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WO2024092160A2 - Protéases bifonctionnelles et leurs utilisations - Google Patents

Protéases bifonctionnelles et leurs utilisations Download PDF

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Publication number
WO2024092160A2
WO2024092160A2 PCT/US2023/077959 US2023077959W WO2024092160A2 WO 2024092160 A2 WO2024092160 A2 WO 2024092160A2 US 2023077959 W US2023077959 W US 2023077959W WO 2024092160 A2 WO2024092160 A2 WO 2024092160A2
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WIPO (PCT)
Prior art keywords
human
domain
proteolytic
polypeptide
bifunctional
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WO2024092160A3 (fr
Inventor
Yiqun Bai
Liam Michael CASEY
John Daniel MENDLEIN
Ayse Jane MUNIZ
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Flagship Pioneering Innovations VII Inc
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Flagship Pioneering Innovations VII Inc
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Publication of WO2024092160A3 publication Critical patent/WO2024092160A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the disclosure relates to compositions and methods for making and using bifunctional proteases (e.g., targeted sheddases, proteases, or amyloid beta (Ap)-degrading enzymes)) that cleave polypeptides in a targeted manner.
  • bifunctional proteases e.g., targeted sheddases, proteases, or amyloid beta (Ap)-degrading enzymes
  • Protein dysfunction impairs the activity of cells, organs, and tissues of the body. Because the affected proteins often arise from different sources, the development of effective treatments to ameliorate the effects of protein dysfunction has been challenging. Therefore, there is a need for novel therapeutics targeting dysfunctional proteins.
  • compositions and methods for making and using bifunctional macromolecules that cleave molecules in a targeted manner describe the compositions and methods for making and using bifunctional macromolecules that cleave molecules in a targeted manner.
  • the disclosure provides a bifunctional macromolecule that incudes (a) a targeting domain that binds a target molecule and (b) a proteolytic domain, wherein upon binding of the targeting domain to the target molecule the proteolytic domain cleaves a peptide bond in a substrate polypeptide.
  • the proteolytic domain is a sheddase domain.
  • the proteolytic domain is derived from a serine protease.
  • the proteolytic domain is mesotrypsin, MMP2, MMP7, MMP9, MMP12, MMP14, cathepsin-B (CTSB), cathepsin-D (CTSD), or kallikrein 7 (KLK7) or a fragment or derivative thereof.
  • the proteolytic domain is an amyloid beta (Ap)-degrading enzyme.
  • the disclosure provides a bifunctional macromolecule that incudes (a) a targeting domain that binds a target molecule and (b) a proteolytic domain derived from a naturally occurring protease with a primary biological function of cleaving bonds in the ectodomain of membrane-associated polypeptides, wherein upon binding of the targeting domain to the target molecule the proteolytic domain cleaves a peptide bond in a substrate polypeptide.
  • the disclosure provides a bifunctional macromolecule that incudes (a) a targeting domain that binds a target molecule and (b) a proteolytic domain having at least 85% sequence identity to the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9, wherein upon binding of the targeting domain to the target molecule the proteolytic domain cleaves a peptide bond in a substrate polypeptide.
  • the proteolytic domain is the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9.
  • the disclosure provides a bifunctional macromolecule that incudes (a) a targeting domain that binds a target molecule and (b) a proteolytic domain having at least 85% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4, wherein upon binding of the targeting domain to the target molecule the proteolytic domain cleaves a peptide bond in a substrate polypeptide.
  • the proteolytic domain is the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the disclosure provides a bifunctional macromolecule comprising (a) a targeting domain that binds a target molecule; and (b) a proteolytic domain derived from a naturally occurring protease with a primary biological function of cleaving bonds in an amyloid protein aggregate, wherein upon binding of the targeting domain to the target molecule the protease cleaves a peptide bond in an amyloid protein aggregate.
  • the disclosure provides a bifunctional macromolecule comprising (a) a targeting domain that binds a target molecule; and (b) a proteolytic domain having at least 85% sequence identity to the proteolytic domain of human MMP2, human MMP9, human MMP14, human CTSB, human CTSD, or human KLK7, wherein upon binding of the targeting domain to the target molecule the proteolytic domain cleaves a peptide bond in an amyloid protein aggregate.
  • the proteolytic domain is the proteolytic domain of human MMP2, human MMP9, human MMP14, human CTSB, human CTSD, or human KLK7.
  • the target molecule and the substrate polypeptide are the same molecule. In some embodiments of any of the foregoing aspects, the target molecule and the substrate polypeptide are not the same molecule.
  • the targeting domain includes a polypeptide.
  • the targeting domain polypeptide includes a monoclonal antibody, Fab, F(ab’)2, scFv with or without an Fc region, immunoprotein, heavy chain variable region and a light chain variable region, affibody, diabody, triabody, tetrabody, knottin, atrimer, avimer, cys-knot, fynomer, kunitz domain, Obody, nanobody, an Fc fusion protein, anticalin, affimer types I or II, FN3 scaffold, centyrinTM, or DARPin.
  • the targeting domain includes a glycan.
  • the targeting domain includes a polynucleotide.
  • the substrate polypeptide is a soluble polypeptide. In some embodiments of any of the foregoing aspects, the substrate polypeptide is a membrane-associated polypeptide.
  • the target molecule is intracellular. In some embodiments of any of the foregoing aspects, the target molecule is extracellular.
  • the disclosure provides a bifunctional macromolecule including (a) a targeting domain that binds a membrane-embedded target polypeptide and (b) a proteolytic domain, wherein upon binding of the targeting domain to the target polypeptide the proteolytic domain cleaves a peptide bond in the target polypeptide.
  • the disclosure provides a bifunctional macromolecule including (a) a targeting domain that binds a membrane-embedded target molecule and (b) a proteolytic domain, wherein upon binding of the targeting domain to the target molecule the proteolytic domain cleaves a peptide bond in a substrate polypeptide.
  • the disclosure provides a bifunctional macromolecule that incudes (a) a targeting domain that binds a target molecule and (b) a proteolytic domain, wherein upon binding of the targeting domain to the target molecule the proteolytic domain cleaves a peptide bond in a membrane-embedded substrate polypeptide.
  • the disclosure provides a bifunctional macromolecule that incudes (a) a targeting domain that binds a target molecule and (b) a proteolytic domain, wherein upon binding of the targeting domain to the target molecule the proteolytic domain cleaves a peptide bond in a substrate polypeptide, wherein the substrate polypeptide is heterologous to the proteolytic domain.
  • the disclosure provides a bifunctional macromolecule that incudes (a) a targeting domain that binds a soluble target polypeptide and (b) a sheddase domain, wherein upon binding of the targeting domain to the target polypeptide the sheddase domain cleaves a peptide bond in the target polypeptide.
  • the disclosure provides a bifunctional macromolecule that incudes (a) a targeting domain that binds a soluble target molecule and (b) a sheddase domain, wherein upon binding of the targeting domain to the target molecule the sheddase domain cleaves a peptide bond in a substrate polypeptide.
  • the disclosure provides a bifunctional macromolecule that incudes (a) a targeting domain that binds a target molecule and (b) a sheddase domain, wherein upon binding of the targeting domain to the target molecule the sheddase domain cleaves a peptide bond in a soluble substrate polypeptide.
  • the target molecule and the substrate polypeptide are the same molecule. In some embodiments of any of the foregoing aspects, the target polypeptide and the substrate polypeptide are not the same polypeptide.
  • the disclosure provides a pharmaceutical composition that includes the bifunctional macromolecule of any of the foregoing aspects.
  • the disclosure provides one or more nucleic acids encoding the bifunctional macromolecule of any of the foregoing aspects.
  • the nucleic acids may be, e.g., linear nucleic acids, circular nucleic acids, and/or modified nucleic acids.
  • the nucleic acid is an RNA.
  • the disclosure provides a vector encoding the bifunctional macromolecule of any of the foregoing aspects.
  • the disclosure provides a host cell that includes the bifunctional macromolecule or the nucleic acid of any of the foregoing aspects.
  • the host cell is a eukaryotic cell.
  • the host cell is a mammalian cell.
  • the disclosure provides a pharmaceutical composition that includes the bifunctional macromolecule of any of the foregoing aspects or the nucleic acid of any of the foregoing aspects.
  • the pharmaceutical composition further includes one or more pharmaceutically acceptable carriers, diluents, and/or excipients.
  • the disclosure provides a method of treating a disorder in a subject in need thereof.
  • This method includes the step of administering to the subject an effective amount of the bifunctional macromolecule of any of the foregoing aspects or the pharmaceutical composition of any of the foregoing aspects.
  • the disclosure provides a method of altering the function of a membrane- associated polypeptide.
  • This method includes the step of contacting the cell with the bifunctional macromolecule of any of the foregoing aspects or the pharmaceutical composition of any of the foregoing aspects.
  • the disclosure provides a method of cleaving a target polypeptide.
  • This method includes the step of contacting the target polypeptide with the bifunctional macromolecule of any of the foregoing aspects or the pharmaceutical composition of any of the foregoing aspects.
  • the disclosure provides a method of cleaving a heterologous polypeptide.
  • This method includes the step of contacting the heterologous target molecule with a bifunctional macromolecule that includes (a) a targeting domain that binds a target polypeptide and (b) a proteolytic domain, wherein upon binding of the targeting domain to the target polypeptide the proteolytic domain cleaves a peptide bond in the heterologous polypeptide.
  • the target polypeptide and the heterologous polypeptide are the same polypeptide.
  • the target polypeptide and the heterologous polypeptide are not the same polypeptide.
  • the heterologous target is not a membrane-associated polypeptide.
  • the proteolytic domain is a sheddase domain.
  • the proteolytic domain is derived from a naturally occurring protease with a primary biological function of cleaving bonds in the ectodomain of membrane-associated proteins.
  • the proteolytic domain has at least 85% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain is a proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the disclosure provides a method of cleaving a membrane-associated polypeptide.
  • This method includes the step of contacting the membrane-embedded polypeptide with a macromolecule including (a) a targeting domain that binds a target polypeptide and (b) a proteolytic domain, wherein upon binding of the targeting domain to the target polypeptide the proteolytic domain cleaves a peptide bond in the membrane-associated polypeptide wherein the proteolytic domain is not a sheddase domain.
  • the membrane-associated polypeptide is selected from the group consisting of an ion channel, an aquaporin, a protein transporter, a glucose transporter, or a membrane-associated receptor.
  • the disclosure provides a bifunctional macromolecule comprising (a) a targeting domain that binds a target molecule, wherein the target molecule is an amyloid, amyloid beta (Ap), TNFa, IL-4, or IL-13; and (b) a proteolytic domain comprising human MMP2, human MMP7, human MMP9, human MMP12, human MMP14, human CTSB, human CTSD or human KLK7 or a fragment or derivative thereof, wherein upon binding of the targeting domain to the target molecule the protease cleaves a peptide bond.
  • a targeting domain that binds a target molecule, wherein the target molecule is an amyloid, amyloid beta (Ap), TNFa, IL-4, or IL-13
  • a proteolytic domain comprising human MMP2, human MMP7, human MMP9, human MMP12, human MMP14, human CTSB, human CTSD or human KLK7 or a fragment or derivative thereof, wherein upon binding of
  • FIG. 1 A is a schematic diagram showing the design of a bifunctional molecule comprising the pro-enzyme form of an enzyme, a linker, a binding domain (e.g., a VHH or a receptor ectodomain), and hemagglutinin (HA) and polyhistidine (HIS) tags.
  • a binding domain e.g., a VHH or a receptor ectodomain
  • HA hemagglutinin
  • HIS polyhistidine
  • FIG. 1B is a diagram showing a bifunctional molecule comprising mesotrypsin, a linker, and a TNFa binding domain (e.g., a VHH or TNFR1 (or the ectodomain thereof)).
  • a bifunctional molecule comprising mesotrypsin, a linker, and a TNFa binding domain (e.g., a VHH or TNFR1 (or the ectodomain thereof)).
  • FIG. 1C is a graph showing levels of TNFa signaling measured in TNFa reporter cells treated with a reaction mixture comprising a mesotrypsin-TNFR1 bifunctional molecule, mesotrypsin, or a catalytically inactive form of the mesotrypsin-TNFR1 bifunctional molecule.
  • the test proteins were incubated with 50 pM TNFa overnight before the reaction mixture was added to the reporter cells.
  • 1 D is a set of western blots showing levels of TNFa (18 kDa intact form and cleavage products) in reaction mixtures in which TNFa was incubated with the indicated test proteins (bifunctional molecules comprising mesotrypsin (meso) and a TNFa binding domain selected from TNFR1 , VHH1 , VHH2, and VHH3) for 30 seconds, 5 minutes, 60 minutes, or overnight (o/n).
  • test proteins bifunctional molecules comprising mesotrypsin (meso) and a TNFa binding domain selected from TNFR1 , VHH1 , VHH2, and VHH3
  • Mesotrypsin is shown as a control.
  • FIG. 2A is a schematic diagram showing the design of a bifunctional molecule comprising the pro-enzyme form of an enzyme, a linker, a binding domain (e.g., a Designed Ankyrin Repeat Protein (DARPin)), and hemagglutinin (HA) and polyhistidine (HIS) tags.
  • DARPin Designed Ankyrin Repeat Protein
  • HIS polyhistidine
  • FIG. 2B is a diagram showing a bifunctional molecule comprising MMP7 or MMP12, a linker, and an anti-IL-13 DARPin.
  • Fig. 2C is a bar graph showing the reaction rate (arbitrary fluorescence units (AFU) per minute) of proteolytic activity of (i) a bifunctional molecule comprising MMP7 and an anti-IL-13 DARPin and (ii) bifunctional molecule comprising MMP12 and an anti-IL-13 DARPin against a fluorogenic peptide at the indicated timepoints.
  • AFU arbitrary fluorescence units
  • Fig. 2D is a bar graph showing the reaction rate (AFU per minute) of proteolytic activity against a fluorogenic peptide in a serum control at the indicated timepoints.
  • Fig. 3 is a set of western blots showing levels of the indicated constructs (antibody targets noted below each blot) and auto-cleavage products thereof. Constructs were incubated at 5 nM and 37°C and measured by western blot after 30 seconds, 2 hours, or 16 hours.
  • Fig. 4A is a set of schematic diagrams showing enzymatic constructs comprising an enzyme and a targeting domain.
  • Fig. 4B is a chart showing proteolytic activity of the indicated proteins or protein complexes over time.
  • RFU relative fluorescence units.
  • APMA 4-aminophenylmercuric acetate.
  • Fig. 4C is a chart showing proteolytic activity of the indicated proteins or protein complexes over time.
  • Fig. 4D is a chart showing proteolytic activity of the indicated proteins or protein complexes over time.
  • Fig. 4E is a chart showing proteolytic activity of the indicated proteins or protein complexes over time.
  • Upper panel pH 7.4; lower panel: pH 3.5.
  • Fig. 4F is a chart showing proteolytic activity of the indicated proteins or protein complexes over time.
  • alkenyl refers to acyclic monovalent straight or branched chain hydrocarbon groups of containing one, two, or three carbon-carbon double bonds.
  • alkenyl groups include ethenyl, prop-1 -enyl, prop-2-enyl, 1 -methylethenyl, but-1 -enyl, but-2-enyl, but-3-enyl, 1 -methylprop-1 -enyl, 2-methylprop-1 -enyl, and 1 -methylprop-2-enyl.
  • Alkenyl groups may be optionally substituted as defined herein for alkyl.
  • alkenylene refers to a divalent alkenyl group.
  • An optionally substituted alkenylene is an alkenylene that is optionally substituted as described herein for alkenyl.
  • alkyl refers to an acyclic straight or branched chain saturated hydrocarbon group, which, when unsubstituted, has from 1 to 12 carbons, unless otherwise specified. In certain preferred embodiments, unsubstituted alkyl has from 1 to 6 carbons.
  • alkylene refers to a divalent alkyl group.
  • An optionally substituted alkylene is an alkylene that is optionally substituted as described herein for alkyl.
  • alkynyl refers to monovalent straight or branched chain hydrocarbon groups of from two to six carbon atoms containing at least one carbon-carbon triple bond and is exemplified by ethynyl, 1 -propynyl, and the like.
