Compositions and methods

Abstract

This specification provides polymers that conditionally induce cellular effector functions (e.g., biological or therapeutic activity) based on the presence of disease signature ligands, compositions containing the same, and methods of using the same. In some embodiments, the cellular effector function includes therapeutic activity. In some embodiments, the cellular effector function includes disease activity. In some embodiments of any of the above embodiments, the cellular effector function is biological activity. In some embodiments, the cellular effector function is therapeutic activity. In some embodiments, the cellular effector function is disease activity.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 495,509, filed on April 11, 2023, and U.S. Provisional Patent Application No. 63 / 613,644, filed on December 21, 2023, the entire contents of each of which are incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. A copy of the XML created on April 9, 2024, is named 51661 - 005WO4_Sequence_Listing_4_9_24 and is 43,890 bytes in size.

Background Art

[0003] There is a need in the art for polymers that conditionally induce cell effector functions (e.g., biological or therapeutic activities) based on the presence of disease signature ligands and methods of using the same.

Summary of the Invention

Means for Solving the Problems

[0004] In a first aspect, herein provided is a polymer complex comprising two polymers, each polymer comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein: (a) the FBD specifically binds to a disease signature ligand in a biological sample; (b) the SBD specifically binds to an effector ligand in the biological sample and induces a cell effector function upon binding to the effector ligand; the two polymers are conjugated to each other; and induction of the effector function by the polymer complex is conditional upon each of the two polymers binding to the disease signature ligand.

[0005] In a second aspect, the Specified Polymer Complex is provided, comprising two polymers, each polymer comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain: (a) the FBD specifically binds to a disease signature ligand in a biological sample; (b) the SBD specifically binds to an effector ligand in a biological sample, and upon binding to the effector ligand, induces cellular effector function; the two polymers are conjugated to each other; and the induction of effector function by the polymer complex is conditional on each of the two polymers binding to the disease signature ligand, wherein the two polymers are identical.

[0006] In a third aspect, the Specified Polymer Complex is provided, comprising a pair of polymers, each polymer independently comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain: (a) the FBD of each polymer specifically binds to a disease signature ligand in a biological sample; (b) the first member of the pair of polymers comprises a second binding domain 1 (SBD1) that specifically binds to a first effector ligand in a biological sample; (c) the second member of the pair of polymers comprises a second binding domain 2 (SBD2) that specifically binds to a second effector ligand in a biological sample; SBD1 and SBD2 induce cellular effector function when they bind to the first and second effector ligands; the first member and the second member of the pair of polymers are conjugated to each other; and the induction of effector function by the polymer complex is conditional on each of the pair of polymers binding to the disease signature ligand.

[0007] In a fourth aspect, the present invention provides for a polymeric complex comprising a pair of polymers, each polymer independently comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain: (a) the first member of the pair of polymers comprises a first binding domain 1 (FBD1) which specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) The second member of the pair of polymers comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of a disease signature ligand in a biological sample; (c) SBD specifically binds to an effector ligand in a biological sample, and upon binding to the effector ligand, induces cellular effector function; the two polymers are conjugated to each other; and the induction of effector function by the polymer complex is conditional on each of the two polymers binding to the disease signature ligand.

[0008] In some embodiments of the first to fourth aspects, the conjugation is a covalent bond. In some embodiments, the covalent conjugation includes a chemical linker or a polypeptide linker. In some embodiments, the covalent conjugation includes a disulfide bond between polymers.

[0009] In some embodiments of the first to fourth aspects, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a complementary moiety of a pair, where each polymer contains one member of the pair. In some embodiments, the complementary moiety of the pair is biotin and avidin; barnase and burster; a complementary aptamer of the pair; or a complementary polypeptide of the pair. In some embodiments, the complementary polypeptide pair is a pair of manipulated Fc fragments, e.g., a knobs-into-holes pair.

[0010] In some embodiments of any of the above-described aspects, the conjugation stereoorients the two polymers to enable conditional co-binding of a disease signal and an effector ligand.

[0011] In some embodiments, non-covalent conjugation is mediated by the binding of FBD to disease signature ligands.

[0012] In some embodiments of the second to fourth aspects, the conjugation stereoorients the first member and the second member of the pair of polymers to enable conditional co-binding of disease signaling and effector ligands.

[0013] In some embodiments, non-covalent conjugation is mediated by the binding of FBD to disease signature ligands.

[0014] In a fifth aspect, the Specified Polymer Complex is provided, comprising two polymers, each polymer comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain: (a) the FBD specifically binds to a disease signature ligand in a biological sample; (b) the SBD specifically binds to an effector ligand in a biological sample, and upon binding to the effector ligand, induces cellular effector function; the two polymers are not conjugated to each other in the absence of the disease signature ligand; and the induction of effector function by the polymer complex is conditional on each of the two polymers binding to the disease signature ligand.

[0015] In a sixth aspect, the Specified Polymer Complex is provided, comprising a pair of polymers, each polymer independently comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain: (a) the first member of the pair of polymers comprises a first binding domain 1 (FBD1) that specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) the second member of the pair of polymers comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of a disease signature ligand in a biological sample; (c) the SBD specifically binds to an effector ligand in a biological sample, and upon binding to the effector ligand, induces cellular effector function; the two polymers are not conjugated to each other in the absence of the disease signature ligand; and the induction of effector function by the polymer complex is conditional on each of the two polymers binding to the disease signature ligand.

[0016] In some embodiments, the first member of a pair of polymers and the second member of a pair of polymers are conjugated non-covalently to each other in the presence of a disease signature ligand, and the non-covalent conjugation is mediated by the disease signature ligand.

[0017] In a seventh aspect, the Specified Polymer provides a polymer comprising two FBDs linked to two SBDs by a linker domain, wherein: (a) the FBDs specifically bind to a disease signature ligand in a biological sample; (b) the SBDs specifically bind to an effector ligand in a biological sample, and upon binding to the effector ligand, induce cellular effector function; and the polymer can adopt a configuration that allows the two SBDs to bind to the effector ligand in the presence of the disease signature ligand, wherein the induction of effector function by the polymer is conditional on each of the two FBDs binding to the disease signature ligand.

[0018] In the eighth aspect, the Specified Polymer provides a polymer comprising FBD1 and FBD2 linked to two SBDs by a linker domain, wherein (a) FBD1 specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) FBD2 specifically binds to a second epitope of the disease signature ligand in a biological sample; and (c) SBDs specifically bind to an effector ligand in a biological sample, and upon binding to the effector ligand, induce cellular effector function; the polymer can adopt a configuration that allows the two SBDs to bind to the effector ligand in the presence of the disease signature ligand, and the induction of effector function by the polymer is conditional on each of FBD1 and FBD2 binding to the disease signature ligand.

[0019] In a ninth aspect, the Specified Information provides a polymer comprising two FBDs linked to SBD1 and SBD2 by a linker domain, wherein (a) the FBDs specifically bind to a disease signature ligand in a biological sample; (b) SBD1 specifically binds to a first effector ligand in a biological sample; (c) SBD2 specifically binds to a second effector ligand in a biological sample; SBD1 and SBD2 induce cellular effector function upon binding to the first and second effector ligands; and the polymer can adopt a configuration that allows SBD1 and SBD2 to bind to the first and second effector ligands in the presence of the disease signature ligands, wherein the induction of effector function by the polymer is conditional on each of the two FBDs binding to the disease signature ligand.

[0020] In a tenth aspect, the Specified Polymer is provided, comprising FBD1 and FBD2 linked to SBD1 and SBD2 by linker domains, wherein (a) FBD1 specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) FBD2 specifically binds to a second epitope of the disease signature ligand in a biological sample; (c) SBD1 specifically binds to a first effector ligand in a biological sample; (d) SBD2 specifically binds to a second effector ligand in a biological sample; SBD1 and SBD2 induce cellular effector function upon binding to the first and second effector ligands; the polymer can adopt a configuration that enables the binding of SBD1 and SBD2 to the first and second effector ligands in the presence of the disease signature ligand, and the induction of effector function by the polymer is conditional on each of FBD1 and FBD2 binding to the disease signature ligand.

[0021] In certain embodiments described above, the first binding domain and the second binding domain are covalently linked (for example, by a small molecule, a peptide (for example, as a single polypeptide chain comprising the first and second binding domains), or a combination thereof).

[0022] In some embodiments of any of the above embodiments, the disease signature ligand comprises a protein, peptide, or small molecule. In some embodiments, the protein comprises a soluble protein or an insoluble protein. In some embodiments of any of the above embodiments, the disease signature ligand is a protein, peptide, or small molecule. In some embodiments, the protein is a soluble protein or an insoluble protein.

[0023] In some embodiments of any of the above embodiments, the disease signature ligand includes a cytokine. In some embodiments, the disease signature ligand is a cytokine. In some embodiments, the cytokine is an interleukin, interferon, growth factor, chemokine, member of the TNF family, or VEGF. In some embodiments, the disease signature ligand is an interleukin. In some embodiments, the interleukin is IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15, IL-17, or IL-23; the interferon is IFN-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 chemotactic protein-1 (MCP-1) or interferon-γ-inducible protein 10 (IP-10); the member of the TNF family is TNF-α; or VEGF is VEGF-A or VEGF-B.

[0024] In some embodiments of any of the above embodiments, the disease signature ligand includes a neurotransmitter.

[0025] In some embodiments of any of the above embodiments, the disease signature ligand includes a cell surface receptor, a surface antigen, a membrane-bound protein, an extracellular matrix component, or an integrin.

[0026] In some embodiments of any of the above embodiments, the disease signature ligand includes an autoantigen of the organism from which the biological sample originates. In some embodiments, the autoantigen is an anti-drug antibody (ADA), an autoantibody, or a tumor marker.

[0027] In some embodiments of any of the above embodiments, the disease signature ligand includes a nucleic acid.

[0028] In some embodiments of any of the above embodiments, the disease signature ligand includes carbohydrates, lipids, peptides, nucleosides, or combinations thereof.

[0029] In some embodiments of any of the above embodiments, the disease signature ligand includes a hormone, an amino acid derivative, a steroid, or an eicosanoid.

[0030] In some embodiments of any of the above embodiments, the disease signature ligand includes a non-self antigen. In some embodiments, the disease signature ligand is a non-self antigen. In some embodiments, the disease signature ligand is a virus, a bacterium, or a fragment or antigen thereof.

[0031] In some embodiments of any of the above-described aspects, the disease signature ligand is a polymer. In some embodiments, the disease signature ligand is a dimer, trimer, or tetramer.

[0032] In some embodiments of any of the above-described aspects, FBD, FBD1, or FBD2 comprises a polypeptide that specifically binds to a disease signature ligand.

[0033] In some embodiments, the polypeptide comprises an antibody or a fragment thereof. In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is scFv, monospecific tandem scFv (taFv), bispecific taFv, VHH, VNAR, Fab, monospecific single-stranded diabody, bispecific single-stranded diabody, or biaffinity retargeted antibody (DART).

[0034] In some embodiments, the polypeptide comprises an antibody mimetic. In some embodiments, the polypeptide is an antibody mimetic. In some embodiments, the antibody mimetic is an afibody, affilin, afimer, afitin, alphabody, anticarin, lipocalin, avimer, DARPin, finomer, gastric body, notottin, Kunitz domain peptide, monobody, fibronectin type III domain (FN3) based binder, nanoantibody, nanoCLAMP, optimizer, lipibody, pronectin, centinlin, obody, peptide aptamer, synthetic peptide, or variable lymphocyte receptor (VLR).

[0035] In some embodiments, the polypeptide includes an endogenous binding domain. In some embodiments, the endogenous binding domain includes a cell receptor domain, an enzyme domain, a variable lymphocyte receptor (VLR) domain, an extracellular receptor domain, a nuclear hormone receptor ligand-binding domain, or a DNA-binding domain. In some embodiments, the polypeptide is an endogenous binding domain. In some embodiments, the endogenous binding domain is a cell receptor domain, an enzyme domain, a variable lymphocyte receptor (VLR) domain, an extracellular receptor domain, a nuclear hormone receptor ligand-binding domain, or a DNA-binding domain.

[0036] In some embodiments of any of the above-described aspects, FBD, FBD1, or FBD2 comprises an oligonucleotide that specifically binds to a disease signature ligand. In some embodiments, the oligonucleotide is a nucleic acid aptamer. In some embodiments, the nucleic acid aptamer is a DNA aptamer.

[0037] In some embodiments of any of the above-described models, FBD, FBD1, or FBD2 comprises a chemical molecule that specifically binds to a disease signature ligand.

[0038] In some embodiments of any of the above-described aspects, FBD, FBD1, or FBD2 has affinity for two or more disease signature portions.

[0039] In some embodiments of any of the above embodiments, the effector ligand comprises a protein or a peptide.

[0040] In some embodiments of any of the above embodiments, the effector ligand includes a cell surface receptor or an intracellular receptor. In some embodiments, the cell surface receptor includes a catalytic receptor, or the intracellular receptor is a nuclear hormone receptor. In some embodiments, the catalytic receptor includes 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 includes 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. In some embodiments of any of the above embodiments, the effector ligand includes a cell surface receptor or an intracellular receptor. In some embodiments, the cell surface receptor is a catalytic receptor, or the intracellular receptor is a nuclear hormone receptor. In some embodiments, the catalytic receptor is a receptor tyrosine kinase (RTK), a receptor serine / threonine kinase (RSK), a type 1 cytokine receptor, a type 2 cytokine receptor, or a tumor necrosis factor (TNF) superfamily receptor. In some embodiments, the RTK is VEGFR, the RSK is TGFBR2, the type 1 cytokine receptor is IL-2R, the type 2 cytokine receptor is IL10R, or the TNF superfamily receptor is TNFR2 or 4-1BB.

[0041] In some embodiments of the first, second, fifth, seventh, and eighth aspects, the SBD comprises an agonist of the effector ligand. In some embodiments, the effector ligand must be homodimerized to exert its cellular effector function. In some embodiments, the effector ligand is homodimerized in the presence of a polymer complex or a polymer and a disease signature ligand.

[0042] In some embodiments of the third, fourth, ninth, and tenth aspects, the first effector ligand and the second effector ligand must be associated to exert cellular effector function. In some embodiments, the first effector ligand and the second effector ligand are associated in the presence of a polymer complex or a polymer and a disease signature ligand. In some embodiments, the association is heterodimerization.

[0043] In some embodiments of any of the above embodiments, the cell effector function includes biological activity. In some embodiments, the cell effector function includes therapeutic activity. In some embodiments, the cell effector function includes disease activity. In some embodiments of any of the above embodiments, the cell effector function is biological activity. In some embodiments, the cell effector function is therapeutic activity. In some embodiments, the cell effector function is disease activity.

[0044] In some embodiments of any of the above-described aspects, SBD, SBD1, or SBD2 comprises a polypeptide that specifically binds to an effector ligand.

[0045] In some embodiments, the polypeptide comprises an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof comprises scFv, monospecific taFv, bispecific taFv, VHH, VNAR, Fab, monospecific single-stranded diabody, bispecific single-stranded diabody, or DART. In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is scFv, monospecific taFv, bispecific taFv, VHH, VNAR, Fab, monospecific single-stranded diabody, bispecific single-stranded diabody, or DART.

[0046] In some embodiments, the polypeptide comprises an antibody mimetic. In some embodiments, the antibody mimetic comprises an afibody, afirin, afimer, afitin, alphabody, anticarin, lipocalin, avimer, DARPin, finomer, gastric body, notottin, Kunitz domain peptide, monobody, FN3-based binder, nanoantibody, nanoCLAMP, optimizer, lipibody, pronectin, centinlin, obody, peptide aptamer, synthetic peptide, or VLR. In some embodiments, the polypeptide is an antibody mimetic. In some embodiments, the antibody mimetic is an afibody, affilin, afimer, afitin, alphabody, anticarin, lipocalin, avimer, DARPin, finomer, gastric body, notottin, Kunitz domain peptide, monobody, FN3-based binder, nanoantibody, nanoCLAMP, optimizer, lipibody, pronectin, centinlin, obody, peptide aptamer, synthetic peptide, or VLR.

[0047] In some embodiments, the polypeptide includes an endogenous binding domain. In some embodiments, the endogenous binding domain includes the ligand of an effector ligand or a fragment thereof. In some embodiments, the endogenous binding domain includes a virus-binding protein or a fragment thereof. In some embodiments, the polypeptide is the endogenous binding domain. In some embodiments, the endogenous binding domain is the ligand of an effector ligand or a fragment thereof. In some embodiments, the endogenous binding domain is a virus-binding protein or a fragment thereof.

[0048] In some embodiments of any of the above configurations, SBD, SBD1, or SBD2 includes an oligonucleotide that specifically binds to an effector ligand. In some embodiments, the oligonucleotide is a nucleic acid aptamer. In some embodiments, the nucleic acid aptamer is a DNA aptamer.

[0049] In some embodiments of any of the above-described aspects, the FBD includes a chemical molecule that specifically binds to a disease signature ligand.

[0050] In some embodiments of the third, fourth, ninth, and tenth aspects, SBD1 includes a first portion of the coupling, and SBD2 includes a second portion of the coupling.

[0051] In some embodiments of the third, fourth, ninth, and tenth aspects, SBD1 specifically binds to a first component of the heterodimer receptor, and SBD2 specifically binds to a second component of the heterodimer receptor.

[0052] In some embodiments of the third, fourth, ninth, and tenth aspects, SBD1 includes a first component of the dimer portion, and SBD2 includes a second component of the dimer portion.

[0053] In some embodiments of the third, fourth, ninth, and tenth aspects, SBD1 comprises a first fragment of the polypeptide chain, and SBD2 is a second fragment of the polypeptide chain. In some embodiments, the polypeptide chain comprises a hormone, cytokine, or growth factor. In some embodiments of the third, fourth, ninth, and tenth aspects, SBD1 is a first fragment of the polypeptide chain, and SBD2 is a second fragment of the polypeptide chain. In some embodiments, the polypeptide chain is a hormone, cytokine, or growth factor.

[0054] In some embodiments of the third, fourth, ninth, and tenth aspects, SBD1 and SBD2 are designed to have reduced affinity for each other.

[0055] In some embodiments of any of the above embodiments, the polymer includes a reporter portion. In some embodiments, the reporter portion includes 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 β-lactamase.

[0056] In some embodiments of any of the above embodiments, the polymer complex or polymer 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 one or more GS linkers. In some embodiments, the GS linker comprises one or more GS(G) n S) m Linker or one or more (G n S) m Includes a linker. In some embodiments, the GS linker is one or more (G4S) m Includes linker.

[0057] In some embodiments of any of the above-described models, the polymer includes a polypeptide.

[0058] In some of the embodiments described above, the polymer is a polypeptide.

[0059] In some embodiments of any of the above embodiments, the biological sample includes an extract, fluid, fraction, cells, tissue, or subject.

[0060] In some embodiments of any of the above configurations, the polymer or one or both members of a pair of polymers include a leader sequence. In some embodiments, the leader sequence includes a secretion signal.

[0061] In some embodiments of any of the above-described aspects, the polymer complex or one or both polymers of the polymer complex include a half-life extension portion. In some embodiments, the half-life extension portion is an Fc domain or a fragment thereof, an albumin domain or a fragment thereof, or polyethylene glycol (PEG) or a modified derivative thereof.

[0062] In another embodiment, nucleic acids encoding any one of the first, fifth, seventh, eighth, ninth, and tenth embodiments are provided herein.

[0063] In another embodiment, this specification provides a pair of nucleic acids encoding a pair of polymers according to the second, third, fourth, or sixth embodiment.

[0064] In some embodiments, nucleic acids are RNA or DNA.

[0065] In some embodiments, nucleic acids are formulated using a delivery platform.

[0066] In some embodiments, the delivery platform is a lipid-based carrier or vector delivery system. In some embodiments, the lipid-based carrier is lipid nanoparticles (LNPs). In some embodiments, the vector delivery system includes or is derived from adenoviruses, aneroviruses, AAVs, or lentiviruses.

[0067] In another embodiment, when the nucleic acid is formulated together with a carrier as described herein, a nucleic acid encoding a polymer according to any one of the first, fifth, seventh, eighth, ninth, and tenth embodiments is provided.

[0068] In another embodiment, this specification provides a pair of nucleic acids encoding a pair of polymers according to the second, third, fourth, or sixth embodiment, which are formulated together with a carrier.

[0069] In some embodiments, the nucleic acid is RNA or DNA.

[0070] In some embodiments, the carrier is a lipid-based carrier. In some embodiments, the lipid-based carrier is an LNP.

[0071] In another embodiment, the Disclosure provides a vector comprising a nucleic acid as described above.

[0072] In another embodiment, the Disclosure provides a vector or pair of vectors comprising any pair of nucleic acids as described above.

[0073] In some embodiments, the vector or pair of vectors is formulated together with a carrier.

[0074] In another embodiment, the Disclosure provides a host cell comprising any one of the nucleic acids, paired nucleic acids, vectors, or paired vectors described above.

[0075] In some embodiments, the polymer complex, polymer, nucleic acid, or paired nucleic acid has a purity of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or a purity of more than 99%.

[0076] In some embodiments, polymeric complexes, polymers, nucleic acids, or paired nucleic acids are manufactured in accordance with U.S. Food and Drug Administration (FDA) Good Manufacturing Practices (GMP), Good Clinical Practices (GCP), and / or Good Laboratory Practices (GLP) standards.

[0077] In another embodiment, the present disclosure provides a polymeric complex comprising two polymers, each polymer comprising a VEGF-binding domain linked to a 4-1BB-binding domain, the 4-1BB-binding domain inducing cellular effector function upon binding to 4-1BB; the two polymers being conjugated to each other; and the induction of effector function by the polymeric complex being conditional on each of the two polymers binding to VEGF. See, for example, the drawings and examples of this specification.

[0078] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a complementary part of a pair, and each polymer contains one member of that pair. In some embodiments, the complementary polypeptide pair is a pair of Fc fragments. In some embodiments, the Fc fragment pair is a knob-into-hole pair. In some embodiments, the two polymers are identical.

[0079] In another embodiment, the disclosure provides a polymeric complex comprising two polymers, each polymer comprising a VEGF-binding domain linked to a 4-1BB-binding domain, the 4-1BB-binding domain inducing cellular effector function upon binding to 4-1BB; the two polymers are not conjugated to each other in the absence of VEGF; and the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to VEGF. In some embodiments, the first member of the pair of polymers and the second member of the pair of polymers are non-covalently conjugated to each other in the presence of VEGF, and the non-covalent conjugation is mediated by VEGF. See, for example, the drawings and examples of this specification.

[0080] In some embodiments, the polymer or one or both members of a pair of polymers include a half-life extension portion. In some embodiments, the half-life extension portion is an Fc domain or a fragment thereof.

[0081] In some embodiments of either of the two embodiments described above, the VEGF-binding domain is an anti-VEGF scFv. In some embodiments, the VEGF-binding domain is a VEGF receptor trap.

[0082] In some embodiments of either of the two above-described aspects, the 4-1BB binding domain is an anti-4-1BB scFv.

[0083] In some embodiments, one of the polymers includes SEQ ID NOs. 36 and 38.

[0084] In some embodiments, one of the polymers includes SEQ ID NOs. 37 and 38.

[0085] In some embodiments, one of the polymers includes SEQ ID NOs: 36 and 39.

[0086] In some embodiments, one of the polymers includes SEQ ID NOs. 37 and 39.

[0087] In some embodiments, both polymers include SEQ ID NOs. 36 and 38.

[0088] In some embodiments, both polymers include SEQ ID NOs. 37 and 38.

[0089] In some embodiments, both polymers include SEQ ID NOs. 36 and 39.

[0090] In some embodiments, both polymers include SEQ ID NOs. 37 and 39.

[0091] In another embodiment, the present disclosure provides a polymeric complex comprising two polymers, each polymer comprising a TGFb-binding domain linked to a TpoR-binding domain, the TpoR-binding domain inducing cellular effector function upon binding to TpoR; the two polymers being conjugated to each other; and the induction of effector function by the polymeric complex being conditional on each of the two polymers binding to TGFb. See, for example, the drawings and examples of this specification.

[0092] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a complementary part of a pair, and each polymer contains one member of that pair. In some embodiments, the complementary polypeptide pair is a pair of Fc fragments. In some embodiments, the Fc fragment pair is a knob-into-hole pair. In some embodiments, the two polymers are identical.

[0093] In another embodiment, the disclosure provides a polymeric complex comprising two polymers, each polymer comprising a TGFb-binding domain linked to a TpoR-binding domain, the TpoR-binding domain inducing cellular effector function upon binding to TpoR; the two polymers are not conjugated to each other in the absence of TGFb; and the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to TGFb. In some embodiments, the first member of the pair of polymers and the second member of the pair of polymers are non-covalently conjugated to each other in the presence of TGFb, and the non-covalent conjugation is mediated by TGFb. See, for example, the drawings and examples of this specification.

[0094] In some embodiments, the polymer or one or both members of a pair of polymers include a half-life extension portion. In some embodiments, the half-life extension portion is an Fc domain or a fragment thereof.

[0095] In some embodiments of either of the two embodiments described above, the TGFb-binding domain is anti-TGFb scFv.

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

[0097] In another embodiment, the disclosure provides a polymeric complex comprising two polymers, each polymer comprising an IL-8 binding domain linked to a TpoR binding domain, wherein the TpoR binding domain induces cellular effector function upon binding to TpoR; the two polymers are conjugated to each other; and the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to IL-8. See, for example, the drawings and examples of this specification.

[0098] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a complementary part of a pair, and each polymer contains one member of that pair. In some embodiments, the complementary polypeptide pair is a pair of Fc fragments. In some embodiments, the Fc fragment pair is a knob-into-hole pair. In some embodiments, the two polymers are identical.

[0099] In another embodiment, the disclosure provides a polymeric complex comprising two polymers, each polymer comprising an IL-8 binding domain linked to a TpoR binding domain, the TpoR binding domain inducing cellular effector function upon binding to TpoR; the two polymers are not conjugated to each other in the absence of IL-8; and the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to IL-8. In some embodiments, the first member of the pair of polymers and the second member of the pair of polymers are non-covalently conjugated to each other in the presence of IL-8, and the non-covalent conjugation is mediated by IL-8. See, for example, the drawings and examples herein.

[0100] In some embodiments, the polymer or one or both members of a pair of polymers include a half-life extension portion. In some embodiments, the half-life extension portion is an Fc domain or a fragment thereof.

[0101] In some embodiments of either of the two embodiments described above, the IL-8 binding domain is anti-IL8 scFv.

[0102] In some embodiments of either of the two embodiments described above, the TpoR binding domain is an anti-TpoR scFv.

[0103] In another embodiment, the present disclosure provides a polymeric complex comprising two polymers, each polymer comprising a TGFb-binding domain linked to an IL2R-binding domain, the IL2R-binding domain inducing cellular effector function upon binding to IL2R; the two polymers being conjugated to each other; and the induction of effector function by the polymeric complex being conditional on each of the two polymers binding to TGFb. See, for example, the drawings and examples of this specification.

[0104] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a complementary part of a pair, and each polymer contains one member of that pair. In some embodiments, the complementary polypeptide pair is a pair of Fc fragments. In some embodiments, the Fc fragment pair is a knob-into-hole pair. In some embodiments, the two polymers are identical.

[0105] In another embodiment, the disclosure provides a polymeric complex comprising two polymers, each polymer comprising a TGFb-binding domain linked to an IL2R-binding domain, the IL2R-binding domain inducing cellular effector function upon binding to IL2R; the two polymers are not conjugated to each other in the absence of TGFb; and the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to TGFb. In some embodiments, the first member of the pair of polymers and the second member of the pair of polymers are non-covalently conjugated to each other in the presence of TGFb, and the non-covalent conjugation is mediated by TGFb. See, for example, the drawings and examples of this specification.

[0106] In some embodiments, the polymer or one or both members of a pair of polymers include a half-life extension portion. In some embodiments, the half-life extension portion is an Fc domain or a fragment thereof.

[0107] In some embodiments of either of the two embodiments described above, the TGFb-binding domain is anti-TGFb scFv.

[0108] In some embodiments of either of the two embodiments described above, the IL2R binding domain includes the N-terminus of IL2. In some embodiments of either of the two embodiments described above, the IL2R binding domain includes the C-terminus of IL2.

[0109] In another embodiment, the present disclosure provides a polymeric complex comprising two polymers, each polymer comprising an IFNg-binding domain linked to an IL10R-binding domain, wherein the IL10R-binding domain induces cellular effector function upon binding to IL10R; the two polymers are conjugated to each other; and the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to IFNg. See, for example, the drawings and examples of this specification.

[0110] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a complementary part of a pair, and each polymer contains one member of that pair. In some embodiments, the complementary polypeptide pair is a pair of Fc fragments. In some embodiments, the Fc fragment pair is a knob-into-hole pair. In some embodiments, the two polymers are identical.

[0111] In another embodiment, the disclosure provides a polymeric complex comprising two polymers, each polymer comprising an IFNg-binding domain linked to an IL10R-binding domain, the IL10R-binding domain inducing cellular effector function upon binding to IL10R; the two polymers are not conjugated to each other in the absence of TGFb; and the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to IFNg. In some embodiments, the first member of the pair of polymers and the second member of the pair of polymers are non-covalently conjugated to each other in the presence of IFNg, and the non-covalent conjugation is mediated by IFNg. See, for example, the drawings and examples herein.

