WO2024092160A2

WO2024092160A2 - Bifunctional proteases and uses thereof

Info

Publication number: WO2024092160A2
Application number: PCT/US2023/077959
Authority: WO
Inventors: Yiqun Bai; Liam Michael CASEY; John Daniel MENDLEIN; Ayse Jane MUNIZ
Original assignee: Flagship Pioneering Innovations VII Inc
Current assignee: Flagship Pioneering Innovations VII Inc
Priority date: 2022-10-26
Filing date: 2023-10-26
Publication date: 2024-05-02
Anticipated expiration: 2025-04-26
Also published as: WO2024092160A3

Abstract

The compositions and methods described herein are useful for generating and using bifunctional proteases (e.g., targeted sheddases, proteases, or amyloid beta (Aβ)-degrading enzymes)) that cleave polypeptides in a targeted manner.

Description

BIFUNCTIONAL PROTEASES AND USES THEREOF

Sequence Listing

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 25, 2023, is named 51660-002W02_Sequence_Listing_10_25_23, and is 101 ,123 bytes in size.

Field of the Invention

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.

Background of the Invention

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.

Summary of the Invention

The disclosure describes the compositions and methods for making and using bifunctional macromolecules that cleave molecules in a targeted manner.

In one aspect, 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. In some embodiments of the foregoing aspect, the proteolytic domain is a sheddase domain. In some embodiments of any of the foregoing aspects, the proteolytic domain is derived from a serine protease. In some embodiments, 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. In some embodiments, the proteolytic domain is an amyloid beta (Ap)-degrading enzyme.

In another aspect, 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.

In another aspect, 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. In some embodiments of the foregoing aspect, 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.

In another aspect, 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. In some embodiments of the foregoing aspect, 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.

In another aspect, 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.

In another aspect, 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. In some embodiments, the proteolytic domain is the proteolytic domain of human MMP2, human MMP9, human MMP14, human CTSB, human CTSD, or human KLK7.

In some embodiments of any of the foregoing aspects, 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.

In some embodiments of any of the foregoing aspects, the targeting domain includes a polypeptide. In some embodiments of the foregoing aspect, 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, centyrin™, or DARPin. In some embodiments of any of the foregoing aspects, the targeting domain includes a glycan.

In some embodiments of any of the foregoing aspects, the targeting domain includes a polynucleotide.

In some embodiments of any of the foregoing aspects, the substrate polypeptide is a soluble polypeptide. In some embodiments of any of the foregoing aspects, the substrate polypeptide is a membrane-associated polypeptide.

In some embodiments of any of the foregoing aspects, the target molecule is intracellular. In some embodiments of any of the foregoing aspects, the target molecule is extracellular.

In another aspect, 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.

In another aspect, 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.

In another aspect, 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.

In another aspect, 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.

In another aspect, 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.

In another aspect, 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.

In another aspect, 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. In some embodiments of any of the foregoing aspects, 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.

In another aspect, the disclosure provides a pharmaceutical composition that includes the bifunctional macromolecule of any of the foregoing aspects.

In another aspect, 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. In some embodiments of the foregoing aspect, the nucleic acid is an RNA.

In another aspect, the disclosure provides a vector encoding the bifunctional macromolecule of any of the foregoing aspects.

In another aspect, the disclosure provides a host cell that includes the bifunctional macromolecule or the nucleic acid of any of the foregoing aspects. In some embodiments of the foregoing aspect, the host cell is a eukaryotic cell. In some embodiments of the foregoing aspect, the host cell is a mammalian cell.

In another aspect, 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. In some embodiments of the foregoing aspect, the pharmaceutical composition further includes one or more pharmaceutically acceptable carriers, diluents, and/or excipients.

In another aspect, 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.

In another aspect, 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.

In another aspect, 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.

In another aspect, 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. In some embodiments of the foregoing aspect, the target polypeptide and the heterologous polypeptide are the same polypeptide. In some embodiments of the foregoing aspect, the target polypeptide and the heterologous polypeptide are not the same polypeptide. In some embodiments of any of the foregoing aspects, the heterologous target is not a membrane-associated polypeptide. In some embodiments of any of the foregoing aspects, the proteolytic domain is a sheddase domain.

In some embodiments of any of the foregoing aspects, 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. In some embodiments of the foregoing aspect, 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. In some embodiments of the foregoing aspect, 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.

In one aspect, 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. In some embodiments of the foregoing aspect, 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.

In another aspect, 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.

Brief Description of the Drawings

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.

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)).

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. Fig. 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). 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.

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.

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.

Definitions

As used herein, the term “alkenyl” refers to acyclic monovalent straight or branched chain hydrocarbon groups of containing one, two, or three carbon-carbon double bonds. Non-limiting examples of the 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. As used herein, the term “alkenylene” refers to a divalent alkenyl group. An optionally substituted alkenylene is an alkenylene that is optionally substituted as described herein for alkenyl.

As used herein, the term “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. Alkyl groups are exemplified by methyl; ethyl; n- and iso-propyl; n-, sec-, iso- and tert-butyl; neopentyl, and the like, and may be optionally substituted, valency permitting, with one, two, three, or, in the case of alkyl groups of two carbons or more, four or more substituents independently selected from the group consisting of: amino; alkoxy; aryl; aryloxy; azido; cycloalkyl; cycloalkoxy; cycloalkenyl; cycloalkynyl; halo; heterocyclyl; (heterocyclyl)oxy; heteroaryl; hydroxy; nitro; thiol; silyl; cyano; alkylsulfonyl; alkylsulfinyl; alkylsulfenyl; =0; =S; -C(O)R or -SO2R, where R is amino; and =NR’, where R’ is H, alkyl, aryl, or heterocyclyl. Each of the substituents may itself be unsubstituted or, valency permitting, substituted with unsubstituted substituent(s) defined herein for each respective group.

As used herein, the term “alkylene” refers to a divalent alkyl group. An optionally substituted alkylene is an alkylene that is optionally substituted as described herein for alkyl.

As used herein, the term “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.

As used herein, the term “alkynylene” refers to a divalent alkynyl group. An optionally substituted alkynylene is an alkynylene that is optionally substituted as described herein for alkynyl.

As used herein, the term “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.

As used herein, the term “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. 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. 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.

As used herein, the term “dispersion forces” refers to weak attractive forces between nonpolar molecules.

The term “glycan” 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. For example, cellulose is a glycan (or more specifically a glucan) composed of p-1 ,4-linked D-glucose, and 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.

As used herein, the term “heterologous target” refers to a polypeptide that is not a natural substrate of proteases containing the recited proteolytic domain in vivo.

As used herein, the term “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.

As used herein, the term “hydrophobic-hydrophobic interactions” refers to interactions between lipophilic moieties to form intermolecular aggregates or intramolecular interactions (particularly in aqueous based environments).

