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WO2025079025A1 - Ides-rapamycin conjugate in aav gene therapy - Google Patents

Ides-rapamycin conjugate in aav gene therapy Download PDF

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Publication number
WO2025079025A1
WO2025079025A1 PCT/IB2024/059972 IB2024059972W WO2025079025A1 WO 2025079025 A1 WO2025079025 A1 WO 2025079025A1 IB 2024059972 W IB2024059972 W IB 2024059972W WO 2025079025 A1 WO2025079025 A1 WO 2025079025A1
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Prior art keywords
ides
protein
composition
conjugate
seq
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French (fr)
Inventor
Tayeba KHAN
Jon A. Kenniston
Madhusudan NATARAJAN
Deborah Palliser
Hong Myung Lee
Elise RICE
Alexander L. Watters
Nikolaos PAPAIOANNOU
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Takeda Pharmaceutical Co Ltd
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Takeda Pharmaceutical Co Ltd
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Publication of WO2025079025A1 publication Critical patent/WO2025079025A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Neutralizing antibodies (NAb) against the AAV vectors pose a major challenge to gene therapy.
  • Neutralizing antibodies block liver transduction and vector re-administration.
  • Neutralizing antibodies can also be induced from rAAV dosing, which in turn creates the additional challenge of re-dosing patients that have received rAAV gene therapy.
  • Current strategies aimed at overcoming anti-AAV antibodies often involve immunosuppression and are not efficient in removing pre-existing antibodies.
  • Imlifidase is another strategy for use as an immunomodulatory therapy to remove anti-AAV NAbs.
  • IdeS is a naturally occurring cysteine protease derived from Streptococcus pyogenes, which has been demonstrated to rapidly and specifically cleave all subclasses of human IgG at the lower hinge region (von Pawel-Rammingen, EMBO J.2002, 21(7):1607-15; PMID 11927545).
  • IdeS is of bacterial origin, it is immunogenic in people (Winstedt 2015, PLoS One 201510(7): e0132011; PMID 26177518).
  • the present invention provides, among other things, a conjugate of an IgG degrading protease, e.g., Immunoglobulin G (IgG) degrading enzyme of the Streptococcus pyogenes (IdeS) protein or IgG-degrading enzyme/MAC-1 (IdeZ) protein, with an immunosuppressant molecule, e.g., rapamycin or sirolimus, for immunosuppression, for example, in gene therapy.
  • IgG degrading protease e.g., Immunoglobulin G (IgG) degrading enzyme of the Streptococcus pyogenes (IdeS) protein or IgG-degrading enzyme/MAC-1 (IdeZ) protein
  • an immunosuppressant molecule e.g., rapamycin or sirolimus
  • the present invention provides, among other things, a conjugate comprising an Immunoglobulin G (IgG)-degrading enzyme of Streptococcus pyogenes (IdeS) protein or IgG-degrading enzyme/Mac-1 (IdeZ) protein, and an immunosuppressant molecule.
  • IgG Immunoglobulin G
  • the IgG-degrading enzyme is conjugated to at least 1 immunosuppressant molecule.
  • the IgG-degrading enzyme is conjugated to at least 2 immunosuppressant molecules. In some embodiments, the IgG- degrading enzyme is conjugated to at least 3 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to at least 4 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to at least 5 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to at least 6 immunosuppressant molecules. In some embodiments, the IgG- degrading enzyme is conjugated to at least 7 immunosuppressant molecules.
  • the IgG-degrading enzyme is conjugated to at least 8 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to at least 9 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to at least 10 immunosuppressant molecules. In some embodiments, the IgG- degrading enzyme is conjugated to 10 or more immunosuppressant molecules. [0010] In some embodiments, the IgG-degrading enzyme is conjugated to 1 immunosuppressant molecule. In some embodiments, the IgG-degrading enzyme is conjugated to 2 immunosuppressant molecules.
  • the IgG-degrading enzyme is conjugated to 3 immunosuppressant molecules. In some embodiments, the IgG- degrading enzyme is conjugated to 4 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 5 immunosuppressant molecules. In some Attorney Docket No. MIL-035WO1 embodiments, the IgG-degrading enzyme is conjugated to 6 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 7 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 8 immunosuppressant molecules.
  • the IgG-degrading enzyme is conjugated to 9 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 10 immunosuppressant molecules. [0011] In some embodiments, the IdeS is conjugated to at least 1 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 2 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 3 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 4 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 5 immunosuppressant molecules.
  • the IdeS is conjugated to at least 6 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 7 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 8 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 9 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 10 immunosuppressant molecules. [0012] In some embodiments, the IdeS is conjugated to 1 immunosuppressant molecule. In some embodiments, the IdeS is conjugated to 2 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 3 immunosuppressant molecules.
  • the IdeS is conjugated to 4 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 5 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 6 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 7 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 8 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 9 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 10 immunosuppressant molecules. [0013] In some embodiments, the IdeZ is conjugated to at least 1 immunosuppressant molecules.
  • the IdeZ is conjugated to at least 2 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to at least 3 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to at least 4 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to at least 5 immunosuppressant Attorney Docket No. MIL-035WO1 molecules. In some embodiments, the IdeZ is conjugated to at least 6 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to at least 7 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to at least 8 immunosuppressant molecules.
  • the IdeZ is conjugated to at least 9 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to at least 10 immunosuppressant molecules. [0014] In some embodiments, the IdeZ is conjugated to 1 immunosuppressant molecule. In some embodiments, the IdeZ is conjugated to 2 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 3 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 4 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 5 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 6 immunosuppressant molecules.
  • the IdeZ is conjugated to 7 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 8 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 9 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 10 immunosuppressant molecules. [0015] In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:1 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:2 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:3 ratio.
  • the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:4 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:5 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:6 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:7 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:8 ratio.
  • the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:9 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:10 ratio. Attorney Docket No. MIL-035WO1 [0016] In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:1 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:2 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:3 ratio.
  • the IdeS and the immunosuppressant molecule are conjugated in at least a 1:4 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:5 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:6 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:7 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:8 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:9 ratio.
  • the IdeS and the immunosuppressant molecule are conjugated in at least a 1:10 ratio. [0017] In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:1 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:2 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:3 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:4 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:5 ratio.
  • the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:6 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:7 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:8 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:9 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:10 ratio.
  • the IgG-degrading enzyme is conjugated to the immunosuppressant molecules via surface amino acid residues on the IgG-degrading enzyme. In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecules via a surface lysine residue on the IgG-degrading enzyme. [0019] In some embodiments, the IdeS is conjugated to the immunosuppressant molecule via a surface lysine residue on IdeS. In some embodiments, the IdeS is conjugated to the immunosuppressant molecules via surface lysine residues on IdeS. Attorney Docket No.
  • the IdeZ is conjugated to the immunosuppressant molecule via a surface lysine residue on IdeZ. In some embodiments, the IdeZ is conjugated to the immunosuppressant molecules via surface lysine residues on IdeZ. [0021] In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecule via a C-terminus on the IgG-degrading enzyme. In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecule via a N-terminus on the IgG-degrading enzyme.
  • the IdeS is conjugated to the immunosuppressant molecule via a C-terminus on the IdeS. In some embodiments, the IdeS is conjugated to the immunosuppressant molecule via a N-terminus on the IdeS. [0023] In some embodiments, the IdeZ is conjugated to the immunosuppressant molecule via a C-terminus on the IdeZ. In some embodiments, the IdeZ is conjugated to the immunosuppressant molecule via a N-terminus on the IdeZ. [0024] In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecule via a cleavable linker.
  • the IgG- degrading enzyme is conjugated to the immunosuppressant molecule via a non-cleavable linker.
  • the IdeS is conjugated to the immunosuppressant molecule via a cleavable linker. In some embodiments, the IdeS is conjugated to the immunosuppressant molecule via a non-cleavable linker.
  • the IdeZ is conjugated to the immunosuppressant molecule via a cleavable linker. In some embodiments, the IdeZ is conjugated to the immunosuppressant molecule via a non-cleavable linker.
  • the IdeS comprises at least 70% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100% identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 75% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 80% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 85% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1.
  • the IdeS comprises at Attorney Docket No. MIL-035WO1 least 90% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 95% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises 100% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. [0028] In some embodiments, the IdeZ comprises at least 70% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51.
  • the IdeZ comprises at least 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100% identity to the wild-type IdEZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 75% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 80% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ protein comprises at least 85% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51.
  • the IdeZ comprises at least 90% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 95% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises 100% identity to the wild-type IdeZ protein of SEQ ID NO: 51. [0029] In some embodiments, the IdeS protein comprises an R39G mutation relative to the wild-type IdeS protein.
  • the IdeS protein is a variant that further comprises one or more mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises at least one mutation selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • the IdeS protein further comprises an E154D mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an Y155H mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an Y155Q mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an F161H mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an F161Y mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an L164H mutation relative to the wild-type IdeS protein.
  • the IdeS protein further comprises an F297T mutation relative to the Attorney Docket No. MIL-035WO1 wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an F297N mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an V300R mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an A303E mutation relative to the wild-type IdeS protein.
  • the IdeS protein is a variant that further comprises one mutation selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises between 2-10 mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • the IdeS protein is a variant that further comprises two mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises three mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • the IdeS protein is a variant that further comprises four mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises five mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • the IdeS protein is a variant that further comprises six mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises seven mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • the IdeS protein is a variant that further comprises eight mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises nine mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • the IdeS protein is a variant that further comprises the combination of mutations consisting of E154D, Y155H, Y155Q, Attorney Docket No. MIL-035WO1 F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • the IdeS protein is a variant comprising a D30-T49 deletion relative to wild-type IdeS protein.
  • the IdeS protein is a variant comprising a DSFSANCEIRYSEVTPYHVT (SEQ ID NO: 50) deletion.
  • the wild-type IdeS protein has 100% identity to SEQ ID NO: 1.
  • the IdeS protein comprises at least 80% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises 80-85%, 85-90%, 95-100% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 2. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 3. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 4.
  • the IdeS protein comprises at least 80% identity to SEQ ID NO: 5. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 6. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 7. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 8. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 9. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 10. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 11. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 12.
  • the IdeS protein comprises at least 80% identity to SEQ ID NO: 13. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 14. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 15. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 16. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 17. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 18. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 19. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 20.
  • the IdeS protein comprises at least 80% identity to SEQ ID NO: 21. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 22. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 23. In some Attorney Docket No. MIL-035WO1 embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 24. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 25. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 26. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 27.
  • the IdeS protein comprises at least 80% identity to SEQ ID NO: 28. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 29. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 30. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 31. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 32. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 33. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 34. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 35.
  • the IdeS protein comprises at least 80% identity to SEQ ID NO: 36. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 37. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 38. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 39. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 40. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 41. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 42. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 43.
  • the IdeS protein comprises at least 80% identity to SEQ ID NO: 44. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 45. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 46. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 47. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 48. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 49. [0035] In some embodiments, the IdeS protein comprises at least 90% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49.
  • the IdeS protein comprises at least 90% identity to SEQ ID NO: 2. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 3. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 4. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 5. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 6. In some embodiments, the IdeS protein comprises at Attorney Docket No. MIL-035WO1 least 90% identity to SEQ ID NO: 7. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 8. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 9.
  • the IdeS protein comprises at least 90% identity to SEQ ID NO: 10. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 11. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 12. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 13. In some embodiments, the IdeS comprises at least 90% identity to SEQ ID NO: 14. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 15. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 16. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 17.
  • the IdeS comprises at least 90% identity to SEQ ID NO: 18. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 19. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 20. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 21. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 22. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 23. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 24. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 25.
  • the IdeS protein comprises at least 90% identity to SEQ ID NO: 26. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 27. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 28. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 29. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 30. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 31. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 32. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 33.
  • the IdeS protein comprises at least 90% identity to SEQ ID NO: 34. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 35. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 36. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 37. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 38. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 39. In some embodiments, the IdeS protein comprises at least 90% identity to Attorney Docket No. MIL-035WO1 SEQ ID NO: 40.
  • the IdeS protein comprises at least 90% identity to SEQ ID NO: 41. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 42. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 43. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 44. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 45. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 46. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 47. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 48.
  • the IdeS protein comprises at least 90% identity to SEQ ID NO: 49. [0036] In some embodiments, the IdeS protein comprises at least 100% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 2. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 3. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 4. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 5. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 6. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 7.
  • the IdeS protein comprises at least 100% identity to SEQ ID NO: 8. In some embodiments, the IdeS comprises at least 100% identity to SEQ ID NO: 9. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 10. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 11. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 12. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 13. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 14. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 15.
  • the IdeS protein comprises at least 100% identity to SEQ ID NO: 16. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 17. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 18. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 19. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 20. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 21. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 22. In some embodiments, the IdeS protein Attorney Docket No.
  • MIL-035WO1 comprises at least 100% identity to SEQ ID NO: 23.
  • the IdeS protein comprises at least 100% identity to SEQ ID NO: 24.
  • the IdeS protein comprises at least 100% identity to SEQ ID NO: 25.
  • the IdeS protein comprises at least 100% identity to SEQ ID NO: 26.
  • the IdeS protein comprises at least 100% identity to SEQ ID NO: 27.
  • the IdeS protein comprises at least 100% identity to SEQ ID NO: 28.
  • the IdeS protein comprises at least 100% identity to SEQ ID NO: 29.
  • the IdeS protein comprises at least 100% identity to SEQ ID NO: 30.
  • the IdeS protein comprises at least 100% identity to SEQ ID NO: 31. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 32. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 33. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 34. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 35. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 36. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 37. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 38.
  • the IdeS protein comprises at least 100% identity to SEQ ID NO: 39. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 40. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 41. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 42. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 43. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 44. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 45. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 46.
  • the IdeS protein comprises at least 100% identity to SEQ ID NO: 47. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 48. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 49. [0037] In some embodiments, the immunosuppressant molecule is rapamycin, an analog or mimetic thereof. In some embodiments, the immunosuppressant molecule is rapamycin. In some embodiments, the immunosuppressant molecule is an analog of rapamycin. In some embodiments, the immunosuppressant molecule is a mimetic of rapamycin. Attorney Docket No.
  • the rapamycin analog is selected from the group consisting of deforolimus, everolimus, and temsirolimus. In some embodiments, the rapamycin analog is deforolimus. In some embodiments, the rapamycin analog is everolimus. In some embodiments, the rapamycin analog is temsirolimus. [0039] In some embodiments, the rapamycin mimetic is isoliquiritigenin or withaferin A. In some embodiments, the rapamycin mimetic is isoliquiritigenin. In some embodiments, the rapamycin mimetic is withaferin A.
  • IdeS is conjugated to a rapamycin-linker payload selected from the group consisting of any one of: [0041] In some embodiments, the IdeS is conjugated to rapamycin linker payload Compound 1 (FIG.2A). In some embodiments, the IdeS is conjugated to rapamycin linker payload Compound 2 (FIG.2B). In some embodiments, the IdeS is conjugated to rapamycin linker payload Compound 4 (FIG.2D). Attorney Docket No. MIL-035WO1 [0042] In some embodiments, Compound 1, Compound 2, or Compound 4 are monomeric compounds. In some embodiments, Compound 1 is a monomeric compound.
  • Compound 2 is a monomeric compound.
  • Compound 4 is a monomeric compound.
  • a composition comprising a plurality of conjugates described herein.
  • the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of between 1 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition comprises one or more immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition comprises up to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 1 immunosuppressant molecule per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 2 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 3 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 4 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 5 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 6 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 7 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 8 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some Attorney Docket No.
  • the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. [0044] In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of between 1 to 10 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition comprises one or more immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition comprises up to 10 immunosuppressant molecules per IdeS molecule.
  • the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 1 immunosuppressant molecule per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 2 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 3 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 4 immunosuppressant molecules per IdeS molecule.
  • the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 5 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 6 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 7 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 8 immunosuppressant molecules per IdeS molecule.
  • the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 9 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 10 immunosuppressant molecules per IdeS molecule. [0045] In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 10 Attorney Docket No. MIL-035WO1 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9 or 8 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 8 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 7 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 6 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 5 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 4 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 3 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 2 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 8 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 7 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 6 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 5 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 4 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 3 immunosuppressant molecules per molecule of IgG-degrading enzyme. [0047] In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 3 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 3 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 3 to 8 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 3 to 7 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 3 to 6 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 3 to 5 immunosuppressant molecules per Attorney Docket No. MIL-035WO1 molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 3 to 4 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 4 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 4 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 4 to 8 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 4 to 7 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 4 to 6 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 4 to 5 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 5 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 5 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 5 to 8 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 5 to 7 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 5 to 6 immunosuppressant molecules per molecule of IgG-degrading enzyme. Attorney Docket No. MIL-035WO1 [0050] In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 6 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 6 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 6 to 8 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 6 to 7 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 7 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 7 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 7 to 8 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 8 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 8 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. [0053] In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 9 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme.
  • the composition comprises less than 5% unconjugated IgG-degrading enzyme. In some embodiments, the composition comprises less than 1% unconjugated IgG-degrading enzyme. In some embodiments, the composition comprises less than 0.1% unconjugated IgG-degrading enzyme. In some embodiments, the composition Attorney Docket No. MIL-035WO1 comprises less than 5% unconjugated IdeS. In some embodiments, the composition comprises less than 1% unconjugated IdeS. In some embodiments, the composition comprises less than 0.1% unconjugated IdeS.
  • the present invention provides a method of reducing an immune response in a subject, comprising administering to the subject in need thereof, a therapeutically effective dose of the composition described herein.
  • the present invention provides a method of reducing an immune response in a subject, wherein the method comprises administering to the subject in need thereof, a therapeutically effective dose of a composition comprising a conjugate of an IdeS or IdeZ protein and an immunosuppressant molecule.
  • the immune response generates one or more antibodies.
  • the antibody is IgG.
  • the antibody is IgG1, IgG2, IgG3 or IgG4.
  • the antibody is IgG1. In some embodiments, the antibody is IgG2. In some embodiments, the antibody is IgG3. In some embodiments, the antibody is IgG4. In some embodiments, the IgG2 antibody is IgG2a or IgG2b. In some embodiments, the IgG2 antibody is IgG2a. In some embodiments, the IgG2 antibody is IgG2b. [0058] In some embodiments, the composition is administered with a gene therapy vector or a replacement enzyme. In some embodiments, the composition is administered with a gene therapy vector. In some embodiments, the composition is administered with a replacement enzyme. [0059] In some embodiments, the composition is administered prior to administration of the gene therapy vector or the replacement enzyme.
  • the composition is administered prior to administration of a gene therapy vector. In some embodiments, the composition is administered prior to administration of the replacement enzyme. [0060] In some embodiments, the composition is administered at least 3 days, 5 days, or 7 days prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered at least 3 days prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 5 days prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 7 days prior to administration of the gene therapy vector. Attorney Docket No. MIL-035WO1 In some embodiments, the composition is administered at least 3 days prior to administration of the replacement enzyme.
  • the composition is administered at least 5 days prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 7 days prior to administration of the replacement enzyme. [0061] In some embodiments, the composition is administered between 1 day to 3 days prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered at least 1 day prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 2 days prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 3 days prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 1 day prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 2 days prior to administration of the replacement enzyme.
  • the composition is administered at least 3 days prior to administration of the replacement enzyme. [0062] In some embodiments, the composition is administered at least 1 hour, 3 hours, 5 hours, 10 hours, 12 hours, 16 hours, or 24 hours prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered at least 1 hour to 3 hours, 3 to 5 hours, 5 to 10 hours, 10 to 12 hours, 12 to 16 hours, or 16 to 24 hours, or any discrete intervening quantity therebetween, prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered at least 1 hour prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 3 hours prior to administration of the gene therapy vector.
  • the composition is administered at least 5 hours prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 10 hours prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 12 hours prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 16 hours prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 24 hours prior to administration of the replacement enzyme.
  • Attorney Docket No. MIL-035WO1 [0063] In some embodiments, the composition is administered at least 1 hour prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 3 hours prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 5 hours prior to administration of the replacement enzyme.
  • the composition is administered at least 10 hours prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 12 hours prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 16 hours prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 24 hours prior to administration of the replacement enzyme. [0064] In some embodiments, the composition is administered concurrently with administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered concurrently with administration of the gene therapy vector. In some embodiments, the composition is administered concurrently with administration of the replacement enzyme. [0065] In some embodiments, the gene therapy vector is an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • the gene therapy vector is an adenovirus vector. In some embodiments, the gene therapy vector is a lentiviral vector. [0066] In some embodiments, the gene therapy vector encodes a protein that elicits production of IgG antibodies. In some embodiments, the protein is a heterologous protein. [0067] In some embodiments the protein that elicits production of IgG antibodies is an AAV capsid protein. In some embodiments, the protein that elicits production of IgG antibodies is a lentiviral envelope protein. [0068] In some embodiments, provided by the present invention is a method of increasing transduction efficiency of a gene therapy vector, the method comprising administering the composition described herein.
  • the replacement enzyme is a lysosomal enzyme.
  • the lysosomal enzyme is selected from an acid phosphatase, an cid phosphodiesterase, an acid ribonuclease, an acid deoxyribonuclease, a ⁇ - Galactosidase, an ⁇ -Glucosidase, an ⁇ -Mannosidase, a ⁇ - Glucoronidase, a lysozymes, a hyaluronidase, anarylsulphatase, a cathepsin, a collagenase, a peptidase, an esterase, a Attorney Docket No.
  • the replacement enzyme is an arylsulfatase A or iduronate-2-sulfatase. In some embodiments, the replacement enzyme is arylsulfatase A. In some embodiments, the replacement enzyme is iduronate-2-sulfatase. In some embodiments, the replacement enzyme is a lysosomal enzyme from Table 4. [0072] In some embodiments, the subject receives an organ transplant.
  • provided by the present invention is a method of reducing organ transplant rejection comprising administering the composition described herein.
  • the composition is administered prior to the organ transplant.
  • IgG cleavage into scIgG, F(ab) 2 , and Fc fragments.
  • IgG is cleaved to scIgG.
  • administration of the composition comprising the conjugate to a subject reduces induction of a T cell response relative to a control.
  • the control is the level of a T cell response in the same subject treated with unconjugated IdeS. [0078] In some embodiments the control is the level of a T cell response in a different subject treated with unconjugated IdeS. [0079] In some embodiments, the T cell response is reduced by 10%-20%, 20%-50%, 50%-80% or 80%-95%, or any intervening quantity therebetween, relative to the control. In some embodiments, the T cell response is reduced by 10%-20% relative to the control. In some embodiments, the T cell response is reduced by 20%-50% relative to the control. In some embodiments, the T cell response is reduced by 50-80% relative to the control.
  • the T cell response is reduced by 80-95% relative to the control. In some embodiments, the T cell response is reduced by at least 10% relative to the control. In some embodiments, the T cell response is reduced by at least 20% relative to the control. In some embodiments, the T cell response is reduced by at least 30% relative to the control. In some embodiments, the T cell response is reduced by at least 40% relative to the control. In some Attorney Docket No. MIL-035WO1 embodiments, the T cell response is reduced by at least 50% relative to the control. In some embodiments, the T cell response is reduced by at least 60% relative to the control. In some embodiments, the T cell response is reduced by at least 70% relative to the control.
  • the T cell response is reduced by at least 80% relative to the control. In some embodiments, the T cell response is reduced by at least 90% relative to the control. In some embodiments, the T cell response is reduced by at least 95% relative to the control. In some embodiments, the T cell response is reduced by at least 99% relative to the control. [0080] In some embodiments, the T cell response is reduced by greater than 95% relative to the control. In some embodiments the T cell response is reduced by greater than 96% relative to the control. In some embodiments the T cell response is reduced by greater than 97% relative to the control. In some embodiments the T cell response is reduced by greater than 98% relative to the control. In some embodiments, the T cell response is reduced by greater than 99% relative to the control.
  • administration of the composition comprising the conjugate reduces induction of inflammatory cytokines relative to the control.
  • the inflammatory cytokines are IFN-gamma, IL-17 alpha, IL-2, IL-5, IL-6, TNF-alpha, and/or monocyte chemoattractant protein-1 (MCP-1).
  • MCP-1 monocyte chemoattractant protein-1
  • the inflammatory cytokines are IFN-gamma.
  • the inflammatory cytokines are IL-17 alpha.
  • the inflammatory cytokines are IL-2.
  • the inflammatory cytokines are IL-5.
  • the inflammatory cytokines are IL-6. In some embodiments, the inflammatory cytokines are TNF-alpha. In some embodiments, the inflammatory cytokines are MCP-1. [0083] In some embodiments, the subject has cancer or an autoimmune disease. In some embodiments, the subject has cancer. In some embodiments, the subject has an autoimmune disease. [0084] In some embodiments, the composition is administered concurrently with or prior to adoptive cell transfer therapy. In some embodiments, the composition is administered concurrently with adoptive cell transfer therapy. In some embodiments, the composition is administered prior to adoptive cell transfer therapy. [0085] In some embodiments, the composition is administered concurrently with or prior to ex vivo cell therapy.
  • the composition is administered Attorney Docket No. MIL-035WO1 concurrently with ex vivo cell therapy. In some embodiments, the composition is administered prior to ex vivo cell therapy.
  • FIG.1A is a schematic that illustrates an IdeS protein, wherein amino acids 1-29 denote a signal peptide; amino acids 1-40 denote a putative signal sequence, amino acids 214-216 denote an RGF motif that is important for ligand recognition and amino acids C94 and H224 are catalytic residues.
  • FIG.1B is a schematic that illustrates sortase mediated conjugation of rapamycin linker-payloads to IdeS.
  • FIG.1C is a schematic that illustrates stochastic lysine conjugation of rapamycin linker payloads to IdeS.
  • FIG.2A to FIG.2D depicts the structures of exemplary rapamycin linker-payloads for IdeS-rapamycin conjugation.
  • FIG.2A depicts the structure of Compound 1, comprising a non-cleavable linker.
  • FIG.2B depicts the structure of Compound 2, comprising a disulfide- cleavable linker.
  • FIG.2C depicts the structure of Compound 3, which is a non-rapamycin linker control.
  • FIG.2D depicts the structure of Compound 4, which is a disulfide-cleavable linker, NHS ester.
  • FIG.3A to FIG.3D depict quality analysis of the conjugation products.
  • FIG.3A shows SEC and LC-QTOF results for IdeS-rapamycin conjugate with sortase-mediated conjugation to Compound 1.
  • FIG.3B shows SEC and LC-QTOF results for IdeS-rapamycin conjugate with sortase-mediated conjugation to Compound 2.
  • FIG.3C shows SEC and LC- QTOF results for IdeS-rapamycin conjugate with sortase-mediated conjugation to Compound 3.
  • FIG.3D shows SDS-PAGE gel and Coomassie staining results for IdeS-rapamycin conjugates made with stochastic lysine conjugation chemistry.
  • FIG.4A to FIG.4B show results from enzymatic activity assays.
  • FIG.4A is an SDS- PAGE gel and Coomassie staining of results from an IgG cleavage assay to determine IdeS enzyme activity in IdeS-rapamycin sortase-mediated conjugates.
  • FIG.4B is a SDS-PAGE gel and Coomassie staining of results from an IgG cleavage assay to determine IdeS enzyme activity in IdeS-rapamycin stochastic lysine conjugates.
  • FIG.5 shows T cell proliferation response and percent positive donor response rate for human PBMCs incubated with IdeS or IdeS + rapamycin.
  • FIG.6 shows T cell proliferation response and percent positive donor response rate for human PBMCs incubated with IdeS or IdeS-rapamycin sortase-mediated conjugates.
  • FIG.7A to FIG.7B show immune response after incubation of human PBMCs with IdeS, rapamycin, or IdeS-rapamycin conjugates.
  • FIG.7A shows T cell proliferation response and percent positive donor response rate for human PBMCs incubated with IdeS or IdeS- rapamycin stochastic lysine conjugates.
  • FIG.7B is an inflammatory cytokine panel for IdeS and IdeS-rapamycin stochastic lysine conjugates.
  • FIG.8 is an overview of synthesis for rapamycin linker-payload Compound 1.
  • FIG.9A to FIG.9D show the synthesis steps for rapamycin linker-payload Compound 1.
  • FIG.9A shows synthesis step 1 for rapamycin linker-payload Compound 1.
  • FIG.9B shows synthesis step 2 for rapamycin linker-payload Compound 1.
  • FIG.9C shows synthesis step 3 for rapamycin linker-payload Compound 1.
  • FIG.9D shows synthesis step 4 for rapamycin linker-payload Compound 1.
  • FIG.10 is an overview of synthesis for rapamycin linker-payload Compound 2.
  • FIG.11A to FIG.11E show the synthesis steps for rapamycin linker-payload Compound 2.
  • FIG.11A shows synthesis step 1 for rapamycin linker-payload Compound 2.
  • FIG.11B shows synthesis step 2 for rapamycin linker-payload Compound 2.
  • FIG.11C shows synthesis step 3 for rapamycin linker-payload Compound 2.
  • FIG.11D shows synthesis step 4 for rapamycin linker-payload Compound 2.
  • FIG.11E shows synthesis step 5 for rapamycin linker-payload Compound 2.
  • FIG.12 is an overview of synthesis for rapamycin linker-payload Compound 4.
  • FIG.13A to FIG.13E show the synthesis steps for rapamycin linker-payload Compound 4.
  • FIG.13A shows synthesis step 1 for rapamycin linker-payload Compound 4.
  • FIG.13B shows synthesis step 2 for rapamycin linker-payload Compound 4.
  • FIG.13C shows synthesis step 3 for rapamycin linker-payload Compound 4.
  • FIG.13D shows synthesis step 4 for rapamycin linker-payload Compound 4.13E shows synthesis step 5 for rapamycin linker-payload Compound 4.
  • Adeno-associated virus As used herein, the terms “adeno-associated virus” or “AAV” or “recombinant AAV” (“rAAV”) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV (see, e.g., Fields et al. Virology, volume 2, chapter 69 (4 th ed. Lippincott-Raven Publishers); Gao et al., J.
  • AAV can infect both dividing and non-dividing cells and can be present in an extrachromosomal state without integrating into the host cell genome.
  • AAV vectors are commonly used in gene therapy, and in some embodiments, AAV vectors are engineered.
  • the AAV vectors can be engineered through any methods known in the art. For example, in some embodiments, AAV capsids are engineered through protein engineering methods.
  • Administering As used herein, the terms “administering” or “introducing” are used interchangeably in the context of delivering rAAV vectors encoding an antibody into a subject, by a method or route which results in efficient delivery of the rAAV vector.
  • the terms “administering” and “introducing” are also used interchangeably in the context of delivering, for example and without limitation, enzyme replacement treatment, adoptive cell therapy, ex vivo cell therapy, IgG degrading enzymes, immunosuppressants, and IdeS- rapamycin conjugates.
  • Various methods are known in the art for administering treatments, including, for example and without limitation, intravenously, subcutaneously, and transdermally.
  • Transdermal administration of an rAAV vector can be performed by use of a “gene gun” or biolistic particle delivery system.
  • the rAAV vectors are administered via non-viral lipid nanoparticles.
  • Adoptive Cell Therapy refers to the transfer of cells into a patient in need thereof.
  • the cells can be derived Attorney Docket No. MIL-035WO1 and propagated from the patient in need or could have been obtained from a non-patient donor.
  • the cell is an immune cell, such as a lymphocyte.
  • Antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • Ig immunoglobulin
  • Antibodies include IgG, IgG2, IgG3, IgG4, IgG2a, and IgG2b. Antibodies include antibody fragments. Antibodies also include, but are not limited to, polyclonal, monoclonal, chimeric dAb (domain antibody), single chain, F ab , F ab’ , F (ab’)2 fragments, scFvs. [0105] Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value.
  • the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • Enzyme Replacement Therapy As used herein, the term “enzyme replacement therapy (ERT)” refers to any therapeutic strategy that corrects an enzyme deficiency by providing the missing enzyme.
  • the composition When used in combination with the composition, the composition reduces the level of antibodies against the recombinant, replacement enzyme in serum.
  • Ex vivo As used herein, the term “ex vivo” means a process in which cells are removed from a living organism and are propagated outside the organism (e.g., in a test tube, in a culture bag, in a bioreactor).
  • Immune Response As used herein, the term “immune response” refers to both active and passive immune response to an unknown antigen. For example, in some embodiments, IdeS may induce an immune response in the body, and IdeS-rapamycin conjugates may reduce said immune response as measured by, for example and without limitations, T cell proliferation or inflammatory cytokine induction.
  • in vivo refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell- based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
  • Linker As used herein: the term “linker” refers to, in a conjugate, a compound connecting the IgG degrading enzyme to the immunosuppressant molecule.
  • Linker-Payload refers to the rapamycin-linker compound to be conjugated to the IgG degrading enzyme.
  • Polypeptide refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of ordinary skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. As is known to those skilled in the art, polypeptides may be processed and/or modified.
  • Protein The term “protein” as used herein refers to one or more polypeptides that function as a discrete unit.
  • polypeptide and “protein” may be used interchangeably. If the discrete functional unit is comprised of more than one polypeptide that physically associate with one another, the term “protein” refers to the multiple polypeptides that are physically coupled and function together as the discrete unit.
  • Recombinant means that compositions have been manipulated or engineered in a fashion that generally does not occur Attorney Docket No. MIL-035WO1 in nature.
  • Recombinant can be used as a modifier of a, for example, viral vector, such as, without limitation, a recombinant AAV (rAAV) vector. It can also be used as a modifier of sequences such as recombinant polynucleotides or polypeptides.
  • a particular example of a recombinant AAV vector would be where a nucleic acid that is not normally present in a wild-type AAV genome (heterologous polynucleotide) is inserted within a viral genome.
  • a nucleic acid e.g., gene
  • Subject refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate).
  • a human includes pre- and post-natal forms.
  • a subject is a human being.
  • a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
  • the term “subject” is used herein interchangeably with “individual” or “patient.”
  • a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • therapeutically effective amount As used herein, the term “therapeutically effective amount” of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.
  • Treating refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease. Attorney Docket No.
  • Compounds of the present invention include those described generally herein, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated.
  • the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.
  • aliphatic or “aliphatic group”, as used herein, means a straight- chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle”, “cycloaliphatic”, or “cycloalkyl”), that has a single point of attachment to the rest of the molecule.
  • aliphatic groups contain 1-6 aliphatic carbon atoms.
  • aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms.
  • “cycloaliphatic” refers to a monocyclic C 3 -C 6 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is Attorney Docket No. MIL-035WO1 not aromatic, that has a single point of attachment to the rest of the molecule.
  • Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
  • heteroatom means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR ⁇ (as in N-substituted pyrrolidinyl)).
  • unsaturated as used herein, means that a moiety has one or more units of unsaturation.
  • bivalent C 1-15 (or C 1-6 ) saturated or unsaturated, straight or branched, hydrocarbon chain refers to bivalent alkylene, alkenylene, and alkynylene chains that are straight or branched as defined herein.
  • alkylene refers to a bivalent alkyl group.
  • An “alkylene chain” is a polymethylene group, i.e., -(CH 2 ) n -, wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3.
  • a substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.
  • alkenylene refers to a bivalent alkenyl group.
  • a substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.
  • halogen means F, Cl, Br, or I.
  • aryl used alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic or bicyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 7 ring members.
  • aryl may be used interchangeably with the term “aryl ring.”
  • aryl refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, Attorney Docket No.
  • MIL-035WO1 naphthyl, anthracyl and the like which may bear one or more sub stituents.
  • aryl is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.
  • heteroatom refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.
  • Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl.
  • heteroaryl and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring.
  • Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3 (4H)-one.
  • heteroaryl group may be mono- or bicyclic.
  • heteroaryl may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted.
  • heteroarylkyl refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.
  • heterocycle As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably and refer to a stable 5- to 7- membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above.
  • nitrogen includes a substituted nitrogen.
  • a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen Attorney Docket No.
  • MIL-035WO1 the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or + NR (as in N-substituted pyrrolidinyl).
  • a heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted.
  • saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl.
  • heterocycle used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl.
  • a heterocyclyl group may be mono- or bicyclic.
  • heterocyclylalkyl refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.
  • partially unsaturated refers to a ring moiety that includes at least one double or triple bond.
  • partially unsaturated is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.
  • compounds of the invention may contain “optionally substituted” moieties.
  • substituted means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
  • an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
  • Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; -(CH 2 ) 0-4 R ⁇ ; -(CH 2 ) 0-4 OR ⁇ ; - O(CH 2 ) 0-4 R ⁇ , -O-(CH 2 ) 0-4C (O)OR ⁇ ; -(CH 2 ) 0-4 CH(OR ⁇ ) 2 ; -(CH 2 ) 0-4 SR ⁇ ; -(CH 2 ) 0-4 Ph, which may be substituted with R ⁇ ; -(CH 2 ) 0-4 O(CH 2 ) 0-1 Ph which may be substituted with R ⁇ ; - CH ⁇ CHPh, which may be substituted with R ⁇ ; -(CH 2 ) 0-4 O(CH 2 ) 0-1 -pyridyl which may be substituted with R ⁇ ;
  • Suitable monovalent substituents on R ⁇ are independently halogen, -(CH 2 ) 0-2 R ⁇ , -(haloR ⁇ ), -(CH 2 ) 0-2 OH, -(CH 2 ) 0-2 OR ⁇ , -(CH 2 ) 0-2 CH(OR ⁇ ) 2 ; - O(haloR ⁇ ), -CN, -N 3 , -(CH 2 ) 0-2 C(O)R ⁇ , -(CH 2 ) 0-2 C(O)OH, -(CH 2 ) 0-2C (O)OR ⁇ , -(CH 2 ) 0-2 SR ⁇ , - (CH 2 ) 0-2 SH, -(CH 2 ) 0-2 NH 2 , -(CH 2 ) 0-2 NHR ⁇ ,
  • Suitable divalent substituents on a saturated carbon atom of R ⁇ include ⁇ O and ⁇ S.
  • Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: ⁇ O, ⁇ S, ⁇ NNR * 2, ⁇ NNHC(O)R * , wherein each independent occurrence of R * is selected from hydrogen, C 1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR * 2) 2-3 O-, wherein each independent occurrence of R * is selected from hydrogen, C 1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on the aliphatic group of R* include halogen, -R ⁇ , - (haloR ⁇ ), -OH, -OR ⁇ , -O(haloR ⁇ ), -CN, -C(O)OH, -C(O)OR ⁇ , -NH2, -NHR ⁇ , -NR ⁇ 2, or - NO 2 , wherein each R ⁇ is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C 1-4 aliphatic, -CH 2 Ph, -O(CH 2 ) 0-1 Ph, or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include -R ⁇ , -NR 2, -C(O)R ⁇ , -C(O)OR ⁇ , -C(O)C(O)R ⁇ , -C(O)CH 2 C(O)R ⁇ , -S(O) 2 R ⁇ , - S(O) 2 NR ⁇ 2, -C(S)NR ⁇ 2, -C(NH)NR ⁇ 2, or -N(R ⁇ )S(O) 2 R ⁇ ; wherein each R ⁇ is independently hydrogen, C 1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R ⁇ ,
  • Suitable substituents on the aliphatic group of R ⁇ are independently halogen, - R ⁇ , -(halon ⁇ ), -OH, -OR ⁇ , -O(haloR ⁇ ), -CN, -C(O)OH, -C(O)OR ⁇ , -NH 2 , -NHR ⁇ , -NR ⁇ 2, or - Attorney Docket No.
  • each R ⁇ is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C 1-4 aliphatic, -CH 2 Ph, -O(CH 2 ) 0-1 Ph, or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference.
  • Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pect
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 alkyl) 4 salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.
  • MIL-035WO1 MIL-035WO1
  • structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.
  • structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon are within the scope of this invention.
  • Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention.
  • the present invention provides, among other things, a conjugate of an IgG degrading protease, e.g., Immunoglobulin G (IgG) degrading enzyme of the Streptococcus pyogenes (IdeS) protein or IgG-degrading enzyme/MAC-1 (IdeZ) protein, with rapamycin for immunosuppression, for example, in gene therapy.
  • IgG degrading protease e.g., Immunoglobulin G (IgG) degrading enzyme of the Streptococcus pyogenes (IdeS) protein or IgG-degrading enzyme/MAC-1 (IdeZ) protein
  • IdeS Streptococcus pyogenes
  • IdeZ IgG-degrading enzyme/MAC-1
  • the present invention provides, among other things, a conjugate comprising an Immunoglobulin G (IgG)-degrading enzyme of Streptococcus pyogenes (IdeS) protein or IgG-degrading enzyme/Mac-1 (IdeZ) protein, and an immunosuppressant molecule.
  • IdeS Immunoglobulin G
  • IdeZ Idegrading enzyme/Mac-1
  • the present invention provides, among other things, a conjugate comprising an Immunoglobulin G (IgG)-degrading enzyme, and an immunosuppressant molecule.
  • IgG-degrading enzymes include, but are not limited to IdeS, IdeZ, MAC2, IdeZ2, IdeE, IdeE2, IdeP, and MMP, among others.
  • the immunosuppressant molecule is rapamycin, an analog (e.g., deforolimus, everlimus, or temsirolimus) or a mimetic (e.g., isoliquiritigenin or withaferin A) thereof.
  • Attorney Docket No. MIL-035WO1 [0148]
  • the present invention provides, among other things, a method of reducing an immune response in a subject, the method comprising administering to the subject in need thereof, a therapeutically effective dose of a composition comprising a conjugate of: (a) an IdeS or an IdeZ protein; and (b) an immunosuppressant molecule.
  • a method of reducing an immune response by administration of the conjugate of the present invention has many therapeutic applications, for example, in treatment of cancer or autoimmune disease, in reducing rejection to organ transplants, and increasing benefits or reducing side-effects of therapeutic agents, increasing transduction and/or efficacy of gene therapy by reducing an immune response directed to a gene therapy vector or replacement enzyme in enzyme replacement therapy. Accordingly, it is contemplated that administration of an IdeS-rapamycin conjugate reduces immune response and increases efficacy of gene therapy (e.g.
  • Immunoglobulin G degrading enzymes are any polypeptides comprising IgG cleavage or degradation activity. They include, for example and without limitation, proteases and glycosidases. Proteases are enzymes that degrade or digest proteins and are also designated peptidases, proteinases, peptide hydrolases, or proteolytic enzymes.
  • Glycosidases are enzymes that hydrolyze glycosidic bonds in complex sugars.
  • Proteases used in the present invention include without limitation, exo-type proteases, also known as exoproteases or exopeptidases, that hydrolyze peptide bonds located towards the N-terminal end or the C-terminal end, and endo-type proteases, also known as endoprotease or endopeptidase, that hydrolyze internal peptide bonds in polypeptide chains.
  • endoproteases without limitation, include IdeS, IdeZ, IgdE, IdeMC, trypsin, chymotrypsin, papain, and pepsin.
  • Glycosidases used in the present invention include without limitation, exoglycosidases and endoglycosidases. Glycosidases cleave and release glycans or oligosaccharides from glycoproteins such as antibodies. Exoglycosidases include, without limitation, N-acetylglucosaminidase, fucosidase, galactosidase, glucosidase, mannosidase, neuraminidase, and xylosidase.
  • endoglycosidases include, without limitation, EndoS, EndoD, endoglycosidase H, Endo F1, Endo F2, and Endo F3.
  • IdeS Attorney Docket No. MIL-035WO1 Cysteine proteases, for example, from bacteria including without limitation, Streptococcus pyogenes, Streptococcus equi, Mycoplasma canis, Streptococcus agalactiae, or Streptococcus pseudoporcinus are used in the present invention.
  • IdeS The protein IdeS was first identified as a secreted immunogenic protein of Streptococcus pyogenes (Lei et al., 2000) and identified as a novel cysteine protease capable of cleaving IgG, therefore denoted IdeS (Ig degrading enzyme of S. pyogenes) (von Pawel-Rammingen et al., 2002). IdeS cleaves all human IgG subtypes, but no other Ig isotypes. In addition, IdeS carries out efficient cleavage of IgG from other species including monkey, sheep, rabbit as well as some activity towards murine and porcine IgG.
  • the IdeS comprises at least 70% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100% identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 75% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 80% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 85% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1.
  • the IdeS comprises at least 90% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 95% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises 100% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1.
  • amino acids 1-29 denote signal peptide
  • amino acids 1-40 identify a putative signal sequence
  • amino acids 214-216 denote an RGF motif which is important for ligand recognition
  • C94-H224 denote catalytic residues. [0153] Table 1. IdeS Wild-Type Protein Sequence.
  • IdeS wild-type protein MRKRCYSTSAVVLAAVTLFALSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPA KFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEE HPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSAKHLGVFPDHVIDMF INGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIK KELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGV NSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 1) [0154]
  • the IgG degrading enzyme used in this invention is a variant of IdeS.
  • the IgG degrading enzyme used in this invention is a deimmunized variant of IdeS.
  • the IdeS protein variant comprises Attorney Docket No. MIL-035WO1 pep7.
  • the IdeS protein is a variant that comprises one or more mutations at amino acid residues 39, 154, 155, 161, 164, 297, 300 and 303 of the wild-type IdeS of SEQ ID NO: 1.
  • the IdeS protein has a mutation at amino acid position 39 of the wild-type IdeS of SEQ ID NO: 1.
  • the IdeS protein has a mutation at position 154 of the wild-type IdeS of SEQ ID NO: 1.
  • the IdeS protein has a mutation at position 155 of the wild-type IdeS of SEQ ID NO: 1. In some embodiments, the IdeS protein has a mutation at position 161 of the wild-type IdeS of SEQ ID NO: 1. In some embodiments, the IdeS protein has a mutation at position 297 of the wild-type IdeS of SEQ ID NO: 1. In some embodiments, the IdeS protein has a mutation at position 300 of the wild-type IdeS of SEQ ID NO: 1. In some embodiments, the IdeS protein has a mutation at position 303 of the wild-type IdeS of SEQ ID NO: 1.
  • the IdeS protein comprises at least 80% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises 80-85%, 85-90%, 95-100% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises at least 85% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises at least 90% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises at least 95% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49.
  • the IdeS protein comprises at least 100% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. [0156] In some embodiments, the IdeS protein comprises an R39G mutation relative to the wild-type IdeS protein. [0157] In some embodiments, the IdeS protein is a variant that further comprises one or more mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • the IdeS protein is a variant that further comprises at least one mutation selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • the IdeS protein further comprises an E154D mutation relative to the wild-type IdeS protein.
  • the IdeS protein further comprises an Y155H mutation relative to the wild-type IdeS protein.
  • the IdeS protein further comprises an Y155Q mutation relative to the wild-type IdeS protein.
  • the IdeS protein further comprises an F161H mutation Attorney Docket No.
  • the IdeS protein further comprises an F161Y mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an L164H mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an F297T mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an F297N mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an V300R mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an A303E mutation relative to the wild-type IdeS protein.
  • the IdeS protein is a variant that further comprises one mutation selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises between 2-10 mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • the IdeS protein is a variant that further comprises two mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises three mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • the IdeS protein is a variant that further comprises four mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises five mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • the IdeS protein is a variant that further comprises six mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises seven mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • the IdeS protein is a variant that further comprises eight mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • the IdeS protein is a variant that further comprises nine mutations selected Attorney Docket No. MIL-035WO1 from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • the IdeS protein is a variant that further comprises the combination of mutations consisting of E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein.
  • Exemplary IdeS variants are described in Table 2. Table 2.
  • the IdeS protein is a variant comprising a D30-T49 deletion relative to wild-type IdeS protein.
  • the IdeS protein is a variant comprising a DSFSANCEIRYSEVTPYHVT (SEQ ID NO: 50) deletion.
  • Exemplary IdeS deletion variants are described in Table 3. Table 3. Variants of IdeS with a Deletion from D30 to T49 Relative to Wild-Type E154D-F297T-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFDYFKEKAFPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYTVGVNSAGKVAISAKEIKEDN
  • the IdeZ recognizes all human, sheep, monkey, and rabbit IgG subclasses, cleaving specifically at a single recognition site below the hinge region, to yield a homogenous pool of F(ab ⁇ )2 and Fc fragments.
  • IdeZ protease more effectively cleaves murine IgG2a and IgG3 than IdeS.
  • the IdeZ comprises at least 70% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100% identity to the wild-type IdEZ protein of SEQ ID NO: 51.
  • the IdeZ comprises at least 75% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 80% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ protein comprises at least 85% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 90% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 95% sequence identity to the wild-type Attorney Docket No. MIL-035WO1 IdeZ protein of SEQ ID NO: 51.
  • the IdeZ comprises 100% identity to the wild-type IdeZ protein of SEQ ID NO: 51.
  • the protease includes a modified variant of IdeZ. Table 4. IdeZ Wild-Type Protein Sequence.
  • the Immunoglobulin G-degrading enzyme is a MAC2 protein. In some embodiments, the Immunoglobulin G-degrading enzyme is an IdeZ2 protein. In some embodiments, the Immunoglobulin G-degrading enzyme is an IdeE protein. In some embodiments, the Immunoglobulin G-degrading enzyme is an IdeE2 protein. In some embodiments, the Immunoglobulin G-degrading enzyme is an IdeP protein. In some embodiments, the Immunoglobulin G-degrading enzyme is an MMP protein.
  • Immunosuppressants or immunosuppressive drugs can be classified into four groups: glucocorticoids, cytostatics, antibodies, drugs acting on immunophilins, and other drugs such as interferons, opiates INF binding proteins, mycophenolate, and FTY720, among others.
  • An exemplary class of immunosuppressant drugs comprises those drugs that act on immunophilins, which are high-affinity, specific binding proteins having physiological significance.
  • Two distinct families of immunophilins are presently known: cyclophilins and macrophilins, the latter of which specifically bind, for example, rapamycin (sirolimus, RAPAMUNE®), and tacrolimus (FK506, PROGRAF®).
  • Immunosuppressant drugs that act on immunophilin include, for example, cyclosporin (including cyclosporin A, cyclosporin B, cyclosporin C, cyclosporin D, cyclosporin E, cyclosporin F, cyclosporin G, cyclosporin H, cyclosporin I), everolimus (RAD, CERTICAN®), and deforolimus (AP23573, MK-8669), among others.
  • cyclosporin including cyclosporin A, cyclosporin B, cyclosporin C, cyclosporin D, cyclosporin E, cyclosporin F, cyclosporin G, cyclosporin H, cyclosporin I
  • everolimus RAD, CERTICAN®
  • deforolimus AP23573, MK-8669
  • the binding can be either covalent attachment such as by a direct connection, e.g., a chemical bond between the immunosuppressant drug and the conjugative moiety or between the immunosuppressant drug and the conjugative moiety and a linking group, or non-covalent attachment involving specific binding between complementary specific binding pair (sbp) members that are attached to the immunosuppressant drug and the conjugative moiety of the conjugate.
  • a direct connection e.g., a chemical bond between the immunosuppressant drug and the conjugative moiety or between the immunosuppressant drug and the conjugative moiety and a linking group
  • non-covalent attachment involving specific binding between complementary specific binding pair (sbp) members that are attached to the immunosuppressant drug and the conjugative moiety of the conjugate.
  • the present disclosure provides conjugates of an immunoglobulin G- degrading enzyme (e.g., IdeS, IdeZ) and one or more immunosuppressant molecules.
  • the immunosuppressant molecule is
  • the immunosuppressant molecule is rapamycin. In some embodiments, the immunosuppressant molecule is a rapamycin analog. In some embodiments, the rapamycin analog is selected from the group consisting of deforolimus, everolimus, and temsirolimus. In some embodiments, the rapamycin analog is deforolimus. In some embodiments, the rapamycin analog is everolimus. In some embodiments, the rapamycin analog is temsirolimus. In some embodiments, the rapamycin mimetic is isoliquiritigenin or withaferin A. In some embodiments, the rapamycin mimetic is isoliquiritigenin.
  • the rapamycin mimetic is withaferin A.
  • IdeS-Rapamcyin Conjugates [0168] In some aspects, the present disclosure provides, among other things, a conjugate comprising an Immunoglobulin G (IgG)-degrading enzyme of Streptococcus pyogenes (IdeS) protein or IgG-degrading enzyme/Mac-1 (IdeZ) protein, and an immunosuppressant molecule. [0169] In some embodiments, the IgG-degrading enzyme is conjugated to up to 10 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 1 immunosuppressant molecule.
  • the IgG-degrading enzyme is conjugated to 2 immunosuppressant molecules. In some embodiments, the IgG- degrading enzyme is conjugated to 3 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 4 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 5 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 6 immunosuppressant Attorney Docket No. MIL-035WO1 molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 7 immunosuppressant molecules.
  • the IgG-degrading enzyme is conjugated to 8 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 9 immunosuppressant molecules. In some embodiments, the IgG- degrading enzyme is conjugated to 10 immunosuppressant molecules. [0170] In some embodiments, the IdeS is conjugated to up to 10 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 1 immunosuppressant molecule. In some embodiments, the IdeS is conjugated to 2 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 3 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 4 immunosuppressant molecules.
  • the IdeS is conjugated to 5 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 6 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 7 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 8 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 9 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 10 immunosuppressant molecules. [0171] In some embodiments, the IdeZ is conjugated to up to 10 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 1 immunosuppressant molecule.
  • the IdeZ is conjugated to 2 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 3 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 4 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 5 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 6 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 7 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 8 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 9 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 10 immunosuppressant molecules.
  • the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:1 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:2 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:3 ratio. In some embodiments, the IgG-degrading enzyme and the Attorney Docket No. MIL-035WO1 immunosuppressant molecule are conjugated in at least a 1:4 ratio.
  • the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:5 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:6 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:7 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:8 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:9 ratio.
  • the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:10 ratio. [0173] In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:1 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:2 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:3 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:4 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:5 ratio.
  • the IdeS and the immunosuppressant molecule are conjugated in at least a 1:6 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:7 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:8 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:9 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:10 ratio. [0174] In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:1 ratio.
  • the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:2 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:3 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:4 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:5 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:6 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:7 ratio.
  • the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:8 ratio. In some embodiments, the IdeZ and the Attorney Docket No. MIL-035WO1 immunosuppressant molecule are conjugated in at least a 1:9 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:10 ratio. [0175] In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecules via surface amino acid residues on the IgG-degrading enzyme.
  • the IgG-degrading enzyme is conjugated to the immunosuppressant molecules via a surface lysine residue on the IgG-degrading enzyme.
  • A is an Immunoglobulin G-degrading enzyme
  • B is an immunosuppressant molecule
  • L 1 is a covalent bond, or an optionally substituted C 1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the chain are independently and optionally replaced with -O-, -S-, S(O) 2 -, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R)-, -N(R)C(O)-, -Cy 1 -, or -Cy 2 -, wherein each -Cy 1 - is independently an optionally substituted bivalent ring selected from 3
  • L 3 is a covalent bond or an optionally substituted C 1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the chain are independently and optionally replaced with -O-, -S-, - N(R)-, -S(O) 2 -, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R)-, -N(R)C(O)-, or - P(O)(R)-, each R is independently hydrogen, or an optionally substituted group selected from C1-6 aliphatic, phenyl, 4-7 membered saturated or partially unsaturated heterocyclic having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, m is an integer greater than 0, and wherein L 1 is connected to one or more heteroatoms selected from nitrogen, oxygen, and sulfur present
  • A is an Immunoglobulin G-degrading enzyme.
  • the Immunoglobulin G-degrading enzyme is an IdeS protein.
  • the Immunoglobulin G-degrading enzyme is an IdeZ protein.
  • B is an immunosuppressant macrolide.
  • B is an immunosuppressant macrophilin.
  • the immunosuppressant molecule is rapamycin (or sirolimus): (rapamycin or sirolimus), or a pharmaceutically acceptable salt thereof. In the present disclosure, rapamycin and sirolimus are used interchangeably.
  • the immunosuppressant molecule is everolimus: (everolimus), or a pharmaceutically acceptable salt thereof.
  • the immunosuppressant molecule is deforolimus: Attorney Docket No. MIL-035WO1 (deforolimus), or a pharmaceutically acceptable salt thereof.
  • the immunosuppressant molecule is temsirolimus: (temsirolimus), or a pharmaceutically acceptable salt thereof.
  • L 1 is a covalent bond, or an optionally substituted C 1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the chain are independently and optionally replaced with -O-, -S-, S(O)2-, C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R)-, -N(R)C(O)-, -Cy 1 -, or -Cy 2 -, wherein each -Cy 1 - is independently an optionally substituted bivalent ring selected from 3-7 membered saturated or partially unsaturated monocyclic carbocyclylene, monocyclic arylene, 4-10 membered saturated or partially unsaturated monocyclic heterocyclylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 5-10 membered saturated or partially unsaturated monocyclic heteroarylene having 1-3 heteroatoms independently selected from nitrogen, oxygen,
  • MIL-035WO1 polycyclic carbocyclylene, polycyclic arylene, 4-10 membered saturated or partially unsaturated polycyclic heterocyclylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 5-20 membered saturated or partially unsaturated polycyclic heteroarylene having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • L 1 is a covalent bond
  • L 1 is an optionally substituted C 1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the chain are independently and optionally replaced with -O-, -S-, S(O) 2 -, C(O)-, -C(O)O-, - OC(O)-, -C(O)N(R)-, -N(R)C(O)-, -Cy 1 -, or -Cy 2 -.
  • -Cy 1 - is independently an optionally substituted bivalent ring selected from 3-7 membered saturated or partially unsaturated monocyclic carbocyclylene, monocyclic arylene, 4-10 membered saturated or partially unsaturated monocyclic heterocyclylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 5-10 membered saturated or partially unsaturation
  • -Cy 2 - is independently an optionally substituted bivalent ring selected from 3-7 membered saturated or partially unsaturated polycyclic carbocyclylene, polycyclic arylene, 4-10 membered saturated or partially unsaturated polycyclic heterocyclylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 5-20 membered saturated or partially unsaturated polycyclic heteroarylene having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • L 2 is a covalent bond, wherein n is an integer between 1-170, or an optionally substituted C 1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the chain are independently and optionally replaced with -O-, -S-, S(O) 2 -, - C(O)-, -C(O)O-, -OC(O)-, - C(O)N(R)-, or -N(R)C(O)-.
  • L 2 is a covalent bond.
  • L 2 is wherein n is an integer between 1-170 or a molecule weight average of 1kDa-15kDa
  • L 2 is an optionally substituted C 1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the Attorney Docket No. MIL-035WO1 chain are independently and optionally replaced with -O-, -S-, S(O) 2 -, - C(O)-, -C(O)O-, - OC(O)-, -C(O)N(R)-, or -N(R)C(O)-.
  • L 3 is a covalent bond or an optionally substituted C 1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the chain are independently and optionally replaced with -O-, -S-, -N(R)-, -S(O) 2 -, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R)-, -N(R)C(O)-, or -P(O)(R)-.
  • L 3 is a covalent bond.
  • L 3 is an optionally substituted C 1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the chain are independently and optionally replaced with -O-, -S-, -N(R)-, -S(O) 2 -, -C(O)-, - C(O)O-, -OC(O)-, -C(O)N(R)-, -N(R)C(O)-, or -P(O)(R)-.
  • m is an integer greater than 0.
  • m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. [0206] In some embodiments, the present disclosure provides a conjugate of Formula III: Attorney Docket No. MIL-035WO1 III, wherein A, L 1 , L 2 , L 3 , and m are as described above and wherein Z is a point of attachment to L 3 or H.
  • the present disclosure provides a conjugate of Formula III: wherein A, L 1 , L 2 , L 3 , and m are as described above.
  • the present disclosure provides a conjugate of Formula IIa: Attorney Docket No. MIL-035WO1 IIIa, wherein A, L 1 , L 2 , L 3 , and m are as described above.
  • the present disclosure provides a conjugate of Formula IIIb: IIIb, wherein A, L 1 , L 2 , L 3 , and m are as described above.
  • the present disclosure provides a conjugate of Formula IV: Attorney Docket No.
  • the present disclosure provides a conjugate of Formula V, wherein A and m are as described above.
  • the present disclosure provides a conjugate of Formula Attorney Docket No. MIL-035WO1 VI wherein A and m are as described above.
  • Conjugation [0213] Exemplary IgG degrading enzyme and immunosuppressant conjugates, for example, an IdeS-rapamycin conjugate of the present disclosure is manufactured by one or more of the methods described herein, for example, sortase-mediated conjugation, lysine conjugation, cysteine conjugation, among others.
  • the conjugation is via a linker.
  • the IgG degrading enzyme is conjugated to the immunosuppressant molecule via a surface lysine residue on IdeS. In some embodiments, the IgG degrading enzyme is conjugated to the immunosuppressant molecule via a surface lysine residue on IdeS. In some embodiments, the IdeZ is conjugated to the immunosuppressant molecule via a surface lysine residue on IdeZ. [0215] In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecule via a C-terminus on the IgG-degrading enzyme.
  • the IgG-degrading enzyme is conjugated to the immunosuppressant molecule via a N-terminus on the IgG-degrading enzyme.
  • the IdeS is conjugated to the immunosuppressant molecule via a C-terminus on the IdeS.
  • the IdeS is conjugated to the immunosuppressant molecule via a N-terminus on the IdeS.
  • the IdeZ is conjugated to the immunosuppressant Attorney Docket No. MIL-035WO1 molecule via a C-terminus on the IdeZ.
  • the IdeZ is conjugated to the immunosuppressant molecule via a N-terminus on the IdeZ.
  • the IgG-degrading enzyme is conjugated to the immunosuppressant molecule via a cleavable linker.
  • the IgG- degrading enzyme is conjugated to the immunosuppressant molecule via a non-cleavable linker.
  • the IdeS is conjugated to the immunosuppressant molecule via a cleavable linker.
  • the IdeS is conjugated to the immunosuppressant molecule via a non-cleavable linker.
  • the IdeZ is conjugated to the immunosuppressant molecule via a cleavable linker. In some embodiments, the IdeZ is conjugated to the immunosuppressant molecule via a non-cleavable linker.
  • Sortase-Mediated Conjugation e.g., Sortase A
  • Sortases are transpeptidases that recognize a core amino acid motif (e.g., LPXTG) and selectively cleave the amide bond between the threonine and glycine residues. The resulting intermediate can then undergo nucleophilic attack by an oligo glycine that can include additional functional groups for further modification and/or site- specific conjugation.
  • Sortase nucleophiles may be equipped with any of the functionalities that previously have been used in the sortase reaction, including, but not limited to, “click handles” for performing click conjugations.
  • sortase Click Chemistry refers to a class of reactions including The Huisgen 1,3- dipolar cycloaddition (e.g., the Cu(I)-catalyzed stepwise variant, often referred to simply as the “click reaction”; see, e.g., Tornoe et al., Journal of Organic Chemistry (2002) 67: 3057- 3064). Copper and ruthenium are the commonly used catalysts in the reaction.