[0112] In some embodiments, the polymer or one or both members of a pair of polymers include a half-life extension portion. In some embodiments, the half-life extension portion is an Fc domain or a fragment thereof.

[0113] In some embodiments of either of the two above-described aspects, the IFNg binding domain is anti-IFNg scFv.

[0114] In some embodiments of either of the two embodiments described above, the IL10R binding domain includes an anti-IL10R VHH.

[0115] In some embodiments of either of the two above-described aspects, the IL10R binding domain includes IL10.

[0116] In another embodiment, the present disclosure provides a polymeric complex comprising two polymers, each polymer comprising an IL6-binding domain linked to an IL10R-binding domain, wherein the IL10R-binding domain induces cellular effector function upon binding to IL10R; the two polymers are conjugated to each other; and the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to IL6. See, for example, the drawings and examples of this specification.

[0117] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a complementary part of a pair, and each polymer contains one member of that pair. In some embodiments, the complementary polypeptide pair is a pair of Fc fragments. In some embodiments, the Fc fragment pair is a knob-into-hole pair. In some embodiments, the two polymers are identical.

[0118] In another embodiment, the disclosure provides a polymeric complex comprising two polymers, each polymer comprising an IL6-binding domain linked to an IL10R-binding domain, the IL10R-binding domain inducing cellular effector function upon binding to IL10R; the two polymers are not conjugated to each other in the absence of IL6; and the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to IL6. In some embodiments, the first member of the pair of polymers and the second member of the pair of polymers are non-covalently conjugated to each other in the presence of IL6, and the non-covalent conjugation is mediated by IL6. See, for example, the drawings and examples of this specification.

[0119] In some embodiments, the polymer or one or both members of a pair of polymers include a half-life extension portion. In some embodiments, the half-life extension portion is an Fc domain or a fragment thereof.

[0120] In some embodiments of either of the two embodiments described above, the IL6 binding domain is anti-IL6 VHH.

[0121] In some embodiments of either of the two embodiments described above, the IL10R binding domain includes an anti-IL10R VHH.

[0122] In some embodiments of either of the two above-described aspects, the IL10R binding domain includes IL10.

[0123] In another embodiment, the disclosure provides a polymeric complex comprising two polymers, each polymer comprising an IL8-binding domain linked to a CD3-binding domain, wherein the CD3-binding domain induces cellular effector function upon binding to CD3; the two polymers are conjugated to each other; and the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to IL8. See, for example, the drawings and examples of this specification.

[0124] In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a complementary part of a pair, and each polymer contains one member of that pair. In some embodiments, the complementary polypeptide pair is a pair of Fc fragments. In some embodiments, the Fc fragment pair is a knob-into-hole pair. In some embodiments, the two polymers are identical.

[0125] In another embodiment, the disclosure provides a polymeric complex comprising two polymers, each polymer comprising an IL8-binding domain linked to a CD3-binding domain, the CD3-binding domain inducing cellular effector function upon binding to CD3; the two polymers are not conjugated to each other in the absence of IL8; and the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to IL8. In some embodiments, the first member of the pair of polymers and the second member of the pair of polymers are non-covalently conjugated to each other in the presence of IL8, and the non-covalent conjugation is mediated by IL8. See, for example, the drawings and examples herein.

[0126] In some embodiments, the polymer or one or both members of a pair of polymers include a half-life extension portion. In some embodiments, the half-life extension portion is an Fc domain or a fragment thereof.

[0127] In some embodiments of either of the two embodiments described above, the IL8 binding domain is anti-IL8 scFv.

[0128] In some embodiments of either of the two embodiments described above, the CD3-binding domain includes anti-CD3 VH.

[0129] In another embodiment, the disclosure provides a polymer comprising a VEGF-binding domain linked to a 4-1BB-binding domain. See, for example, the drawings and examples of this specification.

[0130] In some embodiments, the polymer includes a half-life extension portion. In some embodiments, the half-life extension portion is an Fc domain or a fragment thereof.

[0131] In some embodiments, the VEGF-binding domain is an anti-VEGF scFv. In some embodiments, the VEGF-binding domain is a VEGF receptor trap.

[0132] In some embodiments, the 4-1BB binding domain is an anti-4-1BB scFv.

[0133] In some embodiments, the polymers include SEQ ID NOs. 36 and 38.

[0134] In some embodiments, the polymers include SEQ ID NOs. 37 and 38.

[0135] In some embodiments, the polymers include SEQ ID NOs: 36 and 39.

[0136] In some embodiments, the polymers include SEQ ID NOs. 37 and 39.

[0137] In another embodiment, the disclosure provides a polymer comprising a TGFb-binding domain linked to a TpoR-binding domain. See, for example, the drawings and examples of this specification.

[0138] In some embodiments, the polymer includes a half-life extension portion. In some embodiments, the half-life extension portion is an Fc domain or a fragment thereof.

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

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

[0141] In another embodiment, the disclosure provides polymers comprising an IL-8 binding domain linked to a TpoR binding domain. See, for example, the drawings and examples of this specification.

[0142] In some embodiments, the polymer includes a half-life extension portion. In some embodiments, the half-life extension portion is an Fc domain or a fragment thereof.

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

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

[0145] In another embodiment, the disclosure provides a polymer comprising a TGFb-binding domain linked to an IL2R-binding domain. See, for example, the drawings and examples of this specification.

[0146] In some embodiments, the polymer includes a half-life extension portion. In some embodiments, the half-life extension portion is an Fc domain or a fragment thereof.

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

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

[0149] In another embodiment, the disclosure provides a polymer comprising an IFNg-binding domain linked to an IL10R-binding domain. See, for example, the drawings and examples of this specification.

[0150] In some embodiments, the polymer includes a half-life extension portion. In some embodiments, the half-life extension portion is an Fc domain or a fragment thereof.

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

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

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

[0154] In another embodiment, the disclosure provides a polymer comprising an IL6-binding domain linked to an IL10R-binding domain. See, for example, the drawings and examples of this specification.

[0155] In some embodiments, the polymer includes a half-life extension portion. In some embodiments, the half-life extension portion is an Fc domain or a fragment thereof.

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

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

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

[0159] In another embodiment, the disclosure provides polymers comprising IL8-binding domains linked to CD3-binding domains. See, for example, the drawings and examples of this specification.

[0160] In some embodiments, the polymer includes a half-life extension portion. In some embodiments, the half-life extension portion is an Fc domain or a fragment thereof.

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

[0162] In some embodiments, the CD3-binding domain includes anti-CD3 VH.

[0163] In some embodiments, the Disclosure provides compositions comprising polymeric complexes, polymers, nucleic acids, or pairs of nucleic acids provided herein.

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

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

[0166] In some embodiments, the Disclosure provides pharmaceutical compositions comprising a polymeric complex, polymer, nucleic acid, or pair of nucleic acids provided herein, and a pharmaceutically acceptable excipient.

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

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

[0169] In some embodiments, the Disclosure provides a method for preparing a pharmaceutical composition, comprising combining a polymeric complex, polymer, nucleic acid, or pair of nucleic acids provided herein with a pharmaceutically acceptable excipient to prepare the pharmaceutical composition.

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

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

[0172] In another embodiment, the present disclosure provides a method comprising providing access to a cell for any one of the polymeric complexes, polymers, nucleic acids, pairs of nucleic acids, compositions, or pharmaceutical compositions described above.

[0173] In another aspect, the disclosure provides a use in the manufacture of a pharmaceutical to provide access to cells of any one of the polymeric complexes, polymers, nucleic acids, paired nucleic acids, compositions, or pharmaceutical compositions described above.

[0174] In another embodiment, the Disclosure provides any one of the above embodiments of a polymeric complex, polymer, nucleic acid, paired nucleic acid, composition, or pharmaceutical composition for use in providing access to cells.

[0175] In another embodiment, the present disclosure provides a method for modulating the state of a cell, comprising providing access to a cell of any one of the above-described polymeric complexes, polymers, nucleic acids, paired nucleic acids, compositions, or pharmaceutical compositions, thereby modulating the state of the cell.

[0176] In another aspect, the disclosure provides the use in the manufacture of a pharmaceutical for modulating the cellular state of any one of the polymeric complexes, polymers, nucleic acids, paired nucleic acids, compositions, or pharmaceutical compositions described above.

[0177] In another embodiment, the present disclosure provides any one of the above embodiments of a polymeric complex, polymer, nucleic acid, paired nucleic acid, composition, or pharmaceutical composition for use in modulating the state of cells.

[0178] In another aspect, the present disclosure provides a method for inducing cellular effector function in a cell, comprising providing access to a cell of any one of the above-described polymer complexes, polymers, nucleic acids, pairs of nucleic acids, compositions, or pharmaceutical compositions, thereby inducing cellular effector function in the cell.

[0179] In another aspect, the disclosure provides the use in the manufacture of a pharmaceutical for inducing cellular effector function in cells of any one of the polymeric complexes, polymers, nucleic acids, pairs of nucleic acids, compositions, or pharmaceutical compositions described above.

[0180] In another embodiment, the Disclosure provides any one of the above embodiments of a polymeric complex, polymer, nucleic acid, pair of nucleic acids, composition, or pharmaceutical composition for use in inducing cellular effector function in cells.

[0181] In some embodiments, cells are present in the subject, and a polymeric complex, polymer, nucleic acid, paired nucleic acid, composition, or pharmaceutical composition is administered in a therapeutically effective dose.

[0182] In some embodiments, subjects have or are suspected of having a disease or disorder characterized by abnormal levels of disease signature targets, and optionally, subjects are pre-determined to have abnormal levels of disease signature targets.

[0183] In another aspect, the Disclosure provides a method for determining the state of a cell, comprising providing access to a cell of any one of the polymeric complexes, or a polymer, nucleic acid, paired nucleic acid, composition, or pharmaceutical composition described in the above aspects, detecting the presence of a reporter domain, and thereby determining the state of the cell.

[0184] In another aspect, the disclosure provides a use in the manufacture of a pharmaceutical for determining the cellular state of any one of the polymeric complexes, polymers, nucleic acids, pairs of nucleic acids, compositions, or pharmaceutical compositions described above.

[0185] In another embodiment, the present disclosure provides any one of the above embodiments of a polymeric complex, polymer, nucleic acid, paired nucleic acid, composition, or pharmaceutical composition for use in determining the state of a cell.

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

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

[0188] In another embodiment, the present disclosure provides a polymeric complex comprising two polymers, each comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD specifically binds to a disease signature ligand in a biological sample; (b) the SBD specifically binds to an effector ligand in a biological sample, and upon binding to the effector ligand, induces cellular effector function; the two copies of the polymer are conjugated to each other; and the induction of effector function by the polymeric complex is conditional on each copy of the polymer binding to the disease signature ligand for use in inducing cellular effector function in cells.

[0189] In another aspect, the present disclosure relates to a method for inducing cellular effector function in cells, comprising a polymer complex comprising a pair of polymers, each independently containing a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein: (a) the FBD of each polymer specifically binds to a disease signature ligand in a biological sample; (b) the first member of the pair of polymers contains a second binding domain 1 (SBD1) that specifically binds to a first effector ligand in a biological sample; and (c) the second member of the pair of polymers The present invention provides a method comprising contacting a polymer complex with a cell, wherein the polymer complex comprises a second binding domain 2 (SBD2) that specifically binds to a second effector ligand in a biological sample; SBD1 and SBD2 induce cellular effector function when they bind to a first effector ligand and a second effector ligand; the first member and the second member of the pair of polymers are conjugated to each other; and the induction of effector function by the polymer complex is conditional on each of the pair of polymers binding to a disease signature ligand.

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

[0191] In another embodiment, the disclosure provides a polymeric complex comprising a pair of polymers, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain, wherein (a) the FBD of each polymer specifically binds to a disease signature ligand in a biological sample; (b) the first member of the pair of polymers comprises a second binding domain 1 (SBD1) that specifically binds to a first effector ligand in a biological sample; (c) the second member of the pair of polymers comprises a second binding domain 2 (SBD2) that specifically binds to a second effector ligand in a biological sample; SBD1 and SBD2 induce cellular effector function when bound to the first and second effector ligands; the first and second members of the pair of polymers are conjugated to each other; and the induction of effector function by the polymeric complex is conditional on each of the pair of polymers binding to the disease signature ligand for use in inducing cellular effector function in cells.

[0192] In another aspect, the disclosure provides a method for inducing cellular effector function in cells, comprising a polymer comprising two FBDs linked to two SBDs by a linker domain: (a) the FBDs specifically bind to a disease signature ligand in a biological sample; (b) the SBDs specifically bind to an effector ligand in a biological sample, and upon binding to the effector ligand, induce cellular effector function; and the polymer can adopt a configuration that allows the two SBDs to bind to the effector ligand in the presence of the disease signature ligand, wherein the induction of effector function by the polymer is conditional on each of the two FBDs binding to the disease signature ligand, and the method comprises contacting the polymer with cells.

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

[0194] In another embodiment, the disclosure provides a polymer comprising two FBDs linked to two SBDs by a linker domain, wherein (a) the FBDs specifically bind to a disease signature ligand in a biological sample; (b) the SBDs specifically bind to an effector ligand in a biological sample, and upon binding to the effector ligand, induce cellular effector function; and the polymer can adopt a configuration that allows the two SBDs to bind to the effector ligand in the presence of the disease signature ligand, wherein the induction of effector function by the polymer is conditional on each of the two FBDs binding to the disease signature ligand for use in inducing cellular effector function in cells.

[0195] In another aspect, the disclosure provides a method for inducing cellular effector function in cells, comprising a polymer comprising two FBDs linked to SBD1 and SBD2 by linker domains, wherein (a) the FBDs specifically bind to a disease signature ligand in a biological sample; (b) SBD1 specifically binds to a first effector ligand in a biological sample; (c) SBD2 specifically binds to a second effector ligand in a biological sample; SBD1 and SBD2 induce cellular effector function upon binding to the first and second effector ligands; the polymer can adopt a configuration that allows SBD1 and SBD2 to bind to the first and second effector ligands in the presence of the disease signature ligands, and the induction of effector function by the polymer is conditional on each of the two FBDs binding to the disease signature ligand, comprising contacting the polymer with cells.

[0196] In another aspect, the disclosure provides the use of a polymer comprising two FBDs linked to SBD1 and SBD2 by a linker domain, wherein (a) the FBDs specifically bind to a disease signature ligand in a biological sample; (b) SBD1 specifically binds to a first effector ligand in a biological sample; (c) SBD2 specifically binds to a second effector ligand in a biological sample; SBD1 and SBD2 induce cellular effector function upon binding to the first and second effector ligands; the polymer can adopt a configuration that allows SBD1 and SBD2 to bind to the first and second effector ligands in the presence of the disease signature ligands, and the induction of effector function by the polymer is conditional on each of the two FBDs binding to the disease signature ligand in the manufacture of a pharmaceutical for inducing cellular effector function in cells.

[0197] In another embodiment, the disclosure provides a polymer comprising two FBDs linked to SBD1 and SBD2 by a linker domain, wherein (a) the FBDs specifically bind to a disease signature ligand in a biological sample; (b) SBD1 specifically binds to a first effector ligand in a biological sample; (c) SBD2 specifically binds to a second effector ligand in a biological sample; SBD1 and SBD2 induce cellular effector function upon binding to the first and second effector ligands; the polymer can adopt a configuration that allows SBD1 and SBD2 to bind to the first and second effector ligands in the presence of the disease signature ligands, and the induction of effector function by the polymer is conditional on each of the two FBDs binding to the disease signature ligand for use in inducing cellular effector function in cells.

[0198] Other features and advantages of the present invention will be evident from the following modes for carrying out the invention and the claims.

[0199] definition As used herein, the term “polymer” refers to a macromolecule (e.g., a molecule having a size greater than 1000 daltons (1 kDa)) comprising one or more polypeptides, oligonucleotides, chemicals, lipids, and / or carbohydrate moieties. In some embodiments, the polymer is a recombinant protein (e.g., a fusion protein).

[0200] As used herein, the term “multimer” refers to a molecule composed of at least two subunits (e.g., at least two subunits comprising one or more polypeptides, oligonucleotides, chemicals, lipids, and / or carbohydrate moieties). Examples of multimers include homomultimers and heteromultimers. A “homomultimer” is a multimer consisting of two or more identical or substantially identical subunits (e.g., two or more macromolecules). Examples of homomultimers include homodimers (containing two identical or substantially identical subunits), homotrimers (containing three identical or substantially identical subunits), and homotetramers (containing four identical or substantially identical subunits). “Substantially identical subunits” include subunits having amino acid sequence differences that do not significantly affect the function of the subunit, for example, that do not significantly affect the affinity of the subunit to one or more ligands. A “heteromultimer” is a multimer consisting of two or more non-identical subunits. Examples of heteropolymers include heterodimers (containing a first and second subunit, e.g., a non-identical polymer pair), heterotrimers (containing one copy of the first subunit and two copies of the second subunit), and homotetramers (containing two copies or versions of each of the first and second subunits). Examples of polymers include higher-order polymers, such as hexamers, heptamers, octamers, noumers, and decamers.

[0201] As used herein, the term “binding domain” refers to any domain that has specific affinity for a ligand. Binding domains include, but are not limited to, polypeptides (e.g., antibodies or their fragments (e.g., scFv, monospecific tandem scFv (taFv), bispecific taFv, VHH, VNAR, Fab, monospecific single-stranded diabody, bispecific single-stranded diabody, or biaffinity retargeted antibody (DART))), antibody mimetics (e.g., afibody, affin, affimer, afitin, alphabody, anticarin, lipocalin, avimer, DARPin, finomer, gastric body, notottin, Kunitz domain peptide, monobody, fibronectin type III domain (FN3)). Examples include )-based binders, nanoantibodies, nanoCLAMPs, optimizers, lipibodies, pronectin, centinlin, oligodies, peptide aptamers, synthetic peptides, or variable lymphocyte receptors (VLRs), endogenous binding domains or their variants or derivatives (e.g., cell receptor domains, enzyme domains, variable lymphocyte receptor (VLR) domains, extracellular receptor domains, nuclear hormone receptor ligand-binding domains, DNA-binding domains, or receptor traps), oligonucleotides (e.g., nucleic acid aptamers (e.g., DNA aptamers)), and chemical molecules, as well as combinations thereof.

[0202] As used herein, the term “ligand” refers to any part of a molecule that may have affinity for a binding domain as described herein. Ligands include, but are not limited to, chemical parts, parts of molecules, molecules (e.g., allergens or toxins), polymers (e.g., polypeptides, nucleic acids, or carbohydrates), post-translational modified states of polymers (e.g., polymers that have been phosphorylated, glycosylated, acylated, alkylated, etc.), higher-order polymer structures (e.g., complexes of two or more polypeptides), cells (e.g., cancer cells), parts of cells (e.g., tumor antigens), receptors on the surface of cells, pathogens (e.g., viruses or parts of viruses; bacteria or parts of bacteria; fungi or parts of fungi; or parasites or parts of parasites), or tissue types.

[0203] 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, e.g., a human). Disease signature ligands can be proteins, such as soluble proteins, insoluble proteins, monomeric proteins, and polymeric proteins. Examples of 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 (ADAs), autoantibodies, nucleic acids, carbohydrates, lipids, peptides, nucleosides, hormones, viruses, bacteria, fungi, or their fragments or antigens.

[0204] The term "effector ligand" refers to a ligand that, when bound by a binding domain, such as the polymer of the present invention, can impart cellular effector function. Disease signature ligands include proteins and peptides, such as cell surface receptors (e.g., catalytic receptors, e.g., receptor tyrosine kinase (RTK), receptor serine / threonine kinase (RSK), type 1 cytokine receptor, type 2 cytokine receptor, tumor necrosis factor (TNF) superfamily receptors (e.g., TNFR) 2 Examples include 4-1BB, or nuclear hormone receptors.

[0205] As used herein, the term "associated with" a disease, disorder, or condition refers to a causal or correlational relationship between the entity of the disease, disorder, or condition and its occurrence or severity in the subject. For example, if a target is associated with a disease, disorder, or condition, the target may be the causative agent of the disease, disorder, or condition. For example, a virus may be the causative agent of a viral infection, a bacterium may be the causative agent of a bacterial infection, a fungus may be the causative agent of a fungal infection or a parasitic infection, a cancer cell may be the causative agent of cancer, a toxin may be the causative agent of toxicity, or an allergen may be the causative agent of an allergic reaction. A target associated with a disease, disorder, or condition may correlate, either similarly or alternately, with an increased likelihood of the occurrence or increased severity of the disease or condition.

[0206] As used herein, the term “carrier” means a compound, composition, reagent, or molecule that facilitates the stability, transport, or delivery of a composition (e.g., a polymer or pair of polymers as described herein) to a subject, tissue, or cell. Non-limiting examples of carriers include carbohydrate carriers (e.g., anhydride-modified phytoglycogen or glycogen-type materials), nanoparticles (e.g., encapsulated or covalently bonded nanoparticles to cyclic or linear polyribonucleotides), liposomes, fusosomes, ex vivo differentiated reticulocytes, exosomes, protein carriers (e.g., proteins covalently bonded to polyribonucleotides), and cationic carriers (e.g., cationic lipopolymer transfection agents).

[0207] As used herein, the terms “disease,” “disorder,” and “condition” refer, respectively, to a health condition below optimal, such as a condition diagnosed or treated by a medical professional, or typically such a condition.

[0208] As used herein, the term “pharmaceutically acceptable excipient” means a pharmaceutically acceptable material or vehicle, such as a liquid or solid filter, diluent, excipient, solvent or encapsulating material, that is useful for formulating polymer complexes, polymers, nucleic acids or paired nucleic acids for pharmaceutical or therapeutic use.

[0209] As used herein, the term “polynucleotide” means one or more nucleic acid subunits, or molecules containing nucleotides, and can be used interchangeably with “nucleic acid” or “oligonucleotide.” A polynucleotide may contain one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. A nucleotide may contain a nucleoside and at least one, two, three, four, five, six, seven, eight, nine, or more phosphate (PO3) groups. A nucleotide may contain a nucleic acid base, a five-carbon sugar (either 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 polymer containing multiple ribonucleotides polymerized via phosphodiester bonds. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose. The polynucleotides provided herein may contain one or more modified nucleotides.

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

[0211] As used herein, “polypeptide” most often means a polymer of amino acid residues (natural or unnatural, including D, L, or combinations thereof) linked together by peptide bonds. As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides may include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologues, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. Polypeptides may be single molecules or multimolecular complexes such as dimers, trimers, or tetramers. They may also include single-chain or multi-chain polypeptides, such as antibodies or insulin, and may associate or link. Most commonly, disulfide bonds are found in multi-chain polypeptides. The term polypeptide may also apply to amino acid polymers, in which one or more amino acid residues are artificial chemical analogs of corresponding natural amino acids.

[0212] As used herein, the term “sequence identity” is determined by the alignment of two peptide sequences or two nucleotide sequences using a global or local alignment algorithm. The sequences may then be referred to as “substantially identical” or “essentially similar” if they share at least a certain minimum percentage of sequence identity (for example, if they are optimally aligned by the GAP or BESTFIT program using default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences across their entire length, maximizing the number of matches and minimizing the number of gaps. Generally, GAP default parameters are used, with a gap creation penalty of 50 (nucleotides) / 8 (protein) and a gap extension penalty of 3 (nucleotides) / 2 (protein). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is ​​BLOSUM62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignment and scores for percentage sequence identity can be determined using computer programs such as the GCG Wisconsin Package, version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or EmbossWin version 2.10.0 (using the "needle" program). Alternatively, the identity percentage can be determined by searching a database using algorithms such as FASTA or BLAST. Sequence identity refers to sequence identity across the entire length of the sequence.

[0213] A "signal sequence" or "leader sequence" refers to a polypeptide sequence, for example, 10 to 30 amino acids long, located at the N-terminus of a polypeptide sequence of a newly synthesized protein that targets the polypeptide sequence in the secretory pathway.

[0214] As used herein, the term “specifically binds” refers to a preferential interaction between a binding domain and its target or ligand (such as binding between an antibody and an antigen or epitope), which can determine the presence of a target or ligand in the presence of a heterogeneous population of molecules, including biomolecules. For example, a binding domain that specifically binds to a ligand (e.g., an antibody that specifically binds to an antigen or epitope) may be a binding domain that binds to this ligand with higher affinity, higher binding activity, more readily and / or for a longer duration than a binding domain that binds to other ligands. In one embodiment, the degree of binding of a binding domain to an unrelated molecule (non-ligand) is less than about 10% of the binding of the binding domain to the ligand, as can be measured using a suitable assay. In certain embodiments, a binding domain that specifically binds to a ligand may have a dissociation constant (K D The ) is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. Specific binding may include, but is not required, exclusive binding. In one embodiment, "specific binding" refers to a binding in which the binding domain binds to a specific ligand (e.g., a polypeptide or antigen or epitope on a specific polypeptide) without substantially binding to any other ligand (e.g., a polypeptide or polypeptide antigen or epitope).

