in any one of claims 1-50, wherein the first binding domain, FBD1 or FBD2 comprises a chemical molecule that specifically binds to the disease characteristic ligand.

63. The macromolecular pair of any one of claims 5 and 7-62, wherein the second binding domain of the first member of the macromolecular pair is a first portion of a binding moiety, and the second binding domain of the second member of the macromolecular pair is a second portion of a binding moiety.

64. A macromolecular pair as described in any one of claims 5 and 7-62, wherein the second binding domain of the first member of the macromolecule specifically binds to the first component of the heterodimeric receptor, and the second binding domain of the second member specifically binds to the second component of the heterodimeric receptor.

65. The macromolecular pair of any one of claims 5 and 7-62, wherein the second binding domain of the first member of the macromolecular pair is a first component of a dimer portion, and the second binding domain of the second member is a second component of a dimer portion.

66. The macromolecular pair of any one of claims 5 and 7-62, wherein the second binding domain of the first member of the macromolecular pair is a first fragment of a polypeptide chain, and the second binding domain of the second member is a second fragment of the polypeptide chain.

67. The macromolecule pair of claim 66, wherein the polypeptide chain is a hormone, a cytokine or a growth factor.

68. The macromolecular pair of any one of claims 5 and 7-67, wherein the second binding domain of the first member of the macromolecular pair and the second binding domain of the second member of the macromolecular pair have been engineered to have reduced affinity for each other.

69. The macromolecule or macromolecule pair of any one of claims 1-68, wherein the macromolecule comprises a reporter portion.

70. The macromolecule or macromolecule pair of claim 69, wherein the reporter moiety is an affinity tag, a fluorescent marker, a radioactive marker or a chromogenic marker.

71. The macromolecule or macromolecule pair of claim 70, wherein the affinity tag is a FLAG affinity tag or the chromogenic marker is luciferase or β-lactamase.

72. The macromolecule or macromolecule pair of any one of claims 1-71, further comprising one or more linker domains.

73. The macromolecule or macromolecule pair of claim 72, wherein the one or more linker domains are peptide linkers.

74. The macromolecule or macromolecule pair of claim 73, wherein the peptide linkers comprise GS linkers.

75. The macromolecule or macromolecule pair of claim 74, wherein the GS linkers comprise GS (G n S) m Connector.

76. The macromolecule or macromolecule pair of claim 74, wherein the GS linkers comprise (G n S) m Connector.

77. The macromolecule or macromolecule pair of any one of claims 1-31, 36-58, and 63-76, wherein the macromolecule is a polypeptide.

78. The macromolecule or macromolecule pair of any one of claims 1-77, wherein the biological sample is an extract, a fluid, a fraction, a cell, a tissue or a subject.

79. The macromolecule or macromolecule pair of any one of claims 1-78, wherein the macromolecule, or one or both members of the macromolecule pair, comprises a leader sequence.

80. The macromolecule or macromolecule pair of claim 79, wherein the leader sequence comprises a secretion signal.

81. The macromolecule or macromolecule pair of any one of claims 1-80, wherein the macromolecule, or one or both members of the macromolecule pair, comprises a half-life extending moiety.

82. The macromolecule or macromolecule pair of claim 81, wherein the half-life extending moiety is an Fc domain or a fragment thereof, an albumin domain or a fragment thereof, or polyethylene glycol (PEG) or a modified derivative thereof.

83. A nucleic acid encoding the macromolecule of any one of claims 1-4 and 9-82.

84. A nucleic acid pair encoding the macromolecular pair of any one of claims 5-83.

85. The nucleic acid or nucleic acid pair of claim 83 or 84, wherein the nucleic acid is RNA or DNA.

86. The nucleic acid or nucleic acid pair of any one of claims 83-85, wherein the nucleic acid is formulated with a delivery platform.

87. The nucleic acid or nucleic acid pair of claim 86, wherein the delivery platform is a lipid-based carrier or vector delivery system.

88. nucleic acid or nucleic acid pair as described in claim 87, wherein the lipid-based carrier is a lipid nanoparticle (LNP).