  • the use of copper as a catalyst results in the formation of 1,4-regioisomer whereas ruthenium results in formation of the 1,5-regioisomer; other cycloaddition reactions, such as the Diels-Alder reaction; other Nucleophilic addition to small strained rings like epoxides and aziridines; Attorney Docket No. MIL-035WO1 nucleophilic addition to activated carbonyl groups; and addition reactions to carbon-carbon double or triple bonds.
  • click chemistry handle refers to a reactant, or a reactive group, that can partake in a click chemistry reaction.
  • a strained alkyne e.g., a cyclooctyne
  • click chemistry reactions require at least two molecules comprising click chemistry handles that can react with each other.
  • click chemistry handle pairs that are reactive with each other are sometimes referred to herein as partner click chemistry handles.
  • an azide is a partner click chemistry handle to a cyclooctyne or any other alkyne.
  • Suitable click chemistry handles are known to those of skill in the art. Exemplary click chemistry handles suitable for use according to some aspects of this invention are described below.
  • the nucleophilic ⁇ -amino group of lysine residues on the immunoglobulin G- degrading enzyme may be chemically conjugated under a variety of conditions known to those of skill in the art. For instance, linkers bearing activated ester groups, e.g., N- hydroxysuccinimidyl (NHS) or sulfo-NHS esters, and imido esters, e.g., Traut’s reagent, readily react with lysine residues to form amide or amidine bonds.
  • NHS N- hydroxysuccinimidyl
  • imido esters e.g., Traut’s reagent
  • linkers bearing malimido groups react readily to form maleimide-thiol conjugates.
  • linkers [0222]
  • the terms “linking” and “conjugating” are used interchangeably an each refer to the covalent or non-covalent attachment of two or more moieties comprising a immunosuppressant molecule and an immunoglobulin G-degrading enzyme. In some aspects the linking or conjugating can comprise a linker.
  • the linker comprises L 1 , L 2 , and/or L3 .
  • one or more of the components contains a functional group suitable for attachment to one or more of the other components.
  • the functional groups suitable for attaching the components may be carbonyl functionalities, both oxocarbonyl, e.g., aldehyde, and non-oxocarbonyl (including nitrogen and sulfur analogs) e.g., carboxy, amidine, amidate, thiocarboxy and thionocarboxy.
  • oxo include active halogen, diazo, mercapto, olefin, particularly activated olefin, amino, phosphoro and the like. Of particular interest are activated esters or alkylating agents. Details of techniques for attaching molecules to one another may be found, for example, in Matthews, et al., Anal. Biochem. (1985) 151:205-209; Engelhardt, et al., European Patent Application No.0302175 and U.S. Pat. No.3,817,837, the relevant disclosure of which is incorporated herein by reference in its entirety. Attorney Docket No. MIL-035WO1 [0224] In certain aspects, the linker can contain a heterobifunctional group.
  • heterobifunctional group refers to a chemical moiety that connects the linker of which it is a part to the binding moiety. Heterobifunctional groups are characterized as having different reactive groups at either end of the chemical moiety. Attachment to the immunoglobulin G-degrading enzyme can be accomplished through chemical or enzymatic conjugation, or a combination of both. Chemical conjugation involves the controlled reaction of accessible amino acid residues on the surface of the binding moiety with a reaction handle on the heterobifunctional group.
  • Examples of chemical conjugation include, but are not limited to, lysine amide coupling, cysteine coupling, and coupling via a non-natural amino acid incorporated by genetic engineering, wherein non-natural amino acid residues with a desired reaction handle are installed onto immunoglobulin G-degrading enzyme.
  • an enzyme mediates the coupling of the linker with an accessible amino residue on the binding moiety.
  • Examples of enzymatic conjugation include, but are not limited to, transpeptidation using sortase, transpeptidation using microbial transglutaminase, and N-glycan engineering. Chemical conjugation and enzymatic conjugation may also be used sequentially.
  • Non-cleavable and cleavable linkers [0226]
  • linker e.g., L 1 , L 2 , and/or L 3
  • non-cleavable linker is any chemical moiety that is capable of linking the immunoglobulin G-degrading enzyme to the immunosuppressant molecule in a stable, covalent manner and does not fall under the categories defined herein as “cleavable linkers”.
  • non-cleavable linkers are substantially resistant to acid-induced cleavage, light-induced cleavage, bioreductive cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage.
  • “Substantially resistant to cleavage” means that the chemical bond in the linker or adjoining the linker in at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% of the conjugate population remains non-cleavable by an acid, a photolabile- cleaving agent, a bioreductive agent, a peptidase, an esterase, or a chemical or a physiological compound that cleaves the chemical bond (for example, a disulfide bond) in a cleavable linker, for within a Attorney Docket No. MIL-035WO1 few hours to several days of treatment with any of the agents described above.
  • the linker is not susceptible to acid-induced cleavage, photo-induced cleavage, bioreductive cleavage, enzymatic cleavage, or the like, at conditions under which the immunosuppressant molecule and/or the immunoglobulin G-degrading enzyme can remain active.
  • non-cleavable linkers include, but are not limited to, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) linkers, succinimide thioether linkers, and linkers such as: , wherein p is an integer from 1 to 10.
  • the linker is cleavable.
  • the linker is susceptible to acid-induced cleavage, photo-induced cleavage, bioreductive cleavage, enzymatic cleavage, or the like, at conditions under which the immunosuppressant molecule and/or the immunoglobulin G-degrading enzyme remain active.
  • the cleavable linker can be cleaved enzymatically. In some embodiments, the cleavable linker is cleaved by a protease, peptidase, esterase, beta- gluroronidase, glycosidase, phosphodiesterase, phosphatase, pyrophosphatase, or lipase. [0231] In some aspects, the cleavable linker can be cleaved by a protease. Examples of proteases include, but are not limited to, cathepsin B, VAGP tetrapeptide, and the like.
  • the cleavable linker contains a peptide.
  • the peptide is the site of cleavage of the linker, thereby facilitating release of the drug upon exposure to intracellular proteases, such as lysosomal enzymes.
  • Peptides can be designed and optimized for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.
  • Examples of peptides having two amino acids include, but are not limited to, alanine-alanine (ala-ala), valine-alanine (val-ala), Attorney Docket No.
  • valine-citrulline vc or val-cit
  • alanine-phenylalanine af or ala-phe
  • phenylalanine-lysine fk or phe-lys
  • phenylalanine- homolysine phe-homolys
  • Ne-val-cit N-methyl-valine-citrulline
  • Examples of peptides having three amino acids include, but are not limited to, glycine-valine-citrulline (gly-val-cit), aspartic acid-valine-citrulline (asp-val-cit), alanine- alanine-asparagine (ala-ala-asn), alanine- phenylalanine-lysine (ala-phe-lys), glycine-glycine- phenylalanine (gly-gly-phe), and glycine- glycine-glycine-glycine (gly-gly-gly).
  • peptides having four amino acids include, but are not limited to, glycine-glycine-valine- citrulline (gly-gly-val-cit) and glycine-glycine-phenylalanine- glycine (gly-gly-phe-gly).
  • the amino acid combinations above can also be present in the reverse order (i.e., cit-val).
  • the peptides of the present disclosure can comprise L- or D- isomers of amino acid residues.
  • naturally-occurring amino acid refers to Ala, Asp, Asx, Cit, Cys, Glu, Phe, Glx, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr.
  • D- designates an amino acid having the “D” (dextrorotary) configuration, as opposed to the configuration in the naturally occurring (“L-”) amino acids.
  • the amino acids described herein can be purchased commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.
  • the linker is bioreducible. Bioreducible linkers take advantage of the difference in reduction potential in the intracellular compartment versus plasma.
  • linker is a bioreducible linker selected from: .
  • the linker is acid cleavable. Acid-cleavable linkers are specifically designed to remain stable at the neutral pH of blood circulation, but undergo hydrolysis and release the cytotoxic drug in the acidic environment of the cellular compartments. Attorney Docket No.
  • the linker is a click-to-release linker, where release of the immunosuppressant molecule is chemically triggered by a tetrazine or related compound.
  • IdeS is conjugated to a rapamycin-linker payload selected from the group consisting of any one of: [0239]
  • Compound 1, Compound 2 or Compound 4 are monomeric compounds.
  • Compositions and Kits [0240] In some embodiments, provided by the present disclosure is a composition comprising a plurality of conjugates described herein.
  • the plurality of conjugates is homogenous, each conjugate comprising a uniform number of between 1-6 immunosuppressant molecules per IdES molecule. In some embodiments, the plurality of conjugates is heterogenous, each conjugate comprising a varying number of between 1-6 immunosuppressant molecules per IdES molecule.
  • Attorney Docket No. MIL-035WO1 [0241] In some embodiments, the composition comprises less than 5% unconjugated IdES. In some embodiments, the composition comprises less than 1% unconjugated IdES. In some embodiments, the composition comprises less than 0.1% unconjugated IdES.
  • the present invention further provides kits, that include packaging material and one or more components therein.
  • a kit typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein.
  • a kit can contain a collection of such components, e.g., a nucleic acid, recombinant vector, gene therapy (e.g., AAV, lentivirus) vector, replacement enzyme, and a IgG degrading enzyme conjugated to an immunosuppressant molecule as described in the compositions of the present invention, e.g. IdeS-rapamcyin that degrades or digests antibodies.
  • a kit refers to a physical structure housing one or more components of the kit.
  • Packaging material can maintain sterile components, and be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.).
  • Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics.
  • Labels or inserts can include information identifying manufacturer, lot numbers, manufacture location and date, expiration dates.
  • Labels or inserts can include information on a disease for which a kit component may be used.
  • Labels or inserts can include instructions for the clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen.
  • Labels or inserts can include information on any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, complications or reactions, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition.
  • Methods of Treatment and Use of Compositions [0244] In some aspects, provided herein is a method of reducing an immune response in a subject, the method comprising administering to the subject in need thereof, a therapeutically effective dose of a composition comprising a conjugate of: (a) an IdES or an IdEZ protein; and Attorney Docket No. MIL-035WO1 (b) an immunosuppressant molecule.
  • the immune response generates one or more antibodies.
  • the antibody is IgG.
  • the antibody is IgG1, IgG2, IgG3 or IgG4.
  • the IgG2 antibody is IgG2a or IgG2b.
  • the composition is administered with a gene therapy vector or a replacement enzyme. In some embodiments, the composition is administered prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered at least 3 days, 5 days or 7 days prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered between 1 day to 3 days prior to administration of the gene therapy vector or the replacement enzyme.
  • the composition is administered at least 1 hour, 3 hours, 5 hours, 10 hours, 12 hours, 16 hours, or 24 hours prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered concurrently with administration of the gene therapy vector or the replacement enzyme.
  • the gene therapy vector is an adeno-associated virus (AAV) vector. In some embodiments, the gene therapy vector is an adenovirus vector. In some embodiments, the gene therapy vector is a lentiviral vector. In some embodiments, the gene therapy vector encodes a protein that elicits production of IgG antibodies. In some embodiments, the protein is a heterologous protein.
  • the protein that elicits production of IgG antibodies is an AAV capsid protein. In some embodiments, the protein that elicits production of IgG antibodies is a lentiviral envelope protein. In some embodiments, provided herein is a method of increasing transduction efficiency of a gene therapy vector, the method comprising administering the composition described in the present disclosure.
  • the replacement enzyme is a lysosomal enzyme. In some embodiments, the replacement enzyme is arylsulfatase A or iduronate-2-sulfatase.
  • the subject receives an organ transplant. In some embodiments, provided herein is a method of reducing organ transplant rejection, comprising administering the composition described herein.
  • the composition is administered prior to the organ transplant.
  • Attorney Docket No. MIL-035WO1 [0247]
  • IgG is cleaved to scIgG, F(ab)2 and Fc fragments.
  • IgG upon administration of the composition, IgG is cleaved to scIgG.
  • administration of the composition comprising the conjugate to a subject reduces induction of a T-cell response relative to a control.
  • the control is the level of a T-cell response in the same or a different subject treated with unconjugated IdES.
  • the T cell response is reduced by 10%-20%, 20%-50%, 50%-80% or 80%-95%, or any intervening quantity thereof, relative to the control. In some embodiments, the T cell response is reduced by greater than 95% relative to the control. In some embodiments, the T cell response is reduced by greater than 99% relative to the control. In some embodiments, administration of the composition comprising the conjugate reduces induction of inflammatory cytokines relative to the control. In some embodiments, the inflammatory cytokines are IFN-gamma, IL-17 alpha, IL-2, IL-5, IL-6, TNF-alpha and/or MCP-1.
  • the present invention improves the efficacy and derivable benefit to a subject of a therapy or a therapeutic agent, while minimizing immunogenic responses.
  • Administration of the composition to the subject in need of therapy can safely eliminate Fc receptor binding by all or substantially all IgG molecules in the serum of the subject, such that neutralizing antibodies directed to therapeutic are substantially reduced or eliminated.
  • Reducing Immune Response [0250]
  • the therapeutic agent is a recombinant biologic comprising a vector comprising a heterologous nucleic acid encoding one or more recombinant proteins.
  • the vector is a recombinant virus vector, such as, for example and without limitation, a recombinant AAV vector, a lentiviral vector, or an adenovirus vector.
  • the recombinant biologic is a replacement enzyme.
  • the therapy is, for example and without limitation, adoptive cell therapy or ex vivo cell therapy.
  • the composition reduces immune response by neutralizing antibodies, promoting clearance or degradation of an antibody against the recombinant biologic or the drug entity.
  • the composition reduces Attorney Docket No. MIL-035WO1 immune response by reducing cytokine induction.
  • the composition reduces immune response by decreasing T cell proliferation.
  • the composition reduces neutralizing antibodies to a recombinant biologic or a drug entity wherein the antibodies comprise IgG (including IgG1, IgG2a, IgG2b, IgG3, and IgG4).
  • the antibodies comprise IgG.
  • the antibodies include those that would bind to the conjugate.
  • the antibodies include those that would bind the IgG degrading enzyme.
  • the composition reduces inflammatory cytokine induction to a therapy or therapeutic agent.
  • the composition reduces induction of inflammatory cytokines that include, for example and without limitation, IFN- gamma, IL-17 alpha, IL-2, IL-5, IL-6, TNF-alpha and/or MCP-1.
  • Gene Therapy is gene therapy.
  • the present invention reduces immunogenicity and increases safety and efficacy of gene therapy in treating a variety of diseases by administering a combination of the conjugate together with a gene therapy vector, either prior to gene therapy or concurrently.
  • the gene therapy vector is a viral vector, including, for example and without limitation, AAV vectors, lentiviral vectors, and adenovirus vectors.
  • the viral vector is without limitation, helper-dependent adenoviral, hybrid adenoviral, herpes simplex virus, poxvirus, Epstein-Barr virus, vaccinia virus, and human cytomegalovirus vector, including recombinant versions thereof.
  • Gene therapy vectors provide a means for delivering nucleic acids into a broad range of cells, including dividing and non-dividing cells. Gene therapy vectors can be employed to deliver a nucleic acid of interest to a cell in vitro, e.g., for ex vivo gene therapy.
  • the vectors are additionally useful in a method of delivering a nucleic acid to a subject in need thereof, e.g., to express an immunogenic or therapeutic polypeptide or a functional RNA, thus the polypeptide or functional RNA can be produced in vivo in the subject.
  • the subject is in need of the polypeptide because the subject has a deficiency of the polypeptide, e.g., a lysosomal storage enzyme.
  • Gene therapy vectors are used to treat and/or prevent any disease state for which it is beneficial to deliver a therapeutic polypeptide or functional nucleic acid.
  • Illustrative disease states include, but are not limited to: cystic fibrosis (cystic fibrosis Attorney Docket No.
  • MIL-035WO1 transmembrane regulator protein and other diseases of the lung, hemophilia A (Factor VIII), hemophilia B (Factor IX), thalassemia (B-globin). anemia (erythropoietin) and other blood disorders, Alzheimer’s disease (GDF; neprilysin), multiple sclerosis (B-interferon).
  • Parkinson’s disease glial-cell line derived neurotrophic factor [GDNF]
  • Huntington’s disease RNAi to remove repeats
  • amyotrophic lateral sclerosis epilepsy
  • epilepsy galanin, neurotrophic factors
  • cancer endostatin, angiostatin, TRAIL, FAS-ligand, cytokines including interferons
  • RNAi including RNAi against VEGF or the multiple drug resistance gene product
  • diabetes mellitus insulin
  • muscular dystrophies including Duchenne (dystrophin, mini-dystrophin, insulin-like growth factor I, a sarcoglycan [e.g., a, b, g], RNAi against myostatin, myostatin propeptide, follistatin, activin type II soluble receptor, anti inflammatory polypeptides such as the Ikappa B dominant mutant, sarcospan, utrophin, mini- utrophin, RNAi against splice
  • WO/2006/021724 antibodies or antibody fragments against myostatin or myostatin propeptide
  • Becker Gaucher disease (glucocerebrosidase), Hurler’s disease (a-L- iduronidase), adenosine deaminase deficiency (adenosine deaminase), glycogen storage diseases (e.g., Fabry disease [a-galactosidase] and Pompe disease [lysosomal acid a- glucosidase]) and other metabolic defects, congenital emphysema (al -antitrypsin), Lesch- Nyhan Syndrome (hypoxanthine guanine phosphoribosyl transferase), Niemann-Pick disease (sphingomyelinase), Tays Sachs disease (lysosomal hexosaminidase A), Maple Syrup Urine Disease (branched-chain keto acid dehydrogenase),
  • MIL-035WO1 2 intimal hyperplasia (e.g., by delivering enos, inos), improve survival of heart transplants (superoxide dismutase), AIDS (soluble CD4), muscle wasting (insulin-like growth factor I), kidney deficiency (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors such as IRAP and TNFa soluble receptor), hepatitis (a-interferon), LDL receptor deficiency (LDL receptor), hyperammonemia (ornithine transcarbamylase), Krabbe’s disease (galactocerebrosidase), Batten’s disease, spinal cerebral ataxias including SCA1, SCA2 and SCA3, phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, and the like.
  • the invention can further be used following organ transplantation to increase the success of the transplant and/or to reduce the negative side effects of organ transplantation or adjunct therapies (e.g., by administering immunosuppressant agents or inhibitory nucleic acids to block cytokine production).
  • organ transplantation or adjunct therapies e.g., by administering immunosuppressant agents or inhibitory nucleic acids to block cytokine production.
  • bone morphogenic proteins including BNP 2, 7, etc., RANKL and/or VEGF
  • the nucleic acid delivery vectors may also be employed to provide a functional nucleic acid to a cell in vitro or in vivo. Expression of the functional nucleic acid in the cell, for example, can diminish expression of a particular target protein by the cell.
  • nucleic acid can be administered to decrease expression of a particular protein in a subject in need thereof.
  • the nucleic acid delivery vectors can also be used for the purpose of evaluating safety (spread, toxicity, immunogenicity, etc.).
  • Treating Autoimmune Disorder [0258]
  • administration of the composition removes or reduces the effect of antibodies in a subject.
  • the subject has an autoimmune disease.
  • the antibodies are pathogenic autoantibodies.
  • administration of the composition is used to treat an autoimmune disorder.
  • the autoimmune disorder is, for example and without limitation, Addison’s disease, Anti-GBM glomerulonephritis, Anti-neutrophil cytoplasmic antibody-associated vasculitides, Anti-phospholipid antibody syndrome, Autoimmune bullous skin diseases, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune neutropenia, Bullous pemphigoid, Celiac disease, Chronic utricaria, Complete congenital heart block, Diabetes type 1A, Essential mixed cryoglobulinemia, Goodpasture’s syndrome, Graves’ disease, Guillain-Barre syndrome, Hemophilia-acquired FVIII deficiency, Idiopathic thrombocytopenic purpura, Lambert-Eaton myasthenic syndrome, Mixed connective tissue Attorney Docket No.
  • administering is to treat cancer.
  • administration is in combination with another therapy or therapeutic agent.
  • the therapy administered in combination with the composition is an antibody.
  • the cancer is, for example and without limitation, Acute lymphoblastic leukemia, Acute myeloid leukemia, Adrenocortical carcinoma, AIDS- related cancers, AIDS-related lymphoma, Anal cancer, Appendix cancer, Astrocytoma, childhood cerebellar or cerebral, Basal cell carcinoma, Bile duct cancer, extrahepatic, Bladder cancer, Bone cancer, Osteosarcoma/Malignant fibrous histiocytoma, Brainstem glioma, Brain cancer, Brain tumor, cerebellar astrocytoma, Brain tumor, cerebral astrocytoma/malignant glioma, Brain tumor, ependymoma, Brain tumor, medulloblastoma, Brain tumor, supratentorial primitive neuroectodermal tumors, Brain tumor, visual pathway and hypothalamic glioma, Breast cancer, Bronchial adenomas/carcinoids, Burkitt lymphoma
  • MIL-035WO1 Pancreas Kaposi sarcoma, Kidney cancer (renal cell cancer), Laryngeal Cancer, Leukemias, Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia), Leukemia, acute myeloid (also called acute myelogenous leukemia), Leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia), Leukemia, chronic myelogenous (also called chronic myeloid leukemia), Leukemia, hairy cell, Lip and Oral Cavity Cancer, Liposarcoma, Liver Cancer (Primary), Lung Cancer, Non-Small Cell ,Lung Cancer, Small Cell, Lymphomas, Lymphoma, AIDS-related, Lymphoma, Burkitt, Lymphoma, cutaneous T-Cell, Lymphoma, Hodgkin, Lymphomas, Non-Hodgkin (an old classification of all lymphomas except Hodgkin's), Lymphoma
  • the therapy is an organ transplant.
  • the organ transplant would otherwise be ineffective due to the action of anti- donor IgG antibodies present in the serum of the subject.
  • the organ is, for example and without limitation, the kidney, liver, hear, pancreas, lung, or small intestine.
  • the subject to be treated is sensitized or highly sensitized.
  • sensitized it is meant that the subject has developed antibodies to human major histocompatibility (MHC) antigens (also known as human leukocyte antigens (HLA)).
  • MHC major histocompatibility
  • HLA human leukocyte antigens
  • Anti-MHC antibodies are usually present in subjects that have been previously sensitized by blood transfusion, previous transplantation, or pregnancy.
  • the presence of high titer antibodies against MHC antigens of the potential donor is a risk to transplantation due to the risk of antibody-mediated rejection. Sensitization to donor MHC antigens impedes the identification of suitable donors.
  • Administration of the present invention allows rapid and safe removal of donor specific antigens in a potential transplant recipient. Administering the composition prior to transplantation effectively desensitizes the subject, thereby allowing transplant and avoiding antibody-mediated rejection.
  • the composition is administered concurrently with or prior to adoptive cell transfer therapy. In some embodiments, the composition is administered concurrently with or prior to ex vivo cell therapy.
  • administration of the present invention improved the benefit to a subject of a therapy or a therapeutic agent.
  • the conjugate is administered to the subject to reduce Fc receptor binding of serum IgG molecules in the subject.
  • the therapy or therapeutic agent is administered subsequently Attorney Docket No. MIL-035WO1 to the conjugate.
  • the therapy or therapeutic agent is administered concurrently with the conjugate.
  • Enzyme Replacement Therapy [0267]
  • the replacement enzyme is a lysosomal enzyme.
  • the replacement enzyme is arylsulfatase A or iduronate-2-sulfatase.
  • replacement enzymes suitable for the present invention may include any enzyme that can act to replace at least partial activity of the deficient or missing lysosomal enzyme in a lysosomal storage disease to be treated.
  • a replacement enzyme is capable of reducing accumulated substance in lysosomes or that can rescue or ameliorate one or more lysosomal storage disease symptoms.
  • compositions according to the present invention are used to treat lysosomal storage diseases, including, but not limited to, aspartylglucosaminuria, cholesterol ester storage disease, Wolman disease, cystinosis, Danon disease, Fabry disease, Farber lipogranulomatosis, Farber disease, fucosidosis, galactosialidosis types I/II, Gaucher disease types I/II/III, globoid cell leukodystrophy, Krabbe disease, glycogen storage disease II, Pompe disease, GM1-gangliosidosis types I/II/III, GM2-gangliosidosis type I, Tay Sachs disease, GM2-gangliosidosis type II, Sandhoff disease, GM2-gangliosidosis, ⁇ -mannosidosis types I/II, beta.-mannosidosis, metachromatic leukodystrophy, mucolipidosis type I, sialidosis types I/II, mu
  • lysosomal enzymes deficient in the above exemplary diseases are known to those of skill in the art, some of which are exemplified in Table 4 below.
  • a Attorney Docket No. MIL-035WO1 suitable replacement enzyme may be any lysosomal enzyme known to be associated with the lysosomal storage disease to be treated.
  • a suitable replacement enzyme is an enzyme selected from the enzyme listed in Table 4 above.
  • MIL-035WO1 Sanfilippo D (MPS IIID) Acetyltransferase N- Heparan Sulfate Acetylglucosamine-6- Sulfatase Morquio B (MPS IVB) ⁇ -Galactosidase Keratan Sulfate Maroteaux-Lamy (MPS VI) Arylsulfatase B Dermatan Sulfate Sly Syndrome (MPS VII) ⁇ -Glucuronidase ⁇ -Mannosidosis ⁇ -Mannosidase Mannose/Oligosaccharides ⁇ -Mannosidosis ⁇ -Mannosidase Mannose/Oligosaccharides Fucosidosis ⁇ -L-Fucosidase Fucosyl Oligosaccharides Aspartyl-glucosaminuria N-Aspartyl- ⁇ - Aspartylglucosamine Glucosaminidase Asparagines Sialidosis (Mucolipidosis I) ⁇ -
  • a replacement enzyme suitable for the invention may have a modified sequence having substantial homology or identify to the wild-type or naturally-occurring sequence (e.g., having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% sequence identity to the wild-type or naturally-occurring sequence).
  • a replacement enzyme suitable for the present invention may be produced by any available means. For example, replacement enzymes are recombinantly produced by utilizing a host cell system engineered to express a replacement enzyme-encoding nucleic acid. Alternatively or additionally, replacement enzymes are produced by activating endogenous genes.
  • replacement enzymes are partially or fully prepared by chemical synthesis. Alternatively or additionally, replacement enzymes are purified from natural sources.
  • IdeS The C-terminus of IdeS was engineered with an LPETG tag for sortase recognition and a His6 tag for purification. Using sortase, the Gly-His6 tag was replaced with a nucleophile containing glycine and an azide.
  • IdeS was then treated with, in this embodiment, one of the dibenzocyclooctyne (DBCO) linker-payload constructs, for example, Compound 1, 2, or 3 shown in FIG.2A, FIG.2B, and FIG.2C, for a strained alkyne-azide cycloaddition (SPAAC) reaction to form the IdeS-rapamycin C-terminal conjugates.
  • DBCO dibenzocyclooctyne
  • Compound 1 and 2 are rapamycin linker-payloads while Compound 3 is a control linker-payload without rapamycin. Since substitution of rapamycin analogs is common at the C40, the 40-OH was selected as the position for linker attachment via an ester bond to form the rapamycin linker-payloads.
  • the linker for Compound 1 contains 5 polyethylene glycol (PEG) units between the rapamycin and DBCO. Under physiological conditions, Compound 1 linker-payload is not expected to be cleaved.
  • the linker for Compound 2 contains a disulfide bond between 5 PEG units and rapamycin that is expected to be cleaved by intracellular reducing agents such as cysteine and glutathione. Cleavage of the disulfide bond results in formation of a catabolite with a shorter linker attached to the 40- Attorney Docket No. MIL-035WO1 OH of rapamycin by an ester bond.
  • Compound 3 is a control with only 6 PEG units and no rapamycin.
  • IdeS-rapamycin conjugates were characterized with Size-Exclusion Chromatography (SEC) and Quadrupole Time of Flight Mass Spectrometer (QTOF-MS).
  • This example illustrates the methodology used to conjugate IdeS and rapamycin through, for example, stochastic lysine conjugation chemistry on surface lysine residues.
  • stochastic lysine conjugation chemistry was utilized to non-specifically crosslink multiple rapamycin linker-payloads to surface lysine residues on IdeS as shown in FIG.1C.
  • linker-payload Compound 4 a compound with electrophilic N-hydroxysuccinimide (NHS) ester and polyethylene glycol (PEG) with an average molecular weight of 10 kDa.
  • Compound 4 contains a disulfide bond between the PEG units and rapamycin that is expected to be readily cleaved under physiological conditions.
  • the IdeS-rapamycin conjugate DoL distribution and protein concentration were determined by SDS-PAGE gel and Coomassie staining.
  • This example illustrates the effect of Rapamycin Conjugation on the enzymatic activity of IdeS.
  • enzymatic activity of IdeS was measured by a human IgG- cleavage assay. Results were assessed using SDS-PAGE gel and Coomassie staining. Un- cleaved IgG has a MW of 150 kDa. Cleavage products scIgG, F(ab’) 2 , and Fc appear as bands on the gel at around 125 kDa, 100 kDa, and 25 kDa, respectively.
  • Example 4 Effect of Rapamycin on IdeS-Induced Immune Activation.
  • This example illustrates the effect of rapamycin on the immune response induced by IdeS in human PBMCs.
  • human PBMCs were collected from 20 healthy donors. PBMCs were then incubated with IdeS or IdeS + rapamycin (Table 5) for 5 to 8 days. [0289] Table 5. Donor Response to IdeS, Rapamycin, and IdeS-Rapamycin Conjugates.
  • human PBMCs were collected from 20 healthy donors. PBMCs were then incubated with IdeS or IdeS-rapamycin conjugates for 5 to 8 days.
  • the non-cleavable IdeS-rapamycin (Compound 1) sortase-mediated conjugate, the disulfide cleavable IdeS-rapamycin (Compound 2) sortase-mediated conjugate, and the IdeS-PEG were each incubated with human PBMCs from one set of donors (Table 6).
  • the disulfide cleavable IdeS-rapamycin (Compound 4) lysine conjugate was also tested with a different set of donors (Table 6).
  • the disulfide cleavable IdeS-rapamycin conjugates also reduced the positive donor response rate from 40% to 15% and 0% for the sortase- mediated (Compound 2) conjugate and lysine (Compound 4) conjugate, respectively.
  • the disulfide cleavable IdeS-rapamycin (Compound 4) lysine conjugate also decreased cytokine induction across a panel of inflammatory cytokines (FIG.7).
  • IdeS was encoded in DNA with a C-terminal SMAC tag and a His6 tag or just a C-terminal His6 tag and an N-terminal mammalian secretion signal. The sequence was then cloned into a mammalian expression vector.
  • the expression vector was transiently transfected into CHO-K1 cells by electroporation. The transfected cells were then cultured for 7 days at 32°C with 5 % humidity. Attorney Docket No.
  • MIL-035WO1 For exemplary expression in HEK293 cells, the expression vector was transfected into Expi293 cells with LipofectamineTM. Transfected cells were then cultured for 6 days at 37°C with 8% humidity. [0301] Exemplary IdeS protein features are denoted in the schematic shown in FIG. 1A. [0302] IdeS-His6 and IdeS-SMAC-His6 were purified from cell culture media. Supernatants were harvested, and the clarified supernatants were loaded onto an exemplary His-tag purification column, for example, Roche cOmpleteTM His-Tag Purification Resin.