[0215] As used herein, the terms “to treat” or “to treat” refer to a therapeutic treatment of a disease or disorder in a subject (e.g., an infectious disease, cancer, toxicity, or allergic reaction). The effects of the treatment may include reversing, alleviating, reducing the severity, curing, inhibiting progression, reducing the likelihood of recurrence of the disease or disorder, stabilizing (i.e., preventing exacerbation) the state of the disease or disorder, and / or preventing the spread of the disease or disorder, compared to the state and / or condition of the disease or disorder in the absence of the therapeutic treatment. [Brief explanation of the drawing]

[0216] [Figure 1] This is a schematic diagram showing the domain structure of a fusion protein for exerting effector function on a second target (e.g., a biological effector ligand) in the presence of a first target (e.g., a disease signaling ligand). From the N-terminus to the C-terminus, the fusion protein includes a leader polypeptide; a single-stranded variable fragment (scFv) containing a heavy-chain variable domain (VH) and a light-chain variable domain (VL) linked by a (G4S)3 linker (the VH and VL domains have affinity for the first target); a (G4S)n linker; VH and VL domains having affinity for the second target after polymerization with a second molecule having the same VH and VL domains (the VH and VL domains are linked by a G4S linker); and a FLAG affinity tag. [Figure 2] This is a schematic diagram showing the domain structure of a pair of fusion proteins for exerting effector function on a second target (e.g., a biological effector ligand) conditionally on the presence of a first target (e.g., a disease signaling ligand). From the N-terminus to the C-terminus, the fusion protein comprises a leader polypeptide; a FLAG affinity tag; scFv containing VH and VL domains linked by a (G4S)3 linker (the VH and VL domains have affinity for the first target); a (G4S)n linker; and a conditional effector domain A (in the first fusion protein) or a conditional effector domain B (in the second fusion protein). [Figure 3]This is a schematic diagram showing the domain structure of a pair of fusion proteins for exerting effector function on a second target (e.g., a biological effector ligand) in the presence of a first target (e.g., a disease signaling ligand). From the N-terminus to the C-terminus, the fusion protein includes a leader polypeptide; scFv containing VH and VL domains linked by a (G4S)3 linker (the VH and VL domains have affinity for the first target); a (G4S)n linker; VHH having affinity for the first portion of the second target (in the first fusion protein ("anti-a")) or VHH having affinity for the second portion of the second target (in the second fusion protein ("anti-b")); and a FLAG affinity tag. [Figure 4] This is a schematic diagram showing the domain structure of a pair of fusion proteins for exerting effector function on a second target (e.g., a biological effector ligand) in the presence of a first target (e.g., a disease signaling ligand). From the N-terminus to the C-terminus, the fusion protein includes: a leader polypeptide; the VH domain (in the first fusion protein) or VL domain (in the second fusion protein) of the scFv (the VH and VL domains of the scFv have affinity for the second target when assembled as a functional scFv, and the VH and VL domains have low affinity for each other); a GS(G4S)3 linker; an scFv containing the VH and VL domains linked by the (G4S)3 linker (the VH and VL domains have affinity for the first target); and a FLAG affinity tag. [Figure 5]Figure 5A: A schematic diagram showing the domain structure of the fusion protein that modulates the TPO receptor (TpoR) in the presence of TGFβ1 (TGFb). From the N-terminus to the C-terminus, the fusion protein contains a mouse immunoglobulin κ variable 3 (MS IgKVIII) leader polypeptide; anti-TGFβ1 scFv containing VH and VL linked by a (G4S)3 linker; a (G4S)n linker; anti-TpoR scFv containing VH and VL linked by a (G4S)3 linker; and a FLAG affinity tag. Figure 5B: A schematic diagram showing two copies of the fusion protein from Figure 5A bound to TGFb and TpoR. It shows the membrane orientation and downstream signaling partners of TpoR. Figure 5C: A graph showing secreted alkaline phosphatase (SEAP) activity (gray) in the culture supernatant of TpoR SEAP reporter cells stimulated overnight with a constant amount of the fusion protein from Figure 5A and various concentrations of TGFb. The native TPO cytokine is shown as a positive control against constitutive activity (black). [Figure 6] Figure 6A: A schematic diagram showing the domain structure of the fusion protein that modulates TpoR in the presence of interleukin-8 (IL-8). From the N-terminus to the C-terminus, the fusion protein contains an Ms IgKVIII leader polypeptide; an anti-IL-8 scFv containing VH and VL linked by a (G4S)3 linker; an (G4S)n linker; an anti-TpoR scFv containing VH and VL linked by a (G4S)3 linker; and a FLAG affinity tag. Figure 6B: A schematic diagram showing two copies of the fusion protein from Figure 6A bound to IL-8 and TpoR. It shows the membrane orientation and downstream signaling partners of TpoR. Figure 6C: A graph showing the SEAP activity (gray) in the culture supernatant of TpoR SEAP reporter cells stimulated overnight with a constant amount of the fusion protein from Figure 6A and various concentrations of IL-8. The native TPO cytokine is shown as a positive control against constitutive activity (black). [Figure 7]Figure 7A: Schematic diagram showing the domain structures of a pair of complementary fusion proteins (fusion proteins (a) and (b)) that regulate human interleukin-2 receptor (IL-2R) activity in the presence of TGFb. Fusion protein (a) contains, from N-terminus to C-terminus, an anti-TGFβ1 scFv containing VH and VL linked by a (G4S)3 linker; a (G4S)n linker; and a conditional IL-2R effector domain A (IL-2(N-terminus)). Fusion protein (b) contains, from N-terminus to C-terminus, an anti-TGFβ1 scFv containing VH and VL linked by a (G4S)3 linker; a (G4S)n linker; and a conditional IL-2R effector domain B (IL-2(C-terminus)). Figure 7B: Schematic diagrams showing the fusion proteins (a) and (b) from Figure 7A bound to IL-2R components IL2Rβ and IL2RγC, respectively, in the absence (left) or presence (right) of TGFb. These diagrams illustrate the membrane orientation and downstream signaling partners of IL-2R. Figure 7C: Graph showing SEAP activity in the culture supernatant of IL-2 SEAP reporter cells stimulated overnight with a fixed amount of the fusion proteins (a) and (b) from Figure 7A, as well as various concentrations of TGFb (gray squares). The native IL-2 cytokine is shown as a positive control for constitutive activity (black circles). The TGFβ cytokine is shown as a negative control for conditional activity (gray triangles). [Figure 8-1]Figure 8A: A schematic diagram showing the domain structure of a pair of fusion proteins for exerting effector function on a second target (e.g., a biological effector ligand) subject to the presence of a first target (e.g., a disease signaling ligand), wherein the fusion protein, from N-terminus to C-terminus, comprises: a leader polypeptide; scFv containing VH and VL domains linked by a (G4S)3 linker (the VH and VL domains have affinity for the first target); a (G4S)n linker; VHH having affinity for the first portion of the second target (in the first fusion protein "anti-a") or VHH having affinity for the second portion of the second target (in the second fusion protein "anti-b"); and a FLAG affinity tag (left); and a series of schematic diagrams showing pairs of fusion proteins such that the first target is IFNγ (IFNg) and the second target is the IL-10 receptor (IL-10R) (right). Figure 8B: A schematic diagram showing the domain structure of a pair of fusion proteins for exerting effector function on a second target (e.g., a biological effector ligand) subject to the presence of a first target (e.g., a disease signaling ligand), wherein the pair of fusion proteins self-assemble as a heterodimer, with the fusion protein having a leader polypeptide from the N-terminus to the C-terminus; and a VHH having affinity for the first portion of the second target (in the first fusion protein "anti-a") or the second portion of the second target (the second fusion protein A schematic diagram showing a VHH having affinity for the substance "antib"; a (G4S)2 linker; an scFv containing VH and VL domains linked by a (G4S)3 linker (the VH and VL domains have affinity for the first target); a (G4S)n linker; a human IgG1 crystalline fragment region (Fc region) with a "knob" mutation (in the first fusion protein), or a human IgG1 Fc region with a "hole" mutation (in the second fusion protein) and a 6× histidine (His) affinity tag (left); and a schematic diagram showing such a pair (right) of a fusion protein where the second target is IL-10R. [Figure 8-2]Figure 8C: A schematic diagram showing the domain structure of a fusion protein for exerting effector function on a second target (e.g., a biological effector ligand) subject to the presence of a first target (e.g., a disease signaling ligand), wherein from the N-terminus to the C-terminus, the fusion protein comprises: a leader polypeptide; a VHH having affinity for the first portion of the second target (in the first fusion protein "anti-a"); a (G4S)2 linker; and an scFv containing VH and VL domains linked by a (G4S)3 linker (the VH and VL domains having affinity for the first target). ;(G4S)n linker; a second copy of scFv containing VH and VL domains linked by a (G4S)3 linker (the VH and VL domains have affinity for the first target); (G4S)n linker; VHH having affinity for the second portion of the second target (in the second fusion protein "anti-b"); and a polyhistidine affinity tag (top left); (b) a schematic diagram including a fusion protein like (a) with the order of the anti-a and anti-bVHH domains reversed (bottom left); and a schematic diagram showing such a fusion protein where the second target is IL-10R (right). [Figure 8-3] Figure 8D: A schematic diagram showing the fusion protein pair from Figure 8A bound to IFNg and the IL-10 receptor components IL-10Ra and IL-10Rb. It shows the membrane orientation and downstream signaling partner of IL-10R. Figure 8E: A graph showing the SEAP activity in the culture supernatant of IL-10 SEAP reporter cells stimulated overnight with 10 nM of the fusion protein pair from Figure 8A and various concentrations of IFNg (gray squares). IL-10 cytokines are shown as a positive control for constitutive activity (black circles). IFNg cytokines are shown as a negative control for conditional activity (gray triangles). [Figure 8-4]Figure 8F: A schematic diagram of a pair of variants of the fusion protein pair shown in Figure 8B, where the first target is IFNg and the second target is IL-10R, and the linker between the Fc hinge and the IFNg-bound scFv consists of either 5 amino acid residues (P482+P483; left) or 10 amino acid residues (P484+P485; right). Figure 8G: A graph showing the SEAP activity in the culture supernatant of IL-10 SEAP reporter cells stimulated overnight with a fixed amount of the fusion protein from Figure 8F and various concentrations of IFNg. [Figure 9] Figure 9A: A schematic diagram showing the domain structure of a pair of fusion proteins that regulate IL-10R activity in the presence of IL-6. The first fusion protein contains, from N-terminus to C-terminus, a mouse IgKVIII leader peptide; an anti-IL10Ra VHH antibody; a (G4S)2 linker; a first anti-IL-6 VHH antibody; a (G4S)n linker; and a polyhistidine tag. The second fusion protein contains, from N-terminus to C-terminus, a mouse IgKVIII leader peptide; an anti-IL10Rb VHH antibody; a (G4S)2 linker; a second anti-IL-6 VHH antibody; a (G4S)n linker; and a polyhistidine tag. Figure 9B: A schematic diagram showing the pair of fusion proteins from Figure 9A bound to IL-6 and IL-10Ra and IL-10Rb, which are components of the IL-6 and IL-10 receptors. It shows the membrane orientation and downstream signaling partners of IL-10R. Figure 9C: This graph shows the SEAP activity in the culture supernatant of IL-10 SEAP reporter cells stimulated overnight with a fixed amount of the paired fusion protein from Figure 9A and various concentrations of IL-6 (gray circles). IL-10 cytokines are shown as a positive control for constitutive activity (black squares). IL-6 cytokines are shown as a negative control for conditional activity (gray triangles). [Figure 10]Figure 10A: A schematic diagram showing the domain structure of a pair of complementary fusion proteins that regulate differentiated cluster 3 (CD3) activity in the presence of IL-8. The first fusion protein contains, from N-terminus to C-terminus, a mouse IgKVIII leader peptide; an anti-CD3 VH domain of scFv; a GS(G4S) linker; an anti-IL-8 scFv; and a FLAG affinity tag. The second fusion protein contains, from N-terminus to C-terminus, a mouse IgKVIII leader peptide; an anti-CD3 VL domain of scFv; a GS(G4S) linker; an anti-IL-8 scFv; and a FLAG affinity tag. Figure 10B: A schematic diagram showing the pair of fusion proteins from Figure 10A bound to IL-8 and CD3. It shows the membrane orientation of CD3 and its downstream signaling partners. Figure 10C: This bar graph shows the luciferase activity (by NFAT-Luc reporter assay) in the culture supernatant of NFAT-Luc Jurkat reporter cells stimulated for 24 hours with a fixed amount of the paired fusion protein shown in Figure 10A, in or without 25 nM IL-8. Full-length reference anti-CD3 scFv is shown as a constitutively active positive control. [Figure 11]Figure 11A: A schematic diagram showing the domain structure of a pair of fusion proteins that regulate CD3 activity in the presence of IL-8, wherein the pair of fusion proteins comprises, from N-terminus to C-terminus, a leader polypeptide; an anti-CD3 VH domain of scFv; a GS(G4S) linker; an anti-IL-8 scFv; (G4S)n linker; an Fc region with a "knob" mutation; and a polyhistidine tag, and the second fusion protein comprises a leader polypeptide; an anti-CD3 VL domain of scFv; a GS(G4S) linker; an anti-IL-8 scFv; (G4S)n linker; an Fc region with a "hole" mutation; and a polyhistidine tag. Figure 11B: A schematic diagram showing the domain structure of a pair of fusion proteins that regulate CD3 activity in the presence of IL-8, wherein the fusion protein includes, from N-terminus to C-terminus, a leader polypeptide; a VH domain having affinity for the first portion of CD3; an (G4S)2 linker; an scFv containing VH and VL domains linked by a (G4S)3 linker (the VH and VL domains have affinity for IL-8); an (G4S)n linker; a second copy of the scFv containing VH and VL domains linked by a (G4S)3 linker (the VH and VL domains have affinity for IL-8); an (G4S)n linker; a VL domain having affinity for the second portion of CD3; and a polyhistidine affinity tag (top); and a schematic diagram showing a fusion protein like (a), where the order of the anti-CD3 VH and VL domains is reversed (bottom). [Figure 12-1]Figure 12A: A schematic diagram showing a modified version of the paired fusion proteins shown in Figures 8B and 8F, where the first target is IFNg and the second target is IL-10R, and the linker length between the Fc hinge and the IFNg-binding scFv is regulated. Figure 12B: A schematic diagram showing a modified version of the paired fusion proteins shown in Figures 8B and 8F, where the first target is IFNg and the second target is IL-10R, and the linker length between the IFNg-binding scFv and the IL-10R-binding VHH is regulated. Figure 12C: A schematic diagram showing a modified version of the paired fusion proteins shown in Figures 8B and 8F, where the first target is IFNg and the second target is IL-10R, and the IL-10R-binding VHH is replaced with an scFv agonist for IL-10R. Figure 12D: A schematic diagram showing a modified version of the paired fusion proteins shown in Figures 8B and 8F, where the first target is IFNg, the second target is IL-10R, and IL10R-binding VHH is replaced with monomeric IL-10 cytokine. [Figure 12-2]Figure 12E: A schematic diagram showing a modified version of the paired fusion proteins shown in Figures 8B and 8F, where the first target is IFNg and the second target is IL-10R, and the knob-into-hole Fc region and IFNg-binding scFv are replaced with knob-into-hole full-length IgG having an IFNg-binding Fab region. Figure 12F: A schematic diagram showing a modified version of the paired fusion proteins shown in Figures 8B and 8F, where the first target is IFNg and the second target is IL-10R, and the fusion protein is fused to a full-length IgG antibody at the N-terminus of the heavy chain. Figure 12G: A schematic diagram showing a modified version of the paired fusion proteins shown in Figures 8B and 8F, where the first target is IFNg and the second target is IL-10R, and the fusion protein is fused to a full-length IgG antibody at the N-terminus of the light chain. Figure 12H: A schematic diagram showing a modified version of the paired fusion proteins shown in Figures 8B and 8F, where the first target is IFNg and the second target is IL-10R, and the fusion protein is fused to the full-length IgG antibody at the C-terminus of the light chain. Figure 12I: A schematic diagram showing a modified version of the paired fusion proteins shown in Figures 8B and 8F, where the first target is IFNg and the second target is IL-10R, and the fusion protein is fused to the full-length IgG antibody at the C-terminus of the heavy chain Fc region. [Figure 13] Figure 13A: A schematic diagram showing the domain structure of a fusion protein that regulates 4-1BB activity in the presence of VEGF (VEGF / 4-1BB fusion protein construct), the fusion protein containing, from N-terminus to C-terminus, a leader polypeptide; anti-4-1BB scFv; (G4S)n linker; anti-IL-8 scFv; VEGF binder; and an Fc region. Figure 13B: A schematic diagram showing the fusion protein of Figure 13A in its unbound form and bound to VEGF and 4-1BB receptors on the surface of T cells. Left: Monovalent construct of the fusion protein containing a monomeric Fc region. Right: Bivalent construct of the fusion protein containing a dimerizable Fc region. [Figure 14]Figure 14A: A graph showing the induction factor of luminescence in Jurcut T cells expressing 4-1BB and NFκB-driven luciferase reporter genes, treated with a VEGF / 4-1BB fusion protein construct ("fusion protein construct") containing a urelumab-based effector domain, a VEGF receptor trap-based binding domain, and a monovalent Fc region, either alone or in the presence of human VEGF-A, mouse VEGF-A, or mouse VEGF-B. Urelumab reference monoclonal antibody (mAb) is shown as a control. Figure 14B: A graph showing the induction factor of luminescence in Jurcut T cells expressing 4-1BB and NFκB-driven luciferase reporter genes, treated with a VEGF / 4-1BB fusion protein construct containing a urelumab-based effector domain, a VEGF receptor trap-based binding domain, and a bivalent Fc region, either alone or in the presence of human VEGF-A or mouse VEGF-A. Urelumab reference monoclonal antibody (mAb) is shown as a control. [Figure 15] Figure 15A: Graph showing the induction factor of luminescence in Jurcut T cells expressing 4-1BB and NFκB-driven luciferase reporter genes, treated with a VEGF / 4-1BB fusion protein construct containing an utomirumab-based effector domain, VEGF-binding domain (anti-VEGF scFv), and monovalent Fc region, either alone or in the presence of human VEGF-A or mouse VEGF-A. Utomilumab reference mAb is shown as a control. Figure 15B: Graph showing the induction factor of luminescence in Jurcut T cells expressing 4-1BB and NFκB-driven luciferase reporter genes, treated with a VEGF / 4-1BB fusion protein construct containing an utomirumab-based effector domain, VEGF-binding domain (receptor trap; "VEGF-trap"), and bivalent Fc region, either alone or in the presence of human VEGF-A or mouse VEGF-A. Utomilumab reference mAb is shown as a control. [Figure 16-1]Figure 16A: A schematic diagram showing the design of a lymphopenia-induced proliferation study designed to evaluate in vivo T cell proliferation after intravenous (IV) administration of the VEGF / 4-1BB fusion protein construct ("4-1BB SB") in the presence or absence of VEGF. CFSE: Carboxyfluorescein succinimidyl ester. Figure 16B: A plot showing the percentage of T cells in each of the indicated treatment groups (see Table 4) that underwent rapid lymphopenia-induced proliferation (LIP) on day 7, as measured by dilution of CFSE dye. [Figure 16-2] Figure 16C: A plot showing the percentage of donor-derived T cells that were effector T cells after treatment in each of the indicated treatment groups (see Table 4). Figure 16D: A plot showing the percentage of donor-derived T cells that were naive T cells in each of the indicated treatment groups (see Table 4). [Figure 17-1] Figure 17A: A schematic diagram showing the design of a tumor efficacy study designed to evaluate the antitumor activity of the VEGF / 4-1BB fusion protein construct anti-4-1BBAxVEGF-Trap (bivalent) ("VEGF-4-1bb SB") at doses of 10, 3, 1, and 0.5 milligrams (MPK) per kilogram of body weight in mice with MC38 tumors. Figure 17B: A plot showing tumor size (mm3) over time in mice with MC38 tumors treated with various doses of anti-4-1BBAxVEGF-Trap (bivalent), urelumab comparator, or hIgG4 isotype control. [Figure 17-2] Figure 17C: A series of stacked bar charts showing the percentage of mice that experienced a partial response (PR) (black bars; tumor size was smaller than the mean tumor size in the isotype control group) or a complete response (CR) (gray bars; the mouse had no tumor after treatment) on day 17 after treatment with a specified dose of anti-4-1BBAxVEGF-Trap (bivalent) ("SB"), urelumab comparator, PD1 inhibitor ("PD1"), or a PD1 inhibitor in combination with anti-4-1BBAxVEGF-Trap (bivalent) (low dose). [Figure 18] This plot shows the tumor size (mm3) over time in mice with MC38 tumors that were tumor-free after treatment with anti-4-1BBAxVEGF-Trap (bivalent) or urelumab comparator, and were rechallenged with a lethal dose of MC38 tumor cells compared to treated naive mice. [Figure 19] Figure 19A: Schematic diagram showing mice transplanted with either a “cold” tumor cell that does not secrete human IFNg or a “hot” tumor cell that secretes human IFNg. Both tumor cells possess a pSTAT3-driven luciferase reporter system capable of reading IL-10R signaling activity. Figure 19B: Schematic diagram showing the design of the assay for conditional IL-10R activity. Hot or cold tumor cells were inoculated into NSG mice. When the tumors reached a diameter of 5 mm, an IFNg / IL-10R fusion protein construct was injected. IL-10R activity was evaluated by luciferase injection 24 hours after administration of the IFNg / IL-10 fusion protein construct. Constitutively active IL-10 protein was provided as a control. [Figure 20] Figure 20A: A bar graph showing the quantification of luciferase signaling in mice with IFNg-positive "hot" tumors or IFNg-negative "cold" tumors treated with an IFNg / IL-10 fusion protein construct. Figure 20B: A bar graph showing the quantification of luciferase signaling in mice with IFNg-positive "hot" tumors or IFNg-negative "cold" tumors treated with a constitutively active divalent IL-10 construct. [Figure 21]Figure 21A: A pair of graphs showing the results of purification of the dimeric anti-4-1BBAxVEGF-trap (divalent) construct. The upper graph shows that the protein product from the first peak was eluted with 96.5% monodispersion and at a size consistent with the stoichiometry of the dimer (approximately 130 kDa). The lower graph shows the movement of gel filtration standards to generate elution time estimates for various protein sizes. POI: Peak of interest. Figure 21B: Image showing the dimeric stoichiometry of the dimeric anti-4-1BBAxVEGF-trap (divalent) construct confirmed by SDS-PAGE. Under non-reducing conditions (lane "NR"), the polymeric complex runs as a dimer. Under reducing conditions (lane "R"), the polymeric complex disintegrates into its constitutive monomeric subunits as expected. The estimated monomer size is approximately 65 kDa, and the dimer is approximately 130 kDa. The molecular weight standard is in the left lane. [Modes for carrying out the invention]

[0217] This specification features polymers, polymers thereof, compositions containing the same, and methods of using the same, which conditionally induce cellular effector functions (e.g., biological or therapeutic activity) based on the presence of disease signature ligands.

[0218] I. Composition A. Polymers and polymer composites i. Homopolymer polymer complexes In one embodiment, the Specified Polymer Complex is provided, comprising two polymers, each containing a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the FBD specifically binds to a disease signature ligand in a biological sample; (b) the SBD specifically binds to an effector ligand in a biological sample, and upon binding to the effector ligand, induces cellular effector function; the two polymers are conjugated to each other; and the induction of effector function by the Polymer Complex is conditional on each of the two polymers binding to the disease signature ligand (for example, the Polymer Complex does not induce effector function in the absence of the disease signature ligand; substantially does not induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0219] An exemplary first binding domain and disease signature ligand is provided in Section IB of this specification. An exemplary second binding domain and effector ligand is provided in Section IC of this specification.

[0220] In some embodiments, the conjugation between two polymers is covalent. For example, in some embodiments, the two polymers are conjugated by a chemical linker or a polypeptide linker. In other embodiments, the conjugation between two polymers is non-covalent. For example, in some embodiments, the non-covalent conjugation is mediated by a pair of complementary parts, where each polymer includes one of the members of the pair (e.g., linked by a covalent or non-covalent bond). The pair of complementary parts may be, for example, biotin and avidin; barnase and burster; a pair of complementary aptamers; or a pair of complementary polypeptides. (For example, a pair of Fc fragments (e.g., a pair of manipulated Fc fragments). For example, in some embodiments, the complementary part of the pair is a Knob-into-hole Fc pair (for example, one polymer contains an Fc region having a "Knob" mutation (e.g., conjugated covalently or non-covalently), and the other polymer contains an Fc region having a "hole" mutation (e.g., conjugated covalently or non-covalently). A Knob-into-hole Fc pair is, for example, Xu et This is described in al., mAbs, 7(1):231-242, 2015. As a general principle, conjugations (e.g., covalent or non-covalent conjugations) facilitate the co-binding of disease signal and effector ligands conditionally in the presence of a disease signature ligand, for example, a linker or a pair of complementary moieties is configured to sterically orient two copies of the polymer to enable the conditional co-binding of disease signal and effector ligands. In some embodiments, non-covalent conjugations are mediated by a disease signature ligand (e.g., two polymers are conjugated only in the presence of a disease signature ligand).

[0221] In another embodiment, the Spectral In some embodiments, the first member of a pair of polymers and the second member of a pair of polymers are conjugated non-covalently to each other in the presence of a disease signature ligand, and the non-covalent conjugation is mediated by the disease signature ligand.

[0222] In some embodiments, the two polymers are identical (e.g., identical in amino acid sequence and / or nucleotide sequence). Alternatively, the two polymers may contain a first binding domain and a second binding domain that are not identical in sequence but are substantially identical. For example, the two polymers may contain binding domains that are identical in sequence (e.g., amino acid sequence and / or nucleotide sequence), or they may have different sequences but have substantially the same affinity for disease signature ligands or effector ligands.

[0223] In some embodiments of the present invention, the second binding domain is an antibody or antibody fragment that does not bind to an effector ligand when the two polymers do not bind to a disease signature ligand. For example, in some embodiments, the second binding domain comprises a heavy chain variable domain (VH domain) and a light chain variable domain (VL domain) having affinity for a second target, and the VH and VL domains are linked by a short linker (e.g., a G4S linker) that does not allow intrachain pairing of the VH and VL domains. In some embodiments, the second binding domain is a diabody.

[0224] In one exemplary embodiment, each of the two polymers has a first binding domain from the N-terminus to the C-terminus, comprising a single-stranded variable fragment (scFv) containing a heavy-chain variable domain (VH) and a light-chain variable domain (VL) linked by a leader polypeptide (G4S)3 linker (the VH and VL domains have affinity for disease signaling ligands); (G4S) n The fusion protein comprises a linker; a second binding domain containing VH and VL domains with affinity for the effector ligand, linked by a G4S linker; and an antibody construct containing a FLAG affinity tag.

[0225] In another aspect of the present invention, a complex is provided herein comprising a polymer complex described herein, which is complexed with one or both of a disease signature ligand and an effector ligand.

[0226] In some embodiments, the polymer is a polypeptide. In other embodiments, the polymer comprises one or more non-polypeptide components, including, for example, one or more nucleic acids or chemical components, as further described below.

[0227] In some embodiments, the polymer 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.

[0228] Furthermore, this specification provides a polymeric complex comprising three or more (e.g., three, four, five, or more than five) polymers, each containing a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the FBDs specifically bind to disease signature ligands in a biological sample; (b) the SBDs specifically bind to effector ligands in a biological sample, and upon binding to the effector ligands, induce cellular effector function; the three or more polymers are conjugated to one another; and the induction of effector function by the polymeric complex is conditional on each of the three or more polymers binding to the disease signature ligand (e.g., the polymeric complex does not induce effector function in the absence of the disease signature ligand; substantially does not induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0229] ii. Heteropolymer polymer complexes In another embodiment, this specification provides a polymer complex comprising a pair of polymers, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the FBD of each polymer specifically binds to a disease signature ligand in a biological sample; (b) the first member of the pair of polymers comprises a second binding domain 1 (SBD1) that specifically binds to a first effector ligand in a biological sample; and (c) the second member of the pair of polymers comprises a second binding domain 2 (SBD2) that specifically binds to a second effector ligand in a biological sample; and SBD1 and SBD2 bind to the first effector ligand A polymeric complex is provided that, upon binding to a first effector ligand and a second effector ligand, induces cellular effector function; the first member and the second member of the pair of polymers are conjugated to each other; and the induction of effector function by the polymer is conditional on each of the pair of polymers binding to a disease signature ligand (for example, the polymeric complex does not induce effector function in the absence of the disease signature ligand; substantially does not induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0230] An exemplary first binding domain and disease signature ligand is provided in Section IB of this specification. An exemplary second binding domain and effector ligand is provided in Section IC of this specification.

[0231] In some embodiments, the conjugation between two polymers is covalent. For example, in some embodiments, the two polymers are conjugated by a chemical linker or a polypeptide linker. In other embodiments, the conjugation between two polymers is non-covalent. For example, in some embodiments, the non-covalent conjugation is mediated by a pair of complementary moieties, where each polymer includes one of the members of the pair (e.g., linked by a covalent or non-covalent bond). The pair of complementary moieties may be, for example, biotin and avidin; barnase and burster; a pair of complementary aptamers; or a pair of complementary polypeptides. (For example, a pair of Fc fragments (e.g., a pair of manipulated Fc fragments). For example, in some embodiments, the complementary part of the pair is a Knob-into-hole Fc pair (for example, one polymer contains an Fc region having a "Knob" mutation (e.g., conjugated covalently or non-covalently), and the other polymer contains an Fc region having a "hole" mutation (e.g., conjugated covalently or non-covalently). A Knob-into-hole Fc pair is, for example, Xu et This is described in al., mAbs, 7(1):231-242, 2015. As a general principle, conjugations (e.g., covalent or non-covalent conjugations) facilitate the co-binding of disease signal and effector ligands conditionally in the presence of a disease signature ligand, for example, a linker or a pair of complementary moieties is configured to sterically orient two copies of the polymer to enable the conditional co-binding of disease signal and effector ligands. In some embodiments, non-covalent conjugations are mediated by a disease signature ligand (e.g., two polymers are conjugated only in the presence of a disease signature ligand).

[0232] In another embodiment, as described herein, a polymeric complex comprising a pair of polymers, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD): (a) the first member of the pair of polymers comprises a first binding domain 1 (FBD1) that specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) the second member of the pair of polymers comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of a disease signature ligand in a biological sample; (c) the SBD specifically binds to an effector ligand in a biological sample. A polymeric complex is provided in which, upon binding to an effector ligand, it induces cellular effector function; the two polymers are not conjugated to each other in the absence of a disease signature ligand; and the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to the disease signature ligand (e.g., the polymeric complex does not induce effector function in the absence of the disease signature ligand; it substantially does not induce effector function in the absence of the disease signature ligand, or it preferentially induces effector function in the presence of the disease signature ligand). In some embodiments, the first member of the pair of polymers and the second member of the pair of polymers are non-covalently conjugated to each other in the presence of a disease signature ligand, and the non-covalent conjugation is mediated by the disease signature ligand.

[0233] In another aspect of the present invention, a complex is provided herein comprising a polymer complex described herein, which is complexed with one, two, or all three of the disease signature ligand, the first effector ligand, and the second effector ligand.

[0234] In some embodiments, each member of the paired polymer is a polypeptide. In other embodiments, one or both of the paired polymers include one or more non-polypeptide components, including, for example, one or more nucleic acids or chemical components, as further described below.

[0235] In another embodiment, the Specified Polymer Complex is provided, comprising a pair of polymers, each containing an FBD linked to an SBD, wherein (a) the FBD of the first member of the pair of polymers specifically binds to a first portion of a disease signature ligand in a biological sample; (b) the FBD of the second member of the pair of polymers specifically binds to a second portion of the disease signature ligand in a biological sample; (c) the SBD of each polymer specifically binds to an effector ligand in a biological sample, and upon binding to the effector ligand, induces cellular effector function; the first member and the second member of the pair of polymers are conjugated to each other; and the induction of effector function by the polymers is conditional on each member of the polymer complex binding to the disease signature ligand (for example, the polymer complex does not induce effector function in the absence of the disease signature ligand; substantially does not induce effector function in the absence of the disease signature ligand; or preferentially induces effector function in the presence of the disease signature ligand).

[0236] In another aspect of the present invention, a polymer complex is provided herein comprising a set of three polymers, each containing an FBD linked to an SBD, wherein (a) the FBD of each polymer specifically binds to a disease signature ligand in a biological sample; (b) a first member of the polymer set comprises SBD1, which specifically binds to a first effector ligand in a biological sample; (c) a second member of the polymer set comprises SBD2, which specifically binds to a second effector ligand in a biological sample; (d) a third member of the polymer set comprises a second binding domain 3 (SBD3), which specifically binds to a third effector ligand in a biological sample; SBD1, SBD2, and SBD3 induce cellular effector function upon binding to the first, second, and third effector ligands; the first member of a pair of polymers and the second member of a pair of polymers are conjugated to each other; and the induction of effector function by the polymer complex is conditional on each member of the set of three polymers binding to the disease signature ligand. In some embodiments, the disease signature ligand is a trimer.

[0237] In some embodiments, one or both members of a pair of polymers, or one, two, or all three of a set of three polymers, further include 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.

