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WO2025030053A1 - Nouvelles protéases d'igg et leurs procédés d'utilisation - Google Patents

Nouvelles protéases d'igg et leurs procédés d'utilisation Download PDF

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WO2025030053A1
WO2025030053A1 PCT/US2024/040614 US2024040614W WO2025030053A1 WO 2025030053 A1 WO2025030053 A1 WO 2025030053A1 US 2024040614 W US2024040614 W US 2024040614W WO 2025030053 A1 WO2025030053 A1 WO 2025030053A1
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amino acid
seq
polypeptide variant
acid substitutions
acid sequence
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Spencer MITCHELL
Sasha ROSE
Chris BAILEY-KELLOGG
Karl GRISWOLD
Marion SETLIFF
Lynda TUBERTY
Arielle DORFMAN
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Insmed Inc
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Insmed Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/2201—Streptopain (3.4.22.10)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • Immunoglobulin G-degrading enzyme of Streptococcus pyogenes cleaves all subclasses of human IgG in the hinge region with a high degree of specificity. IdeS also catalyzes the heavy chain cleavage of some subclasses of IgG in various animals.
  • Pathogenic IgG antibodies contribute to the pathogenesis of a number of autoimmune conditions as well as acute and chronic transplant rejection.
  • preexisting antibodies to gene therapy vectors in some cases limit the patients’ ability to receive such therapy. To be able to effectively remove such antibodies is therefore an important clinical challenge.
  • Streptococcus pyogenes is a bacterial pathogen that is responsible for common infections like tonsillitis and strep throat, and as such, most humans have encountered IdeS and are likely to have anti-IdeS antibodies in the bloodstream.
  • IdeS-specific antibodies have been detected in serum samples from random human subjects (likely due to prior streptococcal infections), as well as in intravenous immunoglobulin (IV-Ig) preparations, which are preparations of IgG extracted from the pooled serum of thousands of donors. Even if a subject does not possess IdeS-specific antibodies prior to an initial administration of IdeS, it is likely that such antibodies will be produced subsequent to administration.
  • IdeS is an immunogenic protein
  • the immune system of the patient receiving IdeS will often respond to it.
  • the immune reaction may involve the production of antibodies specific for IdeS.
  • the immune response to IdeS in general, and the production of IdeS-specific anti-drug antibodies (AD As) in particular, may result in (i) the efficacy of IdeS being reduced, e.g., due to ADA binding, and/or (ii) an undesirable or even harmful complications, such as a hyper-inflammatory response triggered by immune complexes of ADA and IdeS.
  • the present invention addresses the need for novel IgG proteases by providing novel proteases and methods for using the same.
  • the present disclosure provides a variant of the IdeS IgG protease (“IdeS variant”).
  • the IdeS IgG protease comprises an amino acid sequence set forth in SEQ ID NO:2.
  • the IdeS variant comprises (i) one or more amino acid mutations at one or more of the following ammo acid positions of SEQ ID NO:2: S3, F4, A6, E9, 110, R11, Y12, Y18, V46, A47, N48, 154, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, G296; and (ii) an amino acid sequence that is at least about 75%, at least about 80%,
  • the IdeS variant is at least about 30% as active as IdeS in an IgG protease enzymatic assay. In a further embodiment, the IdeS variant is at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% as active as IdeS in an IgG protease enzymatic assay.
  • the one or more mutations in the IdeS variant in one embodiment, comprise from about five to about twenty, from about 10 to about 20, from about 12 to about 20, from about 15 to about 20 amino acid substitutions. In another embodiment, the one or more mutations in the IdeS variant comprise from about 10 to about 35, or from about 24 to about 29, amino acid substitutions.
  • the IdeS variant comprises one or more amino acid substitutions set forth in Table 1 disclosed herein.
  • the IdeS variant does not have amino acid mutations at the following amino acid positions of SEQ ID NO:2: K56, C66, H234, D256 and D258.
  • the amino acids at positions 56, 66, 234, 256 and 258 are wild type IdeS residues.
  • the IdeS variant does not have amino acid mutations at one or more of the following amino acid positions of SEQ ID NO:2: A32, N33, T35, Q36, F41, D84, R88, E91, N102, Ml 06, N117, Hl 18, E170, S195, N197, K213, S245, K250, A261, K286, S306.
  • the amino acids at the aforementioned numbered positions are wild type IdeS residues.
  • the IdeS variant comprises one or more amino acid substitutions of SEQ ID NO:2 selected from the group consisting of S3N, F4I, A6S, E9I, HOT, one of R1 ID and RUT, Y12N, Y18K, V46D, A47E, N48G, I54T, one of T57D, T57K, T57N, T57R, T57L, and T57Q, N59D, one of G60S and G60T, K61R, E92R, H93Y, E104R, L120T, K123A, F125W, E126S, one of Y135H and Y135N, T138A, S159K, one of T161W, T161G, and T161Y, N162D, T185G, D188T, F199L, K200R, E201N, N203T, G222A, L223I, one of V230A and V230S, N233S, N2
  • the IdeS variant comprises a set of amino acid substitutions relative to SEQ ID NO:2, selected from one of the following: (i) HOT, R11D, Y12N, Y18K and V46D; (ii) R1 ID, Y12N, Y18K, V46D and A47E; (m) Y12N, Y18K, V46D, A47E and T57K; (iv) Y18K, V46D, A47E, T57K, H93Y, E104R and L120T; (v) HOT, R11D, Y12N, A275S, V294A, and G296E; (vi) HOT, R1 ID, F125W, E126S, Y135H, A275W, V294A, and G296E; (vn) HOT, R1 ID, Y12N, Y18K, K123A, F125W, E126S, V294A, and G296E;
  • the IdeS variant comprises a set of amino acid substitutions relative to SEQ ID NO:2, selected from one of the following:
  • the IdeS variant comprises a set of amino acid substitutions relative to SEQ ID NO: 2, selected from one of the sets of amino acid substitutions set forth in Tables 2, 3 and 4, disclosed herein.
  • the set of amino acid substitutions is set forth in Table 2.
  • the set of amino acid substitutions is set forth in Table 3.
  • the set of amino acid substitutions is set forth in Table 4.
  • the IdeS variant does not have one or more amino acid substitutions of SEQ ID NO:2 selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, N102G, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, K213Q, N233G, S245A, N246E, K250E, A261P, K286E, and S306I.
  • the IdeS variant comprises an amino acid sequence selected from one of SEQ ID NOS: 1492, 1697, 1700, 1701, 1702, 1703, 1704, 1705, 1713, 1720, 1722, 1723, 1724, 1772, 1774, 1775, 1793, 1795, 1797, 1798, and 1949.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence set forth in one of SEQ ID NOS: 1492, 1697, 1700, 1701, 1702, 1703, 1704, 1705, 1713, 1720, 1722, 1723, 1724, 1772, 1774, 1775, 1793, 1795, 1797, 1798, and 1949, and the IdeS variant comprises the amino acid substitutions present in the reference amino acid sequence relative to SEQ ID NO:2.
  • the IdeS variant comprises a first set of amino acid substitutions of SEQ ID NO:2 and a second set of amino acid substitutions of SEQ ID NO:2.
  • the first set of amino acid substitutions of SEQ ID NO:2 consists of Y12N, Y18K, A47E, H93Y, E104R, L120T, E126S, Y135N, S159K, V230S, and A275S.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 18 amino acid substitutions selected from the group consisting of S3N, F4I, A6S, E9I, HOT, RUT, N48G, I54T, one of T57K, T57N, T57R, T57L, and T57Q, N59D, one of G60S and G60T, K61R, E92R, T138A, one of T161G and T161Y, D188T, F199L, K200R, E201N, N203T, G222A, L223I, N233S, N246R, N273G, D288G, and Q293R.
  • the IdeS variant comprising the first set and second set of amino acid substitutions of SEQ ID NO:2 has an amino acid sequence that is at least about 88%, at least about 89%, at least about 90%, at least about 91%, or at least about 92% identical to SEQ ID NO:2. In one embodiment, the IdeS variant comprising the first set and second set of amino acid substitutions of SEQ ID NO:2 does not have amino acid mutations at the following amino acid positions of SEQ ID NO:2: K56, C66, H234, D256 and D258.
  • the IdeS variant comprising the first set and second set of amino acid substitutions of SEQ ID NO: 2 does not have amino acid mutations at one or more of the following amino acid positions of SEQ ID NO:2: A32, N33, T35, Q36, F41, D84, R88, E91, N102, Ml 06, N117, H118, E170, S195, N197, K213, S245, K250, A261, K286, S306.
  • the IdeS variant does not have one or more amino acid substitutions of SEQ ID NO:2 selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, N102G, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, K213Q, N233G, S245A, N246E, K250E, A261P, K286E, and S306I.
  • an IgG protease comprising an amino acid sequence selected from one of SEQ ID NOS: 27-302.
  • an IgG protease variant of one of SEQ ID NOS: 27-302 is provided, wherein the variant comprises an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical, to the amino acid sequence of the respective IgG protease from which the variant is derived.
  • the IgG protease comprises an amino acid sequence selected from one of SEQ ID NOS: 36, 37, 39, 41, 50, 51, 54, 56, 60, 61, 65, 67, 137, 140, 144, 150, 159, 285, 286, 287, 288, 289, 292, 293, 294, 295, 296, 297 and 298.
  • a variant of an IgG protease comprising an amino acid sequence of one of SEQ ID NOS: 36, 37, 39, 41, 50, 51, 54, 56, 60, 61, 65, 67, 137, 140, 144, 150, 159, 285, 286, 287, 288, 289, 292, 293, 294, 295, 296, 297 and 298 is provided, wherein the variant comprises an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical, to the amino acid sequence of the respective IgG protease from which the variant is derived.
  • an IgG protease comprising an amino acid sequence selected from one of SEQ ID NOS: 54, 140, 293, 294, 295, 297 and 298 is provided.
  • a variant of an IgG protease comprising an amino acid sequence of one of SEQ ID NOS: 54, 140, 293, 294, 295, 297 and 298 is provided, wherein the variant comprises an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical, to the amino acid sequence of the respective IgG protease from which the variant is derived.
  • N142 variant a variant of the N142 IgG protease (“N142 variant”) is provided.
  • the N142 IgG protease comprises the amino acid sequence of SEQ ID NO:297.
  • the N142 variant comprises (i) one or more amino acid mutations at one or more of the following ammo acid positions of SEQ ID NO:297: 110, Rl l, Y12, V15, 135, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, K129, R157, S159, L160, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300; and (ii) an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 98% identical to SEQ ID NO:297.
  • the N142 variant is at least about 30% as active as IdeS in an IgG protease enzymatic assay. In a further embodiment, the N142 variant is at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% as active as IdeS in an IgG protease enzymatic assay.
  • the N142 variant does not have amino acid mutations at one or more of the following amino acid positions of SEQ ID NO:297: A32, N33, Q36, F41, D84, R88, E91, E92, H93, Ml 06, N117, Hl 18, El 26, El 70, N197, N203, K213, L223, S245, K286, S306.
  • the one or more amino acid mutations in the N142 variant relative to SEQ ID NO:297 comprise one or more of the following amino acid substitutions: one of I10A and I10Q, RUT, Y12K, one of V15H and VI 5K, 135T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G, L120T, Y127L, K129G, R157G, S159G, L160W, one of V230G, V230R and V230S, N233S, K250S, A261K, F269N, S274D, one of A275F, A275S, and A275W, one of D288H and D288K, Q293K, V294A, L300T.
  • the one or more mutations in the N142 variant in one embodiment, comprise from about five to about twenty five, from about five to about twenty, from about 10 to about 20, from about 12 to about 20, from about 15 to about 20, from about 16 to about 25, or from about 16 to about 17, amino acid substitutions.
  • the N142 variant comprises a set of amino acid substitutions relative to SEQ ID NO:297, selected from one of the following: (i) I10A, V15H, I35T, and Y52F; (ii) I10A, V15H, I35T, and V46S; (m) I10A, V15H, I35T, and V46D; (iv) I10A, V15H, I35T, V46D, and T57K; (v) V230S, A261K, S274D, A275F, and D288H; (vi) L120T, V230R, A261K, S274D, and A275F; (vh) V230G, A261K, S274D, A275F, and D288H; (vm) Il 0A, VI 5H, V230S, A261K, and S274D; (ix) I10A, V15H, 135T, N48H,
  • the N142 variant comprises a set of amino acid mutations relative to SEQ ID NO:297, selected from one of the sets of amino acid substitutions set forth in Tables 6 and 7 disclosed herein.
  • the N142 variant disclosed herein does not have one or more amino acid substitutions of SEQ ID NO:297 selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, T57L, D84E, R88N, E91K, E92K, H93Y, M106L, N117D, H118S, E126D, N162D, E170R, N197Y, F199L, N203T, K213Q, L223I, N233G, S245A, A261P, K286E, and S306I.
  • the N142 variant comprises an amino acid sequence selected from one of SEQ ID NOS: 859, 868, 925, 936, and 942.
  • the N142 variant comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOS:859, 868, 925, 936, and 942, and the N142 variant comprises the amino acid substitutions present in the reference amino acid sequence relative to the amino acid sequence of SEQ ID NO: 297.
  • the variant is a variant of an IgG protease comprising an amino acid sequence set forth in one of SEQ ID NOS: 27-302.
  • the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 293 (the IgG protease comprising the amino acid sequence of SEQ ID NO:293 is also referred to herein as N123).
  • the N123 variant comprises (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 293: Y12, L31, E34, F36, R37, Y38, N39, 144, A60, D133, N162, F274, A279, H280, V299, (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 293.
  • the N123 variant (iii) is at least about 25% as active as IdeS in an IgG protease enzymatic assay.
  • the N123 variant is at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% as active as IdeS in an IgG protease enzymatic assay.
  • the variant comprises an amino acid sequence that is at least about 90% or at least about 95% identical to SEQ ID NO:293, and includes one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 293: Y12, L31, E34, F36, R37, Y38, N39, 144, A60, D133, N162, F274, A279, H280, V299.
  • the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 298 (the IgG protease comprising the amino acid sequence of SEQ ID NO:298 is also referred to herein as N30).
  • the N30 variant comprises (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 298: 110, Rl l, Y12, V15, Y18, V46, N48, Y52, F101, N102, Q105, L120, F125, E126, Y127, F133, Y156, R157, S159, V230, R231, N233, D258, A261, V270, A275, F269, (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 298.
  • the N30 variant (iii) is at least about 25% as active as IdeS in an IgG protease enzymatic assay.
  • the N30 variant is at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% as active as IdeS in an IgG protease enzymatic assay.