  • the column was washed with phosphate buffered saline (PBS), pH 7.4, supplemented with 2 M sodium chloride. PBS with 500 mM imidazole was used for elution. Eluted IdeS-His6 and IdeS-SMAC-His6 were further purified by gel filtration chromatography, for example, using Superdex® 200 prep grade equilibrated with PBS. The purified protein was then aliquoted and stored at -80°C. [0303] The sequence integrity was confirmed by intact mass spectrometry (iMS). Enzymatic activity was confirmed using an IgG cleavage assay using Homogenous Time- Resolved Fluorescence (HTRF) assay to detect intact human IgG.
  • HTRF Homogenous Time- Resolved Fluorescence
  • the rapamycin linker-payload is Compound 1 (FIG.2A), [(1R,2R,4S)-4-[(2R)-2-[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)- 1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo- 11,36-dioxa-4-azatricyclo[30.3.1.04,9] hexatriaconta-16,24,26,28-tetraen-12-yl] propyl]-2- methoxy-cyclohexyl] 4-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[[[3-(2-azatricyclo[10.4.0.04,9] hexadeca- 1(12),
  • step 1 of synthesis (FIG.9A)
  • rapamycin (Int-1, 8.23 g, 9 mmol) was combined with succinic anhydride (9 g, 90 mmol) and an immobilized lipase biocatalyst, for example, Novozym® 435 (23.5 g) in toluene (250 mL) and stirred for 2 days at room temperature.
  • the resin was filtered and washed with dichloromethane (DCM, 2 x 100 mL).
  • DCM dichloromethane
  • step 2 of synthesis a solution of Int-2 (1.52 g, 1.5 mmol) in DCM (25 mL) was added to N’-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU, 903 mg, 3 mmol) followed by triethanolamine (TEA, 455 g, 4.5 mmol). The mixture was stirred for 2 hours at room temperature and then diluted with water (30 mL) and extracted with DCM (2 x 30 mL).
  • TSTU N’-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate
  • TEA triethanolamine
  • step 3 of synthesis (FIG.9C)
  • Int-3 (646 mg, 0.58 mmol)
  • Int-4 (FIG. 10C, 217 mg, 0.64 mmol) were solubilized in dimethylformamide (DMF, 6 mL) before N,N- Diisopropylethylamine (DIPEA, 224 mg, 1.74 mmol) was added.
  • DIPEA N,N- Diisopropylethylamine
  • step 4 of synthesis Int-5 (1.00 eq, 20 mg, 0.0153 mmol), Int-6 (FIG.10C, 1.20 eq, 5.1 mg, 0.0183 mmol), and (1H-Benzotriazol-1-yloxy)(dimethylamino)- N,N-dimethylmethaniminium tetrafluoroborate (TBTU, 1.5 eq, 7.4 mg, 0.0229 mmol) were placed into solution in DMF (0.489 mL). N,N-diisopropylethylamine (2.40 eq, 0.0064 mL, Attorney Docket No.
  • the rapamycin linker-payload is Compound 2 (FIG.2B), [(1R,2R,4S) -4-[(2R)-2- [(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy -19,30- dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4- azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxy-
  • step 1 of synthesis (FIG.11A)
  • TEA (455 mg, 4.5 mmol) was then added, and the mixture was stirred for 16 hours at room temperature.
  • the reaction mixture was diluted with NaHCO 3 (5% aq., 30 mL) and extracted with DCM (2 x 30 mL).
  • Int-8 (738 mg, 1.5 mmol) was solubilized in DCM (15 mL).2-((2-aminoethyl)disulfaneyl)-N- tritylethan-1-amine (711 mg, 1.8 mmol) and triethylamine (455 mg, 4.5 mmol) were then added to the solution, and the reaction was stirred for 16 hours at room temperature. After dilution with water (40 mL) and adjustment to pH 3 with formic acid, the product was Attorney Docket No. MIL-035WO1 extracted with DCM (3 x 40 mL).
  • step 3 of synthesis (FIG.11C) was added to Int-9 (940 mg, 1.2 mmol) in a mixed solvent of trifluoroacetic acid and DCM (1:10, 22 mL). The mixture was stirred for 1 hour at room temperature.
  • step 5 of synthesis Int-11 (10 mg, 0.00707 mmol), Int-6 (1.20 eq, 2.3 mg, 0.00849 mmol), and TBTU (1.50 eq, 3.4 mg, 0.0106 mmol) were solubilized in DMF (0.226 mL). N,N-diisopropylethylamine. (2.40 eq, 0.0030 mL, 0.0170 mmol) was added, and the reaction was stirred for 1 hour at room temperature. A second 10 mg reaction was set up and stirred for 1 hour at room temperature. The two 10 mg reactions were combined, diluted with EtOAc, and washed with water.
  • the rapamycin linker-payload is Compound 4.
  • the overall synthesis is summarized in FIG.12. [0322]
  • step 1 of synthesis (FIG.13A) 2-[(2-Aminoethyl)disulfanyl]ethylamine hydrochloride (Int-12, 5.00 g, 22.2 mmol, 2 HCl, 1 eq) was dissolved in water (20 mL) and 1,4-dioxane (20 mL.
  • Triethylamine (3.94 g, 38.9 mmol, 5.42 mL, 1.75 eq) was added followed by a solution of trityl chloride (2.10 g, 7.55 mmol, 0.34 eq) in 1,4-dioxane (20 mL), which was added over 1 hour.
  • the reaction was stirred under nitrogen for 48 hours at room temperature.
  • the organic solvent was evaporated, and then the reaction was diluted with water (100 mL).
  • the aqueous layer was extracted with dichloromethane (3 x 100 mL).
  • the combined organic layers were washed with NaHCO 3 (sat, 150 mL) and dried over MgSO 4 .
  • step 2 of synthesis (FIG.13B)
  • Int-13 (195 mg, 494 ⁇ mol, 5 eq) was combined with NHS-PEG10k-COOH (Int-14, 1.00 g, 98.7 ⁇ mol, 1 eq, supplied by Biopharma PEG, HE023017-10K) and dissolved in dichloromethane (8 mL).
  • Triethylamine (20.0 mg, 197 ⁇ mol, 27.5 ⁇ L, 2 eq) was added, and the reaction was shaken in a 50 mL falcon tube overnight at room temperature. The reaction was split into two falcon tubes and precipitated by adding diethyl ether to each falcon tube and centrifuging for 5 minutes at 3900 RPM. The diethyl ether was decanted off, and the residue in each tube was dissolved in dichloromethane (4 mL). Diethyl ether (45 mL) was added to each tube, and the mixtures were centrifuged for 5 minutes at 3900 RPM to settle the solids.
  • step 3 of synthesis (FIG.13C) Int-15 (978 mg, 94 ⁇ mol, 1 eq) was dissolved in a mixture of dichloromethane (3 mL), trifluoroacetic acid (3 mL), and triethylsilane (107 mg, 917 ⁇ mol, 9.7 eq). The reaction was then shaken for 2 hours at room temperature in a falcon tube (50 mL).
  • the reaction was split into 2 falcon tubes and diethyl ether (45 mL) was added to each tube. The solid was separated by centrifugation for 5 minutes at 3900 RPM. The diethyl ether was decanted off, and the residue in each tube was dissolved with dichloromethane (4 mL). The product was then precipitated with diethyl ether (45 mL in each tube) and centrifuged for 5 minutes at 3900 RPM. The diethyl ether was again Attorney Docket No. MIL-035WO1 decanted, and the remaining solids were dried overnight under vacuum to yield 950 mg Int- 16 (FIG.14C, 93.5 ⁇ mol), yielding 99.5%.
  • step 4 of synthesis (FIG.13D)
  • two falcon tues with Int-3 (312 mg, 280 ⁇ mol, 3 eq)
  • Int-16 950 mg, 93.5 ⁇ mol, 1 eq
  • triethylamine 102 mg, 1.01 mmol, 141 ⁇ L, 11 eq
  • dichloromethane 8 mL
  • the reactions were stirred overnight at room temperature.
  • Diethyl ether (45 mL) was then added to each reaction tube.
  • the solids were settled using centrifugation for 5 minutes at 3900 RPM. After decanting the solvent, the solids in each tube were re-dissolved in dichloromethan (3 mL).
  • step 5 of synthesis (FIG.13E) Int-17 (550 mg, 49.3 ⁇ mol, 1 eq) was dissolved in dichloromethane (4 mL). TSTU (33.0 mg, 110 ⁇ mol, 2.2 eq) and DIPEA (14.3 mg, 111 ⁇ mol, 19.3 ⁇ L, 2.3 eq) was added. The reaction was stirred for 2 hours at room temperature. Diethyl ether (15 mL) was added and the reaction was centrifuged for 5 minutes at 3900 RPM.
  • This example describes the exemplary methodology of conjugating rapamycin linker payloads to IdeS using sortase-mediated conjugation and SPAAC click chemistry.
  • a solution of IdeS with LPETG- His6 tags at the C-terminus in phosphate buffered saline (PBS), pH 7.4 was further diluted with PBS, pH 7.4, until the protein concentration was 40 ⁇ M.
  • PBS phosphate buffered saline
  • CaCl 2 (4 mM aqueous solution, Attorney Docket No.
  • MIL-035WO1 100 equivalents (eq)) was added to the solution with 2-amino-N-(2-azidoethyl)acetamide (800 ⁇ M in 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 150 mM NaCl, pH 7.4, with 0.1% dimethylsulfoxide (DMSO), 20 eq) followed by a 2 ⁇ M solution of Sortase A Q60-K206-P94S-D160N-D165A-K196 His6 (50 mM HEPES, 150 mM NaCl, pH 7.4, 0.05 eq). The mixture was stirred for 45 minutes at room temperature.
  • 2-amino-N-(2-azidoethyl)acetamide 800 ⁇ M in 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 150 mM NaCl,
  • the product was purified by CapturemTM nickel resin followed by dialysis to give IdeS-LPETGG-NH(CH 2 ) 2 - azide.
  • IdeS-LPETGG-NH(CH 2 ) 2 - azide 40-60 ⁇ M in PBS
  • DMSO DMSO
  • the protein conjugate was deglycosylated using PNGaseF (New England BioLabs, catalog number P0704L) prior to the LC-QTOF analysis.
  • PNGaseF New England BioLabs, catalog number P0704L

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Abstract

The present invention, provides among other things, a conjugate of an IgG degrading protease, e.g., Immunoglobulin G (IgG) degrading enzyme of the S. pyogenes (IdeS) protein or IgG-degrading enzyme/MAC-1 (IdeZ) protein, with an immunosuppressant molecule, e.g., rapamycin, for immunosuppression in applications such as gene therapy. The present disclosure also provides methods of synthesis of conjugates and methods of conjugation (i.e. sortase-mediated conjugation) of payloads to IdeS or IdeZ for use in such applications.

Description

Attorney Docket No. MIL-035WO1 IDES-RAPAMYCIN CONJUGATE IN AAV GENE THERAPY CROSS REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority to U.S. Provisional Application No. 63/590,195 filed on October 13, 2023; the contents of which are incorporated herein by reference for all purposes. INCOPORATION BY REFERENCE OF SEQUENCE LISTING [0002] The present application is being filed along with a Sequence Listing submitted electronically in XML format. The Sequence Listing file “MIL-035WO1_SL.xml” is created on September 27, 2024, which is 65,536 bytes in size; the contents of which are incorporated herein for all purpose. BACKGROUND [0003] Neutralizing antibodies (NAb) against the AAV vectors (e.g. directed to the AAV capsid) pose a major challenge to gene therapy. Neutralizing antibodies block liver transduction and vector re-administration. Reduced therapeutic efficacy from even low NAb titers, in combination with the high prevalence of pre-existing NAbs, results in many patients being ineligible for rAAV gene therapy. Neutralizing antibodies can also be induced from rAAV dosing, which in turn creates the additional challenge of re-dosing patients that have received rAAV gene therapy. [0004] Current strategies aimed at overcoming anti-AAV antibodies often involve immunosuppression and are not efficient in removing pre-existing antibodies. [0005] Imlifidase (IdeS) is another strategy for use as an immunomodulatory therapy to remove anti-AAV NAbs. IdeS is a naturally occurring cysteine protease derived from Streptococcus pyogenes, which has been demonstrated to rapidly and specifically cleave all subclasses of human IgG at the lower hinge region (von Pawel-Rammingen, EMBO J.2002, 21(7):1607-15; PMID 11927545). [0006] However, since IdeS is of bacterial origin, it is immunogenic in people (Winstedt 2015, PLoS One 201510(7): e0132011; PMID 26177518). Many people already have pre-existing antibodies against IdeS and those antibodies get further induced by IdeS administration. Attorney Docket No. MIL-035WO1 SUMMARY OF INVENTION [0007] The present invention provides, among other things, a conjugate of an IgG degrading protease, e.g., Immunoglobulin G (IgG) degrading enzyme of the Streptococcus pyogenes (IdeS) protein or IgG-degrading enzyme/MAC-1 (IdeZ) protein, with an immunosuppressant molecule, e.g., rapamycin or sirolimus, for immunosuppression, for example, in gene therapy. Conjugating exemplary bacterial IgG proteases such as IdeS or IdeZ to the immunosuppressant rapamycin reduces immune stimulation due to bacterial protease alone and increases the efficacy of gene therapy. [0008] In some aspects, the present invention provides, among other things, a conjugate comprising an Immunoglobulin G (IgG)-degrading enzyme of Streptococcus pyogenes (IdeS) protein or IgG-degrading enzyme/Mac-1 (IdeZ) protein, and an immunosuppressant molecule. [0009] In some embodiments, the IgG-degrading enzyme is conjugated to at least 1 immunosuppressant molecule. In some embodiments, the IgG-degrading enzyme is conjugated to at least 2 immunosuppressant molecules. In some embodiments, the IgG- degrading enzyme is conjugated to at least 3 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to at least 4 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to at least 5 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to at least 6 immunosuppressant molecules. In some embodiments, the IgG- degrading enzyme is conjugated to at least 7 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to at least 8 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to at least 9 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to at least 10 immunosuppressant molecules. In some embodiments, the IgG- degrading enzyme is conjugated to 10 or more immunosuppressant molecules. [0010] In some embodiments, the IgG-degrading enzyme is conjugated to 1 immunosuppressant molecule. In some embodiments, the IgG-degrading enzyme is conjugated to 2 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 3 immunosuppressant molecules. In some embodiments, the IgG- degrading enzyme is conjugated to 4 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 5 immunosuppressant molecules. In some Attorney Docket No. MIL-035WO1 embodiments, the IgG-degrading enzyme is conjugated to 6 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 7 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 8 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 9 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 10 immunosuppressant molecules. [0011] In some embodiments, the IdeS is conjugated to at least 1 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 2 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 3 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 4 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 5 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 6 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 7 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 8 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 9 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to at least 10 immunosuppressant molecules. [0012] In some embodiments, the IdeS is conjugated to 1 immunosuppressant molecule. In some embodiments, the IdeS is conjugated to 2 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 3 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 4 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 5 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 6 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 7 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 8 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 9 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 10 immunosuppressant molecules. [0013] In some embodiments, the IdeZ is conjugated to at least 1 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to at least 2 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to at least 3 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to at least 4 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to at least 5 immunosuppressant Attorney Docket No. MIL-035WO1 molecules. In some embodiments, the IdeZ is conjugated to at least 6 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to at least 7 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to at least 8 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to at least 9 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to at least 10 immunosuppressant molecules. [0014] In some embodiments, the IdeZ is conjugated to 1 immunosuppressant molecule. In some embodiments, the IdeZ is conjugated to 2 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 3 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 4 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 5 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 6 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 7 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 8 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 9 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 10 immunosuppressant molecules. [0015] In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:1 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:2 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:3 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:4 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:5 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:6 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:7 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:8 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:9 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:10 ratio. Attorney Docket No. MIL-035WO1 [0016] In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:1 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:2 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:3 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:4 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:5 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:6 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:7 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:8 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:9 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:10 ratio. [0017] In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:1 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:2 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:3 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:4 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:5 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:6 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:7 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:8 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:9 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:10 ratio. [0018] In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecules via surface amino acid residues on the IgG-degrading enzyme. In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecules via a surface lysine residue on the IgG-degrading enzyme. [0019] In some embodiments, the IdeS is conjugated to the immunosuppressant molecule via a surface lysine residue on IdeS. In some embodiments, the IdeS is conjugated to the immunosuppressant molecules via surface lysine residues on IdeS. Attorney Docket No. MIL-035WO1 [0020] In some embodiments, the IdeZ is conjugated to the immunosuppressant molecule via a surface lysine residue on IdeZ. In some embodiments, the IdeZ is conjugated to the immunosuppressant molecules via surface lysine residues on IdeZ. [0021] In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecule via a C-terminus on the IgG-degrading enzyme. In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecule via a N-terminus on the IgG-degrading enzyme. [0022] In some embodiments, the IdeS is conjugated to the immunosuppressant molecule via a C-terminus on the IdeS. In some embodiments, the IdeS is conjugated to the immunosuppressant molecule via a N-terminus on the IdeS. [0023] In some embodiments, the IdeZ is conjugated to the immunosuppressant molecule via a C-terminus on the IdeZ. In some embodiments, the IdeZ is conjugated to the immunosuppressant molecule via a N-terminus on the IdeZ. [0024] In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecule via a cleavable linker. In some embodiments, the IgG- degrading enzyme is conjugated to the immunosuppressant molecule via a non-cleavable linker. [0025] In some embodiments, the IdeS is conjugated to the immunosuppressant molecule via a cleavable linker. In some embodiments, the IdeS is conjugated to the immunosuppressant molecule via a non-cleavable linker. [0026] In some embodiments, the IdeZ is conjugated to the immunosuppressant molecule via a cleavable linker. In some embodiments, the IdeZ is conjugated to the immunosuppressant molecule via a non-cleavable linker. [0027] In some embodiments, the IdeS comprises at least 70% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100% identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 75% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 80% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 85% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at Attorney Docket No. MIL-035WO1 least 90% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 95% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises 100% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. [0028] In some embodiments, the IdeZ comprises at least 70% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100% identity to the wild-type IdEZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 75% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 80% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ protein comprises at least 85% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 90% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 95% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises 100% identity to the wild-type IdeZ protein of SEQ ID NO: 51. [0029] In some embodiments, the IdeS protein comprises an R39G mutation relative to the wild-type IdeS protein. [0030] In some embodiments, the IdeS protein is a variant that further comprises one or more mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises at least one mutation selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an E154D mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an Y155H mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an Y155Q mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an F161H mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an F161Y mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an L164H mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an F297T mutation relative to the Attorney Docket No. MIL-035WO1 wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an F297N mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an V300R mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an A303E mutation relative to the wild-type IdeS protein. [0031] In some embodiments, the IdeS protein is a variant that further comprises one mutation selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises between 2-10 mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises two mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises three mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises four mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises five mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises six mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises seven mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises eight mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises nine mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises the combination of mutations consisting of E154D, Y155H, Y155Q, Attorney Docket No. MIL-035WO1 F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. [0032] In some embodiments, the IdeS protein is a variant comprising a D30-T49 deletion relative to wild-type IdeS protein. In some embodiments, the IdeS protein is a variant comprising a DSFSANCEIRYSEVTPYHVT (SEQ ID NO: 50) deletion. [0033] In some embodiments, the wild-type IdeS protein has 100% identity to SEQ ID NO: 1. [0034] In some embodiments, the IdeS protein comprises at least 80% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises 80-85%, 85-90%, 95-100% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 2. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 3. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 4. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 5. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 6. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 7. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 8. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 9. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 10. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 11. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 12. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 13. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 14. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 15. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 16. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 17. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 18. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 19. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 20. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 21. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 22. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 23. In some Attorney Docket No. MIL-035WO1 embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 24. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 25. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 26. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 27. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 28. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 29. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 30. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 31. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 32. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 33. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 34. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 35. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 36. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 37. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 38. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 39. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 40. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 41. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 42. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 43. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 44. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 45. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 46. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 47. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 48. In some embodiments, the IdeS protein comprises at least 80% identity to SEQ ID NO: 49. [0035] In some embodiments, the IdeS protein comprises at least 90% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 2. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 3. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 4. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 5. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 6. In some embodiments, the IdeS protein comprises at Attorney Docket No. MIL-035WO1 least 90% identity to SEQ ID NO: 7. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 8. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 9. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 10. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 11. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 12. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 13. In some embodiments, the IdeS comprises at least 90% identity to SEQ ID NO: 14. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 15. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 16. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 17. In some embodiments, the IdeS comprises at least 90% identity to SEQ ID NO: 18. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 19. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 20. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 21. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 22. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 23. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 24. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 25. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 26. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 27. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 28. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 29. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 30. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 31. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 32. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 33. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 34. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 35. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 36. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 37. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 38. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 39. In some embodiments, the IdeS protein comprises at least 90% identity to Attorney Docket No. MIL-035WO1 SEQ ID NO: 40. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 41. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 42. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 43. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 44. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 45. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 46. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 47. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 48. In some embodiments, the IdeS protein comprises at least 90% identity to SEQ ID NO: 49. [0036] In some embodiments, the IdeS protein comprises at least 100% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 2. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 3. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 4. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 5. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 6. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 7. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 8. In some embodiments, the IdeS comprises at least 100% identity to SEQ ID NO: 9. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 10. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 11. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 12. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 13. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 14. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 15. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 16. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 17. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 18. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 19. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 20. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 21. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 22. In some embodiments, the IdeS protein Attorney Docket No. MIL-035WO1 comprises at least 100% identity to SEQ ID NO: 23. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 24. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 25. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 26. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 27. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 28. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 29. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 30. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 31. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 32. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 33. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 34. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 35. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 36. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 37. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 38. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 39. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 40. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 41. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 42. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 43. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 44. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 45. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 46. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 47. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 48. In some embodiments, the IdeS protein comprises at least 100% identity to SEQ ID NO: 49. [0037] In some embodiments, the immunosuppressant molecule is rapamycin, an analog or mimetic thereof. In some embodiments, the immunosuppressant molecule is rapamycin. In some embodiments, the immunosuppressant molecule is an analog of rapamycin. In some embodiments, the immunosuppressant molecule is a mimetic of rapamycin. Attorney Docket No. MIL-035WO1 [0038] In some embodiments the rapamycin analog is selected from the group consisting of deforolimus, everolimus, and temsirolimus. In some embodiments, the rapamycin analog is deforolimus. In some embodiments, the rapamycin analog is everolimus. In some embodiments, the rapamycin analog is temsirolimus. [0039] In some embodiments, the rapamycin mimetic is isoliquiritigenin or withaferin A. In some embodiments, the rapamycin mimetic is isoliquiritigenin. In some embodiments, the rapamycin mimetic is withaferin A. [0040] In some embodiments, IdeS is conjugated to a rapamycin-linker payload selected from the group consisting of any one of:
Figure imgf000015_0001