[0238] iii. Polymers containing two FBDs and two SBDs In another embodiment, the Specified Information provides a polymer comprising two first binding domains (FBDs) linked to two second binding domains (SBDs), wherein (a) the FBDs specifically bind to a disease signature ligand in a biological sample; (b) the SBDs specifically bind to an effector ligand in a biological sample, and upon binding to the effector ligand, induce cellular effector function; and the polymer can adopt a configuration that allows the binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, wherein the induction of effector function by the polymer is conditional on each of the two FBDs binding to the disease signature ligand for use in inducing cellular effector function in cells (for example, the polymer does not induce effector function in the absence of the disease signature ligand; substantially does not induce effector function in the absence of the disease signature ligand, or preferentially induces effector function in the presence of the disease signature ligand).

[0239] An exemplary first binding domain and disease signature ligand is provided in Section IB of this specification. An exemplary second binding domain and effector ligand is provided in Section IC of this specification.

[0240] In some embodiments, the two FBDs are identical (for example, identical in amino acid sequence and / or nucleotide sequence). Alternatively, the two FBDs may have different sequences but have substantially identical affinity for the disease signature ligand.

[0241] In some embodiments, the two SBDs are identical (for example, identical in amino acid sequence and / or nucleotide sequence). Alternatively, the two SBDs may have different sequences but have substantially the same affinity for the effector ligand.

[0242] In some embodiments of the present invention, the SBD is an antibody or antibody fragment that does not bind to an effector ligand when two FBDs of the polymer do not bind to a disease signature ligand. For example, in some embodiments, the SBD comprises a heavy chain variable domain (VH domain) and a light chain variable domain (VL domain) having affinity for a second target, and the VH and VL domains are linked by a short linker (e.g., a G4S linker) that does not allow intra-chain pairing of the VH and VL domains. In some embodiments, the second binding domain is a diabody.

[0243] In another aspect of the present invention, provided herein is a complex comprising a polymer described herein complexed with one or both of a disease signature ligand and an effector ligand.

[0244] In some embodiments, the polymer is a polypeptide (e.g., comprising a single polypeptide chain). In other embodiments, the polymer comprises one or more non-polypeptide components, e.g., one or more nucleic acids or chemical constituents, as further described below.

[0245] In some embodiments, the polymer 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.

[0246] iv. A polymer comprising two FBDs, one SBD1, and one SBD2 In another aspect, provided herein is a polymer comprising two non-identical second binding domains (SBDs), e.g., a first second binding domain (SBD1) and a second second binding domain (SBD2), linked to two first binding domains (FBDs), wherein (a) the FBDs specifically bind to a disease signature ligand in a biological sample; (b) SBD1 specifically binds to a first effector ligand in the biological sample; (c) SBD2 specifically binds to a second effector ligand in the biological sample; SBD1 and SBD2 induce a cellular effector function upon binding to the first effector ligand and the second effector ligand; the polymer can adopt a conformation that allows binding of SBD1 and SBD2 to the first effector ligand and the second effector ligand in the presence of the disease signature ligand, and induction of the effector function by the polymer is conditional upon each of the two FBDs binding to the disease signature ligand (e.g., the polymer does not induce an effector function in the absence of the disease signature ligand; does not substantially induce an effector function in the absence of the disease signature ligand, or preferentially induces an effector function in the presence of the disease signature ligand).

[0247] Exemplary first binding domains and disease signature ligands are provided in Section IB herein. Exemplary second binding domains and effector ligands are provided in Section IC herein.

[0248] In some embodiments of the present invention, the SBD (e.g., SBD1 and / or SBD2) is an antibody or antibody fragment that does not bind to an effector ligand when two high molecular weight FBDs do not bind to a disease signature ligand. For example, in some embodiments, the SBD (e.g., SBD1 and / or SBD2) comprises a heavy chain variable domain (VH domain) and a light chain variable domain (VL domain) having affinity for a second target, and the VH and VL domains are linked by a short linker (e.g., a G4S linker) that does not allow intrachain pairing of the VH and VL domains. In some embodiments, the SBD (e.g., SBD1 and / or SBD2) is a diabody.

[0249] In another aspect of the present invention, a complex comprising the polymer described herein, complexed with one or both of a disease signature ligand and an effector ligand, is provided herein.

[0250] In some embodiments, the polymer is a polypeptide (e.g., comprising a single polypeptide chain). In other embodiments, the polymer comprises one or more non-polypeptide components, including, for example, one or more nucleic acids or chemical components, as further described below.

[0251] In some embodiments, the polymer 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.

[0252] v. Nucleic acids, vectors, and host cells In another aspect of the present invention, one or more nucleic acids (e.g., one or more RNA molecules or DNA molecules) encoding one or more of the above-mentioned polymers (e.g., a pair of nucleic acids encoding one or more of the above-mentioned pair of polymers (e.g., a pair of RNA molecules or a pair of DNA molecules)). The one or more nucleic acids may be cyclic or linear. The one or more nucleic acids may be formulated together with a carrier and / or delivery platform, e.g., a lipid-based carrier (e.g., lipid nanoparticles (LNPs)) and / or a vector delivery system (e.g., adenovirus, adeno-associated virus (AAV), anerovirus or lentivirus). Further examples of lipid-based carriers that may be used in the present invention are provided in Section I(I) herein. For example, in some aspects, one or more nucleic acids (e.g., one or more RNA molecules or DNA molecules, e.g., cyclic or linear RNA molecules or DNA molecules) encoding one or more of the above-mentioned polymers are provided, which are formulated together with a carrier, e.g., a lipid carrier, e.g., LNPs. In some aspects, the nucleic acid comprises one or more modified nucleotides.

[0253] Furthermore, vectors (e.g., plasmids or viral vectors) comprising or encoding any of the above nucleic acids are provided herein. The vectors may be formulated with a carrier, such as a carrier suitable for delivery to target cells (e.g., mammalian cells), such as a lipid-containing carrier, such as an LNP-containing formulation.

[0254] Furthermore, host cells modified to contain the above-mentioned nucleic acids or vectors are provided herein. Suitable host cells include bacterial cells and eukaryotic cells (e.g., mammalian cells). In some embodiments, the nucleic acids or vectors described herein are produced in a host cell and isolated from the host cell.

[0255] B. Disease signature ligand and first binding domain i. Disease signature ligands Disease signature ligands, conjugated by polymers, paired polymers, or polymeric complexes, may be any part (e.g., proteins, peptides, or small molecules) associated with a disease state or disorder of a cell, tissue, or subject (e.g., a mammal, e.g., a human).

[0256] In some embodiments, the disease signature ligand is a protein. In some embodiments, the protein is either a soluble or insoluble protein. For example, the disease signature ligand may be present in a solution in a biological sample (e.g., in the extracellular space) or embedded in a membrane present in the biological sample (e.g., embedded in the cell membrane).

[0257] In some embodiments, the disease signature ligand is a cell surface receptor (e.g., HER2), a cell surface antigen (e.g., prostate-specific membrane antigen (PSMA)), a membrane-bound protein (e.g., ASCT2), an extracellular matrix component (e.g., fibronectin or collagen), or an integrin.

[0258] In some embodiments, the disease signature ligand is a multimeric protein (e.g., a homomultimeric protein), such as a dimeric, trimer, or tetrameric protein (e.g., an immunologically active multimeric protein). For example, in some embodiments comprising a single type of polymer that can form dimers in the presence of the disease signature ligand (e.g., embodiments as described in Section IA(i) of this specification), the disease signature ligand may be dimerized such that each polymer binds to one member of the dimerized disease signature ligand. For example, in some embodiments comprising two identical or substantially identical first binding domains (FBDs) (e.g., embodiments as described in Sections IA(i) and IA(iii) of this specification), the disease signature ligand may be dimerized such that each polymer binds to one member of the dimerized disease signature ligand.

[0259] In other embodiments, the disease signature ligand is a monomeric protein.

[0260] In some embodiments, the disease signature ligand is a cytokine. In some embodiments, the cytokine is an interleukin (e.g., IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15, IL-17, or IL-23); an interferon (e.g., IFN-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 chemotactic protein-1 (MCP-1); or interferon-γ-inducible protein 10 (IP-10, also called CXCL10); or a member of the TNF family (e.g., TNF-α). In some embodiments, the cytokine is multimerized (e.g., dimerized, trimerized, or tetramerized).

[0261] In some embodiments, the disease signature ligand is a neurotransmitter (e.g., serotonin, dopamine, or histamine).

[0262] In some embodiments, the disease signature ligand is a VEGF (e.g., VEGF-A or VEGF-B).

[0263] In some embodiments, the disease signature ligand is an autoantigen of the organism from which the biological sample originates (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), e.g., an ADA that targets a therapeutic agent used to treat a disease condition or disorder of a cell, tissue, or subject. In some embodiments, the autoantigen is an autoantibody. In some embodiments, the autoantigen is a cancer antigen, e.g., a tumor marker.

[0264] In some embodiments, the disease signature ligand is a nucleic acid.

[0265] In some embodiments, the disease signature ligand is a carbohydrate, lipid, peptide, nucleoside, or a combination of the foregoing.

[0266] In some embodiments, the disease signature ligand is a hormone (e.g., a peptide / protein hormone such as insulin, oxytocin, or growth hormone), an amino acid derivative (e.g., melatonin or thyroxine), a steroid (e.g., glucocorticoid), or an eicosanoid (e.g., prostaglandin).

[0267] 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, bacterium, fungus, or a fragment or antigen thereof (e.g., a cell, tissue, or virus, bacterium, or fungus that causes a disease state or disorder in a subject, or a fragment thereof).

[0268] ii. Disease signature ligand binding domain (first binding domain) Each of the polymers provided herein includes at least one first binding domain (FBD) that specifically binds to a disease signature ligand (e.g., binds to a disease signature ligand as described in Section IB(i) above). In some embodiments, the first binding domain is designed or selected to enable binding of additional binding domains to the disease signature ligand (e.g., such that at least two copies of the first binding domain can bind to the disease signature ligand and / or such that the first binding domain of each member of a pair of polymers can bind to the disease signature ligand).

[0269] In some embodiments, the first binding domain includes a polypeptide that specifically binds to a disease signature ligand.

[0270] In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is scFv, monospecific tandem scFv (taFv), bispecific taFv, VHH, VNAR, Fab, monospecific single-stranded diabody, bispecific single-stranded diabody, or biaffinity retargeted antibody (DART).

[0271] In some embodiments, the polypeptide is an antibody mimetic. In some embodiments, the antibody mimetic is an afibody, affilin, afimer, afitin, alphabody, anticarin, lipocalin, avimer, DARPin, finomer, gastric body, notottin, Kunitz domain peptide, monobody, fibronectin type III domain (FN3) based binder, nanoantibody, nanoCLAMP, optimizer, lipibody, pronectin, centinlin, obody, peptide aptamer, synthetic peptide, or variable lymphocyte receptor (VLR).

[0272] In some embodiments, the polypeptide is an endogenous binding domain of the organism from which the biological sample and / or disease signature ligand originates, 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, an extracellular receptor domain, a nuclear hormone receptor ligand binding domain, or a DNA binding domain. For example, in some embodiments, the first binding domain is a polypeptide containing or comprising a receptor for a cytokine (e.g., a multimerized cytokine, e.g., a dimeric, trimer, or tetramer cytokine) or an immunologically active multimer (e.g., an immunologically active dimeric, trimer, or tetramer). In some embodiments, the disease signature ligand is IL-6, and the first binding domain is a polypeptide containing the IL-6 receptor. In some embodiments, the disease signature ligand is TNF-α, and the first binding domain is a polypeptide containing the TNF-α receptor. In some embodiments, the disease signature ligand is VEGF, and the first binding domain includes a receptor trap derived from the VEGF receptor VEGFR3 or VEGFR2 (e.g., aflibercept).

[0273] In some embodiments, the first binding domain includes an oligonucleotide that specifically binds to a disease signature ligand. In some embodiments, the oligonucleotide is a nucleic acid aptamer (e.g., a DNA aptamer).

[0274] In some embodiments, the first binding domain includes a chemical molecule that specifically binds to a disease signature ligand.

[0275] Further binding domains that may be used in the present invention are described, for example, in Zhong and D'Antona, Antibodies, 10(2):13, 2021.

[0276] In some embodiments, the disease signature ligand binding domain has an affinity (K D value) for a disease signature ligand of >10 to >100 pM, <10 nM, >10 nM, or >100 nM, or has a micromolar affinity (e.g., K D of 1 μM or less) for a disease signature ligand. In some aspects, the disease signature ligand binding domain binds to the disease signature ligand with a K D of 1 nM or less.

[0277] In some embodiments of any of the polymers provided herein, the first binding domain has an affinity for two or more disease signature moieties. For example, the first binding domain may include at least two binding moieties as described above, and the at least two binding moieties specifically bind to at least two different disease signature moieties.

[0278] In some embodiments comprising a pair of polymers (e.g., embodiments as described in Sections IA(i) and IA(ii) herein), the first and second members of the pair of polymers have the same sequence (e.g., amino acid sequence and / or nucleotide sequence) or different sequences but include a first binding domain having substantially the same affinity for a disease signature ligand or effector ligand. Alternatively, in other embodiments, the first and second members of the pair of polymers include different first binding domains. For example, in some embodiments, the first binding domain of the first member of the pair of polymers specifically binds to a first epitope or moiety of a disease signature ligand in a biological sample, and the second binding domain of the second member of the pair of polymers specifically binds to a second epitope or moiety of the disease signature ligand in the biological sample.

[0279] For example, in some embodiments, the Disclosure provides a polymeric complex comprising two polymers, each comprising a first binding domain (FBD) conjugated to a second binding domain (SBD), wherein (a) a first member of the pair of polymers comprises a first binding domain 1 (FBD1) that specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) a second member of the pair of polymers comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of a disease signature ligand in a biological sample; (c) the SBD specifically binds to an effector ligand in a biological sample, and upon binding to the effector ligand, induces cellular effector function; the two polymers are conjugated to each other; and the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to the disease signature ligand.

[0280] In further examples, in some embodiments, the present disclosure provides a polymeric complex comprising a pair of polymers, each comprising a first binding domain (FBD) independently linked to a second binding domain (SBD), wherein (a) the first member of the pair of polymers comprises a first binding domain 1 (FBD1) that specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) the second member of the pair of polymers comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of a disease signature ligand in a biological sample; and (c) the first member of the pair of polymers binds to a first effector in a biological sample. (d) comprising a second binding domain 1 (SBD1) that specifically binds to a ligand; (d) a second member of the pair of polymers comprising a second binding domain 2 (SBD2) that specifically binds to a second effector ligand in a biological sample; SBD1 and SBD2 induce cellular effector function when they bind to a first effector ligand and a second effector ligand, respectively; the first member and the second member of the pair of polymers are conjugated to each other; and the induction of effector function by the polymer complex is conditional on each of the pair of polymers binding to a disease signature ligand.

[0281] Similarly, in some embodiments of a polymer containing two FBDs (for example, the embodiments described in sections IA(iii) and IA(iv) of this specification), the two FBDs are either identical in sequence (e.g., amino acid sequence and / or nucleotide sequence) or different in sequence, but have substantially the same affinity for the disease signature ligand or effector ligand. Alternatively, in other embodiments, the polymer contains two different FBDs. For example, in some embodiments, the polymer includes a first FBD that specifically binds to a first epitope or portion of the disease signature ligand in a biological sample, and a second FBD that specifically binds to a second epitope or portion of the disease signature ligand in a biological sample.

[0282] For example, in some embodiments, the Disclosure provides a polymer comprising FBD1 and FBD2 linked to two SBDs, wherein (a) FBD1 specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) FBD2 specifically binds to a second epitope of the disease signature ligand in a biological sample; and (c) SBDs specifically bind to an effector ligand in a biological sample, inducing cellular effector function upon binding to the effector ligand; the polymer can adopt a configuration that allows the two SBDs to bind to the effector ligand in the presence of the disease signature ligand, and the induction of effector function by the polymer is conditional on each of FBD1 and FBD2 binding to the disease signature ligand.

[0283] In further examples, in some embodiments, the present disclosure is a polymer comprising FBD1 and FBD2 linked to SBD1 and SBD2, wherein (a) FBD1 specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) FBD2 specifically binds to a second epitope of the disease signature ligand in a biological sample; (c) SBD1 specifically binds to a first effector ligand in a biological sample; and (d) SBD2 specifically binds to a second effector ligand in a biological sample. The present invention provides a polymer that binds heteromorphically; when SBD1 and SBD2 bind to the first and second effector ligands, they induce cellular effector function; the polymer can adopt a configuration that allows SBD1 and SBD2 to bind to the first and second effector ligands in the presence of a disease signature ligand, and the induction of effector function by the polymer is conditional on each of the two FBDs (FBD1 and FBD2) binding to the disease signature ligand.

[0284] In embodiments including non-identical first binding domains (e.g., first binding domain 1 (FBD1) and first binding domain 2 (FBD2)), the first binding domains may bind to different epitopes of the disease signature ligand, such as partially overlapping or non-overlapping epitopes, so that, for example, the two first binding domains do not sterically interfere with the binding of their respective epitopes on the disease signature ligand; they only partially interfere with the binding of their respective epitopes on the disease signature ligand; or they do not completely interfere with the binding of their respective epitopes on the disease signature ligand. Thus, in some embodiments, the first binding domains may bind to the disease signature ligand simultaneously.

[0285] In some embodiments, which include non-identical first binding domains (e.g., FBD1 and FBD2), the two first binding domains have substantially similar affinities to their respective epitopes (e.g., affinities differing 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, FBD1 and FBD2 have substantially different affinities to their respective epitopes (e.g., affinities that differ by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, e.g., 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2500%, 5000%, 7500%, or 10000%).

[0286] 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 Arg, Lys, Asp, His, or Glu amino acid residues (or combinations thereof) (for example, a binding site enriched with one or more of these residues).

[0287] In one embodiment, the disease signature ligand is TGFb.

[0288] In one embodiment, the disease signature ligand is VEGF.

[0289] In one embodiment, the disease signature ligand is IL-8.

[0290] In one embodiment, the disease signature ligand is IL-6.

[0291] In one embodiment, the disease signature ligand is IFNg.

[0292] In one embodiment, the disease signature ligand is IL-10, and the first binding domain is a polypeptide containing the IL-10 receptor.

[0293] In one embodiment, the disease signature ligand is IL-12, and the first binding domain includes the p40 subunit and / or p35 subunit of IL-12.

[0294] In one embodiment, the disease signature ligand is VEGF, and the first binding domain includes (i) a VEGF neutralizing antibody (e.g., bevacizumab), or (ii) a receptor trap derived from the VEGF receptor VEGFR3 or VEGFR2 (e.g., aflibercept).

[0295] C. Effector ligand and second binding domain i. Effector Ligand The effector ligands, bound by polymers, paired polymers, or polymeric complexes, may be any portion (e.g., a protein or peptide) present in a biological sample that, when bound by the polymer of the present invention, can impart cellular effector function.

[0296] In some embodiments, the effector ligand is a protein or peptide.

[0297] In some embodiments, the effector ligand is a cell surface receptor.

[0298] In some embodiments, the effector ligand is TpoR.

[0299] In some embodiments, the effector ligand is 4-1BB.

[0300] In some embodiments, the effector ligand is IL2R.

[0301] In some embodiments, the effector ligand is IL10R.

[0302] In some embodiments, the effector ligand is CD3.

[0303] In some embodiments, the cell surface receptor is a catalytic receptor, such as a receptor tyrosine kinase (RTK), a receptor serine / threonine kinase (RSK), a type 1 cytokine receptor, a type 2 cytokine receptor, or a tumor necrosis factor (TNF) superfamily receptor (e.g., TNFR2 or 4-1BB). In some embodiments, the cell surface receptor is 4-1BB. In some embodiments, the cell surface receptor is TpoR.

[0304] In some embodiments, RTK is VEGFR and RSK is TGFBR 2 The type 1 cytokine receptor is IL2R, the type 2 cytokine receptor is IL10R, or the TNF superfamily receptor is TNFR2. In some embodiments, the receptor is CD3.

[0305] 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.

[0306] ii. Mechanism of effector ligand activation In some embodiments, the second binding domain (SBD) (e.g., SBD1, SBD2, and / or SBD3) is an agonist of the effector ligand. In other embodiments, the second binding domain is an antagonist of the effector ligand.

[0307] In some embodiments, the effector ligand must be homodimerized to exert cellular effector function. In some embodiments, the effector ligand can be homodimerized and exert cellular effector function at an intensity at least 2, 5, 10, 100, or 1000 times greater in the homodimerized form compared to the monomeric form. In some embodiments, the effector ligand is homodimerized in the presence of a polymer or polymeric complex. In some embodiments, the effector ligand is activated by binding of a polymer or polymeric complex in the absence of its endogenous ligand.

[0308] In some embodiments, the polymer or polymer complex exhibits conditional binding activity, trigger binding activity, and / or dimerization binding activity. For example, in some embodiments, multiple polymers or polymer complexes bind to a disease signature ligand (e.g., a tumor antigen or pathogen surface marker), and proximity leads to increased binding activity, which then activates a potent downstream effect.

[0309] In some embodiments, including a second binding domain that is not identical (e.g., including SBD1 and SBD2) (e.g., embodiments as described in sections IA(ii) and IA(iv) of this specification), the first effector ligand and the second effector ligand need to be associated (e.g., in close proximity and correctly oriented (e.g., heterodimerized)) in order to exert cellular effector function. In some embodiments, the effector ligand can heterodimerize and exert cellular effector function at an intensity at least 2, 5, 10, 100, or 1000 times greater in the heterodimerized form compared to the monomeric form. Therefore, in some embodiments, the first effector ligand and the second effector ligand are associated (e.g., in close proximity and correctly oriented (e.g., heterodimerized)) in the presence of a polymer or polymer complex.

[0310] In other embodiments including a second binding domain that is not identical (e.g., including SBD1 and SBD2) (e.g., embodiments as described in sections IA(ii) and IA(iv) of this specification), the first effector ligand and the second effector ligand must be associated (e.g., in close proximity and correctly oriented (e.g., heterodimerized)) and further associated with one or more additional parts in order to exert cellular effector function. For example, in some embodiments, the first effector ligand and the second effector ligand are members of a receptor complex (e.g., a homotrimeric receptor complex, a heterotrimeric receptor complex, a homotetrameric receptor complex, or a heterotetrameric receptor complex) comprising at least three members.

[0311] In some embodiments of any polymer, paired polymer, and polymer complex provided herein, the disease signature ligand is a soluble protein (e.g., a cytokine), and the effector ligand is a catalytic receptor (e.g., a catalytic receptor that exerts cellular function upon polymerization (e.g., homopolymerization or heteropolymerization)).

[0312] iii. Cell Effector Function In some embodiments, the cellular effector function of disease signature ligands is biological activity.

[0313] In some embodiments, the cellular effector function of disease-signature ligands is therapeutic activity.

[0314] In some embodiments, the cellular effector function of a disease signature ligand is disease activity (e.g., abnormal activity associated with a disease state), and this cellular effector function is suppressed by the binding of a polymerized polymer or a pair of polymers to the effector ligand.

[0315] iv. Effector ligand binding domain (second binding domain) In some embodiments, a second binding domain (SBD) (e.g., SBD1, SBD2, or SBD3) comprises a polypeptide that specifically binds to an effector ligand. In some embodiments, the first binding domain allows for the binding of additional binding domains to a disease signature ligand (e.g., the second binding domain is designed or selected so that at least two copies can bind to the effector ligand).

[0316] In some embodiments, the polypeptide is an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is scFv, monospecific tandem scFv (taFv), bispecific taFv, VHH, VNAR, Fab, monospecific single-stranded diabody, bispecific single-stranded diabody, or biaffinity retargeted antibody (DART).

[0317] In some embodiments, the polypeptide is an antibody mimetic. In some embodiments, the antibody mimetic is an afibody, affilin, afimer, afitin, alphabody, anticarin, lipocalin, avimer, DARPin, finomer, gastric body, notottin, Kunitz domain peptide, monobody, fibronectin type III domain (FN3) based binder, nanoantibody, nanoCLAMP, optimizer, lipibody, pronectin, centinlin, obody, peptide aptamer, synthetic peptide, or variable lymphocyte receptor (VLR).

[0318] In some embodiments, the polypeptide is the endogenous binding domain. In some embodiments, the endogenous binding domain is the ligand or fragment thereof of an effector ligand. In some embodiments, the endogenous binding domain is the virus-binding protein or fragment thereof.

[0319] In some embodiments, the second binding domain includes an oligonucleotide that specifically binds to the effector ligand. In some embodiments, the oligonucleotide is a nucleic acid aptamer (e.g., a DNA aptamer).

[0320] Further binding domains that may be used in the present invention are described, for example, in Zhong and D'Antona, Antibodies, 10(2):13, 2021.

[0321] In some embodiments, the effector ligand-binding domain has an affinity for the effector ligand similar to that of the native effector ligand. In some embodiments, the effector ligand-binding domain has an affinity (K) for the effector ligand in the picomolar (pM) range or <1 μM. D The effector ligand-binding domain has an affinity for an effector ligand of 500 nM or less. In some embodiments, the effector ligand-binding domain comprises two monomers, and the affinity of each monomer to the effector ligand is one to two orders of magnitude higher than the affinity of the effector ligand-binding domain monomers to each other.

[0322] In some embodiments of any polymer provided herein, the second binding domain has affinity for two or more effector ligands. For example, the second binding domain may include at least two binding moieties as described above, where at least two binding moieties specifically bind to at least two different effector moieties.

[0323] The second binding domain of the pair In some embodiments, which include a polymer or pair of polymers (e.g., embodiments described in sections IA(ii) and IA(iv) of this specification) containing a second non-identical binding domain (e.g., SBD1 and SBD2), one of the two SBDs (e.g., SBD1) specifically binds to a first effector ligand in the biological sample, and the other SBD (e.g., SBD2) specifically binds to a second effector ligand in the biological sample.

[0324] For example, in some embodiments, SBD1 is a first part of the coupling portion, and SBD2 is a second part of the coupling portion.

[0325] In another example, one of two SBDs (e.g., SBD1) specifically binds to the first component of a heteromultimer (e.g., heterodimer) receptor, while the other SBD (e.g., SBD2) specifically binds to the second component of the heteromultimer (e.g., heterodimer) receptor.

[0326] In another example, one of the two SBDs (e.g., SBD1) is the first component of the dimer, and the other SBD (e.g., SBD2) is the second component of the dimer.

[0327] In another example, one of the two SBDs (e.g., SBD1) is a first fragment of a polypeptide chain, and the other SBD (e.g., SBD2) is a second fragment of the polypeptide chain. In some embodiments, the polypeptide chain is a hormone, cytokine, or growth factor.

[0328] In some embodiments of any of the above examples, non-identical second binding domains (e.g., SBD1 and SBD2) are manipulated to have reduced affinity to each other. In some embodiments, non-identical second binding domains (e.g., SBD1 and SBD2) are greater than >1 μm, e.g., greater than >5-10 μm, but have an affinity (K) of less than 1 mM (e.g., 10-200 μm). D ) has.

[0329] In some embodiments of any polymer or polymer complex described herein, the second binding domain includes a conditional effector domain.

[0330] D. The first and second binding domains of the pair In some embodiments, the disease signature ligand is TGFβ and the effector ligand is the TPO receptor. For example, in some embodiments, the first binding domain includes a TGFβ binding domain (e.g., an anti-TGFβ1 antibody or antibody fragment, e.g., an anti-TGFβ1 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, e.g., an anti-TpoR heavy chain variable domain (VH) and a light chain variable domain (VL)).

[0331] In some embodiments, the disease signature ligand is IL-8 and the effector ligand is the TPO receptor. For example, in some embodiments, the first binding domain includes an IL-8 binding domain (e.g., an anti-IL-8 antibody or antibody fragment, e.g., anti-IL-8 scFv), and the second binding domain includes a TpoR binding domain (e.g., an anti-TpoR antibody or antibody fragment, e.g., anti-TpoR VH and VL).

[0332] In some embodiments, the disease signature ligand is TGFβ and the effector ligand is the IL-2 receptor (IL-2R). For example, in some embodiments, the first binding domain comprises a TGFβ binding domain (e.g., an anti-TGF-β1 antibody or antibody fragment, e.g., anti-TGF-β1 scFv) and one or more second binding domains comprises IL-2R binding domains (e.g., fragments and / or modified versions of IL-2R). In some embodiments, the disclosure provides a polymer, polymer complex, or pair of polymers comprising two second binding domains containing complementary fragments of IL-2R.

[0333] In some embodiments, the disease signature ligand is IL-8 and the effector ligand is the IL-2 receptor (IL-2R). For example, in some embodiments, the first binding domain comprises an IL-8 binding domain (e.g., an anti-IL-8 antibody or antibody fragment), and one or more second binding domains comprises IL-2R binding domains (e.g., fragments and / or modified versions of IL-2). In some embodiments, the disclosure provides a polymer, polymer complex, or pair of polymers comprising two second binding domains containing complementary fragments of IL-2R.

[0334] In some embodiments, the disease signature ligand is IFNγ and the effector ligand is the IL-10 receptor. For example, in some embodiments, the first binding domain comprises an IFNγ binding domain (e.g., an anti-IFNγ antibody or antibody fragment, e.g., anti-IFNγ scFv), and one or more second binding domains comprises an IL-10 receptor binding domain (e.g., an antibody or antibody fragment that targets one or more components of the IL-10 receptor). In some embodiments, the disclosure provides a polymer, polymer complex, or pair of polymers comprising two second binding domains that target different components of the IL-10 receptor, e.g., IL-10Ra and IL-10Rb.