  • the alkynyl groups may be unsubstituted or substituted (e.g., optionally substituted alkynyl) as defined for alkyl.
  • alkynylene refers to a divalent alkynyl group.
  • An optionally substituted alkynylene is an alkynylene that is optionally substituted as described herein for alkynyl.
  • charge-charge interactions refers to electrostatic interactions between two different atoms in which one atom (the anion) donates its valence electrons to another atom (the cation). This bond is non-directional.
  • disorder associated with protein dysfunction includes disorders characterized by high expression of inflammatory cytokines such as tumor necrosis factor alpha (TNFa), proteins such as programmed cell death protein 1 (PD1 ) or programmed cell death ligand 1 (PD-L1 ), or protein aggregates in a subject relative to a reference, such as a healthy individual of the same age as the subject.
  • TNFa tumor necrosis factor alpha
  • proteins such as programmed cell death protein 1 (PD1 ) or programmed cell death ligand 1 (PD-L1 )
  • protein aggregates in a subject relative to a reference such as a healthy individual of the same age as the subject.
  • Exemplary disorders characterized by high expression of inflammatory cytokines such as TNFa are rheumatoid arthritis, ankylosing spondylitis, Crohn disease, ulcerative colitis, hidradenitis suppurativa, juvenile idiopathic arthritis, plaque psoriasis, psoriatic arthritis, uveitis, neutrophilic dermatosis (e.g., pyoderma gangrenosum, Behcet disease), granulomatosis with polyangiitis (e.g., Wegener granulomatosis), sarcoidosis, pemphigus, multicentric reticulohistiocytosis, and alopecia areata.
  • inflammatory cytokines such as TNFa are rheumatoid arthritis, ankylosing spondylitis, Crohn disease, ulcerative colitis, hidradenitis suppurativa, juvenile idiopathic arthritis, plaque ps
  • Exemplary disorders characterized by high expression of PD1 or PD-L1 are melanoma, non-small cell lung carcinoma (NSCLC), small cell lung carcinoma (SCLC), head and neck squamous cell carcinomas (HNSCC), lymphomas, urothelial carcinoma, bladder cancer, unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) solid tumors, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma, endometrial carcinoma, cancers with high tumor mutational burden (>10 mutations per megabase), squamous cell carcinoma, and breast cancer.
  • Exemplary disorders characterized by protein aggregates are alpha-1 -antitrypsin disorders, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and Huntington’s disease.
  • dispersion forces refers to weak attractive forces between nonpolar molecules.
  • glycocan refers to a polysaccharide, or oligosaccharide. Glycan is also used herein to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide or a proteoglycan. Glycans usually consist solely of O-glycosidic linkages between monosaccharides.
  • cellulose is a glycan (or more specifically a glucan) composed of p-1 ,4-linked D-glucose
  • chitin is a glycan composed of p-1 ,4-linked N-acetyl-D-glucosamine.
  • Glycans can be homo or heteropolymers of monosaccharide residues and can be linear or branched. Glycans can be found attached to proteins as in glycoproteins and proteoglycans. They are generally found on the exterior surface of cells. O- and N-linked glycans are very common in eukaryotes but may also be found, although less commonly, in prokaryotes.
  • N-Linked glycans are attached to the R-group nitrogen (N) of asparagine in the sequon.
  • the sequon may be a Asn-X-Set or Asn-X-Thr sequence, where X is any amino acid except praline.
  • heterologous target refers to a polypeptide that is not a natural substrate of proteases containing the recited proteolytic domain in vivo.
  • hydrogen bonding interactions refers to intermolecular forces that occur between hydrogen atoms that are covalently bonded to small, strongly electronegative elements (such as nitrogen and oxygen) and nonbonding electron pairs on other such electronegative elements.
  • hydrophobic-hydrophobic interactions refers to interactions between lipophilic moieties to form intermolecular aggregates or intramolecular interactions (particularly in aqueous based environments).
  • linker refers to a covalent or noncovalent linkage or connection between two or more components in a fusion protein or a conjugate.
  • a linker provides space, rigidity, and/or flexibility between the two or more components.
  • the linker may join the targeting domain to the proteolytic domain.
  • the linker is a bivalent linker.
  • Linkers may be chemical linkers, which are known to one of skill in the art and are described in detail herein.
  • a linker may be a bond, e.g., a covalent bond.
  • bond refers to a chemical bond, e.g., an amide bond, a disulfide bond, a C-0 bond, a C-N bond, a N-N bond, a C-S bond, or any kind of bond created from a chemical reaction, e.g., chemical conjugation.
  • Molecules that may be used as linkers include at least two functional groups, which may be the same or different, e.g., two carboxylic acid groups, two amine groups, two sulfonic acid groups, a carboxylic acid group and a maleimide group, a carboxylic acid group and an alkyne group, a carboxylic acid group and an amine group, a carboxylic acid group and a sulfonic acid group, an amine group and a maleimide group, an amine group and an alkyne group, or an amine group and a sulfonic acid group.
  • two functional groups which may be the same or different, e.g., two carboxylic acid groups, two amine groups, two sulfonic acid groups, a carboxylic acid group and a maleimide group, a carboxylic acid group and an alkyne group, a carboxylic acid group and an amine group, a carboxylic acid group and a sulfonic acid
  • a chemical linker may include a polyethylene glycol (PEG) polymer, e.g., a PEG2-PEG50, most preferably PEG2, PEG3, PEG4, PEGs, PEGs, PEG?, PEGs, PEG9, or PEG10.
  • PEG polyethylene glycol
  • a linker may include one or more, e.g., 1 -100, 1 -50, 1 -25, 1 - 10, 1 -5, or 1 -3, optionally substituted alkylene, optionally substituted heteroalkylene (e.g., a PEG unit), optionally substituted alkenylene, optionally substituted heteroalkenylene, optionally substituted alkynylene, optionally substituted heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted cycloalkenylene, optionally substituted heterocycloalkenylene, optionally substituted cycloalkynylene, optionally substituted heterocycloalkynylene, optionally substituted arylene, optionally substituted heteroarylene (e.g., pyridine), O, S, NR' (R' is H, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted alkenyl, optionally substituted optionally substituted
  • a linker may include one or more optionally substituted C1 -C20 alkylene, optionally substituted C1 -C20 heteroalkylene (e.g., a PEG unit), optionally substituted C2- C20 alkenylene (e.g., C2 alkenylene), optionally substituted C2-C20 heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20 cycloalkylene (e.g., cyclopropylene, cyclobutylene), optionally substituted C2-C20 heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C6-C14 arylene (e.
  • Linkers may alternately be peptide linkers, such as those described herein. Peptide linkers may also be used to join two small molecules, to join a small molecule monomer or small molecule dimer to a polypeptide, or to join to polypeptides to form a fusion protein.
  • Peptide linkers also known as polypeptide linkers, include any linker than includes two or more amino acid residues.
  • a peptide linker may include 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more amino acid residues, which are joined, for example by peptide bonds.
  • the carboxy terminus of a peptide linker may be covalently conjugated (e.g., by a peptide bond) to a first moiety and the amino terminus of the peptide linker may be covalently conjugated (e.g., by a peptide bond) to a second moiety.
  • a peptide linker may be expressed from a polynucleotide composition or chemically synthesized and subsequently chemically conjugated to a first moiety and a second moiety.
  • a peptide linker may be expressed in tandem, e.g., with a first polypeptide (e.g., a targeting domain) and a second polypeptide (e.g., a proteolytic domain), thereby joining the first polypeptide and the second to form a composition.
  • a first polypeptide e.g., a targeting domain
  • a second polypeptide e.g., a proteolytic domain
  • macromolecule refers to a molecular entity that comprises one or more polypeptides.
  • the chains are covalently or non-covalently bound to one another.
  • the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • non-covalent interactions refers to chemical bonds that do not involve the sharing of pairs of electrons, but rather involve more dispersed variations of electromagnetic interactions.
  • a “polynucleotide,” such as a nucleic acid, is a macromolecule comprising two or more nucleotides.
  • the polynucleotide or nucleic acid may comprise any combination of any nucleotides.
  • the nucleotides can be naturally occurring or artificial.
  • One or more nucleotides in the polynucleotide can be oxidized or methylated.
  • One or more nucleotides in the polynucleotide may be modified, for instance with a label or a tag.
  • a nucleotide typically contains a nucleobase, a sugar and at least one phosphate group.
  • the nucleobase and sugar form a nucleoside.
  • the nucleobase is typically heterocyclic.
  • Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C).
  • the sugar is typically a pentose sugar.
  • Nucleotide sugars include, but are not limited to, ribose and deoxyribose.
  • the nucleotide in the polynucleotide is typically a ribonucleotide or deoxyribonucleotide.
  • the polynucleotide may comprise the following nucleosides: adenosine, uridine, guanosine and cytidine.
  • the polynucleotide may be single stranded or double stranded.
  • the polynucleotide can be a nucleic acid.
  • the polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) or other synthetic polymers with nucleotide side chains.
  • the PNA backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds.
  • the GNA backbone is composed of repeating glycol units linked by phosphodiester bonds.
  • the TNA backbone is composed of repeating threose sugars linked together by phosphodiester bonds.
  • LNA is formed from ribonucleotides as discussed above having an extra bridge connecting the 2' oxygen and 4' carbon in the ribose moiety.
  • proteolytic domain refers to a domain that is capable of catalyzing a chemical reaction that cleaves a peptide bond.
  • sheddase domain refers to a domain derived from a sheddase.
  • Sheddase refers to a polypeptide having protease activity, wherein the polypeptide having protease activity is (i) embedded in a lipid membrane or covalently or non-covalently bound to a moiety that is embedded in a lipid membrane and (ii) cleaves a bond in the ectodomain of a membrane-associated polypeptide.
  • targeting domain refers to a domain that binds a target molecule (e.g., a polypeptide, a polynucleotide, a lipid, glycan, or a polysaccharide).
  • a target molecule e.g., a polypeptide, a polynucleotide, a lipid, glycan, or a polysaccharide.
  • percent identity between two sequences may be determined by the BLAST 2.0 algorithm, which is described in Altschul et al., (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • bifunctional proteases e.g., targeted sheddases, proteases, or amyloiddegrading enzymes (e.g., amyloid beta (Ap)-degrading enzymes)
  • methods of cleaving macromolecules e.g., polypeptides, e.g., target or heterologous peptides.
  • methods of treating disorders associated with protein dysfunction by administering an effective amount of a bifunctional protease to a patient (e.g., a human patient having a disorder associated with protein dysfunction).
  • the bifunctional proteases may, e.g., cleave a macromolecule target at an increased rate and/or for an increased duration relative to a control molecule, e.g., a protease not comprising a targeting domain.
  • Bifunctional proteases of the disclosure include one or more targeting domains and one or more proteolytic domains.
  • a bifunctional protease of the disclosure may include a single targeting domain and a single proteolytic domain, or may include two or more targeting domains or proteolytic domains (e.g., may include two or more copies or versions of the targeting domain and/or may include two or more copies or versions of the proteolytic domain).
  • Targeting domains recognize and selectively bind a molecule of interest.
  • targeting domains of the disclosure bind their target molecules with high affinity.
  • targeting domains of the disclosure bind their target molecules with moderate affinity.
  • the molecule of interest is membrane-embedded.
  • the molecule of interest is not membrane-embedded, e.g., the polypeptide may be soluble.
  • the molecule of interest is in an amyloid protein oligomer, fibril, or plaque.
  • a targeting domain may be any moiety that binds to a target.
  • a targeting domain may be a polypeptide, polysaccharide, polynucleotide, or small molecule (e.g., an organic compound having a molecular weight less than or equal to 1000 Da).
  • the targeting domain may be a receptor or a ligand binding portion of a receptor.
  • the targeting domain may be a ligand that binds to a receptor.
  • the targeting domain binds to an amyloid protein (e.g., a monomer or a protein in an amyloid oligomer, fibril, or plaque).
  • the targeting domain binds to amyloid beta (Ap).
  • the targeting domain may comprise aducanumab, crenezumab, gantenerumab, bapineuzumab (3D6), solanezumab, ponezumab, lecanemab, or a fragment or derivative thereof.
  • Fragments or derivatives of the listed antibodies that may be useful in the invention include those that conserve or substantially conserve the complementarity determining regions (CDRs) and/or the variable regions of the antibody.
  • an exemplary variant of aducanumab that may be used in the invention is an scFv designed from linking the variable chains of aducanumab (e.g., as presented in SEQ ID NO: 17).
  • the targeting domain binds to TNFa.
  • the targeting domain may comprise a receptor protein or a fragment thereof that binds to TNFa (e.g., may comprise an ectodomain of TNF receptor 1 (TNFR1 )), or may comprise an antibody or antibody fragment (e.g., a VHH) that binds to TNFa.
  • TNFR1 ectodomain of TNF receptor 1
  • VHH antibody or antibody fragment
  • the targeting domain binds to IL-4.
  • the targeting domain may comprise a receptor protein or a fragment thereof that binds to IL-4 (e.g., may comprise an ectodomain of IL4Ra), may comprise an antibody or antibody fragment (e.g., a VHH) that binds to IL- 4, or may comprise a Designed Ankyrin Repeat Protein (DARPin) that binds to IL-4.
  • DARPin Designed Ankyrin Repeat Protein
  • the targeting domain binds to IL-13.
  • the targeting domain may comprise a receptor protein or a fragment thereof that binds to IL-13, may comprise an antibody or antibody fragment (e.g., a VHH) that binds to IL-13, or may comprise a DARPin that binds to IL-13.
  • the targeting domain may comprise an amino acid sequence that has at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity (e.g., has 80-85% identity, 85-90% identity, 90-95% identity, or 95-100% identity) to any particular targeting domain sequence provided herein.
  • the targeting domain may comprise all or a portion of the antigen binding fragment of the antibody, e.g., may comprise the CDRs or the full variable domains.
  • the address target binding domain comprises a variant of the antigen binding fragment of the antibody, e.g., a humanized variant or a variant otherwise comprising one or more amino acid substitutions, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions, e.g., about: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20%, or more, divergence from the antibody sequence; in certain embodiments, any substitutions are in the CDRs while in other embodiments any substitutions are outside the CDRs, while in other embodiments, the substitution may be in both CDR and non-CDR sequences.
  • Substitutions in some embodiments may be non-conservative, conservative, highly conservative, or a combination thereof (e.g., as determined according to BLOSSUM62), e.g., conservative or highly conservative substitutions in the CDRs (particularly in paratopic residues, while in some embodiments, any substitutions are outside of paratopic residues) and non-conservative, conservative, highly conservative, or a combination thereof in non-CDR residues.
  • the targeting domain may comprise all or a portion of the region or regions of the binding domain that interact with the target.
  • the binding domain comprises a variant of a binding domain sequence provided herein, e.g., a variant comprising one or more amino acid substitutions, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions, e.g., about: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20%, or more, divergence from the binding domain sequence; in certain embodiments, any substitutions are in the regions of the binding domain that interact with the target, while in other embodiments any substitutions are outside regions of the binding domain that interact with the target, while in other embodiments, the substitution may be in both regions of the binding domain that interact and do not interact with the target.
  • a variant comprising one or more amino acid substitutions e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions, e.g., about: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20%, or more, divergence from the
  • substitutions in some embodiments may be non-conservative, conservative, highly conservative, or a combination thereof, e.g., conservative or highly conservative substitutions in the regions of the binding domain that interact with the target and non-conservative, conservative, highly conservative, or a combination thereof in regions of the binding domain that do not interact with the target.
  • Targeting domains of the disclosure may include a polypeptide.
  • the targeting domain includes a protein ligand or a portion thereof, a receptor or portion thereof, or any other polypeptide that selectively binds a target molecule.
  • the targeting domain includes an antibody or antigen-binding fragment thereof.
  • the targeting domain includes an antigen-binding fragment (e.g., VHH, Fab, F(ab’)2, scFv with or without an Fc region, immunoprotein, heavy chain variable region and a light chain variable region, affibody, diabody, triabody, tetrabody, knottin, atrimer, avimer, cys-knot, fynomer, kunitz domain, Obody, nanobody, an Fc fusion protein, anticalin, affimer types I or II, FN3 scaffold, centyrin, or DARPin).