  • the N30 variant comprises an amino acid sequence that is at least about 90% or at least about 95% identical to SEQ ID NO:298, and includes one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 298: 110, Rl l, Y12, V15, Y18, V46, N48, Y52, F101, N102, Q105, L120, F125, E126, Y127, F133, Y156, R157, S159, V230, R231, N233, D258, A261, V270, A275, F269.
  • the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 294 (the IgG protease comprising the amino acid sequence of SEQ ID NO:294 is also referred to herein as N31).
  • the N31 variant comprises (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 294: 110, Rl l, Y12, E14, V15, Y18, V46, N48, Y52, D130, A132, Y157, N159, Y161, K162, V232, R233, N235, D260, A263, V272, S276, T1 , (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 294.
  • the N31 variant (iii) is at least about 25% as active as IdeS in an IgG protease enzymatic assay.
  • the N31 variant is at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% as active as IdeS in an IgG protease enzymatic assay.
  • the N31 variant comprises an amino acid sequence that is at least about 90% or at least about 95% identical to SEQ ID NO:294, and includes one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 294: 110, Rl l, Y12, E14, V15, Y18, V46, N48, Y52, D130, A132, Y157, N159, Y161, K162, V232, R233, N235, D260, A263, V272, S276, A277.
  • the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 54.
  • the IgG protease variant comprises (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 54: 110, Il l, Y12, E14, Y18, H19V143, Y157, Y161, K162, (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 54.
  • the variant (iii) is at least about 25% as active as IdeS in an IgG protease enzymatic assay.
  • the variant is at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% as active as IdeS in an IgG protease enzymatic assay.
  • the variant comprises an amino acid sequence that is at least about 90% or at least about 95% identical to SEQ ID NO: 54, and includes one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 54: 110, Il l, Y12, E14, Y18, H19V143, Y157, Y161, K162.
  • the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 140.
  • the variant comprises (i) one or more amino acid mutations at one or more ammo acid positions of SEQ ID NO: 140: M10, Rl l, Y12, V15, Y42, V46, A47, N48, Q49, N102, V104, 1113, Y118, L120, D121, K123, Y127, Y156, R157, Y201, Q202, V230, F269, S274, A275, D288, G296, and (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 140.
  • the variant (iii) is at least about 25% as active as IdeS in an IgG protease enzymatic assay. In a further embodiment, the variant is at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% as active as IdeS in an IgG protease enzymatic assay.
  • the variant comprises an amino acid sequence that is at least about 90% or at least about 95% identical to SEQ ID NO: 140 and includes one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 140: M10, Rl l, Y12, V15, Y42, V46, A47, N48, Q49, N102, V104, 1113, Y118, L120, D121, K123, Y127, Y156, R157, Y201, Q202, V230, F269, S274, A275, D288, G296.
  • N144 variant a variant of the N144 IgG protease
  • the N144 IgG protease comprises an amino acid sequence set forth in SEQ ID NO:295.
  • the N144 variant comprises (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO:295: Y12, SI 8, 135, 146, A47, Y52, 154, T55, T57, N59, H93, F101, N102, E104, Q105, L120, F125, E126, Y127, T135, Y156, S159, L175, V183, R200, N201, N205, 1207, V230, R231, N233, A241, N244, E246, A261, Y269, V270, S274, A275, 1280, S281, A282, 1285, D288, V290, and (ii) an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 98% identical to SEQ ID
  • the N144 variant is at least about 30% as active as IdeS in an IgG protease enzymatic assay. In a further embodiment, the N144 variant is at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% as active as IdeS in an IgG protease enzymatic assay.
  • the N144 variant does not have amino acid mutations at one or more of the following ammo acid positions of SEQ ID NO:295: A32, N33, Q36, F41, D84, E91, Ml 06, Hl 18, El 70, SI 95, N197, N203, S245, K286.
  • the one or more mutations in the N144 variant in one embodiment, comprise from about 5 to about 16, from about 10 to about 16, from about 10 to about 30, from about 12 to about 16, from about 12 to about 24, about 12, about 13, about 14, about 21, about 22, about 23, or about 24 amino acid substitutions, compared to SEQ ID NO:295.
  • the N144 variant comprises one or more amino acid substitutions of SEQ ID NO:295 selected from the group consisting of Y12N, S18K, one of I35E, I35K, and I35S, I46D, A47E, I54T, one of T55K, T55H, and T55R, T57L, N59D, H93Y, N102G, E104R, one of L120T and L120S, F125W, T135H, S159K, one of L175A, L175N, and L175S, V183W, one of R200D, R200G and R200T, N201E, N205D, I207T, one of V230H and V230Q, R231W, N233G, A241G, one of E246G and E246K, one of A261K, A261I, A261F, A261G, A261Q, and A261S, one of Y269D,
  • the N144 variant comprises from about 5 to about 16, from about 10 to about 16, from about 10 to about 30, from about 12 to about 16, from about 12 to about 24, about 12, about 13, about 14, about 21, about 22, about 23, or about 24 of the amino acid substitutions of SEQ ID NO:295 selected from the group consisting of Y12N, S18K, one of I35E, I35K, and I35S, I46D, A47E, I54T, one of T55K, T55H, and T55R, T57L, N59D, H93Y, N102G, E104R, one of L120T and L120S, F125W, T135H, S159K, one of L175A, L175N, and L175S, V183W, one of R200D, R200G and R200T, N201E, N205D, I207T, one of V230H and V230Q, R231W, N233G, A241G, one of E246G and E246K,
  • the N144 variant does not have one or more amino acid substitutions of SEQ ID NO:295 selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, F199L, N203T, K213Q, L223I, S245A, N246E, K250E, K286E, and S306I.
  • the N144 variant comprises a set of amino acid substitutions relative to SEQ ID NO:295, selected from one of the following:
  • the N144 variant comprises an amino acid sequence selected from one of SEQ ID NOS: 1118, 1120, 1122, 1123, 1125, 1126, 1127, 1129, 1282, 1962, 1963, 1964, 1965, 1970, 1971, 1983, 2004, 2024, 2028, 2371, 2372, 2373, 2390, 2421, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2437, 2439, 2446, 2450, 2463, 2464, 2476, 2478, 2485, 2486, 2487, 2489, 2491, 2493, 2494, 2511, 2524, 2526, 2528, 2574, and 2575.
  • the N144 variant comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOS: 1118, 1120, 1122, 1123, 1125, 1126, 1127, 1129, 1282, 1962, 1963, 1964, 1965, 1970, 1971, 1983, 2004, 2024, 2028, 2371, 2372, 2373, 2390, 2421, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2437, 2439, 2446, 2450, 2463, 2464, 2476, 2478, 2485, 2486, 2487, 2489, 2491, 2493, 2494, 2511, 2524, 2526, 2528, 2574, and 2575, and the N144 variant comprises the amino acid substitutions present in the reference amino acid sequence relative to the amino acid sequence of SEQ ID NOS: 1118, 1120
  • the N144 variant comprises a first set of amino acid substitutions of SEQ ID NO:295 and a second set of amino acid substitutions of SEQ ID NO:295.
  • the first set of amino acid substitutions of SEQ ID NO:295 consists of Y12N, S18K, I46D, A47E, H93Y, E104R, F125W, T135H, and S159K.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 3 to about 15 amino acid substitutions selected from the group consisting of one of I35K, I35E, and I35S, I54T, one of T55K, T55R, and T55H, T57L, N59D, N102G, one of L120T and L120S, one of L175N, L175A, and L175S, V183W, one of R200Gand R200T, N201E, N205D, one of V230H and V230Q, R231W, N233G, A241G, one of E246K and E246G, one of A261K, A261Q, A261S, A261I, A261F, and A261G, Y269W, V270T, one of S274G and S274D, one of A275W and A275S, I280M, A282G, one of D288T and D288
  • the N144 variant has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or at least about 96% identical to SEQ ID NO:295.
  • the N144 variant comprising the first set and second set of amino acid substitutions of SEQ ID NO:295 does not have amino acid mutations at one or more of the following amino acid positions of SEQ ID NO:295: A32, N33, Q36, F41, D84, E91, Ml 06, Hl 18, E170, S195, N197, N203, S245, K286.
  • the N144 variant does not have one or more amino acid substitutions of SEQ ID NO:295 selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, F199L, N203T, K213Q, L223I, S245A, N246E, K250E, K286E, and S306I.
  • a polypeptide variant in one embodiment, is T cell epitope depleted compared to the protein from which the variant’s amino acid sequence is derived.
  • a fusion protein comprising (i) a first domain comprising one of the polypeptides or variants thereof described herein, and (ii) a second domain.
  • the fusion protein comprises a third domain.
  • the second domain comprises a random coil polypeptide.
  • the second domain comprises an immunoglobin Fragment crystallizable (Fc) domain of the human immunoglobulin class G (IgG).
  • the second domain comprises an albumin protein.
  • a method for treating a patient for a disease mediated in whole or in part by pathogenic immunoglobulin G (IgG) antibodies.
  • the method comprises administering to the patient in need of treatment, one of the polypeptides or variants thereof, or one of the fusion proteins described herein.
  • the disease in one embodiment, is chronic inflammatory demyelinating polyneuropathy (CIDP). In another embodiment, the disease is acute inflammatory demyelinating polyneuropathy (AIDP).
  • a method for preventing or treating antibody-mediated rejection (AMR) of an organ allograft in an organ-transplant patient in need of treatment.
  • the method comprises administering to the patient, one of the polypeptides, or variants thereof, or one of the fusion proteins described herein.
  • Administration in one embodiment, is carried out prior to transplantation of the organ allograft, e.g., from about three weeks to about 1 hour prior to transplantation of the organ allograft.
  • the organ in one embodiment, is a solid organ, e.g., a kidney, small intestine, pancreas, heart, lung or liver.
  • FIG. 1 is a schematic of the PBMC (peripheral blood mononuclear cell) assay used to measure CD4+ T-cell activation and epitopes for N142 and wild type IdeS, as well as their respective select variants.
  • PBMC peripheral blood mononuclear cell
  • FIG. 2 is a graph showing full CD4+ T cell epitope mapping of N142 by the PBMC assay.
  • FIG. 3 is a graph showing targeted CD4+ T cell epitope mapping of select N142 variants as compared to the parent N142 by the PBMC assay.
  • FIG. 4 is a graph showing full CD4+ T cell epitope mapping of wild type IdeS by the PBMC assay.
  • FIG. 5 is a graph showing targeted CD4+ T cell epitope mapping of select IdeS variants as compared to wild type IdeS by the PBMC assay.
  • thermostability refers to a measure of thermostability, e.g., the melting temperature in the unit of degree Celsius, of an IgG protease variant that is identical to, or less than 10%, less than 5%, less than 3%, or less than 1% different from, the same measure of thermostability of the reference IgG protease.
  • sequence comparison For sequence comparison, and to determine the “percent identity” of two peptide, polypeptide or nucleic acid sequences, typically one sequence acts as a reference sequence to which a test sequence(s) is compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul etal. (1990) J.
  • HSPs high scoring sequence pairs
  • T some positive-valued threshold score threshold
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
  • cumulative scores are calculated using a scoring matrix. Extension of the word hits in each direction is halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1992). Proc. Natl. Acad. Sci. USA 89, pp.10915-10919).
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’L Acad. Sci. USA 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • T cell epitope refers to a peptide epitope that binds to an MHCII molecule and subsequently forms a ternary complex with a cognate T cell receptor (TCR).
  • TCR T cell receptor
  • a recombinant IgG protease, or a polynucleotide comprising the same is provided that is “T cell epitope depleted” compared to a counterpart IgG protease protein.
  • a protein is “T cell epitope depleted” if one or more amino acid mutations are present in one or more T cell epitopes such that the T cell epitopes in the IgG protease are depleted, as compared to a counterpart IgG protease protein, e.g., a wild type (WT) IgG protease protein.
  • WT wild type
  • one or more T cell epitopes are “depleted” from an IgG protease variant if a peptide originating from the IgG protease variant is unable to bind an MHCII molecule, or if the peptide binds the MHCII molecule but subsequently does not bind a TCR, e.g., because of the lack of affinity to the MHCII molecule.
  • IgG protease variants are referred to herein in some instances, as a “deimmunized IgG protease variant” or a “T cell epitope depleted” IgG protease variant. The use of such variants and methods of manufacturing the variants are also described herein.
  • variant protein refers to a protein that is distinguished from a counterpart protein, such as the WT form of the protein, on the basis of the presence of one or more amino acid modifications, such as, for example, one or more amino acid substitutions, insertions, deletions, or a combination thereof.
  • variant protein encompasses a full-length form as well as a truncated form of an IgG protease variant, e.g., a catalytically active fragment of an IgG protease variant.
  • mutant gene is a term of the art and refers to a gene that is distinguished from the WT form of the gene on the basis of the presence of one or more nucleic acid modifications, such as, for example, one or more nucleic acid substitutions, insertions, deletions, or a combination thereof. In some embodiments, a mutant gene encodes a variant protein.
  • a mutation in a gene may also be a silent mutation, i.e., a mutation that does not affect the amino acid sequence in the protein that is encoded by the gene.
  • a mutant / variant may occur in nature or may be an engineered mutant / variant.
  • an IgG protease variant is disclosed to be some percent identical to another IgG protease variant (reference variant)
  • the IgG protease variant includes the amino acid mutations present in the reference variant.
  • the IgG protease variant A’ is 90 percent identical to (reference) IgG protease variant A
  • the IgG protease variant A’ also includes the amino acid mutations present in IgG protease variant A.
  • wild type refers to the most prevalent form of an organism, strain, gene, protein, or characteristic as it occurs in nature and/or is distinguished from mutant or variant forms.
  • WT wild type
  • the full-length amino acid sequence of IdeS is publicly available as NCBI Reference Sequence no. WP 010922160.1 and is provided herein as SEQ ID NO: 1.
  • the full-length sequence includes an N-terminal methionine followed by a 28 amino acid secretion signal sequence.
  • the N-terminal methionine and the signal sequence are typically removed to form the mature IdeS protein, and the sequence of which is publicly available as Genbank accession no.
  • the IgG protease variants described herein encompass polypeptides (i) that include the N-terminal methionine and the secretion signal sequence and (ii) those that do not include the N-terminal methionine and the secretion signal sequence.
  • alternative embodiments include IgG protease variants of the same sequences that have the N- terminal methionine residue present. Similarly, if an N-terminal methionine residue is present in an IgG protease variant, alternative embodiments include IgG protease variants of the same sequences that do not include the N-terminal methionine residue. Similarly, with respect to the polypeptides disclosed herein, if a signal sequence (e.g., secretion signal sequence) is not present, alternative embodiments include IgG protease variants of the same sequences that include a signal sequence. Alternatively, if a signal sequence is present in an IgG protease variant, alternative embodiments include IgG protease variants of the same sequences that do not include the signal sequence, and do not include an N-terminal methionine residue.