[0335] In some embodiments, the disease signature ligand is IL-6 and the effector ligand is the IL-10 receptor. For example, in some embodiments, the first binding domain comprises an IL-6 binding domain (e.g., an anti-IL-6 antibody or antibody fragment, e.g., an anti-IL-6 VHH antibody), and one or more second binding domains comprises an IL-10 receptor binding domain (e.g., an antibody or antibody fragment that targets one or more components of the IL-10 receptor). In some embodiments, the disclosure provides a polymer, polymer complex, or pair of polymers comprising two second binding domains that target different components of the IL-10 receptor, e.g., IL-10Ra and IL-10Rb, and / or two first binding domains that target different epitopes of IL-6.

[0336] In some embodiments, the disease signature ligand is TGFβ and the effector ligand is differentiation cluster 3 (CD3). For example, in some embodiments, the first binding domain comprises a TGFβ binding domain (e.g., an anti-TGFβ1 antibody or antibody fragment, e.g., an anti-TGFβ1 single-chain variable fragment (scFv)) and one or more second binding domains comprises CD3 binding domains (e.g., an anti-CD3 antibody or antibody fragment, e.g., an anti-CD3 heavy-chain variable domain (VH) and / or light-chain variable domain (VL)). In some embodiments, the disclosure provides a polymer, polymer complex, or pair of polymers comprising different components of the CD3-targeting moiety, for example, two second binding domains comprising anti-CD3 VH and VL, respectively.

[0337] In some embodiments, the disease signature ligand is IL-8 and the effector ligand is CD3. For example, in some embodiments, the first binding domain comprises an IL-8 binding domain (e.g., an anti-IL-8 antibody or antibody fragment, e.g., an anti-IL-8 single-chain variable fragment (scFv)) and one or more second binding domains comprises CD3 binding domains (e.g., an anti-CD3 antibody or antibody fragment, e.g., anti-CD3 heavy chain VH and / or VL). In some embodiments, the disclosure provides a polymer, polymer complex, or pair of polymers comprising different components of the CD3 targeting moiety, for example, two second binding domains comprising anti-CD3 VH and VL, respectively.

[0338] 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) contains IL-10 or a fragment and / or modified version thereof. In some embodiments, the effector ligand is the IL-10 receptor.

[0339] In some embodiments, the disease signature ligand-binding domain (first binding domain) comprises TNFα, MCP-1, or IL-12 or a fragment and / or modified version thereof, and the effector ligand-binding domain (second binding domain) comprises IL-10 or a fragment and / or modified version thereof. In some embodiments, the effector ligand is the IL-10 receptor.

[0340] In some embodiments, the disease signature ligand is VEGF and the effector ligand is 4-1BB. For example, in some embodiments, the first binding domain includes a VEGF-binding domain (e.g., (i) a VEGF-neutralizing antibody (e.g., bevacizumab) or (ii) a receptor trap derived from the VEGF receptor VEGFR3 or VEGFR2 (e.g., aflibercept)), and the second binding domain includes a 4-1BB-binding domain (e.g., an anti-4-1BB antibody or antibody fragment, e.g., an anti-4-1BB scFv). In some embodiments, the 4-1BB-binding domain is an scFv reformatted from a full-length agonist mAb such as urelumab or utomirumab (e.g., as shown in SEQ ID NOs. 36 and SEQ ID NOs. 37, respectively).

[0341] In one embodiment, the Spectrum Complex is provided, comprising two polymers, each containing a VEGF-binding domain linked to a 4-1BB-binding domain, wherein the 4-1BB-binding domain induces cellular effector function upon binding to 4-1BB; the two polymers are conjugated to each other; and the induction of effector function by the polymer complex is conditional on each of the two polymers binding to VEGF. In some embodiments, the conjugation is non-covalent. In some embodiments, the non-covalent conjugation is mediated by a complementary part of a pair, with each polymer containing one member of the pair. In some embodiments, the complementary polypeptide pair is a pair of Fc fragments. In some embodiments, the Fc fragment pair is a knob-into-hole pair. In some embodiments, the two polymers are identical.

[0342] In another embodiment, the Spectrum provides a polymeric complex comprising two polymers, each containing a VEGF-binding domain linked to a 4-1BB-binding domain, wherein the 4-1BB-binding domain induces cellular effector function upon binding to 4-1BB; the two polymers are not conjugated to each other in the presence of a disease signature ligand; and the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to VEGF. In some embodiments, the first member and the second member of the pair of polymers are non-covalently conjugated to each other in the presence of a disease signature ligand, and the non-covalent conjugation is mediated by the disease signature ligand. In some embodiments, the polymers or one or both members of the pair of polymers contain a half-life extension portion (e.g., an Fc domain or a fragment thereof).

[0343] In some embodiments of any of the above embodiments, the VEGF-binding domain is an anti-VEGF scFv (e.g., as provided in SEQ ID NO: 39). In some embodiments, the VEGF-binding domain is a VEGF receptor trap (e.g., as provided in SEQ ID NO: 38). In some embodiments, the VEGF-binding domain is (i) a VEGF-neutralizing antibody (e.g., bevacizumab), or (ii) a receptor trap derived from the VEGF receptor VEGFR3 or VEGFR2 (e.g., aflibercept).

[0344] In some embodiments of any of the above embodiments, the 4-1BB binding domain is an anti-4-1BB scFv. In some embodiments, the 4-1BB binding domain is an scFv reformatted from a full-length agonist mAb such as urelumab or utomirumab (as shown in, for example, SEQ ID NO: 36 and SEQ ID NO: 37, respectively).

[0345] Exemplary reference polypeptides (e.g., antibodies and their antigen-binding fragments) containing variants of reference sequences can be used in the present invention. For example, "TPO receptor (TpoR) binding polypeptide variants," "IFNγ binding polypeptide variants," "IL-10b binding polypeptide variants," "CD3 binding polypeptide variants," "IL-8 binding polypeptide variants," "TGFb ​​binding polypeptide variants," "IL-6 binding polypeptide variants," and "4-1BB binding polypeptide variants" (e.g., "variants" relating to polypeptides provided by the present invention) include amino acid sequences having, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions relative to a reference sequence (e.g., any of SEQ ID NOs: 1-9, 12-14, and 32-43). In some embodiments, the variants retain the function of the disclosed polypeptide, for example, in assays provided herein, for example, in examples provided herein.

[0346] In certain embodiments, the polypeptide variants provided by the present invention include up to 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, for example, 1 to 3 amino acid substitutions. Various amino acid substitutions are possible for the polypeptide variants provided by the present invention, including substitutions with non-standard amino acids. In some embodiments, the polypeptide variants provided by the present invention retain the functionality of the disclosed polypeptide, for example, in the assays provided herein, for example, in the examples provided herein.

[0347] In some embodiments, the polypeptide variant includes a conserved substitution or a highly conserved substitution with respect to the reference sequence. A “conserved substitution” with respect to the reference sequence means that a given amino acid substitution has a value of 0 or greater in BLOSUM62.

[0348] A "highly conserved substitution" relative to a reference sequence means that a given amino acid substitution has a value of 1 or greater (for example, 2 or greater in some embodiments) in BLOSUM62.

[0349] In certain embodiments, a polypeptide variant provided by the present invention comprises a paratope of the polypeptide provided by the present invention (e.g., an antibody or fragment thereof provided by the present invention), and only highly conserved substitutions are made in the residues of the paratope (e.g., a maximum of 1, 2, 3, 4, or 5 substitutions in the paratope). However, in certain embodiments, no substitutions are made in the paratope, and any substitutions are outside the paratope, and in various embodiments, these non-paratope residues may be highly conserved substitutions, conservative substitutions, non-conservative substitutions, or a combination thereof. For example, in certain embodiments, any substitution of non-paratope residues in the CDR of the variable region is either a conservative substitution or, in some embodiments, a highly conserved substitution. For example, in certain embodiments, the CDR of the polypeptide variant may have a maximum of 1, 2, 3, 4, or 5 substitutions per CDR in residues other than those constituting the paratope, and the substitutions are conservative substitutions or, in more specific embodiments, highly conserved substitutions. In more specific embodiments, non-conservative substitutions can be made outside the CDR, but those skilled in the art will understand that substitutions outside the CDR can be conservative or highly conservative in some embodiments. In specific embodiments, the variant polypeptides provided by the present invention are at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more identical to a reference immunoglobulin light chain variable region or immunoglobulin heavy chain variable region, with full consideration given to the various substitution criteria described above (e.g., substitutions for paratopes, CDRs, non-paratope residues, and non-CDR residues).

[0350] E. Reader, Reporter, and Linker Sections leader In some embodiments of any of the compositions and methods provided herein, the polymer comprises a leader peptide, e.g., a leader peptide that targets the polymer for secretion. The leader peptide can be cleaved from the polymer before polymer formation. In some embodiments, the leader peptide is a mouse immunoglobulin κ variable 3 (IgKVIII) leader peptide (e.g., UniProt ID A0A140T8P0 M1 to G20). Further exemplary leader sequences are provided in Table 1 (SEQ ID NOs: 15-31).

[0351] [Table 1]

[0352] reporter In some embodiments of any of the compositions and methods provided herein, the polymer includes a reporter moiety. For example, in embodiments including a pair of polymers, one or both members of the pair of polymers may include a reporter moiety, or each member of the pair of polymers may include a different reporter moiety.

[0353] Examples of reporter portions include, but are not limited to, affinity tags (e.g., FLAG affinity tags), fluorescent markers, and colorimetric markers. For example, in some embodiments, the reporter portion 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 portion comprises a fragment of bait protein, which is detected by adding an exogenous dye to detect the bait protein.

[0354] In some embodiments comprising a pair of polymers, the first and second members of the pair of polymers include a complementary reporter moiety, for example, a reporter moiety that is detectable (e.g., produces a fluorescent signal) when the first and second members of the pair of polymers form a polymer. For example, the first and second members of the pair of polymers may include a fluorescence resonance energy transfer (FRET) pair (e.g., a near-infrared FRET pair), for example, a peptide-based or protein-based FRET pair member.

[0355] Linker In some embodiments of any of the compositions and methods provided herein, the polymer includes one or more linker domains, for example, a linker domain that connects a first binding domain to a second binding domain, connects one or more subdomains within the first or second binding domain, and / or connects the first or second binding domain to a leader peptide or reporter portion.

[0356] In some embodiments, 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, for example, a GS linker having the GS(GnS)m form or a GS linker having the (GnS)m form (for example, n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (for example, n=1-5 or 5-10, for example, n=4) and m=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (for example, m=1-5, 5-10, or 10-15, for example, m=5)). In some embodiments, the peptide linker is a GS linker having the form (G4S)n (e.g., n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., n=1-5 or 5-10)).

[0357] F. Half-life extension portion Any of the polymers (e.g., polypeptides) provided herein can be modified to alter (e.g., extend) their half-lives (e.g., half-lives in circulation (e.g., in serum)) and / or to induce desired effector functions. For example, in some embodiments, any of the polymers provided herein may include a half-life-extending moiety (e.g., a heterogeneous moiety). Exemplary half-life-extending moieties include polypeptides (e.g., crystalline fragment regions (Fc regions) or fragments or variants thereof, or albumin domains or fragments or variants thereof) and non-polypeptide moieties (e.g., polyethylene glycol (PEG) or modified derivatives thereof).

[0358] In some embodiments, the polypeptides provided herein are modified to include an Fc region that extends the half-life of the polypeptide compared to a polypeptide version that does not include an Fc region. In some embodiments, the Fc region is an IgG isotype Fc region, e.g., an IgG1, IgG2, or IgG4 subtype Fc region (e.g., an Fc region derived from human, mouse, or non-human primate (NHP)). In some embodiments, the Fc region includes one or more Fc effector function silencing mutations (e.g., LALA or LALAPG mutations (mutations at positions L234, L235, G236, N297, or P329 in IgG1)); in other embodiments, the Fc region can induce one or more Fc effector functions. The Fc region may be modified to extend the half-life using one or more mutations that enhance neonatal Fc receptor (FcRn)-based recycling. Further Fc variants that may be used in the present invention include mutant Fc variants previously described for altering Fcγ receptor binding or Fc neonatal receptor binding and recycling, as well as Fc variants comprising glycosylation modifications. Variant Fc regions that may be used in the present invention are provided in Saunders, Frontiers in Immunology, 10: Article 1296, 2019; Delidakis et al., Annual Review of Biomedical Engineering, 24: 249-274, 2022; and Wilkinson et al., PLoS ONE, 16(12): e0260954, 2021.

[0359] In some embodiments, the polypeptides provided herein are modified to include an Fc region that alters Fc gamma receptor binding and / or effector function or Fc neonatal receptor binding and / or recycling. In some embodiments, the polypeptides provided herein are modified to include an Fc region comprising one or more glycosylation modifications.

[0360] In some embodiments, the polymers (e.g., polypeptides) provided herein are modified to include human serum albumin (HSA) or its binder, thereby extending the half-life (e.g., circulating half-life) of the polypeptide compared to the polypeptide version without HSA or its binder. For example, in some embodiments, the polypeptide is directly fused to HSA. In other embodiments, the polypeptide is fused to an HSA binder, such as a short peptide sequence, VHH, or any other antibody or native scaffold that targets HSA.

[0361] In some embodiments, the modification (e.g., a different part) reduces the half-life of the polymer (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%, and 95% compared to the control polymer (e.g., a version of the polymer without the modification). Increase by %, 100% or more (for example, 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%).

[0362] G. Manufacturing and Purity In some embodiments of any of the compositions and methods provided herein, the polymer, polymer pair, polymer complex, nucleic acid, nucleic acid pair, polymer, or composition containing the same is at least 95% pure (for example, at least 95% free of any impurities or undesirable substances). In some embodiments, the polymer, polymer pair, polymer complex, nucleic acid, nucleic acid pair, polymer, or composition containing the same is more than 95% pure, for example, at least 96%, 97%, 98%, or 99% pure, or 100% pure.

[0363] In some embodiments of any of the compositions and methods provided herein, polymers, paired polymers, polymeric complexes, nucleic acids, paired nucleic acids, polymers, or compositions comprising the same are prepared in accordance with one or more International Organization for Standardization (ISO) standards.

[0364] In some embodiments, polymers, paired polymers, polymeric complexes, nucleic acids, nucleic acids, polymers, or compositions containing the same are manufactured in accordance with the Good Manufacturing Practice (GMP), Good Clinical Practice (GCP), and / or Good Laboratory Practice (GLP) standards of the U.S. Food and Drug Administration (FDA).

[0365] H. Biological sample In some embodiments of any of the compositions and methods provided herein, the biological sample comprises an extract, fluid, or fraction (e.g., a fraction containing extracts, fluids, or cells derived from the subject); cells; tissue; or subject (e.g., a mammalian subject, e.g., a human subject).

[0366] In some embodiments of any of the compositions and methods provided herein, the biological sample is a human subject.

[0367] In some embodiments, the biological sample is derived from a vertebrate (e.g., mammal, bird, fish, reptile, or amphibian). In some embodiments, the biological sample is derived from a human (e.g., the subject is human). In other embodiments, the biological sample is derived from a non-human animal (e.g., the subject is a non-human mammal). In embodiments, non-human mammals include non-human primates (e.g., monkeys, apes), ungulates (e.g., cattle, buffalo, sheep, goats, pigs, camels, llamas, alpacas, deer, horses, donkeys), carnivores (e.g., dogs, cats), rodents (e.g., rats, mice), or rabbits (e.g., rabbits). In some embodiments, the biological samples are derived from birds such as members of the order Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., domestic pigeons, doves), or Psittaciformes (e.g., parrots). In some embodiments, the biological samples are derived from invertebrates such as arthropods (e.g., insects, spiders, crustaceans), nematodes, annelids, helminths, or mollusks.

[0368] I. Lipid nanoparticles Compositions (e.g., polymers, paired polymers, polymer complexes, polypeptides, nucleic acids, and compositions comprising the same), methods, and delivery systems provided herein may use any preferred carrier or delivery mode described herein, including lipid nanoparticles (LNPs) in certain embodiments. In some embodiments, lipid nanoparticles include one or more ionic lipids, such as noncationic lipids (e.g., neutral, anionic, or zwitterionic lipids); one or more conjugated lipids (e.g., lipids conjugated to PEG or lipids conjugated to polymers as described in Table 5 of International Publication No. 2019217941, which is incorporated herein by reference); and one or more sterols (e.g., cholesterol).

[0369] Lipids that can be used in nanoparticle formation (e.g., lipid nanoparticles) include, for example, those listed in Table 4 of International Publication No. 2019217941, which is incorporated by reference. For example, lipid-containing nanoparticles may contain one or more of the lipids listed in Table 4 of International Publication No. 2019217941. Lipid nanoparticles may also contain additional elements such as polymers, such as those listed in Table 5 of International Publication No. 2019217941, which is incorporated by reference.

[0370] In some embodiments, the conjugated lipids, if present, include PEG-diacylglycerol (DAG) (e.g., l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), pegylated phosphatidylethanolamine (PEG-PE), and PEG succinate diacylglycerol (PEGS-DAG) (4-0-(2',3'-di(tetradi) This may include canoyloxy)propyl-l-0-(w-methoxy(polyethoxy)ethyl)butanediate (PEG-S-DMG), PEG dialkoxypropyl carbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and one or more of those listed in Table 2 of International Publication No. 2019051289 (incorporated herein by reference), as well as any combination thereof.

[0371] In some embodiments, sterols that may be incorporated into lipid nanoparticles include one or more cholesterol or cholesterol derivatives, such as those incorporated by reference in International Publication No. 2009 / 127060 or U.S. Patent Application Publication No. 2010 / 0130588. Additional exemplary sterols include, but are not limited to, those described in Eygeris et al. (2020)dx.doi.org / 10.1021 / acs.nanolett.0c01386, incorporated herein by reference.

[0372] In some embodiments, lipid particles include ionizable lipids, noncationic lipids, conjugated lipids that inhibit particle aggregation, and sterols. The amounts of these components can be varied independently to achieve desired properties. For example, in some embodiments, lipid particles include about 20 mol% to about 90 mol% of total lipids in the ionizable lipids (in other embodiments, this may be 20-70% (mol), 30-60% (mol), or 40-50% (mol); about 50 mol% to about 90 mol% of total lipids present in the lipid nanoparticles), about 5 mol% to about 30 mol% of total lipids in the noncationic lipids, about 0.5 mol% to about 20 mol% of total lipids in the conjugated lipids, and about 20 mol% to about 50 mol% of total lipids in the sterols. The ratio of total lipids to nucleic acids can be varied as needed. For example, the ratio of total lipids to nucleic acids (mass or weight) may be about 10:1 to about 30:1.

[0373] In some embodiments, the ratio of lipids to nucleic acids (mass / mass ratio; w / w ratio) may range from about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipids and nucleic acids may be adjusted to provide a desired N / P ratio, for example, an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Generally, the overall lipid content of a lipid nanoparticle formulation may range from about 5 mg / mL to about 30 mg / mL.

[0374] Some non-limiting examples of lipid compounds that can be used (e.g., in combination with other lipid components) to form lipid nanoparticles for the delivery of the compositions described herein, for example, nucleic acids described herein (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)) include: [ka]

[0375] In some embodiments, LNPs comprising formula (i) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. [ka]

[0376] In some embodiments, LNPs comprising formula (ii) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. [ka]

[0377] In some embodiments, LNPs comprising formula (iii) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. [ka]

[0378] In some embodiments, LNPs comprising formula (v) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. [ka]

[0379] In some embodiments, LNPs comprising formula (vi) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. [ka]

[0380] In some embodiments, LNPs comprising formula (viii) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. [ka]

[0381] In some embodiments, LNPs comprising formula (ix) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. [ka] [In the formula, X 1 O, NR 1 , or direct bond, X 2 These are C2-5 alkylenes, and X 3 is a C (=O) or direct bond, R 1 is H or Me, and R 3 It is a C1-3 alkyl group, and R 2 is either C1-3 alkyl or R 2 The nitrogen atom to which it is bonded and X 2 Together with 1 to 3 carbon atoms, it forms a 4, 5, or 6-membered ring, or X 1 , NR 1 And R 1 and R2 These, together with the nitrogen atom to which they are bonded, form a 5- or 6-membered ring, or R 2 R 3 And together with the nitrogen atoms to which they are bonded, they form a 5, 6, or 7-membered ring, Y 1 These are C2-12 alkylenes, and Y 2 teeth, [ka] Selected from, where n is 0 to 3, R 4 These are C1-C15 alkyl groups, and Z 1 These are C1-6 alkylenes or direct bonds. Z 2 teeth [ka] (either orientation) or not present, however Z 1 If it is a direct bond, then Z 2 It shall be considered not to exist; R 5 R is a C5-9 alkyl or C6-10 alkoxy, 6 is a C5-9 alkyl or C6-10 alkoxy, W is a methylene or direct bond, R 7 (is H or Me), or a salt thereof, provided that R 3 and R 2 is a C2 alkyl group, X 1 However, O and X 2 However, it is a linear C3 alkylene, X 3 However, C (=O) and Y 1 However, it is a linear Ce alkylene, (Y 2 )nR 4 but, [ka] And R 4 However, it is a linear C5 alkyl, Z 1 However, it is C2 alkylene, Z 2 However, it does not exist, and W is methylene, R 7 However, if H, then R5 and R 6 [This is not Cx alkoxy]

[0382] In some embodiments, LNPs comprising formula (xii) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. [ka]

[0383] In some embodiments, LNPs comprising formula (xi) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. [ka] [In the formula, R= [ka] is

[0384] In some embodiments, the LNP includes compounds of formula (xiii) and formula (xiv). [ka]

[0385] In some embodiments, LNPs comprising formula (xv) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. [ka]

[0386] In some embodiments, LNPs comprising the formulation of formula (xvi) are used to deliver the polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. [ka] [where X= [ka] is

[0387] In some embodiments, lipid compounds are used to form lipid nanoparticles for the delivery of the compositions described herein, for example, nucleic acids described herein (e.g., RNA (e.g., cyclic polynucleotides, linear polyribonucleotides)) are prepared by one of the following reactions: [ka]

[0388] In some embodiments, LNPs comprising formula (xxi) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. In some embodiments, the LNP of formula (xxi) is an LNP described in International Publication No. 2021113777 (e.g., lipids of formula (1), such as the lipids in Table 1 of International Publication No. 2021113777). [ka] [In the formula, Each n is an integer between 2 and 15, independently of the others; L1 and L3 are independently -OC(O)-* or -C(O)O-*, where "*" indicates a connection point to R1 or R3; R1 and R3 are independently oxo, halo, hydroxy, cyano, alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylaminoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, (heterocyclyl)(alkyl)aminoalkyl, heterocyclyl, heteroaryl, alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino, aminoalkylcarbonylamino, aminocarbonylalkylamino, (aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl, alkyloxycarbonyl, A A linear or branched C9-C20 alkyl or C9-C20 alkenyl that is optionally substituted with one or more substituents selected from the group consisting of minocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl, dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl, (alkylaminoalkyl)(alkyl)aminocarbonyl, alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkylsulfonyl, and alkylsulfonalkyl; R2 is selected from the following group: [ka]

[0389] In some embodiments, LNPs comprising formula (xxii) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. In some embodiments, the LNP of formula (xxii) is an LNP described in International Publication No. 2021113777 (e.g., lipids of formula (2), such as the lipids in Table 2 of International Publication No. 2021113777). [ka] [In the formula, Each n is an independent integer between 1 and 15; R1 and R2 are each independently selected from the following group: [ka] R3 is selected from the following group: [ka]

[0390] In some embodiments, LNPs comprising formula (xxiii) are used to deliver polyribonucleotide (e.g., cyclic polyribonucleotides, linear polyribonucleotides) compositions described herein to cells. In some embodiments, the LNP of formula (xxiii) is an LNP described in International Publication No. 2021113777 (e.g., lipids of formula (3), such as the lipids in Table 3 of International Publication No. 2021113777). [ka] [In the formula, X is selected from -O-, -S-, or -OC(O)-*, where * indicates the attachment point to R1; R1 is selected from the following group: [ka] R2 is selected from the following group: [ka]

[0391] In some embodiments, the compositions described herein (e.g., nucleic acids (e.g., cyclic polyribonucleotides, linear polyribonucleotides) or proteins) are provided in LNPs containing ionizable lipids. In some embodiments, the ionizable lipid is, for example, 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate heptadecan-9-yl (SM-102) as described in Example 1 of U.S. Patent No. 9,867,888 (which is incorporated herein by reference in whole). In some embodiments, the ionizable lipid is octadeca-9,12-dienoic acid 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (LP01), as synthesized, for example, in Example 13 of International Publication No. 2015 / 095340 (which is incorporated herein by reference as a whole). In some embodiments, the ionizable lipid is 9-((4-dimethylamino)butanoyl)oxy)heptadecanedienoic acid di((Z)-non-2-en-1-yl) (L319), as synthesized, for example, in Example 7, 8, or 9 of U.S. Patent Application Publication No. 2012 / 0027803 (which is incorporated herein by reference as a whole). 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)azanegiyl)bis(dodecane-2-ol)(C12-200), as synthesized in Examples 14 and 16 of International Publication No. 2010 / 053572 (which is incorporated herein by reference in whole).In some embodiments, the ionizable lipid is the imidazole cholesterol ester (ICE) lipid 3-(1H-imidazole-4-yl)propanoic acid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl, for example, structure (I) derived from International Publication No. 2020 / 106946 (which is incorporated herein by reference in whole).

[0392] In some embodiments, the ionizable lipid may be a cationic lipid that is an ionizable cationic lipid, for example, a cationic lipid that can exist in a pH-dependently positively charged or neutral form, or an amine-containing lipid that can be readily protonated. In some embodiments, the cationic lipid is, for example, a lipid that can be positively charged under physiological conditions. Examples of cationic lipids include one or more positively charged amine groups. In some embodiments, the lipid particles in the formulation include a cationic lipid together with one or more of the following: neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, and lipids conjugated to polymers. In some embodiments, the cationic lipid may be an ionizable cationic lipid. Exemplary cationic lipids as disclosed herein may have an effective pKa greater than 6.0. In embodiments, the lipid nanoparticles may include a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa) from the first cationic lipid. Lipid nanoparticles may include 40–60 mol percent of cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and therapeutic agents, such as nucleic acids (e.g., RNA (e.g., cyclic polyribonucleotides, linear polyribonucleotides)) as described herein, encapsulated within or associated with the lipid nanoparticles. In some embodiments, nucleic acids are combined with cationic lipids. Nucleic acids may be adsorbed onto the surface of LNPs, such as LNPs containing cationic lipids. In some embodiments, nucleic acids may be encapsulated within LNPs, such as LNPs containing cationic lipids. In some embodiments, lipid nanoparticles may include a targeting moiety, such as being coated with a targeted agent. In embodiments, the LNP formulations are biodegradable.In some embodiments, lipid nanoparticles comprising one or more lipids described herein, for example, formulas (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 RNA molecules.

[0393] Examples of ionizable lipids that may be used in lipid nanoparticle formulations are, but are not limited to, those listed in Table 1 of International Publication No. 2019051289, which is incorporated herein by reference. Additional example lipids include, but are not limited to, one or more of the following formulas: X of U.S. Patent Application Publication No. 2016 / 0311759; I of U.S. Patent Application Publication No. 20150376115 or U.S. Patent Application Publication No. 2016 / 0376224; I, II, or III of U.S. Patent Application Publication No. 20160151284; I, IA, II, or IIA of U.S. Patent Application Publication No. 20170210967; U.S. Patent Application Publication No. 20 Ic of 150140070; A of U.S. Patent Application Publication 2013 / 0178541; I of U.S. Patent Application Publication 2013 / 0303587 or U.S. Patent Application Publication 2013 / 0123338; ​​I of U.S. Patent Application Publication 2015 / 0141678; II, III, IV, or V of U.S. Patent Application Publication 2015 / 0239926; I of U.S. Patent Application Publication 2017 / 0119904; International Publication 2017 / 117528 A or II of the Brochure; A of the Specification of U.S. Patent Application Publication No. 2012 / 0149894; A of the Specification of U.S. Patent Application Publication No. 2015 / 0057373; A of the Brochure of International Publication No. 2013 / 116126; A of the Specification of U.S. Patent Application Publication No. 2013 / 0090372; A of the Specification of U.S. Patent Application Publication No. 2013 / 0274523; A of the Specification of U.S. Patent Application Publication No. 2013 / 0274504; A of the Specification of U.S. Patent Application Publication No. 2013 / 0053572; International Publication No. 201 Brochure A of 3 / 016058; Brochure A of International Publication 2012 / 162210; Specification I of U.S. Patent Application Publication 2008 / 042973; Specification I, II, III, or IV of U.S. Patent Application Publication 2012 / 01287670; Specification I or II of U.S. Patent Application Publication 2014 / 0200257; Specification I, II, or III of U.S. Patent Application Publication 2015 / 0203446; Specification I or III of U.S. Patent Application Publication 2015 / 0005363;Sections I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of U.S. Patent Application Publication No. 2014 / 0308304; Sections I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of U.S. Patent Application Publication No. 2013 / 0338210; Sections I, II, III, or IV of International Publication No. 2009 / 132131; Section A of U.S. Patent Application Publication No. 2012 / 01011478; Section I or XXXV of U.S. Patent Application Publication No. 2012 / 0027796; U.S. Patent Application Publication XIV or XVII of U.S. Patent Application Publication No. 2012 / 0058144; XIV or XVII of U.S. Patent Application Publication No. 2013 / 0323269; I of U.S. Patent Application Publication No. 2011 / 0117125; I, II, or III of U.S. Patent Application Publication No. 2011 / 0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of U.S. Patent Application Publication No. 2012 / 0202871; I, II, III, IV, V, VI, VII, VIII, X of U.S. Patent Application Publication No. 2011 / 0076335 XII, XIII, XIV, XV, or XVI; I or II of U.S. Patent Application Publication 2006 / 008378; I of U.S. Patent Application Publication 2013 / 0123338; ​​I or XAYZ of U.S. Patent Application Publication 2015 / 0064242; XVI, XVII, or XVIII of U.S. Patent Application Publication 2013 / 0022649; I, II, or III of U.S. Patent Application Publication 2013 / 0116307; I, II, or III of U.S. Patent Application Publication 2013 / 0116307; U.S. Patent Application Publication 2010 / 00 I or II of U.S. Patent Application No. 62967; IX of U.S. Patent Application Publication No. 2013 / 0189351; I of U.S. Patent Application Publication No. 2014 / 0039032; V of U.S. Patent Application Publication No. 2018 / 0028664; I of U.S. Patent Application Publication No. 2016 / 0317458; I of U.S. Patent Application Publication No. 2013 / 0195920; 5, 6, or 10 of U.S. Patent No. 10,221,127; III-3 of International Publication Brochure No. 2018 / 081480; I-5 or I-8 of International Publication Brochure No. 2020 / 081938;U.S. Patent No. 9,867,888, Specification 18 or 25; U.S. Patent Application Publication 2019 / 0136231, Specification A; International Publication 2020 / 219876, Brochure II; U.S. Patent Application Publication 2012 / 0027803, Specification 1; U.S. Patent Application Publication 2019 / 0240349, Specification OF-02; U.S. Patent No. 10,086,013, Specification 23; Miao et al (2020), cKK-E12 / A6; International Publication 2010 / 053572, Brochure C12-200; Dahlman et al (2017), 7C1; Whitehead et al. Al 304-O13 or 503-O13; TS-P4C2 of U.S. Patent No. 9,708,628; I of International Publication No. 2020 / 106946; and (1), (2), (3), or (4) of International Publication No. 2021 / 113777. Exemplary lipids further include any one of the lipids in Tables 1-16 of International Publication No. 2021 / 113777.