  • the antigen-binding fragment is a VHH.
  • the antigen-binding fragment is a Fab.
  • the antigen-binding fragment is a F(ab’)2. In some embodiments, the antigen-binding fragment is a scFv with an Fc region. In some embodiments, the antigen-binding fragment is a scFv without an Fc region. In some embodiments, the antigen-binding fragment is an immunoprotein. In some embodiments, the antigen-binding fragment is a heavy chain variable region. In some embodiments, the antigen-binding fragment is a light chain variable region. In some embodiments, the antigen-binding fragment is an affibody. In some embodiments, the antigen-binding fragment is a diabody. In some embodiments, the antigenbinding fragment is a triabody.
  • the antigen-binding fragment is a tetrabody. In some embodiments, the antigen-binding fragment is a knottin. In some embodiments, the antigen- binding fragment is an atrimer. In some embodiments, the antigen-binding fragment is an avimer. In some embodiments, the antigen-binding fragment is a cys-knot. In some embodiments, the antigenbinding fragment is a fynomer. In some embodiments, the antigen-binding fragment is a kunitz domain. In some embodiments, the antigen-binding fragment is an Obody. In some embodiments, the antigen-binding fragment is a nanobody.
  • the antigen-binding fragment is an Fc fusion protein. In some embodiments, the antigen-binding fragment is an anticalin. In some embodiments, the antigen-binding fragment is an affimer type I. In some embodiments, the antigenbinding fragment is an affimer type II. In some embodiments, the antigen-binding fragment is a FN3 scaffold. In some embodiments, the antigen-binding fragment is a centyrin. In some embodiments, the antigen-binding fragment is a DARPin.
  • the protein ligand or portion thereof is capable of binding to or binds to a target molecule of interest.
  • the protein ligand or portion thereof is a heat shock protein 90 (Hsp90) inhibitor, a kinase inhibitor, a MDM2 inhibitor, a compound targeting Human bromodomain and extra-terminal domain (BET) bromodomain-containing proteins, a compound targeting cytosolic signaling proteins (e.g., FK506 binding protein (FKBP12)), a histone deacetylase (HDAC) inhibitor, a human lysine methyltransferase inhibitor, an angiogenesis inhibitor, an immunosuppressive compound, a compound targeting the aryl hydrocarbon receptor (AHR), a nuclear protein, an estrogen receptor, an androgen receptor, a glucocorticoid receptor, or a transcription factor (e.g., SWI/SNF-related matrix-associated actin-dependent regulator of chromatin 4 (SMARCA4),
  • Hsp90 heat
  • a kinase to which the protein ligand or portion thereof is capable of binding to or binds to includes, but is not limited to, a tyrosine kinase (e.g., AATK, ABL, ABL2, ALK, AXL, BLK, BMX, BTK, CSF1 R, CSK, DDR1 , DDR2, EGFR, EPHA1 , EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA10, EPHB1 , EPHB2, EPHB3, EPHB4, EPHB6, ERBB2, ERBB3, ERBB4, FER, FES, FGFR1 , FGFR2, FGFR3, FGFR4, FGR, FLT1 , FLT3, FLT4, FRK, FYN, GSG2, HCK, IGF1 R, ILK, INSR, INSRR, IRAK4, ITK, JAK1 , JAK2, JAK3, KDR, KIT,
  • a BET bromodomain-containing protein to which the protein ligand or portion thereof is capable of binding to or binds to includes, but is not limited to, bromodomaincontaining 1 (BRD1 ), BRD2, BRD3, BRD4, BRD5, BRD6, BRD7, BRD8, BRD9, BRD10, and bromodomain testis-specific protein (BRDT).
  • BRD1 bromodomaincontaining 1
  • BRD2, BRD3, BRD4, BRD5, BRD6, BRD7, BRD8, BRD9, BRD10 includes, but is not limited to, bromodomaincontaining 1 (BRD1 ), BRD2, BRD3, BRD4, BRD5, BRD6, BRD7, BRD8, BRD9, BRD10, and bromodomain testis-specific protein (BRDT).
  • BRD1 bromodomaincontaining 1
  • BRD2, BRD3, BRD4, BRD5, BRD6, BRD7, BRD8, BRD9, BRD10 bromodomain testis-specific protein
  • a nuclear protein to which the protein ligand or portion thereof is capable of binding to or binds to includes, but is not limited to, BRD2, BRD3, BRD4, antennapedia homeodomain protein, breast cancer type 1 susceptibility protein (BRCA1 ), BRCA2, CCAAT-Enhanced-Binding Proteins, histones, polycomb-group proteins, high mobility group proteins, telomere binding proteins, Fanconi anemia group A protein (FANCA), FANCD2, FANCE, FANCF, hepatocyte nuclear factors, mitotic arrest deficient 2 (MAD2), nuclear factor kappa B (NF-kB), nuclear receptor coactivators, cyclic adenosine monophosphate response element binding protein binding protein (CREB-binding protein), p55, p107, p130, Rb proteins, p53, c- fos, c-jun, c-mdm2, c-myc, and c-rel.
  • BRD2 breast cancer type 1 susceptibility protein
  • Receptor proteins can bind to corresponding ligands to initiate cellular signaling pathways.
  • Targeting domains provided herein may include receptor proteins or portions thereof.
  • the receptor protein or portion thereof is a cytokine receptor (e.g., a TNF receptor, a CSK receptor, an interleukin (IL) receptor, a chemokine receptor, an interferon (INF) receptor, or a transforming growth factor beta (TGFp) superfamily receptor).
  • cytokine receptor e.g., a TNF receptor, a CSK receptor, an interleukin (IL) receptor, a chemokine receptor, an interferon (INF) receptor, or a transforming growth factor beta (TGFp) superfamily receptor.
  • the receptor protein or portion thereof is a growth factor receptor (e.g., a Wnt receptor, a Tie receptor, a IGF receptor, an EGF receptor, a neutotrophin receptor, an ephrin receptor, a fibroblast growth factor (FGF) receptor, a PDGF receptor, or a VEGF receptor).
  • the receptor protein or portion thereof is a B cell receptor protein, a natural killer cell receptor protein, a T cell receptor protein, a monocyte receptor protein, a stem cell receptor protein, a dendritic cell receptor protein, or a granulocyte receptor protein.
  • the receptor protein or portion thereof is a G- protein coupled receptor (e.g., a 5-hydroxytryptamine receptor, a muscarinic acetylcholine receptor, an adenosine receptor, an adrenoreceptor, an angiotensin receptor, an apelin receptor, a bile acid receptor, a bombesin receptor, a bradykinin receptor, a cannabinoid receptor, a chemerin receptor, a chemokine receptor, a cholecystokinin receptor, a complement peptide receptor, a dopamine receptor, an endothelin receptor, a formylpeptide receptor, a galanin receptor, a ghrelin receptor, a glycoprotein hormone receptor, a gonadotrophin-releasing hormone receptor, a histamine receptor, a hydroxycarboxylic acid receptor, a kisspeptin receptor, a leukotriene receptor, a lysophospholipid receptor
  • the receptor protein or portion thereof is an enzyme-linked receptor (e.g., a receptor tyrosine kinase, a receptor serine/threonine kinase, a receptor guanylyl cyclase, a tyrosine-kinase associated receptor, or a receptor tyrosine phosphatase).
  • the receptor protein or portion thereof is an adhesion receptor (e.g., an integrin, a cadherin, a selectin, or an immunoglobin-like cell adhesion molecule).
  • the receptor protein or portion thereof is a nuclear hormone receptor (e.g., a thyroid hormone receptor, a retinoic acid receptor, a peroxisome proliferator-activated receptor, a vitamin D receptor-like receptor, a hepatocyte nuclear factor-4 receptor, a retinoid X receptor, a tailless-like receptor, an estrogen receptor, a nerve growth factor IB-like receptor, or a germ cell nuclear factor receptor).
  • a nuclear hormone receptor e.g., a thyroid hormone receptor, a retinoic acid receptor, a peroxisome proliferator-activated receptor, a vitamin D receptor-like receptor, a hepatocyte nuclear factor-4 receptor, a retinoid X receptor, a tailless-like receptor, an estrogen receptor, a nerve growth factor IB-like receptor, or a germ cell nuclear factor receptor.
  • a monoclonal antibody may be obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the polypeptide is a monoclonal antibody.
  • Antigen-binding fragments are one or more fragments of an antibody that retain the ability to specifically bind to a target antigen.
  • the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • the antibody fragments can be, e.g., a single-domain antibody (sdAb), Fab, F(ab’)2, scFv, diabody, a triabody, a tetramer, a fynomer, an affibody, an aptamer, an obody, an anticalin, an atrimer, a nanobody, an immunoprotein, an FN3 scaffold, a centyrinTM, a DARPin, or a domain antibody.
  • binding fragments encompassed by the term “antigenbinding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb including VH and VL domains; (vi) a dAb fragment (Ward et al., Nature 341 :544-546, 1989), which consists of a VH domain; (vii) a dAb which consists of a VH or a VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988).
  • scFv single chain Fv
  • These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies.
  • Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some embodiments, by chemical peptide synthesis procedures known in the art.
  • the targeting domain includes an antigen-binding fragment.
  • the antigen-binding fragment is a Fab, F(ab’)2, scFv with or without an Fc region, immunoprotein, heavy chain variable region and a light chain variable region, affibody, diabody, triabody, tetrabody, knottin, atrimer, avimer, cys-knot, fynomer, kunitz domain, Obody, nanobody, an Fc fusion protein, anticalin, affimer types I or II, FN3 scaffold, centyrin, or a DARPin.
  • a Fab is an antibody fragment that has two polypeptide chains, the heavy- and light-chain variable domains, and that also contains the constant domain of the light chain and the first constant domain (CH1 ) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily.
  • the targeting domain includes a Fab.
  • F(ab')2 fragment antibodies are generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving intact some of the hinge region.
  • F(ab')2 fragments have two antigen-binding F(ab) portions linked together by disulfide bonds, and therefore are divalent with a molecular weight of about 110 kDa.
  • Divalent antibody fragments are smaller than whole IgG molecules and enable a better penetration into tissue thus faciliting better antigen recognition in IHC.
  • the use of F(ab')2 fragments also avoids unspecific binding to Fc receptor on live cells or to Protein A/G.
  • the targeting domain includes a F(ab')2.
  • Single-chain Fv antibodies are antibodies in which the variable domains of the heavy chain and the light chain from an antibody have been joined to form one chain.
  • ScFv fragments contain a single polypeptide chain that includes the variable region of an antibody light chain (VL) (e.g., CDR-L1 , CDR-L2, and/or CDR-L3) and the variable region of an antibody heavy chain (VH) (e.g., CDR-H1 , CDR-H2, and/or CDR-H3) separated by a linker.
  • VL antibody light chain
  • VH variable region of an antibody heavy chain
  • the linker that joins the VL and VH regions of a scFv fragment can be a peptide linker composed of proteinogenic amino acids.
  • linkers can be used to increase the resistance of the scFv fragment to proteolytic degradation (e.g., linkers containing D-amino acids), in order to enhance the solubility of the scFv fragment (e.g., hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (e.g., a linker containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the scFv fragment (e.g., linkers containing glycosylation sites).
  • linkers containing D-amino acids e.g., hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues
  • hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine
  • ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019, Flo et al., (Gene 77:51 , 1989); Bird et al., (Science 242:423, 1988); Pantoliano et al., (Biochemistry 30:10117, 1991 ); Milenic et al., (Cancer Research 51 :6363, 1991 ); and Takkinen et al., (Protein Engineering 4:837, 1991 ).
  • the VL and VH domains of a scFv molecule can be derived from one or more antibody molecules.
  • variable regions of the scFv molecules described herein can be modified such that they vary in amino acid sequence from the antibody molecule from which they were derived.
  • nucleotide or amino acid substitutions leading to conservative substitutions or changes at amino acid residues can be made (e.g., in CDR and/or framework residues).
  • mutations are made to CDR amino acid residues to optimize antigen binding using art recognized techniques.
  • ScFv fragments are described, for example, in WO 2011/084714; incorporated herein by reference.
  • the targeting domain includes a scFv without an Fc region.
  • ScFvs may be fused to a fragment crystallizable (Fc) region to enhance the affinity of the scFv with certain cell surface receptors (e.g., Fc receptors) and proteins of the complement system.
  • the targeting domain includes a scFv with an Fc region.
  • Immunoproteins are proteins or peptides that are associated with an immune response.
  • Nonlimiting examples of immunoproteins include T cell receptors (TCRs), antibodies (immunoglobulins), major histocompatibility complex (MHO) proteins, complement proteins, and RNA binding proteins.
  • TCRs T cell receptors
  • antibodies immunoglobulins
  • MHO major histocompatibility complex
  • complement proteins RNA binding proteins.
  • RNA binding proteins include an immunoprotein.
  • the targeting domain includes a heavy chain variable region and/or a light chain variable region.
  • the heavy chain variable region is a segment of an immunoglobulin heavy polypeptide chain.
  • Immunoglobulin molecules comprise four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain comprises a heavy chain variable (VH) region and a heavy chain constant region (CH).
  • the heavy chain constant region comprises three domains, CH1 , CH2 and CH3.
  • Each light chain comprises light chain variable (VL) region and a light chain constant region (CL).
  • the VH and VL regions can be further subdivided into regions of hypervariability, or CDRs, interspersed with regions that are more conserved, termed framework regions (FR).
  • Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3,
  • variable domains differ extensively in sequence among antibodies. Variable regions confer antigen-binding specificity. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR) regions.
  • CDRs complementarity determining regions
  • FR framework
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a p-pleated-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the p-pleated-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al. (1991 ) NIH Publ. No. 91 -3242, Vol. I, pages 647-669).
  • the targeting domain includes an affibody.
  • Affibody molecules are small highly robust proteins with specific affinities to target proteins. They can be designed and used, for example, like aptamers.
  • Affibody molecules in accordance with the invention comprise a backbone derived from an IgG-bind ing domain of Staphlococcal Protein A (Protein A produced by S. aureus).
  • the backbone can be derived from an IgG binding domain comprising the three alpha helices of the IgG-bindi ng domain of Staphlococcal Protein A termed the B domain.
  • the amino acid sequence of the B domain is described in Uhlen et al., J. Biol. Chem. 259: 1695-1702 (1984).
  • the backbone can be derived from the three alpha helices of the synthetic IgG-binding domain known in the art as the Z domain, which is described in Nilsson et al., Protein Eng. 1 : 107-113 (1987).
  • the backbone of an affibody comprises the amino acid sequences of the IgG binding domain with amino acid substitutions at one or more amino acid positions.
  • Affibodies against novel targets are generated by randomizing the 13 amino acid residues in the IgG-binding surface using phage or yeast display techniques. Affinity-matured proteins are produced as either a single 7 kDa domain (monovalent Affibody) or as two tandem 7 kDa domains (bivalent Affibody). In some embodiments, the targeting domain includes an affibody.
  • the targeting domain includes a diabody.
  • Diabodies are bivalent antibodies comprising two polypeptide chains, in which each polypeptide chain includes VH and VL domains joined by a linker that is too short (e.g., a linker composed of five amino acids) to allow for intramolecular association of VH and VL domains on the same peptide chain. This configuration forces each domain to pair with a complementary domain on another polypeptide chain so as to form a homodimeric structure.
  • the targeting domain includes a diabody.
  • the targeting domain includes a triabody.
  • the term “triabody” refers to a trivalent antibody comprising three peptide chains, each of which contains one VH domain and one VL domain joined by a linker that is exceedingly short (e.g., a linker composed of 1 -2 amino acids) to permit intramolecular association of VH and VL domains within the same peptide chain.
  • a linker that is exceedingly short (e.g., a linker composed of 1 -2 amino acids) to permit intramolecular association of VH and VL domains within the same peptide chain.
  • peptides configured in this way typically trimerize so as to position the VH and VL domains of neighboring peptide chains spatially proximal to one another to permit proper folding (see Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993; incorporated herein by reference).
  • the targeting domain includes a triabody.
  • the targeting domain includes a tetrabody.