  • a signal sequence e.g., secretion signal sequence
  • the present disclosure provides a variant of the IdeS IgG protease (“IdeS variant”).
  • the IdeS IgG protease as provided above, comprises an amino acid sequence set forth in SEQ ID NO:2.
  • the IdeS variant comprises (i) one or more amino acid mutations at one or more of the following ammo acid positions of SEQ ID NO: 2: S3, F4, A6, E9, 110, Rl l, Y12, Y18, V46, A47, N48, 154, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, G296, and (ii) an amino acid sequence that is at least about 75% identical
  • the IdeS variant does not have amino acid mutations at the following amino acid positions of SEQ ID NO:2: K56, C66, H234, D256 and D258. In a further embodiment or in another embodiment, the IdeS variant does not have amino acid mutations at one or more of the following amino acid positions of SEQ ID NO:2: A32, N33, T35, Q36, F41, D84, R88, E91, N102, Ml 06, N117, Hl 18, E170, S195, N197, K213, S245, K250, A261, K286, S306.
  • the mutation(s) in the IdeS variant at one or more of the aforementioned amino acid positions eliminates one or more dominant immunogenic T cell epitopes present in the WT IdeS enzyme, such that the IdeS variant is T cell epitope depleted compared to the WT IdeS (i.e., e.g., the polypeptide of SEQ ID NO:2).
  • Amino acid mutations of the present disclosure include, but are not limited to, amino acid exchange(s), insertion(s), deletion(s), addition(s), substitution(s), inversion(s) and/or duplicati on(s). These mutations/modification(s) also include conservative and/or homologous amino acid exchange(s).
  • the one or more mutations in the IdeS variant are one or more amino acid substitutions.
  • the IdeS variant comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid mutations, relative to a wild type IdeS enzyme of SEQ ID NO:2, and the mutations are selected from mutations (e.g., substitutions) at the following amino acid positions of SEQ ID NO: 2: S3, F4, A6, E9, 110, Rl l, Y12, Y18, V46, A47, N48, 154, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288,
  • mutations are selected from
  • two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid mutations are two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid substitutions.
  • the amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO:2 set forth in Table 1.
  • the IdeS variant comprises from about five to about twenty-three amino acid mutations relative to the WT IdeS enzyme of SEQ ID NO:2 and the mutations are selected from mutations at the following amino acid positions of SEQ ID NO:2: S3, F4, A6, E9, 110, Rl l, Y12, Y18, V46, A47, N48, 154, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, G296.
  • the from about five to about twenty -three amino acid mutations are from about five to about twenty- three amino acid substitutions. In a further embodiment, the from about five to about twenty-three amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO:2 set forth in Table 1.
  • the IdeS variant comprises from about 5 to about 20 amino acid mutations, from about 10 to about 20 amino acid mutations, from about 10 to about 18 amino acid mutations, from about 10 to about 16 amino acid mutations, from about 10 to about 14 amino acid mutations, from about 10 to about 13 amino acid mutations, from about 10 to about 12 amino acid mutations, from about 11 to about 20 amino acid mutations, from about 11 to about 13 amino acid mutations, from about 12 to about 20 amino acid mutations, from about 12 to about 14 amino acid mutations, from about 13 to about 20 amino acid mutations, from about 14 to about 20 amino acid mutations, from about 15 to about 20 amino acid mutations, from about 10 to about 35 amino acid mutations, from about 15 to about 30 amino acid mutations, from about 20 to about 30 amino acid mutations, from about 24 to about 35 amino acid mutations, from about 24 to about 32 amino acid mutations, or from about 24 to about 29 amino acid mutations relative to the WT IdeS enzyme of SEQ ID NO:2, and the mutations are
  • each of the amino acid mutations is an amino acid substitution.
  • the amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO:2 set forth in Table 1.
  • the IdeS variant comprises from about 5 to about 20 amino acid mutations relative to the WT IdeS enzyme of SEQ ID NO: 2, wherein the amino acid mutations are selected from mutations at the following amino acid residues of SEQ ID NO:2: S3, F4, A6, E9, 110, Rl l, Y12, Y18, V46, A47, N48, 154, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, G2
  • the IdeS variant comprises from about 10 to about 20 amino acid mutations relative to the WT IdeS enzyme of SEQ ID NO: 2, wherein the amino acid mutations are selected from the following amino acid residues of SEQ ID NO:2: S3, F4, A6, E9, 110, Rl l, Y12, Y18, V46, A47, N48, 154, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, G296.
  • each of the amino acid mutations is an amino acid substitution.
  • the from about 10 to about 20 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO:2
  • the IdeS variant comprises from about 12 to about 20 amino acid mutations relative to the WT IdeS enzyme of SEQ ID NO:2, wherein the amino acid mutations are selected from the following amino acid residues of SEQ ID NO:2: S3, F4, A6, E9, 110, Rl l, Y12, Y18, V46, A47, N48, 154, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, G296.
  • each of the amino acid mutations is an amino acid substitution.
  • the from about 12 to about 20 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO:2
  • the IdeS variant comprises from about 15 to about 20 amino acid mutations relative to the WT IdeS enzyme of SEQ ID NO:2, wherein the amino acid mutations are selected from the following amino acid residues of SEQ ID NO:2: S3, F4, A6, E9, 110, Rl l, Y12, Y18, V46, A47, N48, 154, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, G296.
  • each of the amino acid mutations is an amino acid substitution.
  • the from about 15 to about 20 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO:2
  • the IdeS variant comprises one of the sets of amino acid substitutions of SEQ ID NO:2 as set forth in Table 2 herein.
  • the IdeS variant comprises one of the sets of amino acid substitutions of SEQ ID NO:2 as set forth in Table 3 herein.
  • the IdeS variant comprises one of the sets of amino acid substitutions of SEQ ID NO:2 as set forth in Table 4 herein.
  • the IdeS variant does not have one or more amino acid substitutions of SEQ ID NO:2 selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, N102G, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, K213Q, N233G, S245A, N246E, K250E, A261P, K286E, and S306I.
  • the IdeS variant has an amino acid sequence that is at least about 75% identical to SEQ ID NO:2. In some embodiments, the IdeS variant has an amino acid sequence that is at least about 80% identical to SEQ ID NO:2. In some embodiments, the IdeS variant has an amino acid sequence that is at least about 85% identical to SEQ ID NO:2. In some embodiments, the IdeS variant has an amino acid sequence that is at least about 90% identical to SEQ ID NO:2. In some embodiments, the IdeS variant has an amino acid sequence that is at least about 95% identical to SEQ ID NO:2. In some embodiments, the IdeS variant has an amino acid sequence that is at least about 97% identical to SEQ ID NO:2. In some embodiments, the IdeS variant has an amino acid sequence that is at least about 98% identical to SEQ ID NO:2.
  • the IdeS variant comprises an amino acid sequence set forth in one of SEQ ID NOS: 3 -26. In a further embodiment, the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence set forth in one of SEQ ID NOS: 3 -26, and the IdeS variant comprises the amino acid substitutions present in the reference amino acid sequence relative to the amino acid sequence of SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO:3.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:3, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 3 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:4, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO:4 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO:5.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO NO: 5, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 5 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO:6.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:6, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 6 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO:7.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:7, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 7 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO: 8.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 8, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 8 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO:9.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:9, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 9 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO: 10.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO NO: 10, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 10 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO:11.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 11, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 11 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 12, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 12 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO: 13.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 13, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 13 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO: 14.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 14, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 14 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO: 15.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO NO: 15, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 15 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO: 16.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 16, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 16 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO: 17.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 17, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 17 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO: 18.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO NO: 18, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 18 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 19, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO: 19 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO:20.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:20, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO:20 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO:21.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:21, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO:21 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO:22.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:22, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO:22 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO:23.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:23, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO 23 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO:24.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:24, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO:24 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO:25.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:25, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO:25 relative to SEQ ID NO:2.
  • the IdeS variant comprises the amino acid sequence set forth in SEQ ID NO:26.
  • the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:26, and the IdeS variant comprises the amino acid substitutions present in the amino acid sequence of SEQ ID NO:26 relative to SEQ ID NO:2.
  • the IdeS variant comprises an amino acid sequence set forth in one of SEQ ID NOS: 1491 -1951.
  • the IdeS variant of the disclosure comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence set forth in one of SEQ ID NOS: 1491-1951, and the IdeS variant comprises the amino acid substitutions present in the reference amino acid sequence relative to SEQ ID NO:2.
  • an IdeS variant that is 85% identical to the variant of SEQ ID NO: 1491 includes the amino acid substitutions that are present in SEQ ID NO: 1491 (substitutions relative to SEQ ID NO:2).
  • the IdeS variant comprises an amino acid sequence selected from one of SEQ ID NOS: 1492, 1697, 1700, 1701, 1702, 1703, 1704, 1705, 1713, 1720, 1722, 1723, 1724, 1772, 1774, 1775, 1793, 1795, 1797, 1798, and 1949.
  • the IdeS variant of the disclosure comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence set forth in one of SEQ ID NOS: 1492, 1697, 1700, 1701, 1702, 1703, 1704, 1705, 1713, 1720, 1722, 1723, 1724, 1772, 1774, 1775, 1793, 1795, 1797, 1798, and 1949, and the IdeS variant comprises the amino acid substitutions present in the reference amino acid sequence relative to SEQ ID NO:2.
  • the IdeS variant comprises a first set of amino acid substitutions of SEQ ID NO:2 and a second set of amino acid substitutions of SEQ ID NO:2.
  • the first set of amino acid substitutions of SEQ ID NO:2 consists of Y12N, Y18K, A47E, H93Y, E104R, L120T, E126S, Y135N, S159K, V230S, and A275S.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 18 amino acid substitutions selected from the group consisting of S3N, F4I, A6S, E9I, HOT, RUT, N48G, I54T, one of T57K, T57N, T57R, T57L, and T57Q, N59D, one of G60S and G60T, K61R, E92R, T138A, one of T161G and T161Y, D188T, F199L, K200R, E201N, N203T, G222A, L223I, N233S, N246R, N273G, D288G, and Q293R.
  • the IdeS variant comprising the first set and second set of amino acid substitutions of SEQ ID NO:2 has an amino acid sequence that is at least about 88% identical to SEQ ID NO:2. In another embodiment, the IdeS variant has an amino acid sequence that is at least about 89%, at least about 90%, at least about 91%, or at least about 92% identical to SEQ ID NO:2. In another embodiment, the IdeS variant has an amino acid sequence that is from about 88% to about 92%, from about 89% to about 92%, or from about 90% to about 92% identical to SEQ ID NO:2.
  • the IdeS variant comprising the first set and second set of amino acid substitutions of SEQ ID NO:2 does not have amino acid mutations at the following amino acid positions of SEQ ID NO:2: K56, C66, H234, D256 and D258. In another embodiment, the IdeS variant does not have amino acid mutations at one or more of the following amino acid positions of SEQ ID NO:2: A32, N33, T35, Q36, F41, D84, R88, E91, N102, Ml 06, N117, Hl 18, E170, S195, N197, K213, S245, K250, A261, K286, S306.
  • the IdeS variant does not have one or more amino acid substitutions of SEQ ID NO:2 selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, N102G, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, K213Q, N233G, S245A, N246E, K250E, A261P, K286E, and S306I.
  • the second set of amino acid substitutions of SEQ ID NO: 2 is a set of from about 13 to about 17 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 16 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of from about 13 to about 15 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:2 is a set of 13, 14, 15, 16, 17, or 18 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 18 amino acid substitutions selected from the group consisting of N48G, I54T, one of T57K, T57N, T57R, and T57L, N59D, one of G60S and G60T, K61R, E92R, T138A, one of T161G and T161Y, D188T, F199L, K200R, E201N, N203T, L223I, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 17 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 16 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 15 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:2 is a set of 13, 14, 15, 16, 17, or 18 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 18 amino acid substitutions selected from the group consisting of N48G, I54T, one of T57K and T57N, N59D, one of G60S and G60T, K61R, E92R, T138A, one of T161G and T161 Y, D188T, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 17 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 16 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 15 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:2 is a set of 13, 14, 15, 16, 17, or 18 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 18 amino acid substitutions selected from the group consisting of N48G, I54T, one of T57K and T57N, N59D, G60S, K61R, E92R, T138A, one of T161G and T161Y, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 17 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID N0:2 is a set of from about 13 to about 16 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 15 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:2 is a set of 13, 14, 15, 16, 17, or 18 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 18 amino acid substitutions selected from the group consisting of N48G, I54T, one of T57K and T57N, N59D, G60T, K61R, E92R, T138A, one of T161G and T161Y, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 17 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 16 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 15 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:2 is a set of 13, 14, 15, 16, 17, or 18 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 17 amino acid substitutions selected from the group consisting of N48G, I54T, T57K, N59D, G60S, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 15 amino acid substitutions selected from the group consisting of N48G, I54T, N59D, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of from about 13 to about 14 amino acid substitutions selected from the group consisting of N48G, N59D, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 13 amino acid substitutions consisting of N48G, N59D, K61R, E92R, T138A, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 13 amino acid substitutions consisting of S3N, F4I, A6S, N48G, I54T, T57N, N59D, T138A, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 15 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, T138A, D188T, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 17 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161G, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 16 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 17 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N203T, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 17 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161G, D188T, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 16 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161Y, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 17 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161Y, D188T, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 16 amino acid substitutions consisting of N48G, T57Q, N59D, G60T, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 17 amino acid substitutions consisting of N48G, T57R, N59D, G60T, K61R, E92R, T138A, T161G, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 17 amino acid substitutions consisting of N48G, T57R, N59D, G60T, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N203T, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 18 amino acid substitutions consisting of N48G, T57R, N59D, G60T, K61R, E92R, T138A, T161G, D188T, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 16 amino acid substitutions consisting of N48G, T57R, N59D, G60T, K61R, E92R, T138A, T161Y, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 13 amino acid substitutions consisting of N48G, I54T, T57L, N59D, T138A, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 16 amino acid substitutions consisting of N48G, I54T, T57L, N59D, E92R, T138A, T161G, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 15 amino acid substitutions consisting of N48G, I54T, T57L, N59D, E92R, T138A, T161Y, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 15 amino acid substitutions consisting of N48G, I54T, T57N, N59D, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 17 amino acid substitutions consisting of N48G, I54T, T57N, N59D, E92R, T138A, T161G, D188T, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 13 amino acid substitutions consisting of N48G, I54T, T57N, N59D, E92R, T138A, T161Y, G222A, L223I, N233S, N246R, N273G, and D288G
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 15 amino acid substitutions consisting of N48G, I54T, T57N, N59D, E92R, T138A, T161Y, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.