[0041] In some embodiments, the IdeS is conjugated to rapamycin linker payload Compound 1 (FIG.2A). In some embodiments, the IdeS is conjugated to rapamycin linker payload Compound 2 (FIG.2B). In some embodiments, the IdeS is conjugated to rapamycin linker payload Compound 4 (FIG.2D). Attorney Docket No. MIL-035WO1 [0042] In some embodiments, Compound 1, Compound 2, or Compound 4 are monomeric compounds. In some embodiments, Compound 1 is a monomeric compound. In some embodiments, Compound 2 is a monomeric compound. In some embodiments, Compound 4 is a monomeric compound. [0043] In some embodiments, provided by the present invention is a composition comprising a plurality of conjugates described herein. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of between 1 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition comprises one or more immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition comprises up to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 1 immunosuppressant molecule per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 2 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 3 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 4 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 5 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 6 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 7 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 8 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some Attorney Docket No. MIL-035WO1 embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. [0044] In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of between 1 to 10 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition comprises one or more immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition comprises up to 10 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 1 immunosuppressant molecule per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 2 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 3 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 4 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 5 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 6 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 7 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 8 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 9 immunosuppressant molecules per IdeS molecule. In some embodiments, the plurality of conjugates in the composition is homogenous wherein each conjugate comprises a uniform number of 10 immunosuppressant molecules per IdeS molecule. [0045] In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 10 Attorney Docket No. MIL-035WO1 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9 or 8 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 8 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 7 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 6 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 5 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 4 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 3 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 1 to 2 immunosuppressant molecules per molecule of IgG-degrading enzyme. [0046] In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some Attorney Docket No. MIL-035WO1 embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 8 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 7 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 6 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 5 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 4 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 2 to 3 immunosuppressant molecules per molecule of IgG-degrading enzyme. [0047] In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 3 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 3 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 3 to 8 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 3 to 7 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 3 to 6 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 3 to 5 immunosuppressant molecules per Attorney Docket No. MIL-035WO1 molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 3 to 4 immunosuppressant molecules per molecule of IgG-degrading enzyme. [0048] In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 4 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 4 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 4 to 8 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 4 to 7 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 4 to 6 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 4 to 5 immunosuppressant molecules per molecule of IgG-degrading enzyme. [0049] In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 5 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 5 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 5 to 8 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 5 to 7 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 5 to 6 immunosuppressant molecules per molecule of IgG-degrading enzyme. Attorney Docket No. MIL-035WO1 [0050] In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 6 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 6 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 6 to 8 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 6 to 7 immunosuppressant molecules per molecule of IgG-degrading enzyme. [0051] In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 7 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 7 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 7 to 8 immunosuppressant molecules per molecule of IgG-degrading enzyme. [0052] In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 8 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 8 to 9 immunosuppressant molecules per molecule of IgG-degrading enzyme. [0053] In some embodiments, the plurality of conjugates in the composition is heterogenous wherein each conjugate comprises a varying number of between 9 to 10 immunosuppressant molecules per molecule of IgG-degrading enzyme. [0054] In some embodiments, the composition comprises less than 5% unconjugated IgG-degrading enzyme. In some embodiments, the composition comprises less than 1% unconjugated IgG-degrading enzyme. In some embodiments, the composition comprises less than 0.1% unconjugated IgG-degrading enzyme. In some embodiments, the composition Attorney Docket No. MIL-035WO1 comprises less than 5% unconjugated IdeS. In some embodiments, the composition comprises less than 1% unconjugated IdeS. In some embodiments, the composition comprises less than 0.1% unconjugated IdeS. [0055] In some embodiments, the present invention provides a method of reducing an immune response in a subject, comprising administering to the subject in need thereof, a therapeutically effective dose of the composition described herein. [0056] In some aspects, the present invention provides a method of reducing an immune response in a subject, wherein the method comprises administering to the subject in need thereof, a therapeutically effective dose of a composition comprising a conjugate of an IdeS or IdeZ protein and an immunosuppressant molecule. [0057] In some embodiments, the immune response generates one or more antibodies. In some embodiments, the antibody is IgG. In some embodiments, the antibody is IgG1, IgG2, IgG3 or IgG4. In some embodiments, the antibody is IgG1. In some embodiments, the antibody is IgG2. In some embodiments, the antibody is IgG3. In some embodiments, the antibody is IgG4. In some embodiments, the IgG2 antibody is IgG2a or IgG2b. In some embodiments, the IgG2 antibody is IgG2a. In some embodiments, the IgG2 antibody is IgG2b. [0058] In some embodiments, the composition is administered with a gene therapy vector or a replacement enzyme. In some embodiments, the composition is administered with a gene therapy vector. In some embodiments, the composition is administered with a replacement enzyme. [0059] In some embodiments, the composition is administered prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered prior to administration of a gene therapy vector. In some embodiments, the composition is administered prior to administration of the replacement enzyme. [0060] In some embodiments, the composition is administered at least 3 days, 5 days, or 7 days prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered at least 3 days prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 5 days prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 7 days prior to administration of the gene therapy vector. Attorney Docket No. MIL-035WO1 In some embodiments, the composition is administered at least 3 days prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 5 days prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 7 days prior to administration of the replacement enzyme. [0061] In some embodiments, the composition is administered between 1 day to 3 days prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered at least 1 day prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 2 days prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 3 days prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 1 day prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 2 days prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 3 days prior to administration of the replacement enzyme. [0062] In some embodiments, the composition is administered at least 1 hour, 3 hours, 5 hours, 10 hours, 12 hours, 16 hours, or 24 hours prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered at least 1 hour to 3 hours, 3 to 5 hours, 5 to 10 hours, 10 to 12 hours, 12 to 16 hours, or 16 to 24 hours, or any discrete intervening quantity therebetween, prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered at least 1 hour prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 3 hours prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 5 hours prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 10 hours prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 12 hours prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 16 hours prior to administration of the gene therapy vector. In some embodiments, the composition is administered at least 24 hours prior to administration of the replacement enzyme. Attorney Docket No. MIL-035WO1 [0063] In some embodiments, the composition is administered at least 1 hour prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 3 hours prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 5 hours prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 10 hours prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 12 hours prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 16 hours prior to administration of the replacement enzyme. In some embodiments, the composition is administered at least 24 hours prior to administration of the replacement enzyme. [0064] In some embodiments, the composition is administered concurrently with administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered concurrently with administration of the gene therapy vector. In some embodiments, the composition is administered concurrently with administration of the replacement enzyme. [0065] In some embodiments, the gene therapy vector is an adeno-associated virus (AAV) vector. In some embodiments, the gene therapy vector is an adenovirus vector. In some embodiments, the gene therapy vector is a lentiviral vector. [0066] In some embodiments, the gene therapy vector encodes a protein that elicits production of IgG antibodies. In some embodiments, the protein is a heterologous protein. [0067] In some embodiments the protein that elicits production of IgG antibodies is an AAV capsid protein. In some embodiments, the protein that elicits production of IgG antibodies is a lentiviral envelope protein. [0068] In some embodiments, provided by the present invention is a method of increasing transduction efficiency of a gene therapy vector, the method comprising administering the composition described herein. [0069] In some embodiments, the replacement enzyme is a lysosomal enzyme. [0070] In some embodiments, the lysosomal enzyme is selected from an acid phosphatase, an cid phosphodiesterase, an acid ribonuclease, an acid deoxyribonuclease, a β- Galactosidase, an α-Glucosidase, an α-Mannosidase, a β- Glucoronidase, a lysozymes, a hyaluronidase, anarylsulphatase, a cathepsin, a collagenase, a peptidase, an esterase, a Attorney Docket No. MIL-035WO1 phosholipipase, an arysulfatase, a glycosamine (N-acetyl)-6-sulfatase, and an iduronate 2- sulfatase. [0071] In some embodiments, the replacement enzyme is an arylsulfatase A or iduronate-2-sulfatase. In some embodiments, the replacement enzyme is arylsulfatase A. In some embodiments, the replacement enzyme is iduronate-2-sulfatase. In some embodiments, the replacement enzyme is a lysosomal enzyme from Table 4. [0072] In some embodiments, the subject receives an organ transplant. [0073] In some embodiments, provided by the present invention is a method of reducing organ transplant rejection comprising administering the composition described herein. [0074] In some embodiments, the composition is administered prior to the organ transplant. [0075] In some embodiments, upon administration of the composition, IgG cleavage into scIgG, F(ab)2, and Fc fragments. In some embodiments, upon administration of the composition IgG is cleaved to scIgG. [0076] In some embodiments, administration of the composition comprising the conjugate to a subject reduces induction of a T cell response relative to a control. [0077] In some embodiments, the control is the level of a T cell response in the same subject treated with unconjugated IdeS. [0078] In some embodiments the control is the level of a T cell response in a different subject treated with unconjugated IdeS. [0079] In some embodiments, the T cell response is reduced by 10%-20%, 20%-50%, 50%-80% or 80%-95%, or any intervening quantity therebetween, relative to the control. In some embodiments, the T cell response is reduced by 10%-20% relative to the control. In some embodiments, the T cell response is reduced by 20%-50% relative to the control. In some embodiments, the T cell response is reduced by 50-80% relative to the control. In some embodiments, the T cell response is reduced by 80-95% relative to the control. In some embodiments, the T cell response is reduced by at least 10% relative to the control. In some embodiments, the T cell response is reduced by at least 20% relative to the control. In some embodiments, the T cell response is reduced by at least 30% relative to the control. In some embodiments, the T cell response is reduced by at least 40% relative to the control. In some Attorney Docket No. MIL-035WO1 embodiments, the T cell response is reduced by at least 50% relative to the control. In some embodiments, the T cell response is reduced by at least 60% relative to the control. In some embodiments, the T cell response is reduced by at least 70% relative to the control. In some embodiments, the T cell response is reduced by at least 80% relative to the control. In some embodiments, the T cell response is reduced by at least 90% relative to the control. In some embodiments, the T cell response is reduced by at least 95% relative to the control. In some embodiments, the T cell response is reduced by at least 99% relative to the control. [0080] In some embodiments, the T cell response is reduced by greater than 95% relative to the control. In some embodiments the T cell response is reduced by greater than 96% relative to the control. In some embodiments the T cell response is reduced by greater than 97% relative to the control. In some embodiments the T cell response is reduced by greater than 98% relative to the control. In some embodiments, the T cell response is reduced by greater than 99% relative to the control. [0081] In some embodiments of the method, administration of the composition comprising the conjugate reduces induction of inflammatory cytokines relative to the control. [0082] In some embodiments, the inflammatory cytokines are IFN-gamma, IL-17 alpha, IL-2, IL-5, IL-6, TNF-alpha, and/or monocyte chemoattractant protein-1 (MCP-1). In some embodiments, the inflammatory cytokines are IFN-gamma. In some embodiments, the inflammatory cytokines are IL-17 alpha. In some embodiments, the inflammatory cytokines are IL-2. In some embodiments, the inflammatory cytokines are IL-5. In some embodiments, the inflammatory cytokines are IL-6. In some embodiments, the inflammatory cytokines are TNF-alpha. In some embodiments, the inflammatory cytokines are MCP-1. [0083] In some embodiments, the subject has cancer or an autoimmune disease. In some embodiments, the subject has cancer. In some embodiments, the subject has an autoimmune disease. [0084] In some embodiments, the composition is administered concurrently with or prior to adoptive cell transfer therapy. In some embodiments, the composition is administered concurrently with adoptive cell transfer therapy. In some embodiments, the composition is administered prior to adoptive cell transfer therapy. [0085] In some embodiments, the composition is administered concurrently with or prior to ex vivo cell therapy. In some embodiments, the composition is administered Attorney Docket No. MIL-035WO1 concurrently with ex vivo cell therapy. In some embodiments, the composition is administered prior to ex vivo cell therapy. BRIEF DESCRIPTION OF DRAWINGS [0086] The drawings are for illustration purposes and are in no way limiting. FIG.1A is a schematic that illustrates an IdeS protein, wherein amino acids 1-29 denote a signal peptide; amino acids 1-40 denote a putative signal sequence, amino acids 214-216 denote an RGF motif that is important for ligand recognition and amino acids C94 and H224 are catalytic residues. FIG.1B is a schematic that illustrates sortase mediated conjugation of rapamycin linker-payloads to IdeS. FIG.1C is a schematic that illustrates stochastic lysine conjugation of rapamycin linker payloads to IdeS. [0087] FIG.2A to FIG.2D depicts the structures of exemplary rapamycin linker-payloads for IdeS-rapamycin conjugation. FIG.2A depicts the structure of Compound 1, comprising a non-cleavable linker. FIG.2B depicts the structure of Compound 2, comprising a disulfide- cleavable linker. FIG.2C depicts the structure of Compound 3, which is a non-rapamycin linker control. FIG.2D depicts the structure of Compound 4, which is a disulfide-cleavable linker, NHS ester. [0088] FIG.3A to FIG.3D depict quality analysis of the conjugation products. FIG.3A shows SEC and LC-QTOF results for IdeS-rapamycin conjugate with sortase-mediated conjugation to Compound 1. FIG.3B shows SEC and LC-QTOF results for IdeS-rapamycin conjugate with sortase-mediated conjugation to Compound 2. FIG.3C shows SEC and LC- QTOF results for IdeS-rapamycin conjugate with sortase-mediated conjugation to Compound 3. FIG.3D shows SDS-PAGE gel and Coomassie staining results for IdeS-rapamycin conjugates made with stochastic lysine conjugation chemistry. [0089] FIG.4A to FIG.4B show results from enzymatic activity assays. FIG.4A is an SDS- PAGE gel and Coomassie staining of results from an IgG cleavage assay to determine IdeS enzyme activity in IdeS-rapamycin sortase-mediated conjugates. FIG.4B is a SDS-PAGE gel and Coomassie staining of results from an IgG cleavage assay to determine IdeS enzyme activity in IdeS-rapamycin stochastic lysine conjugates. Attorney Docket No. MIL-035WO1 [0090] FIG.5 shows T cell proliferation response and percent positive donor response rate for human PBMCs incubated with IdeS or IdeS + rapamycin. [0091] FIG.6 shows T cell proliferation response and percent positive donor response rate for human PBMCs incubated with IdeS or IdeS-rapamycin sortase-mediated conjugates. [0092] FIG.7A to FIG.7B show immune response after incubation of human PBMCs with IdeS, rapamycin, or IdeS-rapamycin conjugates. FIG.7A shows T cell proliferation response and percent positive donor response rate for human PBMCs incubated with IdeS or IdeS- rapamycin stochastic lysine conjugates. FIG.7B is an inflammatory cytokine panel for IdeS and IdeS-rapamycin stochastic lysine conjugates. [0093] FIG.8 is an overview of synthesis for rapamycin linker-payload Compound 1. [0094] FIG.9A to FIG.9D show the synthesis steps for rapamycin linker-payload Compound 1. FIG.9A shows synthesis step 1 for rapamycin linker-payload Compound 1. FIG.9B shows synthesis step 2 for rapamycin linker-payload Compound 1. FIG.9C shows synthesis step 3 for rapamycin linker-payload Compound 1. FIG.9D shows synthesis step 4 for rapamycin linker-payload Compound 1. [0095] FIG.10 is an overview of synthesis for rapamycin linker-payload Compound 2. [0096] FIG.11A to FIG.11E show the synthesis steps for rapamycin linker-payload Compound 2. FIG.11A shows synthesis step 1 for rapamycin linker-payload Compound 2. FIG.11B shows synthesis step 2 for rapamycin linker-payload Compound 2. FIG.11C shows synthesis step 3 for rapamycin linker-payload Compound 2. FIG.11D shows synthesis step 4 for rapamycin linker-payload Compound 2. FIG.11E shows synthesis step 5 for rapamycin linker-payload Compound 2. [0097] FIG.12 is an overview of synthesis for rapamycin linker-payload Compound 4. [0098] FIG.13A to FIG.13E show the synthesis steps for rapamycin linker-payload Compound 4. FIG.13A shows synthesis step 1 for rapamycin linker-payload Compound 4. FIG.13B shows synthesis step 2 for rapamycin linker-payload Compound 4. FIG.13C shows synthesis step 3 for rapamycin linker-payload Compound 4. FIG.13D shows synthesis step 4 for rapamycin linker-payload Compound 4.13E shows synthesis step 5 for rapamycin linker-payload Compound 4. Attorney Docket No. MIL-035WO1 DEFINITIONS [0099] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. [0100] As used in the specification and claims, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including a mixture thereof. [0101] Adeno-associated virus (AAV): As used herein, the terms “adeno-associated virus” or “AAV” or “recombinant AAV” (“rAAV”) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV (see, e.g., Fields et al. Virology, volume 2, chapter 69 (4th ed. Lippincott-Raven Publishers); Gao et al., J. Virology 78:6381- 6388 (2004); Mori et al. Virology 330:375-383 (2004)). AAV can infect both dividing and non-dividing cells and can be present in an extrachromosomal state without integrating into the host cell genome. AAV vectors are commonly used in gene therapy, and in some embodiments, AAV vectors are engineered. The AAV vectors can be engineered through any methods known in the art. For example, in some embodiments, AAV capsids are engineered through protein engineering methods. [0102] Administering: As used herein, the terms “administering” or “introducing” are used interchangeably in the context of delivering rAAV vectors encoding an antibody into a subject, by a method or route which results in efficient delivery of the rAAV vector. The terms “administering” and “introducing” are also used interchangeably in the context of delivering, for example and without limitation, enzyme replacement treatment, adoptive cell therapy, ex vivo cell therapy, IgG degrading enzymes, immunosuppressants, and IdeS- rapamycin conjugates. Various methods are known in the art for administering treatments, including, for example and without limitation, intravenously, subcutaneously, and transdermally. Transdermal administration of an rAAV vector, for example, can be performed by use of a “gene gun” or biolistic particle delivery system. In some embodiments, the rAAV vectors are administered via non-viral lipid nanoparticles. [0103] Adoptive Cell Therapy: As used herein, the term “adoptive cell transfer” or “ACT” refers to the transfer of cells into a patient in need thereof. The cells can be derived Attorney Docket No. MIL-035WO1 and propagated from the patient in need or could have been obtained from a non-patient donor. In some embodiments, the cell is an immune cell, such as a lymphocyte. Various cell types can be used for ACT such as a T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells, regulatory T-cells and peripheral blood mononuclear cells. [0104] Antibody: As used herein, the term “antibody” or “Ab” or “Abs” or “mAbs” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. By “specifically binds” or “immunoreacts with,” it is meant that the antibody reacts with one or more regions of an antigen. Antibodies include IgG, IgG2, IgG3, IgG4, IgG2a, and IgG2b. Antibodies include antibody fragments. Antibodies also include, but are not limited to, polyclonal, monoclonal, chimeric dAb (domain antibody), single chain, Fab, Fab’, F(ab’)2 fragments, scFvs. [0105] Approximately or about: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). [0106] Enzyme Replacement Therapy: As used herein, the term “enzyme replacement therapy (ERT)” refers to any therapeutic strategy that corrects an enzyme deficiency by providing the missing enzyme. When used in combination with the composition, the composition reduces the level of antibodies against the recombinant, replacement enzyme in serum. [0107] Ex vivo: As used herein, the term “ex vivo” means a process in which cells are removed from a living organism and are propagated outside the organism (e.g., in a test tube, in a culture bag, in a bioreactor). [0108] Immune Response: As used herein, the term “immune response” refers to both active and passive immune response to an unknown antigen. For example, in some embodiments, IdeS may induce an immune response in the body, and IdeS-rapamycin conjugates may reduce said immune response as measured by, for example and without limitations, T cell proliferation or inflammatory cytokine induction. Attorney Docket No. MIL-035WO1 [0109] Improve, increase, or reduce: As used herein, the terms “improve,” “increase,” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to administration of the composition described herein, or a measurement in a control individual (or multiple control individuals) in the absence of administration of the composition described herein. [0110] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism. [0111] In vivo: As used herein, the term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell- based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems). [0112] Linker: As used herein: the term “linker” refers to, in a conjugate, a compound connecting the IgG degrading enzyme to the immunosuppressant molecule. [0113] Linker-Payload: As used herein, the term “linker-payload” refers to the rapamycin-linker compound to be conjugated to the IgG degrading enzyme. [0114] Polypeptide: The term, “polypeptide,” as used herein refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of ordinary skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. As is known to those skilled in the art, polypeptides may be processed and/or modified. [0115] Protein: The term “protein” as used herein refers to one or more polypeptides that function as a discrete unit. If a single polypeptide is the discrete functioning unit and does not require permanent or temporary physical association with other polypeptides in order to form the discrete functioning unit, the terms “polypeptide” and “protein” may be used interchangeably. If the discrete functional unit is comprised of more than one polypeptide that physically associate with one another, the term “protein” refers to the multiple polypeptides that are physically coupled and function together as the discrete unit. [0116] Recombinant: As used herein, the term “recombinant” means that compositions have been manipulated or engineered in a fashion that generally does not occur Attorney Docket No. MIL-035WO1 in nature. “Recombinant” can be used as a modifier of a, for example, viral vector, such as, without limitation, a recombinant AAV (rAAV) vector. It can also be used as a modifier of sequences such as recombinant polynucleotides or polypeptides. A particular example of a recombinant AAV vector would be where a nucleic acid that is not normally present in a wild-type AAV genome (heterologous polynucleotide) is inserted within a viral genome. An example of which would be where a nucleic acid (e.g., gene) encoding a therapeutic protein or polynucleotide sequence is cloned into a vector. [0117] Subject: As used herein, the term “subject” refers to a human or any non- human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder. [0118] Suffering from: An individual who is “suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and/or condition. [0119] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose. [0120] Treating: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease. Attorney Docket No. MIL-035WO1 [0121] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.9, 4 and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about.” [0122] Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of “or” means “and/or” unless stated otherwise. As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. Compounds and Definitions [0123] Compounds of the present invention include those described generally herein, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference. [0124] The term “aliphatic” or “aliphatic group”, as used herein, means a straight- chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle”, “cycloaliphatic”, or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refers to a monocyclic C3 -C6 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is Attorney Docket No. MIL-035WO1 not aromatic, that has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl. [0125] The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR− (as in N-substituted pyrrolidinyl)). [0126] The term “unsaturated”, as used herein, means that a moiety has one or more units of unsaturation. [0127] As used herein, the term “bivalent C1-15 (or C1-6) saturated or unsaturated, straight or branched, hydrocarbon chain”, refers to bivalent alkylene, alkenylene, and alkynylene chains that are straight or branched as defined herein. [0128] The term “alkylene” refers to a bivalent alkyl group. An “alkylene chain” is a polymethylene group, i.e., -(CH2)n-, wherein n is a positive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group. [0129] The term “alkenylene” refers to a bivalent alkenyl group. A substituted alkenylene chain is a polymethylene group containing at least one double bond in which one or more hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group. [0130] The term “halogen” means F, Cl, Br, or I. [0131] The term “aryl” used alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic or bicyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 7 ring members. The term “aryl” may be used interchangeably with the term “aryl ring.” In certain embodiments of the present invention, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, Attorney Docket No. MIL-035WO1 naphthyl, anthracyl and the like, which may bear one or more sub stituents. Also included within the scope of the term “aryl,” as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like. [0132] The terms “heteroaryl” and “heteroar-”, used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14π electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. The term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3 (4H)-one. A heteroaryl group may be mono- or bicyclic. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted. The term “heteroaralkyl” refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted. [0133] As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably and refer to a stable 5- to 7- membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, Attorney Docket No. MIL-035WO1 the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl). [0134] A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical,” are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. A heterocyclyl group may be mono- or bicyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted. [0135] As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined. [0136] As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. Attorney Docket No. MIL-035WO1 [0137] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; -(CH2)0-4R○; -(CH2)0-4OR○; - O(CH2)0-4R○, -O-(CH2)0-4C(O)OR○; -(CH2)0-4CH(OR○)2; -(CH2)0-4SR○; -(CH2)0-4Ph, which may be substituted with R○; -(CH2)0-4O(CH2)0-1Ph which may be substituted with R○; - CH═CHPh, which may be substituted with R○; -(CH2)0-4O(CH2)0-1-pyridyl which may be substituted with R○; -NO2; -CN; -N3; -(CH2)0-4N(R○)2; -(CH2)0-4N(R○)C(O)R○; - N(R○)C(S)R○; -(CH2)0-4N(R○)C(O)NR○ 2; -N(R○)C(S)NR○ 2; -(CH2)0-4N(R○)C(O)OR○; - N(R○)N(R○)C(O)R○; -N(R○)N(R○)C(O)NR○ 2; -N(R○)N(R○)C(O)OR○; -(CH2)0-4C(O)R○; - C(S)R○; -(CH2)0-4C(O)OR○; -(CH2)0-4C(O)SR○; -(CH2)0-4C(O)OSiR○ 3; -(CH2)0-4OC(O)R○; - OC(O)(CH2)0-4SR-, SC(S)SR○; -(CH2)0-4SC(O)R○; -(CH2)0-4C(O)NR○ 2; -C(S)NR○ 2; - C(S)SR○; -SC(S)SR○, -(CH2)0-4OC(O)NR○ 2; -C(O)N(OR○)R○; -C(O)C(O)R○; - C(O)CH2C(O)R○; -C(NOR○)R○; -(CH2)0-4SSR○; -(CH2)0-4S(O)2R○; -(CH2)0-4S(O)2OR○; - (CH2)0-4OS(O)2R○; -S(O)2NR○ 2; -(CH2)0-4S(O)R○; -N(R○)S(O)2NR○ 2; -N(R○)S(O)2R○; - N(OR○)R○; -C(NH)NR○ 2; -P(O)2R○; -P(O)R○ 2; -OP(O)R○ 2; -OP(O)(OR○)2; SiR○ 3; -(C1-4 straight or branched)alkylene)O-N(R○)2; or -(C1-4 straight or branched alkylene)C(O)O- N(R○)2, wherein each R○ may be substituted as defined below and is independently hydrogen, C1-6 aliphatic, -CH2Ph, -O(CH2)0-1Ph, -CH2-(5-6 membered heteroaryl ring), or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R○, taken together with their intervening atom(s), form a 3-12- membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below. [0138] Suitable monovalent substituents on R○(or the ring formed by taking two independent occurrences of R○ together with their intervening atoms), are independently halogen, -(CH2)0-2R●, -(haloR●), -(CH2)0-2OH, -(CH2)0-2OR●, -(CH2)0-2CH(OR●)2; - O(haloR●), -CN, -N3, -(CH2)0-2C(O)R●, -(CH2)0-2C(O)OH, -(CH2)0-2C(O)OR●, -(CH2)0-2SR●, - (CH2)0-2SH, -(CH2)0-2NH2, -(CH2)0-2NHR●, -(CH2)0-2NR● 2, -NO2, -SiR● 3, -OSiR● 3, - C(O)SR●, -(C1-4 straight or branched alkylene)C(O)OR●, or -SSR● wherein each R● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1-4 aliphatic, -CH2Ph, -O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected Attorney Docket No. MIL-035WO1 from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R○ include ═O and ═S. [0139] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: ═O, ═S, ═NNR* 2, ═NNHC(O)R*,
Figure imgf000038_0001
wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR* 2)2-3O-, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. [0140] Suitable substituents on the aliphatic group of R* include halogen, -R●, - (haloR●), -OH, -OR●, -O(haloR●), -CN, -C(O)OH, -C(O)OR●, -NH2, -NHR●, -NR● 2, or - NO2, wherein each R● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, -CH2Ph, -O(CH2)0-1Ph, or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. [0141] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include -R†, -NR 2, -C(O)R†, -C(O)OR†, -C(O)C(O)R†, -C(O)CH2C(O)R†, -S(O)2R†, - S(O)2NR† 2, -C(S)NR● 2, -C(NH)NR● 2, or -N(R†)S(O)2R†; wherein each R† is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R†, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. [0142] Suitable substituents on the aliphatic group of R† are independently halogen, - R●, -(halon●), -OH, -OR●, -O(haloR●), -CN, -C(O)OH, -C(O)OR●, -NH2, -NHR●, -NR● 2, or - Attorney Docket No. MIL-035WO1 NO2, wherein each R● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, -CH2Ph, -O(CH2)0-1Ph, or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. [0143] As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. [0144] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate. Attorney Docket No. MIL-035WO1 [0145] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention. DETAILED DESCRIPTION [0146] The present invention provides, among other things, a conjugate of an IgG degrading protease, e.g., Immunoglobulin G (IgG) degrading enzyme of the Streptococcus pyogenes (IdeS) protein or IgG-degrading enzyme/MAC-1 (IdeZ) protein, with rapamycin for immunosuppression, for example, in gene therapy. Conjugating exemplary bacterial IgG proteases such as IdeS or IdeZ to the immunosuppressant rapamycin reduces immune stimulation due to bacterial protease alone and increases the efficacy of gene therapy. [0147] In some aspects, the present invention provides, among other things, a conjugate comprising an Immunoglobulin G (IgG)-degrading enzyme of Streptococcus pyogenes (IdeS) protein or IgG-degrading enzyme/Mac-1 (IdeZ) protein, and an immunosuppressant molecule. In some embodiments, the present invention provides, among other things, a conjugate comprising an Immunoglobulin G (IgG)-degrading enzyme, and an immunosuppressant molecule. Exemplary IgG-degrading enzymes, include, but are not limited to IdeS, IdeZ, MAC2, IdeZ2, IdeE, IdeE2, IdeP, and MMP, among others. In some embodiments, the immunosuppressant molecule is rapamycin, an analog (e.g., deforolimus, everlimus, or temsirolimus) or a mimetic (e.g., isoliquiritigenin or withaferin A) thereof. Attorney Docket No. MIL-035WO1 [0148] In some aspects, the present invention provides, among other things, a method of reducing an immune response in a subject, the method comprising administering to the subject in need thereof, a therapeutically effective dose of a composition comprising a conjugate of: (a) an IdeS or an IdeZ protein; and (b) an immunosuppressant molecule. A method of reducing an immune response by administration of the conjugate of the present invention has many therapeutic applications, for example, in treatment of cancer or autoimmune disease, in reducing rejection to organ transplants, and increasing benefits or reducing side-effects of therapeutic agents, increasing transduction and/or efficacy of gene therapy by reducing an immune response directed to a gene therapy vector or replacement enzyme in enzyme replacement therapy. Accordingly, it is contemplated that administration of an IdeS-rapamycin conjugate reduces immune response and increases efficacy of gene therapy (e.g. using AAV, adenovirus vector or lentiviral vector) or enzyme replacement therapy (e.g., lysosomal enzyme therapy, e.g., arylsulfatase A or iduronate-2-sulfatase). Immunoglobulin G Degrading Enzymes [0149] Immunoglobulin G (IgG) degrading enzymes are any polypeptides comprising IgG cleavage or degradation activity. They include, for example and without limitation, proteases and glycosidases. Proteases are enzymes that degrade or digest proteins and are also designated peptidases, proteinases, peptide hydrolases, or proteolytic enzymes. Glycosidases are enzymes that hydrolyze glycosidic bonds in complex sugars. [0150] Proteases used in the present invention, include without limitation, exo-type proteases, also known as exoproteases or exopeptidases, that hydrolyze peptide bonds located towards the N-terminal end or the C-terminal end, and endo-type proteases, also known as endoprotease or endopeptidase, that hydrolyze internal peptide bonds in polypeptide chains. Examples of endoproteases, without limitation, include IdeS, IdeZ, IgdE, IdeMC, trypsin, chymotrypsin, papain, and pepsin. [0151] Glycosidases used in the present invention, include without limitation, exoglycosidases and endoglycosidases. Glycosidases cleave and release glycans or oligosaccharides from glycoproteins such as antibodies. Exoglycosidases include, without limitation, N-acetylglucosaminidase, fucosidase, galactosidase, glucosidase, mannosidase, neuraminidase, and xylosidase. Examples of endoglycosidases include, without limitation, EndoS, EndoD, endoglycosidase H, Endo F1, Endo F2, and Endo F3. IdeS Attorney Docket No. MIL-035WO1 [0152] Cysteine proteases, for example, from bacteria including without limitation, Streptococcus pyogenes, Streptococcus equi, Mycoplasma canis, Streptococcus agalactiae, or Streptococcus pseudoporcinus are used in the present invention. The protein IdeS was first identified as a secreted immunogenic protein of Streptococcus pyogenes (Lei et al., 2000) and identified as a novel cysteine protease capable of cleaving IgG, therefore denoted IdeS (Ig degrading enzyme of S. pyogenes) (von Pawel-Rammingen et al., 2002). IdeS cleaves all human IgG subtypes, but no other Ig isotypes. In addition, IdeS carries out efficient cleavage of IgG from other species including monkey, sheep, rabbit as well as some activity towards murine and porcine IgG. In some embodiments, the IdeS comprises at least 70% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100% identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 75% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 80% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 85% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 90% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises at least 95% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In some embodiments, the IdeS comprises 100% sequence identity to the wild-type IdeS protein of SEQ ID NO: 1. In the sequence described in Table 1, amino acids 1-29 denote signal peptide, amino acids 1-40 identify a putative signal sequence, amino acids 214-216 denote an RGF motif which is important for ligand recognition and C94-H224 denote catalytic residues. [0153] Table 1. IdeS Wild-Type Protein Sequence. IdeS wild-type protein MRKRCYSTSAVVLAAVTLFALSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPA KFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEE HPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSAKHLGVFPDHVIDMF INGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIK KELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGV NSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 1) [0154] In some embodiments, the IgG degrading enzyme used in this invention is a variant of IdeS. In some embodiments, the IgG degrading enzyme used in this invention is a deimmunized variant of IdeS. In