  • tetrabody refers to a tetravalent antibody comprising four peptide chains, each of which contains one VH domain and one VL domain joined by a linker that is exceedingly short (e.g., a linker composed of 1 -2 amino acids) to permit intramolecular association of VH and VL domains within the same peptide chain.
  • linker that is exceedingly short (e.g., a linker composed of 1 -2 amino acids) to permit intramolecular association of VH and VL domains within the same peptide chain.
  • peptides configured in this way typically tetramerize so as to position the VH and VL domains of neighboring peptide chains spatially proximal to one another to permit proper folding.
  • the targeting domain includes a tetrabody.
  • the targeting domain includes a knottin.
  • Knottins are a structural family (typically 30-50 amino acids in length) characterized by a core of antiparallel p-strands stabilized by at least three disulfide bonds. In a characteristic cystine-knot motif, the first and fourth and the second and fifth cysteine residues form disulfide bonds. A disulfide bond formed between the third and sixth cysteine residues passes through these first two disulfides, creating a macrocyclic knot. This disulfide-constrained core confers chemical, thermal and proteolytic stability upon the peptide. Knottins also possess loop regions of variable length and composition that are constrained to the core of antiparallel p-strands.
  • knottin peptides have been shown to tolerate amino acid mutations. In contrast to linear peptides, knottins have been shown to retain their three-dimensional structure and function after boiling or incubation in acid, base and serum.
  • a knottin refers to a member of a family of small proteins, typically 25-50 amino acids in length, that bind to various molecular targets, including proteins, sugars and lipids. Their three- dimensional structure is minimally defined by a particular arrangement of three disulfide bonds. This characteristic topology forms a molecular knot in which one disulfide bond passes through a macrocycle formed by the other two intrachain disulfide bridges. Although their secondary structure content is generally low, knottins share a small triple-stranded antiparallel p-sheet, which is stabilized by the disulfide bond framework.
  • the targeting domain includes a knottin.
  • the targeting domain includes an atrimer.
  • the targeting domain includes an alternative scaffold.
  • alternative scaffold refers to a single chain polypeptidic framework typically of reduced size (e.g., less than about 200 amino acids) that contains a highly structured core associated with variable domains of high conformational tolerance allowing insertions, deletions, or other substitutions. These scaffolds are based either on a conventional Ig backbone or are derived from a completely unrelated protein. These variable domains can be modified to create novel binding interfaces toward any targeted protein.
  • such a scaffold can be derived from Protein A, in particular, the Z- domain thereof (affibodies), lmmE7 (immunity proteins), BPTI/APPI (Kunitz domains), Ras-binding protein AF-6 (PDZ-domains), charybdotoxin (Scorpion toxin), CTLA-4, Min-23 (knottins), lipocalins (anticalins), neokarzi nostatin, a fibronectin domain, an ankyrin consensus repeat domain, or thioredoxin (Skerra, A., “Alternative Non-Antibody Scaffolds for Molecular Recognition,” Curr. Opin. Biotechnol.
  • Non-lg based scaffolds include, but are not limited to, lipocalins (used in “anticalins”), ankyrin repeat (AR) proteins (used in “designed AR proteins” or “DARPins”), fibronectin domain derivatives (used in “adnectin”), and avidity multimers (also known as “avimers”).
  • the targeting domain includes an alternative scaffold.
  • the alternative scaffold is a non-lg based scaffold.
  • Avimers are a class of antibody mimetics which consist of two or more peptide sequences of 30 to 35 amino acids each, which are derived from A- domains of various membrane receptors and which are connected by linker peptides. Binding of target molecules occurs via the A-domain and domains with desired binding specificity can be selected, for example, by phage display techniques.
  • the target specificity of the different A-domains contained in an avimer may, but do not have to be identical (Weidle UH, et al., (2013), Cancer Genomics Proteomics; 10(4): 155-68).
  • the avimer structure can also be used as a protein backbone to generate a suitable non-lg based alternative scaffold.
  • the avimer scaffold is based on oligomerization of A-domains from low- density lipoprotein (LRL) cell surface receptors.
  • the avimer scaffold comprises universally conserved residues of the A-domain, which is about 35 amino acids in length and comprises four loops with three disulfide bridges.
  • the targeting domain includes an avimer.
  • cystine knot superfamily The typical structure seen in the cystine knot superfamily is based on the presence of 6 cysteine residues creating 3 disulphide bonds. Two of the disulphide bonds create a ‘ring-like’ structure, which is penetrated by the third disulphide bond, (Sun et al. 1995). Cystine knot domains are often found with more than 6 cysteine residues. The extra cysteine residues are normally used to create further disulphide bonds within the cystine knot domain or interchain disulphide bonds, during dimerisation.
  • the targeting domain includes a cys-knot.
  • Fynomers are small (approximately 7 kDa) binding proteins that can bind to target antigens with a similar affinity and specificity to antibodies. Fynomers are based on the human Fyn SH3 domain as a scaffold for assembly of binding molecules. The Fyn SH3 domain is a fully human, 63- aa protein that can be produced in bacteria with high yields. Fynomers may be linked together to yield a multispecific binding protein with affinities for two or more different antigen targets. In some embodiments, the targeting domain includes a fynomer.
  • the Kunitz domain is a folding domain of approximately 50-60 residues which forms a central anti-parallel beta sheet and a short C-terminal helix. This characteristic domain comprises six cysteine residues that form three disulfide bonds, resulting in a double-loop structure. Between the N- terminal region and the first beta strand resides the active inhibitory binding loop. This binding loop is disulfide bonded through the P2 Cys residue to the hairpin loop formed between the last two beta strands. Isolated Kunitz domains from a variety of proteinase inhibitors have been shown to have inhibitory activity (e.g., Petersen et al., Eur. J. Biochem. 125:310-316, 1996; Wagner et al., Biochem. Biophys. Res. Comm. 186:1138-1145, 1992; Dennis et al., J. Biol. Chem. 270:25411 -25417, 1995). In some embodiments, the targeting domain includes a Kunitz domain.
  • the targeting domain includes an Obody.
  • Nanobodies are single-chain antibody fragments that contain only a single heavy-chain variable domain. Unlike a traditional, full-length antibody, which includes heavy chains and light chains, each containing a corresponding variable domain (i.e. , a heavy chain variable domain, VH, and a light chain variable domain, VL) having three CDRs, a single-domain antibody only includes one heavy-chain variable domain having a total of three CDRs (referred to herein as CDR-H1 , CDR-H2, and CDR-H3).
  • the targeting domain includes a nanobody.
  • Anticalins are non-immunoglobulin binding proteins based on the human lipocalin scaffold. Anticalin molecules are generated by combinatorial design from natural lipocalins, which are abundant plasma proteins in humans, and reveal a simple, compact fold dominated by a central b- barrel, supporting four structurally variable loops that form a binding site. Reshaping of this loop region results in Anticalin proteins that can recognize and tightly bind a wide range of medically relevant targets, from small molecules to peptides and proteins, as validated by X-ray structural analysis. Their robust format allows for modification in several ways, both as fusion proteins and by chemical conjugation, for example, to tune plasma half-life.
  • Antagonistic Anticalin therapeutics have been developed for systemic administration (e.g., PRS-080: anti-hepcidin) or pulmonary delivery (e.g., PRS- 060/AZD1402: anti-interleukin [IL]-4-Ra).
  • PRS-080 anti-hepcidin
  • PRS- 060/AZD1402 anti-interleukin [IL]-4-Ra
  • Anticalin proteins allow molecular formatting as bi- and even multispecific fusion proteins, especially in combination with antibodies that provide a second specificity.
  • Anticalins an engineered protein scaffold comprising a lipocalin backbone, are a suitable non-lg based alternative scaffolds for use in the binding molecules of the present invention.
  • Lipocalins a family of proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids, are the parental protein structure of anticalins. Lipocalins have limited sequence homology, but share a common tertiary structure architecture based on eight antiparallel b-barrels. These proteins contain four exposed loops built on the rigid beta-barrel structure.
  • the targeting domain includes a lipocalin.
  • the targeting domain includes an affimer type I.
  • the targeting domain includes an affimer type II.
  • Proteins derived from fibronectin III (FN3) domains may be used to generate a suitable non-lg based alternative scaffold.
  • the tenth fibronectin type III domain (FN10) of human fibronectin corresponds to a beta-sandwich with seven beta-strands and three connecting loops showing structural homologies to Ig domains without disulfide bridges.
  • the connecting loops of FN10 each about 15 to 21 amino acids in length, can be randomized and the domains displayed on both phage and yeast to select for a scaffold with the desirable properties.
  • the targeting domain includes a FN3 scaffold. CentyrinTM
  • Centyrins are small, engineered proteins derived from FN domains of a human protein, Tenascin C (TNC), found in the extracellular matrix of various tissues. CentryrinsTM contain the consensus sequence of FN3 domains of TNC. CentyrinTM scaffolds have loops (i.e., DE, BC, and FG) that have structural homology to antibody variable domains (i.e., CDR1 , CDR2 and CDR3), and are small (about 10 kDa), simple, and highly stable single domain proteins that do not contain cysteine, disulfides or glycosylated residues.
  • loops i.e., DE, BC, and FG
  • antibody variable domains i.e., CDR1 , CDR2 and CDR3
  • the targeting domain includes a centyrinTM scaffold.
  • AR proteins are another suitable non-lg based alternative scaffold that can act as a targeting domain of the present invention.
  • AR proteins comprise a 33 amino acid protein motif consisting of two alpha helices separated by loops, which repeats mediate protein — protein interactions.
  • Designed Ankyrin Repeat Proteins (DARPins) comprise an engineered protein scaffold resulting from rational design strategies (e.g., multiple sequence alignments and statistical analysis) based on human AR proteins.
  • DARPins can be generated using combinatorial AR libraries constructed based on the 33 amino acid AR motif with seven randomized positions.
  • DARPin libraries are preferentially screened using ribosome display, and library members typically are well produced in E. coll, do not aggregate, and display high thermodynamic stability.
  • DARPins contain two to four of these motifs flanked by N- and C-terminal capping motifs to shield hydrophobic regions and allow increased solubility.
  • the targeting domain includes a DARPin.
  • Targeting domains of the disclosure may include glycan moieties.
  • the glycan moiety is a polysaccharide.
  • the glycan moiety is an oligosaccharide.
  • the glycan moiety is the carbohydrate portion of a glycoconjugate (e.g., a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide, or proteoglycan).
  • the glycan moiety is located on the exterior surface of a cell.
  • the glycan moiety is membrane-embedded.
  • the glycan moiety is N-acetylglucosamine (GIcNac).
  • the glycan moiety is N-acetylgalactosamine (GalNac).
  • Targeting domains of the disclosure may include, or be fused to, polynucleotides.
  • the polynucleotides may be naturally occurring or artificial (e.g., synthetic, e.g., peptide nucleic acids (PNAs), glycerol nucleic acids (GNAs), threose nucleic acids (TNAs), locked nucleic acids (LNAs), or other synthetic polymers with nucleotide side chains).
  • PNAs peptide nucleic acids
  • GNAs glycerol nucleic acids
  • TAAs threose nucleic acids
  • LNAs locked nucleic acids
  • One or more nucleotides in the polynucleotide may be modified, for instance with a label or a tag.
  • Proteolytic domains are the catalytically active portion of a protease. Proteolytic domains catalyze chemical reactions that cleave peptide bonds in substrate polypeptides.
  • a proteolytic domain of the disclosure contains all of the requisite properties required for its proteolytic activity, such as for example, the catalytic center.
  • the proteolytic domain comprises or consists of mesotrypsin (e.g., human mesotrypsin), or a fragment or derivative thereof.
  • the proteolytic domain may comprise an amino acid sequence having at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2 or may comprise the amino acid sequence of SEQ ID NO: 2.
  • the proteolytic domain comprises or consists of mesotrypsin and the targeting domain binds to TNFa.
  • the proteolytic domain comprises or consists of mesotrypsin and the targeting domain binds to IL-4.
  • the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds TNFa; and (b) mesotrypsin or a fragment or derivative thereof.
  • the bifunctional protease has at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity (e.g., has 80-85% identity, 85-90% identity, 90-95% identity, or 95-100% identity) to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.
  • the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds IL-4; and (b) mesotrypsin or a fragment or derivative thereof.
  • the bifunctional protease has at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity (e.g., has 80-85% identity, 85- 90% identity, 90-95% identity, or 95-100% identity) to SEQ ID NO: 14.
  • the proteolytic domain comprises or consists of MMP7 (e.g., human MMP7), or a fragment or derivative thereof (e.g., comprises the pro-enzyme form of MMP7).
  • the proteolytic domain may comprise an amino acid sequence having at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity to human MMP7 or may comprise the amino acid sequence of human MMP7.
  • the proteolytic domain comprises or consists of MMP7 and the targeting domain binds to IL-4.
  • the proteolytic domain comprises or consists of MMP7 and the targeting domain binds to IL-13.
  • the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds IL-4; and (b) MMP7 or a fragment or derivative thereof.
  • the bifunctional protease has at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity (e.g., has 80-85% identity, 85-90% identity, 90-95% identity, or 95-100% identity) to SEQ ID NO: 11.
  • the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds IL-13; and (b) MMP7 or a fragment or derivative thereof.
  • the bifunctional protease has at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity (e.g., has 80-85% identity, 85-90% identity, 90-95% identity, or 95-100% identity) to SEQ ID NO: 8 or SEQ ID NO: 15.
  • the proteolytic domain comprises or consists of MMP12 (e.g., human MMP12), or a fragment or derivative thereof (e.g., comprises the pro-enzyme form of MMP12).
  • the proteolytic domain comprises or consists of MMP12 and the targeting domain binds to IL-4.
  • the proteolytic domain comprises or consists of MMP12 and the targeting domain binds to IL-13.
  • the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds IL-4; and (b) MMP12 or a fragment or derivative thereof.
  • the bifunctional protease has at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity (e.g., has 80-85% identity, 85-90% identity, 90-95% identity, or 95-100% identity) to SEQ ID NO: 12.
  • the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds IL-13; and (b) MMP12 or a fragment or derivative thereof.
  • the bifunctional protease has at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity (e.g., has 80-85% identity, 85-90% identity, 90-95% identity, or 95-100% identity) to SEQ ID NO: 9 or SEQ ID NO: 16.
  • the proteolytic domain comprises or consists of MMP2 (e.g., human MMP2), or a fragment or derivative thereof.
  • the proteolytic domain comprises the hemopexin (HEX) domain of MMP2; in other embodiments, the HEX domain is absent.
  • the proteolytic domain comprises or consists of MMP2 and the targeting domain binds to amyloid beta.
  • the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds amyloid beta; and (b) MMP2 or a fragment or derivative thereof.
  • the bifunctional protease has at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity (e.g., has 80-85% identity, 85-90% identity, 90-95% identity, or 95-100% identity) to SEQ ID NO: 31 , SEQ ID NO: 32, or SEQ ID NO: 40.
  • the proteolytic domain comprises or consists of MMP9 (e.g., human MMP9), or a fragment or derivative thereof.
  • the proteolytic domain comprises the HEX domain of MMP9; in other embodiments, the HEX domain is absent.
  • the proteolytic domain comprises or consists of MMP9 and the targeting domain binds to amyloid beta.
  • the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds amyloid beta; and (b) MMP9 or a fragment or derivative thereof.
  • the bifunctional protease has at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity (e.g., has 80-85% identity, 85-90% identity, 90-95% identity, or 95-100% identity) to SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 41 , or SEQ ID NO: 46.
  • the proteolytic domain comprises or consists of MMP14 (e.g., human MMP14), or a fragment or derivative thereof.
  • the proteolytic domain comprises the HEX domain of MMP14; in other embodiments, the HEX domain is absent.
  • the proteolytic domain comprises or consists of MMP14 and the targeting domain binds to amyloid beta.
  • the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds amyloid beta; and (b) MMP14 or a fragment or derivative thereof.
  • the bifunctional protease has at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity (e.g., has 80-85% identity, 85-90% identity, 90-95% identity, or 95-100% identity) to SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 42.
  • the proteolytic domain comprises or consists of cathepsin-B (CTSB) (e.g., human CTSB), or a fragment or derivative thereof.