  • the second set of amino acid substitutions of SEQ ID NO:2 is a set of 13 amino acid substitutions consisting ofE9I, HOT, RUT, N48G, I54T, T57N, N59D, T138A, N233S, N246R, N273G, D288G, and Q293R.
  • the IdeS variant disclosed herein is at least 25% as active as an IdeS enzyme comprising an amino acid sequence set forth in SEQ ID NO:2 in an IgG protease enzymatic assay. In some embodiments, the IdeS variant is at least 30% as active as an IdeS enzyme comprising an amino acid sequence set forth in SEQ ID NO:2 in an IgG protease enzymatic assay.
  • the IdeS variant is at least about 40% as active, at least about 50% as active, at least about 60% as active, at least about 70% as active, at least about 75% as active, at least about 80% as active, at least about 90% as active, at least about 100% as active, at least about 110% as active, at least about 120% as active, as an IdeS enzyme comprising the amino acid sequence of SEQ ID NO:2 in an IgG protease enzymatic assay.
  • the IdeS variant is at least about 25% as active, at least about 40% as active, at least about 50% as active, at least about 60% as active, at least about 70% as active, at least about 80% as active, at least about 90% as active, at least about 100% as active, at least about 110% as active, at least about 120% as active, as an IdeZ enzyme comprising the amino acid sequence of SEQ ID NO: 830 in an IgG protease enzymatic assay.
  • the IgG protease activity in the IgG protease enzymatic assay is measured via an enzyme linked immunosorbent assay comprising digestion of an immobilized antibody substrate.
  • the IdeS variant has one or more T cell epitopes depleted, e.g., two or more, three or more, or four or more T cell epitopes depleted, compared to an IdeS enzyme comprising the amino acid sequence set forth in SEQ ID NO:2.
  • the IdeS variant has substantially the same thermostability as an IdeS enzyme comprising the amino acid sequence set forth in SEQ ID NO:2. In other embodiments, the IdeS variant is more thermostable than an IdeS enzyme comprising the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, thermostability is measured by differential scanning fluorimetry.
  • the IdeS variant has substantially the same thermostability as an IdeZ enzyme comprising the amino acid sequence set forth in SEQ ID NO: 830. In other embodiments, the IdeS variant is more thermostable than an IdeZ enzyme comprising the amino acid sequence set forth in SEQ ID NO: 830. In some embodiments, thermostability is measured by differential scanning fluorimetry.
  • an IgG protease comprising an amino acid sequence selected from one of SEQ ID NOS: 27-302 is provided.
  • an IgG protease variant is provided, wherein the variant is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical, to the amino acid sequence of the respective IgG protease from which the variant is derived.
  • an IgG protease comprising an amino acid sequence selected from one of SEQ ID NOS: 36, 37, 39, 41, 50, 51, 54, 56, 60, 61, 65, 67, 137, 140, 144, 150, 159, 285, 286, 287, 288, 289, 292, 293, 294, 295, 296, 297 and 298 is provided.
  • an IgG protease variant is provided, and the variant comprises an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical, to the amino acid sequence of the respective IgG protease from which the variant is derived.
  • an IgG protease comprising an amino acid sequence selected from one of SEQ ID NOS: 54, 140, 293, 294, 295, 297 and 298 is provided.
  • an IgG protease variant is provided, and the variant comprises an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical, to the amino acid sequence of the respective IgG protease from which the variant is derived.
  • the IgG protease comprises an amino acid sequence set forth in SEQ ID NO: 54.
  • an IgG protease variant is provided, and the variant comprises an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical, to SEQ ID NO: 54.
  • the IgG protease comprises an amino acid sequence set forth in SEQ ID NO: 140.
  • an IgG protease variant is provided, and the variant comprises an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical, to SEQ ID NO: 140.
  • the IgG protease comprises an amino acid sequence set forth in SEQ ID NO:293.
  • an IgG protease variant is provided, and the variant comprises an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical, to SEQ ID NO:293.
  • the IgG protease comprises an amino acid sequence set forth in SEQ ID NO:294.
  • an IgG protease variant is provided, and the variant comprises an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical, to SEQ ID NO:294.
  • the IgG protease comprises an amino acid sequence set forth in SEQ ID NO:295.
  • an IgG protease variant is provided, and the variant comprises an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical, to SEQ ID NO:295.
  • the IgG protease comprises an amino acid sequence set forth in SEQ ID NO:297.
  • an IgG protease variant is provided, and the variant comprises an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical, to SEQ ID NO:297.
  • the IgG protease comprises an amino acid sequence set forth in SEQ ID NO:298.
  • an IgG protease variant is provided, and the variant comprises an amino acid sequence that is at least about 80% identical, at least about 85% identical, at least about 90% identical, or at least about 95% identical, to SEQ ID NO:298.
  • variants of the disclosure include both (i) polypeptides that include the N-terminal methionine and (ii) polypeptides that do not include the N-terminal methionine.
  • a variant of the predicted N142 IgG protease is provided.
  • the N142 IgG protease as provided above, comprises an amino acid sequence set forth in SEQ ID NO:297.
  • the N142 variant in one embodiment, comprises (i) one or more amino acid mutations at the following amino acid positions of SEQ ID NO:297: 110, Rl l, Y12, V15, 135, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, K129, R157, S159, L160, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300, and (n) an ammo acid sequence that is at least about 75% identical to SEQ ID NO: 297.
  • the N142 variant having one or more mutation(s) at one or more of the aforementioned amino acid positions is T cell epitope depleted, as compared to the WT N142 enzyme comprising the amino acid sequence of SEQ ID NO:297.
  • the N142 variant has one or more dominant immunogenic T cell epitopes eliminated, as compared to the immunogenic T cell epitopes present in the WT N142 enzyme comprising the amino acid sequence of SEQ ID NO:297.
  • Amino acid mutations for use herein include, but are not limited to, amino acid exchange(s), insertion(s), deletion(s), addition(s), substitution(s), inversion(s) and/or duplication(s). These mutations/modification(s) also include conservative and/or homologous amino acid exchange(s).
  • the one or more mutations are one or more amino acid substitutions.
  • the N142 variant comprises one or more amino acid mutations at the following ammo acid positions of SEQ ID NO:297: 110, Rl l, Y12, V15, 135, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, L300.
  • the N142 variant does not have amino acid mutations at one or more of the following amino acid positions of SEQ ID NO:297: A32, N33, Q36, F41, D84, R88, E91, E92, H93, Ml 06, N117, Hl 18, El 26, El 70, N197, N203, K213, L223, S245, K286, S306.
  • the N142 variant comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid mutations, relative to the WT N142 enzyme comprising the amino acid sequence of SEQ ID NO:297, and the mutations are selected from mutations at the following amino acid positions of SEQ ID NO:297: 110, Rl l, Y12, V15, 135, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, K129, R157, S159, L160, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300.
  • the mutations are selected from mutations at the following amino acid positions of SEQ ID NO:297: 110, Rl l, Y12, V15, 135, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, SI 59, LI 60, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, L300.
  • the two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid mutations are two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid substitutions.
  • the amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO:297 set forth in Table 5.
  • amino acid substitutions of SEQ ID NO:297 are selected from the group consisting of one of I10A and I10Q, RUT, Y12K, one of V15H and V15K, I35T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G, L120T, Y127L, K129G, R157G, S159G, L160W, one of V230G, V230R and V230S, N233S, K250S, A261K, F269N, S274D, one of A275F, A275S, and A275W, one of D288H and D288K, Q293K, V294A, L300T, and combinations thereof.
  • the N142 variant comprises from about 5 to about 25 amino acid mutations, from about 5 to about 20 amino acid mutations, from about 5 to about 15 amino acid mutations, from about 8 to about 20 amino acid mutation, from about 10 to about 20 amino acid mutations, from about 10 to about 18 amino acid mutations, from about 10 to about 16 amino acid mutations, from about 10 to about 14 amino acid mutations, from about 10 to about 13 amino acid mutations, from about 10 to about 12 amino acid mutations, from about 11 to about 20 amino acid mutations, from about 11 to about 13 amino acid mutations, from about 12 to about 14 amino acid mutations, from about 13 to about 20 amino acid mutations, from about 14 to about 20 amino acid mutations, from about 15 to about 20 amino acid mutations, from about 16 to about 20 amino acid mutations, from about 16 to about 25 amino acid mutations, or from about 16 to about 17 amino acid mutations relative to the WT N142 enzyme comprising the amino acid sequence set forth in SEQ ID NO:297, wherein the amino acid mutations comprise at
  • the amino acid mutations comprise at least five, or at least 10 amino acid mutations at the following ammo acid positions of SEQ ID NO:297: 110, Rl l, Y12, V15, 135, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, L300.
  • each of the amino acid mutations is an amino acid substitution.
  • the at least 5 or at least 10 amino acid substitutions at the aforementioned amino acid positions are selected from at least five or at least 10 amino acid substitutions of SEQ ID NO: 297 set forth in Table 5.
  • the at least five or at least 10 amino acid substitutions of SEQ ID NO:297 are selected from the group consisting of one of I10A and I10Q, RUT, Y12K, one of V15H and V15K, 135T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G, L120T, Y127L, K129G, R157G, S159G, L160W, one ofV230G, V230R and V230S, N233S, K250S, A261K, F269N, S274D, one of A275F, A275S, and A275W, one of D288H and D288K, Q293K, V2
  • the N142 variant comprises from about 5 to about 20 amino acid mutations relative to the WT N142 enzyme of SEQ ID NO:297. In a further embodiment, the from about 5 to about 20 amino acid mutations are from about 5 to about 20 amino acid substitutions relative to the wild type N142 enzyme of SEQ ID NO: 297.
  • the N142 variant comprises from about 5 to about 20 amino acid mutations relative to the WT N142 enzyme of SEQ ID NO:297, wherein the amino acid mutations include from about 5 to about 20 amino acid mutations at the following amino acid residues of SEQ ID NO:297: 110, Rl l, Y12, VI 5, 135, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, K129, R157, S159, L160, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300.
  • the amino acid mutations include from about 5 to about 20 amino acid mutations at the following ammo acid residues of SEQ ID NO:297: 110, Rl l, Y12, V15, 135, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, L300.
  • each of the amino acid mutations is an amino acid substitution.
  • the from about 5 to about 20 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO:297 set forth in Table 5.
  • the from about 5 to about 20 amino acid substitutions of SEQ ID NO:297 are selected from the group consisting of one of 110 A and 110Q, RUT, Y12K, one of VI 5H and V 15K, 135 T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G, L120T, Y127L, K129G, R157G, S159G, L160W, one of V230G, V230R and V230S, N233S, K250S, A261K, F269N, S274D, one of A275F, A275S, and A275W, one of D288H and D288K, Q293K, V294A, L300T, and combinations thereof.
  • the N142 variant comprises from about 5 to about 16 amino acid mutations relative to the wild type N142 enzyme of SEQ ID NO:297. In a further embodiment, the from about 5 to about 16 amino acid mutations are from about 5 to about 16 amino acid substitutions relative to the WT N142 enzyme of SEQ ID NO:297.
  • the N142 variant comprises from about 5 to about 16 amino acid mutations relative to the WT N142 enzyme of SEQ ID NO:297, wherein the amino acid mutations include from about 5 to about 16 amino acid mutations at the following amino acid residues of SEQ ID NO:297: 110, R11, Y12, V15, 135, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, K129, R157, S159, L160, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300.
  • the amino acid mutations include from about 5 to about 16 amino acid mutations at the following ammo acid residues of SEQ ID NO:297: 110, Rl l, Y12, V15, 135, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, L300.
  • each of the amino acid mutations is an amino acid substitution.
  • the from about 5 to about 16 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO:297 set forth in Table 5.
  • the from about 5 to about 16 amino acid substitutions of SEQ ID NO:297 are selected from the group consisting of one of I10A and I10Q, RUT, Y12K, one of V15H and V15K, I35T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G, L120T, Y127L, K129G, R157G, S159G, L160W, one of V230G, V230R and V230S, N233S, K250S, A261K, F269N, S274D, one of A275F, A275S, and A275W, one of D288H and D288K, Q293K, V294A, L300T, and combinations thereof.
  • the N142 variant comprises from about 10 to about 20 amino acid mutations relative to the WT N142 enzyme comprising the amino acid sequence of SEQ ID NO:297.
  • the from about 10 to about 20 amino acid mutations are from about 10 to about 20 amino acid substitutions relative to the WT N142 enzyme of SEQ ID NO:297.
  • the N142 variant comprises from about 10 to about 20 amino acid mutations relative to the WT N142 enzyme of SEQ ID NO:297, wherein the amino acid mutations include from about 10 to about 20 amino acid mutations at the following amino acid residues of SEQ ID NO:297: 110, Rl l, Y12, VI 5, 135, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, K129, R157, S159, L160, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300.
  • the amino acid mutations include from about 10 to about 20 amino acid mutations at the following amino acid residues of SEQ ID NO:297: 110, Rl l, Y12, V15, 135, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, L300.
  • each of the amino acid mutations is an amino acid substitution.
  • the from about 10 to about 20 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO:297 set forth in Table 5.
  • the from about 10 to about 20 amino acid substitutions of SEQ ID NO: 297 are selected from the group consisting of one of I10A and I10Q, RUT, Y12K, one of V15H and VI 5K, 135T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G, L120T, Y127L, K129G, R157G, S159G, L160W, one of V230G, V230R and V230S, N233S, K250S, A261K, F269N, S274D, one of A275F, A275S, and A275W, one of D288H and D288K, Q293K, V294A, L300T, and combinations thereof.
  • the N142 variant comprises from about 12 to about 18 amino acid mutations relative to the WT N142 enzyme of SEQ ID NO:297. In a further embodiment, the from about 12 to about 18 amino acid mutations are from about 12 to about 18 amino acid substitutions relative to the WT N142 enzyme of SEQ ID NO:297.
  • the N142 variant comprises from about 12 to about 18 amino acid mutations at the following amino acid residues of SEQ ID NO:297: 110, Rl l, Y12, VI 5, 135, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, KI 29, R157, SI 59, LI 60, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300.
  • the amino acid mutations comprise from about 12 to about 18 amino acid mutations at the following amino acid residues of SEQ ID NO:297: 110, Rl l, Y12, V15, 135, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, L300.
  • each of the amino acid mutations is an amino acid substitution.