89. The nucleic acid or nucleic acid pair of claim 87, wherein the vector delivery system comprises or is derived from an adenovirus, an anellovirus, an AAV or a lentivirus.

90. A nucleic acid encoding the macromolecule of any one of claims 1-4 and 9-82, wherein the nucleic acid is formulated with a carrier.

91. A nucleic acid pair encoding the macromolecular pair of any one of claims 5-82, wherein the nucleic acid pair is formulated with a carrier.

92. The nucleic acid or nucleic acid pair of claim 90 or 91, wherein the nucleic acid is RNA or DNA.

93. The nucleic acid or nucleic acid pair of claim 90 or 91, wherein the carrier is a lipid-based carrier.

94. nucleic acid or nucleic acid pair as described in claim 93, wherein the lipid-based carrier is LNP.

95. A vector comprising the nucleic acid of claim 83 or 85.

96. A vector or vector pair comprising the nucleic acid pair of claim 84 or 85.

97. The vector or vector pair of claim 95 or 96, wherein the vector or the vector pair is formulated with a carrier.

98. A host cell comprising a nucleic acid or a nucleic acid pair as described in any one of claims 83-94 or a vector or a vector pair as described in any one of claims 95-97.

99. A multimer comprising two or more macromolecules as described in any one of claims 1-4 and 9-82.

100. The multimer of claim 99, wherein each macromolecule comprises the same amino acid sequence.

101. A multimer comprising at least one copy of each of the macromolecular pair described in any one of claims 5-82.

102. The macromolecular complex or macromolecule of any one of claims 1-82, or the nucleic acid or nucleic acid pair of any one of claims 83-94, wherein the macromolecular complex, macromolecule, nucleic acid or nucleic acid pair is more than 95% pure.

103. The macromolecular complex or macromolecule of any one of claims 1-82, or the nucleic acid or nucleic acid pair of any one of claims 83-94, wherein the macromolecular complex, macromolecule, nucleic acid or nucleic acid pair is manufactured according to the Good Manufacturing Practice, Good Clinical Practice and / or Good Non-clinical Research Practice standards of the U.S. Food and Drug Administration.

104. A method comprising providing access to a cell for a macromolecule or a pair of macromolecules as described in any one of claims 1-82, a nucleic acid or a pair of nucleic acids as described in any one of claims 83-94, or a multimer as described in any one of claims 99-101.

105. A method for regulating the state of a cell, the method comprising providing a pathway for a macromolecule or a macromolecule pair as described in any one of claims 1-82, a nucleic acid or a nucleic acid pair as described in any one of claims 83-94, or a multimer as described in any one of claims 99-101 to enter the cell, thereby regulating the state of the cell.

106. A method for inducing a cellular effector function in a cell, the method comprising providing access to the cell for a macromolecule or a macromolecule pair as described in any one of claims 1-82, a nucleic acid or a nucleic acid pair as described in any one of claims 83-94, or a multimer as described in any one of claims 99-101, thereby inducing the cellular effector function in the cell.

107. The method of claim 105 or 106, wherein the cell is in a subject and the macromolecule, nucleic acid, or polymer is administered in a therapeutically effective amount.

108. The method of claim 107, wherein the subject has or is suspected of having a disease or disorder characterized by abnormal levels of the disease-characteristic target, optionally wherein the subject was previously determined to have abnormal levels of the disease-characteristic target.

109. A method for determining the state of a cell, the method comprising providing a pathway for the macromolecule or macromolecule pair, a polymer thereof, or a nucleic acid or nucleic acid pair encoding the macromolecule or macromolecule pair as described in any one of claims 69-71 to enter the cell, and detecting the presence of the reporter domain, thereby determining the state of the cell.

110. A method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a macromolecule comprising a first binding domain linked to a second binding domain, wherein: (a) the first binding domain specifically binds to a disease characteristic ligand in a biological sample containing the cell; and (b) the second binding domain specifically binds to the effector ligand in the biological sample and induces the cell effector function after binding to the effector ligand; The macromolecule is capable of forming a multimer in the presence of the disease characteristic ligand, and the induction of the effector function by the macromolecule is conditional on the binding of each multimer member to the disease characteristic ligand.