The term “linker,” as used herein, refers to a covalent or noncovalent linkage or connection between two or more components in a fusion protein or a conjugate. In some embodiments, a linker provides space, rigidity, and/or flexibility between the two or more components. In some embodiments, the linker may join the targeting domain to the proteolytic domain. In some embodiments, 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. In some embodiments, a linker may be a bond, e.g., a covalent bond. The term “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. 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. In some embodiments, 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 heteroalkenyl, optionally substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted cycloalkenyl, optionally substituted heterocycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heterocycloalkynyl, optionally substituted aryl, or optionally substituted heteroaryl), carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, imino, or combinations thereof. For example, 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.g., C6 arylene), optionally substituted 5-10 membered heteroarylene (e.g., imidazole, pyridine), O, S, NR' (R' is H, optionally substituted C1 - C20 alkyl, optionally substituted C1 -C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2- C20 heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C2-C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C6-C14 aryl, or optionally substituted C3-C15 heteroaryl), carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, imino, or combinations thereof.

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. For example, 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. Alternately, 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.

As used herein, the term “macromolecule” refers to a molecular entity that comprises one or more polypeptides. In instances where the bifunctional macromolecule comprises two polypeptides, the chains are covalently or non-covalently bound to one another.

As used herein, 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.

As used herein, the term “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.

As used herein, 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.

As used herein, the term “proteolytic domain” refers to a domain that is capable of catalyzing a chemical reaction that cleaves a peptide bond.

As used herein, the term “sheddase domain” refers to a domain derived from a sheddase. “Sheddase” as used herein 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.

As used herein, the term “targeting domain” refers to a domain that binds a target molecule (e.g., a polypeptide, a polynucleotide, a lipid, glycan, or a polysaccharide).

As used herein, “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.

Detailed Description

Provided herein are bifunctional proteases (e.g., targeted sheddases, proteases, or amyloiddegrading enzymes (e.g., amyloid beta (Ap)-degrading enzymes))), as well as methods of cleaving macromolecules (e.g., polypeptides, e.g., target or heterologous peptides). Also provided herein are 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.

Targeting Domains

Bifunctional proteases of the disclosure include one or more targeting domains and one or more proteolytic domains. For example, 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. In some embodiments, targeting domains of the disclosure bind their target molecules with high affinity. In some embodiments, targeting domains of the disclosure bind their target molecules with moderate affinity. In some embodiments, the molecule of interest is membrane-embedded. In some embodiments, the molecule of interest is not membrane-embedded, e.g., the polypeptide may be soluble. In some embodiments, 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. For example, in some embodiments, 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). In some embodiments, the targeting domain may be a receptor or a ligand binding portion of a receptor. In some embodiments, the targeting domain may be a ligand that binds to a receptor.

In some aspects, the targeting domain binds to an amyloid protein (e.g., a monomer or a protein in an amyloid oligomer, fibril, or plaque). In some aspects, the targeting domain binds to amyloid beta (Ap). For example, 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. For example, 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).

In some aspects, the targeting domain binds to TNFa. For example, 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.

In some aspects, the targeting domain binds to IL-4. For example, 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.

In some aspects, the targeting domain binds to IL-13. For example, 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.

As a general principle, 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. For example, in aspects in which an antibody sequence is provided, 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. In other embodiments, 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. Similarly, in aspects in which a nonantibody binding domain is provided, the targeting domain may comprise all or a portion of the region or regions of the binding domain that interact with the target. In some embodiments, 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. 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.

Polypeptides

Targeting domains of the disclosure may include a polypeptide. In some embodiments, 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. In some embodiments, the targeting domain includes an antibody or antigen-binding fragment thereof. In some embodiments, 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). In some embodiments, the antigen-binding fragment is a VHH. In some embodiments, the antigen-binding fragment is a Fab. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

Protein Ligands

In some embodiments, the protein ligand or portion thereof is capable of binding to or binds to a target molecule of interest. In some embodiments, 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), SMARCA2, and tripartite motif-containing 24 (TRIM24)).

In some embodiments, 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, KSR1 , LCK, LMTK2, LMTK3, LTK, LYN, MATK, MERTK, MET, MLTK, MST1 R, MUSK, NPR1 , NTRK1 , NTRK2, NTRK3, PDGFRA, PDGFRB, PLK4, PTK2, PTK2B, PTK6, PTK7, RET, ROR1 , ROR2, ROS1 , RYK, SGK493, SRC, SRMS, STYK1 , SYK, TEC, TEK, TEX14, TIE1 , TNK1 , TNK2, TNNI3K, TXK, TYK2, TYRO3, YES1 , or ZAP70), a serine/threonine kinase (e.g., casein kinase 2, protein kinase A, protein kinase B, protein kinase C, Raf kinases, CaM kinases, AKT1 , AKT2, AKT3, ALK1 , ALK2, ALK3, ALK4, Aurora A, Aurora B, Aurora C, CHK1 , CHK2, CLK1 , CLK2, CLK3, DAPK1 , DAPK2, DAPK3, DMPK, ERK1 , ERK2, ERK5, GCK, GSK3, HIPK, KHS1 , LKB1 , LOK, MAPKAPK2, MAPKAPK, MNK1 , MSSK1 , MST1 , MST2, MST4, NDR, NEK2, NEK3, NEK6, NEK7, NEK9, NEK11 , PAK1 , PAK2, PAK3, PAK4, PAK5, PAK6, PIM1 , PIM2, PLK1 , RIP2, RIP5, RSK1 , RSK2, SGK2, SGK3, SIK1 , STK33, TAO1 , TAO2, TGF-p, TLK2, TSSK1 , TSSK2, ULK1 , or ULK2), a cyclin dependent kinase (e.g., CDK1 , CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, or CDK11 ), and a leucine-rich repeat kinase (e.g., LRRK2).

In some embodiments, 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). In some embodiments, 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.

Receptors

Receptor proteins can bind to corresponding ligands to initiate cellular signaling pathways. Targeting domains provided herein may include receptor proteins or portions thereof. In some embodiments, 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). In some embodiments, 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). In some embodiments, 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. In some embodiments, 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, a melanin-concentrating hormone receptor, a melanocortin receptor, a motilin receptor, a neuromedin U receptor, a neuropeptide receptor, an opioid receptor, an opsin receptor, an orexin receptor, an oxoglutarate receptor, a P2Y receptor, a platelet-activating factor receptor, a prokineticin receptor, a prolactin-releasing peptide receptor, a prostanoid receptor, a proteinase-activated receptor, a relaxin family peptide receptor, a somatostatin receptor, a succinate receptor, a tachykinin receptor, a thyrotropin-releasing hormone receptor, a trace amine receptor, an urotensin receptor, a vasopressin receptor, or an oxytocin receptor). In some embodiments, 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). In some embodiments, 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). In some embodiments, 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).