  • the from about 12 to about 18 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO:297 set forth in Table 5.
  • the from about 12 to about 18 amino acid substitutions of SEQ ID NO:297 are selected from the group consisting of one of I10A and I10Q, RUT, Y12K, one of V15H and VI 5K, 135T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G, L120T, Y127L, K129G, R157G, S159G, L160W, one of V230G, V230R and V230S, N233S, K250S, A261K, F269N, S274D, one of A275F, A275S, and A275W, one of D288H and D288K, Q293K, V294A, L300T, and combinations thereof
  • the N142 variant comprises from about 15 to about 18 amino acid mutations relative to the WT N142 enzyme of SEQ ID NO:297. In a further embodiment, the from about 15 to about 18 amino acid mutations are from about 15 to about 18 amino acid substitutions relative to the WT N142 enzyme of SEQ ID NO:297.
  • the N142 variant comprises from about 15 to about 18 amino acid mutations at the following amino acid residues of SEQ ID NO:297: 110, Rl l, Y12, VI 5, 135, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, KI 29, R157, SI 59, LI 60, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300.
  • the amino acid mutations comprise from about 15 to about 18 amino acid mutations at the following amino acid residues of SEQ ID NO:297: 110, Rl l, Y12, V15, 135, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, L300.
  • each of the amino acid mutations is an amino acid substitution.
  • the from about 15 to about 18 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO:297 set forth in Table 5.
  • the from about 15 to about 18 amino acid substitutions of SEQ ID NO:297 are selected from the group consisting of one of I10A and I10Q, RUT, Y12K, one of V15H and VI 5K, 135T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G, L120T, Y127L, K129G, R157G, S159G, L160W, one of V230G, V230R and V230S, N233S, K250S, A261K, F269N, S274D, one of A275F, A275S, and A275W, one of D288H and D288K, Q293K, V294A, L300T, and combinations thereof
  • the N142 variant disclosed herein does not have one or more amino acid substitutions of SEQ ID NO:297 selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, T57L, D84E, R88N, E91K, E92K, H93Y, M106L, N117D, H118S, E126D, N162D, E170R, N197Y, F199L, N203T, K213Q, L223I, N233G, S245A, A261P, K286E, and S306I.
  • the N142 variant comprises one or more amino acid substitutions of SEQ ID NO:297 selected from the group consisting of: I10A, V15H, I35T, V46D, V46S, N48H, Y52F, T57K, N102G, L120T, Y127L, V230G, V230R, V230S, K250S, A261K, S274D, A275F, D288H, and combinations thereof.
  • the one or more amino acid substitutions in the N142 variant in a further embodiment, comprises from about five to about sixteen, from about 8 to about 16, from about 10 to about 16, or from about 12 to about 16, of the aforementioned amino acid substitutions.
  • the N142 variant comprises one of the groups of mutations set forth in Table 6.
  • the N142 variant comprises one or more amino acid substitutions of SEQ ID NO:297 selected from the group consisting of: I10A, V15H, I35T, V46D, V46S, N48H, Y52F, T57K, N102G, L120T, Y127L, V230G, V230R, V230S, K250S, A261K, S274D, A275F, D288H, and combinations thereof.
  • the one or more amino acid substitutions in the N142 variant in one embodiment, comprise from about five to about twenty, from about 10 to about 20, from about 12 to about 20, or from about 15 to about 20 amino acid substitutions at the one or more amino acid positions.
  • the N142 variant comprises one of the groups of mutations set forth in Table 7.
  • the N142 variant comprises one of the sets of mutations set forth in Table 6.
  • the N142 variant comprises one of the sets of mutations set forth in Table 7. [0241] In one embodiment, the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: 110A, V15H, I35T, Y52F (Table 7, Nl).
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: 110A, V15H, I35T, V46S (Table 7, N2).
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: 110A, V15H, I35T, V46D (Table 7, N3).
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: 110A, V15H, I35T, V46D, and T57K (Table 7, N4).
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: 110A, V15H, I35T, Y52F, and T57K (Table 7, N5).
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: 110A, V15H, I35T, Y52F, and N102G (Table 7, N6).
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: L120T, V230R, A261K, S274D, and A275F (Table 7, N7).
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: V230S, A261K, S274D, A275F, and D288H (Table 7, N8).
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: V230G, A261K, S274D, A275F, and D288H (Table 7, N9).
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: I10A, V15H, V230S, A261K, S274D (Table 7, N10).
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: I10A, V15H, 135T, V46D, T57K, L120T, V230R, A261K, S274D, A275F, and D288H (Table 7, Ni l).
  • an N 142 variant comprising (i) the amino acid sequence set forth in SEQ ID NO: 303, or (ii) an amino acid sequence that is at least about 90% identical or 95% identical, to SEQ ID NO:303.
  • the N142 variant comprises the amino acid sequence set forth in SEQ ID NO:303.
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: I10A, V15H, I35T, V46S, T57K, L120T, V230R, A261K, S274D, A275F, and D288H (Table 7, N12).
  • an N142 variant comprising (i) the amino acid sequence set forth in SEQ ID NO: 304, or (ii) an amino acid sequence that is at least about 90% identical or 95% identical, to SEQ ID NO:304.
  • the N142 variant comprises the amino acid sequence set forth in SEQ ID NO:304.
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: I10A, V15H, I35T, N48H, Y52F, T57K, L120T, V230R, A261K, S274D, A275F, and D288H (Table 7, N13).
  • an N142 variant comprising (i) the amino acid sequence set forth in SEQ ID NO: 305, or (ii) an amino acid sequence that is at least about 90% identical or 95% identical, to SEQ ID NO:305.
  • the N142 variant comprises the amino acid sequence set forth in SEQ ID NO:305.
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: I10A, V15H, I35T, Y52F, T57K, L120T, V230R, A261K, S274D, A275F, and D288H (Table 7, N14).
  • an N 142 variant comprising (i) the amino acid sequence set forth in SEQ ID NO: 306, or (ii) an amino acid sequence that is at least about 90% identical or 95% identical, to SEQ ID NO:306.
  • the N142 variant comprises the amino acid sequence set forth in SEQ ID NO:306.
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: I10A, V15H, I35T, Y52F, T57K, L120T, V230R, K250S, A261K, S274D, A275F, and D288H (Table 7, N15).
  • an N142 variant comprising (i) the amino acid sequence set forth in SEQ ID NO: 307, or (ii) an amino acid sequence that is at least about 90% identical or 95% identical, to SEQ ID NO:307.
  • the N142 variant comprises the amino acid sequence set forth in SEQ ID NO:307.
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: I10A, V15H, I35T, Y52F, T57K, L120T, V230S, A261K, S274D, A275F, and D288H (Table 7, N16).
  • an N 142 variant comprising (i) the amino acid sequence set forth in SEQ ID NO: 308, or (ii) an amino acid sequence that is at least about 90% identical or 95% identical, to SEQ ID NO:308.
  • the N142 variant comprises the amino acid sequence set forth in SEQ ID NO:308.
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: 110A, V15H, I35T, Y52F, N102G, L120T, V230G, A261K, S274D, A275F, and D288H (Table 7, N17).
  • an N142 variant comprising (i) the amino acid sequence set forth in SEQ ID NO: 309, or (ii) an amino acid sequence that is at least about 90% identical or 95% identical, to SEQ ID NO:309.
  • the N142 variant comprises the amino acid sequence set forth in SEQ ID NO:309.
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: 110A, V15H, I35T, Y52F, Y127L, V230G, A261K, S274D, A275F, and D288H (Table 7, N18).
  • an N142 variant comprising (i) the amino acid sequence set forth in SEQ ID NO: 310, or (ii) an amino acid sequence that is at least about 90% identical or 95% identical, to SEQ ID NO:310.
  • the N142 variant comprises the amino acid sequence set forth in SEQ ID NO:310.
  • an N142 variant comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOS:303-310.
  • the N142 variant comprises the amino acid substitutions present in the reference amino acid sequence relative to the amino acid sequence of SEQ ID NO:297.
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: I10A, V15H, I35T, N48H, Y52S, T57K, N102G, L120T, S159G, V230R, K250S, A261K, S274D, A275F, D288H, and V294A.
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: I10A, V15H, I35T, N48H, T57K, N102G, L120T, S159G, L160W, V230R, K250S, A261K, S274D, A275F, D288H, and L300T.
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: I10Q, RUT, Y12K, V15K, N48G, Y52S, T57K, L120T, R157G, V230S, K250S, A261K, F269N, A275S, D288K, and Q293K.
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: I10Q, RUT, Y12K, V15K, N48G, T57K, L120T, K129G, R157G, V230S, N233S, K250S, A261K, F269N, A275F, D288K, and Q293K.
  • the N142 variant comprises the following amino acid mutations of SEQ ID NO:297: I10Q, RUT, Y12K, V15K, N48G, T57K, L120T, K129G, R157G, V230S, N233S, K250S, A261K, F269N, A275W, D288K, and Q293K.
  • the N142 variant comprises an amino acid sequence set forth in one of SEQ ID NOS:358-430.
  • the N142 variant comprises an amino acid sequence set forth in one of SEQ ID NOS:431-448.
  • the N142 variant comprises an amino acid sequence set forth in one of SEQ ID NOS:449-485.
  • the N142 variant comprises an amino acid sequence set forth in one of SEQ ID NOS:486-489.
  • the N142 variant comprises an amino acid sequence set forth in one of SEQ ID NOS:490-596.
  • the N142 variant comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOS:358-596, and the N142 variant comprises the amino acid substitutions present in the reference amino acid sequence relative to the amino acid sequence of SEQ ID NO:297.
  • the N142 variant comprises an amino acid sequence set forth in one of SEQ ID NOS: 847- 1005.
  • the N 142 variant comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOS: 847- 1005, and the N142 variant comprises the amino acid substitutions present in the reference amino acid sequence relative to the amino acid sequence of SEQ ID NO:297.
  • the N142 variant comprises an amino acid sequence selected from one of SEQ ID NOS: 859, 868, 925, 936, and 942.
  • the N142 variant comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOS:859, 868, 925, 936, and 942, and the N142 variant comprises the amino acid substitutions present in the reference amino acid sequence relative to the amino acid sequence of SEQ ID NO: 297.
  • the N142 variant is at least 25% as active as an IdeS enzyme comprising an amino acid sequence set forth in SEQ ID NO:2 in an IgG protease enzymatic assay. In some embodiments, the N142 variant is at least 30% as active as an IdeS enzyme comprising an amino acid sequence set forth in SEQ ID NO:2 in an IgG protease enzymatic assay.
  • the N142 variant is at least about 40% as active, at least about 50% as active, at least about 60% as active, at least about 70% as active, at least about 75% as active, at least about 80% as active, at least about 90% as active, at least about 100% as active, at least about 110% as active, at least about 120% as active, as an IdeS enzyme comprising the amino acid sequence of SEQ ID NO:2 in an IgG protease enzymatic assay.
  • the N142 variant is at least about 40% as active, at least about 50% as active, at least about 60% as active, at least about 70% as active, at least about 80% as active, at least about 90% as active, at least about 100% as active, at least about 110% as active, at least about 120% as active, as an IdeZ enzyme comprising the amino acid sequence of SEQ ID NO:830 in an IgG protease enzymatic assay.
  • the IgG protease activity in the IgG protease enzymatic assay is measured via an enzyme linked immunosorbent assay comprising digestion of an immobilized antibody substrate.
  • the N142 variant has one or more T cell epitopes depleted, e.g., two or more, three or more, or four or more T cell epitopes depleted, compared to the polypeptide of the amino acid sequence of SEQ ID NO: 297.
  • the N142 variant has substantially the same thermostability as an IdeS enzyme comprising the amino acid sequence set forth in SEQ ID NO:2. In other embodiments, the N142 variant is more thermostable than an IdeS enzyme comprising the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, thermostability is measured by differential scanning fluorimetry.
  • the N142 variant has substantially the same thermostability as an IdeZ enzyme comprising the amino acid sequence set forth in SEQ ID NO: 830. In other embodiments, the N142 variant is more thermostable than an IdeZ enzyme comprising the amino acid sequence set forth in SEQ ID NO: 830. In some embodiments, thermostability is measured by differential scanning fluorimetry.
  • the N142 variant provided herein has an amino acid sequence that is at least about 75% identical to SEQ ID NO:297. In some embodiments, the N142 variant has an amino acid sequence that is at least about 80% identical to SEQ ID NO:297. In some embodiments, the N142 variant has an amino acid sequence that is at least about 85% identical to SEQ ID NO:297. In some embodiments, the N142 variant has an amino acid sequence that is at least about 90% identical to SEQ ID NO:297. In some embodiments, the N142 variant has an amino acid sequence that is at least about 95% identical to SEQ ID NO:297. In some embodiments, the N142 variant has an amino acid sequence that is at least about 97% identical to SEQ ID NO:297. In some embodiments, the N142 variant has an amino acid sequence that is at least about 98% identical to SEQ ID NO:297.
  • the present disclosure provides an IgG protease variant of one of SEQ ID NOS:27-302.
  • the IgG protease variant in one embodiment, has an amino acid sequence that is at least about 75% identical to the amino acid sequence of the counterpart WT enzyme selected from one of SEQ ID NOS: 27-302 from which the IgG protease variant is derived.
  • the amino acid sequence of the IgG protease variant is at least about 80% identical to the amino acid sequence of the counterpart WT enzyme, or at least about 85% identical to the amino acid sequence of the counterpart WT enzyme, or at least about 90% identical to the amino acid sequence of the counterpart WT enzyme, or at least about 95% identical to the amino acid sequence of the counterpart WT enzyme, at least about 97% identical, or at least about 98% identical to the amino acid sequence of the counterpart WT enzyme.
  • the IgG protease variant is T cell epitope depleted, as compared to the counterpart WT enzyme.
  • the variant has one or more dominant immunogenic T cell epitopes eliminated, as compared to the immunogenic T cell epitopes present in the counterpart WT enzyme.
  • the IgG protease variant is a variant of an enzyme comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 36, 37, 39, 41, 50, 51, 54, 56, 60, 61, 65, 67, 137, 140, 144, 150, 159, 285, 286, 287, 288, 289, 292, 293, 294, 295, 296, 297 and 298.
  • the IgG protease variant is a variant of an amino acid sequence selected from the group consisting of SEQ ID NO: 54, 140, 293, 294, 295, 297, and 298.
  • the IgG protease variant comprises an amino acid sequence set forth in one of SEQ ID NOS: 318-829.
  • the IgG protease variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90% at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence set forth in one of SEQ ID NOS: 318-829, and the IgG protease variant comprises the amino acid substitutions present in the reference amino acid sequence relative to the reference enzyme’s counterpart enzyme.