111. A method of inducing a cellular effector function in a cell, the method comprising contacting the cell with a pair of macromolecules, each macromolecule independently comprising a first binding domain linked to a second binding domain, wherein: (a) the first binding domain of each macromolecule specifically binds to a disease characteristic ligand in a biological sample containing the cell; (b) the second binding domain of the first member of the macromolecular pair specifically binds to the first effector ligand in the biological sample; and (c) the second binding domain of the second member of the macromolecular pair specifically binds to a second effector ligand in the biological sample; wherein the second binding domain of the first member of the macromolecular pair and the second binding domain of the second member of the macromolecular pair induce the cellular effector function upon binding to the first effector ligand and the second effector ligand; Wherein the macromolecule pair is capable of forming a heteromultimer 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 heteromultimer member to the disease characteristic ligand.

112. A macromolecule pair, each macromolecule comprising a first binding domain linked to a second binding domain, wherein: (a) the first binding domain of the first member of the macromolecular pair specifically binds to a first portion of a disease characteristic ligand in a biological sample; (b) the first binding domain of the second member of the macromolecular pair specifically binds to the second portion of the disease characteristic ligand in the biological sample; and (c) the second binding domain of each macromolecule specifically binds to an effector ligand in the biological sample and induces a cellular effector function upon binding to the effector ligand; Wherein the macromolecule is capable of forming a heteromultimer 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 heteromultimer member to the disease characteristic ligand.

113. A group consisting of three macromolecules, each macromolecule comprising a first binding domain connected to a second binding domain, wherein: (a) the first binding domain of each macromolecule specifically binds to a disease characteristic ligand in a biological sample; (b) the second binding domain of the first member of the macromolecule group specifically binds to the first effector ligand in the biological sample; (c) the second binding domain of the second member of the macromolecule set specifically binds to a second effector ligand in the biological sample; and (d) the second binding domain of the third member of the macromolecule group specifically binds to a third effector ligand in the biological sample; wherein the second binding domain of the first, second, and third members of the macromolecular pair induces cellular effector function after binding to the first, second, and third effector ligands; Wherein the macromolecule group is capable of forming a heterotrimer 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 heterotrimer member to the disease characteristic ligand.

114. The group of three macromolecules as described in claim 113, wherein the disease-characteristic ligand is a trimer.

115. The macromolecule or macromolecule pair as described in any one of claims 1 and 5-7, wherein the binding of the second binding domain to the effector ligand is conditional on the binding of each multimer member to the disease characteristic ligand.

116. The macromolecule or macromolecule pair as described in any one of claims 1 and 5-7, wherein the binding of the second binding domain to the effector ligand is not conditional on the binding of each multimer member to the disease characteristic ligand.

117. A plurality of macromolecules, each macromolecule independently comprising a first binding domain linked to a second binding domain, wherein: (a) the first binding domain of each macromolecule specifically binds to a disease characteristic ligand in a biological sample; and (b) the second binding domain of each macromolecule specifically binds to the effector ligand in the biological sample; The plurality of macromolecules are capable of forming a multimer in the presence of the disease characteristic ligand, and the induction of the effector function by the macromolecule is conditional on the binding of the disease characteristic ligand to each multimer member.

118. The plurality of macromolecules of claim 117, wherein the multimer is a homodimer, homotrimer or homotetramer.

119. The plurality of macromolecules of claim 117, wherein the polymer is a heterodimer, heterotrimer or heterotetramer.

120. The plurality of macromolecules of any one of claims 117-119, wherein at least one macromolecule comprises a disease signature ligand that is a protein, peptide, or small molecule.

121. A plurality of macromolecules as described in any one of claims 117-120, wherein the binding of the second binding domain to the effector ligand is conditional on the binding of each multimer member to the disease characteristic ligand.

122. The plurality of macromolecules of any one of claims 117-120, wherein the binding of the second binding domain to the effector ligand is not conditional upon every multimeric member binding to the disease characteristic ligand.

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