Monoclonal Antibodies

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. 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. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. In some embodiments, the polypeptide is a monoclonal antibody.

Antigen-binding Fragments

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 centyrin™, a DARPin, or a domain antibody. Examples of 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 of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although 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). 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. In some embodiments, the targeting domain includes an antigen-binding fragment. In some embodiments, 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.

Fab

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. In some embodiments, the targeting domain includes a Fab.

F(ab’)₂

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 (F(ab')2 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. In some embodiments, the targeting domain includes a F(ab')2. scFv

Single-chain Fv antibodies (scFvs) 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. The linker that joins the VL and VH regions of a scFv fragment can be a peptide linker composed of proteinogenic amino acids. Alternative 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). 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. It will also be understood by one of ordinary skill in the art that the 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. For example, in one embodiment, 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). Alternatively or additionally, 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. In some embodiments, 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. In some embodiments, the targeting domain includes a scFv with an Fc region.

Immunoprotein

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. In some embodiments, the targeting domain includes an immunoprotein.

Heavy Chain Variable Regions and Light Chain Variable Regions

In some embodiments, 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, CDR3, FR4.

Certain portions of the 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. 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).

Affibody

In some embodiments, 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). Alternatively, 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.

Diabody

In some embodiments, 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. In some embodiments, the targeting domain includes a diabody.

Triabody

In some embodiments, 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. In order to fold into their native structure, 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). In some embodiments, the targeting domain includes a triabody.

Tetrabody

In some embodiments, the targeting domain includes a tetrabody. The term “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. In order to fold into their native structure, 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. In some embodiments, the targeting domain includes a tetrabody.

Knottin

In some embodiments, 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. These loops are important for folding, structural integrity, molecular recognition and biological function. The loop regions of 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. In some embodiments, the targeting domain includes a knottin.

Atrimer

In some embodiments, the targeting domain includes an atrimer.

Alternative Scaffolds

In some embodiments, the targeting domain includes an alternative scaffold. As used herein, the term “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. For example, 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. 18:295-304 (2005); Hosse et al., “A New Generation of Protein Display Scaffolds for Molecular Recognition,” Protein Sci. 15:14-27 (2006); Nicaise et al., “Affinity Transfer by CDR Grafting on a Nonimmunoglobulin Scaffold,” Protein Sci. 13:1882-1891 (2004); Nygren and Uhlen, “Scaffolds for Engineering Novel Binding Sites in Proteins,” Curr. Opin. Struc. Biol. 7:463-469 (1997), all of which are hereby incorporated by reference in their entirety). The structure of alternative scaffolds vary but preferably are of human origin for those developed as therapeutics.

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”). In some embodiments, the targeting domain includes an alternative scaffold. In some embodiments, the alternative scaffold is a non-lg based scaffold.

Avimer

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. Preferably, 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. In some embodiments, the targeting domain includes an avimer.

Cys-knot

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. In some embodiments, the targeting domain includes a cys-knot.

Fynomer

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.

Kunitz Domain

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.

Obody

In some embodiments, the targeting domain includes an Obody.

Nanobody

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). In some embodiments, the targeting domain includes a nanobody.

Anticalin

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). Moreover, 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. In some embodiments, the targeting domain includes a lipocalin.

Affimer Type I

In some embodiments, the targeting domain includes an affimer type I.

Affimer Type II

In some embodiments, the targeting domain includes an affimer type II.

FN3 Scaffold

Proteins derived from fibronectin III (FN3) domains may be used to generate a suitable non-lg based alternative scaffold. For example, 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. In some embodiments, the targeting domain includes a FN3 scaffold. Centyrin™

Centyrins are small, engineered proteins derived from FN domains of a human protein, Tenascin C (TNC), found in the extracellular matrix of various tissues. Centryrins™ contain the consensus sequence of FN3 domains of TNC. Centyrin™ 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. These molecules have excellent biophysical properties (e.g., greater than 100 mg/mL expression, greater than 170 mg/mL solubility, greater than 82° C. melting temperature, low predicted immunogenicity, and stable in serum for more than one month) and can be engineered for improved stability. In some embodiments, the targeting domain includes a centyrin™ scaffold.

DARPin

Ankyrin repeat (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. Preferably, DARPins contain two to four of these motifs flanked by N- and C-terminal capping motifs to shield hydrophobic regions and allow increased solubility. In some embodiments, the targeting domain includes a DARPin.

Glycans

Targeting domains of the disclosure may include glycan moieties. For example, in some embodiments, the glycan moiety is a polysaccharide. In some embodiments, the glycan moiety is an oligosaccharide. In some embodiments, the glycan moiety is the carbohydrate portion of a glycoconjugate (e.g., a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide, or proteoglycan). In some embodiments, the glycan moiety is located on the exterior surface of a cell. In some embodiments, the glycan moiety is membrane-embedded. In some embodiments, the glycan moiety is N-acetylglucosamine (GIcNac). In some embodiments, the glycan moiety is N-acetylgalactosamine (GalNac).

Polynucleotides

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). One or more nucleotides in the polynucleotide may be modified, for instance with a label or a tag.

Proteolytic Domains

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.

Mesotrypsin

In some embodiments, the proteolytic domain comprises or consists of mesotrypsin (e.g., human mesotrypsin), or a fragment or derivative thereof. For example, 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. In some embodiments, the proteolytic domain comprises or consists of mesotrypsin and the targeting domain binds to TNFa. In some embodiments, the proteolytic domain comprises or consists of mesotrypsin and the targeting domain binds to IL-4.

Accordingly, in some embodiments, the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds TNFa; and (b) mesotrypsin or a fragment or derivative thereof. In some embodiments, 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. In other embodiments, the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds IL-4; and (b) mesotrypsin or a fragment or derivative thereof. In some embodiments, 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.

MMP7

In some embodiments, 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). For example, 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. In some embodiments, the proteolytic domain comprises or consists of MMP7 and the targeting domain binds to IL-4. In some embodiments, the proteolytic domain comprises or consists of MMP7 and the targeting domain binds to IL-13.

Accordingly, in some embodiments, the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds IL-4; and (b) MMP7 or a fragment or derivative thereof. In some embodiments, 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. In some embodiments, the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds IL-13; and (b) MMP7 or a fragment or derivative thereof. In some embodiments, 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.

MMP12

In some embodiments, 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). In some embodiments, the proteolytic domain comprises or consists of MMP12 and the targeting domain binds to IL-4. In some embodiments, the proteolytic domain comprises or consists of MMP12 and the targeting domain binds to IL-13.

Accordingly, in some embodiments, the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds IL-4; and (b) MMP12 or a fragment or derivative thereof. In some embodiments, 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. In some embodiments, the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds IL-13; and (b) MMP12 or a fragment or derivative thereof. In some embodiments, 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.