  • CTSB cathepsin-B
  • the proteolytic domain comprises or consists of CTSB and the targeting domain binds to amyloid beta.
  • the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds amyloid beta; and (b) CTSB or a fragment or derivative thereof.
  • the bifunctional protease has at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity (e.g., has 80-85% identity, 85-90% identity, 90-95% identity, or 95-100% identity) to SEQ ID NO: 37 or SEQ ID NO: 43.
  • the proteolytic domain comprises or consists of cathepsin-D (CTSD) (e.g., human CTSD), or a fragment or derivative thereof.
  • CTSD cathepsin-D
  • the proteolytic domain comprises or consists of CTSD and the targeting domain binds to amyloid beta.
  • the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds amyloid beta; and (b) CTSD or a fragment or derivative thereof.
  • the bifunctional protease has at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity (e.g., has 80-85% identity, 85-90% identity, 90-95% identity, or 95-100% identity) to SEQ ID NO: 38, SEQ ID NO: 44, or SEQ ID NO: 47.
  • the proteolytic domain comprises or consists of Kallikrein 7 (KLK7) (e.g., human KLK7), or a fragment or derivative thereof. In some embodiments, the proteolytic domain comprises or consists of KLK7 and the targeting domain binds to amyloid beta.
  • KLK7 Kallikrein 7
  • the targeting domain binds to amyloid beta.
  • the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds amyloid beta; and (b) KLK7 or a fragment or derivative thereof.
  • the bifunctional protease has at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity (e.g., has 80-85% identity, 85-90% identity, 90-95% identity, or 95-100% identity) to SEQ ID NO: 39, SEQ ID NO: 45, or SEQ ID NO: 48.
  • the proteolytic domain may comprise an amino acid sequence that has at least 80%, 85%, 95%, 95%, 96%, 97%, 98%, or 99% identity (e.g., has 80-85% identity, 85-90% identity, 90-95% identity, or 95-100% identity) to any particular proteolytic domain sequence provided herein.
  • the proteolytic domain may comprise all or a portion of the enzymatic active site (e.g., catalytic site) of the proteolytic domain.
  • the proteolytic domain comprises a variant of the proteolytic domain, e.g., a variant comprising one or more amino acid substitutions, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions, e.g., about: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20%, or more, divergence from the proteolytic domain sequence; in certain embodiments, any substitutions are in the enzymatic active site while in other embodiments any substitutions are outside the enzymatic active site, while in other embodiments, the substitution may be in both enzymatic active site and non- enzymatic active site sequences.
  • a variant comprising one or more amino acid substitutions e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions, e.g., about: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20%, or more, divergence from the proteolytic domain
  • Substitutions in some embodiments may be non-conservative, conservative, highly conservative, or a combination thereof (e.g., as determined according to BLOSSUM62), e.g., conservative or highly conservative substitutions in the enzymatic active site (while in some embodiments, any substitutions are outside of the enzymatic active site) and non-conservative, conservative, highly conservative, or a combination thereof in non- enzymatic active site residues.
  • the proteolytic domain may comprise MMP2 or a catalytic site thereof, e.g., as illustrated by the sequences and annotated catalytic sites given in reference sequence NP_004521 .1 ; may comprise ADAM9 or a catalytic site thereof, e.g., as illustrated by the sequences and annotated catalytic sites given in reference sequence NP_003807.1 ; or may comprise PCSK1 or a catalytic site thereof, e.g., as illustrated by the sequences and annotated catalytic sites given in reference sequence NP_000430.3.
  • the proteolytic domain is a sheddase domain. In some embodiments, the proteolytic domain is derived from a naturally occurring protease with a primary biological function of cleaving a bond in the ectodomain of a membrane-associated polypeptide. In some embodiments, the proteolytic domain is derived from a naturally occurring soluble protease.
  • the proteolytic domain is derived from a naturally occurring soluble protease having a biological function of cleaving a bond in the ectodomain of a membrane-associated polypeptide, non- limiting examples of which include MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, and MMP26.
  • the proteolytic domain has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the proteolytic domain of human a disintegrin and metalleoproteinase domain-containing protein 8 (ADAM8), human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human p- site amyloid precursor protein cleaving enzyme 1 (BACE1 ), human BACE2, human site-1 protease, human rhomboid like 1 (RHBDL1 ), human RHBDL2, human RHBDL3, or human RHBDL4.
  • ADAM8 disintegrin and metalleoproteinase domain-containing protein 8
  • BACE1 human BACE2
  • the proteolytic domain has at least 86% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain has at least 87% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain has at least 88% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain has at least 89% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain has at least 90% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain has at least 91% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain has at least 92% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain has at least 93% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain has at least 94% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain has at least 95% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain has at least 96% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain has at least 97% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain has at least 98% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain has at least 99% sequence identity to the proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain is a proteolytic domain of human ADAM8, human ADAM9, human ADAM10, human ADAM12, human ADAM15, human ADAM17, human ADAM19, human ADAM20, human ADAM21 , human ADAM28, human ADAM30, human ADAM33, human BACE1 , human BACE2, human site-1 protease, human RHBDL1 , human RHBDL2, human RHBDL3, or human RHBDL4.
  • the proteolytic domain is derived from a serine protease.
  • the proteolytic domain has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the proteolytic domain of human proprotein convertase subtilisin/kexin type 1 (PCSK1 ), human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human paired amino acids converting enzyme 4 (PACE4), human PCSK7, or human PCSK9.
  • the proteolytic domain has at least 86% sequence identity to the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9. In some embodiments, the proteolytic domain has at least 87% sequence identity to the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9. In some embodiments, the proteolytic domain has at least 88% sequence identity to the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9.
  • the proteolytic domain has at least 89% sequence identity to the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9. In some embodiments, the proteolytic domain has at least 90% sequence identity to the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9. In some embodiments, the proteolytic domain has at least 91% sequence identity to the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9.
  • the proteolytic domain has at least 92% sequence identity to the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9. In some embodiments, the proteolytic domain has at least 93% sequence identity to the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9. In some embodiments, the proteolytic domain has at least 94% sequence identity to the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9.
  • the proteolytic domain has at least 95% sequence identity to the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9. In some embodiments, the proteolytic domain has at least 96% sequence identity to the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9. In some embodiments, the proteolytic domain has at least 97% sequence identity to the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9.
  • the proteolytic domain has at least 98% sequence identity to the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9. In some embodiments, the proteolytic domain has at least 99% sequence identity to the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9. In some embodiments, the proteolytic domain is the proteolytic domain of human PCSK1 , human PCSK3, human PCSK2, human furin, human PCSK5, human PCSK6, human PACE4, human PCSK7, or human PCSK9.
  • the proteolytic domain is derived from a non-serine protease. In some embodiments, the proteolytic domain has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the proteolytic domain of cathepsin S, cathepsin L, corin, differentially expressed in squamous cell carcinoma gene 1 (DESC1 ), enteropeptidase, histone acetyltransferase (HAT), HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, matrix metalloproteinase 1 (MMP1 ), MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13
  • the proteolytic domain has at least 86% sequence identity to the proteolytic domain of cathepsin S, cathepsin L, corin, DESC1 , enteropeptidase, HAT, HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MSPL, MT1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , prese
  • the proteolytic domain has at least 87% sequence identity to the proteolytic domain of cathepsin S, cathepsin L, corin, DESC1 , enteropeptidase, HAT, HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MSPL, MT1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , prese
  • the proteolytic domain has at least 88% sequence identity to the proteolytic domain of cathepsin S, cathepsin L, corin, DESC1 , enteropeptidase, HAT, HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MSPL, MT1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , prese
  • the proteolytic domain has at least 89% sequence identity to the proteolytic domain of cathepsin S, cathepsin L, corin, DESC1 , enteropeptidase, HAT, HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MSPL, MT1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , prese
  • the proteolytic domain has at least 90% sequence identity to the proteolytic domain of cathepsin S, cathepsin L, corin, DESC1 , enteropeptidase, HAT, HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MSPL, MT1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , presen
  • the proteolytic domain has at least 91% sequence identity to the proteolytic domain of cathepsin S, cathepsin L, corin, DESC1 , enteropeptidase, HAT, HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MSPL, MT1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , prese
  • the proteolytic domain has at least 92% sequence identity to the proteolytic domain of cathepsin S, cathepsin L, corin, DESC1 , enteropeptidase, HAT, HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MSPL, MT1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , prese
  • the proteolytic domain has at least 93% sequence identity to the proteolytic domain of cathepsin S, cathepsin L, corin, DESC1 , enteropeptidase, HAT, HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MSPL, MT1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , prese
  • the proteolytic domain has at least 94% sequence identity to the proteolytic domain of cathepsin S, cathepsin L, corin, DESC1 , enteropeptidase, HAT, HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MSPL, MT1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , prese
  • the proteolytic domain has at least 95% sequence identity to the proteolytic domain of cathepsin S, cathepsin L, corin, DESC1 , enteropeptidase, HAT, HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MSPL, MT1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , prese
  • the proteolytic domain has at least 96% sequence identity to the proteolytic domain of cathepsin S, cathepsin L, corin, DESC1 , enteropeptidase, HAT, HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MSPL, MT1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , prese
  • the proteolytic domain has at least 97% sequence identity to the proteolytic domain of cathepsin S, cathepsin L, corin, DESC1 , enteropeptidase, HAT, HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MSPL, MT1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , prese
  • the proteolytic domain has at least 98% sequence identity to the proteolytic domain of cathepsin S, cathepsin L, corin, DESC1 , enteropeptidase, HAT, HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MSPL, MT1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , prese
  • the proteolytic domain has at least 99% sequence identity to the proteolytic domain of cathepsin S, cathepsin L, corin, DESC1 , enteropeptidase, HAT, HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MSPL, MT1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , prese
  • the proteolytic domain is the proteolytic domain of cathepsin S, cathepsin L, corin, DESC1 , enteropeptidase, HAT, HAT-like 4, HAT-like 5, hepsin, legumain, matriptase, matriptase-2, matriptase-3, meprin p, MMP1 , MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11 , MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, MSPL, MT1 -MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , presenilin-1 , prese
  • the proteolytic domain is derived from a serine or cysteine protease domain. In some embodiments, the proteolytic domain has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • trypsin 1 trypsin 2
  • caspase-3 caspase-4
  • caspase-5 caspase-6
  • caspase-7 caspase-8
  • caspase-9
  • the proteolytic domain has at least 86% sequence identity to the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • the proteolytic domain has at least 87% sequence identity to the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • the proteolytic domain has at least 88% sequence identity to the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • the proteolytic domain has at least 89% sequence identity to the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • the proteolytic domain has at least 90% sequence identity to the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • the proteolytic domain has at least 91% sequence identity to the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • the proteolytic domain has at least 92% sequence identity to the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • the proteolytic domain has at least 93% sequence identity to the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • the proteolytic domain has at least 94% sequence identity to the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • the proteolytic domain has at least 95% sequence identity to the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • the proteolytic domain has at least 96% sequence identity to the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • the proteolytic domain has at least 97% sequence identity to the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • the proteolytic domain has at least 98% sequence identity to the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • the proteolytic domain has at least 99% sequence identity to the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • the proteolytic domain is the proteolytic domain of trypsin 1 , trypsin 2, chymotrypsin, mesotrypsin, granzyme B, caspase-1 , caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11 , caspase-12, caspase-13, or elastase.
  • the proteolytic domain upon binding of the targeting domain to a target molecule (e.g., target polypeptide), cleaves a peptide bond in a substrate peptide.
  • a substrate may be essentially any peptide capable of being cleaved by a contemplated bifunctional macromolecule.
  • the target polypeptide and the substrate polypeptide are the same polypeptide.
  • the target polypeptide and the substrate polypeptide are not the same polypeptide.
  • the substrate polypeptide is heterologous to the proteolytic domain.
  • the substrate polypeptide is soluble.
  • the substrate polypeptide is membrane-embedded.
  • the substrate polypeptide is membrane-associated.
  • the membrane-associated polypeptide is an ion channel, an aquaporin, a protein transporter, a glucose transporter, or a membrane-associated receptor.
  • the membrane-associated polypeptide is an ion channel.
  • the membrane-associated polypeptide is an aquaporin.
  • the membrane-associated polypeptide is a protein transporter.
  • the membrane-associated polypeptide is a glucose transporter.
  • the membrane-associated polypeptide is a membrane-associated receptor.
  • the substrate polypeptide may be an extracellular substrate.
  • the substrate polypeptide may be an intracellular substrate.
  • the substrate polypeptide is a membrane protein.
  • membrane proteins include CD1 , CD3D, CD3E, CD274, CTLA4 , ERBB2, SLC5A8, CCR5, EPCAM.
  • the substrate polypeptide is a single-pass membrane protein, such as membrane-bound tumor necrosis factor alpha (TNFa) or transforming growth factor beta (TGF-p).
  • the substrate polypeptide is a multi-pass membrane protein, such as proteins with two or more transmembrane domains.
  • the substrate polypeptide is a membrane protein having multiple complexes.
  • the membrane protein having multiple complexes is the sodium transporter Na v 1 .7.
  • the substrate polypeptide is a facilitative transporter.
  • the facilitative transporter is selected from the major facilitative superfamily (MFS) of solute carriers.
  • the facilitative transporter is cystine/glutamate antiporter xCT.
  • the substrate polypeptide is a membrane receptor.
  • the membrane receptor is selected from epidermal growth factor receptor (EGFR), IL-2Ry, IL-4R, IL-23R, programmed cell death protein 1 (PD-1 ), and programmed cell death protein ligand 1 (PD-L1 ).
  • the substrate polypeptide is a membrane ligand.
  • the membrane ligand is PD-L1 , transforming growth factor alpha (TGFa), p-selectin glycoprotein ligand-1 .
  • the substrate polypeptide is an ATP-binding cassette (ABC) transporter.
  • the ABC transporter is cystic fibrosis transmembrane conductance regulator (CFTR).
  • the substrate polypeptide is an ATPase.
  • the ATPase is HK-ATPase.
  • the substrate polypeptide is a co-stimulatory molecule.
  • the substrate polypeptide is a co-inhibitory molecule.
  • the extracellular substrate is a cytokine.
  • the cytokine is TGFp, TNFa, interleukin 1 (IL-1 ), IL-1 p, IL-2, IL-4, IL-6, IL-13, interferon alpha-1 IFNA1 , or IFN-y.
  • the extracellular substrate is a cytokine fragment, such as IL-12p or IL- 230.
  • the extracellular substrate is a chemokine.
  • the chemokine is CXC motif chemokine ligand 8 (CXCL-8), CC motif chemokine ligand 2 (CCL2), CCL3, CCL4, CCL5, CCL11 , or CXCL10).
  • the extracellular substrate is a blood protein, such as Von Willebrand factor (VWF).
  • VWF Von Willebrand factor
  • the extracellular substrate is a protein precursor such as a pro-protein or pro-peptide.
  • the protein precursor is pro-insulin or pro-thrombin.
  • the extracellular substrate is an extracellular matrix protein.
  • the extracellular matrix protein is collage, fibronectin, elastin, integrin, or vitronectin.
  • the extracellular substrate is a protein aggregate.
  • the protein aggregate is composed of beta-amyloid, Z-alpha-1 antitrypsin, alpha- synuclein, FUS RNA binding proten (FUS), TAR DNA-binding protein 43 (TDP-43), superoxide dismutase 1 (SOD-1 ), or huntingtin (HTT).
  • the extracellular substrate is a protease.
  • the protease is alpha-1 -antitrypsin or elastase.
  • the extracellular substrate is a pathogen-associated protein.
  • the pathogen-associated protein is a protein associated with viral pathogenesis, such as spike protein or hemagglutinin. In some embodiments, the pathogen-associated protein is associated with bacterial, fungal, or parasite pathogenesis and immune activation, such as lipopolysaccharide.
  • the extracellular substrate is an immunoglobulin, such as an autoreactive antibody. In some embodiments, the extracellular substrate is a protein associated with cholesterol particles, such as an apoliprotein.
  • the extracellular substrate is a prion or prion-like protein, such as scrapie-associated prion protein (PrPSc), mitochondrial antiviral signaling protein (MAVS), receptor interacting protein 1 (RIP1 ), or RIP3.