[0394] In some embodiments, the ionizable lipid is, for example, MC3-butanoic acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino) (DLin-MC3-DMA or MC3) as described in Example 9 of International Publication No. 2019051289A9 (which is incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is, for example, lipid ATX-002 as described in Example 10 of International Publication No. 2019051289A9 (which is incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is (13Z,16Z)-A,A-dimethyl-3-nonyldocosa-13,16-diene-l-amine (compound 32), as described in Example 11 of International Publication No. 2019051289A9 (which is incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is compound 6 or compound 22, as described in Example 12 of International Publication No. 2019051289A9 (which is incorporated herein by reference in its entirety).

[0395] Examples of noncationic lipids include distearoyl-sn-glycerol-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoylphosphatidyl POPE, dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (16-O-monomethylPE, etc.), dimethyl-phosphatidylethanolamine (16-O-dimethylPE, etc.), l8 -l-transPE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), diylcoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphate Examples include, but are not limited to, sphatidylglycerol (POPG), dierydoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof.It is 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 having a C10-C24 carbon chain, such as lauroyl, myristoyl, pimitoyl, stearoyl, or oleoyl. Additional exemplary lipids include, but are not limited to, those described in Kim et al. (2020)dx.doi.org / 10.1021 / acs.nanolett.0c01386, which are incorporated herein by reference in certain embodiments. Such lipids include plant lipids (e.g., DGTS) that have been found to improve mRNA-mediated hepatic transfection in some embodiments.

[0396] Other examples of non-cationic lipids suitable for use in lipid nanoparticles include, but are not limited to, nonphospholipids such as stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecylsterate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, and sphingomyelin. Other non-cationic lipids are described in International Publication No. 2017 / 099823 or U.S. Patent Application Publication No. 2018 / 0028664, the contents of which are incorporated herein by reference in their entirety.

[0397] In some embodiments, the noncationic lipid is oleic acid or a compound of formula I, II, or IV of U.S. Patent Application Publication 2018 / 0028664, which is incorporated herein by reference as a whole. The noncationic lipid may constitute, for example, 0 to 30% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the noncationic lipid content is 5 to 20% (mol) or 10 to 15% (mol) of the total lipids present in the lipid nanoparticles. In embodiments, the molar ratio of ionizable lipids to neutral lipids is in the range of 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).

[0398] In some embodiments, the lipid nanoparticles do not contain any phospholipids.

[0399] In some embodiments, lipid nanoparticles may further contain components such as sterols to provide membrane integration. One exemplary sterol that may be used in lipid nanoparticles is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 53-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestanol, cholestenone, 5α-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog, e.g., cholesteryl-(4'-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT International Publication 2009 / 127060 and U.S. Patent Application Publication 2010 / 0130588, each of which is incorporated herein by reference as a whole.

[0400] In some embodiments, membrane-integrating components such as sterols may constitute 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 constitute 20-50% (mol) or 30-40% (mol) of the total lipid content of the lipid nanoparticles.

[0401] In some embodiments, lipid nanoparticles may include polyethylene glycol (PEG) or conjugated lipid molecules. Generally, these are used to inhibit aggregation of lipid nanoparticles and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymeric lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxypolyethylene glycol)-conjugated lipid.

[0402] Examples of PEG-lipid conjugates include PEG-diacylglycerol (DAG) (e.g., l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), pegylated phosphatidylethanolamine (PEG-PE), and PEG-succinate diacylglycerol (PEGS-DAG) (4-O-(2',3'-di(tetradecanoyloxy)propyl-l-O-(w-methoxypropyl Examples of PEG-lipid conjugates include, but are not limited to, cy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEG-dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEG-lipid conjugates are, for example, U.S. Patent No. 5,885,613, U.S. Patent No. 6,283, and U.S. Patent No. 6,283, respectively, the entire contents of which are incorporated herein by reference. Specifications 7,59l, U.S. Patent Application Publication No. 2003 / 0077829, U.S. Patent Application Publication No. 2003 / 0077829, U.S. Patent Application Publication No. 2005 / 0175682, U.S. Patent Application Publication No. 2008 / 0020058, U.S. Patent Application Publication No. 2011 / 0117125, U.S. Patent Application Publication No. 2010 / 0130588, U.S. Patent Application Publication No. 2016 / 0376224, U.S. Patent Application Publication No. 2017 / 0119904, and U.S. Patent Application Publication No. 099823 As described in the specification, in some embodiments, the PEG-lipid is a compound of formula III, III-aI, III-a-2, III-b-1, III-b-2, or V of U.S. Patent Application Publication 2018 / 0028664, the contents of which are incorporated herein by reference as a whole. In some embodiments, the PEG-lipid is of formula II of U.S. Patent Application Publication 20150376115 or U.S. Patent Application Publication 2016 / 0376224, the contents of which are incorporated herein by reference as a whole.In some embodiments, the PEG-DAA conjugate may be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmitoyloxypropyl, or PEG-distearyloxypropyl. The PEG-lipids may be PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol(l-[8'-(cholest-5-ene-3[β]-oxy)carboxamide-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB(3,4-ditetradioxide) The PEG-lipid may be one or more of coxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether and 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG, 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises a structure selected from the following: [ka]

[0403] In some embodiments, lipids conjugated with molecules other than PEG may also be used as a substitute for PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymeric lipid (GPL) conjugates may be used as a substitute for or in addition to PEG-lipids.

[0404] Exemplary conjugated lipids, namely 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 International Publication Brochure 2019051289A9, the entire contents of which are incorporated herein by reference.

[0405] In some embodiments, PEG or conjugated lipids may constitute 0-20% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the PEG or conjugated lipid content is 0.5-10% or 2-5% (mol) of the total lipids present in the lipid nanoparticles. The molar ratios of ionizable lipids, noncationic lipids, sterols, and PEG / conjugated lipids may vary as needed. For example, the lipid particles may contain 30-70% ionizable lipids by moles or total weight of the composition, 0-60% cholesterol by moles or total weight of the composition, 0-30% noncationic lipids by moles or total weight of the composition, and 1-10% conjugated lipids by moles or total weight of the composition. Preferably, the composition contains 30-40% ionizable lipids by moles or total weight of the composition, 40-50% cholesterol by moles or total weight of the composition, and 10-20% noncationic lipids by moles or total weight of the composition. In some other embodiments, the composition comprises 50-75% ionizable lipids by mole or total weight of the composition, 20-40% cholesterol by mole or total weight of the composition, 5-10% noncationic lipids by mole or total weight of the composition, and 1-10% conjugated lipids by mole or total weight of the composition. The composition may also contain 60-70% ionizable lipids by mole or total weight of the composition, 25-35% cholesterol by mole or total weight of the composition, and 5-10% noncationic lipids by mole or total weight of the composition. The composition may also contain up to 90% ionizable lipids by mole or total weight of the composition and 2-15% noncationic lipids by mole or total weight of the composition.The formulation may also contain, for example, 8-30% ionizable lipids by mole or total weight of the composition, 5-30% noncationic lipids by mole or total weight of the composition, and 0-20% cholesterol by mole or total weight of the composition; 4-25% ionizable lipids by mole or total weight of the composition, 4-25% noncationic lipids by mole or total weight of the composition, 2-25% cholesterol by mole or total weight of the composition, 10-35% conjugate lipids by mole or total weight of the composition, and 5% cholesterol by mole or total weight of the composition; or the composition may contain The lipid nanoparticle formulation may contain ionizable lipids in a molar or total weight of 2-30% of the composition, noncationic lipids in a molar or total weight of 2-30% of the composition, cholesterol in a molar or total weight of 1-15% of the composition, conjugated lipids in a molar or total weight of 2-35% of the composition, and cholesterol in a molar or total weight of 1-20% of the composition; or up to 90% of the composition's molar or total weight of ionizable lipids and 2-10% of the composition's molar or total weight of noncationic lipids, or 100% of the composition's molar or total weight of cationic lipids. In some embodiments, the lipid particle formulation contains ionizable lipids, phospholipids, cholesterol, and PEGylated lipids in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation contains ionizable lipids, cholesterol, and PEGylated lipids in a molar ratio of 60:38.5:1.5.

[0406] In some embodiments, the lipid particles include ionizable lipids, noncationic lipids (e.g., phospholipids), sterols (e.g., cholesterol), and PEGylated lipids, where the molar ratio of lipids is in the range of 20 to 70 molar percent with respect to ionizable lipids, with a target of 40 to 60; the molar percentage of noncationic lipids is in the range of 0 to 30, with a target of 0 to 15; the molar percentage of sterols is in the range of 20 to 70, with a target of 30 to 50; and the molar percentage of PEGylated lipids is in the range of 1 to 6, with a target of 2 to 5.

[0407] In some embodiments, the lipid particles contain ionizable lipids / noncationic lipids / sterols / conjugated lipids in a molar ratio of 50:10:38.5:1.5.

[0408] In one embodiment, the disclosure provides a lipid nanoparticle formulation comprising phospholipids, lecithin, phosphatidylcholine, and phosphatidylethanolamine.

[0409] In some embodiments, one or more additional compounds may also be included. These compounds may be administered separately, or the additional compounds may be included in the lipid nanoparticles of the present invention. In other words, the lipid nanoparticles may contain, in addition to nucleic acids, other compounds or at least one second nucleic acid different from the first. The 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, peptidomimetic compounds, nucleic acids, nucleic acid analogs and derivatives, extracts made from biomaterials, or any combination thereof.

[0410] In some embodiments, the LNP comprises a biodegradable ionizable lipid. In some embodiments, the LNP comprises octadeca-9,l2-dienoate (9Z,l2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl, also known as (9Z,l2Z)-octadeca-9,l2-dienoate (9Z,l2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl, or another ionizable lipid. For example, see International Publication No. 2019 / 067992, International Publication No. 2017 / 173054, International Publication No. 2015 / 095340, and International Publication No. 2014 / 136086, and the lipids in the references provided therein. In some embodiments, the terms cationic and ionizable in the context of LNP lipids are synonymous; for example, ionizable lipids are pH-dependently cationic.

[0411] In some embodiments, the average LNP diameter of an LNP formulation may be between nm at 10 s and nm at 100 s, for example, as measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of an LNP formulation may be between approximately 40 nm and approximately 150 nm, such as approximately 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the LNP formulation may be approximately 50 nm to 100 nm, approximately 50 nm to 90 nm, approximately 50 nm to 80 nm, approximately 50 nm to 70 nm, approximately 50 nm to 60 nm, approximately 60 nm to 100 nm, approximately 60 nm to 90 nm, approximately 60 nm to 80 nm, approximately 60 nm to 70 nm, approximately 70 nm to 100 nm, approximately 70 nm to 90 nm, approximately 70 nm to 80 nm, approximately 80 nm to 100 nm, approximately 80 nm to 90 nm, or approximately 90 nm to 100 nm. In some embodiments, the average LNP diameter of the LNP formulation may be approximately 70 nm to 100 nm. In certain embodiments, the average LNP diameter of the LNP formulation may be approximately 80 nm. In some embodiments, the average LNP diameter of the LNP formulation may be approximately 100 nm. In some embodiments, the average LNP diameter of the LNP formulation is approximately 1 mm to 500 mm, 5 mm to 200 mm, 10 mm to 100 mm, 20 mm to 80 mm, 25 mm to 60 mm, 30 mm to 55 mm, 35 mm to 50 mm, or 38 mm to 42 mm.

[0412] LNPs may be relatively homologous in some examples. The polydispersity index can be used to indicate the uniformity of LNPs, e.g., the particle size distribution of lipid nanoparticles. Small polydispersity indices (e.g., less than 0.3) generally indicate a narrow particle size distribution. LNPs may have polydispersity indices ranging from about 0 to about 0.25, e.g., 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of LNPs may be about 0.10 to about 0.20.

[0413] The zeta potential of LNPs can be used to indicate the electrokinetic potential of a composition. In some embodiments, the zeta potential can explain the surface charge of the LNPs. Lipid nanoparticles with relatively low positive or negative charges are generally desirable because species with higher charges may interact unnecessarily with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the LNP may be approximately -10mV to approximately +20mV, approximately -10mV to approximately +15mV, approximately -10mV to approximately +10mV, approximately -10mV to approximately +5mV, approximately -10mV to approximately 0mV, approximately -10mV to approximately -5mV, approximately -5mV to approximately +20mV, approximately -5mV to approximately +15mV, approximately -5mV to approximately +10mV, approximately -5mV to approximately +5mV, approximately -5mV to approximately 0mV, approximately 0mV to approximately +20mV, approximately 0mV to approximately +15mV, approximately 0mV to approximately +10mV, approximately 0mV to approximately +5mV, approximately +5mV to approximately +20mV, approximately +5mV to approximately +15mV, or approximately +5mV to approximately +10mV.

[0414] The efficiency of protein and / or nucleic acid encapsulation describes the amount of protein and / or nucleic acid encapsulated or otherwise associated with the prepared LNP relative to the initial amount provided. The encapsulation efficiency is preferably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing lipid nanoparticles before and after the breakdown of lipid nanoparticles with one or more organic solvents or surfactants. Anion exchange resins can be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence can be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in a solution. With respect to the lipid nanoparticles described herein, the encapsulation efficiency of proteins and / or nucleic acids may be at least 50%, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0415] LNP may optionally comprise one or more coatings. In some embodiments, LNP may be formulated in capsules, films, or tablets having the coatings. Capsules, films, or tablets containing the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0416] Characterization of additional exemplary lipids, formulations, methods, and LNPs is taught in International Publications 2020 / 061457 and 2021 / 113777, each of which is incorporated herein by reference as a whole. Further characterization of exemplary lipids, formulations, methods, and LNPs is taught in Hou et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater (2021).doi.org / 10.1038 / s41578-021-00358-0, which is incorporated herein by reference as a whole (see, for example, the exemplary lipids and lipid derivatives in Figure 2 of Hou et al.).

[0417] In some embodiments, cell lipofection is performed in vitro or ex vivo using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection reagent (Mirus Bio). In certain embodiments, LNPs are formulated using GenVoy_ILM ionized lipid mix (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulations and their in vivo use being taught in their entirety in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), which are incorporated herein by reference.

[0418] CRISPR-Cas systems, such as LNP formulations optimized for the delivery of Cas9-gRNA RNP, gRNA, and Cas9 mRNA, are described in International Publication No. 2019067992 and International Publication No. 2019067910, both of which are incorporated by reference, and are useful for the delivery of cyclic and linear polyribonucleotides as described herein.

[0419] Additional specific LNP formulations useful for the delivery of nucleic acids (e.g., cyclic polyribonucleotides, linear polyribonucleotides) are described in U.S. Patent No. 8,158601 and U.S. Patent No. 8,168775, both of which are incorporated by reference, and include formulations used in Patisirane, which are marketed under the name ONPATTRO.

[0420] Exemplary doses 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 doses of AAV containing polyribonucleotides (e.g., cyclic polyribonucleotides, linear polyribonucleotides) may include about 10 11 , 10 12 , 10 13 , and 10 14 May include MOI in vg / kg.

[0421] II.How to use In some embodiments, the Specified herein provides methods for providing any of the polymers, paired polymers, or polymeric complexes described herein; nucleic acids or paired nucleic acids; or polymers to approach cells (for example, methods for inducing cellular effector functions, methods for regulating cellular states, and / or methods for treating diseases).

[0422] In some embodiments, the Specified Use of any of the polymers, paired polymers, or polymeric complexes; nucleic acids or paired nucleic acids; or polymers described herein in the manufacture of agents for accessing cells (e.g., agents for use in methods of inducing cellular effector functions, regulating cellular states, and / or treating diseases) is provided herein.

[0423] In some embodiments, the Specified herein provides any polymer, paired polymer, or polymeric complex; nucleic acid or paired nucleic acid; or polymer for use in approaching cells (for example, as agents for use in methods of inducing cellular effector functions, regulating cellular states, and / or treating diseases).

[0424] A. Methods for inducing cell effector function In one embodiment, the Specified Information provides a method for inducing cellular effector function in cells, comprising a polymeric complex (e.g., the polymeric complex described in Section IA(i)) comprising two polymers, each comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein (a) the FBD specifically binds to a disease signature ligand in a biological sample; (b) the SBD specifically binds to an effector ligand in the biological sample, and upon binding to the effector ligand, induces cellular effector function; the two copies of the polymer are conjugated to each other; and the induction of effector function by the polymeric complex is conditional on each copy of the polymer binding to the disease signature ligand, comprising contacting the polymeric complex with cells.

[0425] In another embodiment, the Specified Method for Inducing Cellular Effector Function in Cells, comprising a polymeric complex (e.g., the polymeric complex described in Section IA(ii)) comprising a pair of polymers, each independently comprising a first binding domain (FBD) linked to a second binding domain (SBD), wherein: (a) the FBD of each polymer specifically binds to a disease signature ligand in a biological sample; (b) the first member of the pair of polymers comprises a second binding domain 1 (SBD1) that specifically binds to a first effector ligand in a biological sample; and (c) the pair of polymers A method is provided comprising contacting a cell with a polymer complex, wherein the second member of the polymer comprises a second binding domain 2 (SBD2) that specifically binds to a second effector ligand in a biological sample; SBD1 and SBD2 induce cellular effector function when they bind to a first effector ligand and a second effector ligand; the first member and the second member of the pair of polymers are conjugated to each other; and the induction of effector function by the polymer complex is conditional on each of the pair of polymers binding to a disease signature ligand.

[0426] In another aspect, the Specified Description provides a method for inducing cellular effector function in cells, comprising a polymer (e.g., a polymer complex as described in Section IA(iii)) comprising two FBDs linked to two SBDs, wherein: (a) the FBDs specifically bind to a disease signature ligand in a biological sample; (b) the SBDs specifically bind to an effector ligand in a biological sample, and upon binding to the effector ligand, induce cellular effector function; and the polymer can be configured to allow the binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and the induction of effector function by the polymer is provided, provided that each of the two FBDs binds to the disease signature ligand, the polymer comprising contacting the cell.

[0427] In another embodiment, this specification provides a method for inducing cellular effector function in cells, comprising a polymer (e.g., a polymer complex as described in Section IA(iv)) comprising two FBDs linked to SBD1 and SBD2, wherein: (a) the FBDs specifically bind to a disease signature ligand in a biological sample; (b) SBD1 specifically binds to a first effector ligand in a biological sample; (c) SBD2 specifically binds to a second effector ligand in a biological sample; SBD1 and SBD2 induce cellular effector function upon binding to the first and second effector ligands; the polymer can adopt a configuration that allows SBD1 and SBD2 to bind to the first and second effector ligands in the presence of the disease signature ligands, and the induction of effector function by the polymer is provided, provided that each of the two FBDs binds to the disease signature ligand, and the method comprises contacting the polymer with cells.

[0428] In some embodiments, the use of any of the polymers, paired polymers, or polymeric complexes described herein; nucleic acids or paired nucleic acids; or polymers in the manufacture of agents for inducing cellular effector functions in cells is provided herein.

[0429] In some embodiments, the Specified Polymers or Polymeric Complexes; Nucleic Acids or Pairs of Nucleic Acids; or Polymers are provided herein for use in inducing cellular effector functions in cells.

[0430] In some embodiments of any of the above-described aspects, cells are present in the subject, and a polymer, polymer complex, or nucleic acid is administered in a therapeutically effective dose.

[0431] In some embodiments, subjects have or are suspected of having a disease or disorder characterized by abnormal levels of disease signature targets, and optionally, subjects are pre-determined to have abnormal levels of disease signature targets.

[0432] In some embodiments, the cells are T cells. In some embodiments, the effector ligand is 4-1BB and the cells are T cells. In some embodiments, the cell effector function is T cell proliferation. In some embodiments, the cell effector function is antitumor activity.

[0433] B. Methods for regulating the state of cells In another embodiment, the Specified provides a method for modulating the state of a cell, which includes bringing any of the polymers or polymer complexes or nucleic acids or pairs of nucleic acids described herein close to the cell (for example, disease signature ligands and effector ligands bound by a polymer or polymer complex are present on or in close proximity to the cell), thereby modulating the state of the cell.

[0434] In some embodiments, the use of any of the polymers, paired polymers, or polymeric complexes described herein; nucleic acids or paired nucleic acids; or polymers in the manufacture of agents for modulating the state of cells is provided herein.

[0435] In some embodiments, the Specified herein provides any polymer or polymeric complex; nucleic acid or paired nucleic acid; or polymer for use in modulating the state of cells.

[0436] In some embodiments of any of the above-described aspects, cells are present in the subject, and a polymer, polymer complex, or nucleic acid is administered in a therapeutically effective dose.

[0437] In some embodiments, subjects have or are suspected of having a disease or disorder characterized by abnormal levels of disease signature targets, and optionally, subjects are pre-determined to have abnormal levels of disease signature targets.

[0438] C. Methods for determining the state of cells In another embodiment, the Specified provides a method for determining the state of a cell, comprising: providing access to the cell of a polymer or polymeric complex containing a reporter domain, or a nucleic acid or pair of nucleic acids encoding the same as described herein (for example, disease signature ligands and effector ligands bound by a polymer or polymeric complex are present on or in close proximity to the cell); detecting the presence of the reporter domain; and thereby determining the state of the cell.

[0439] In some embodiments, the use of any of the polymers, paired polymers, or polymeric complexes described herein; nucleic acids or paired nucleic acids; or polymers in the manufacture of agents for determining the state of cells is provided herein.

[0440] In some embodiments, the Specified herein provides any polymer or polymeric complex; nucleic acid or paired nucleic acid; or polymer for use in determining the state of a cell.

[0441] In some embodiments of any of the above-described aspects, cells are present in the subject, and a polymer, polymer complex, or nucleic acid is administered in a therapeutically effective dose.

[0442] In some embodiments, the subject has a disease or disorder characterized by abnormal levels of disease signature targets.

[0443] III. Dimeric Polymer Assemblies A. Composition In one embodiment, the Specified herein provides a composition comprising a polymer complex (for example, a polymer complex described herein), wherein the polymer complex comprises a dimer, and at least about 90% of the dimer is monodisperse.

[0444] In some embodiments, at least about 92% of the dimer is monodisperse. In some embodiments, at least about 93% of the dimer is monodisperse. In some embodiments, at least about 94% of the dimer is monodisperse. In some embodiments, at least about 95% of the dimer is monodisperse. In some embodiments, at least about 96% of the dimer is monodisperse. In some embodiments, at least about 97% of the dimer is monodisperse. In some embodiments, at least about 98% of the dimer is monodisperse. In some embodiments, at least about 99% of the dimer is monodisperse.

[0445] In another embodiment, the Specified provides a composition comprising a polymeric composite (e.g., a polymeric composite described herein), wherein the polymeric composite comprises two polymers that can be dimerized, and the composition is at least about 90% monodisperse in a size consistent with the dimerized polymeric composite.

[0446] In some embodiments, the composition is at least about 92% monodisperse, at least about 93% monodisperse, at least about 94% monodisperse, at least about 95% monodisperse, at least about 96% monodisperse, at least about 97% monodisperse, at least about 98% monodisperse, or at least about 99% monodisperse (e.g., at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% monodisperse) in size consistent with the dimerized polymer complex.

[0447] The composition may be produced from one or more cell lines that produce one or more components of a polymer complex (e.g., a cell line that produces a single polymer that forms a homodimer, a cell line that expresses two polymers that form a heterodimer, or two cell lines that each express one polymer in a pair that form a heterodimer). For example, a cell line may be transiently or stably transfected with a plasmid encoding a secreted form of one or more polymers. The composition may be purified before evaluating its monodispersity. Purification may include, for example, clarifying the culture supernatant of the cell line (e.g., via a two-step purification scheme using protein A affinity chromatography followed by polishing using cation exchange chromatography).

[0448] In some embodiments, the percentage monodispersity is determined by size exclusion chromatography (SEC), as described, for example, in the examples provided herein. In some embodiments, the percentage monodispersity is determined by analytical SEC. In some embodiments, the percentage monodispersity is determined by analytical SEC using a Superdex® 200 column.

[0449] In some embodiments, the size of the polymer composite coincides with the size of the dimer, as described, for example, in the examples provided herein.

[0450] In some embodiments, the polymer complex comprises two polymers, each containing a VEGF-binding domain linked to a 4-1BB-binding domain.

[0451] In some embodiments, the VEGF-binding domain is anti-VEGF scFv.

[0452] In some embodiments, the VEGF-binding domain is a VEGF receptor trap.

[0453] In some embodiments, the 4-1BB binding domain is an anti-4-1BB scFv.

[0454] In some embodiments, the polymeric composite comprises 4-1BB X VEGF-TRAP.

[0455] In some embodiments, the polymer composite comprises 4-1BB X VEGF-scFV.

[0456] In some embodiments, the polymer complex comprises two polymers, each containing a TGFb-binding domain linked to a TpoR-binding domain.

[0457] In some embodiments, the TGFb-binding domain is anti-TGFb scFv.

[0458] In some embodiments, the TpoR-binding domain is an anti-TpoR scFv.

[0459] In some embodiments, the polymer complex comprises two polymers, each containing an IL-8 binding domain linked to a TpoR binding domain.

[0460] In some embodiments, the IL-8 binding domain is anti-IL-8 scFv.

[0461] In some embodiments, the TpoR-binding domain is an anti-TpoR scFv.

[0462] In some embodiments, the polymer complex comprises two polymers, each containing a TGFb-binding domain linked to an IL2R-binding domain.

[0463] In some embodiments, the TGFb-binding domain is anti-TGFb scFv.

[0464] In some embodiments, the IL2R binding domain includes the N-terminus of IL2. In some embodiments, the IL2R binding domain includes the C-terminus of IL2.

[0465] In some embodiments, the polymer complex comprises two polymers, each containing an IFNg-binding domain linked to an IL10R-binding domain.

[0466] In some embodiments, the IFNg binding domain is anti-IFNg scFv.

[0467] In some embodiments, the IL10R binding domain includes an anti-IL10R VHH.

[0468] In some embodiments, the IL10R binding domain includes IL10.

[0469] In some embodiments, the polymer complex comprises two polymers, each containing an IL6-binding domain linked to an IL10R-binding domain.

[0470] In some embodiments, the IL6 binding domain is anti-IL6 VHH.

[0471] In some embodiments, the IL10R binding domain includes an anti-IL10R VHH.

[0472] In some embodiments, the IL10R binding domain includes IL10.

[0473] In some embodiments, the polymer complex comprises two polymers, each containing an IL8-binding domain linked to a CD3-binding domain.

[0474] In some embodiments, the IL8 binding domain is anti-IL8 scFv.

[0475] In some embodiments, the CD3-binding domain includes anti-CD3 VH.

[0476] B. Method In one aspect, the present disclosure relates to a method for preparing a pharmaceutical composition, (a) preparing or obtaining a composition (e.g., a sample thereof) comprising a polymer complex (e.g., a polymer complex as described herein); (b) Measuring or having measured the percentage of polymer in a monodisperse composition (or a sample thereof); (c) If the percentage of polymers in the monodisperse composition (or the sample thereof) in the sample is above a predetermined threshold, the composition shall be formulated as a pharmaceutical composition or the composition shall not be formulated This provides a method that includes this.