MMP2

In some embodiments, the proteolytic domain comprises or consists of MMP2 (e.g., human MMP2), or a fragment or derivative thereof. In some embodiments, the proteolytic domain comprises the hemopexin (HEX) domain of MMP2; in other embodiments, the HEX domain is absent. In some embodiments, the proteolytic domain comprises or consists of MMP2 and the targeting domain binds to amyloid beta.

Accordingly, in some embodiments, the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds amyloid beta; and (b) MMP2 or a fragment or derivative thereof. In some embodiments, 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.

MMP9

In some embodiments, the proteolytic domain comprises or consists of MMP9 (e.g., human MMP9), or a fragment or derivative thereof. In some embodiments, the proteolytic domain comprises the HEX domain of MMP9; in other embodiments, the HEX domain is absent. In some embodiments, the proteolytic domain comprises or consists of MMP9 and the targeting domain binds to amyloid beta.

Accordingly, in some embodiments, the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds amyloid beta; and (b) MMP9 or a fragment or derivative thereof. In some embodiments, 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.

MMP14

In some embodiments, the proteolytic domain comprises or consists of MMP14 (e.g., human MMP14), or a fragment or derivative thereof. In some embodiments, the proteolytic domain comprises the HEX domain of MMP14; in other embodiments, the HEX domain is absent. In some embodiments, the proteolytic domain comprises or consists of MMP14 and the targeting domain binds to amyloid beta.

Accordingly, in some embodiments, the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds amyloid beta; and (b) MMP14 or a fragment or derivative thereof. In some embodiments, 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.

CTSB

In some embodiments, the proteolytic domain comprises or consists of cathepsin-B (CTSB) (e.g., human CTSB), or a fragment or derivative thereof. In some embodiments, the proteolytic domain comprises or consists of CTSB and the targeting domain binds to amyloid beta.

Accordingly, in some embodiments, the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds amyloid beta; and (b) CTSB or a fragment or derivative thereof. In some embodiments, 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.

CTSD

In some embodiments, the proteolytic domain comprises or consists of cathepsin-D (CTSD) (e.g., human CTSD), or a fragment or derivative thereof. In some embodiments, the proteolytic domain comprises or consists of CTSD and the targeting domain binds to amyloid beta.

Accordingly, in some embodiments, the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds amyloid beta; and (b) CTSD or a fragment or derivative thereof. In some embodiments, 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. KLK7

In some embodiments, 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.

Accordingly, in some embodiments, the disclosure provides a bifunctional protease comprising (a) a targeting domain that binds amyloid beta; and (b) KLK7 or a fragment or derivative thereof. In some embodiments, 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.

As a general principle, 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. For example, the proteolytic domain may comprise all or a portion of the enzymatic active site (e.g., catalytic site) of the proteolytic domain. In some embodiments, 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. 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.

A skilled artisan would be able to determine the enzymatic active site or sites of a given proteolytic domain, e.g., based on knowledge in the art. For example, 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.

In some embodiments, 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. For example, in some embodiments, 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.

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 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the proteolytic domain is derived from a 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 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21 , MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, mosaic serine protease large-form (MSPL), membrane type 1 matrix metalloproteinase (MT1 -MMP), MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, PCSK4, PCSK5, polyserase-1 , presenilin-1 , presenilin-2, spinesin, signal peptide peptidase (SPP), SPP-like 2A (SPPL2a), SPPL2b, SPPL2c, SPPL3, transmembrane protease serine 2 (TMPRSS2), TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y-secretase. In some embodiments, 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 , presenilin-1 , presenilin-2, spinesin, SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y-secretase. In some embodiments, 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 , presenilin-1 , presenilin-2, spinesin, SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y- secretase. In some embodiments, 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 , presenilin-1 , presenilin-2, spinesin, SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y-secretase. In some embodiments, 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 , presenilin-1 , presenilin-2, spinesin, SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y- secretase. In some embodiments, 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 , presenilin-1 , presenilin-2, spinesin, SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y-secretase. In some embodiments, 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 , presenilin-1 , presenilin-2, spinesin, SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y- secretase. In some embodiments, 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 , presenilin-1 , presenilin-2, spinesin, SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y-secretase. In some embodiments, 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 , presenilin-1 , presenilin-2, spinesin, SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y- secretase. In some embodiments, 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 , presenilin-1 , presenilin-2, spinesin, SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y-secretase. In some embodiments, 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 , presenilin-1 , presenilin-2, spinesin, SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y- secretase. In some embodiments, 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 , presenilin-1 , presenilin-2, spinesin, SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y-secretase. In some embodiments, 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 , presenilin-1 , presenilin-2, spinesin, SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y- secretase. In some embodiments, 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 , presenilin-1 , presenilin-2, spinesin, SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y-secretase. In some embodiments, 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 , presenilin-1 , presenilin-2, spinesin, SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y- secretase. In some embodiments, 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 , presenilin-2, spinesin, SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS11 A, or TMPRSS13, Y-secretase.

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

Substrate Polypeptide

In some embodiments, upon binding of the targeting domain to a target molecule (e.g., target polypeptide), the proteolytic domain cleaves a peptide bond in a substrate peptide. A substrate may be essentially any peptide capable of being cleaved by a contemplated bifunctional macromolecule. In some embodiments, the target polypeptide and the substrate polypeptide are the same polypeptide. In some embodiments, the target polypeptide and the substrate polypeptide are not the same polypeptide. In some embodiments, the substrate polypeptide is heterologous to the proteolytic domain. In some embodiments, the substrate polypeptide is soluble. In some embodiments, the substrate polypeptide is membrane-embedded. In some embodiments, the substrate polypeptide is membrane-associated. In some embodiments, the membrane-associated polypeptide is an ion channel, an aquaporin, a protein transporter, a glucose transporter, or a membrane-associated receptor. In some embodiments, the membrane-associated polypeptide is an ion channel. In some embodiments, the membrane-associated polypeptide is an aquaporin. In some embodiments, the membrane-associated polypeptide is a protein transporter. In some embodiments, the membrane- associated polypeptide is a glucose transporter. In some embodiments, the membrane-associated polypeptide is a membrane-associated receptor. In some embodiments, the substrate polypeptide may be an extracellular substrate. In some embodiments, the substrate polypeptide may be an intracellular substrate.