  • the extracellular substrate is a hormone.
  • the hormone is insulin, testosterone, or progesterone.
  • the extracellular substrate is an extracellular nucleic acid.
  • a non-limiting example of an intracellular substrate is a histone protein.
  • the bifunctional protease comprises a proteolytic domain that is capable of degrading amyloid beta (Ap).
  • Ap amyloid beta
  • the proteolytic domain is MMP2, MMP9, MMP14, Cathepsin-B (CTSB), Cathepsin-D (CTSD), Kallikrein 7 (KLK7), neprilysin (NEP), or insulin-degrading enzyme (IDE) (e.g., human MMP2, MMP9, MMP14, CTSB, CTSD, KLK7, NEP, or IDE), or a fragment or derivative (e.g., functionally active fragment or derivative) thereof.
  • CTSB Cathepsin-B
  • CSD Cathepsin-D
  • KLK7 Kallikrein 7
  • NEP insulin-degrading enzyme
  • IDE insulin-degrading enzyme
  • the bifunctional protease further comprises a targeting domain with affinity for amyloid beta.
  • a targeting domain with affinity for amyloid beta is provided in Table 2, below.
  • the targeting domain may comprise aducanumab, crenezumab, gantenerumab, bapineuzumab (3D6), solanezumab, ponezumab, lecanemab, or a fragment or derivative thereof.
  • the bifunctional protease degrades amyloid beta monomers, oligomers, fibrils, and/or plaques (e.g., is capable of active aggregate dissolution of beta amyloid fibrils).
  • the bifunctional protease may degrade amyloid beta monomers, oligomers, fibrils, and/or plaques at an increased rate and/or for an increased duration relative to a control molecule, e.g., a protease not comprising a targeting domain.
  • Linkers provide space, rigidity, and/or flexibility between two or more components of a bifunctional molecule of the disclosure.
  • Proteolytic domains and targeting domains of the disclosure may be joined by a linker.
  • the linker includes any peptide linker that includes two or more amino acid residues.
  • a peptide linker may include 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more amino acid residues, which are joined, for example by peptide bonds.
  • the carboxy terminus of a peptide linker may be covalently conjugated (e.g., by a peptide bond) to a first domain and the amino terminus of the peptide linker may be covalently conjugated (e.g., by a peptide bond) to a second domain, thereby conjugating the first moiety and the second moiety and allowing for space and/or flexibility between the first domain and the second domain.
  • a peptide linker may be expressed from a polynucleotide construct or chemically synthesized and subsequently chemically conjugated to a first domain and a second domain. Alternately, a peptide linker may be expressed in tandem with a first polypeptide and a second polypeptide, thereby joining the first polypeptide and the second polypeptide to form a fusion protein.
  • a linker can be a simple covalent bond, e.g., a peptide bond, a synthetic polymer, e.g., a polyethylene glycol (PEG) polymer, or any kind of bond created from a chemical reaction, e.g., chemical conjugation.
  • a linker is a peptide bond
  • the carboxylic acid group at the C- terminus of one protein domain can react with the amino group at the N-terminus of another protein domain in a condensation reaction to form a peptide bond.
  • the peptide bond can be formed from synthetic means through a conventional organic chemistry reaction well-known in the art, or by natural production from a host cell, wherein a polynucleotide sequence encoding the DNA sequences of both proteins, e.g., in tandem series can be directly transcribed and translated into a contiguous polypeptide encoding both proteins by the necessary molecular machineries, e.g., DNA polymerase and ribosome, in the host cell.
  • a polynucleotide sequence encoding the DNA sequences of both proteins e.g., in tandem series can be directly transcribed and translated into a contiguous polypeptide encoding both proteins by the necessary molecular machineries, e.g., DNA polymerase and ribosome, in the host cell.
  • a linker is a synthetic polymer, e.g., a PEG polymer
  • the polymer can be functionalized with reactive chemical functional groups at each end to react with the terminal amino acids at the connecting ends of two proteins.
  • a linker is synthesized by means of a chemical reaction
  • chemical functional groups e.g., amines, carboxylic acids, esters, azides, or other functional groups commonly used in the art
  • the two functional groups can then react to through synthetic chemistry means to form a chemical bond, thus connecting the two proteins together.
  • Such chemical conjugation procedures are routine for those skilled in the art.
  • a linker between a first moiety (e.g., a targeting domain) and a second moiety (e.g., a proteolytic domain) can be a polypeptide including 2-200 amino acids (e.g., 4- 175, 5-150, 10-125, 20-100, 25-75, or 50 amino acids).
  • a linker is a polypeptide containing at least 12 amino acids, such as 12-200 amino acids (e.g., 13-175, 14-150, 15- 125, 20-100, 25-75, or 50 amino acids).
  • a linker is a polypeptide containing 12-50 amino acids (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids).
  • Suitable peptide linkers are known in the art, and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine.
  • a linker can contain motifs, e.g., multiple or repeating motifs, of GS, GGS, GGGGS (SEQ ID NO: 52), GGSG, or SGGG.
  • a peptide linker (e.g., Li and L2) is a peptide linker including the amino acid sequence of any one of (GS)x, (GGS)x, (GGGGS)x (SEQ ID NO: 52)x, (GGSG)x, (SGGG)x, wherein x is an integer from 1 to 50 (e.g., 1 -40, 1 -30, 1 -20, 1 -10, or 1 -5).
  • the peptide linker is GGGGSGGGGS (SEQ ID NO: 53), GGGGSGGGGSGGGGS (SEQ ID NO: 54); or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 55).
  • a linker can contain 2 to 12 amino acids including motifs of GS, e.g., GS, GSGS, GSGSGS (SEQ ID NO: 56), GSGSGSGS (SEQ ID NO: 57), GSGSGSGSGS (SEQ ID NO: 58), or GSGSGSGSGSGSGS (SEQ ID NO: 59).
  • a linker can contain 3 to 12 amino acids including motifs of GGS, e.g., GGS, GGSGGS (SEQ ID NO: 60), GGSGGSGGS (SEQ ID NO: 61 ), and GGSGGSGGSGGS (SEQ ID NO: 62).
  • a linker can contain 4 to 20 amino acids including motifs of GGSG, e.g., GGSGGGSG (SEQ ID NO: 63), GGSGGGSGGGSG (SEQ ID NO: 64), GGSGGGSGGGSGGGSG (SEQ ID NO: 65), or GGSGGGSGGGSGGGSG (SEQ ID NO: 66).
  • a linker can contain motifs of GGGGS (SEQ ID NO: 52), e.g., GGGGSGGGGS (SEQ ID NO: 53) or GGGGSGGGGSGGGGS (SEQ ID NO: 54).
  • a linker is SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 67).
  • a peptide linker contains only glycine residues, e.g., at least 4 glycine residues (e.g., 4-200, 4-180, 4-160, 4-140, 4-120, 4-100, 4-90, 4-80, 4-70, 4-60, 4-50, 4-40, 4-30, 4- 20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11 , 4-10, 4-9, 4-8, 4-7, 4-6 or 4-5 glycine residues) (e.g., 4-200, 6-200, 8-200, 10-200, 12-200, 14-200, 16-200, 18-200, 20-200, 30-200, 40- 200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200, or 190- 200 glycine residues) (e.g., at least 5, 10, 25, 50, 100, 150, or 200 glycine residues) (
  • a linker has 4-30 glycine residues (e.g., 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 glycine residues).
  • a linker containing only glycine residues may not be glycosylated (e.g., O-linked glycosylation, also referred to as O-glycosylation) or may have a decreased level of glycosylation (e.g., a decreased level of O- glycosylation) (e.g., a decreased level of O-glycosylation with glycans such as xylose, mannose, sialic acids, fucose (Fuc), and/or galactose (Gal) (e.g., xylose)) as compared to, e.g., a linker containing one or more serine residues (e.g., SGGGSGGGSGGGSGGG
  • a linker containing only glycine residues may not be O-glycosylated (e.g., O-xylosylation) or may have a decreased level of O-glycosylation (e.g., a decreased level of O- xylosylation) as compared to, e.g., a linker containing one or more serine residues (e.g., SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 67)).
  • a linker containing only glycine residues may not undergo proteolysis or may have a decreased rate of proteolysis as compared to, e.g., a linker containing one or more serine residues (e.g., SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 67)).
  • a linker can contain motifs of GGGG, e.g., GGGGGGGG (SEQ ID NO: 68), GGGGGGGGGGGG (SEQ ID NO: 69), GGGGGGGGGGGGGG (SEQ ID NO: 70), or GGGGGGGGGGGGGGGGGG (SEQ ID NO: 71 ).
  • a linker can contain motifs of GGGGG (SEQ ID NO: 72), e.g., GGGGGGGGGG (SEQ ID NO: 73), or GGGGGGGGGGGGGGG (SEQ ID NO: 74).
  • a linker is GGGGGGGGGGGGGGGGGG (SEQ ID NO: 71).
  • a linker can also contain amino acids other than glycine and serine, e.g., GENLYFQSGG (SEQ ID NO: 75), SACYCELS (SEQ ID NO: 76), RSIAT (SEQ ID NO: 77), RPACKIPNDLKQKVMNH (SEQ ID NO: 78), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 79), AAANSSIDLISVPVDSR (SEQ ID NO: 80), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGSGGGS (SEQ ID NO: 81).
  • GENLYFQSGG SEQ ID NO: 75
  • SACYCELS SEQ ID NO: 76
  • RSIAT SEQ ID NO: 77
  • RPACKIPNDLKQKVMNH SEQ ID NO: 78
  • GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG SEQ ID NO: 79
  • a 12- or 20-amino acid peptide linker is used to connect a targeting domain and a proteolytic domain in tandem series, the 12- and 20-amino acid peptide linkers consisting of sequences GGGSGGGSGGGS (SEQ ID NO: 82) and SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 67), respectively.
  • an 18- amino acid peptide linker consisting of sequence GGSGGGSGGGSGGGSGGS (SEQ ID NO: 83) may be used.
  • a linker provides space, rigidity, and/or flexibility between two or more components of the fusion protein or conjugate.
  • a linker may be a bond, e.g., a covalent bond, e.g., an amide bond, a disulfide bond, a C-0 bond, a C-N bond, a N-N bond, a C-S bond, or any kind of bond created from a chemical reaction, e.g., chemical conjugation.
  • a linker includes no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1- 16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1- 240, or 1-250 atom(s);250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom(s)).
  • a linker includes no more than 250 nonhydrogen atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1- 45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1- 150, 1 -160, 1 -170, 1 -180, 1 -190, 1 -200, 1 -210, 1 -220, 1 -230, 1 -240, or 1 -250 non-hydrogen atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8,
  • the backbone of a linker includes no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1- 60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom(s)).
  • the “backbone” of a linker refers to the atoms in the linker that together form the shortest path from one part of the conjugate to another part of the conjugate.
  • the atoms in the backbone of the linker are directly involved in linking one part of the conjugate to another part of the conjugate.
  • hydrogen atoms attached to carbons in the backbone of the linker are not considered as directly involved in linking one part of the conjugate to another part of the conjugate.
  • Molecules that may be used to make linkers include at least two functional groups, e.g., two carboxylic acid groups.
  • the divalent linker may contain two carboxylic acids, in which the first carboxylic acid may form a covalent linkage with one component in the conjugate and the second carboxylic acid may form a covalent linkage (e.g., a C-S bond or a C-N bond) with another component in the conjugate.
  • dicarboxylic acid molecules may be used as linkers (e.g., a dicarboxylic acid linker).
  • linkers e.g., a dicarboxylic acid linker.
  • Examples of dicarboxylic acids molecules that may be used to form linkers include, but are not limited to,
  • n is an integer from 1 to 20 (e.g., n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18,
  • dicarboxylic acid molecules such as the ones described herein, may be further functionalized to contain one or more additional functional groups.
  • the linking group may include a moiety including a carboxylic acid moiety and an amino moiety that are spaced by from 1 to 25 atoms. Examples of such linking groups include, but are not limited to, wherein n is an integer from 1 to 20 (e.g., n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20).
  • a linking group may include a moiety including a carboxylic acid moiety and an amino moiety, such as the ones described herein, may be further functionalized to contain one or more additional functional groups. Such linking groups may be further functionalized, for example, to provide an attachment point to a first moiety or a second moiety.
  • the linking group may include a moiety including two or amino moieties (e.g., a diamino moiety) that are spaced by from 1 to 25 atoms.
  • Examples of such linking groups include, but are not limited to,
  • n is an integer from 1 to 20 (e.g., n is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20).
  • a linking group may include a diamino moiety, such as the ones described herein, may be further functionalized to contain one or more additional functional groups. Such diamino linking groups may be further functionalized, for example, to provide an attachment point to a first moiety or a second moiety.
  • a molecule containing an azide group may be used to form a linker, in which the azide group may undergo cycloaddition with an alkyne to form a 1 ,2,3-triazole linkage.
  • a molecule containing an alkyne group may be used to form a linker, in which the alkyne group may undergo cycloaddition with an azide to form a 1 ,2,3-triazole linkage.
  • a molecule containing a maleimide group may be used to form a linker, in which the maleimide group may react with a cysteine to form a C-S linkage.
  • a molecule containing one or more haloalkyl groups may be used to form a linker, in which the haloalkyl group may form a covalent linkage, e.g., C-N and C-0 linkage.
  • a linker may include a synthetic group derived from, e.g., a synthetic polymer (e.g., a PEG polymer).
  • a linker may include one or more amino acid residues.
  • a linker may be an amino acid sequence (e.g., a 1 -25 amino acid, 1 -10 amino acid, 1 -9 amino acid, 1 -8 amino acid, 1 -7 amino acid, 1 -6 amino acid, 1 -5 amino acid, 1 -4 amino acid, 1 -3 amino acid, 1 -2 amino acid, or 1 amino acid sequence).
  • a linker may include one or more optionally substituted C1 -C20 alkylene, optionally substituted C1 -C20 heteroalkylene (e.g., a PEG unit), optionally substituted C2-C20 alkenylene (e.g., C2 alkenylene), optionally substituted C2-C20 heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20 cycloalkylene (e.g., cyclopropylene, cyclobutylene), optionally substituted C2-C20 heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C6-
  • the targeting domain and proteolytic domain may be covalently attached or associated by non-covalent means.
  • the moieties can be covalently attached as by fusion of two protein domains, with or without intervening sequences, to form a single polypeptide chain, or through derivation of the amino or carboxy terminus, or a sidechain of a polypeptide chain.
  • the targeting moiety and catalytic domain are produced as a cotranslational fusion by expression of a single recombinant nucleic acid construct.
  • the various moieties may also be associated by non-covalent interactions, such as between protein domains or by way of interaction with a common cross-linking ligand.
  • the proteolytic domain and the targeting domain are associated with each other by non-covalent interactions (e.g., hydrogen bonding interactions, charge-charge interactions, hydrophobic-hydrophobic interactions, or dispersion forces).
  • the proteolytic domain and the targeting domain are associated with each other by hydrogen bonding interactions.
  • the proteolytic domain and the targeting domain are associated with each other by charge-charge interactions.
  • the proteolytic domain and the targeting domain are associated with each other by hydrophobic- hydrophobic interactions.
  • the proteolytic domain and the targeting domain are associated with each other by dispersion forces.
  • Covalent conjugation of two or more components in a conjugate using a linker may be accomplished using well-known organic chemical synthesis techniques and methods.
  • Complementary functional groups on two components may react with each other to form a covalent bond.
  • Examples of complementary reactive functional groups include, but are not limited to, e.g., maleimide and cysteine, amine and activated carboxylic acid, thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and alkene and tetrazine.
  • Site-specific conjugation to a polypeptide may accomplished using techniques known in the art. Exemplary techniques for site-specific conjugation are provided in Agarwall. P., et al. Bioconjugate Chem. 26:176-192 (2015).
  • amino-reactive acylating groups include, e.g., (i) an isocyanate and an isothiocyanate; (ii) a sulfonyl chloride; (iii) an acid halide; (iv) an active ester, e.g., a nitrophenylester or N-hydroxysuccinimidyl ester; (v) an acid anhydride, e.g., a mixed, symmetrical, or N- carboxyanhydride; (vi) an acylazide; and (vii) an imidoester. Aldehydes and ketones may be reacted with amines to form Schiff’s bases, which may be stabilized through reductive amination.