some embodiments, the IdeS protein variant comprises Attorney Docket No. MIL-035WO1 pep7. In some embodiments, the IdeS protein is a variant that comprises one or more mutations at amino acid residues 39, 154, 155, 161, 164, 297, 300 and 303 of the wild-type IdeS of SEQ ID NO: 1. In some embodiments, the IdeS protein has a mutation at amino acid position 39 of the wild-type IdeS of SEQ ID NO: 1. In some embodiments, the IdeS protein has a mutation at position 154 of the wild-type IdeS of SEQ ID NO: 1. In some embodiments, the IdeS protein has a mutation at position 155 of the wild-type IdeS of SEQ ID NO: 1. In some embodiments, the IdeS protein has a mutation at position 161 of the wild-type IdeS of SEQ ID NO: 1. In some embodiments, the IdeS protein has a mutation at position 297 of the wild-type IdeS of SEQ ID NO: 1. In some embodiments, the IdeS protein has a mutation at position 300 of the wild-type IdeS of SEQ ID NO: 1. In some embodiments, the IdeS protein has a mutation at position 303 of the wild-type IdeS of SEQ ID NO: 1. [0155] In some embodiments, the IdeS protein comprises at least 80% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises 80-85%, 85-90%, 95-100% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises at least 85% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises at least 90% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises at least 95% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. In some embodiments, the IdeS protein comprises at least 100% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. [0156] In some embodiments, the IdeS protein comprises an R39G mutation relative to the wild-type IdeS protein. [0157] In some embodiments, the IdeS protein is a variant that further comprises one or more mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises at least one mutation selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an E154D mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an Y155H mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an Y155Q mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an F161H mutation Attorney Docket No. MIL-035WO1 relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an F161Y mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an L164H mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an F297T mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an F297N mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an V300R mutation relative to the wild-type IdeS protein. In some embodiments, the IdeS protein further comprises an A303E mutation relative to the wild-type IdeS protein. [0158] In some embodiments, the IdeS protein is a variant that further comprises one mutation selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises between 2-10 mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises two mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises three mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises four mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises five mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises six mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises seven mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises eight mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises nine mutations selected Attorney Docket No. MIL-035WO1 from E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. In some embodiments, the IdeS protein is a variant that further comprises the combination of mutations consisting of E154D, Y155H, Y155Q, F161H, F161Y, L164H, F297T, F297N, V300R, and A303E relative to the wild-type IdeS protein. Exemplary IdeS variants are described in Table 2. Table 2. Variants of IdeS Modified with pep7 E154D-F297T-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFDYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYTVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 2) E154D-F297N-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFDYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 3) E154D-V300R-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFDYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGRNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 4) E154D-A303E-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFDYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGRNSEGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 5) Y155H-F297T-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEHFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYTVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 6) Attorney Docket No. MIL-035WO1 Y155H-F297N-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEHFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 7) Y155H-V300R-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEHFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGRNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 8) Y155H-A303E-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEHFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGRNSEGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 9) Y155Q-F297T-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEQFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYTVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 10) Y155Q-F297N-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEQFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 11) Y155Q-V300R-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEQFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGRNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 12) Attorney Docket No. MIL-035WO1 Y155Q-A303E-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEQFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGRNSEGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 13) F161H-F297T-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEYFKEKAHPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYTVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 14) F161H-F297N-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEYFKEKAHPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 15) F161H-V300R-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEYFKEKAHPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGRNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 16) F161H-A303E-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEYFKEKAHPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGRNSEGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 17) F161Y-F297T-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEYFKEKAYPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYTVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 18) Attorney Docket No. MIL-035WO1 F161Y-F297N-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEYFKEKAYPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 19) F161Y-V300R-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEYFKEKAYPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGRNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 20) F161Y-A303E-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEYFKEKAYPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGRNSEGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 21) L164H-F297T-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEYFKEKAYPYHSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYTVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 22) L164H-F297N-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEYFKEKAYPYHSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 23) L164H-V300R-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEYFKEKAYPYHSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGRNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 24) Attorney Docket No. MIL-035WO1 L164H-A303E-pep7 DSFSANQEIGYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKD DLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSK LFEYFKEKAYPYHSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVF TRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS NGNLKAIYVTDSDSNASIGMKKYNVGRNSEGKVAISAKEIKEDNIGAQVLGLFTLSTGQDS WNQTN (SEQ ID NO: 25) [0159] In some embodiments, the IdeS protein is a variant comprising a D30-T49 deletion relative to wild-type IdeS protein. In some embodiments, the IdeS protein is a variant comprising a DSFSANCEIRYSEVTPYHVT (SEQ ID NO: 50) deletion. Exemplary IdeS deletion variants are described in Table 3. Table 3. Variants of IdeS with a Deletion from D30 to T49 Relative to Wild-Type E154D-F297T-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFDYFKEKAFPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYTVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 26) E154D-F297N-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFDYFKEKAFPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 27) E154D-V300R-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFDYFKEKAFPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGRNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 28) E154D-A303E-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFDYFKEKAFPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM Attorney Docket No. MIL-035WO1 KKYNVGRNSEGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 29) Y155H-F297T-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEHFKEKAFPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYTVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 30) Y155H-F297N-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEHFKEKAFPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 31) Y155H-V300R-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEHFKEKAFPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGRNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 32) Y155H-A303E-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEHFKEKAFPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGRNSEGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 33) Y155Q-F297T-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEQFKEKAFPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYTVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 34) Y155Q-F297N-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEQFKEKAFPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL Attorney Docket No. MIL-035WO1 KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 35) Y155Q-V300R-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEQFKEKAFPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGRNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 36) Y155Q-A303E-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEQFKEKAFPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGRNSEGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 37) F161H-F297T-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAHPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYTVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 38) F161H-F297N-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAHPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 39) F161H-V300R-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAHPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGRNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 40) F161H-A303E-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAHPYLSTKHLGV Attorney Docket No. MIL-035WO1 FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGRNSEGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 41) F161Y-F297T-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAYPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYTVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 42) F161Y-F297N-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAYPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 43) F161Y-V300R-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAYPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGRNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 44) F161Y-A303E-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAYPYLSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGRNSEGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 45) L164H-F297T-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAYPYHSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYTVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 46) L164H-F297N-D30-T49del Attorney Docket No. MIL-035WO1 SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAYPYHSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 47) L164H-V300R-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAYPYHSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGRNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 48) L164H-A303E-D30-T49del SVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAYPYHSTKHLGV FPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNL KEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGM KKYNVGRNSEGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 49) IdeZ [0160] In certain embodiments, the IgG degrading enzyme comprises the endopeptidase from Streptococcus equi, IgG-degrading enzyme/MAC-1 (IdeZ). IdeZ recognizes all human, sheep, monkey, and rabbit IgG subclasses, cleaving specifically at a single recognition site below the hinge region, to yield a homogenous pool of F(ab´)2 and Fc fragments. In some embodiments, IdeZ protease more effectively cleaves murine IgG2a and IgG3 than IdeS. [0161] In some embodiments, the IdeZ comprises at least 70% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100% identity to the wild-type IdEZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 75% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 80% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ protein comprises at least 85% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 90% sequence identity to the wild-type IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises at least 95% sequence identity to the wild-type Attorney Docket No. MIL-035WO1 IdeZ protein of SEQ ID NO: 51. In some embodiments, the IdeZ comprises 100% identity to the wild-type IdeZ protein of SEQ ID NO: 51. [0162] In some embodiments, the protease includes a modified variant of IdeZ. Table 4. IdeZ Wild-Type Protein Sequence. IdeZ wild-type protein MKKQSFTHSRKPKFGMRKLSIGLASCMLGMMFLTTSHVSGEVVEVWPYGQNPNGKTEILSQ TEDSESSQRLRDIEDFQAEKKMQNVVYTKWLDGVDVKDHDFRKIVDGNIAYYATPLLNGRG FYDINKDFNRDSDKCAAAVAANMFHYWLDINRDNVDRFLRQNPEKHGIIELPDGQLKLSDF LNTYESDHGYRDKSKLFDFISNNFNGPVWTDKLLDNYINGYAYNYKYGRTIEDPTKNTSKI NFFKEVFNEKILTNNHSIRNQNEFSVLLSEALYTGKAIGLSYGPAGLRHSLGHIISVWGAD LDADGNVVAIYVTDSDDKKLTIGDERVGLKRYKISTDDENRLRLTAYEETHNTGGQIRGLW TLDTGKYAWADYFDKTEQTGTDQAEQ (SEQ ID NO: 51) [0163] In some embodiments, the protease includes a modified variant of IdeZ. [0164] In some embodiments, the Immunoglobulin G-degrading enzyme is a MAC2 protein. In some embodiments, the Immunoglobulin G-degrading enzyme is an IdeZ2 protein. In some embodiments, the Immunoglobulin G-degrading enzyme is an IdeE protein. In some embodiments, the Immunoglobulin G-degrading enzyme is an IdeE2 protein. In some embodiments, the Immunoglobulin G-degrading enzyme is an IdeP protein. In some embodiments, the Immunoglobulin G-degrading enzyme is an MMP protein. Immunosuppressants [0165] Immunosuppressants or immunosuppressive drugs can be classified into four groups: glucocorticoids, cytostatics, antibodies, drugs acting on immunophilins, and other drugs such as interferons, opiates INF binding proteins, mycophenolate, and FTY720, among others. An exemplary class of immunosuppressant drugs comprises those drugs that act on immunophilins, which are high-affinity, specific binding proteins having physiological significance. Two distinct families of immunophilins are presently known: cyclophilins and macrophilins, the latter of which specifically bind, for example, rapamycin (sirolimus, RAPAMUNE®), and tacrolimus (FK506, PROGRAF®). Immunosuppressant drugs that act on immunophilin include, for example, cyclosporin (including cyclosporin A, cyclosporin B, cyclosporin C, cyclosporin D, cyclosporin E, cyclosporin F, cyclosporin G, cyclosporin H, cyclosporin I), everolimus (RAD, CERTICAN®), and deforolimus (AP23573, MK-8669), among others. Attorney Docket No. MIL-035WO1 [0166] Immunosuppressant drug conjugates comprise an immunosuppressant drug and a conjugative moiety bound together, optionally through a linking group, to form a single structure. The binding can be either covalent attachment such as by a direct connection, e.g., a chemical bond between the immunosuppressant drug and the conjugative moiety or between the immunosuppressant drug and the conjugative moiety and a linking group, or non-covalent attachment involving specific binding between complementary specific binding pair (sbp) members that are attached to the immunosuppressant drug and the conjugative moiety of the conjugate. [0167] The present disclosure provides conjugates of an immunoglobulin G- degrading enzyme (e.g., IdeS, IdeZ) and one or more immunosuppressant molecules. In some embodiments, the immunosuppressant molecule is rapamycin (sirolimus), an analog or a mimetic thereof. In some embodiments, the immunosuppressant molecule is rapamycin. In some embodiments, the immunosuppressant molecule is a rapamycin analog. In some embodiments, the rapamycin analog is selected from the group consisting of deforolimus, everolimus, and temsirolimus. In some embodiments, the rapamycin analog is deforolimus. In some embodiments, the rapamycin analog is everolimus. In some embodiments, the rapamycin analog is temsirolimus. In some embodiments, the rapamycin mimetic is isoliquiritigenin or withaferin A. In some embodiments, the rapamycin mimetic is isoliquiritigenin. In some embodiments, the rapamycin mimetic is withaferin A. IdeS-Rapamcyin Conjugates [0168] In some aspects, the present disclosure provides, among other things, a conjugate comprising an Immunoglobulin G (IgG)-degrading enzyme of Streptococcus pyogenes (IdeS) protein or IgG-degrading enzyme/Mac-1 (IdeZ) protein, and an immunosuppressant molecule. [0169] In some embodiments, the IgG-degrading enzyme is conjugated to up to 10 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 1 immunosuppressant molecule. In some embodiments, the IgG-degrading enzyme is conjugated to 2 immunosuppressant molecules. In some embodiments, the IgG- degrading enzyme is conjugated to 3 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 4 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 5 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 6 immunosuppressant Attorney Docket No. MIL-035WO1 molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 7 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 8 immunosuppressant molecules. In some embodiments, the IgG-degrading enzyme is conjugated to 9 immunosuppressant molecules. In some embodiments, the IgG- degrading enzyme is conjugated to 10 immunosuppressant molecules. [0170] In some embodiments, the IdeS is conjugated to up to 10 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 1 immunosuppressant molecule. In some embodiments, the IdeS is conjugated to 2 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 3 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 4 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 5 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 6 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 7 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 8 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 9 immunosuppressant molecules. In some embodiments, the IdeS is conjugated to 10 immunosuppressant molecules. [0171] In some embodiments, the IdeZ is conjugated to up to 10 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 1 immunosuppressant molecule. In some embodiments, the IdeZ is conjugated to 2 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 3 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 4 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 5 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 6 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 7 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 8 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 9 immunosuppressant molecules. In some embodiments, the IdeZ is conjugated to 10 immunosuppressant molecules. [0172] In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:1 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:2 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:3 ratio. In some embodiments, the IgG-degrading enzyme and the Attorney Docket No. MIL-035WO1 immunosuppressant molecule are conjugated in at least a 1:4 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:5 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:6 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:7 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:8 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:9 ratio. In some embodiments, the IgG-degrading enzyme and the immunosuppressant molecule are conjugated in at least a 1:10 ratio. [0173] In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:1 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:2 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:3 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:4 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:5 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:6 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:7 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:8 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:9 ratio. In some embodiments, the IdeS and the immunosuppressant molecule are conjugated in at least a 1:10 ratio. [0174] In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:1 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:2 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:3 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:4 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:5 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:6 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:7 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:8 ratio. In some embodiments, the IdeZ and the Attorney Docket No. MIL-035WO1 immunosuppressant molecule are conjugated in at least a 1:9 ratio. In some embodiments, the IdeZ and the immunosuppressant molecule are conjugated in at least a 1:10 ratio. [0175] In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecules via surface amino acid residues on the IgG-degrading enzyme. In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecules via a surface lysine residue on the IgG-degrading enzyme. [0176] In one aspect, the present disclosure provides a conjugate of Formula I:
Figure imgf000058_0001
I, wherein: A is an Immunoglobulin G-degrading enzyme, B is an immunosuppressant molecule, L1 is a covalent bond, or an optionally substituted C1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the chain are independently and optionally replaced with -O-, -S-, S(O)2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R)-, -N(R)C(O)-, -Cy1-, or -Cy2-, wherein each -Cy1- is independently an optionally substituted bivalent ring selected from 3-7 membered saturated or partially unsaturated monocyclic carbocyclylene, monocyclic arylene, 4-10 membered saturated or partially unsaturated monocyclic heterocyclylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 5-10 membered saturated or partially unsaturated monocyclic heteroarylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, each -Cy2- is independently an optionally substituted bivalent ring selected from 3-7 membered saturated or partially unsaturated polycyclic carbocyclylene, polycyclic arylene, 4-10 membered saturated or partially unsaturated polycyclic heterocyclylene having 1- Attorney Docket No. MIL-035WO1 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 5-20 membered saturated or partially unsaturated polycyclic heteroarylene having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and wherein L2 is a covalent bond,
Figure imgf000059_0001
wherein n is an integer between 1-170 or a molecule weight average of 1kDa-15kDa, or an optionally substituted C1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the chain are independently and optionally replaced with -O-, -S-, -S(O)2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R)-, or - N(R)C(O)-. L3 is a covalent bond or an optionally substituted C1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the chain are independently and optionally replaced with -O-, -S-, - N(R)-, -S(O)2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R)-, -N(R)C(O)-, or - P(O)(R)-, each R is independently hydrogen, or an optionally substituted group selected from C1-6 aliphatic, phenyl, 4-7 membered saturated or partially unsaturated heterocyclic having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, m is an integer greater than 0, and wherein L1 is connected to one or more heteroatoms selected from nitrogen, oxygen, and sulfur present in the immunosuppressant molecule, and wherein L3 is connected to one or more heteroatoms selected from nitrogen, oxygen, and sulfur present in the Immunoglobulin G-degrading enzyme. [0177] In some embodiments, A is an Immunoglobulin G-degrading enzyme. In some embodiments the Immunoglobulin G-degrading enzyme is an IdeS protein. In some embodiments the Immunoglobulin G-degrading enzyme is an IdeZ protein. Attorney Docket No. MIL-035WO1 [0178] In some embodiments, B is an immunosuppressant macrolide. In some embodiment, B is an immunosuppressant macrophilin. [0179] In some embodiments, the immunosuppressant molecule is rapamycin (or sirolimus):
Figure imgf000060_0001
(rapamycin or sirolimus), or a pharmaceutically acceptable salt thereof. In the present disclosure, rapamycin and sirolimus are used interchangeably. [0180] In some embodiments, the immunosuppressant molecule is everolimus:
Figure imgf000060_0002
(everolimus), or a pharmaceutically acceptable salt thereof. [0181] In some embodiments, the immunosuppressant molecule is deforolimus: Attorney Docket No. MIL-035WO1
Figure imgf000061_0001
(deforolimus), or a pharmaceutically acceptable salt thereof. [0182] In some embodiments, the immunosuppressant molecule is temsirolimus:
Figure imgf000061_0002
(temsirolimus), or a pharmaceutically acceptable salt thereof. [0183] In some embodiments, L1 is a covalent bond, or an optionally substituted C1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the chain are independently and optionally replaced with -O-, -S-, S(O)2-, C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R)-, -N(R)C(O)-, -Cy1-, or -Cy2-, wherein each -Cy1- is independently an optionally substituted bivalent ring selected from 3-7 membered saturated or partially unsaturated monocyclic carbocyclylene, monocyclic arylene, 4-10 membered saturated or partially unsaturated monocyclic heterocyclylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 5-10 membered saturated or partially unsaturated monocyclic heteroarylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and each -Cy2- is independently an optionally substituted bivalent ring selected from 3-7 membered saturated or partially unsaturated Attorney Docket No. MIL-035WO1 polycyclic carbocyclylene, polycyclic arylene, 4-10 membered saturated or partially unsaturated polycyclic heterocyclylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 5-20 membered saturated or partially unsaturated polycyclic heteroarylene having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. [0184] In some embodiments, L1 is a covalent bond [0185] In some embodiments, L1 is an optionally substituted C1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the chain are independently and optionally replaced with -O-, -S-, S(O)2-, C(O)-, -C(O)O-, - OC(O)-, -C(O)N(R)-, -N(R)C(O)-, -Cy1-, or -Cy2-., [0186] In some embodiments, -Cy1- is independently an optionally substituted bivalent ring selected from 3-7 membered saturated or partially unsaturated monocyclic carbocyclylene, monocyclic arylene, 4-10 membered saturated or partially unsaturated monocyclic heterocyclylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 5-10 membered saturated or partially unsaturated monocyclic heteroarylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. [0187] In some embodiments, -Cy2- is independently an optionally substituted bivalent ring selected from 3-7 membered saturated or partially unsaturated polycyclic carbocyclylene, polycyclic arylene, 4-10 membered saturated or partially unsaturated polycyclic heterocyclylene having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 5-20 membered saturated or partially unsaturated polycyclic heteroarylene having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. [0188] In some embodiments,
Figure imgf000062_0001
[0189] In some embodiments,
Figure imgf000062_0002
Attorney Docket No. MIL-035WO1 [0190] In some
Figure imgf000063_0001
[0191] In some embodiments, -
Figure imgf000063_0002
[0192] In some embodiments, -
Figure imgf000063_0003
[0193] In some embodiments, -
Figure imgf000063_0004
[0194] In some embodiments, -
Figure imgf000063_0005
[0195]
Figure imgf000063_0007
some embodiments, L2 is a covalent bond,
Figure imgf000063_0006
wherein n is an integer between 1-170, or an optionally substituted C1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the chain are independently and optionally replaced with -O-, -S-, S(O)2-, - C(O)-, -C(O)O-, -OC(O)-, - C(O)N(R)-, or -N(R)C(O)-. [0196] In some embodiments, L2 is a covalent bond. [0197] In some embodiments, L2 is
Figure imgf000063_0008
wherein n is an integer between 1-170 or a molecule weight average of 1kDa-15kDa [0198] In some embodiments, L2 is an optionally substituted C1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the Attorney Docket No. MIL-035WO1 chain are independently and optionally replaced with -O-, -S-, S(O)2-, - C(O)-, -C(O)O-, - OC(O)-, -C(O)N(R)-, or -N(R)C(O)-. [0199] In some embodiments, L3 is a covalent bond or an optionally substituted C1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the chain are independently and optionally replaced with -O-, -S-, -N(R)-, -S(O)2-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R)-, -N(R)C(O)-, or -P(O)(R)-. [0200] In some embodiments, L3 is a covalent bond. [0201] In some embodiments, L3 is an optionally substituted C1-15 bivalent straight or branched saturated or unsaturated hydrocarbon chain wherein 1-10 methylene units of the chain are independently and optionally replaced with -O-, -S-, -N(R)-, -S(O)2-, -C(O)-, - C(O)O-, -OC(O)-, -C(O)N(R)-, -N(R)C(O)-, or -P(O)(R)-. [0202] In some embodiments,
Figure imgf000064_0001
[0203] In some embodiments,
Figure imgf000064_0002
[0204] In some embodiments,
Figure imgf000064_0003
[0205] In some embodiments, m is an integer greater than 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. [0206] In some embodiments, the present disclosure provides a conjugate of Formula III: Attorney Docket No. MIL-035WO1
Figure imgf000065_0001
III, wherein A, L1, L2, L3, and m are as described above and wherein Z is a point of attachment to L3 or H. [0207] In some embodiments, the present disclosure provides a conjugate of Formula III:
Figure imgf000065_0002
wherein A, L1, L2, L3, and m are as described above. [0208] In some embodiments, the present disclosure provides a conjugate of Formula IIa: Attorney Docket No. MIL-035WO1
Figure imgf000066_0001
IIIa, wherein A, L1, L2, L3, and m are as described above. [0209] In some embodiments, the present disclosure provides a conjugate of Formula IIIb:
Figure imgf000066_0002
IIIb, wherein A, L1, L2, L3, and m are as described above. [0210] In some embodiments, the present disclosure provides a conjugate of Formula IV: Attorney Docket No. MIL-035WO1
Figure imgf000067_0001
IV, wherein A and m are as described above. [0211] In some embodiments, the present disclosure provides a conjugate of Formula
Figure imgf000067_0002
V, wherein A and m are as described above. [0212] In some embodiments, the present disclosure provides a conjugate of Formula Attorney Docket No. MIL-035WO1
Figure imgf000068_0001
VI wherein A and m are as described above. Conjugation [0213] Exemplary IgG degrading enzyme and immunosuppressant conjugates, for example, an IdeS-rapamycin conjugate of the present disclosure is manufactured by one or more of the methods described herein, for example, sortase-mediated conjugation, lysine conjugation, cysteine conjugation, among others. In some embodiments, the conjugation is via a linker. [0214] In some embodiments, the IgG degrading enzyme is conjugated to the immunosuppressant molecule via a surface lysine residue on IdeS. In some embodiments, the IgG degrading enzyme is conjugated to the immunosuppressant molecule via a surface lysine residue on IdeS. In some embodiments, the IdeZ is conjugated to the immunosuppressant molecule via a surface lysine residue on IdeZ. [0215] In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecule via a C-terminus on the IgG-degrading enzyme. In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecule via a N-terminus on the IgG-degrading enzyme. In some embodiments, the IdeS is conjugated to the immunosuppressant molecule via a C-terminus on the IdeS. In some embodiments, the IdeS is conjugated to the immunosuppressant molecule via a N-terminus on the IdeS. In some embodiments, the IdeZ is conjugated to the immunosuppressant Attorney Docket No. MIL-035WO1 molecule via a C-terminus on the IdeZ. In some embodiments, the IdeZ is conjugated to the immunosuppressant molecule via a N-terminus on the IdeZ. [0216] In some embodiments, the IgG-degrading enzyme is conjugated to the immunosuppressant molecule via a cleavable linker. In some embodiments, the IgG- degrading enzyme is conjugated to the immunosuppressant molecule via a non-cleavable linker. In some embodiments, the IdeS is conjugated to the immunosuppressant molecule via a cleavable linker. In some embodiments, the IdeS is conjugated to the immunosuppressant molecule via a non-cleavable linker. In some embodiments, the IdeZ is conjugated to the immunosuppressant molecule via a cleavable linker. In some embodiments, the IdeZ is conjugated to the immunosuppressant molecule via a non-cleavable linker. Sortase-Mediated Conjugation [0217] Sortases (e.g., Sortase A) are transpeptidases that recognize a core amino acid motif (e.g., LPXTG) and selectively cleave the amide bond between the threonine and glycine residues. The resulting intermediate can then undergo nucleophilic attack by an oligo glycine that can include additional functional groups for further modification and/or site- specific conjugation. Sortase nucleophiles may be equipped with any of the functionalities that previously have been used in the sortase reaction, including, but not limited to, “click handles” for performing click conjugations. sortase
Figure imgf000069_0001
Click Chemistry [0218] Click chemistry refers to a class of reactions including The Huisgen 1,3- dipolar cycloaddition (e.g., the Cu(I)-catalyzed stepwise variant, often referred to simply as the “click reaction”; see, e.g., Tornoe et al., Journal of Organic Chemistry (2002) 67: 3057- 3064). Copper and ruthenium are the commonly used catalysts in the reaction. The use of copper as a catalyst results in the formation of 1,4-regioisomer whereas ruthenium results in formation of the 1,5-regioisomer; other cycloaddition reactions, such as the Diels-Alder reaction; other Nucleophilic addition to small strained rings like epoxides and aziridines; Attorney Docket No. MIL-035WO1 nucleophilic addition to activated carbonyl groups; and addition reactions to carbon-carbon double or triple bonds. [0219] The term “click chemistry handle,” as used herein, refers to a reactant, or a reactive group, that can partake in a click chemistry reaction. For example, a strained alkyne, e.g., a cyclooctyne, is a click chemistry handle, since it can partake in a strain-promoted cycloaddition. In general, click chemistry reactions require at least two molecules comprising click chemistry handles that can react with each other. Such click chemistry handle pairs that are reactive with each other are sometimes referred to herein as partner click chemistry handles. For example, an azide is a partner click chemistry handle to a cyclooctyne or any other alkyne. Suitable click chemistry handles are known to those of skill in the art. Exemplary click chemistry handles suitable for use according to some aspects of this invention are described below.
Figure imgf000070_0001
Stochastic Lysine Conjugation [0220] The nucleophilic ε-amino group of lysine residues on the immunoglobulin G- degrading enzyme may be chemically conjugated under a variety of conditions known to those of skill in the art. For instance, linkers bearing activated ester groups, e.g., N- hydroxysuccinimidyl (NHS) or sulfo-NHS esters, and imido esters, e.g., Traut’s reagent, readily react with lysine residues to form amide or amidine bonds. Attorney Docket No. MIL-035WO1
Figure imgf000071_0001
Stochastic Cysteine Conjugation [0221] The nucleophilic sulfur of cysteine resides on the immunoglobulin G- degrading enzyme may be chemically conjugated under a variety of conditions known to those of skill in the art. For instance, linkers bearing malimido groups react readily to form maleimide-thiol conjugates.
Figure imgf000071_0002