  • the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 293 [N123].
  • the IgG protease variant comprises (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 293: Y12, L31, E34, F36, R37, Y38, N39, 144, A60, D133, N162, F274, A279, H280, V299, and (n) an ammo acid sequence that is at least about 80% identical to SEQ ID NO: 293.
  • the IgG protease variant (iii) is at least about 25% as active as IdeS in an IgG protease enzymatic assay. In a further embodiment, the IgG protease variant is at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% as active as IdeS in an IgG protease enzymatic assay. In even a further embodiment, the variant has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95% or at least about 98% identical to SEQ ID NO:293.
  • the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 298 [N30],
  • the variant comprises (i) one or more amino acid mutations at one or more ammo acid positions of SEQ ID NO: 298: 110, R11, Y12, V15, Y18, V46, Y52, F101, N102, Q105, L120, F125, E126, Y127, Y156, R157, S159, V230, R231, N233, A261, F269, V270, A275, F269, (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 298.
  • the IgG protease variant (iii) is at least about 25% as active as IdeS in an IgG protease enzymatic assay. In a further embodiment, the IgG protease variant is at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% as active as IdeS in an IgG protease enzymatic assay. In even a further embodiment, the variant has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95% or at least about 98% identical to SEQ ID NO:298.
  • the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 294 [N31],
  • the IgG protease variant comprises (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 294: 110, Rl l, Y12, E14, V15, Y18, V46, N48, Y52, D130, A132, Y157, N159, Y161, K162, V232, R233, N235, D260, A263, V272, S276, T1 , (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 294.
  • the IgG protease variant (iii) is at least about 25% as active as IdeS in an IgG protease enzymatic assay. In a further embodiment, the IgG protease variant is at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% as active as IdeS in an IgG protease enzymatic assay. In even a further embodiment, the variant has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95% or at least about 98% identical to SEQ ID NO:294.
  • the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 54.
  • the IgG protease variant comprises (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 54: 110, Il l, Y12, E14, Y18, H19, VI 43, Y157, Y161, KI 62, (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 54.
  • the IgG protease variant (iii) is at least about 25% as active as IdeS in an IgG protease enzymatic assay.
  • the IgG protease variant is at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% as active as IdeS in an IgG protease enzymatic assay.
  • the variant has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95% or at least about 98% identical to SEQ ID NO: 54.
  • the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 140.
  • the IgG protease variant comprises (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 140: M10, Rl l, Y12, V15, Y42, V46, A47, N48, Q49, N102, V104, 1113, Y118, L120, D121, K123, Y127, Y156, R157, Y201, Q202, V230, F269, S274, A275, D288, G296, (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 140.
  • the IgG protease variant (iii) is at least about 25% as active as IdeS in an IgG protease enzymatic assay. In a further embodiment, the IgG protease variant is at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% as active as IdeS in an IgG protease enzymatic assay. In even a further embodiment, the IgG protease variant has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95% or at least about 98% identical to SEQ ID NO: 140.
  • the present disclosure provides a variant of the N144 IgG protease.
  • the N144 IgG protease as provided above, comprises an amino acid sequence set forth in SEQ ID NO:295.
  • the N144 variant comprises (i) one or more amino acid mutations at one or more ammo acid positions of SEQ ID NO:295: Y12, SI 8, 135, 146, A47, Y52, 154, T55, T57, N59, H93, F101, N102, E104, Q105, L120, F125, E126, Y127, T135, Y156, S159, L175, VI 83, R200, N201, N205, 1207, V230, R231, N233, A241, N244, E246, A261, Y269, V270, S274, A275, 1280, S281, A282, 1285, D288, V290, and (ii) an amino acid sequence that is at least about 75% identical to SEQ ID NO: 295.
  • the N144 variant does not have amino acid mutations at one or more of the following ammo acid positions of SEQ ID NO:295: A32, N33, Q36, F41, D84, E91, Ml 06, Hl 18, El 70, SI 95, N197, N203, S245, K286.
  • the mutation(s) in the N144 variant at one or more of the aforementioned amino acid positions eliminates one or more dominant immunogenic T cell epitopes present in the wild type N144, such that the N144 variant is T cell epitope depleted compared to the wild type N144.
  • Amino acid mutations of the present disclosure include, but are not limited to, amino acid exchange(s), insertion(s), deletion(s), addition(s), substitution(s), inversion(s) and/or duplicati on(s). These mutations/modification(s) also include conservative and/or homologous amino acid exchange(s).
  • the one or more mutations in the N144 variant are one or more amino acid substitutions.
  • the one or more amino acid substitutions of SEQ ID NO:295 in the N144 variant are selected from the group consisting of Y12N, S18K, one of I35E, 135K, and I35S, I46D, A47E, I54T, one of T55K, T55H, and T55R, T57L, N59D, H93Y, N102G, E104R, one of L120T and L120S, F125W, T135H, S159K, one of L175A, L175N, and L175S, V183W, one of R200D, R200G and R200T, N201E, N205D, I207T, one of V230H and V230Q, R231W, N233G, A241G, one of E246G and E246K, one of A261K, A261I, A261F, A261G, A261Q, and A261S, one of Y269D,
  • the N144 variant comprises two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid mutations, relative to the WT N144 comprising the amino acid sequence of SEQ ID NO:295, and the mutations are selected from mutations at the following amino acid positions of SEQ ID NO: 295: Y12, S18, 135, 146, A47, Y52, 154, T55, T57, N59, H93, F101, N102, E104, Q105, L120, F125, E126, Y127, T135, Y156, S159, L175, V183, R200, N201, N205, 1207, V230, R231, N233, A241, N244, E246, A261, Y269, V270, S274, A275, 1280, S281, A282, 1285, D
  • the two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid mutations are two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid substitutions.
  • amino acid substitutions of SEQ ID NO:295 are selected from the group consisting of selected from the group consisting of Y12N, S18K, one of I35E, I35K, and I35S, I46D, A47E, I54T, one of T55K, T55H, and T55R, T57L, N59D, H93Y, N102G, E104R, one of L120T and L120S, F125W, T135H, S159K, one of L175A, L175N, and L175S, V183W, one of R200D, R200G and R200T, N201E, N205D, I207T, one of V230H and V230Q, R231W, N233G, A241G, one of E246G and E246K, one of A261K, A261I, A261F, A261G, A261Q, and A261S, one of Y269D, Y
  • the N144 variant comprises from about 10 to about 30 amino acid mutations, from about 10 to about 25 amino acid mutations, from about 12 to about 24 amino acid mutations, from about 12 to about 20 amino acid mutations, from about 12 to about 16 amino acid mutations, from about 21 to about 24 amino acid mutations, or 12, 13, 14, 21, 22, 23, or 24 amino acid mutations relative to the WT N144 comprising the amino acid sequence of SEQ ID NO:295, and the mutations are selected from mutations at the following amino acid positions of SEQ ID NO:295: Y12, S18, 135, 146, A47, Y52, 154, T55, T57, N59, H93, F101, N102, E104, Q105, L120, F125, E126, Y127, T135, Y156, S159, L175, V183, R200, N201, N205, 1207, V230, R231, N233, A241, N244, E246, A261, Y269, V270, S27
  • each of the amino acid mutations is an amino acid substitution.
  • the amino acid substitutions of SEQ ID NO:295 are selected from the group consisting of Y12N, S18K, one of I35E, 135K, and I35S, I46D, A47E, I54T, one of T55K, T55H, and T55R, T57L, N59D, H93Y, N102G, E104R, one of L120T and L120S, F125W, T135H, S159K, one of L175A, L175N, and L175S, V183W, one of R200D, R200G and R200T, N201E, N205D, I207T, one of V230H and V230Q, R231W, N233G, A241G, one of E246G and E246K, one of A261K, A261I, A261F, A261G, A261Q, and A261
  • the N144 variant does not have one or more amino acid substitutions of SEQ ID NO:295 selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, F199L, N203T, K213Q, L223I, S245A, N246E, K250E, K286E, and S306I.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I46D, A47E, T55H, H93Y, E104R, L120T, F125W, T135H, S159K, and A275S.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I46D, A47E, T55H, H93Y, E104R, L120T, F125W, T135H, S159K, A261I, and A275S.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261F, and A275S.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261G, and A275S.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261Q, and A275S.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261S, and A275S.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I46D, A47E, T55R, H93Y, E104R, L120S, F125W, T135H, S159K, A261K, and A275S.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I46D, A47E, T55R, H93Y, E104R, L120T, F125W, T135H, S159K, A261K, and A275S.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, and A275S.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N201E, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261Q, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135E, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, A261S, Y269W, A275W, A282G, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135E, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, A261S, Y269W, A275W, A282G, and D288T.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135E, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N201E, N233G, E246K, A261Q, Y269W, A275W, A282G, and D288T.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35E, I46D, A47E, T55R, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, A261K, Y269W, A275W, A282G, and D288T.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35E, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N201E, R231W, E246G, Y269W, A275W, A282G, and D288T.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35E, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, LI 75 A, N205D, N233G, E246K, Y269W, A275W, A282G, and D288T.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135K, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135K, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261S, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135K, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246K, A261S, Y269W, S274G, A275S, A282G, and D288K.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, R200G, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N201E, N233G, E246G, V270T, A275W, I280M, and
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N201E, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246G, A261K, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, R200T, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, 135K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175S, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, LI 75 A, N205D, V230H, E246K, Y269W, A275W, A282G, and D288T.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, V183W, N205D, N233G, E246K, Y269W, S274D, A275S, A282G, and D288T.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, V183W, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, V230H, E246K, A261K, Y269W, S274G, A275S, A282G, and D288T.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, V230Q, E246G, A261K, Y269W, S274G, A275S, A282G, and D288T.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246K, A261K, Y269W, S274G, A275S, A282G, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, Y269W, S274G, A275S, A282G, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, Y269W, A275W, A282G, and D288T.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246G, Y269W, S274G, A275S, A282G, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, Y269W, S274G, A275S, A282G, and D288K.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261K, Y269W, S274D, A275S, A282G, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261K, Y269W, S274D, A275S, A282G, and D288K.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175S, N205D, N233G, E246G, Y269W, A275W, A282G, and D288T.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35S, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35S, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35S, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261Q, V270T, A275W, I280M, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35S, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, V230Q, A241G, Y269W, A275W, A282G, and V290F.
  • the N144 variant comprises the following amino acid substitutions of SEQ ID NO:295: Y12N, S18K, I35S, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, Y269W, A275W, A282G, and V290F.
  • the N144 variant comprises an amino acid sequence set forth in one of SEQ ID NOS: 597-657.
  • the N144 variant comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOS: 597-657, and the N144 variant comprises the amino acid substitutions present in the reference amino acid sequence relative to the amino acid sequence of SEQ ID NO: 295.
  • the N144 variant comprises an amino acid sequence set forth in one of SEQ ID NOS:1006-1490 and 1952-2590.
  • the N144 variant comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOS: 1006-1490 and 1952-2590, and the N144 variant comprises the amino acid substitutions present in the reference amino acid sequence relative to the amino acid sequence of SEQ ID NO:295.
  • the N144 variant comprises an amino acid sequence selected from one of SEQ ID NOS: 1118, 1120, 1122, 1123, 1125, 1126, 1127, 1129, 1282, 1962, 1963, 1964, 1965, 1970, 1971, 1983, 2004, 2024, 2028, 2371, 2372, 2373, 2390, 2421, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2437, 2439, 2446, 2450, 2463, 2464, 2476, 2478, 2485, 2486, 2487, 2489, 2491,
  • the N144 variant comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOS: 1118, 1120, 1122, 1123, 1125, 1126, 1127, 1129, 1282, 1962, 1963, 1964, 1965, 1970, 1971, 1983, 2004, 2024, 2028, 2371, 2372, 2373, 2390, 2421, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2437, 2439, 2446, 2450, 2463, 2464, 2476, 2478, 2485, 2486, 2487, 2489, 2491, 2493, 2494, 2511, 2524, 2526, 2528, 2574, and 2575, and the N144 variant comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 1118, 1120, 1122, 1123, 11
  • the N144 variant comprises an amino acid sequence selected from one of SEQ ID NOS: 1118, 1120, 1122, 1123, 1125, 1126, 1127, 1129, 1282, 1970, 1971, 1983, 2004, 2024, 2028, 2390, 2421, 2446, 2450, 2463, 2464, 2476, 2478, 2485, 2486, 2487, 2489, 2491, 2493,
  • the N144 variant comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOS:1118, 1120, 1122, 1123, 1125, 1126, 1127, 1129, 1282, 1970, 1971, 1983, 2004, 2024, 2028, 2390, 2421, 2446, 2450, 2463, 2464, 2476, 2478, 2485, 2486, 2487, 2489, 2491, 2493, 2494, 2511, 2574, and 2575, and the N144 variant comprises the amino acid substitutions present in the reference amino acid sequence relative to the amino acid sequence of SEQ ID NO:295.
  • the N144 variant comprises a first set of amino acid substitutions of SEQ ID NO:295 and a second set of amino acid substitutions of SEQ ID NO:295.
  • the first set of amino acid substitutions of SEQ ID NO:295 consists of Y12N, S18K, I46D, A47E, H93Y, E104R, F125W, T135H, and S159K.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 3 to about 15 amino acid substitutions selected from the group consisting of one of I35K, I35E, and I35S, I54T, one of T55K, T55R, and T55H, T57L, N59D, N102G, one of L120T and L120S, one of L175N, L175A, and L175S, V183W, one of R200Gand R200T, N201E, N205D, one of V230H and V230Q, R231W, N233G, A241G, one of E246K and E246G, one of A261K, A261Q, A261S, A261I, A261F, and A261G, Y269W, V270T, one of S274G and S274D, one of A275W and A275S, I280M, A282G, one of D288T and D288
  • the N144 variant comprising the first set and second set of amino acid substitutions of SEQ ID NO:295 does not have amino acid mutations at one or more of the following ammo acid positions of SEQ ID NO:295: A32, N33, Q36, F41, D84, E91, Ml 06, Hl 18, E170, S195, N197, N203, S245, K286.
  • the N144 variant does not have one or more amino acid substitutions of SEQ ID NO: 295 selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, F199L, N203T, K213Q, L223I, S245A, N246E, K250E, K286E, and S306I.