In some embodiments, the substrate polypeptide is a membrane protein. Non-limiting examples of membrane proteins include CD1 , CD3D, CD3E, CD274, CTLA4 , ERBB2, SLC5A8, CCR5, EPCAM. In some embodiments, 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). In some embodiments, the substrate polypeptide is a multi-pass membrane protein, such as proteins with two or more transmembrane domains. In some embodiments, the substrate polypeptide is a membrane protein having multiple complexes. In some embodiments, the membrane protein having multiple complexes is the sodium transporter Na_v1 .7. In some embodiments, the substrate polypeptide is a facilitative transporter. In some embodiments, the facilitative transporter is selected from the major facilitative superfamily (MFS) of solute carriers. In some embodiments, the facilitative transporter is cystine/glutamate antiporter xCT. In some embodiments, the substrate polypeptide is a membrane receptor. In some embodiments, 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 ). In some embodiments, the substrate polypeptide is a membrane ligand. In some embodiments, the membrane ligand is PD-L1 , transforming growth factor alpha (TGFa), p-selectin glycoprotein ligand-1 . In some embodiments, the substrate polypeptide is an ATP-binding cassette (ABC) transporter. In some embodiments, the ABC transporter is cystic fibrosis transmembrane conductance regulator (CFTR). In some embodiments, the substrate polypeptide is an ATPase. In some embodiments, the ATPase is HK-ATPase. In some embodiments, the substrate polypeptide is a co-stimulatory molecule. In some embodiments, the substrate polypeptide is a co-inhibitory molecule.

In some embodiments, the extracellular substrate is a cytokine. In some embodiments, 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. In some embodiments, the extracellular substrate is a cytokine fragment, such as IL-12p or IL- 230. In some embodiments, the extracellular substrate is a chemokine. In some embodiments, the chemokine is CXC motif chemokine ligand 8 (CXCL-8), CC motif chemokine ligand 2 (CCL2), CCL3, CCL4, CCL5, CCL11 , or CXCL10). In some embodiments, the extracellular substrate is a blood protein, such as Von Willebrand factor (VWF). In some embodiments, the extracellular substrate is a protein precursor such as a pro-protein or pro-peptide. In some embodiments, the protein precursor is pro-insulin or pro-thrombin. In some embodiments, the extracellular substrate is an extracellular matrix protein. In some embodiments, the extracellular matrix protein is collage, fibronectin, elastin, integrin, or vitronectin. In some embodiments, the extracellular substrate is a protein aggregate. In some embodiments, 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). In some embodiments, the extracellular substrate is a protease. In some embodiments, the protease is alpha-1 -antitrypsin or elastase. In some embodiments, the extracellular substrate is a pathogen-associated protein. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the extracellular substrate is a hormone. In some embodiments, the hormone is insulin, testosterone, or progesterone. In some embodiments, the extracellular substrate is an extracellular nucleic acid.

A non-limiting example of an intracellular substrate is a histone protein.

Proteolytic Domains that Degrade Amyloid Beta and Bifunctional Proteases Comprising the Same

In some embodiments, the bifunctional protease comprises a proteolytic domain that is capable of degrading amyloid beta (Ap). Non-limiting examples of such proteolytic domains are provided in Table 1 , below. For example, in some embodiments, 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.

In some embodiments, the bifunctional protease further comprises a targeting domain with affinity for amyloid beta. Non-limiting examples of such targeting domains are provided in Table 2, below. For example, the targeting domain may comprise aducanumab, crenezumab, gantenerumab, bapineuzumab (3D6), solanezumab, ponezumab, lecanemab, or a fragment or derivative thereof.

Accordingly, in some aspects, the bifunctional protease degrades amyloid beta monomers, oligomers, fibrils, and/or plaques (e.g., is capable of active aggregate dissolution of beta amyloid fibrils). For example, 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.

Table 1. Enzymes with natural ability to degrade Ap amyloid

Table 2. Binders, epitopes, and affinities against Ap

Linkers

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. In some embodiments, the linker includes any peptide linker that includes two or more amino acid residues. For example, 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. In the case that 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. Specifically, 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.

In the case that 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.

In the case that 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, can be attached synthetically to the C-terminus of one protein and the N-terminus of another protein, respectively. 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.

Peptide Linkers

In the present disclosure, 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). In some embodiments, 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). In some embodiments, 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. In certain embodiments, a linker can contain motifs, e.g., multiple or repeating motifs, of GS, GGS, GGGGS (SEQ ID NO: 52), GGSG, or SGGG. In preferred embodiments, 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). In some embodiments, the peptide linker is GGGGSGGGGS (SEQ ID NO: 53), GGGGSGGGGSGGGGS (SEQ ID NO: 54); or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 55). In some embodiments, 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 GSGSGSGSGSGS (SEQ ID NO: 59). In certain other embodiments, 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). In yet other embodiments, 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 GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 66). In other embodiments, a linker can contain motifs of GGGGS (SEQ ID NO: 52), e.g., GGGGSGGGGS (SEQ ID NO: 53) or GGGGSGGGGSGGGGS (SEQ ID NO: 54). In certain embodiments, a linker is SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 67).

In some embodiments, 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). In certain embodiments, 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). In some embodiments, 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., SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 67)).

In some embodiments, 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)).

In some embodiments, 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)).

In some embodiments, a linker can contain motifs of GGGG, e.g., GGGGGGGG (SEQ ID NO: 68), GGGGGGGGGGGG (SEQ ID NO: 69), GGGGGGGGGGGGGGGG (SEQ ID NO: 70), or GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 71 ). In some embodiments, a linker can contain motifs of GGGGG (SEQ ID NO: 72), e.g., GGGGGGGGGG (SEQ ID NO: 73), or GGGGGGGGGGGGGGG (SEQ ID NO: 74). In some embodiments, a linker is GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 71).

In other embodiments, 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 GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 81).

In some embodiments in the present disclosure, 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. In other embodiments, an 18- amino acid peptide linker consisting of sequence GGSGGGSGGGSGGGSGGS (SEQ ID NO: 83) may be used.

Chemical Linkers

In some embodiments, a linker provides space, rigidity, and/or flexibility between two or more components of the fusion protein or conjugate. In some embodiments, 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. In some embodiments, 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)). In some embodiments, 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, 7, 6, 5, 4, 3, 2, or 1 nonhydrogen atom(s)). In some embodiments, 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. For examples, 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. In some embodiments of a divalent linker, 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.

In some embodiments, dicarboxylic acid molecules may be used as 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,

wherein 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,

19, or 20).

Other examples of dicarboxylic acids molecules that may be used to form linkers include, but are not limited to,

In some embodiments, dicarboxylic acid molecules, such as the ones described herein, may be further functionalized to contain one or more additional functional groups. In some embodiments, 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).

In some embodiments, 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.

In some embodiments, 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,

In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

In some embodiments, a linker may include a synthetic group derived from, e.g., a synthetic polymer (e.g., a PEG polymer). In some embodiments, a linker may include one or more amino acid residues. In some embodiments, 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). In some embodiments, a linker (L or L’) 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.g., C6 arylene), optionally substituted 5-10 membered heteroarylene (e.g., imidazole, pyridine), O, S, NR' (R' is H, optionally substituted C1 -C20 alkyl, optionally substituted C1 -C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C2-C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C6-C14 aryl, or optionally substituted C3-C15 heteroaryl), P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino.