[0477] In another aspect, the present disclosure relates to a method for preparing a pharmaceutical composition, (a) preparing or obtaining a composition (e.g., a sample thereof) comprising a polymer complex (e.g., a polymer complex as described herein); (b) The percentage monodispersity of polymers in the composition (or a sample thereof) has been measured or has been measured; (c) If the percentage monodispersity of polymers in the composition (or a sample thereof) is above a predetermined threshold, the composition is formulated as a pharmaceutical composition or is deemed to have been formulated. This provides a method that includes this.

[0478] In some embodiments of the above-described model, formulating the composition as a pharmaceutical composition or formulating the composition includes combining the composition with one or more pharmaceutically acceptable excipients.

[0479] In some embodiments, the polymer is a dimer.

[0480] In some embodiments, a predetermined threshold is such that at least about 90% of the dimer is monodisperse. In some embodiments, a predetermined threshold is such that at least about 92% of the dimer is monodisperse. In some embodiments, a predetermined threshold is such that at least about 93% of the dimer is monodisperse. In some embodiments, a predetermined threshold is such that at least about 94% of the dimer is monodisperse. In some embodiments, a predetermined threshold is such that at least about 95% of the dimer is monodisperse. In some embodiments, a predetermined threshold is such that at least about 96% of the dimer is monodisperse. In some embodiments, a predetermined threshold is such that at least about 97% of the dimer is monodisperse. In some embodiments, a predetermined threshold is such that at least about 98% of the dimer is monodisperse. In some embodiments, a predetermined threshold is such that at least about 99% of the dimer is monodisperse.

[0481] In some embodiments, a given threshold is a monodispersity of at least about 90%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% (e.g., a monodispersity of at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) in size that matches the dimerized polymer complex.

[0482] In some embodiments, the percentage monodispersity is determined by size exclusion chromatography (SEC), as described, for example, in the examples provided herein. In some embodiments, the percentage monodispersity is determined by analytical SEC. In some embodiments, the percentage monodispersity is determined by analytical SEC using a Superdex® 200 column.

[0483] In some embodiments, the size of the polymer composite coincides with the size of the dimer, as described, for example, in the examples provided herein.

[0484] In some embodiments, the polymer complex comprises two polymers, each containing a VEGF-binding domain linked to a 4-1BB-binding domain.

[0485] In some embodiments, the VEGF-binding domain is anti-VEGF scFv.

[0486] In some embodiments, the VEGF-binding domain is a VEGF receptor trap.

[0487] In some embodiments, the 4-1BB binding domain is an anti-4-1BB scFv.

[0488] In some embodiments, the polymeric composite comprises 4-1BB X VEGF-TRAP.

[0489] In some embodiments, the polymer composite comprises 4-1BB X VEGF-scFV.

[0490] In some embodiments, the polymer complex comprises two polymers, each containing a TGFb-binding domain linked to a TpoR-binding domain.

[0491] In some embodiments, the TGFb-binding domain is anti-TGFb scFv.

[0492] In some embodiments, the TpoR-binding domain is an anti-TpoR scFv.

[0493] In some embodiments, the polymer complex comprises two polymers, each containing an IL-8 binding domain linked to a TpoR binding domain.

[0494] In some embodiments, the IL-8 binding domain is anti-IL-8 scFv.

[0495] In some embodiments, the TpoR-binding domain is an anti-TpoR scFv.

[0496] In some embodiments, the polymer complex comprises two polymers, each containing a TGFb-binding domain linked to an IL2R-binding domain.

[0497] In some embodiments, the TGFb-binding domain is anti-TGFb scFv.

[0498] In some embodiments, the IL2R binding domain includes the N-terminus of IL2. In some embodiments, the IL2R binding domain includes the C-terminus of IL2.

[0499] In some embodiments, the polymer complex comprises two polymers, each containing an IFNg-binding domain linked to an IL10R-binding domain.

[0500] In some embodiments, the IFNg binding domain is anti-IFNg scFv.

[0501] In some embodiments, the IL10R binding domain includes an anti-IL10R VHH.

[0502] In some embodiments, the IL10R binding domain includes IL10.

[0503] In some embodiments, the polymer complex comprises two polymers, each containing an IL6-binding domain linked to an IL10R-binding domain.

[0504] In some embodiments, the IL6 binding domain is anti-IL6 VHH.

[0505] In some embodiments, the IL10R binding domain includes an anti-IL10R VHH.

[0506] In some embodiments, the IL10R binding domain includes IL10.

[0507] In some embodiments, the polymer complex comprises two polymers, each containing an IL8-binding domain linked to a CD3-binding domain.

[0508] In some embodiments, the IL8 binding domain is anti-IL8 scFv.

[0509] In some embodiments, the CD3-binding domain includes anti-CD3 VH.

[0510] IV. Other Embodiments Some embodiments of the technology described herein may be defined according to any of the following numbered embodiments:

[0511] 1. A polymeric complex comprising two polymers, each polymer comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain: (a) FBD specifically binds to disease signature ligands in biological samples; (b) SBD specifically binds to effector ligands in biological samples, and upon binding to effector ligands, induces cellular effector function; Two polymers are conjugated together; A polymeric complex in which the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to a disease signature ligand.

[0512] 2. A polymeric complex comprising a pair of polymers, each polymer independently comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain: (a) The first member of the pair of polymers comprises a first binding domain 1 (FBD1) that specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) The second member of the pair of polymers comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of the disease signature ligand in the biological sample; (c) SBD specifically binds to effector ligands in biological samples, and upon binding to effector ligands, it induces cellular effector function; Two polymers are conjugated together; A polymeric complex in which the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to a disease signature ligand.

[0513] 3. The polymer composite according to Embodiment 1 or 2, wherein the conjugation is a covalent bond.

[0514] 4. The polymeric composite according to Embodiment 3, wherein the covalent conjugation comprises a chemical linker or a polypeptide linker.

[0515] 5. The polymer composite according to Embodiment 1 or 2, wherein the conjugation is non-covalent.

[0516] 6. The polymeric composite according to Embodiment 5, wherein the non-covalent conjugation is mediated by complementary parts of a pair, and each polymer comprises one member of the pair.

[0517] 7. The polymeric complex according to Embodiment 6, wherein the complementary moieties of the pair are biotin and avidin; barnase and burster; a complementary aptamer of the pair; or a complementary polypeptide of the pair.

[0518] 8. The polymeric complex according to Embodiment 7, wherein the pair of complementary polypeptides are the pair of manipulated Fc fragments.

[0519] 9. The polymeric composite according to Embodiment 8, wherein the pair of manipulated Fc fragments are knob-into-hole pairs.

[0520] 10. A polymeric complex according to any one of embodiments 4 and 6-9, wherein the conjugation sterically orients two polymers to enable conditional co-binding of a disease signal and an effector ligand.

[0521] 11. The polymeric complex according to Embodiment 5, wherein the non-covalent conjugation is mediated by a disease signature ligand.

[0522] 12. A polymer composite according to any one of Embodiments 1, 3-5, 10, and 11, wherein the two polymers are identical.

[0523] 13. A polymeric complex comprising a pair of polymers, each polymer independently comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain: (a) The FBD of each polymer specifically binds to disease signature ligands in the biological sample; (b) The first member of the pair of polymers comprises a second binding domain 1 (SBD1) that specifically binds to a first effector ligand in the biological sample; (c) The second member of the pair of polymers includes a second binding domain 2 (SBD2) that specifically binds to a second effector ligand in the biological sample; When SBD1 and SBD2 bind to the first and second effector ligands, they induce cellular effector function; The first member of the paired polymer and the second member of the paired polymer are conjugated to each other; A polymeric complex in which the induction of effector function by the polymeric complex is conditional on each of the paired polymers binding to a disease signature ligand.

[0524] 14. A polymeric complex comprising a pair of polymers, each polymer independently comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain: (a) The first member of the pair of polymers comprises a first binding domain 1 (FBD1) that specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) The second member of the pair of polymers comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of the disease signature ligand in the biological sample; (c) The first member of the pair of polymers comprises a second binding domain 1 (SBD1) that specifically binds to a first effector ligand in the biological sample; (d) The second member of the pair of polymers includes a second binding domain 2 (SBD2) that specifically binds to a second effector ligand in the biological sample; When SBD1 and SBD2 bind to the first and second effector ligands, they induce cellular effector function; The first member of the paired polymer and the second member of the paired polymer are conjugated to each other; A polymeric complex in which the induction of effector function by the polymeric complex is conditional on each of the paired polymers binding to a disease signature ligand.

[0525] 15. The polymer composite according to Embodiment 13 or 14, wherein the conjugation is covalent.

[0526] 16. The polymeric composite according to Embodiment 15, wherein the covalent conjugation comprises a chemical linker or a polypeptide linker.

[0527] 17. The polymeric composite according to Embodiment 13 or 14, wherein the conjugation is non-covalent.

[0528] 18. The polymeric composite according to Embodiment 17, wherein the non-covalent conjugation is mediated by complementary parts of a pair, and each polymer comprises one member of the pair.

[0529] 19. The polymeric complex according to Embodiment 18, wherein the complementary moieties of the pair are biotin and avidin; barnase and burster; a complementary aptamer of the pair; or a complementary polypeptide of the pair.

[0530] 20. The polymeric complex according to Embodiment 19, wherein the pair of complementary polypeptides are a pair of manipulated Fc fragments.

[0531] 21. The polymeric composite according to Embodiment 20, wherein the pair of manipulated Fc fragments are knob-into-hole pairs.

[0532] 22. A polymeric complex according to any one of embodiments 13, 14, and 18-21, wherein the conjugation stereoorients the first member and the second member of the pair of polymers to enable conditional co-binding of disease signaling and effector ligands.

[0533] 23. The polymeric complex according to Embodiment 18, wherein the non-covalent conjugation is mediated by a disease signature ligand.

[0534] 24. A polymeric complex comprising two polymers, each polymer comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain: (a) FBD specifically binds to disease signature ligands in biological samples; (b) SBD specifically binds to effector ligands in biological samples, and upon binding to effector ligands, induces cellular effector function; The two polymers are not conjugated to each other in the absence of disease signature ligands; A polymeric complex in which the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to a disease signature ligand.

[0535] 25. A polymeric complex comprising a pair of polymers, each polymer independently comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain: (a) The first member of the pair of polymers comprises a first binding domain 1 (FBD1) that specifically binds to a first epitope of a disease signature ligand in a biological sample; (b) The second member of the pair of polymers comprises a first binding domain 2 (FBD2) that specifically binds to a second epitope of the disease signature ligand in the biological sample; (c) SBD specifically binds to effector ligands in biological samples, and upon binding to effector ligands, it induces cellular effector function; The two polymers are not conjugated to each other in the absence of disease signature ligands; A polymeric complex in which the induction of effector function by the polymeric complex is conditional on each of the two polymers binding to a disease signature ligand.

[0536] 26. The polymer complex according to Embodiment 24 or 25, wherein a first member of a pair of polymers and a second member of a pair of polymers are non-covalently conjugated to each other in the presence of a disease signature ligand, and the non-covalent conjugation is mediated by the disease signature ligand.

[0537] 27. A polymer comprising two FBDs linked to two SBDs by one or more linker domains: (a) FBD specifically binds to disease signature ligands in biological samples; (b) SBD specifically binds to effector ligands in biological samples, and upon binding to effector ligands, induces cellular effector function; A polymer that can adopt a stereochemical configuration that enables the binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, wherein the induction of effector function by the polymer is conditional on each of the two FBDs binding to the disease signature ligand.

[0538] 28. A polymer comprising FBD1 and FBD2 linked to two SBDs by one or more linker domains: (a) FBD1 specifically binds to the first epitope of the disease signature ligand in the biological sample; (b) FBD2 specifically binds to a secondary epitope of the disease signature ligand in the biological sample; (c) SBD specifically binds to effector ligands in biological samples, and upon binding to effector ligands, it induces cellular effector function; A polymer that can adopt a stereochemistry that allows two SBDs to bind to effector ligands in the presence of a disease signature ligand, and the induction of effector function by the polymer is conditional on each of FBD1 and FBD2 binding to the disease signature ligand.

[0539] 29. A polymer comprising two FBDs linked to SBD1 and SBD2 by one or more linker domains: (a) FBD specifically binds to disease signature ligands in biological samples; (b) SBD1 specifically binds to the first effector ligand in the biological sample; (c) SBD2 specifically binds to a second effector ligand in the biological sample; When SBD1 and SBD2 bind to the first and second effector ligands, they induce cellular effector function; A polymer that can adopt a stereochemistry that allows SBD1 and SBD2 to bind to a first effector ligand and a second effector ligand in the presence of a disease signature ligand, wherein the induction of effector function by the polymer is conditional on each of the two FBDs binding to the disease signature ligand.

[0540] 30. A polymer comprising FBD1 and FBD2 linked to SBD1 and SBD2 by one or more linker domains: (a) FBD1 specifically binds to the first epitope of the disease signature ligand in the biological sample; (b) FBD2 specifically binds to a secondary epitope of the disease signature ligand in the biological sample; (c) SBD1 specifically binds to the first effector ligand in the biological sample; (d) SBD2 specifically binds to a second effector ligand in the biological sample; When SBD1 and SBD2 bind to the first and second effector ligands, they induce cellular effector function; A polymer that can adopt a stereochemistry that allows SBD1 and SBD2 to bind to a first effector ligand and a second effector ligand in the presence of a disease signature ligand, and the induction of effector function by the polymer is conditional on each of FBD1 and FBD2 binding to the disease signature ligand.

[0541] 31. A polymeric complex or polymer according to any one of Embodiments 1 to 30, wherein the disease signature ligand is a protein, peptide, or small molecule.

[0542] 32. The polymeric complex or polymer according to Embodiment 31, wherein the protein is a soluble protein or an insoluble protein.

[0543] 33. A polymeric complex or polymer according to any one of Embodiments 1 to 30, wherein the disease signature ligand is a cytokine.

[0544] 34. The polymeric complex or polymer according to Embodiment 33, wherein the cytokine is an interleukin, interferon, growth factor, chemokine, or a member of the TNF family.

[0545] 35. A polymeric complex or polymer according to Embodiment 34, wherein the interleukin is IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15, IL-17, or IL-23; the interferon is IFN-γ; the growth factor is transforming growth factor β (TGF-β), granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GCSF), epidermal growth factor (EGF), erythropoietin (EPO), or VEGF; the chemokine is monocyte chemotactic protein-1 (MCP-1) or interferon-γ-inducible protein 10 (IP-10); or the member of the TNF family is TNF-α.

[0546] 36. A polymeric complex or polymer according to any one of Embodiments 1 to 30, wherein the disease signature ligand is a neurotransmitter.

[0547] 37. A polymeric complex or polymer according to any one of Embodiments 1 to 30, wherein the disease signature ligand is a cell surface receptor, a surface antigen, a membrane-bound protein, an extracellular matrix component, or an integrin.

[0548] 38. A polymeric complex or polymer according to any one of Embodiments 1 to 30, wherein the disease signature ligand is an autoantigen of an organism from which the biological sample originates.

[0549] 39. The polymeric complex or polymer according to Embodiment 38, wherein the autoantigen is an anti-drug antibody (ADA), an autoantibody, or a tumor marker.

[0550] 40. A polymeric complex or polymer according to any one of Embodiments 1 to 30, wherein the disease signature ligand is a nucleic acid.

[0551] 41. A polymeric complex or polymer according to any one of Embodiments 1 to 30, wherein the disease signature ligand is a carbohydrate, lipid, peptide, nucleoside, or a combination thereof.

[0552] 42. A polymeric complex or polymer according to any one of Embodiments 1 to 30, wherein the disease signature ligand is a hormone, an amino acid derivative, a steroid, or an eicosanoid.

[0553] 43. A polymeric complex or polymer according to any one of Embodiments 1 to 30, wherein the disease signature ligand is a non-self antigen.

[0554] 44. The polymeric complex or polymer according to Embodiment 43, wherein the disease signature ligand is a virus, a bacterium, or a fragment or antigen thereof.

[0555] 45. A polymeric complex or polymer according to any one of Embodiments 1 to 44, wherein the disease signature ligand is a polymer.

[0556] 46. ​​The polymeric complex or polymer according to Embodiment 45, wherein the disease signature ligand is a dimer, trimer, or tetramer.

[0557] 47. A polymeric complex or polymer according to any one of Embodiments 1 to 46, wherein FBD, FBD1, or FBD2 comprises a polypeptide that specifically binds to a disease signature ligand.

[0558] 48. The polymeric complex or polymer according to Embodiment 47, wherein the polypeptide is an antibody or a fragment thereof.

[0559] 49. The polymeric complex or polymer according to Embodiment 48, wherein the antibody or fragment thereof is scFv, monospecific tandem scFv (taFv), bispecific taFv, VHH, VNAR, Fab, monospecific single-stranded diabody, bispecific single-stranded diabody, or biaffinity retargeting antibody (DART).

[0560] 50. A polymeric complex or polymer according to Embodiment 47, which is a polypeptide or antibody mimetic.

[0561] 51. The polymeric complex or polymer according to Embodiment 50, wherein the antibody mimetic is an afibody, affin, afimer, afitin, alphabody, anticarin, lipocalin, avimer, DARPin, finomer, gastric body, notottin, Kunitz domain peptide, monobody, fibronectin type III domain (FN3) based binder, nanoantibody, nanoCLAMP, optimer, lipibody, pronectin, centinline, obody, peptide aptamer, synthetic peptide, or variable lymphocyte receptor (VLR).

[0562] 52. The polymeric complex or polymer according to Embodiment 47, wherein the polypeptide is an endogenous binding domain.

[0563] 53. The polymer complex or polymer according to Embodiment 52, wherein the endogenous binding domain is a cell receptor domain, an enzyme domain, a variable lymphocyte receptor (VLR) domain, an extracellular receptor domain, a nuclear hormone receptor ligand binding domain, or a DNA binding domain.

[0564] 54. A polymeric complex or polymer according to any one of Embodiments 1 to 46, wherein FBD, FBD1, or FBD2 comprises an oligonucleotide that specifically binds to a disease signature ligand.

[0565] 55. The polymeric complex or polymer according to Embodiment 54, wherein the oligonucleotide is a nucleic acid aptamer.

[0566] 56. The polymeric complex or polymer according to Embodiment 55, wherein the nucleic acid aptamer is a DNA aptamer.

[0567] 57. A polymeric complex or polymer according to any one of Embodiments 1 to 46, wherein FBD, FBD1, or FBD2 comprises a chemical molecule that specifically binds to a disease signature ligand.

[0568] 58. A polymeric complex or polymer according to any one of Embodiments 1 to 57, wherein FBD, FBD1, or FBD2 has affinity for two or more disease signature moieties.

[0569] 59. A polymeric complex or polymer according to any one of Embodiments 1 to 58, wherein the effector ligand is a protein or a peptide.

[0570] 60. A polymeric complex or polymer according to any one of Embodiments 1 to 59, wherein the effector ligand is a cell surface receptor or an intracellular receptor.

[0571] 61. The polymer complex or polymer according to Embodiment 60, wherein the cell surface receptor is a catalytic receptor, or the intracellular receptor is a nuclear hormone receptor.

[0572] 62. The polymeric complex or polymer according to Embodiment 61, wherein the catalytic receptor is a receptor tyrosine kinase (RTK), a receptor serine / threonine kinase (RSK), a type 1 cytokine receptor, a type 2 cytokine receptor, or a tumor necrosis factor (TNF) superfamily receptor.

[0573] 63. RTK is VEGFR, and RSK is TGFBR 2 The type 1 cytokine receptor is IL-2R and the type 2 cytokine receptor is IL-10R, or the TNF superfamily receptor is TNFR 2 Alternatively, the polymer composite or polymer according to Embodiment 62, which is 4-1BB.

[0574] 64. A polymeric complex or polymer according to any one of embodiments 1 to 12, 24, 26 to 28, and 31 to 63, wherein SBD is an agonist of an effector ligand.

[0575] 65. The polymeric complex or polymer according to Embodiment 64, wherein the effector ligand must be homodimerized in order to exert its cellular effector function.

[0576] 66. The polymer complex or polymer according to Embodiment 65, wherein the effector ligand is homodimerized in the presence of the polymer complex or polymer and a disease signature ligand.

[0577] 67. A polymeric complex or polymer according to any one of embodiments 13-23 and 29-63, wherein the first effector ligand and the second effector ligand must be associated to exert cellular effector function.

[0578] 68. The polymer complex or polymer according to Embodiment 67, wherein the first effector ligand and the second effector ligand are associated in the presence of the polymer complex or polymer and a disease signature ligand.

[0579] 69. A polymeric composite or polymer according to embodiment 67 or 68, wherein the association is heterodimerization.

[0580] 70. A polymeric complex or polymer according to any one of Embodiments 1 to 69, wherein the cell effector function is biologically active.

[0581] 71. A polymeric complex or polymer according to any one of Embodiments 1 to 70, wherein the cell effector function is therapeutic activity.

[0582] 72. A polymeric complex or polymer according to any one of Embodiments 1 to 70, wherein the cell effector function is disease activity.

[0583] 73. A polymeric complex or polymer according to any one of Embodiments 1 to 72, wherein SBD, SBD1, or SBD2 comprises a polypeptide that specifically binds to an effector ligand.

[0584] 74. The polymeric complex or polymer according to Embodiment 73, wherein the polypeptide is an antibody or a fragment thereof.

[0585] 75. The polymeric complex or polymer according to Embodiment 74, wherein the antibody or fragment thereof is scFv, single-specific taFv, bispecific taFv, VHH, VNAR, Fab, single-specific single-stranded diabody, bispecific single-stranded diabody, or DART.

[0586] 76. A polymeric complex or polymer according to Embodiment 73, which is a polypeptide or antibody mimetic.

[0587] 77. The polymeric complex or polymer according to Embodiment 76, wherein the antibody mimetic is an afibody, affin, afimer, afitin, alphabody, anticalin, lipocalin, avimer, DARPin, finomer, gastric body, notottin, Kunitz domain peptide, monobody, FN3-based binder, nanoantibody, nanoCLAMP, optimer, lipibody, pronectin, centinline, obody, peptide aptamer, synthetic peptide, or VLR.

[0588] 78. The polymeric complex or polymer according to Embodiment 73, wherein the polypeptide is an endogenous binding domain.

[0589] 79. The polymeric complex or polymer according to Embodiment 78, wherein the endogenous binding domain is a ligand or fragment thereof of an effector ligand.

[0590] 80. The polymeric complex or polymer according to Embodiment 78, wherein the endogenous binding domain is a virus-binding protein or a fragment thereof.

[0591] 81. A polymeric complex or polymer according to any one of Embodiments 1 to 72, wherein SBD, SBD1, or SBD2 comprises an oligonucleotide that specifically binds to an effector ligand.

[0592] 82. The polymeric complex or polymer according to Embodiment 81, wherein the oligonucleotide is a nucleic acid aptamer.

[0593] 83. The polymeric complex or polymer according to Embodiment 82, wherein the nucleic acid aptamer is a DNA aptamer.

[0594] 84. A polymeric complex or polymer according to any one of Embodiments 1 to 72, wherein FBD, FBD1, or FBD2 comprises a chemical molecule that specifically binds to a disease signature ligand.

[0595] 85. A polymer composite according to any one of embodiments 13-23, 29-63, and 67-84, wherein SBD1 is a first portion of the binding site and SBD2 is a second portion of the binding site.

[0596] 86. A polymeric complex according to any one of embodiments 13-23, 29-63, and 67-84, wherein SBD1 specifically binds to a first component of the heterodimer receptor and SBD2 specifically binds to a second component of the heterodimer receptor.

[0597] 87. A polymer composite according to any one of embodiments 13-23, 29-63, and 67-84, wherein SBD1 is the first component of the dimer portion and SBD2 is the second component of the dimer portion.

[0598] 88. A polymeric complex according to any one of embodiments 13-23, 29-63, and 67-84, wherein SBD1 is a first fragment of the polypeptide chain and SBD2 is a second fragment of the polypeptide chain.

[0599] 89. The polymer complex according to Embodiment 88, wherein the polypeptide chain is a hormone, cytokine, or growth factor.

[0600] 90. A polymeric composite according to any one of embodiments 13-23, 29-63, and 67-89, wherein SBD1 and SBD2 are manipulated to reduce their affinity for each other.

[0601] 91. A polymeric composite or polymer according to any one of Embodiments 1 to 90, wherein the polymer includes a reporter moiety.

[0602] 92. The polymer composite or polymer according to Embodiment 91, wherein the reporter portion includes an affinity tag, a fluorescent marker, a radioactive marker, or a chromogenic marker.

[0603] 93. The polymer complex or polymer according to Embodiment 92, wherein the affinity tag is a FLAG affinity tag, or the color marker is luciferase or β-lactamase.

[0604] 94. A polymeric composite or polymer according to any one of Embodiments 1 to 93, further comprising one or more linker domains.

[0605] 95. A polymeric complex or polymer according to Embodiment 94, wherein one or more linker domains are peptide linkers.

[0606] 96. The polymeric complex or polymer according to Embodiment 95, wherein the peptide linker comprises one or more GS linkers.

[0607] 97. GS linker has one or more GS(G n S) m A polymer composite or polymer according to Embodiment 96, comprising a linker.

[0608] 98. GS linker has one or more (G n S) m A polymer composite or polymer according to Embodiment 96, comprising a linker.

[0609] 99. A polymeric composite or polymer according to any one of Embodiments 1 to 53, 58 to 80, and 85 to 98, wherein the polymer is a polypeptide.

[0610] 100. A polymeric complex or polymer according to any one of Embodiments 1 to 99, wherein the biological sample is an extract, fluid, fraction, cell, tissue, or subject.

[0611] 101. A polymeric composite or polymer according to any one of embodiments 1 to 100, wherein one or both members of the polymer or a pair of polymers include a leader sequence.

[0612] 102. A polymeric complex or polymer according to Embodiment 101, wherein the leader sequence includes a secretion signal.

[0613] 103. A polymer composite or polymer according to any one of Embodiments 1 to 102, wherein one or both members of the polymer or a pair of polymers include a half-life extension portion.

[0614] 104. The polymeric composite or polymer according to Embodiment 103, wherein the half-life extension portion is an Fc domain or a fragment thereof, an albumin domain or a fragment thereof, or polyethylene glycol (PEG) or a modified derivative thereof.

[0615] 105. A nucleic acid encoding a polymer according to any one of Embodiments 1, 3-12, and 24-104.

[0616] 106. A pair of nucleic acids encoding a pair of polymers as described in Embodiment 2 and any one of 13 to 23.

[0617] 107. The nucleic acid or pair of nucleic acids according to Embodiment 105 or 106, wherein the nucleic acid is RNA or DNA.

[0618] 108. A nucleic acid or a pair of nucleic acids according to any one of embodiments 105 to 107, wherein the nucleic acid is formulated using a delivery platform.

[0619] 109. The nucleic acid or pair of nucleic acids according to Embodiment 108, wherein the delivery platform is a lipid-based carrier or vector delivery system.

[0620] 110. The nucleic acid or pair of nucleic acids according to Embodiment 109, wherein the lipid-based carrier is lipid nanoparticles (LNPs).

[0621] 111. The nucleic acid or pair of nucleic acids according to Embodiment 109, wherein the vector delivery system includes or is derived from an adenovirus, anerovirus, AAV, or lentivirus.

[0622] 112. A nucleic acid encoding a polymer according to any one of Embodiments 1, 3-12, and 24-104, wherein the nucleic acid is formulated together with a carrier.

[0623] 113. A pair of nucleic acids encoding a pair of polymers according to Embodiment 2 and any one of Embodiments 13-23, wherein the pair of nucleic acids are formulated together with a carrier.

[0624] 114. The nucleic acid or pair of nucleic acids according to Embodiment 112 or 113, wherein the nucleic acid is RNA or DNA.

[0625] 115. The nucleic acid or pair of nucleic acids according to Embodiment 112 or 113, wherein the carrier is a lipid-based carrier.

[0626] 116. The nucleic acid or pair of nucleic acids according to Embodiment 115, wherein the lipid-based carrier is an LNP.

[0627] 117. A vector comprising the nucleic acid described in Embodiment 105 or 107.

[0628] 118. A vector or pair of vectors comprising a pair of nucleic acids as described in Embodiment 106 or 107.

[0629] 119. The vector or vector pair according to Embodiment 117 or 118, wherein the vector or vector pair is formulated together with a carrier.

[0630] 120. A host cell containing a nucleic acid or pair of nucleic acids as described in any one of embodiments 105 to 116, or a vector or pair of vectors as described in any one of embodiments 117 to 119.

[0631] 121. A polymer complex, polymer, nucleic acid, or pair of nucleic acids according to any one of Embodiments 1 to 104, wherein the polymer complex, polymer, nucleic acid, or pair of nucleic acids has a purity of more than 95%.

[0632] 122. A polymeric complex, polymer, nucleic acid, or paired nucleic acid described in any one of Embodiments 1 to 104, manufactured in accordance with the Good Manufacturing Practice (GMP), Good Clinical Practice (GCP), and / or Good Laboratory Practice (GLP) standards of the U.S. Food and Drug Administration (FDA), or a nucleic acid or paired nucleic acid described in any one of Embodiments 105 to 116.