  • the N144 variant comprising the first set and second set of amino acid substitutions of SEQ ID NO:295 has an amino acid sequence that is at least about 90% identical to SEQ ID NO:295. In another embodiment, the N144 variant has an amino acid sequence that is at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or at least about 96% identical to SEQ ID NO: 295. In another embodiment, the N144 variant has an amino acid sequence that is from about 90% to about 96%, or from about 92% to about 96% identical to SEQ ID NO:295. In one embodiment, the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 3 to about 5 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 12 to about 15 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 5 to about 12 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 5 to about 15 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 3 to about 15 amino acid substitutions selected from the group consisting of one of I35K and 135E, I54T, T55K, T57L, N59D, N102G, L120T, L175N, N205D, R231W, N233G, one of E246K and E246G, one of A261K and A261Q, Y269W, V270T, one of A275W and A275S, I280M, A282G, D288T, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 3 to about 5 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 12 to about 15 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 5 to about 12 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 5 to about 15 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 3 to about 15 amino acid substitutions selected from the group consisting of I35K, I54T, T55K, T57L, N59D, N102G, L120T, L175N, N205D, N233G, E246K, Y269W, V270T, one of A275W and A275S, I280M, A282G, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 3 to about 5 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 12 to about 15 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 5 to about 12 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 5 to about 15 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 3 to about 13 amino acid substitutions selected from the group consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 3 to about 5 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 12 to about 13 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of from about 5 to about 13 amino acid substitutions.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 3 amino acid substitutions consisting of T55H, L120T, and A275S.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 4 amino acid substitutions consisting of T55H, L120T, A261I, and A275S.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 4 amino acid substitutions consisting of T55K, L120T, A261F, and A275S.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 4 amino acid substitutions consisting of T55K, L120T, A261G, and A275S.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 4 amino acid substitutions consisting of T55K, L120T, A261Q, and A275S.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 4 amino acid substitutions consisting of T55K, L120T, A261S, and A275S.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 4 amino acid substitutions consisting of T55R, L120S, A261K, and A275S.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 4 amino acid substitutions consisting of T55R, L120T, A261K, and A275S.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 5 amino acid substitutions consisting of I54T, T57L, N59D, L120T, and A275S.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35E, T55K, N102G, L120T, L175N, N201E, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35E, T55K, N102G, L120T, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35E, T55K, N102G, L120T, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting ofI35E, T55K, N102G, L120T, L175N, N205D, R231W, E246K, A261Q, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 12 amino acid substitutions consisting of I35E, T55K, L120T, L175A, N205D, N233G, E246K, A261S, Y269W, A275W, A282G, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 12 amino acid substitutions consisting of I35E, T55K, L120T, L175A, N205D, N233G, E246K, A261S, Y269W, A275W, A282G, and D288T.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 12 amino acid substitutions consisting of I35E, T55K, L120T, L175N, N201E, N233G, E246K, A261Q, Y269W, A275W, A282G, and D288T.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 12 amino acid substitutions consisting of I35E, T55R, L120T, L175A, N205D, N233G, E246K, A261K, Y269W, A275W, A282G, and D288T.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35E, I54T, T57L, N59D, L120T, L175A, N201E, R231W, E246G, Y269W, A275W, A282G, and D288T.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35E, I54T, T57L, N59D, L120T, L175A, N205D, N233G, E246K, Y269W, A275W, A282G, and D288T.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35K, T55K, N102G, L120T, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35K, T55K, N102G, L120T, L175N, N205D, N233G, E246G, A261S, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35K, T55K, N102G, L120T, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35K, T55K, L120T, L175N, N205D, N233G, E246K, A261S, Y269W, S274G, A275S, A282G, and D288K.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175A, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, R200G, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N201E, N233G, E246G, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N201E, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N205D, N233G, A241G, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N205D, R231W, E246G, A261K, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N205D, R231W, E246K, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N205D, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, R200T, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175S, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175A, N205D, V230H, E246K, Y269W, A275W, A282G, and D288T.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175A, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, V183W, N205D, N233G, E246K, Y269W, S274D, A275S, A282G, and D288T.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, V183W, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, V230H, E246K, A261K, Y269W, S274G, A275S, A282G, and D288T.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, V230Q, E246G, A261K, Y269W, S274G, A275S, A282G, and D288T.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, N233G, E246K, A261K, Y269W, S274G, A275S, A282G, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, N233G, A241G, Y269W, S274G, A275S, A282G, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, N233G, A241G, Y269W, A275W, A282G, and D288T.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, R231W, E246G, Y269W, S274G, A275S, A282G, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, R231W, E246K, Y269W, S274G, A275S, A282G, and D288K.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, R231W, E246K, A261K, Y269W, S274D, A275S, A282G, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, R231W, E246K, A261K, Y269W, S274D, A275S, A282G, and D288K.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175S, N205D, N233G, E246G, Y269W, A275W, A282G, and D288T.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35S, T55K, N102G, L120T, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35S, T55K, N102G, L120T, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35S, T55K, N102G, L120T, L175N, N205D, R231W, E246K, A261Q, V270T, A275W, I280M, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35S, I54T, T57L, N59D, L120T, L175N, N205D, V230Q, A241G, Y269W, A275W, A282G, and V290F.
  • the second set of amino acid substitutions of SEQ ID NO:295 is a set of 13 amino acid substitutions consisting of I35S, I54T, T57L, N59D, L120T, L175N, N205D, R231W, E246K, Y269W, A275W, A282G, and V290F.
  • the IgG protease variant of the disclosure e.g., a N144 variant of the disclosure, is at least 25% as active as an IdeS enzyme comprising an amino acid sequence set forth in SEQ ID NO:2 in an IgG protease enzymatic assay. In some embodiments, the IgG protease variant of the disclosure, e.g., a N144 variant of the disclosure, is at least 30% as active as an IdeS enzyme comprising an amino acid sequence set forth in SEQ ID NO:2 in an IgG protease enzymatic assay.
  • the IgG protease variant of the disclosure is at least about 40% as active, at least about 50% as active, at least about 60% as active, at least about 70% as active, at least about 75% as active, at least about 80% as active, at least about 90% as active, at least about 100% as active, at least about 110% as active, at least about 120% as active, as an IdeS enzyme comprising the amino acid sequence of SEQ ID NO:2 in an IgG protease enzymatic assay.
  • the IgG protease variant of the disclosure is at least about 25% as active, at least about 40% as active, at least about 50% as active, at least about 60% as active, at least about 70% as active, at least about 80% as active, at least about 90% as active, at least about 100% as active, at least about 110% as active, at least about 120% as active, as an IdeZ enzyme comprising the amino acid sequence of SEQ ID NO: 830 in an IgG protease enzymatic assay.
  • the IgG protease activity in the IgG protease enzymatic assay is measured via an enzyme linked immunosorbent assay comprising digestion of an immobilized antibody substrate.
  • the N144 variant has one or more T cell epitopes depleted, e.g., two or more, three or more, or four or more T cell epitopes depleted, compared to the polypeptide of the amino acid sequence of SEQ ID NO:295.
  • the IgG protease variant of the disclosure e.g., a N144 variant of the disclosure, has substantially the same thermostability as an IdeS enzyme comprising the amino acid sequence set forth in SEQ ID NO:2.
  • the IgG protease variant of the disclosure e.g., a N144 variant of the disclosure, is more thermostable than an IdeS enzyme comprising the amino acid sequence set forth in SEQ ID NO:2.
  • thermostability is measured by differential scanning fluorimetry.
  • the IgG protease variant of the disclosure e.g., a N144 variant of the disclosure, has substantially the same thermostability as an IdeZ enzyme comprising the amino acid sequence set forth in SEQ ID NO: 830.
  • the IgG protease variant of the disclosure e.g., a N144 variant of the disclosure, is more thermostable than an IdeZ enzyme comprising the amino acid sequence set forth in SEQ ID NO: 830.
  • thermostability is measured by differential scanning fluorimetry.
  • Melting temperatures of the polypeptides described herein can be determined by methods known to those of ordinary skill in the art.
  • a thermal shift assay also referred to as differential scanning fluorimetry (DSF)
  • DSF differential scanning fluorimetry
  • Tm melting temperature
  • a thermal shift assay to determine the Tm of a polypeptide of the invention a solution comprising the protein is combined with a dye, for example, SYPROTM Orange, heated in a quantitative polymerase chain reaction (qPCR) instrument.
  • qPCR quantitative polymerase chain reaction
  • IgG protease e.g., an IgG protease variant provided herein, or a fusion protein comprising the same
  • an enzyme-linked immunosorbent assay ELISA
  • individual purified IgG proteases of the invention are added individually to wells of an assay plate, e.g., a 96-well plate, and anti-human IgG(Fc)-HRP (horseradish peroxidase added).
  • TMB 3,3’,5,5’-Tetramethylbenzidine
  • the polypeptide is PEGylated, i.e., the polypeptide is covalently conjugated to polyethylene glycol (PEG), e.g., at one or more lysine residues.
  • PEG polyethylene glycol
  • An IgG protease, an IgG protease variant, or a fusion protein comprising the same may be conjugated to any desired number of PEG or monomethoxypoly(ethylene glycol) (mPEG) molecules, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.
  • an IgG protease, an IgG protease variant, or a fusion protein comprising the same is conjugated to from about 8 to about 11 PEG or mPEG molecules, about 9 PEG or mPEG molecules, or about 10 PEG or mPEG molecules.
  • the average total molecular weight of the PEG moiety of a PEGylated IgG protease, IgG protease variant, or fusion protein is about 10 kDa to about 150 kDa, about 30 kDa to 120 kDa, or about 50 kDa to about 100 kDa.
  • the IgG protease or IgG protease variant disclosed herein, or a fusion protein comprising the same is covalently bonded to PEG, e.g., mPEG, via a biocompatible linking group, using methods known in the art, as described, for example, by Park et al, Anti cancer Res., 1981, 1:373-376; and Zaplipsky and Lee, Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, ed., 1992, Plenum Press, New York, Chapter 21, the disclosures of each of which are incorporated by reference herein in their entireties.
  • a biocompatible linking group is non-toxic and can be used in vitro or in vivo without causing injury, sickness, disease, or death.
  • PEG can be bonded to the linking group, for example, via an ether bond, an ester bond, a thiol bond or an amide bond.
  • Suitable biocompatible linking groups include, for example, an ester group, an amide group, an imide group, a carbamate group, a carboxyl group, a hydroxyl group, a carbohydrate, a succinimide group, an epoxide group, an oxycarbonylimidazole group, a nitro phenyl group, a trysylate group, an aldehyde group, an isocyanate group, a vinylsulfone group, a tyrosine group, a cysteine group, a histidine group or a primary amine.
  • the IgG protease or IgG protease variant disclosed herein, or fusion protein comprising the same is conjugated to PEG without a linking group, e.g., mPEG, through an amino group, a sulfhydral group, a hydroxyl group or a carboxyl group of the IgG protease, IgG protease variant, or the fusion protein.
  • PEG is conjugated to one or more lysine residues on the IgG protease, IgG protease variant, or the fusion protein.
  • PEG is conjugated to one or more cysteine residues on the IgG protease, IgG protease variant, or the fusion protein. In one embodiment, PEG is conjugated to one or more serine residues on the IgG protease, IgG protease variant, or the fusion protein. In yet another embodiment, PEG is conjugated to one or more lysine residues, one or more cysteine residues, one or more serine residues, or a combination of any of the foregoing.
  • a fusion protein comprising two or more domains, and/or a polynucleotide encoding the same.
  • One of the domains is an IgG protease domain, i.e., the domain comprising one of the IgG protease polypeptides or IgG protease variant polypeptides described herein.
  • the second domain in one embodiment, is a domain that increases the half-life of the IgG protease, e.g., an immunoglobin Fragment crystallizable (Fc) domain, and/or a random coil polypeptide domain.
  • a “fusion protein” refers to a protein composed of a plurality of polypeptide components (or polypeptide “domains”), that while typically unjoined in their native state, are joined by their respective N-terminus and C-terminus through a peptide linkage to form a single continuous polypeptide.
  • a fusion protein is produced via recombinant expression of a single DNA sequence comprising the fusion protein domains.
  • a “domain” relates to any region/part of an amino acid sequence that is capable of autonomously adopting a specific structure and/or function.
  • a “domain” may represent a functional domain or a structural domain.
  • a fusion protein of the present invention comprises at least one IgG protease domain and (i) at least one domain forming a random coil conformation (e.g., a PAS polypeptide domain), (ii) a domain that increases the half-life of the IgG protease, e.g., an Fc domain or an albumin domain.
  • the IgG protease fusion protein of the present invention in one embodiment, comprises two domains.
  • a fusion protein of the disclosure comprises three or more domains.
  • the three or more domains in one embodiment, comprise: (i) an IgG protease domain, (ii) a domain that increases the half-life of the IgG protease, and (iii) a random coil domain.
  • the three or more domains in another embodiment, comprise: an IgG protease domain, and two domains that increases the half-life of the IgG protease, e.g., where a domain that increases the half-life of the IgG protease is N-terminal to the IgG protease and C-terminal to the IgG protease.
  • the three or more domains in even another embodiment, comprise: an IgG protease domain, and two random coil domains, e.g., where a random coil domain is N-terminal to the IgG protease and C- terminal to the IgG protease.
  • the domains of the fusion proteins provided herein can be linked directly or through an amino acid or peptide linker.
  • the amino acid spacer linker in one embodiment, is one amino acid long, two amino acids long, three amino acids long, four amino acids long, or five amino acids long.
  • the amino acid spacer sequence is from about two amino acids long to about 5 amino acids long.
  • the amino acid spacer sequence is from about two amino acids long to about 4 amino acids long.
  • the amino acid spacer sequence is two amino acids long.
  • the spacer sequence is Gly-Ser.
  • the IgG protease polypeptide domain is conjugated to a second domain, in one preferred embodiment, at the DNA level via operably linking the IgG protease DNA sequence to a DNA sequence encoding the second domain, such that the DNA sequences are present within one open reading frame, followed by recombinant expression of the continuous DNA sequence encoding the first and second domains.
  • the linking of the two domains at the DNA level obviates the need for in vitro coupling or modification steps to achieve conjugate synthesis, which is needed, for example, for the coupling of polyethylene glycol (PEG) to an IgG protease, in other embodiments described herein.
  • PEG polyethylene glycol
  • IgG protease fusion proteins may be a combination of two, three or four or more different domains. IgG protease fusion proteins can also include fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; as well as fusion proteins that include additional sequences for purification of the fusion protein (e.g., a polyhistidine tag).
  • An IgG protease fusion protein in one embodiment, comprises a protease sensitive cleavage site, an affinity tag such as the polyhistidine tag or the Strep-tag, a signal peptide, retention peptide, a targeting peptide like a membrane translocation peptide or additional effector domains like antibody fragments for tumor targeting associated with an anti-tumor toxin or an enzyme for prodrug-activation etc.