The targeting domain and proteolytic domain may be covalently attached or associated by non-covalent means. For instance, 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. In certain preferred embodiments, 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.

In some embodiments, 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). For example, in some embodiments, the proteolytic domain and the targeting domain are associated with each other by hydrogen bonding interactions. In some embodiments, the proteolytic domain and the targeting domain are associated with each other by charge-charge interactions. In some embodiments, the proteolytic domain and the targeting domain are associated with each other by hydrophobic- hydrophobic interactions. In some embodiments, the proteolytic domain and the targeting domain are associated with each other by dispersion forces.

Conjugation Chemistries

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).

Other examples of functional groups capable of reacting with amino groups include, e.g., alkylating and acylating agents. Representative alkylating agents include: (i) an a-haloacetyl group, e.g., XCH2CO- (where X=Br, Cl, or I); (ii) a N-maleimide group, which may react with amino groups either through a Michael type reaction or through acylation by addition to the ring carbonyl group; (iii) an aryl halide, e.g., a nitrohaloaromatic group; (iv) an alkyl halide; (v) an aldehyde or ketone capable of Schiff’s base formation with amino groups; (vi) an epoxide, e.g., an epichlorohydrin and a bisoxirane, which may react with amino, sulfhydryl, or phenolic hydroxyl groups; (vii) a chlorine- containing of s-triazine, which is reactive towards nucleophiles such as amino, sufhydryl, and hydroxyl groups; (viii) an aziridine, which is reactive towards nucleophiles such as amino groups by ring opening; (ix) a squaric acid diethyl ester; and (x) an a-haloalkyl ether.

Examples of 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.

It will be appreciated that certain functional groups may be converted to other functional groups prior to reaction, for example, to confer additional reactivity or selectivity. Examples of methods useful for this purpose include conversion of amines to carboxyls using reagents such as dicarboxylic anhydrides; conversion of amines to thiols using reagents such as N-acetylhomocysteine thiolactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane, or thiol-containing succinimidyl derivatives; conversion of thiols to carboxyls using reagents such as a -haloacetates; conversion of thiols to amines using reagents such as ethylenimine or 2-bromoethylamine; conversion of carboxyls to amines using reagents such as carbodiimides followed by diamines; and conversion of alcohols to thiols using reagents such as tosyl chloride followed by transesterification with thioacetate and hydrolysis to the thiol with sodium acetate. In some embodiments, a linker of the disclosure, is conjugated (e.g., by any of the methods described herein) to a fusion protein. In some embodiments of the disclosure, the linker is conjugated by way of: (a) a thiourea linkage (i.e., -NH(C=S)NH-) to a lysine; (b) a carbamate linkage (i.e., - NH(C=O)-O) to a lysine; (c) an amine linkage by reductive amination (i.e., -NHCH2) to a lysine; (d) an amide (i.e., -NH-(C=0)CH2) to a lysine; (e) a cysteine-maleimide conjugate between a maleimide of the linker to a cysteine; (f) an amine linkage by reductive amination (i.e., -NHCH2) between the linker and a carbohydrate; (g) a rebridged cysteine conjugate, wherein the linker is conjugated to two cysteines; (h) an oxime linkage between the linker and a carbohydrate; (i) an oxime linkage between the linker and an amino acid residue; (j) an azido linkage between the linker; (k) direct acylation of a linker; or (I) a thioether linkage between the linker.

In some aspects, 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. For example, in some embodiments, a pair of Fc regions is used to link a targeting domain to a proteolytic domain. In other embodiments, a pair of Fc regions is used to link two or more moieties comprising both a targeting domain and a proteolytic domain.

Dimerization Selectivity

In the present disclosure, 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. Specifically, 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. In a dimerization selectivity module, 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.

In some embodiments, 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. To selectively form an Fc 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.

In other embodiments, 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. In some embodiments, 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. In one example, an Fc domain monomer containing a positively-charged amino acid substitution, e.g., D356K or E357K, and an Fc domain monomer containing a negatively-charged amino acid substitution, e.g., K370D or K370E, may selectively combine to form an Fc domain through favorable electrostatic steering of the charged amino acids. In another example, 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. In some embodiments, 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. In some cases, 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). In further embodiments, 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. For example, 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. In some cases, 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.

Furthermore, other methods used to promote the formation of 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.

Engineered Cavities and Engineered Protuberances

The use of engineered cavities and engineered protuberances (or the “knob-into-hole” strategy) is described by Carter and co-workers (Ridgway et al., Protein Eng. 9:617-612, 1996; Atwell et al., J Mol Biol. 270:26-35, 1997; Merchant et al., Nat Biotechnol. 16:677-681 , 1998). The knob and hole interaction favors heterodimer formation, whereas the knob-knob and the hole-hole interaction hinder homodimer formation due to steric clash and deletion of favorable interactions. The “knob-into- hole” technique is also disclosed in U.S. Pat. No. 5,731 ,168.

In the present disclosure, 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. Specifically, 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. In some embodiments, 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. In other embodiments, an engineered cavity in one CH3 antibody constant domain is created to better accommodate an original amino acid in another CH3 antibody constant domain. In yet other embodiments, 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. Specifically, some dimerization selectivity modules (e.g., heterodimerizing selectivity modules) (described further above) contain engineered cavities such as Y407V mutation in the CH3 antibody constant domain. Similarly, an engineered protuberance can be constructed by replacing amino acids containing smaller side chains with amino acids containing larger side chains. Specifically, some dimerization selectivity modules (e.g., heterodimerizing selectivity modules) (described further above) contain engineered protuberances such as T366W mutation in the CH3 antibody constant domain. In the present disclosure, engineered cavities and engineered protuberances are also combined with inter-CH3 domain disulfide bond engineering to enhance heterodimer formation. In one example, 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. In another example, 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.

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

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.

In the present disclosure, electrostatic steering is used to control the dimerization of Fc domain monomers and the formation of Fc constructs. In particular, to control the dimerization of Fc domain monomers using electrostatic steering, 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. In some embodiments, 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. In other embodiments, 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. By introducing charged amino acids to the interacting CH3 antibody constant domains, 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.

In some embodiments, to create a dimerization selectivity module including reversed charges that can selectively form dimers of Fc domain monomers as controlled by the electrostatic steering effects, the two Fc domain monomers may be selectively formed through heterodimerization or homodimerization.