Linkers [0222] As used herein, the terms “linking” and “conjugating” are used interchangeably an each refer to the covalent or non-covalent attachment of two or more moieties comprising a immunosuppressant molecule and an immunoglobulin G-degrading enzyme. In some aspects the linking or conjugating can comprise a linker. In some embodiments the linker comprises L1, L2, and/or L3. [0223] For covalent attachment of the components of a conjugate, that is the immunosuppressant molecule and the immunoglobulin G-degrading enzyme and/or linker, one or more of the components contains a functional group suitable for attachment to one or more of the other components. The functional groups suitable for attaching the components may be carbonyl functionalities, both oxocarbonyl, e.g., aldehyde, and non-oxocarbonyl (including nitrogen and sulfur analogs) e.g., carboxy, amidine, amidate, thiocarboxy and thionocarboxy. Alternative functionalities of oxo include active halogen, diazo, mercapto, olefin, particularly activated olefin, amino, phosphoro and the like. Of particular interest are activated esters or alkylating agents. Details of techniques for attaching molecules to one another may be found, for example, in Matthews, et al., Anal. Biochem. (1985) 151:205-209; Engelhardt, et al., European Patent Application No.0302175 and U.S. Pat. No.3,817,837, the relevant disclosure of which is incorporated herein by reference in its entirety. Attorney Docket No. MIL-035WO1 [0224] In certain aspects, the linker can contain a heterobifunctional group. In the present disclosure, the term “heterobifunctional group” refers to a chemical moiety that connects the linker of which it is a part to the binding moiety. Heterobifunctional groups are characterized as having different reactive groups at either end of the chemical moiety. Attachment to the immunoglobulin G-degrading enzyme can be accomplished through chemical or enzymatic conjugation, or a combination of both. Chemical conjugation involves the controlled reaction of accessible amino acid residues on the surface of the binding moiety with a reaction handle on the heterobifunctional group. Examples of chemical conjugation include, but are not limited to, lysine amide coupling, cysteine coupling, and coupling via a non-natural amino acid incorporated by genetic engineering, wherein non-natural amino acid residues with a desired reaction handle are installed onto immunoglobulin G-degrading enzyme. [0225] In enzymatic conjugation, an enzyme mediates the coupling of the linker with an accessible amino residue on the binding moiety. Examples of enzymatic conjugation include, but are not limited to, transpeptidation using sortase, transpeptidation using microbial transglutaminase, and N-glycan engineering. Chemical conjugation and enzymatic conjugation may also be used sequentially. For example, enzymatic conjugation can also be used for installing unique reaction handles on the immunoglobulin G-degrading enzyme to be utilized in subsequent chemical conjugation. Non-cleavable and cleavable linkers [0226] In certain aspects, linker (e.g., L1, L2, and/or L3) is non-cleavable. As used here, the term “non- cleavable linker” is any chemical moiety that is capable of linking the immunoglobulin G-degrading enzyme to the immunosuppressant molecule in a stable, covalent manner and does not fall under the categories defined herein as “cleavable linkers”. Thus, non-cleavable linkers are substantially resistant to acid-induced cleavage, light-induced cleavage, bioreductive cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. “Substantially resistant to cleavage” means that the chemical bond in the linker or adjoining the linker in at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% of the conjugate population remains non-cleavable by an acid, a photolabile- cleaving agent, a bioreductive agent, a peptidase, an esterase, or a chemical or a physiological compound that cleaves the chemical bond (for example, a disulfide bond) in a cleavable linker, for within a Attorney Docket No. MIL-035WO1 few hours to several days of treatment with any of the agents described above. In certain aspects the linker is not susceptible to acid-induced cleavage, photo-induced cleavage, bioreductive cleavage, enzymatic cleavage, or the like, at conditions under which the immunosuppressant molecule and/or the immunoglobulin G-degrading enzyme can remain active. [0227] A person of ordinary skill in the art would readily distinguish non-cleavable from cleavable linkers. [0228] Examples of non-cleavable linkers include, but are not limited to, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) linkers, succinimide thioether linkers, and linkers such as:
Figure imgf000073_0001
Figure imgf000073_0002
, wherein p is an integer from 1 to 10. [0229] In some embodiments, the linker is cleavable. In some embodiments, the linker is susceptible to acid-induced cleavage, photo-induced cleavage, bioreductive cleavage, enzymatic cleavage, or the like, at conditions under which the immunosuppressant molecule and/or the immunoglobulin G-degrading enzyme remain active. [0230] In some embodiments, the cleavable linker can be cleaved enzymatically. In some embodiments, the cleavable linker is cleaved by a protease, peptidase, esterase, beta- gluroronidase, glycosidase, phosphodiesterase, phosphatase, pyrophosphatase, or lipase. [0231] In some aspects, the cleavable linker can be cleaved by a protease. Examples of proteases include, but are not limited to, cathepsin B, VAGP tetrapeptide, and the like. [0232] In certain aspects, the cleavable linker contains a peptide. In some aspects, the peptide is the site of cleavage of the linker, thereby facilitating release of the drug upon exposure to intracellular proteases, such as lysosomal enzymes. Peptides can be designed and optimized for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease. Examples of peptides having two amino acids include, but are not limited to, alanine-alanine (ala-ala), valine-alanine (val-ala), Attorney Docket No. MIL-035WO1 valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); phenylalanine- homolysine (phe-homolys); and N-methyl-valine-citrulline (Me-val-cit). Examples of peptides having three amino acids include, but are not limited to, glycine-valine-citrulline (gly-val-cit), aspartic acid-valine-citrulline (asp-val-cit), alanine- alanine-asparagine (ala-ala-asn), alanine- phenylalanine-lysine (ala-phe-lys), glycine-glycine- phenylalanine (gly-gly-phe), and glycine- glycine-glycine (gly-gly-gly). Examples of peptides having four amino acids include, but are not limited to, glycine-glycine-valine- citrulline (gly-gly-val-cit) and glycine-glycine-phenylalanine- glycine (gly-gly-phe-gly). The amino acid combinations above can also be present in the reverse order (i.e., cit-val). [0233] The peptides of the present disclosure can comprise L- or D- isomers of amino acid residues. The term “naturally-occurring amino acid” refers to Ala, Asp, Asx, Cit, Cys, Glu, Phe, Glx, Gly, His, lie, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr. “D-” designates an amino acid having the “D” (dextrorotary) configuration, as opposed to the configuration in the naturally occurring (“L-”) amino acids. The amino acids described herein can be purchased commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art. [0234] In some embodiments, the linker is bioreducible. Bioreducible linkers take advantage of the difference in reduction potential in the intracellular compartment versus plasma. Reduced glutathione presented in tumor cells’ cytoplasm is up to 1000-fold higher than that present in normal cells’ cytoplasm, and the tumor cells also contain enzymes which can contribute to reduction in cellular compartments. The linkers keep conjugates intact during systemic circulation, and are selectively cleaved by the high intracellular concentration of glutathione, releasing the immunosuppressant molecule. [0235] In some embodiments, linker is a bioreducible linker selected from:
Figure imgf000074_0001
. [0236] In some embodiments, the linker is acid cleavable. Acid-cleavable linkers are specifically designed to remain stable at the neutral pH of blood circulation, but undergo hydrolysis and release the cytotoxic drug in the acidic environment of the cellular compartments. Attorney Docket No. MIL-035WO1 [0237] In some embodiments, the linker is a click-to-release linker, where release of the immunosuppressant molecule is chemically triggered by a tetrazine or related compound. [0238] In some embodiments, IdeS is conjugated to a rapamycin-linker payload selected from the group consisting of any one of:
Figure imgf000075_0001
[0239] In some embodiments, Compound 1, Compound 2 or Compound 4 are monomeric compounds. Compositions and Kits [0240] In some embodiments, provided by the present disclosure is a composition comprising a plurality of conjugates described herein. In some embodiments, the plurality of conjugates is homogenous, each conjugate comprising a uniform number of between 1-6 immunosuppressant molecules per IdES molecule. In some embodiments, the plurality of conjugates is heterogenous, each conjugate comprising a varying number of between 1-6 immunosuppressant molecules per IdES molecule. Attorney Docket No. MIL-035WO1 [0241] In some embodiments, the composition comprises less than 5% unconjugated IdES. In some embodiments, the composition comprises less than 1% unconjugated IdES. In some embodiments, the composition comprises less than 0.1% unconjugated IdES. [0242] The present invention further provides kits, that include packaging material and one or more components therein. A kit typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein. A kit can contain a collection of such components, e.g., a nucleic acid, recombinant vector, gene therapy (e.g., AAV, lentivirus) vector, replacement enzyme, and a IgG degrading enzyme conjugated to an immunosuppressant molecule as described in the compositions of the present invention, e.g. IdeS-rapamcyin that degrades or digests antibodies. [0243] A kit refers to a physical structure housing one or more components of the kit. Packaging material can maintain sterile components, and be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.). Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics. Labels or inserts can include information identifying manufacturer, lot numbers, manufacture location and date, expiration dates. Labels or inserts can include information on a disease for which a kit component may be used. Labels or inserts can include instructions for the clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. Labels or inserts can include information on any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, complications or reactions, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition. Methods of Treatment and Use of Compositions [0244] In some aspects, provided herein is a method of reducing an immune response in a subject, the method comprising administering to the subject in need thereof, a therapeutically effective dose of a composition comprising a conjugate of: (a) an IdES or an IdEZ protein; and Attorney Docket No. MIL-035WO1 (b) an immunosuppressant molecule. In some embodiments, the immune response generates one or more antibodies. In some embodiments, the antibody is IgG. In some embodiments, the antibody is IgG1, IgG2, IgG3 or IgG4. In some embodiments, the IgG2 antibody is IgG2a or IgG2b. [0245] In some embodiments, the composition is administered with a gene therapy vector or a replacement enzyme. In some embodiments, the composition is administered prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered at least 3 days, 5 days or 7 days prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered between 1 day to 3 days prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered at least 1 hour, 3 hours, 5 hours, 10 hours, 12 hours, 16 hours, or 24 hours prior to administration of the gene therapy vector or the replacement enzyme. In some embodiments, the composition is administered concurrently with administration of the gene therapy vector or the replacement enzyme. In some embodiments, the gene therapy vector is an adeno-associated virus (AAV) vector. In some embodiments, the gene therapy vector is an adenovirus vector. In some embodiments, the gene therapy vector is a lentiviral vector. In some embodiments, the gene therapy vector encodes a protein that elicits production of IgG antibodies. In some embodiments, the protein is a heterologous protein. In some embodiments, the protein that elicits production of IgG antibodies is an AAV capsid protein. In some embodiments, the protein that elicits production of IgG antibodies is a lentiviral envelope protein. In some embodiments, provided herein is a method of increasing transduction efficiency of a gene therapy vector, the method comprising administering the composition described in the present disclosure. In some embodiments, the replacement enzyme is a lysosomal enzyme. In some embodiments, the replacement enzyme is arylsulfatase A or iduronate-2-sulfatase. [0246] In some embodiments, the subject receives an organ transplant. In some embodiments, provided herein is a method of reducing organ transplant rejection, comprising administering the composition described herein. In some embodiments, the composition is administered prior to the organ transplant. Attorney Docket No. MIL-035WO1 [0247] In some embodiments, upon administration of the composition, IgG is cleaved to scIgG, F(ab)2 and Fc fragments. In some embodiments, upon administration of the composition, IgG is cleaved to scIgG. [0248] In some embodiments, administration of the composition comprising the conjugate to a subject reduces induction of a T-cell response relative to a control. In some embodiments, the control is the level of a T-cell response in the same or a different subject treated with unconjugated IdES. In some embodiments, the T cell response is reduced by 10%-20%, 20%-50%, 50%-80% or 80%-95%, or any intervening quantity thereof, relative to the control. In some embodiments, the T cell response is reduced by greater than 95% relative to the control. In some embodiments, the T cell response is reduced by greater than 99% relative to the control. In some embodiments, administration of the composition comprising the conjugate reduces induction of inflammatory cytokines relative to the control. In some embodiments, the inflammatory cytokines are IFN-gamma, IL-17 alpha, IL-2, IL-5, IL-6, TNF-alpha and/or MCP-1. [0249] The present invention improves the efficacy and derivable benefit to a subject of a therapy or a therapeutic agent, while minimizing immunogenic responses. Administration of the composition to the subject in need of therapy can safely eliminate Fc receptor binding by all or substantially all IgG molecules in the serum of the subject, such that neutralizing antibodies directed to therapeutic are substantially reduced or eliminated. Reducing Immune Response [0250] This disclosure provides compositions comprising conjugates that reduce immune response against a therapy or therapeutic agent in a subject. In some embodiments, the therapeutic agent is a recombinant biologic comprising a vector comprising a heterologous nucleic acid encoding one or more recombinant proteins. In some embodiments, the vector is a recombinant virus vector, such as, for example and without limitation, a recombinant AAV vector, a lentiviral vector, or an adenovirus vector. In some embodiments, the recombinant biologic is a replacement enzyme. In some embodiments, the therapy is, for example and without limitation, adoptive cell therapy or ex vivo cell therapy. [0251] In some embodiments, the composition reduces immune response by neutralizing antibodies, promoting clearance or degradation of an antibody against the recombinant biologic or the drug entity. In some embodiments, the composition reduces Attorney Docket No. MIL-035WO1 immune response by reducing cytokine induction. In some embodiments, the composition reduces immune response by decreasing T cell proliferation. [0252] In some embodiments, the composition reduces neutralizing antibodies to a recombinant biologic or a drug entity wherein the antibodies comprise IgG (including IgG1, IgG2a, IgG2b, IgG3, and IgG4). In some embodiments, the antibodies comprise IgG. In some embodiments, the antibodies include those that would bind to the conjugate. In some embodiments, the antibodies include those that would bind the IgG degrading enzyme. [0253] In some embodiments, the composition reduces inflammatory cytokine induction to a therapy or therapeutic agent. In some embodiments, the composition reduces induction of inflammatory cytokines that include, for example and without limitation, IFN- gamma, IL-17 alpha, IL-2, IL-5, IL-6, TNF-alpha and/or MCP-1. Gene Therapy [0254] In some embodiments, the therapy is gene therapy. The present invention reduces immunogenicity and increases safety and efficacy of gene therapy in treating a variety of diseases by administering a combination of the conjugate together with a gene therapy vector, either prior to gene therapy or concurrently. In some embodiments, the gene therapy vector is a viral vector, including, for example and without limitation, AAV vectors, lentiviral vectors, and adenovirus vectors. In some embodiments, the viral vector is without limitation, helper-dependent adenoviral, hybrid adenoviral, herpes simplex virus, poxvirus, Epstein-Barr virus, vaccinia virus, and human cytomegalovirus vector, including recombinant versions thereof. [0255] Gene therapy vectors provide a means for delivering nucleic acids into a broad range of cells, including dividing and non-dividing cells. Gene therapy vectors can be employed to deliver a nucleic acid of interest to a cell in vitro, e.g., for ex vivo gene therapy. The vectors are additionally useful in a method of delivering a nucleic acid to a subject in need thereof, e.g., to express an immunogenic or therapeutic polypeptide or a functional RNA, thus the polypeptide or functional RNA can be produced in vivo in the subject. In some embodiments, the subject is in need of the polypeptide because the subject has a deficiency of the polypeptide, e.g., a lysosomal storage enzyme. [0256] Gene therapy vectors are used to treat and/or prevent any disease state for which it is beneficial to deliver a therapeutic polypeptide or functional nucleic acid. Illustrative disease states include, but are not limited to: cystic fibrosis (cystic fibrosis Attorney Docket No. MIL-035WO1 transmembrane regulator protein) and other diseases of the lung, hemophilia A (Factor VIII), hemophilia B (Factor IX), thalassemia (B-globin). anemia (erythropoietin) and other blood disorders, Alzheimer’s disease (GDF; neprilysin), multiple sclerosis (B-interferon). Parkinson’s disease (glial-cell line derived neurotrophic factor [GDNF]), Huntington’s disease (RNAi to remove repeats), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factors), and other neurological disorders, cancer (endostatin, angiostatin, TRAIL, FAS-ligand, cytokines including interferons; RNAi including RNAi against VEGF or the multiple drug resistance gene product), diabetes mellitus (insulin), muscular dystrophies including Duchenne (dystrophin, mini-dystrophin, insulin-like growth factor I, a sarcoglycan [e.g., a, b, g], RNAi against myostatin, myostatin propeptide, follistatin, activin type II soluble receptor, anti inflammatory polypeptides such as the Ikappa B dominant mutant, sarcospan, utrophin, mini- utrophin, RNAi against splice junctions in the dystrophin gene to induce exon skipping [see, e.g., WO/2003/095647], antisense against U7 snRNAs to induce exon skipping, see, e.g.. WO/2006/021724], and antibodies or antibody fragments against myostatin or myostatin propeptide) and Becker, Gaucher disease (glucocerebrosidase), Hurler’s disease (a-L- iduronidase), adenosine deaminase deficiency (adenosine deaminase), glycogen storage diseases (e.g., Fabry disease [a-galactosidase] and Pompe disease [lysosomal acid a- glucosidase]) and other metabolic defects, congenital emphysema (al -antitrypsin), Lesch- Nyhan Syndrome (hypoxanthine guanine phosphoribosyl transferase), Niemann-Pick disease (sphingomyelinase), Tays Sachs disease (lysosomal hexosaminidase A), Maple Syrup Urine Disease (branched-chain keto acid dehydrogenase), retinal degenerative diseases (and other diseases of the eye and retina; e.g., PDGF for macular degeneration), diseases of solid organs such as brain (including Parkinson’s Disease [GDNF], astrocytomas [endostatin, angiostatin and/or RNAi against VEGF], glioblastomas [endostatin, angiostatin and/or RNAi against VEGF]), liver, kidney, heart including congestive heart failure or peripheral artery disease (PAD) (e.g., by delivering protein phosphatase inhibitor I (1-1), serca2a, zinc finger proteins that regulate the phospholamban gene, Barkct, P2-adrenergic receptor, P2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), S100A1, parvalbumin, adenylyl cyclase type 6, a molecule that effects G-protein coupled receptor kinase type 2 knockdown such as a truncated constitutively active bARKct; calsarcin, RNAi against phospholamban; phospholamban inhibitory or dominant-negative molecules such as phospholamban S16E, etc.), arthritis (insulin-like growth factors), joint disorders (insulin-like growth factor 1 and/or Attorney Docket No. MIL-035WO1 2), intimal hyperplasia (e.g., by delivering enos, inos), improve survival of heart transplants (superoxide dismutase), AIDS (soluble CD4), muscle wasting (insulin-like growth factor I), kidney deficiency (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors such as IRAP and TNFa soluble receptor), hepatitis (a-interferon), LDL receptor deficiency (LDL receptor), hyperammonemia (ornithine transcarbamylase), Krabbe’s disease (galactocerebrosidase), Batten’s disease, spinal cerebral ataxias including SCA1, SCA2 and SCA3, phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, and the like. The invention can further be used following organ transplantation to increase the success of the transplant and/or to reduce the negative side effects of organ transplantation or adjunct therapies (e.g., by administering immunosuppressant agents or inhibitory nucleic acids to block cytokine production). As another example, bone morphogenic proteins (including BNP 2, 7, etc., RANKL and/or VEGF) can be administered with a bone allograft, for example, following a break or surgical removal in a cancer patient. [0257] The nucleic acid delivery vectors may also be employed to provide a functional nucleic acid to a cell in vitro or in vivo. Expression of the functional nucleic acid in the cell, for example, can diminish expression of a particular target protein by the cell. Accordingly, functional nucleic acid can be administered to decrease expression of a particular protein in a subject in need thereof. The nucleic acid delivery vectors can also be used for the purpose of evaluating safety (spread, toxicity, immunogenicity, etc.). Treating Autoimmune Disorder [0258] In some embodiments, administration of the composition removes or reduces the effect of antibodies in a subject. In some embodiments, the subject has an autoimmune disease. In some embodiments, the antibodies are pathogenic autoantibodies. In some embodiments, administration of the composition is used to treat an autoimmune disorder. [0259] In some embodiments, the autoimmune disorder is, for example and without limitation, Addison’s disease, Anti-GBM glomerulonephritis, Anti-neutrophil cytoplasmic antibody-associated vasculitides, Anti-phospholipid antibody syndrome, Autoimmune bullous skin diseases, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune neutropenia, Bullous pemphigoid, Celiac disease, Chronic utricaria, Complete congenital heart block, Diabetes type 1A, Essential mixed cryoglobulinemia, Goodpasture’s syndrome, Graves’ disease, Guillain-Barre syndrome, Hemophilia-acquired FVIII deficiency, Idiopathic thrombocytopenic purpura, Lambert-Eaton myasthenic syndrome, Mixed connective tissue Attorney Docket No. MIL-035WO1 disease, Multiple myeloma, Myasthenia gravis, Myocarditis, dilated cardiomyopathy, Primary biliary cirrhosis, Primary progressive multiple sclerosis, rheumatic hear disease, Rheumatoid arthritis, Sjogren syndrome, SLE including lupus nephritis, Stiff-person syndrome, or Transplant rejection. Treating Cancer [0260] In some embodiments, administration of the composition is to treat cancer. In some embodiments, administration is in combination with another therapy or therapeutic agent. In some embodiments, the therapy administered in combination with the composition is an antibody. [0261] In some embodiments, the cancer is, for example and without limitation, Acute lymphoblastic leukemia, Acute myeloid leukemia, Adrenocortical carcinoma, AIDS- related cancers, AIDS-related lymphoma, Anal cancer, Appendix cancer, Astrocytoma, childhood cerebellar or cerebral, Basal cell carcinoma, Bile duct cancer, extrahepatic, Bladder cancer, Bone cancer, Osteosarcoma/Malignant fibrous histiocytoma, Brainstem glioma, Brain cancer, Brain tumor, cerebellar astrocytoma, Brain tumor, cerebral astrocytoma/malignant glioma, Brain tumor, ependymoma, Brain tumor, medulloblastoma, Brain tumor, supratentorial primitive neuroectodermal tumors, Brain tumor, visual pathway and hypothalamic glioma, Breast cancer, Bronchial adenomas/carcinoids, Burkitt lymphoma, Carcinoid tumor, Carcinoid tumor, gastrointestinal, Carcinoma of unknown primary, Central nervous system lymphoma, Cerebellar astrocytoma, Cerebral astrocytoma/Malignant glioma, Cervical cancer, Chronic lymphocytic leukemia, Chronic myelogenous leukemia Chronic myeloproliferative disorders, Colon Cancer, Cutaneous T -cell lymphoma, Desmoplastic small round cell tumor, Endometrial cancer, Ependymoma, Esophageal cancer, Ewing's sarcoma in the Ewing family of tumors, Extracranial germ cell tumor, Childhood, Extragonadal Germ cell tumor, Extrahepatic bile duct cancer, Eye Cancer, Intraocular melanoma, Eye Cancer, Retinoblastoma, Gallbladder cancer, Gastric (Stomach) cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal stromal tumor (GIST), Germ cell tumor: extracranial, extragonadal, or ovarian, Gestational trophoblastic tumor, Glioma of the brain stem, Glioma, Childhood Cerebral Astrocytoma, Glioma, Childhood Visual Pathway and Hypothalamic, Gastric carcinoid, Hairy cell leukemia, Head and neck cancer, Heart cancer, Hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, Hypothalamic and visual pathway glioma, Intraocular Melanoma, Islet Cell Carcinoma (Endocrine Attorney Docket No. MIL-035WO1 Pancreas), Kaposi sarcoma, Kidney cancer (renal cell cancer), Laryngeal Cancer, Leukemias, Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia), Leukemia, acute myeloid (also called acute myelogenous leukemia), Leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia), Leukemia, chronic myelogenous (also called chronic myeloid leukemia), Leukemia, hairy cell, Lip and Oral Cavity Cancer, Liposarcoma, Liver Cancer (Primary), Lung Cancer, Non-Small Cell ,Lung Cancer, Small Cell, Lymphomas, Lymphoma, AIDS-related, Lymphoma, Burkitt, Lymphoma, cutaneous T-Cell, Lymphoma, Hodgkin, Lymphomas, Non-Hodgkin (an old classification of all lymphomas except Hodgkin's), Lymphoma, Primary Central Nervous System, Macroglobulinemia, Waldenstrom, Malignant Fibrous Histiocytoma of Bone/Osteosarcoma, Medulloblastoma, Melanoma, Melanoma, Intraocular (Eye), Merkel Cell Carcinoma, Mesothelioma, Adult Malignant, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Mouth Cancer, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Diseases, Myelogenous Leukemia, Chronic, Myeloid Leukemia, Adult Acute, Myeloid Leukemia, Childhood Acute, Myeloma, Multiple (Cancer of the Bone-Marrow), Myeloproliferative Disorders, Nasal cavity and paranasal sinus cancer, Nasopharyngeal carcinoma, Neuroblastoma, Non-Hodgkin lymphoma, Non- small cell lung cancer, Oral Cancer, Oropharyngeal cancer, Osteosarcoma/malignant fibrous histiocytoma of bone, Ovarian cancer, Ovarian epithelial cancer (Surface epithelial-stromal tumor), Ovarian germ cell tumor, Ovarian low malignant potential tumor, Pancreatic cancer, Pancreatic cancer, islet cell, Paranasal sinus and nasal cavity cancer, Parathyroid cancer, Penile cancer, Pharyngeal cancer, Pheochromocytoma, Pineal astrocytoma, Pineal germinoma, Pineoblastoma and supratentorial primitive neuroectodermal tumors, Pituitary adenoma, Plasma cell neoplasia/Multiple myeloma, Pleuropulmonary blastoma, Primary central nervous system lymphoma, Prostate cancer, Rectal cancer, Renal cell carcinoma (kidney cancer), Renal pelvis and ureter, transitional cell cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma, Ewing family of tumors, Kaposi Sarcoma, Sarcoma, soft tissue, Sarcoma, uterine, Sezary syndrome, Skin cancer (nonmelanoma), Skin cancer (melanoma), Skin carcinoma, Merkel cell, Small cell lung cancer, Small intestine cancer, Soft tissue sarcoma, Squamous cell carcinoma, Squamous neck cancer with occult primary, metastatic, Stomach cancer, Supratentorial primitive neuroectodermal tumor, T-Cell lymphoma, cutaneous - see Mycosis Fungoides and Sezary syndrome, Testicular cancer, Throat cancer, Attorney Docket No. MIL-035WO1 Thymoma, Thymoma and Thymic carcinoma, Thyroid cancer, Thyroid cancer, Transitional cell cancer of the renal pelvis and ureter, Trophoblastic tumor, Ureter and renal pelvis, transitional cell cancer Urethral cancer, Uterine cancer, endometrial, Uterine sarcoma, Vaginal cancer, Visual pathway and hypothalamic glioma, Vulvar cancer, Waldenstrom macroglobulinemia and Wilms tumor (kidney cancer). Reducing Transplant Rejection and Improving Organ Transplant [0262] In some embodiments, the therapy is an organ transplant. In some embodiments, the organ transplant would otherwise be ineffective due to the action of anti- donor IgG antibodies present in the serum of the subject. In some embodiments, the organ is, for example and without limitation, the kidney, liver, hear, pancreas, lung, or small intestine. [0263] In some embodiments, the subject to be treated is sensitized or highly sensitized. By “sensitized,” it is meant that the subject has developed antibodies to human major histocompatibility (MHC) antigens (also known as human leukocyte antigens (HLA)). Anti-MHC antibodies are usually present in subjects that have been previously sensitized by blood transfusion, previous transplantation, or pregnancy. [0264] The presence of high titer antibodies against MHC antigens of the potential donor is a risk to transplantation due to the risk of antibody-mediated rejection. Sensitization to donor MHC antigens impedes the identification of suitable donors. Administration of the present invention allows rapid and safe removal of donor specific antigens in a potential transplant recipient. Administering the composition prior to transplantation effectively desensitizes the subject, thereby allowing transplant and avoiding antibody-mediated rejection. Increasing Benefits of Therapeutic Agents [0265] In some embodiments, the composition is administered concurrently with or prior to adoptive cell transfer therapy. In some embodiments, the composition is administered concurrently with or prior to ex vivo cell therapy. [0266] In some embodiments, administration of the present invention improved the benefit to a subject of a therapy or a therapeutic agent. In some embodiments, the conjugate is administered to the subject to reduce Fc receptor binding of serum IgG molecules in the subject. In some embodiments, the therapy or therapeutic agent is administered subsequently Attorney Docket No. MIL-035WO1 to the conjugate. In some embodiments, the therapy or therapeutic agent is administered concurrently with the conjugate. Enzyme Replacement Therapy [0267] In some embodiments, the replacement enzyme is a lysosomal enzyme. In some embodiments, the replacement enzyme is arylsulfatase A or iduronate-2-sulfatase. [0268] As used herein, replacement enzymes suitable for the present invention may include any enzyme that can act to replace at least partial activity of the deficient or missing lysosomal enzyme in a lysosomal storage disease to be treated. In some embodiments, a replacement enzyme is capable of reducing accumulated substance in lysosomes or that can rescue or ameliorate one or more lysosomal storage disease symptoms. [0269] The methods and compositions according to the present invention are used to treat lysosomal storage diseases, including, but not limited to, aspartylglucosaminuria, cholesterol ester storage disease, Wolman disease, cystinosis, Danon disease, Fabry disease, Farber lipogranulomatosis, Farber disease, fucosidosis, galactosialidosis types I/II, Gaucher disease types I/II/III, globoid cell leukodystrophy, Krabbe disease, glycogen storage disease II, Pompe disease, GM1-gangliosidosis types I/II/III, GM2-gangliosidosis type I, Tay Sachs disease, GM2-gangliosidosis type II, Sandhoff disease, GM2-gangliosidosis, α-mannosidosis types I/II, beta.