  • a linker of the disclosure is conjugated (e.g., by any of the methods described herein) to a fusion protein.
  • a linker of the disclosure comprises a pair of Fc regions (e.g., comprises two Fc domain monomers with compatible dimerization selectivity modules, e.g., one CH3 antibody constant domain containing an engineered cavity and the other CH3 antibody constant domain containing an engineered protuberance, combine to form a protuberance-into-cavity pair of Fc domain monomers).
  • Fc domains comprising engineered protuberances and engineered cavities are described in further detail below.
  • a pair of Fc regions is used to link a targeting domain to a proteolytic domain.
  • a pair of Fc regions is used to link two or more moieties comprising both a targeting domain and a proteolytic domain.
  • a dimerization selectivity module is the part of the Fc domain monomer that facilitates the preferred pairing of two Fc domain monomers to form an Fc domain.
  • a dimerization selectivity module is that part of the CH3 antibody constant domain of an Fc domain monomer which includes amino acid substitutions positioned at the interface between interacting CH3 antibody constant domains of two Fc domain monomers.
  • the amino acid substitutions make favorable the dimerization of the two CH3 antibody constant domains as a result of the compatibility of amino acids chosen for those substitutions.
  • the ultimate formation of the favored Fc domain is selective over other Fc domains which form from Fc domain monomers lacking dimerization selectivity modules or with incompatible amino acid substitutions in the dimerization selectivity modules.
  • This type of amino acid substitution can be made using conventional molecular cloning techniques well-known in the art, such as QuikChange® mutagenesis.
  • a dimerization selectivity module includes an engineered cavity (described further herein) in the CH3 antibody constant domain. In other embodiments, a dimerization selectivity module includes an engineered protuberance (described further herein) in the CH3 antibody constant domain.
  • two Fc domain monomers with compatible dimerization selectivity modules e.g., one CH3 antibody constant domain containing an engineered cavity and the other CH3 antibody constant domain containing an engineered protuberance, combine to form a protuberance-into-cavity pair of Fc domain monomers.
  • Engineered protuberances and engineered cavities are examples of heterodimerizing selectivity modules, which can be made in the CH3 antibody constant domains of Fc domain monomers in order to promote favorable heterodimerization of two Fc domain monomers that have compatible heterodimerizing selectivity modules.
  • an Fc domain monomer with a dimerization selectivity module containing positively-charged amino acid substitutions and an Fc domain monomer with a dimerization selectivity module containing negatively-charged amino acid substitutions may selectively combine to form an Fc domain through the favorable electrostatic steering (described further herein) of the charged amino acids.
  • an Fc domain monomer may include one of the following positively-charged and negatively-charged amino acid substitutions: K392D, K392E, D399K, K409D, K409E, K439D, and K439E.
  • an Fc domain monomer containing a positively-charged amino acid substitution e.g., D356K or E357K
  • an Fc domain monomer containing a negatively-charged amino acid substitution e.g., K370D or K370E
  • an Fc domain monomer containing E357K and an Fc domain monomer containing K370D may selectively combine to form an Fc domain through favorable electrostatic steering of the charged amino acids.
  • reverse charge amino acid substitutions may be used as heterodimerizing selectivity modules, wherein two Fc domain monomers containing different, but compatible, reverse charge amino acid substitutions combine to form a heterodimeric Fc domain.
  • An unmodified Fc domain monomer can be a naturally occurring human Fc domain monomer or a wild-type (WT) human Fc domain monomer.
  • An Fc domain monomer can be a naturally occurring human Fc domain monomer comprising a hinge, a CH2 domain, and a CH3 domain; or a variant thereof having up to 16 (e.g., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16) amino acid modifications (e.g., single amino acid modifications) to accommodate or promote directed dimerization.
  • the Fc domain includes at least one amino acid modification, wherein the amino acid modifications alter one or more of (i) binding affinity to one or more Fc receptors, (ii) effector functions, (iii) the level of Fc domain sulfation, (iv) half-life, (v) protease resistance, (vi) Fc domain stability, and/or (vii) susceptibility to degradation (e.g., when compared to the unmodified Fc domain).
  • an Fc domain monomer containing (i) at least one reverse charge mutation and (ii) at least one engineered cavity or at least one engineered protuberance may selectively combine with another Fc domain monomer containing (i) at least one reverse charge mutation and (ii) at least one engineered protuberance or at least one engineered cavity to form an Fc domain.
  • an Fc domain monomer containing reversed charge mutation K370D and engineered cavities Y349C, T366S, L368A, and Y407V and another Fc domain monomer containing reversed charge mutation E357K and engineered protuberances S354C and T366W may selectively combine to form an Fc domain.
  • the Fc domain includes no more than 16 amino acid modifications (e.g., no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16 amino acid modifications in the CH3 domain).
  • the formation of such Fc domains is promoted by the compatible amino acid substitutions in the CH3 antibody constant domains.
  • Two dimerization selectivity modules containing incompatible amino acid substitutions e.g., both containing engineered cavities, both containing engineered protuberances, or both containing the same charged amino acids at the CH3-CH3 interface, will not promote the formation of a heterodimeric Fc domain.
  • Fc domains with defined Fc domain monomers include, without limitation, the LUZ-Y approach (U.S. Patent Application Publication No. WO2011034605) which includes C-terminal fusion of a monomer a-helices of a leucine zipper to each of the Fc domain monomers to allow heterodimer formation, as well as strandexchange engineered domain (SEED) body approach (Davis et al., Protein Eng Des Sei. 23:195-202, 2010) that generates Fc domains with heterodimeric Fc domain monomers, each including alternating segments of IgA and IgG CH3 sequences.
  • SEED strandexchange engineered domain
  • engineered cavities and engineered protuberances are used in the preparation of the Fc constructs described herein.
  • An engineered cavity is a void that is created when an original amino acid in a protein is replaced with a different amino acid having a smaller side-chain volume.
  • An engineered protuberance is a bump that is created when an original amino acid in a protein is replaced with a different amino acid having a larger side-chain volume.
  • the amino acid being replaced is in the CH3 antibody constant domain of an Fc domain monomer and is involved in the dimerization of two Fc domain monomers.
  • an engineered cavity in one CH3 antibody constant domain is created to accommodate an engineered protuberance in another CH3 antibody constant domain, such that both CH3 antibody constant domains act as dimerization selectivity modules (e.g., heterodimerizing selectivity modules) (described above) that promote or favor the dimerization of the two Fc domain monomers.
  • an engineered cavity in one CH3 antibody constant domain is created to better accommodate an original amino acid in another CH3 antibody constant domain.
  • an engineered protuberance in one CH3 antibody constant domain is created to form additional interactions with original amino acids in another CH3 antibody constant domain.
  • An engineered cavity can be constructed by replacing amino acids containing larger side chains such as tyrosine or tryptophan with amino acids containing smaller side chains such as alanine, valine, or threonine.
  • some dimerization selectivity modules e.g., heterodimerizing selectivity modules
  • engineered cavities such as Y407V mutation in the CH3 antibody constant domain.
  • an engineered protuberance can be constructed by replacing amino acids containing smaller side chains with amino acids containing larger side chains.
  • some dimerization selectivity modules e.g., heterodimerizing selectivity modules
  • contain engineered protuberances such as T366W mutation in the CH3 antibody constant domain.
  • engineered cavities and engineered protuberances are also combined with inter-CH3 domain disulfide bond engineering to enhance heterodimer formation.
  • an Fc domain monomer containing engineered cavities Y349C, T366S, L368A, and Y407V may selectively combine with another Fc domain monomer containing engineered protuberances S354C and T366W to form an Fc domain.
  • an Fc domain monomer containing engineered cavity Y349C and an Fc domain monomer containing engineered protuberance S354C may selectively combine to form an Fc domain.
  • Other engineered cavities and engineered protuberances, in combination with either disulfide bond engineering or structural calculations (mixed HA-TF) are included, without limitation, in Table 3.
  • Replacing an original amino acid residue in the CH3 antibody constant domain with a different amino acid residue can be achieved by altering the nucleic acid encoding the original amino acid residue.
  • the upper limit for the number of original amino acid residues that can be replaced is the total number of residues in the interface of the CH3 antibody constant domains, given that sufficient interaction at the interface is still maintained.
  • Electrostatic steering is the utilization of favorable electrostatic interactions between oppositely charged amino acids in peptides, protein domains, and proteins to control the formation of higher ordered protein molecules.
  • a method of using electrostatic steering effects to alter the interaction of antibody domains to reduce for formation of homodimer in favor of heterodimer formation in the generation of bi-specific antibodies is disclosed in U.S. Patent Application Publication No. 2014-0024111.
  • electrostatic steering is used to control the dimerization of Fc domain monomers and the formation of Fc constructs.
  • one or more amino acid residues that make up the CH3-CH3 interface are replaced with positively- or negatively-charged amino acid residues such that the interaction becomes electrostatically favorable or unfavorable depending on the specific charged amino acids introduced.
  • a positively-charged amino acid in the interface such as lysine, arginine, or histidine, is replaced with a negatively-charged amino acid such as aspartic acid or glutamic acid.
  • a negatively-charged amino acid in the interface is replaced with a positively-charged amino acid.
  • the charged amino acids may be introduced to one of the interacting CH3 antibody constant domains, or both.
  • dimerization selectivity modules (described further above) are created that can selectively form dimers of Fc domain monomers as controlled by the electrostatic steering effects resulting from the interaction between charged amino acids.
  • the two Fc domain monomers may be selectively formed through heterodimerization or homodimerization.
  • an Fc domain monomer may include one of the following positively-charged and negatively-charged amino acid substitutions: D356K, D356R, E357K, E357R, K370D, K370E, K392D, K392E, D399K, K409D, K409E, K439D, and K439E.
  • an Fc domain monomer containing a positively-charged amino acid substitution e.g., D356K or E357K
  • an Fc domain monomer containing a negatively- charged amino acid substitution e.g., K370D or K370E
  • an Fc domain monomer containing E357K and an Fc domain monomer containing K370D may selectively combine to form an Fc domain through favorable electrostatic steering of the charged amino acids.
  • two of the three Fc domains may be formed by the heterodimerization of two Fc domain monomers, as promoted by the electrostatic steering effects.
  • heterodimeric Fc domain refers to an Fc domain that is formed by the heterodimerization of two Fc domain monomers, wherein the two Fc domain monomers contain different reverse charge mutations (heterodimerizing selectivity modules) that promote the favorable formation of these two Fc domain monomers.
  • Bifunctional proteases e.g., targeted sheddases
  • a bifunctional protease described herein can be prepared by recombinant expression of genes in a host cell.
  • a host cell can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the desired bifunctional protease, such that the bifunctional protease is expressed in the host cell and, optionally, secreted into the medium in which the host cells are cultured, from which medium the bifunctional proteases can be recovered.
  • Standard recombinant DNA methodologies are used to obtain bifunctional protease genes, incorporate these genes into recombinant expression vectors, and introduce the vectors into host cells, such as those described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989), Current Protocols in Molecular Biology (Ausubel et al., eds., Greene Publishing Associates, 1989), and in U.S. Patent No. 4,816,397; the disclosures of each of which are incorporated herein by reference.
  • Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into the genome of a cell (e.g., a eukaryotic or prokaryotic cell). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration.
  • viral vectors examples include a retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., Measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox).
  • adenovirus e.g., Ad5, Ad26,
  • viruses useful for delivering polynucleotides encoding bifunctional proteases described herein include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
  • murine leukemia viruses include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses.
  • vectors are described, for example, in McVey et al., (U.S. Patent. No. 5,801 ,030); the disclosures of each of which are incorporated herein by reference.
  • Non-viral vectors such as plasmids
  • plasmids include, but are not limited to prokaryotic and eukaryotic vectors (e.g., yeast- and bacteria-based plasmids), as well as plasmids for expression in mammalian cells.
  • prokaryotic and eukaryotic vectors e.g., yeast- and bacteria-based plasmids
  • plasmids for expression in mammalian cells e.g., yeast- and bacteria-based plasmids
  • Methods of introducing the vectors into a host cell and isolating and purifying the expressed protein are also well known in the art (e.g., Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989)).
  • bifunctional proteases e.g., targeted sheddases
  • expression of bifunctional proteases is performed in eukaryotic cells, e.g., mammalian host cells, for high secretion of a properly folded and immunologically active bifunctional proteases.
  • eukaryotic cells e.g., mammalian host cells
  • Exemplary mammalian host cells for expressing the recombinant bifunctional protease described herein include Chinese Hamster Ovary (CHO cells) (including DHFR CHO cells, described in Urlaub and Chasin (1980, Proc. Natl. Acad. Sci.
  • DHFR selectable marker e.g., as described in Kaufman and Sharp (1982, Mol. Biol. 159:601 -621 ), NSO myeloma cells, COS cells, 293 cells, and SP2/0 cells.
  • Additional cell types that may be useful for the expression of bifunctional proteases and fragments thereof include bacterial cells, such as BL-21 (DE3) E. Co// cells, which can be transformed with vectors containing foreign DNA according to established protocols.
  • Additional eukaryotic cells that may be useful for expression of bifunctional proteases include yeast cells, such as auxotrophic strains of S. cerevisiae, which can be transformed and selectively grown in incomplete media according to established procedures known in the art.
  • the bifunctional proteases are produced by culturing the host cells for a period of time sufficient to allow for expression of the bifunctional protease in the host cells or secretion of the bifunctional protease into the culture medium in which the host cells are grown.
  • Bifunctional proteases can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact bifunctional proteases. Also included herein are methods in which the above procedure is varied according to established protocols known in the art.
  • a bifunctional protease e.g., a sheddase
  • it can be purified by any method known in the art, such as a method useful for purification of a bifunctional protease molecule, for example, by chromatography, centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the bifunctional proteases described herein or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification or to produce therapeutic conjugates.
  • a bifunctional protease if desired, can be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques in Biochemistry and Molecular Biology (Work and Burdon, eds., Elsevier, 1980); incorporated herein by reference), or by gel filtration chromatography, such as on a SuperdexTM 75 column (Pharmacia Biotech AB, Uppsala, Sweden).
  • compositions of the disclosure can be administered not only as proteins but also in the form of nucleic acids.
  • This section provides exemplary nucleic acids that may be used to deliver proteins of the disclosure to a subject (e.g., a subject suffering from a disorder associated with protein dysfunction described herein).
  • These nucleic acids e.g., RNAs, such as mRNAs, circular RNAs, or self-amplifying RNAs
  • RNAs such as mRNAs, circular RNAs, or self-amplifying RNAs
  • RNAs such as mRNAs, circular RNAs, or self-amplifying RNAs
  • nucleic acid molecules of the disclosure may include one or more alterations.
  • a nucleotide, nucleoside, or polynucleotide such as the nucleic acids of the invention (e.g., an RNA or an oligonucleotide)
  • alteration or, as appropriate, “alternative” refer to alteration with respect to A, G, U or C ribonucleotides.
  • the alterations may be various distinct alterations.
  • the coding region, the flanking regions, and/or the terminal regions may contain one, two, or more (optionally different) nucleoside or nucleotide alterations.
  • an alternative polynucleotide introduced to a cell may exhibit reduced degradation in the cell, as compared to an unaltered polynucleotide.
  • the polynucleotides can include any useful alteration, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage, or to the phosphodiester backbone).
  • alterations e.g., one or more alterations are present in each of the sugar and the internucleoside linkage.
  • Alterations according to the present invention may be alterations of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs) (e.g., the substitution of the 2’OH of the ribofuranosyl ring to 2’H), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof. Additional alterations are described herein.
  • nucleic acid molecule introduced into the cell may be degraded intracellularly.
  • degradation of a nucleic acid molecule may be preferable if precise timing of protein production is desired.
  • the invention provides an alternative nucleic acid molecule containing a degradation domain, which is capable of being acted on in a directed manner within a cell.
  • the polynucleotides can optionally include other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers, vectors, etc.).
  • the polynucleotides may include one or more messenger RNAs (mRNAs) having one or more alternative nucleoside or nucleotides (i.e., mRNA molecules).