[0633] 123. A method comprising providing access to a cell for a polymer complex or polymer according to any one of Embodiments 1 to 104, or for a nucleic acid or pair of nucleic acids according to any one of Claims 105 to 116.

[0634] 124. A method for regulating the state of a cell, comprising providing access to a cell to a polymer complex or polymer according to any one of embodiments 1 to 104 or to a nucleic acid or pair of nucleic acids according to any one of claims 105 to 116, and thereby regulating the state of the cell.

[0635] 125. A method for inducing cellular effector function in a cell, comprising providing access to a cell to a polymer complex or polymer according to any one of embodiments 1 to 104 or to a nucleic acid or pair of nucleic acids according to any one of claims 105 to 116, thereby inducing cellular effector function in the cell.

[0636] 126. The method according to Embodiment 124 or 125, wherein cells are present in the subject, and a polymer complex, polymer, nucleic acid, or paired nucleic acid is administered in a therapeutically effective dose.

[0637] 127. The method according to Embodiment 126, wherein the subject has or is suspected of having a disease or disorder characterized by abnormal levels of a disease signature target, and the subject has been selectively determined in advance to have abnormal levels of a disease signature target.

[0638] 128. A method for determining the state of a cell, comprising providing access to a cell of a polymer complex or polymer, or nucleic acid or pair of nucleic acids encoding the same, as described in any one of embodiments 91 to 93; detecting the presence of a reporter domain; and thereby determining the state of the cell.

[0639] 129. A method for inducing cellular effector function in cells, comprising a polymeric complex comprising two polymers, each polymer comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain: (a) FBD specifically binds to disease signature ligands in biological samples; (b) SBD specifically binds to effector ligands in biological samples, and upon binding to effector ligands, induces cellular effector function; Two copies of the polymer are conjugated together; A method for inducing effector function by a polymer complex, in which each copy of the polymer binds to a disease signature ligand.

[0640] 130. A method for inducing cellular effector function in cells, comprising a polymeric complex comprising a pair of polymers, each polymer independently comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain: (a) The FBD of each polymer specifically binds to disease signature ligands in the biological sample; (b) The first member of the pair of polymers comprises a second binding domain 1 (SBD1) that specifically binds to a first effector ligand in the biological sample; (c) The second member of the pair of polymers includes a second binding domain 2 (SBD2) that specifically binds to a second effector ligand in the biological sample; When SBD1 and SBD2 bind to the first and second effector ligands, they induce cellular effector function; The first member of the paired polymer and the second member of the paired polymer are conjugated to each other; A method comprising contacting a polymer complex with a cell, wherein the induction of effector function by the polymer complex is conditional on each of the paired polymers binding to a disease signature ligand.

[0641] 131. A method for inducing cellular effector function in cells, comprising a polymer containing two FBDs linked to two SBDs by one or more linker domains: (a) FBD specifically binds to disease signature ligands in biological samples; (b) SBD specifically binds to effector ligands in biological samples, and upon binding to effector ligands, induces cellular effector function; A method comprising contacting a cell with a polymer, wherein the polymer can adopt a configuration that enables the binding of the two SBDs to the effector ligand in the presence of the disease signature ligand, and the induction of effector function by the polymer is conditional on each of the two FBDs binding to the disease signature ligand.

[0642] 132. A method for inducing cellular effector function in cells, comprising a polymer containing two FBDs linked to SBD1 and SBD2 by one or more linker domains: (a) FBD specifically binds to disease signature ligands in biological samples; (b) SBD1 specifically binds to the first effector ligand in the biological sample; (c) SBD2 specifically binds to a second effector ligand in the biological sample; When SBD1 and SBD2 bind to the first and second effector ligands, they induce cellular effector function; A method comprising contacting a cell with a polymer, wherein the polymer can adopt a configuration that enables the binding of SBD1 and SBD2 to a first effector ligand and a second effector ligand in the presence of a disease signature ligand, and the induction of effector function by the polymer is conditional on each of the two FBDs binding to the disease signature ligand.

[0643] 133. A polymeric complex comprising two polymers, each polymer containing a VEGF-binding domain linked to a 4-1BB-binding domain by a linker domain, wherein the 4-1BB-binding domain induces cellular effector function upon binding to 4-1BB; Two polymers are conjugated together; A polymer complex in which the induction of effector function by the polymer complex is conditional on each of the two polymers binding to VEGF.

[0644] 134. The polymer composite according to Embodiment 133, wherein the conjugation is non-covalent.

[0645] 135. The polymeric composite according to Embodiment 134, wherein the non-covalent conjugation is mediated by complementary parts of a pair, and each polymer comprises one member of the pair.

[0646] 136. The polymeric complex according to Embodiment 135, wherein the pair of complementary polypeptides are the pair of Fc fragments.

[0647] 137. The polymeric composite according to Embodiment 136, wherein the pair of Fc fragments are a knob-into-hole pair.

[0648] 138. A polymer composite according to any one of embodiments 133 to 136, wherein the two polymers are identical.

[0649] 139. A polymeric complex comprising two polymers, each polymer containing a VEGF-binding domain linked to a 4-1BB-binding domain by a linker domain, wherein the 4-1BB-binding domain induces cellular effector function upon binding to 4-1BB; The two polymers are not conjugated to each other in the absence of disease signature ligands; A polymer complex in which the induction of effector function by the polymer complex is conditional on each of the two polymers binding to VEGF.

[0650] 140. The polymer complex according to Embodiment 139, wherein a first member of a pair of polymers and a second member of a pair of polymers are non-covalently conjugated to each other in the presence of a disease signature ligand, and the non-covalent conjugation is mediated by the disease signature ligand.

[0651] 141. A polymer composite according to Embodiment 139 or 140, wherein one or both members of the polymer or a pair of polymers include a half-life extension portion.

[0652] 142. The polymeric composite according to Embodiment 103, wherein the half-life extension portion is an Fc domain or a fragment thereof.

[0653] 143. A polymeric complex according to any one of embodiments 133 to 142, wherein the VEGF-binding domain is anti-VEGF scFv.

[0654] 144. A polymeric complex according to any one of embodiments 133 to 142, wherein the VEGF-binding domain is a VEGF receptor trap.

[0655] A polymeric complex according to any one of embodiments 133 to 144, wherein the 145.4-1BB binding domain is anti-4-1BB scFv.

[0656] 146. A polymeric complex comprising two polymers, each polymer containing an IFNg-binding domain linked to an IL-10R-binding domain by a linker domain, wherein the IL-10R-binding domain induces cellular effector function upon binding to IL-10R; Two polymers are conjugated together; A polymer complex in which the induction of effector function by the polymer complex is conditional on each of the two polymers binding to IFNg.

[0657] 147. The polymeric composite according to Embodiment 146, wherein the conjugation is non-covalent.

[0658] 148. The polymeric composite according to Embodiment 147, wherein the non-covalent conjugation is mediated by complementary moieties of a pair, and each polymer comprises one member of the pair.

[0659] 149. The polymeric complex according to Embodiment 148, wherein the pair of complementary polypeptides are the pair of Fc fragments.

[0660] 150. The polymeric composite according to Embodiment 149, wherein the pair of Fc fragments are knob-into-hole pairs.

[0661] 151. A polymer composite according to any one of embodiments 146 to 150, wherein the two polymers are identical.

[0662] 152. A polymeric complex comprising two polymers, each polymer containing an IFNg-binding domain linked to an IL-10R-binding domain by a linker domain, wherein the IL-10R-binding domain induces cellular effector function upon binding to IL-10R; The two polymers are not conjugated to each other in the absence of disease signature ligands; A polymer complex in which the induction of effector function by the polymer complex is conditional on each of the two polymers binding to IFNg.

[0663] 153. The polymer complex according to Embodiment 152, wherein a first member of a pair of polymers and a second member of a pair of polymers are non-covalently conjugated to each other in the presence of a disease signature ligand, and the non-covalent conjugation is mediated by the disease signature ligand.

[0664] 154. A polymer composite according to Embodiment 152 or 153, wherein one or both members of the polymer or a pair of polymers include a half-life extension portion.

[0665] 155. The polymeric composite according to Embodiment 154, wherein the half-life extension portion is an Fc domain or a fragment thereof.

[0666] 156. A polymer complex according to any one of embodiments 146 to 155, wherein the IFNg binding domain is anti-IFNg scFv.

[0667] 157. A polymer complex according to any one of embodiments 146 to 156, wherein the IL-10R binding domain is IL-10. [Examples]

[0668] The following are examples of the method of the present invention. Considering the overview provided above, it will be understood that various other embodiments may be implemented.

[0669] [Table 2]

[0670] Example 1. Protein complex that modulates TPO receptor activity conditioned on TGF-β1 or IL-8. This example describes the expression, purification, and characterization of (i) a protein complex that modulates the thrombopoietin (TPO) receptor conditioned on the presence of transforming growth factor β1 (TGF-β1), and (ii) a protein complex that modulates the TPO receptor conditioned on the presence of interleukin-8 (IL-8). A generalized version of the fusion protein is shown in Figure 1.

[0671] A. Fusion protein TGF-β1-TPO fusion protein This embodiment provides a protein complex (referred to herein as the "TGFb-TPO fusion protein") comprising two identical fusion proteins that modulate the TPO receptor in the presence of TGF-β1 (TGFb). Each fusion protein consists of (N-terminus to C-terminus): mouse immunoglobulin κ variable 3 (IgKVIII) leader peptide (UniProt ID A0A140T8P0, M1 to G20); anti-TGF-β1 single-strand variable fragment (scFv) (PDB 4KV5; SEQ ID NOs. 13 and 14; Table 2); (G4S). n The construct includes a linker (n=2, 3, or 5), as well as anti-TPO receptor heavy chain variable domains (VH) and light chain variable domains (VL) (SEQ ID NOs. 1 and 2 or SEQ ID NOs. 32 and 33, respectively; Table 2), linked by a G4S linker (Figure 5A). The mechanism of action of the proposed construct is shown in Figure 5B: TGFb recruits two copies of the fusion protein by binding to their TGFb targeting domains, thereby aligning and binding the TpoR effector domain in close proximity to induce TpoR activity.

[0672] Selectively, each fusion protein further includes a FLAG affinity tag following the anti-TPO receptors VH and VL.

[0673] IL-8-TPO fusion protein This embodiment provides a protein complex (referred to herein as the "IL-8-TPO fusion protein") comprising two identical fusion proteins that modulate the TPO receptor in the presence of IL-8. Each fusion protein is (N-terminus to C-terminus): mouse immunoglobulin κ variable 3 (IgKVIII) leader peptide (UniProt ID A0A140T8P0, M1 to G20); anti-IL-8 scFv (PDB 6WZM; SEQ ID NO: 12; Table 2); (G4S) n The construct includes a linker (n=2, 3, or 5), as well as anti-TPO receptor heavy chain variable domains (VH) and light chain variable domains (VL) (SEQ ID NOs. 1 and 2 or SEQ ID NOs. 32 and 33, respectively; Table 2), linked by a G4S linker (Figure 6A). The mechanism of action of the proposed construct is shown in Figure 6B: IL-8 recruits two copies of the fusion protein by binding to their IL-8 targeting domains, thereby dimerizing the TpoR effector domain in close proximity and inducing TpoR activity.

[0674] Selectively, each fusion protein further includes a FLAG affinity tag following the anti-TPO receptors VH and VL.

[0675] Expression and Purification The fusion protein is expressed via transient transfection of human embryonic kidney 293 (HEK293) cells under the control of a cytomegalovirus (CMV) promoter. The secreted protein is analyzed by Western blotting or ELISA using an anti-FLAG capture antibody to assess expression and quality. The fusion protein is purified, if necessary, using a FLAG capture step followed by size exclusion chromatography (SEC) polishing. Expression is expected to yield a strong band at the predicted molecular weight by Western blotting, with minimal product-related variants.

[0676] B. In vitro assay to evaluate the effect of fusion proteins on the TPO signaling pathway in the presence or absence of soluble TPO or TGF-β1. HEK-BLUE® TPO cells (InvivoGen catalog code: hkb-tpo) are used to evaluate the activity of the fusion protein using the manufacturer's specified protocol unless otherwise indicated. Briefly, HEK-BLUE® TPO cells are transiently transfected with a fusion protein expression construct (as described in Example 1A) to induce autocrine secretion of the fusion protein in the assay. Alternatively, the supernatant from HEK293 cells transiently expressing the fusion protein is applied to HEK-BLUE® TPO cells as a test sample. A confirmation assay is performed using FLAG-purified fusion protein material. Activation of the JAK2 / STAT5 pathway by secretory alkaline phosphatase (SEAP) production is compared between the positive control (administration of purified recombinant human TPO; transient transfection to express and secrete human TPO as autocrine), the negative control (administration of recombinant human interleukin-2 (IL-2); pseudotransfection plasmid), and the test sample (fusion protein variant). HEK-BLUE® TPO cells expressing the fusion protein are incubated overnight in the presence or absence of TPO, and in the presence or absence of TGF-β1 (in the case of TGFb-TPO fusion protein) or IL-8 (in the case of IL-8-TPO fusion protein). Functional fusion protein samples are further evaluated by dose titration of the fusion protein sample in the presence of TGF-β1 or IL-8 to assess EC50, or by assessing IC50 in the presence of TGF-β1 and TPO or IL-8. The fusion protein is expected to activate TPO receptor signaling conditioned on the presence of TGF-β1 (TGFb-TPO fusion protein) or IL-8 (IL-8-TPO fusion protein).

[0677] Figure 5C shows the dose-response relationship between TGFb concentration and activity of TGFb-TPO fusion proteins (containing the anti-TPO receptor VH and VL sequences of SEQ ID NO: 32 and SEQ ID NO: 33, respectively). TpoR SEAP reporter cells were stimulated overnight with a fixed amount of TGFb-TPO fusion protein and various concentrations of TGFb, and subsequently, SEAP activity in the culture supernatant was measured.

[0678] Figure 6C shows the dose-response relationship between IL-8 concentration and activity of IL-8-TPO fusion proteins (containing the anti-TPO receptor VH and VL sequences of SEQ ID NO: 32 and SEQ ID NO: 33, respectively). TpoR SEAP reporter cells were stimulated overnight with a fixed amount of IL-8-TPO fusion protein and various concentrations of IL-8, and subsequently, SEAP activity in the culture supernatant was measured.

[0679] C. Characterization of TPO receptor agonism by fusion protein vs. natural agonist Cell samples for profiling and comparing gene expression between cells treated with fusion proteins or soluble ligands were collected after the overnight incubation step of the assay described in Example 1B. RNA was extracted and purified using the Qiagen RNeasy kit (catalog no. 74104) with a threshold RNA Quality Number > 7. RNA libraries for gene expression profiling were prepared using the Roche KAPA HyperPrep kit and sequenced using Illumina NextSeq to generate 40-nucleotide pair terminal reads. The sequenced reads were quality-checked, aligned, counted, and analyzed using standard procedures. See, for example, Kukurba and Montgomery, RNA sequencing and analysis. Cold Spring Harbor Protocols, 2015(11), doi:10.1101 / pdb.top084970.

[0680] [Table 3]

[0681] [Table 4]

[0682] Example 2. Protein complex that agonizes IL-2R activity conditioned on TGF-β1 or IL-8. This example describes the expression, purification, and characterization of (i) protein complexes that stimulate human interleukin-2 receptor (IL-2R) activity conditioned on TGF-β1, and (ii) protein complexes that stimulate human IL-2R activity conditioned on interleukin-8 (IL-8; also known as CXCL8). Generalized versions of the paired fusion proteins are shown in Figure 2.

[0683] A. Fusion protein TGFβ1-IL-2R fusion protein pair This embodiment provides a protein complex composed of two complementary fusion proteins (fusion proteins (a) and (b)) that regulate the IL-2 receptor (IL-2R) in the presence of TGF-β1. Each fusion protein is (N-terminus to C-terminus): mouse IgKVIII leader peptide (UniProt ID A0A140T8P0, M1 to G20); anti-TGF-β1 scFv (PDB 4KV5; SEQ ID NOs. 13 and 14; Table 2); (G4S) n The fusion protein comprises a linker (n=10, 15, or 25); and (a) a conditional IL-2R effector domain A (SEQ ID NO: 3; Table 2) or (b) a conditional IL-2R effector domain B (SEQ ID NO: 4; Table 2) (Figure 7A). The proposed mechanism of action of the complementary fusion protein is shown in Figure 7B. TGFb recruits each of the fusion proteins by binding to their TGFb targeting domains, thereby dimerizing and positioning the IL-2R effector domains in close proximity to induce IL-2R activity.

[0684] Optionally, each fusion protein also includes a FLAG affinity tag between the leader peptide and the anti-TGF-β1 scFv.

[0685] IL-8-IL-2R fusion protein pair This embodiment also describes a protein complex comprising two complementary fusion proteins that modulate the IL-2 receptor in the presence of IL-8, each containing an anti-IL-8 scFv (PDB 6WZM, SEQ ID NO: 12; Table 2).

[0686] Expression and Purification The fusion protein is expressed and purified as described in Example 1A by either 1) transfecting it with equal amounts of each complementary construct (fusion protein (a) and (b)), or 2) independently transfecting, purifying, and pre-mixing equal amounts of fusion protein (a) and (b).

[0687] In vitro assay for IL-2R agonism in cells in the presence or absence of B.TGF-β1 Using HEK-BLUE® CD122 / CD132 cells (InvivoGen catalog code: hkb-il2bg), fusion protein activity is evaluated using the manufacturer's specified protocol, unless otherwise indicated and as described in Example 1B. Activation of the IL-2 signaling pathway via SEAP production is compared between a positive control (administration of purified recombinant human IL-2; transient transfection to express and secrete human IL-2 as autocrine), a negative control (administration of recombinant human TPO; pseudotransfection plasmid), and test samples (fusion proteins (a) and (b) and their variants). HEK-BLUE® CD122 CD132 cells expressing the fusion proteins are incubated overnight in the presence or absence of IL-2 and in the presence or absence of TGF-β1. Functional fusion protein samples are further evaluated by performing dose titration of the fusion protein samples in the presence of TGF-β1 to assess EC50. The fusion proteins (a) and (b), administered in combination, are expected to activate IL-2 signaling, which is conditioned on the presence of TGF-β1.

[0688] Figure 7C shows the dose-response relationship between fusion proteins (a) and (b) and TGFb concentration. IL-2 reporter cells were stimulated overnight with a fixed amount of fusion proteins (a) and (b) and various concentrations of TGFb, followed by measurement of SEAP activity in the culture supernatant.

[0689] C. Characterization of TGFR agonism by fusion protein vs. natural agonist A transcriptome comparison between the fusion protein and the soluble ligand is performed as described in Example 1C.

[0690] Example 3. Protein complex that agonizes IL-10R activity conditioned on IFN-γ or IL-6. This example describes the expression, purification, and characterization of proteins and protein complexes that stimulate the human interleukin-10 receptor (IL-10 receptor) in the presence of interferon-γ (IFN-γ) or interleukin-6 (IL-6).

[0691] A. Fusion protein IFN-γ-IL-10 fusion protein pair This embodiment provides a protein complex composed of two complementary fusion proteins (fusion proteins (c) and (d)) that modulate the IL-10 receptor in the presence of IFN-γ. Each fusion protein is (N-terminus to C-terminus): mouse IgKVIII leader peptide (UniProt ID A0A140T8P0 M1 to G20); anti-IFN-γ scFv (PDB 1T3F; SEQ ID NO: 5; Table 2); (G4S) nThe fusion protein comprises a linker (n=1, 2, or 3) and an anti-IL10Ra VHH antibody (SEQ ID NO: 6; Table 2) (fusion protein (c)) or an anti-IL10Rb VHH antibody (SEQ ID NO: 7; Table 2) (fusion protein (d)) (Figure 8A). A generalized version of the complementary fusion protein is shown in Figure 3. Optionally, each fusion protein further includes a FLAG affinity tag following 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 8D: IFN-γ recruits each of the fusion proteins by binding to their IFN-γ targeting domains, thus dimerizing and positioning the IL-10R binding effector domains in close proximity to induce IL-10R activity.

[0692] The fusion protein is expressed and purified as described in Example 1A by either 1) transfecting it with equal amounts of each complementary construct (fusion protein (c) and (d)), or 2) independently transfecting, purifying, and pre-mixing equal amounts of fusion protein (c) and (d).

[0693] Self-assembling IFN-γ-IL-10 fusion protein pair This embodiment also provides a protein complex composed of two complementary fusion proteins (fusion protein (g) and (h)) that regulate the IL-10 receptor in the presence of IFN-γ, wherein the two complementary fusion proteins self-assemble as a heterodimer. Each fusion protein is (N-terminus to C-terminus): mouse IgKVIII leader peptide (UniProt ID A0A140T8P0 M1 to G20); anti-IL10Ra VHH antibody (SEQ ID NO: 6; Table 2) (fusion protein (g)) or anti-IL10Rb VHH antibody (SEQ ID NO: 7; Table 2) (fusion protein (h)); (G4S) n Linker (n=2); anti-IFN-γ scFv (SEQ ID NO: 5; Table 2); (G4S) nThe variants include a linker (n=1, 2, 3, or 4) and a human IgG1 Fc having a knob mutation (SEQ ID NO: 10; Table 2) or a hole mutation (SEQ ID NO: 11; Table 2) (Figure 8B). Figure 8F shows a variant of the fusion protein in which the linker between the Fc hinge and the IFNg-bound scFv consists of a pair of amino acid residues (construct P482+P483) or a pair of amino acid residues (P484+P485).

[0694] Optionally, each fusion protein further includes a polyhistidine (His) affinity tag.

[0695] The fusion protein is expressed by transfecting with equal amounts of each complementary construct (fusion protein (g) and (h)). These strands spontaneously form heterodimers via their knob-into-hole Fc domains to produce a single molecule having one copy of the anti-IL10Ra domain and one copy of the IL10Rb domain, which can be purified via either protein A or Ni-NTA resin.

[0696] Variations of IFN-γ-IL-10 fusion protein pairs Variations of the above protein complex are shown in Figures 12A to 12I. Figure 12A shows a variation in which the linker length between the Fc hinge and the IFNg-binding scFv is regulated (increased or decreased). Figure 12B shows a variation in which the linker length between the IFNg-binding scFv and the IL10R-binding VHH is regulated (increased or decreased). Figures 12B and 12C show variations in which the IL10R-binding VHH is replaced with scFv agonists for IL-10R and monomeric IL-10 cytokines, respectively. Figure 12E shows a variation in which the Fc region and IFNg-binding scFv of the knob-into-hole protein are replaced with full-length IgG of the knob-into-hole protein that has its Fab region encoding IFNg binding. Figures 12F to 12I show variants in which each fusion protein (excluding the knob-into-hole Fc region) is fused to the full-length IgG antibody at the N-terminus of the heavy chain; the N-terminus of the light chain; the C-terminus of the light chain; or the C-terminus of the heavy chain Fc region.

[0697] Single polypeptide IFN-γ-IL-10 fusion protein This embodiment also provides a protein that modulates the IL-10 receptor in the presence of IFN-γ, and consists of a single polypeptide chain encoded by the following (N-terminus to C-terminus): mouse IgKVIII leader peptide (UniProt ID A0A140T8P0 M1 to G20); anti-IL10Ra VHH antibody (SEQ ID NO: 6; Table 2); anti-IFN-γ scFv (SEQ ID NO: 5; Table 2); (G4S) n Linker (n=2, 3, 4, 5, or 6); second copy of anti-IFN-γ scFv (SEQ ID NO: 5; Table 2); and anti-IL10Rb VHH antibody (SEQ ID NO: 7; Table 2) (Figure 8C). Alternatively, the positions of anti-IL10Ra VHH antibody (SEQ ID NO: 6; Table 2) and anti-IL10Rb VHH antibody (SEQ ID NO: 7; Table 2) may be reversed (Figure 8C). Optionally, the protein further includes a histidine (His) affinity tag.

[0698] The fusion protein can be expressed as described in Example 1A and purified using Ni-NTA resin.

[0699] IL-6-IL-10 fusion protein pair This embodiment also provides a protein complex composed of two complementary fusion proteins that modulate the IL-10 receptor in the presence of IL-6. Each fusion protein consists of (N-terminus to C-terminus): mouse IgKVIII leader peptide (UniProt ID A0A140T8P0 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: 34; first fusion protein) or second anti-IL-6 VHH antibody (SEQ ID NO: 35; second fusion protein) (the first and second anti-IL-6 VHH antibodies bind to different epitopes on IL-6); (G4S) n Linker; and polyhistidine tag included (Figure 9A). Alternatively, the binding domain may be swapped so that (i) the first fusion protein contains a first anti-IL-6 VHH antibody and a second anti-IL-6 VHH antibody (SEQ ID NO: 35), and (ii) the second fusion protein contains a second anti-IL-6 VHH antibody and a first anti-IL-6 VHH antibody (SEQ ID NO: 34). The mechanism of action of the proposed construct is shown in Figure 9B: IL-6 recruits each of the fusion proteins by binding to their IL-6 targeting domains, thereby dimerizing and positioning the IL-10R binding effector domains in close proximity to induce IL-10R activity.

[0700] B. In vitro assay for IL-10 agonism in cells in the presence or absence of IFN-γ or IL-6. Using HEK-BLUE® IL-10 cells (InvivoGen catalog code: hkb-il10), fusion protein activity is evaluated using the manufacturer's standard protocol, unless otherwise indicated and as described in Example 1B. JAK1 / STAT3 pathway activation via SEAP production is compared between a positive control (administration of purified recombinant human IL-10; transient transfection to express and secrete human IL-10 as autocrine), a negative control (administration of recombinant human TPO; pseudotransfection plasmid), and test samples (fusion proteins or fusion protein pairs and their variants as described herein). HEK-BLUE® IL-10 cells expressing the fusion protein are incubated overnight in the presence or absence of IL-10, or in the presence or absence of IFN-γ (in the case of IFN-γ-IL-10 fusion protein) or IL-6 (in the case of IL-6-IL-10 fusion protein). Functional fusion protein samples are further evaluated by dose titration of the fusion protein sample in the presence of IFN-γ or IL-6 to assess the EC50, or by assessing the IC50 in the presence of IFN-γ or IL-6 and IL-10. The fusion protein or fusion protein pair (administered in combination) is expected to activate IL-10 receptor signaling, condit...

Claims

1. A polymeric complex comprising two polymers, each polymer comprising a first binding domain (FBD) linked to a second binding domain (SBD) by a linker domain: (a) The FBD specifically binds to disease signature ligands in the biological sample; (b) The SBD specifically binds to the effector ligand in the biological sample, and upon binding to the effector ligand, induces cellular effector function; The two polymers are conjugated together; A polymer complex in which the induction of the effector function by the polymer complex is conditional on each of the two polymers binding to the disease signature ligand.

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

3. The polymer composite according to claim 1 or 2, wherein the conjugation is a covalent bond.

4. The polymer composite according to claim 1 or 2, wherein the conjugation is a non-covalent bond.

5. The polymeric complex or polymer according to any one of claims 1 to 4, wherein the disease signature ligand is a protein, peptide, or small molecule.

6. The polymeric complex or polymer according to any one of claims 1 to 5, wherein the FBD, FBD1, or FBD2 comprises a polypeptide that specifically binds to the disease signature ligand.

7. The polymeric complex or polymer according to claim 6, wherein the polypeptide is an antibody or a fragment thereof.

8. The polymeric complex or polymer according to any one of claims 1 to 7, wherein the effector ligand is a protein or a peptide.

9. A polymeric composite or polymer according to any one of claims 1 to 8, further comprising one or more additional linker domains.

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

11. The polymer composite or polymer according to any one of claims 1 to 10, wherein the polymer is a polypeptide.

12. A nucleic acid encoding the polymer according to any one of claims 1 to 11.

13. A pair of nucleic acids encoding a pair of polymers according to claim 2.

14. The nucleic acid or pair of nucleic acids according to claim 12 or 13, wherein the nucleic acid is RNA or DNA.

15. A nucleic acid encoding a polymer according to any one of claims 1 to 11, wherein the nucleic acid is formulated together with a carrier.

16. A vector comprising the nucleic acid according to claim 12, 14, or 15.

17. A vector comprising a pair of nucleic acids as described in claim 13 or 14, or a pair of vectors.

18. A host cell comprising a nucleic acid or pair of nucleic acids according to any one of claims 12 to 15, or a vector or pair of vectors according to any one of claims 16 or 17.

19. A method comprising providing access to a cell for a polymer complex or polymer according to any one of claims 1 to 11, or for a nucleic acid or pair of nucleic acids according to any one of claims 12 to 15.

20. A polymeric complex comprising two polymers, each polymer containing a VEGF-binding domain linked to a 4-1BB-binding domain by a linker domain, wherein the 4-1BB-binding domain induces cellular effector function upon binding to 4-1BB; The two polymers are conjugated together; A polymer complex in which the induction of the effector function by the polymer complex is conditional on each of the two polymers binding to VEGF.

21. A polymeric complex comprising two polymers, each polymer containing an IFNg-binding domain linked to an IL-10R-binding domain by a linker domain, wherein the IL-10R-binding domain induces cellular effector function upon binding to IL-10R; The two polymers are conjugated together; A polymer complex in which the induction of the effector function by the polymer complex is conditional on each of the two polymers binding to IFNg.

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