-mannosidosis, metachromatic leukodystrophy, mucolipidosis type I, sialidosis types I/II, mucolipidosis types II/III, I-cell disease, mucolipidosis type IIIC pseudo- Hurler polydystrophy, mucopolysaccharidosis type I, mucopolysaccharidosis type II, mucopolysaccharidosis type IIIA, Sanfilippo syndrome, mucopolysaccharidosis type TIM, mucopolysaccharidosis type IIIC, mucopolysaccharidosis type IIID, mucopolysaccharidosis type IVA, Morquio syndrome, mucopolysaccharidosis type IVB, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, Sly syndrome, mucopolysaccharidosis type IX, multiple sulfatase deficiency, neuronal ceroid lipofuscinosis, CLN1 Batten disease, CLN2 Batten disease, Niemann-Pick disease types A/B, Niemann-Pick disease type C1, Niemann-Pick disease type C2, pycnodysostosis, Schindler disease types I/II, Gaucher disease and sialic acid storage disease, among others. The genetic etiology, clinical manifestations, and molecular biology of the lysosomal storage diseases are detailed in Scriver et al., eds., The Metabolic and Molecular Basis of Inherited Disease, 7th Ed., Vol. II, McGraw Hill, (1995). Thus, the lysosomal enzymes deficient in the above exemplary diseases are known to those of skill in the art, some of which are exemplified in Table 4 below. In some embodiments, a Attorney Docket No. MIL-035WO1 suitable replacement enzyme may be any lysosomal enzyme known to be associated with the lysosomal storage disease to be treated. In some embodiments, a suitable replacement enzyme is an enzyme selected from the enzyme listed in Table 4 above. [0270] Table 4. Enzyme Replacement Therapy to Treat Exemplary Lysosomal Storage Diseases Associated with Enzyme Deficiencies Disease Enzyme Deficiency Substance Stored Pompe Disease Acid- α1, 4- Glucosidase Glycogen α-1-4-linked oligosaccharides GM1 Gangliodsidosis β-Galactosidase GM1 Gangliosides Tay-Sachs Disease β-Hexosaminidase A GM2 Ganglioside GM2 Gangliosidosis: AB GM2 Activator Protein GM2 Ganglioside Variant Sandhoff Disease β-Hexosaminidase A&B GM2 Ganglioside Fabry Disease α-Galactosidase A Globosides Gaucher Disease Glucocerebrosidase Glucosylceramide Metachromatic Arylsulfatase A Sulphatides Leukodystrophy Krabbe Disease Galactosylceramidase Galactocerebroside Niemann Pick, Types A & Acid Sphingomyelinase Sphingomyelin B Niemann Pick, Type C Cholesterol Esterification Sphingomyelin Defect Niemann Pick, Type D Unknown Sphingomyelin Farber Disease Acid Ceramidase Ceramide Wolman Disease Acid Lipase Cholesteryl Esters Hurler Syndrome (MPS IH) α-L-Iduronidase Heparan and Dermatan Sulfates Scheie Syndrome (MPS IS) α-L-Iduronidase Heparan and Dermatan Sulfates Hurler-Scheie Syndrome α-L-Iduronidase Heparan and Dermatan (MPS IS) Sulfates Hunter Syndrome (MPS II) Iduronate Sulfatase Heparan and Dermatan Sulfates Sanfilippo A (MPS IIIA) Heparan N-Sulfatase Heparan Sulfate Sanfilippo B (MPS IIIB) α-N-Acetylglucosaminidase Heparan Sulfate Sanfilippo C (MPS IIIC) Acetyl-CoA-Glucosaminide Heparan Sulfate Attorney Docket No. MIL-035WO1 Sanfilippo D (MPS IIID) Acetyltransferase N- Heparan Sulfate Acetylglucosamine-6- Sulfatase Morquio B (MPS IVB) β-Galactosidase Keratan Sulfate Maroteaux-Lamy (MPS VI) Arylsulfatase B Dermatan Sulfate Sly Syndrome (MPS VII) β-Glucuronidase α-Mannosidosis α-Mannosidase Mannose/Oligosaccharides β-Mannosidosis β-Mannosidase Mannose/Oligosaccharides Fucosidosis α-L-Fucosidase Fucosyl Oligosaccharides Aspartyl-glucosaminuria N-Aspartyl-β- Aspartylglucosamine Glucosaminidase Asparagines Sialidosis (Mucolipidosis I) α-Neuraminidase Sialyloligosaccharides Galactosialidosis (Goldberg Lysosomal Protective Sialyloligosaccharides Syndrome) Protein Deficiency Schindler Disease α-N-Acetyl- Galactosaminidase Mucolipidosis II (I-Cell N-Acetylglucosamine-1- Heparan Sulfate Disease) Phosphotransferase Mucolipidosis III (Pseudo- Same as ML II Hurler Polydystrophy) Cystinosis Cystine Transport Protein Free Cystine Salla Disease Sialic Acid Transport Free Sialic Acid and Protein Glucuronic Acid Infantile Sialic Acid Sialic Acid Transport Free Sialic Acid and Storage Disease Protein Glucuronic Acid Infantile Neuronal Ceroid Palmitoyl-Protein Lipofuscins Lipofuscinosis Thioesterase Mucolipidosis IV Unknown Gangliosides & Hyaluronic Acid Prosaposin Saposins A, B, C or D [0271] In some embodiments, a replacement enzyme suitable for the invention may have a wild-type or naturally occurring sequence. In some embodiments, a replacement enzyme suitable for the invention may have a modified sequence having substantial homology or identify to the wild-type or naturally-occurring sequence (e.g., having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% sequence identity to the wild-type or naturally-occurring sequence). Attorney Docket No. MIL-035WO1 [0272] A replacement enzyme suitable for the present invention may be produced by any available means. For example, replacement enzymes are recombinantly produced by utilizing a host cell system engineered to express a replacement enzyme-encoding nucleic acid. Alternatively or additionally, replacement enzymes are produced by activating endogenous genes. Alternatively or additionally, replacement enzymes are partially or fully prepared by chemical synthesis. Alternatively or additionally, replacement enzymes are purified from natural sources. EXAMPLES Example 1. Production of IdeS-Rapamycin Conjugates through Sortase-Mediated Conjugation. [0273] This example illustrates the methods to conjugate IdeS and rapamycin through, for example, sortase-mediated conjugation chemistry on the C-terminus of IdeS. [0274] To site specifically crosslink a single rapamycin linker-payload to the C- terminus of IdeS in this embodiment, sortase-mediated conjugation was performed, for example, as described in FIG.1B. The C-terminus of IdeS was engineered with an LPETG tag for sortase recognition and a His6 tag for purification. Using sortase, the Gly-His6 tag was replaced with a nucleophile containing glycine and an azide. [0275] IdeS was then treated with, in this embodiment, one of the dibenzocyclooctyne (DBCO) linker-payload constructs, for example, Compound 1, 2, or 3 shown in FIG.2A, FIG.2B, and FIG.2C, for a strained alkyne-azide cycloaddition (SPAAC) reaction to form the IdeS-rapamycin C-terminal conjugates. [0276] In this example, Compound 1 and 2 are rapamycin linker-payloads while Compound 3 is a control linker-payload without rapamycin. Since substitution of rapamycin analogs is common at the C40, the 40-OH was selected as the position for linker attachment via an ester bond to form the rapamycin linker-payloads. The linker for Compound 1 contains 5 polyethylene glycol (PEG) units between the rapamycin and DBCO. Under physiological conditions, Compound 1 linker-payload is not expected to be cleaved. The linker for Compound 2 contains a disulfide bond between 5 PEG units and rapamycin that is expected to be cleaved by intracellular reducing agents such as cysteine and glutathione. Cleavage of the disulfide bond results in formation of a catabolite with a shorter linker attached to the 40- Attorney Docket No. MIL-035WO1 OH of rapamycin by an ester bond. Compound 3 is a control with only 6 PEG units and no rapamycin. [0277] IdeS-rapamycin conjugates were characterized with Size-Exclusion Chromatography (SEC) and Quadrupole Time of Flight Mass Spectrometer (QTOF-MS). As shown in FIG.3, the SEC resulted in a single peak for each conjugate, indicating a high purity with conjugates in monomeric form with minimal aggregation. MS analysis demonstrated that each conjugate had a major peak at the expected molecular weight. The molecular weight indicated a degree-of-labeling (DoL) of 1 and confirmed the loss of the His6 tag and addition of the linker-payload during conjugation. Over 95% of the initial IdeS was conjugated and purified. [0278] Overall, the results indicate successful production of 3 different IdeS- rapamycin conjugates each with the expected singular rapamycin attached to the C-terminus of IdeS. Example 2. Production of IdeS-Rapamycin Conjugates through Stochastic Lysine Conjugation. [0279] This example illustrates the methodology used to conjugate IdeS and rapamycin through, for example, stochastic lysine conjugation chemistry on surface lysine residues. [0280] In this embodiment, stochastic lysine conjugation chemistry was utilized to non-specifically crosslink multiple rapamycin linker-payloads to surface lysine residues on IdeS as shown in FIG.1C. Surface lysine residues were reacted with linker-payload Compound 4 from FIG.2, a compound with electrophilic N-hydroxysuccinimide (NHS) ester and polyethylene glycol (PEG) with an average molecular weight of 10 kDa. Compound 4 contains a disulfide bond between the PEG units and rapamycin that is expected to be readily cleaved under physiological conditions. [0281] The IdeS-rapamycin conjugate DoL distribution and protein concentration were determined by SDS-PAGE gel and Coomassie staining. As shown in FIG.4, a heterogenous mixture of conjugates with different molecular weights (MWs) was produced due to the non-specific nature of stochastic lysine conjugation and varying numbers of rapamycin linker Compound 4 attached to each IdeS protein. Each shift in MW in FIG.3 represents an additional rapamycin linker (~11 kDa). The weighted average DoL of the IdeS conjugate with Compound 4 was calculated to be around 2. Attorney Docket No. MIL-035WO1 [0282] Overall, the results indicated successful production of a heterogenous mixture of IdeS-rapamycin conjugates with an average of 2 rapamycin to 1 IdeS molecule. Example 3. Effect of Rapamycin Conjugation on IdeS Enzymatic Activity. [0283] This example illustrates the effect of rapamycin conjugation on the enzymatic activity of IdeS. [0284] In this example, enzymatic activity of IdeS was measured by a human IgG- cleavage assay. Results were assessed using SDS-PAGE gel and Coomassie staining. Un- cleaved IgG has a MW of 150 kDa. Cleavage products scIgG, F(ab’)2, and Fc appear as bands on the gel at around 125 kDa, 100 kDa, and 25 kDa, respectively. [0285] As shown in FIG.4, unconjugated IdeS rapidly cleaved IgG with cleavage products detected within 30 seconds of reaction. The IdeS-PEG control (Compound 3 linker) and non-cleavable IdeS-rapamycin (Compound 1) sortase-mediated conjugate had similar activity to unconjugated IdeS. The disulfide cleavable IdeS-rapamycin (Compound 2) sortase-mediated conjugate was active with a slightly slower rate as indicated by the slower decrease in IgG band size. The disulfide cleavable IdeS-rapamycin (Compound 4) lysine conjugate was slightly active. [0286] Overall, the results indicated that most of the IdeS-rapamycin conjugates retained their enzymatic activity as compared to unconjugated IdeS. Example 4. Effect of Rapamycin on IdeS-Induced Immune Activation. [0287] This example illustrates the effect of rapamycin on the immune response induced by IdeS in human PBMCs. [0288] Briefly, human PBMCs were collected from 20 healthy donors. PBMCs were then incubated with IdeS or IdeS + rapamycin (Table 5) for 5 to 8 days. [0289] Table 5. Donor Response to IdeS, Rapamycin, and IdeS-Rapamycin Conjugates. Donors 1 2 3 4 5 6 7 8 9 1 1 1 1 1 1 1 1 1 1 2 Positive 0 1 2 3 4 5 6 7 8 9 0 Donor Response Rate IdeS, 19 nM P P P P P P P P 40 IdeS, 6 nM P P P P P P P P 40 IdeS, 2 nM P P P P P P P P 40 IdeS, Free Rapa-mycin 0 19 nM 95 nM Free Rapa-mycin 0 19 nM Attorney Docket No. MIL-035WO1 Free Rapa-mycin P 5 1.9 nM Free Rapa-mycin P P P P P P P 35 0.19 nM Free Rapa-mycin P P P P P P P P 40 0.038 nM Free Rapa-mycin P P P P P P P 35 0.019 nM Free Rapa-mycin P P P P P P P 35 0.0038 nM IdeS-PEG6 (C-3, control), P P P P P P P 35 19 nM IdeS-PEG5-Rapamycin P P P P P P 30 (C-1, non-cleavable), 19 nM IdeS-PEG5-Rapamycin P P P 15 (C-2, disulfide cleavable), 19 nM Herceptin™ P P 10 CEFT P P P P P P P P P P 50 KLH P P P P P P P P P P P P P P P P P P P 95 [0290] As shown in FIG.5, IdeS induced a similar T cell proliferation response at all dose levels, 2 nM, 6 nM, and 19 nM, with 40% of donors having a positive T cell response (Table 5). Co-incubation of IdeS and rapamycin revealed a dose dependent decline in T cell proliferation response as rapamycin dosage increased. A reduction in the positive donor response rate was also seen with 0.19 nM, 1.9 nM, and 19 nM rapamycin resulting in 35%, 5%, and 0% positive donor response rate, respectively. [0291] Overall, the results indicated that addition of rapamycin reduced the T cell response to IdeS in a dose dependent manner. Example 5. Effect of Rapamycin Conjugation on IdeS-Induced Immune Activation. [0292] This example illustrates the effect of rapamycin conjugation on the immune response induced by IdeS in human PBMCs. [0293] Briefly, human PBMCs were collected from 20 healthy donors. PBMCs were then incubated with IdeS or IdeS-rapamycin conjugates for 5 to 8 days. In this example, the non-cleavable IdeS-rapamycin (Compound 1) sortase-mediated conjugate, the disulfide cleavable IdeS-rapamycin (Compound 2) sortase-mediated conjugate, and the IdeS-PEG were each incubated with human PBMCs from one set of donors (Table 6). The disulfide cleavable IdeS-rapamycin (Compound 4) lysine conjugate was also tested with a different set of donors (Table 6). Attorney Docket No. MIL-035WO1 [0294] Table 6. Donor Response for IdeS-Rapamycin Conjugates Donors 1 2 3 4 5 6 7 8 9 1 1 1 1 1 1 1 1 1 1 2 Positive 0 1 2 3 4 5 6 7 8 9 0 Donor Response Rate IdeS, 600 nM P P P P P P P P P 45 IdeS-Rapamycin 0 (C-4, disulfide cleavable), 600 nM IdeS, 170 nM P P P P P P P P 40 IdeS-Rapamycin 0 (C-4, disulfide cleavable), 170 nM Herceptin™ P P 10 CEFT P P P P P 25 KLH P P P P P P P P P P P P P P P P 80 [0295] Both disulfide cleavable IdeS-rapamycin conjugates tested decreased T cell proliferation response (FIG.6) compared to unconjugated IdeS and Herceptin®, the negative clinical control with low immunogenicity. The disulfide cleavable IdeS-rapamycin conjugates also reduced the positive donor response rate from 40% to 15% and 0% for the sortase- mediated (Compound 2) conjugate and lysine (Compound 4) conjugate, respectively. The disulfide cleavable IdeS-rapamycin (Compound 4) lysine conjugate also decreased cytokine induction across a panel of inflammatory cytokines (FIG.7). [0296] Overall, the results showed that conjugation of IdeS with a cleavable rapamycin linker decreased human immune response as shown by lower T cell proliferation, reduced positive donor response rate, and decreased cytokine induction. Example 6. Production of Recombinant IdeS. [0297] This example describes the methodology used for production of recombinant IdeS in both HEK293 and CHO cells. [0298] In this embodiment, IdeS was encoded in DNA with a C-terminal SMAC tag and a His6 tag or just a C-terminal His6 tag and an N-terminal mammalian secretion signal. The sequence was then cloned into a mammalian expression vector. [0299] For exemplary expression in CHO cells, the expression vector was transiently transfected into CHO-K1 cells by electroporation. The transfected cells were then cultured for 7 days at 32°C with 5 % humidity. Attorney Docket No. MIL-035WO1 [0300] For exemplary expression in HEK293 cells, the expression vector was transfected into Expi293 cells with Lipofectamine™. Transfected cells were then cultured for 6 days at 37°C with 8% humidity. [0301] Exemplary IdeS protein features are denoted in the schematic shown in FIG. 1A. [0302] IdeS-His6 and IdeS-SMAC-His6 were purified from cell culture media. Supernatants were harvested, and the clarified supernatants were loaded onto an exemplary His-tag purification column, for example, Roche cOmplete™ His-Tag Purification Resin. The column was washed with phosphate buffered saline (PBS), pH 7.4, supplemented with 2 M sodium chloride. PBS with 500 mM imidazole was used for elution. Eluted IdeS-His6 and IdeS-SMAC-His6 were further purified by gel filtration chromatography, for example, using Superdex® 200 prep grade equilibrated with PBS. The purified protein was then aliquoted and stored at -80°C. [0303] The sequence integrity was confirmed by intact mass spectrometry (iMS). Enzymatic activity was confirmed using an IgG cleavage assay using Homogenous Time- Resolved Fluorescence (HTRF) assay to detect intact human IgG. [0304] Overall, this example demonstrated purification of recombinant IdeS protein. Example 7. Synthesis of Rapamycin Linker-Payload Compound 1. [0305] This example describes exemplary synthesis of Compound 1, a rapamycin linker-payload prepared for sortase-mediated conjugation. [0306] In this embodiment, the rapamycin linker-payload is Compound 1 (FIG.2A), [(1R,2R,4S)-4-[(2R)-2-[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)- 1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo- 11,36-dioxa-4-azatricyclo[30.3.1.04,9] hexatriaconta-16,24,26,28-tetraen-12-yl] propyl]-2- methoxy-cyclohexyl] 4-[2-[2-[2-[2-[2-[2-[2-[[3-(2-azatricyclo[10.4.0.04,9] hexadeca- 1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl)-3-oxo-propyl] amino]-2-oxo-ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethylamino]-4-oxo-butanoate. The overall synthesis is summarized in FIG.8. Intermediates were verified by ESI-MS. [0307] In this example, in step 1 of synthesis (FIG.9A), rapamycin (Int-1, 8.23 g, 9 mmol) was combined with succinic anhydride (9 g, 90 mmol) and an immobilized lipase biocatalyst, for example, Novozym® 435 (23.5 g) in toluene (250 mL) and stirred for 2 days at room temperature. Subsequently, the resin was filtered and washed with dichloromethane (DCM, 2 x 100 mL). The combined organic solvent was removed in vacuo and then purified Attorney Docket No. MIL-035WO1 by silica gel chromatography. The product (Int-2), 4-(((1R,2R,4S)-4-((R)-2- ((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,27-dihydroxy-10,21- dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo- 1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetracosahydro-3H- 23,27-epoxypyrido[2,1-c][1]oxa[4]azacyclohentriacontin-3-yl) propyl)-2- methoxycyclohexyl) oxy)-4-oxobutanoic acid, had a yield of 76% with 7.0 g produced. [0308] In step 2 of synthesis (FIG.9B), a solution of Int-2 (1.52 g, 1.5 mmol) in DCM (25 mL) was added to N’-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU, 903 mg, 3 mmol) followed by triethanolamine (TEA, 455 g, 4.5 mmol). The mixture was stirred for 2 hours at room temperature and then diluted with water (30 mL) and extracted with DCM (2 x 30 mL). The organic layer of extraction was concentrated to a residue and purified with Prep-TLC to produce Int-3, (1R,2R,4S)-4-((R)-2- ((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,27-dihydroxy-10,21- dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo- 1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetracosahydro-3H- 23,27-epoxypyrido[2,1-c][1]oxa[4]azacyclohentriacontin-3-yl)propyl)-2-methoxycyclohexyl (2,5-dioxopyrrolidin-1-yl) succinate, with a yield of 48% and production of 0.8 g. [0309] In step 3 of synthesis (FIG.9C), Int-3 (646 mg, 0.58 mmol) and Int-4 (FIG. 10C, 217 mg, 0.64 mmol) were solubilized in dimethylformamide (DMF, 6 mL) before N,N- Diisopropylethylamine (DIPEA, 224 mg, 1.74 mmol) was added. The mixture was stirred for 3 hours at room temperature then concentrated into a residue and purified by preparatory HPLC. By this process240 mg of Int-5, 2-(2-(2-(4-(((1R,2R,4S)-4-((R)-2- ((3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R, 27R,34aS)-9,27-dihydroxy-10,21- dimethoxy-6,8,12,14,20,26-hexamethyl-1,5,11,28,29-pentaoxo- 1,4,5,6,9,10,11,12,13,14,21,22,23,24,25,26,27,28,29,31,32,33,34,34a-tetracosahydro-3H- 23,27-epoxypyrido[2,1-c][1]oxa[4]azacyclohentriacontin-3-yl) propyl)-2-methoxy- cyclohexyl) oxy)-4-oxobutanamido) ethoxy) ethoxy) acetic acid, was produced, providing a yield of about 35%. [0310] In step 4 of synthesis (FIG.9D), Int-5 (1.00 eq, 20 mg, 0.0153 mmol), Int-6 (FIG.10C, 1.20 eq, 5.1 mg, 0.0183 mmol), and (1H-Benzotriazol-1-yloxy)(dimethylamino)- N,N-dimethylmethaniminium tetrafluoroborate (TBTU, 1.5 eq, 7.4 mg, 0.0229 mmol) were placed into solution in DMF (0.489 mL). N,N-diisopropylethylamine (2.40 eq, 0.0064 mL, Attorney Docket No. MIL-035WO1 0.0367 mmol) was added, and the reaction was stirred for 1 hour at room temperature. The reaction was then partitioned between ethyl acetate (EtOAc) and water. The aqueous layer was again extracted with EtOAc. The combined organic layers were washed with saturated NaHCO3, brine, dried over Na2SO4, filtered, and concentrated. The product, Compound 1, was purified by silica gel column chromatography and yielded 19 mg, providing a yield of about 78%. [0311] Overall, by the method provided in this example, rapamycin linker-payload Compound 1 was synthesized to a high yield. Example 8. Synthesis of Rapamycin Linker-Payload Compound 2. [0312] This example describes the exemplary synthesis of Compound 2, a rapamycin linker-payload prepared for sortase-mediated conjugation. [0313] In this embodiment, the rapamycin linker-payload is Compound 2 (FIG.2B), [(1R,2R,4S) -4-[(2R)-2- [(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy -19,30- dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4- azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxy- cyclohexyl] 4-[2-[2-[2-[2-[2-[2-[2-[2-[[3-(2-azatricyclo[10.4.0.04,9]hexadeca- 1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl)-3-oxo-propyl]amino]-2-oxo- ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxycarbonylamino]ethyldisulfanyl]ethylamino]-4- oxo-butanoate. The overall synthesis is summarized in FIG.10. [0314] In step 1 of synthesis (FIG.11A), a mixture of tert-butyl 17-hydroxy- 3,6,9,12,15-pentaoxahepta-decanoate (Int-7, 528 mg, 1.5 mmol) and bis(2,5-dioxopyrrolidin- 1-yl)carbonate (576 mg, 2.25 mmol) were solubilized in DCM. TEA (455 mg, 4.5 mmol) was then added, and the mixture was stirred for 16 hours at room temperature. The reaction mixture was diluted with NaHCO3 (5% aq., 30 mL) and extracted with DCM (2 x 30 mL). The combined organic phases were concentrated to give a residue of tert-butyl 1-((2,5- dioxopyrrolidin-1-yl)oxy)-1-oxo-2,5,8,11,14,17-hexaoxanonadecan-19-oate (Int-8, 738 mg, 1.5 mmol). [0315] Without purification, Int-8 was used in step 2 of synthesis (FIG.11B). Int-8 (738 mg, 1.5 mmol) was solubilized in DCM (15 mL).2-((2-aminoethyl)disulfaneyl)-N- tritylethan-1-amine (711 mg, 1.8 mmol) and triethylamine (455 mg, 4.5 mmol) were then added to the solution, and the reaction was stirred for 16 hours at room temperature. After dilution with water (40 mL) and adjustment to pH 3 with formic acid, the product was Attorney Docket No. MIL-035WO1 extracted with DCM (3 x 40 mL). The organic phases were then combined and concentrated to produce tert-butyl 10-oxo-1,1,1-triphenyl-11,14,17,20,23,26-hexaoxa-5,6-dithia-2,9- diazaoctacosan-28-oate (Int-9) with a yield of 940 mg or 81%. [0316] In step 3 of synthesis (FIG.11C), triethylsilane (14 mg) was added to Int-9 (940 mg, 1.2 mmol) in a mixed solvent of trifluoroacetic acid and DCM (1:10, 22 mL). The mixture was stirred for 1 hour at room temperature. Afterwards, the reaction mixture was diluted with water (40 mL) and extracted with DCM (3 x 40 mL). The combined organic phases were concentrated to give 460 mg of 1-amino-8-oxo-9,12,15,18,21,24-hexaoxa-3,4- dithia-7-azahexacosan-26-oic acid (Int-10) with a yield of 81% [0317] In step 4 of synthesis (FIG.11D), DIPEA (159 mg, 1.23 mmol) was added to a mixture of Int-3 (460 mg, 0.41 mmol) and Int-10 (214 mg, 0.45 mmol). The mixture was stirred for 3 hours at room temperature. After concentration and purification by preparatory HPLC, 200 mg of 2-[2-[2-[2-[2-[2-[2-[2-[[4-[(1R,2R,4S)-4-[(2R)-2- [(1R,9S,12S,15R,16E,18R,19R,21R,23S, 24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-19,30- dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4- azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxy- cyclohexoxy]-4-oxo-butanoyl]amino]ethyldisulfanyl] ethylcarbamoyloxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]acetic acid (Int-11) with a yield of 33%. [0318] In step 5 of synthesis (FIG.11E), Int-11 (10 mg, 0.00707 mmol), Int-6 (1.20 eq, 2.3 mg, 0.00849 mmol), and TBTU (1.50 eq, 3.4 mg, 0.0106 mmol) were solubilized in DMF (0.226 mL). N,N-diisopropylethylamine. (2.40 eq, 0.0030 mL, 0.0170 mmol) was added, and the reaction was stirred for 1 hour at room temperature. A second 10 mg reaction was set up and stirred for 1 hour at room temperature. The two 10 mg reactions were combined, diluted with EtOAc, and washed with water. The aqueous layer was again extracted with EtOAc. The organics were combined and washed with saturated NaHCO3, brine, dried over Na2SO4, filtered, and concentrated. Purification was performed by silica gel column chromatography and produced 19 mg of Compound 2, yielding 78%. [0319] Overall, by the method provided in this example, rapamycin linker-payload Compound 2 was synthesized to a high yield. Example 9. Synthesis of Rapamycin Linker-Payload Compound 4. [0320] This example describes the exemplary synthesis of Compound 4, a rapamycin linker-payload prepared for stochastic lysine conjugation. Attorney Docket No. MIL-035WO1 [0321] In this embodiment, the rapamycin linker-payload is Compound 4. The overall synthesis is summarized in FIG.12. [0322] In step 1 of synthesis (FIG.13A), 2-[(2-Aminoethyl)disulfanyl]ethylamine hydrochloride (Int-12, 5.00 g, 22.2 mmol, 2 HCl, 1 eq) was dissolved in water (20 mL) and 1,4-dioxane (20 mL. Triethylamine (3.94 g, 38.9 mmol, 5.42 mL, 1.75 eq) was added followed by a solution of trityl chloride (2.10 g, 7.55 mmol, 0.34 eq) in 1,4-dioxane (20 mL), which was added over 1 hour. The reaction was stirred under nitrogen for 48 hours at room temperature. The organic solvent was evaporated, and then the reaction was diluted with water (100 mL). The aqueous layer was extracted with dichloromethane (3 x 100 mL). The combined organic layers were washed with NaHCO3 (sat, 150 mL) and dried over MgSO4. The solvent was then evaporated, and the crude was purified with by silica gel chromatography to produce 2.05 g (5.20 mmol) of 2-[2- (tritylamino)ethyldisulfanyl]ethanamine (Int-13), yielding 68.8%. [0323] In step 2 of synthesis (FIG.13B), Int-13 (195 mg, 494 μmol, 5 eq) was combined with NHS-PEG10k-COOH (Int-14, 1.00 g, 98.7 μmol, 1 eq, supplied by Biopharma PEG, HE023017-10K) and dissolved in dichloromethane (8 mL). Triethylamine (20.0 mg, 197 μmol, 27.5 μL, 2 eq) was added, and the reaction was shaken in a 50 mL falcon tube overnight at room temperature. The reaction was split into two falcon tubes and precipitated by adding diethyl ether to each falcon tube and centrifuging for 5 minutes at 3900 RPM. The diethyl ether was decanted off, and the residue in each tube was dissolved in dichloromethane (4 mL). Diethyl ether (45 mL) was added to each tube, and the mixtures were centrifuged for 5 minutes at 3900 RPM to settle the solids. The diethyl ehter was decanted off, and the solids were dried overnight under vacuum to yield 976 mg of Int-15 (FIG.14B, 93.8 μmol), yielding 95.0%. [0324] In step 3 of synthesis (FIG.13C), Int-15 (978 mg, 94 μmol, 1 eq) was dissolved in a mixture of dichloromethane (3 mL), trifluoroacetic acid (3 mL), and triethylsilane (107 mg, 917 μmol, 9.7 eq). The reaction was then shaken for 2 hours at room temperature in a falcon tube (50 mL). The reaction was split into 2 falcon tubes and diethyl ether (45 mL) was added to each tube. The solid was separated by centrifugation for 5 minutes at 3900 RPM. The diethyl ether was decanted off, and the residue in each tube was dissolved with dichloromethane (4 mL). The product was then precipitated with diethyl ether (45 mL in each tube) and centrifuged for 5 minutes at 3900 RPM. The diethyl ether was again Attorney Docket No. MIL-035WO1 decanted, and the remaining solids were dried overnight under vacuum to yield 950 mg Int- 16 (FIG.14C, 93.5 μmol), yielding 99.5%. [0325] In step 4 of synthesis (FIG.13D), two falcon tues with Int-3 (312 mg, 280 μmol, 3 eq), Int-16 (950 mg, 93.5 μmol, 1 eq) and triethylamine (102 mg, 1.01 mmol, 141 μL, 11 eq) were dissolved in dichloromethane (8 mL). The reactions were stirred overnight at room temperature. Diethyl ether (45 mL) was then added to each reaction tube. The solids were settled using centrifugation for 5 minutes at 3900 RPM. After decanting the solvent, the solids in each tube were re-dissolved in dichloromethan (3 mL). Diethyl ether (45 mL) was again added to each tube, and the solids were settled using centrifugation for 5 minutes at 3900 RPM. The solid was dried under vacuum for 2 hours then dissolved in water (30 mL each). This solution was added to two dialysis chambers (15 mL in each, 3500 MWC, Slide- A-Lyzer™ G2 Dialysis Cassettes, Thermofisher, Catalog No 87724) and placed into separate beakers containing HPLC grate water (1 L). After 2 hours dialysis chambers were placed in separate beaker containing fresh HPLC water. This was repeated after another 2 hours, before leaving the dialysis overnight in fresh HPLC water (1 L). Total amount of HPLC water used: 3 L for each Cassette. The product solutions were combined and freeze-dried overnight to yield 800 mg of Int-17 (FIG.13D, 71.7 μmol), yielding 76.7%. [0326] In step 5 of synthesis (FIG.13E), Int-17 (550 mg, 49.3 μmol, 1 eq) was dissolved in dichloromethane (4 mL). TSTU (33.0 mg, 110 μmol, 2.2 eq) and DIPEA (14.3 mg, 111 μmol, 19.3 μL, 2.3 eq) was added. The reaction was stirred for 2 hours at room temperature. Diethyl ether (15 mL) was added and the reaction was centrifuged for 5 minutes at 3900 RPM. The solevent was decanted and the solid was re-dissolved in DCM (1 mL). This was re-precipitated using diethyl ether (15 mL) and then centrifuged for 5 minutes at 3900 RPM. The solvent was again decanted, and the resulting solids were dried under vacuum to yield Compound 4 (500 m, 44.4 μmol), yielding 90.1%. [0327] Overall, by the method provided in this example, rapamycin linker-payload Compound 4 was synthesized to a high yield. Example 10. Sortase-Mediated Conjugation of Rapamycin Linker-Payloads to IdeS. [0328] This example describes the exemplary methodology of conjugating rapamycin linker payloads to IdeS using sortase-mediated conjugation and SPAAC click chemistry. [0329] To perform the sortase mediated conjugation, a solution of IdeS with LPETG- His6 tags at the C-terminus in phosphate buffered saline (PBS), pH 7.4, was further diluted with PBS, pH 7.4, until the protein concentration was 40 μM. CaCl2 (4 mM aqueous solution, Attorney Docket No. MIL-035WO1 100 equivalents (eq)) was added to the solution with 2-amino-N-(2-azidoethyl)acetamide (800 μM in 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 150 mM NaCl, pH 7.4, with 0.1% dimethylsulfoxide (DMSO), 20 eq) followed by a 2 μM solution of Sortase A Q60-K206-P94S-D160N-D165A-K196 His6 (50 mM HEPES, 150 mM NaCl, pH 7.4, 0.05 eq). The mixture was stirred for 45 minutes at room temperature. The product was purified by Capturem™ nickel resin followed by dialysis to give IdeS-LPETGG-NH(CH2)2- azide. [0330] To conjugate Compound 1 to IdeS, a solution of IdeS-LPETGG- NH(CH2)2- azide (40-60 μM in PBS) was added to a 5 mM solution of Compound 1 (10 eq) in DMSO (<10% of total solvent volume). The solution was stirred for 2 to 5 days at room temperature. The product was then purified to yield 25% of the bioconjugate. The protein conjugate was deglycosylated using PNGaseF (New England BioLabs, catalog number P0704L) prior to the LC-QTOF analysis. [0331] To conjugate Compound 2 to IdeS, a solution of IdeS-LPETGG- NH(CH2)2- azide (40-60 μM in PBS) was further diluted to 30-50 μM with propylene glycol. A 5 mM solution of Compound 2 (10 eq) in DMSO (<10% of total solvent volume) was added and the resulting solution was stirred for 2 to 5 days at room temperature. The product was then purified to yield 10%. [0332] To conjugate Compound 3 to IdeS, a solution of IdeS-LPETGG- NH(CH2)2- azide (40-60 μM in PBS) was added to a 5 mM solution of Compound 3 (purchased from BroadPHarm, catalog number BP-25746, 10 eq) in DMSO (<10% of total solvent volume). The solution was stirred overnight at room temperature. The product was then purified to yield 42%. The protein conjugate was deglycosylated using PNGaseF (New England BioLabs, catalog number P0704L) prior to the LC-QTOF analysis. [0333] Overall, this method prepared IdeS-rapamycin conjugates with Compound 1, Compound 2, and Compound 3. Example 11. Stochastic Lysine Conjugation of Rapamycin Linker-Payloads to IdeS. [0334] This example describes the stochastic lysine conjugation methodology to conjugate Compound 4 to IdeS. [0335] In this example, a solution of IdeS in PBS, pH 7.4, (~180 μM) was cooled to 4°C. A solution of Compound 4 (15-30 eq to IdeS) was diluted with DMSO (<10% final reaction solution) to ~26 μM, and the mixture was stirred for 1 to 3 minutes at 4°C. The mixture was then brought to the same volume as the IdeS solution using PBS, pH 7.4 and Attorney Docket No. MIL-035WO1 then added to the IdeS solution. The mixture was shaken overnight at 4°C. The product was purified until all unconjugated Compound 4 was removed, yielding 30%. [0336] Overall, this method prepared IdeS-rapamycin conjugates with Compound 4.

Claims

Attorney Docket No. MIL-035WO1 CLAIMS 1. A conjugate comprising: (a) an Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) protein or IgG-degrading enzyme/Mac-1 (IdeZ) protein; and (b) an immunosuppressant molecule. 2. The conjugate of claim 1, wherein the IdeS is conjugated to up to 6 immunosuppressant molecules. 3. The conjugate of any one of the preceding claims, wherein the IdeS and the immunosuppressant molecule are in at least 1:1 ratio. 4. The conjugate of any one of the preceding claims, wherein the IdeS and the immunosuppressant molecule are in at least 1:2 ratio. 5. The conjugate of any one of the preceding claims, wherein the IdeS is conjugated to the immunosuppressant molecule via a surface lysine residue on IdeS. 6. The conjugate of any one of the preceding claims, wherein the IdeS is conjugated to the immunosuppressant molecule via the C-terminus on IdeS. 7. The conjugate of any one of the preceding claims, wherein the conjugate is cleavable. 8. The conjugate of any one of the preceding claims, wherein the conjugate is non- cleavable. 9. The conjugate of any one of the preceding claims, wherein the IdeS comprises at least 70% sequence identity to a wild-type IdeS protein of SEQ ID NO: 1. 10. The conjugate of claim 1, wherein the IdeZ comprises at least 70% sequence identity to a wild-type IdeZ protein of SEQ ID NO: 51. 11. The conjugate of claim 9, wherein the IdeS protein comprises a R39G mutation relative to the wild-type IdeS protein. 12. The conjugate of claim 11, wherein the IdeS protein is a variant that further comprises one or more of mutations selected from E154D, Y155H, Y155Q, F161H, F161Y, Attorney Docket No. MIL-035WO1 L164H, F297T, F297N, V300R, and A303E, or comprises a D30-T49 deletion relative to the wild-type IdeS protein. 13. The conjugate of any one of the preceding claims, wherein the wild-type IdeS protein has 100% identity to SEQ ID NO: 1. 14. The conjugate of any one of the preceding claims, wherein the IdeS protein comprises at least 80% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. 15. The conjugate of any one of the preceding claims, wherein the IdeS protein comprises at least 90% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. 16. The conjugate of any one of the preceding claims, wherein the IdeS protein comprises 100% identity to any one of SEQ ID NO: 2 to SEQ ID NO: 49. 17. The conjugate of any one of the preceding claims, wherein the immunosuppressant molecule is rapamycin, an analog or a mimetic thereof. 18. The conjugate of claim 17, wherein the rapamycin analog is selected from the group consisting of deforolimus, everlimus, and temsirolimus. 19. The conjugate of claim 17, wherein the rapamycin mimetic is isoliquiritigenin or withaferin A. 20. The conjugate of any one of the preceding claims, wherein IdeS is conjugated to a rapamycin-linker payload selected from the group consisting of any one of:
Figure imgf000102_0001
(Compound 1), Attorney Docket No. MIL-035WO1
Figure imgf000103_0001
4). 21. The conjugate of claim 20, wherein Compound 1, Compound 2 or Compound 4 are monomeric compounds. 22. A composition comprising a plurality of conjugates of any one of the preceding claims. 23. The composition of claim 22, wherein the plurality of conjugates is homogenous, each conjugate comprising a uniform number of between 1 to 10 immunosuppressant molecules per IdeS molecule. 24. The composition of claim 22, wherein the plurality of conjugates is heterogenous, each conjugate comprising a varying number of between 1 to 10 immunosuppressant molecules per IdeS molecule. 25. The composition of any one of the preceding claims, wherein the composition comprises less than 5% unconjugated IdeS. 26. The composition of any one of the preceding claims, wherein the composition comprises less than 1% unconjugated IdeS. 27. The composition of any one of the preceding claims, wherein the composition comprises less than 0.1% unconjugated IdeS. Attorney Docket No. MIL-035WO1 28. A method of reducing an immune response in a subject, the method comprising administering to the subject in need thereof, a therapeutically effective dose of the composition of any one of the preceding claims. 29. A method of reducing an immune response in a subject, the method comprising administering to the subject in need thereof, a therapeutically effective dose of a composition comprising a conjugate of: (a) an IdeS or an IdeZ protein; and (b) an immunosuppressant molecule. 30. The method of any one of claims 28 or 29, wherein the immune response generates one or more antibodies. 31. The method of claim 30, wherein the antibody is IgG. 32. The method of claim 31, wherein the antibody is IgG1, IgG2, IgG3 or IgG4. 33. The method of claim 32, wherein the IgG2 antibody is IgG2a or IgG2b. 34. The method of any one of the preceding claims, wherein the composition is administered with a gene therapy vector or a replacement enzyme. 35. The method of claim 34, wherein the composition is administered prior to administration of the gene therapy vector or the replacement enzyme. 36. The method of claim 35, wherein the composition is administered at least 3 days, 5 days or 7 days prior to administration of the gene therapy vector or the replacement enzyme. 37. The method of claim 35, wherein the composition is administered between 1 day to 3 days prior to administration of the gene therapy vector or the replacement enzyme. 38. The method of claims 35, wherein the composition is administered at least 1 hour, 3 hours, 5 hours, 10 hours, 12 hours, 16 hours, or 24 hours prior to administration of the gene therapy vector or the replacement enzyme. Attorney Docket No. MIL-035WO1 39. The method of claim 34, wherein the composition is administered concurrently with administration of the gene therapy vector or the replacement enzyme. 40. The method of any one of the preceding claims, wherein the gene therapy vector is an adeno-associated virus (AAV) vector. 41. The method of any one of the preceding claims, wherein the gene therapy vector is an adenovirus vector. 42. The method of any one of the preceding claims, wherein the gene therapy vector is a lentiviral vector. 43. The method of any one of claims 40-42, wherein the gene therapy vector encodes a protein that elicits production of IgG antibodies. 44. The method of claim 43, wherein the protein is a heterologous protein. 45. The method of any one of the preceding claims, wherein the protein that elicits production of IgG antibodies is an AAV capsid protein. 46. The method of any one of the preceding claims, wherein the protein that elicits production of IgG antibodies is a lentiviral envelope protein. 47. A method of increasing transduction efficiency of a gene therapy vector, the method comprising administering the composition of any one of the preceding claims. 48. The method of any one of claims 34-39, wherein the replacement enzyme is a lysosomal enzyme. 49. The method of claim 48, wherein the replacement enzyme is arylsulfatase A or iduronate-2-sulfatase. 50. The method of any one of claims 28 or 29, wherein the subject receives an organ transplant. 51. A method of reducing organ transplant rejection, comprising administering the composition of any one of the preceding claims. Attorney Docket No. MIL-035WO1 52. The method of claim 50 or 51, wherein the composition is administered prior to the organ transplant. 53. The method of any one of the preceding claims, wherein upon administration of the composition, IgG is cleaved to scIgG, F(ab)2 and Fc fragments. 54. The method of any one of the preceding claims, wherein upon administration of the composition, IgG is cleaved to scIgG. 55. The method of any one of the preceding claims, wherein administration of the composition comprising the conjugate to a subject reduces induction of a T-cell response relative to a control. 56. The method of claim 55, wherein the control is the level of a T-cell response in the same or a different subject treated with unconjugated IdeS. 57. The method of claim 55 or 56, wherein the T cell response is reduced by 10%-20%, 20%-50%, 50%-80% or 80%-95%, or any intervening quantity therebetween, relative to the control. 58. The method of any one of claims 55 to 57, wherein the T cell response is reduced by greater than 95% relative to the control. 59. The method of any one of claims 55 to 58, wherein the T cell response is reduced by greater than 99% relative to the control. 60. The method of any one of the preceding claims, wherein administration of the composition comprising the conjugate reduces induction of inflammatory cytokines relative to the control. 61. The method of claim 60, wherein the inflammatory cytokines are IFN-gamma, IL-17 alpha, IL-2, IL-5, IL-6, TNF-alpha and/or MCP-1. 62. The method of any one of the preceding claims, wherein the subject has cancer or an autoimmune disease. 63. The method of any one of the preceding claims, wherein the composition is administered concurrently with or prior to adoptive cell transfer therapy. Attorney Docket No. MIL-035WO1 64. The method of any one of the preceding claims, wherein the composition is administered concurrently with or prior to ex vivo cell therapy.
PCT/IB2024/059972 2023-10-13 2024-10-11 Ides-rapamycin conjugate in aav gene therapy Pending WO2025079025A1 (en)

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