Heterodimerization of Fc Domain Monomers

Heterodimerization of Fc domain monomers can be promoted by introducing different, but compatible, mutations in the two Fc domain monomers. In some embodiments, 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. In one example, an Fc domain monomer containing a positively-charged amino acid substitution, e.g., D356K or E357K, and an Fc domain monomer containing a negatively- charged amino acid substitution, e.g., K370D or K370E, may selectively combine to form an Fc domain through favorable electrostatic steering of the charged amino acids. In another example, 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. For example, in an Fc construct having three Fc domains, two of the three Fc domains may be formed by the heterodimerization of two Fc domain monomers, as promoted by the electrostatic steering effects. A “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.

Nucleic Acids and Expression Systems

Bifunctional proteases (e.g., targeted sheddases) described herein can be prepared by any of a variety of established techniques. For instance, a bifunctional protease described herein can be prepared by recombinant expression of genes in a host cell. To express a bifunctional protease recombinantly, 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.

Vectors for Expression of Bifunctional Proteases

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. Examples of viral vectors 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). Other viruses useful for delivering polynucleotides encoding bifunctional proteases described herein include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of 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). Other examples 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. Other examples of 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, are also well known in the art and 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. 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)).

Host Cells for Expression of Proteins

It is possible to express the bifunctional proteases (e.g., targeted sheddases) described herein in either prokaryotic or eukaryotic host cells. In some embodiments, 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. 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. USA 77:4216-4220), used with a 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. When recombinant expression vectors encoding bifunctional protease genes are introduced into mammalian host cells, 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. Once a bifunctional protease (e.g., a sheddase) described herein has been produced by recombinant expression, 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. Further, 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.

Once isolated, 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 Superdex™ 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

Nucleic Acids as Agents for Delivering Proteins

The 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) may be used as therapeutic agents to express proteins of the disclosure as a therapy to treat a target disease.

The nucleic acid molecules of the disclosure may include one or more alterations. Herein, in a nucleotide, nucleoside, or polynucleotide (such as the nucleic acids of the invention (e.g., an RNA or an oligonucleotide)), the terms “alteration” or, as appropriate, “alternative” refer to alteration with respect to A, G, U or C ribonucleotides.

The alterations may be various distinct alterations. In some embodiments, where the nucleic acid is an mRNA, the coding region, the flanking regions, and/or the terminal regions may contain one, two, or more (optionally different) nucleoside or nucleotide alterations. In some embodiments, 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). In certain embodiments, 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.

In certain embodiments, it may be desirable for a nucleic acid molecule introduced into the cell to be degraded intracellularly. For example, degradation of a nucleic acid molecule may be preferable if precise timing of protein production is desired. Thus, in some embodiments, 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.). In some embodiments, the polynucleotides may include one or more messenger RNAs (mRNAs) having one or more alternative nucleoside or nucleotides (i.e., mRNA molecules). In some embodiments, the polynucleotides may include one or more oligonucleotides having one or more alternative nucleoside or nucleotides. In some embodiments, a composition of the invention includes an mRNA and/or one or more oligonucleotides having one or more alternative nucleoside or nucleotides.

Methods of Treatment

Provided herein are methods for treating disorders associated with protein dysfunction in a patient (e.g., a human patient) in need thereof (e.g., 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). In some embodiments, the patient is administered a bifunctional protease, e.g., a sheddase, protease, or amyloid-degrading enzyme (e.g., Ap-degrading enzymes) of the disclosure.

Pharmaceutical Composition

Any of the compositions disclosed herein (e.g., a composition that includes a bifunctional protease of the disclosure) 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.

For instance, pharmaceutical 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. For example, 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).

Pharmaceutical 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. 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.

Routes of Administration

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. In some embodiments, 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.

Examples

Example 1. Preparation and Characterization of a Bifunctional Molecule, MMP7-6xG4S- TNFR1(2.6)

This Example describes the construction and testing of a bifunctional molecule with targeted proteolytic activity against the protein target tumor necrosis factor alpha (TNFa). In this example, 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.

The bifunctional molecule was expressed by transfecting human embryonic kidney (HEK) 293 cells. HEK 293 cells were plated at 2x10⁵ 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.

Western blotting of HEK 293 supernatants was conducted to confirm the secretion of the bifunctional molecules. Samples were taken 96 hours. The medium was removed from the cells. 80 pL was mixed with 20 pl of 5xSDS sample buffer. For protein expression analysis, samples in SDS loading buffer were loaded on to a NuPage 4-12% Bis Tris gel. The gel was run in MOPS running buffer. Proteins were transferred to PVDF membrane using transfer buffer. The blot was blocked in TBST with 5% milk powder overnight at 4°C. Blot was then incubated in TBST/5% milk with anti-HA- HRP antibody overnight at 4°C. 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.

Example 2. Preparation and Characterization of Bifunctional Molecules Comprising Mesotrypsin and a TNFa-Binding Domain

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). To produce the bifunctional molecule, 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.

To assess the activity of the bifunctional molecule, 50 pM TNFa was incubated with various doses (dose response) of the bifunctional molecule (mesotrypsin-TNFR1 ) overnight, and the reaction mixture was added to HEK-BLUE™ TNF-a reporter cell lines to measure inhibition of target protein activity (Fig. 1 C). Mesotrypsin and a catalytically inactive form of the bifunctional molecule were provided as controls. Mesotrypsin and the bifunctional molecule (mesotrypsin-TNFR1 ) were activated with enterokinase and were therefore catalytically active; promesotrypsin-TNFR1 had not been activated with enterokinase, so was catalytically inactive. 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. Next, 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.

To test the activity of these molecules, 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. The resulting mixture was western blotted with an antibody against TNFa (Fig. 1 D). Mesotrypsin was provided as a control. As shown in Fig. 1 D, 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 (VHH3, in the uM binding range) 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.

Table 4. Sequences Relating to Bifunctional Molecules Comprising Mesotrypsin and a TNFa-

Binding Domain

Example 3. Preparation and Characterization of Bifunctional Molecules Comprising MMP7 or MMP12 and an IL-13-Binding Domain

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. To produce the bifunctional molecules, 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.

Each of the bifunctional molecules was incubated at 18 nM in 98% human serum. Proteolytic activity against a fluorogenic peptide (Mca-KPLGL-Dpa-AR-NH2) was tested at the noted times (Fig. 2C). Serum was heat-treated at 56°C for 30 minutes, as per standard cell-culture protocols). 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. Fig. 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). Next, 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. In each molecule, 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). Autocatalysis of the bifunctional molecules was assessed. Each construct was incubated at 5 nM and 37°C and measured by western blot after 30 seconds, 2 hours, or 16 hours with an antibody against its catalytic domain to detect changes in molecular weight resulting from autocatalytic cleavage events (Fig. 3). A catalytically inactive form of the mesotrypsin bifunctional molecule was included as a negative control. As shown in Fig. 3, autocatalysis of proteolytic fusion proteins can be mitigated and minimized by selection of enzymatic domains and binder domains that are compatible against self-proteolysis.