  • the polynucleotides may include one or more oligonucleotides having one or more alternative nucleoside or nucleotides.
  • a composition of the invention includes an mRNA and/or one or more oligonucleotides having one or more alternative nucleoside or nucleotides.
  • a patient e.g., a human patient
  • a human patient having a disorder associated with protein dysfunction e.g., a disease or disorder associated with the formation of amyloids, e.g., a disease or disorder associated with the formation of amyloid beta plaques
  • the patient is administered a bifunctional protease, e.g., a sheddase, protease, or amyloid-degrading enzyme (e.g., Ap-degrading enzymes) of the disclosure.
  • a bifunctional protease e.g., a sheddase, protease, or amyloid-degrading enzyme (e.g., Ap-degrading enzymes) of the disclosure.
  • compositions disclosed herein may be a pharmaceutical composition.
  • Pharmaceutical compositions can be prepared using methods known in the art.
  • Pharmaceutical compositions described herein may contain a bifunctional protease of the disclosure in combination with one or more pharmaceutically acceptable carriers, excipients, diluents, or stabilizers.
  • compositions described herein can be prepared using physiologically acceptable carriers, excipients, or stabilizers, and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions.
  • the compositions can also be prepared so as to contain the active agent (e.g., a bifunctional protease) at a desired concentration.
  • a pharmaceutical composition described herein may contain at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100%) active agent by weight (w/w).
  • compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the active agent having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers typically employed in the art, e.g., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives.
  • optional pharmaceutically acceptable carriers e.g., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives.
  • Such additives must be nontoxic to the recipients at the dosages and concentrations employed.
  • Pharmaceutically acceptable carriers that can be incorporated into a pharmaceutical composition described herein may include dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxyl benzoate, talc, magnesium stearate, and mineral oils.
  • a pharmaceutical composition described herein may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative.
  • Bifunctional proteases of the disclosure, and nucleic acids encoding the same can be administered to a subject (e.g., a human patient) by a variety of routes.
  • the bifunctional protease or nucleic acid is administered to the subject intravenously, subcutaneously, intramuscularly, parenterally, intrathecally, intracerebroventricularly, transdermally, or orally.
  • the most suitable route for administration in any given case will depend on the particular therapeutic agent administered, the patient, pharmaceutical formulation methods, and various patientspecific parameters, such as the patient’s age, body weight, sex, severity of the disease being treated, the patient’s diet, and the patient’s excretion rate.
  • This Example describes the construction and testing of a bifunctional molecule with targeted proteolytic activity against the protein target tumor necrosis factor alpha (TNFa).
  • the bifunctional molecule was generated by fusing an enzymatic domain from MMP7, attached by an amino acid linker, to a receptor ectodomain having affinity for TNFa (TNFR1 ).
  • the TNFa-degrading bifunctional molecule was composed of three connected components: a catalytic domain, a linker, and a binding domain.
  • the N-terminal catalytic domain was derived from the protease matrix metalloproteinase-7 (MMP7). This domain contains residues 1 -267 of human MMP7 (Uniprot: P09237), which includes a signal peptide, a propeptide domain, and a catalytic domain.
  • the catalytic domain was connected to the binding domain using an amino acid linker 6xGGGGS (6xG4S) (SEQ ID NO: 84).
  • the TNFa receptor 1 ectodomain subunit 2.6 (TNFR1 2.6) was used as the TNFa binding domain.
  • the bifunctional molecule was cloned into a pTwist plasmid backbone on a cytomegalovirus (CMV) constitutive promoter, with additional components including an HA and His tag, as well as a mCherry reporter downstream of a T2A ribosome skip site.
  • CMV cytomegalovirus
  • the bifunctional molecule was expressed by transfecting human embryonic kidney (HEK) 293 cells.
  • HEK 293 cells were plated at 2x10 5 cells per well in 6 well plates (2ml of medium per well) and incubated at 37°C/5% CO2 overnight. Immediately before transfection the medium was removed from the cells and replaced with 2 mL of fresh medium.
  • Transfection mixtures contained: 200 pL serum free medium, 4.5 pg DNA, 13.5 pL transfection reagent. Plates were incubated at 37°C/5% CO2 for 5 hours. Medium was removed and replaced with 2 mL of serum free medium and the plates incubated at 37°C/5% CO2.
  • the expression of constructs was measured by fluorescent microscopy for mCherry which is co-expressed by the plasmids.
  • the blot was then washed 3 x 10 minutes in TBST, incubated with ECL reagent, and images were captured on a SynGene G:Box.
  • the secretion of bifunctional constructs was confirmed with ELISA (against His tag) which was also used to quantify the amount of construct in the media.
  • Enzymatic activity of the supernatant containing the bifunctional molecules was confirmed using fluorogenic substrate Mca-KPLGL-Dpa-AR-NH2 with a plate reader delivering excitation at 320 nm and reading emission at 405 nm. Proteolytic activity was tested over time (10-30 minutes).
  • the bifunctional molecules were expressed by the HEK cells as measured by the presence of mCherry co-expression.
  • the western blots and ELISAs confirmed that the constructs were translated and secreted into the cell culture supernatants.
  • the fluorescence generated by the fluorogenic peptide confirmed that the secreted bifunctional molecules were enzymatically active.
  • a bifunctional molecule (SEQ ID NO: 3) was generated by fusing the pro-enzyme form of mesotrypsin (SEQ ID NO: 2) with a 6XG4S amino acid linker to a TNFa-binding domain (SEQ ID NO: 1 ) (TNFR1 ; 2.6 subunit of ectodomain of TNF receptor 1 ) (Figs. 1 A and 1 B).
  • SEQ ID NO: 1 TNFa-binding domain
  • plasmid DNA encoding the bifunctional molecule was transfected into HEK293T cells, and supernatants were collected after 96 hours and quantified using an anti-HIS ELISA.
  • the bifunctional molecule (mesotrypsin- TNFR1 ) was about 100-fold more potent than either the protease (mesotrypsin) alone or the bifunctional molecule with an inactive protease (Fig. 1C), indicating that the binder and protease act cooperatively to inhibit the activity of the TNFa target.
  • bifunctional molecules as described above were generated with the TNFa-binding domains VHH1 , VHH2, and VHH3 (5m2i, 5m2j, and 5m2m; nanobodies with different affinities for TNFa). These bifunctional molecules are presented in SEQ ID NOs: 4, 5, and 6, respectively.
  • TNFa 65 nM of TNFa was incubated with 1 -2 nM of test protein (bifunctional molecule comprising TNFR1 , VHH1 , VHH2, or VHH3) for 30 seconds, 5 minutes, 60 minutes, or overnight.
  • test protein bifunctional molecule comprising TNFR1 , VHH1 , VHH2, or VHH3
  • the resulting mixture was western blotted with an antibody against TNFa (Fig. 1 D).
  • Mesotrypsin was provided as a control.
  • constructs comprising the same protease and linker, but having different TNFa binding affinity and binding epitopes, have different proteolytic activity profiles against TNFa, as demonstrated by the cleavage fragments in the western blots.
  • the construct comprising the TNFa-binding domain with the lowest affinity had a cleavage profile similar to the mesotrypsin protease with no binding domain.
  • the higher-affinity constructs, (VHH2 and TNFR1 ; 130 and 380 pM binders) had more rapid cleavage, and the moderate binder (VHH1 , at 540 pM) had the most rapid and most extensive cleavage productivity. This indicates that the optimal binding affinity for a targeted proteolytic degrader may not be the highest possible affinity.
  • Bifunctional molecules were generated by fusing the pro-enzyme form of MMP7 or MMP12 with a 6XG4S amino acid linker to a DARPin binder with affinity against IL-13 (SEQ ID NO: 7) (Figs. 2A and 2B).
  • a bifunctional molecule comprising MMP7 and the anti-IL-13 DARPin is shown in SEQ ID NO: 8;
  • a bifunctional molecule comprising MMP12 and the anti-IL-13 DARPin is shown in SEQ ID NO: 9.
  • plasmid DNA encoding the bifunctional molecule was transfected into HEK293T cells, and supernatants were collected after 96 hours, enriched by nickel affinity, and quantified using an anti-HIS ELISA.
  • Fig. 2C illustrates that, despite the vast quantities or protease inhibitors in human serum, proteolytic protein fusions can be designed with proteases that retain their activity in serum for hours.
  • 2D shows a negative control: the fluorogenic peptide was added to human serum in the absence of the bifunctional molecules to confirm that fluorogenic activity was not coming from endogenous enzymes (e.g., endogenous enzymes in the matrix).
  • bifunctional molecules comprising MMP7, MMP12, or mesotrypsin (as described above) and (i) an anti-IL-4 DARPin (SEQ ID NO: 10) or (ii) the IL-4Ra ectodomain (SEQ ID NO: 13) were generated.
  • a bifunctional molecule comprising MMP7 and the anti-IL-4 DARPin is shown in SEQ ID NO: 11 .
  • a bifunctional molecule comprising MMP12 and the anti-IL-4 DARPin is shown in SEQ ID NO: 12.
  • a bifunctional molecule comprising mesotrypsin and the IL-4Ra ectodomain is shown in SEQ ID NO: 14.
  • a bifunctional molecule comprising MMP7 and the IL-4Ra ectodomain is shown in SEQ ID NO: 15.
  • a bifunctional molecule comprising MMP12 and the IL-4Ra ectodomain is shown in SEQ ID NO: 16.
  • the enzymatic domain was at the N-terminus, linked to the binder by a 6XG4S linker, and the indicated binder domain was at the C-terminus (Figs. 1 A and 2A).
  • Enzymatic constructs comprising an enzyme and a targeting domain, as shown in Fig. 4A, were designed and tested.
  • the targeting domain comprised an scFv based on the anti-amyloid beta antibody aducanumab (SEQ ID NO: 17; scFv designed from linking the variable chains of aducanumab). This scFv binds amyloid beta aggregates.
  • Enzymes included MMP2, MMP14, MMP9, Cathepsin-B (CTSB), Cathepsin-D (CTSD), and Kallikrein 7 (KLK7). Specific constructs and components thereof are shown in Table 6.
  • a bifunctional molecule comprising (i) an enzyme and (ii) an scFv is shown in Fig. 4A (“enzyme-scFv”).
  • HEX indicates that the natural MMP hemopexin domain was preserved; where HEX is omitted, there was not a hemopexin domain.
  • a bifunctional molecule comprising (i) an MMP enzyme comprising its natural hemopexin domain and (ii) an scFv is shown in Fig. 4A (“MMP-HEX-scFv contains hemopexin domain”).
  • a bifunctional molecule comprising MMP2 and the aducanumab scFv is shown in SEQ ID NO: 31 (MMP2-scFv).
  • a bifunctional molecule comprising MMP2 (comprising the natural MMP hemopexin domain) and the aducanumab scFv is shown in SEQ ID NO: 32 (MMP2-HEX-scFv).
  • a bifunctional molecule comprising MMP9 and the aducanumab scFv is shown in SEQ ID NO: 33 (MMP9-scFv).
  • a bifunctional molecule comprising full-length MMP9 (comprising the HEX domain) and the aducanumab scFv is shown in SEQ ID NO: 34 (MMP9-HEX-scFv).
  • a bifunctional molecule comprising MMP14 and the aducanumab scFv is shown in SEQ ID NO: 35 (MMP14-scFv).
  • a bifunctional molecule comprising full-length MMP14 (comprising the HEX domain) and the aducanumab scFv is shown in SEQ ID NO: 36.
  • a bifunctional molecule comprising CTSB and the aducanumab scFv is shown in SEQ ID NO: 37.
  • a bifunctional molecule comprising CTSD and the aducanumab scFv is shown in SEQ ID NO: 38 (CTSD-scFv).
  • KLK7 and the aducanumab scFv is shown in SEQ ID NO: 39 (KLK7-scFv).
  • Additional enzymatic constructs comprised (i) two versions or copies of the enzyme; (ii) two versions or copies of the scFv, and (iii) Fc regions linking the enzymes and scFvs (Fig. 4A).
  • the Fc domain was based on Human lgG4-Fc from pFUSE-lnvivogen (SEQ ID NO: 30). Constructs were produced in which (a) the enzyme is N-terminal and the scFv is C-terminal and (b) the scFv is N- terminal and the enzyme is C-terminal.
  • a bifunctional molecule comprising the MMP2 catalytic domain, an Fc region, and the aducanumab scFv (N-terminal to C-terminal) is shown in SEQ ID NO: 40.
  • a bifunctional molecule comprising the MMP9 catalytic domain, an Fc region, and the aducanumab scFv is shown in SEQ ID NO: 41 (MMP9-Fc-scFv).
  • a bifunctional molecule comprising the MMP14 catalytic domain, an Fc region, and the aducanumab scFv is shown in SEQ ID NO: 42.
  • a bifunctional molecule comprising CTSB, an Fc region, and the aducanumab scFv is shown in SEQ ID NO: 43.
  • a bifunctional molecule comprising CTSD, an Fc region, and the aducanumab scFv is shown in SEQ ID NO: 44 (CTSD-Fc-ScFv).
  • a bifunctional molecule comprising KLK7, an Fc region, and the aducanumab scFv is shown in SEQ ID NO: 45 (KLK7-Fc-scFv).
  • a bifunctional molecule comprising the aducanumab scFv, an Fc region, and MMP9 (N-terminal to C-terminal) is shown in SEQ ID NO: 46 (scFv-Fc-MMP9).
  • a bifunctional molecule comprising the aducanumab scFv, an Fc region, and CTSD is shown in SEQ ID NO: 47 (scFv-Fc- CTSD).
  • SEQ ID NO: 48 scFv-Fc-KLK7.
  • Molecules consisting of only the enzyme (e.g., only MMP2, MMP14, or MMP9) were used as controls. Enzyme sequences are shown in Table 7.
  • plasmid DNA encoding the bifunctional molecule was transfected into HEK293T cells, and supernatants were collected after 96 hours, enriched by nickel affinity, and quantified using an anti-HIS ELISA.
  • MMP2, MMP14, and MMP9 constructs were tested at 10 nM with 10 uM Mca-PLGL- Dpa-AR-NH2 fluorogenic peptide substrate and monitored using a plate reader (Figs. 4B-4D).
  • Cathepsin D constructs were pre-incubated in a mixture at a 1 :1 ratio with 2x activation buffer (0.1 M NaOAc, 0.2M NaCI, pH 3.5) or PBS (pH 7.4), and incubated for 30 minutes at 37°C. Then samples were mixed 1 :1 with 25 mM MES, pH 5, containing 60uM Mca-PLGL-Dpa-AR- NH2 fluorogenic peptide substrate, and monitored using a plate reader (Fig. 4E).
  • 2x activation buffer 0.1 M NaOAc, 0.2M NaCI, pH 3.5
  • PBS pH 7.4
  • thermolysin (1 :50) in 0.5M Tris, 0.1 M CaCl2, 1 .5M NaCI and incubated for 2 hours at 37°C.
  • the thermolysin was inactivated with 0.5M EDTA (1 :10).
  • the resulting mixture was combined 1 :1 in PBS containing 20 uM Mca-RPKPVE-Nval- WRK(Dnp)-NH2 fluorogenic substrate and monitored using a plate reader (Fig. 4F).
  • MMP2 and MMP9 constructs were activated with 4-aminophenylmercuric acetate (APMA).
  • MMP14 constructs were naturally activated during protein production via endogenous furin in the cell’s expression and secretion process. Cathepsin was naturally activated, and activation was enhanced with lower pH.
  • Kallikrein constructs were activated with thermolysin, and unactivated controls were untreated with thermolysin. Controls in Figs. 4B-4E indicate that supernatants from untransfected cells were used.
  • product forms from several enzymes including enzyme-binder, enzyme-Fc-binder, and binder-Fc-enzyme, were designed, produced, purified, and tested with confirmed functional enzymatic activity.

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Abstract

Les compositions et les procédés de l'invention sont utiles pour générer et utiliser des protéases bifonctionnelles (par exemple, des sheddases, des protéases ou des enzymes de dégradation de bêta-amyloïde (Aβ) ciblées) qui clivent des polypeptides d'une manière ciblée.
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