Table 5. Sequences Relating to Bifunctional Molecules Comprising MMP7 or MMP12 and an

IL-13-Binding Domain

Example 4. Preparation and Characterization of Bifunctional Molecules Comprising an Amyloid Beta Binding Domain

Enzymatic constructs comprising an enzyme and a targeting domain, as shown in Fig. 4A, were designed and tested. In each construct, 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 (enzymatic construct) comprising (i) an enzyme and (ii) an scFv is shown in Fig. 4A (“enzyme-scFv”).

In Fig. 4A, “HEX” indicates that the natural MMP hemopexin domain was preserved; where HEX is omitted, there was not a hemopexin domain. For example, a bifunctional molecule (enzymatic construct) 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”).

Accordingly, 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). A bifunctional molecule comprising

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.

Accordingly, 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).

Further, 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). A bifunctional molecule comprising the aducanumab scFv, an Fc region, and KLK7 is shown in 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.

To produce the bifunctional molecules, 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.

Proteolytic activity of the protein constructs was tested in fluorogenic peptide assays (Figs. 4B-4F).

The 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).

The kallikrein constructs were activated with 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).

The 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.

As shown in Figs. 4B-4E, 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.

Table 6. Sequences Relating to Bifunctional Molecules Comprising an Amyloid Beta Binding Domain

Table 7. Enzyme Sequences

Other Embodiments

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

Claims

WHAT IS CLAIMED IS:

1 . A bifunctional macromolecule comprising:

(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.

2. The bifunctional macromolecule of claim 1 , wherein the proteolytic domain is a sheddase domain.

3. The bifunctional macromolecule of claim 1 or 2, wherein the proteolytic domain is derived from a serine protease.

4. The bifunctional macromolecule of claim 1 , wherein 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.

5. The bifunctional macromolecule of claim 1 , wherein the proteolytic domain is an amyloid beta (Ap)-degrading enzyme.

6. 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 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.

7. 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 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.

8. The bifunctional macromolecule of claim 5, wherein 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.

9. 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 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.

10. The bifunctional macromolecule of claim 9, wherein 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.

11 . 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.

12. 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.

13. The bifunctional macromolecule of claim 12, wherein the proteolytic domain is the proteolytic domain of human MMP2, human MMP9, human MMP14, human CTSB, human CTSD, or human KLK7.

14. The bifunctional macromolecule of any one of claims 1 -13, wherein the target molecule and the substrate polypeptide are the same molecule.

15. The bifunctional macromolecule of any one of claims 1 -13, wherein the target molecule and the substrate polypeptide are not the same molecule.

16. The bifunctional macromolecule of any one of claims 1 -15, wherein the targeting domain comprises a polypeptide.

17. The bifunctional macromolecule of claim 16, wherein the targeting domain polypeptide comprises a monoclonal antibody, 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.

18. The bifunctional macromolecule of any one of claims 1 -15, wherein the targeting domain comprises a glycan.

19. The bifunctional macromolecule of any one of claims 1 -15, wherein the targeting domain comprises a polynucleotide.

20. The bifunctional macromolecule of any one of claims 1 -19, wherein the substrate polypeptide is a soluble polypeptide.

21 . The bifunctional macromolecule of any one of claims 1 -19, wherein the substrate polypeptide is a membrane-associated polypeptide.

22. The bifunctional macromolecule of any one of claims 1 -21 , wherein the target molecule is intracellular.

23. The bifunctional macromolecule of any one of claims 1 -21 , wherein the target molecule is extracellular.

24. A bifunctional macromolecule comprising:

(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.

25. A bifunctional macromolecule comprising:

(a) a targeting domain that binds a membrane-embedded target molecule; and

26. A bifunctional macromolecule comprising:

(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.

27. A bifunctional macromolecule comprising: (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.

28. A bifunctional macromolecule comprising:

(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.

29. A bifunctional macromolecule comprising:

(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.

30. A bifunctional macromolecule comprising:

(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.

31 . The bifunctional macromolecule of any one of claims 25-27, 29, and 30, wherein the target molecule and the substrate polypeptide are the same molecule.

32. The bifunctional macromolecule of any one of claims 25-27, 29, and 30, wherein the target polypeptide and the substrate polypeptide are not the same polypeptide.

33. A pharmaceutical composition comprising the bifunctional macromolecule of any one of claims 1 -32 and a pharmaceutically acceptable carrier.

34. A nucleic acid encoding the bifunctional macromolecule of any one of claims 1 -17 and 20-33.

35. The nucleic acid of claim 34, wherein the nucleic acid is an RNA.

36. A vector encoding the bifunctional macromolecule of any one of claims 1 -17 and 20-33.

37. A host cell comprising the bifunctional macromolecule of any one of claims 1 -32, the nucleic acid of claim 34 or 35, or the vector of claim 36.

38. The host cell of claim 37, wherein the host cell is a eukaryotic cell.

39. The host cell of claim 38, wherein the eukaryotic cell is a mammalian cell.

40. A pharmaceutical composition comprising the bifunctional macromolecule of any one of claims 1 -32 or the nucleic acid of claim 34 or 35.

41 . The pharmaceutical composition of claim 40, further comprising one or more pharmaceutically acceptable carriers, diluents, and/or excipients.

42. A method of treating a disorder in a subject in need thereof, the method comprising the step of administering to the subject an effective amount of the bifunctional macromolecule of any one of claims 1 to 32 or the pharmaceutical composition of any one of claims 33, 40, and 41 .

43. A method of altering the function of a membrane-associated polypeptide, the method comprising the step of contacting the cell with the bifunctional macromolecule of any one of claims 1 to 32 or the pharmaceutical composition of any one of claims 33, 40, and 41 .

44. A method of cleaving a target polypeptide, the method comprising the step of contacting the target polypeptide with the bifunctional macromolecule of any one of claims 1 to 32 or the pharmaceutical composition of any one of claims 33, 40, and 41 .

45. A method of cleaving a heterologous polypeptide, the method comprising the step of contacting the heterologous target molecule with a bifunctional macromolecule comprising

(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.

46. The method of claim 45, wherein the target polypeptide and the heterologous polypeptide are the same polypeptide.

47. The method of claim 45, wherein the target polypeptide and the heterologous polypeptide are not the same polypeptide.

48. The method of any one of claims 45-47, wherein the heterologous target is not a membrane- associated polypeptide.

49. The method of any one of claims 45-48, wherein the proteolytic domain is a sheddase domain.

50. The method of any one of claims 45-49, wherein 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.

51 . The method of claim 50, wherein 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.

52. The method of claim 50, wherein 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.

53. A method of cleaving a membrane-associated polypeptide, the method comprising the step of contacting the membrane-embedded polypeptide with a macromolecule comprising

(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.

54. The method of claim 53, wherein 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.

55. 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.