  • an affinity tag such as the polyhistidine tag or the Strep-tag
  • a signal peptide retention peptide
  • a targeting peptide like a membrane translocation peptide or additional effector domains like antibody fragments for tumor targeting associated with an anti-tumor toxin or an enzyme for prodrug-activation etc.
  • a fusion protein provided herein comprises a first domain comprising an IgG protease or an IgG protease variant provided herein and a second domain comprising an immunoglobulin fragment crystallizable (Fc) domain.
  • the Fc domain genetic sequence is fused to the IgG protease genetic sequence, and the fusion protein is produced via standard molecular biology techniques.
  • the Fc domain prolongs the serum half-life of proteins. Without wishing to be bound by theory, it is thought that the extended half-life is because of the pH-dependent binding of the neonatal Fc receptor (FcRn). Such binding salvages the protein from being degraded in endosomes.
  • renal clearance of the fusion protein is limited resulting from the increase of mass due to the homodimeric nature of the Fc fusion domain.
  • the Fc region in many cases improves the biophysical properties of its fusion partner, such as the solubility and stability of the protein.
  • high expression, secretion to tissue culture medium, and protein- A affinity purification of Fc-fusion proteins simplify the downstream manufacturing processes.
  • the Fc domain for the fusion protein disclosed herein may comprise a native (or naturally - occurring or wild-type) Fc polypeptide, or a variant Fc polypeptide (e.g., a non-naturally occurring Fc polypeptide), comprising, e.g., 1, 2, 3, 4, 5, 1-5, 1-10 or 5-10 or more amino acid mutations, e.g., substitutions, additions or deletions.
  • a variant Fc polypeptide may comprise an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to a wild-type Fc polypeptide.
  • the Fc polypeptides or variant Fc polypeptides suitable for use in the Fc domain of the fusion protein provided herein are those disclosed in U.S. Patent Application Publication No. US 2024/0229002, incorporated herein by reference in its entirety for all purposes.
  • Exemplary Fc polypeptides and variant Fc polypeptides include those comprising an amino acid sequence selected from one of SEQ ID NOS :2591 -2606 (Table A).
  • Fc polypeptides or variant Fc polypeptides comprising an amino acid sequence having at least about 50%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to their counterpart exemplary Fc polypeptides or variant Fc polypeptides can also be used.
  • the Fc domain of a fusion protein provided herein dimerizes with a second Fc domain, and as such, provides a dimeric protein.
  • the dimeric protein is a homodimer. In another embodiment, the dimeric protein is a heterodimer.
  • the Fc domain in one embodiment, is N-terminal to the IgG protease domain. In another embodiment, the Fc domain is C-terminal to the IgG protease domain. In even another embodiment, the fusion protein comprises an Fc domain N-terminal to the IgG protease domain, and a second Fc domain C-terminal to the IgG protease domain. [0439] The Fc domain, in one embodiment, is an IgGl Fc domain. In another embodiment, the Fc domain, in one embodiment, is an IgG2 Fc domain. In another embodiment, the Fc domain, in one embodiment, is an IgG3 Fc domain. In another embodiment, the Fc domain, in one embodiment, is an IgG4 Fc domain.
  • the IgG protease or IgG protease variant provided herein is part of a fusion protein with an albumin protein domain.
  • the albumin genetic sequence is fused to the IgG protease genetic sequence, and the fusion protein is produced via standard molecular biology techniques.
  • the albumin protein has a circulatory half-life of 19 days, and as such, fusion proteins provided herein comprising an albumin domain have an extended half-life, when compared to the half-life of the IgG protease when the IgG protease is present as a single domain protein.
  • renal clearance of the fusion protein is limited resulting from the increase of mass due to the size of the albumin domain.
  • the albumin domain improves the biophysical properties of the IgG protease, such as the solubility and stability of the protein.
  • the albumin domain in one embodiment, is N-terminal to the IgG protease domain. In another embodiment, the albumin domain is C-terminal to the IgG protease domain.
  • the fusion protein comprises at least two domains: (i) a first domain comprising one of the IgG proteases or the IgG protease variants disclosed herein (IgG protease domain) conjugated to (ii) a second domain comprising a first random coil polypeptide (in some embodiments, referred to a as a “first random coil polypeptide domain”).
  • the IgG protease fusion protein comprises (i) a first domain comprising an IgG protease or an IgG protease variant disclosed herein and (ii) a second domain comprising a random coil polypeptide domain C-terminal to the IgG protease domain.
  • the fusion protein comprises (i) an IgG protease or an IgG protease variant disclosed herein and (ii) a random coil polypeptide domain N-terminal to the IgG protease domain.
  • the fusion protein comprises (i) an IgG protease or an IgG protease variant disclosed herein and (ii) a random coil polypeptide domain C-terminal to the IgG protease domain and (iii) a random coil polypeptide
  • the random coil polypeptide domains comprise PAS polypeptides.
  • random coil relates to a conformation of a polymeric molecule, including amino acid polymers, in which the individual monomeric elements that form the polymeric structure are essentially randomly oriented towards the adjacent monomeric elements while still being chemically bound to said adjacent monomeric elements.
  • a polypeptide or amino acid polymer adopting/having/forming “random coil” conformation substantially lacks a defined secondary and tertiary structure.
  • the nature of polypeptide random coils and their methods of experimental identification are known to the person skilled in the art.
  • the random coil polypeptides suitable for use in the IgG protease fusion proteins disclosed herein are described in International Patent Application Publication No. WO 2024/124142, incorporated herein by reference in its entirety.
  • the IgG protease fusion protein provided herein is a recombinant IgG protease fusion protein.
  • the first domain may be C-terminal to the second domain, or may be N-terminal to the second domain.
  • a linker peptide e.g., Gly-Ser (GS) is present between the IgG protease domain and a C-terminal random coil PAS polypeptide domain (e.g., PAS10, PAS20, or PAS30) or a C-terminal random coil XTEN polypeptide domain.
  • an IgG protease fusion protein comprises a first random coil polypeptide domain.
  • the first random coil polypeptide domain comprises at least about 100 amino acid residues.
  • the first random coil polypeptide domain is a PA polypeptide, a PAS polypeptide, or an XTEN polypeptide.
  • the random coil conformation mediates an increased in vivo and/or in vitro stability of the IgG protease.
  • the random coil polypeptide domain is thought to not adopt a stable structure or function by itself, the biological activity of the IgG protease to which it is conjugated is essentially preserved.
  • the random coil polypeptide domain comprises the two amino acids, proline (Pro) and alanine (Ala).
  • the random coil polypeptide consists of the two amino acids, proline (Pro) and alanine (Ala).
  • the amino acid residues in a polypeptide are all Pro and Ala, or substantially all the amino acids are Pro and Ala in a polypeptide, such a polypeptide is referred to herein as a “PA polypeptide.”
  • an IgG protease comprises a PA polypeptide domain.
  • the PA polypeptide is at least about 100 amino acids long, at least about 150 amino acids long, at least about 200 amino acids long, at least about 250 amino acids long, at least about 300 amino acids long, at least about 350 amino acids long, at least about 400 amino acids long, at least about 450 amino acids long, at least about 500 amino acids long, or at least about 550 amino acids long, e.g., the PA polypeptide is about 200 amino acids long, about 300 amino acids long, about 400 amino acids long, about 500 amino acids long, or about 600 amino acids long.
  • the polypeptide comprises two domains: (i) an IgG protease and (ii) a first random coil polypeptide domain.
  • the random coil polypeptide domain comprises the three amino acids, proline (Pro), alanine (Ala) and serine (Ser).
  • the random coil polypeptide consists of the three amino acids, proline (Pro), alanine (Ala) and serine (Ser).
  • PAS polypeptide where the amino acid residues in a polypeptide are all Pro, Ala and Ser, or substantially all the amino acids are Pro, Ala and Ser in a polypeptide, such a polypeptide is referred to herein as a “PAS polypeptide.”
  • a PAS polypeptide may also be referred to as a PAS domain when the PAS polypeptide is present within a fusion protein.
  • an IgG protease fusion protein comprises a PAS polypeptide and the PAS polypeptide comprises from about 10 to about 30 tandem copies of a PAS sequence comprising Pro, Ala, and Ser, e.g., PAS 10 with 10 tandem copies and a total of about 200 amino acids; PAS20 with 20 tandem copies and a total of about 400 amino acids; and PAS30 with 30 tandem copies and a total of about 600 amino acids.
  • Exemplary amino acid sequences of PAS 10, PAS20, and PAS30 are set forth in SEQ ID NO:831, SEQ ID NO:832, and SEQ ID NO:833, respectively.
  • a PAS amino acid sequence for use in a random coil domain comprises one of the amino acid sequences set forth in Table 8.
  • the random coil polypeptide is an extended recombinant (XTEN) polypeptide.
  • XTEN polypeptide includes six amino acids A, E, G, S and T at varying percentages so as to form a long unstructured hydrophilic amino acid sequence (Pasut, Polymers 2014, 6, 160-178).
  • the XTEN polypeptide in one embodiment, is one of the polypeptides disclosed in U.S. Patent Application Publication No. 2015/0037359, the content of which is incorporated by reference in its entirety for all purposes.
  • the XTEN polypeptide in one embodiment, is at least about 800 amino acids in length consisting of six hydrophilic chemically stable amino acids Ala, Asp, Gly, Pro, Ser, and Thr, in a nonrepetitive manner.
  • the XTEN polypeptide is 864 residues long.
  • the XTEN polypeptide is a fragment of the 864 aa XTEN polypeptide.
  • the XTEN polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 834.
  • XTEN polypeptides may be conjugated to the first domain comprising an IgG protease described herein via chemical conjugation or produced as a fusion protein with the IgG protease.
  • a second domain of the IgG protease fusion protein comprises a PAS polypeptide and comprises an amino acid sequence comprising at least about 100 amino acid residues that form a random coil conformation.
  • the at least about 100 amino acid residues forming the random coil comprise the amino acids proline (Pro), alanine (Ala) and serine (Ser).
  • Pro proline
  • Ala alanine
  • Ser serine
  • all or substantially all of the amino acids are Pro, Ala and Ser.
  • the random coil conformation mediates an increased in vivo and/or in vitro stability of the IgG protease enzyme. Details regarding various types of PAS polypeptides and nucleic acids encoding the same, for use in the present invention, can be found in PCT Publication No. WO 2008/155134, the content of which is incorporated by reference in its entirety for all purposes.
  • the random coil of an IgG protease fusion protein is formed under physiological conditions.
  • the physiological conditions are the parameters that are typically valid for higher forms of life, and in particular mammals, most preferably humans.
  • physiological conditions can be the conditions that are normally found in the body fluids of mammals.
  • the physiological conditions may relate to the corresponding parameters found in the healthy body as well as the parameters as found in sick mammals or human patients. For example, a sick mammal or human patient may have a higher, yet physiological temperature condition when said mammal or said human suffers from fever.
  • buffers in experimental settings e.g., for use in the determination of protein structures, in particular in circular dichroism (CD) measurements and other methods for determining the structural properties of a protein/amino acid stretch
  • solvents and/or excipients for pharmaceutical compositions are considered to represent physiological solutions and/or physiological conditions in vitro.
  • buffers are, e.g., phosphate-buffered saline, Tris buffers, acetate buffers, citrate buffers or similar buffers.
  • the pH of a buffer representing physiological solution conditions is in a range from 6.5 to 8.5, e.g., in a range from 7.0 to 8.0, e.g., in a range from 7.2 to 7.7 and the osmolarity may lie in a range from 10 to 1000 mmol/kg H2O, more particularly in a range from 50 to 500 mmol/kg H2O, e.g., in a range from 200 to 350 mmol/kg H2O.
  • Methods for determining whether an amino acid polymer forms/adopts random coil conformation are known in the art. Such methods include CD spectroscopy, which represents a light absorption spectroscopy method in which the difference in absorbance of right- and left- circularly polarized light by a substance is measured.
  • the secondary structure of a protein can be determined by CD spectroscopy using far-ultraviolet spectra with a wavelength between approximately 190 and 250 nm. At these wavelengths, the different secondary structures commonly found in polypeptides can be analyzed, since a-helix, parallel and anti-parallel P-sheet and random coil conformations each give rise to a characteristic shape and magnitude of the CD spectrum.
  • CD spectrometry the skilled artisan is readily capable of determining whether an amino acid polymer forms/adopts random coil conformation at physiological conditions.
  • Other established biophysical methods include nuclear magnetic resonance (NMR) spectroscopy, absorption spectrometry, infrared and Raman spectrometry, measurement of the hydrodynamic volume via size exclusion chromatography, analytical ultracentrifugation or dynamic/static light scattering as well as measurements of the frictional coefficient or intrinsic viscosity.
  • a random coil polypeptide domain comprises at least about 100 amino acid residues, at least about 150 amino acid residues, at least about 200 amino acid residues, at least about 250 amino acid residues, at least about 300 amino acid residues, at least about 350 amino acid residues, or at least about 400 amino acid residues. In another embodiment, a random coil polypeptide domain comprises maximally about 1000 amino acid residues, maximally about 900 amino acid residues, maximally about 800 amino acid residues, maximally about 700 amino acid residues, or maximally about 600 amino acid residues. In one embodiment, a random coil polypeptide domain comprises maximally about 500 amino acid residues or of maximally about 450 amino acid residues.
  • a random coil polypeptide domain comprises from about 100 to about 3000 amino acid residues. In a further embodiment, the random coil polypeptide domain comprises from about 100 to 1000 amino acid residues. In some embodiments, the random coil polypeptide domain comprises from about 100 to about 800, from about 100 to about 700, from about 100 to about 600, from about 100 to about 500, from about 100 to about 400, or from about 100 to about 300 amino acid residues.
  • a random coil polypeptide domain comprises an amino acid sequence whereby proline residues represent about 4% to about 40% of the random coil polypeptide domain.
  • alanine and serine residues comprise the remaining about 60% to about 96% of the random coil polypeptide domain.
  • the random coil polypeptide domain comprises further amino acids differing from Ala, Ser and Pro as minor constituents.
  • amino constituent means that maximally 10% of the amino acids in a random coil polypeptide domain are different from alanine, serine and proline, e.g., maximally 8% of the amino acids in a random coil polypeptide domain, e.g., maximally 6% of the amino acids in a random coil polypeptide domain, e.g., maximally 5% of the amino acids in a random coil polypeptide domain, maximally 4% of the amino acids in a random coil polypeptide domain, maximally 3% of the amino acids in a random coil polypeptide domain, maximally 2% of the amino acids in a random coil polypeptide domain, maximally 1% of the amino acids in a random coil polypeptide domain are different from Ala, Ser and Pro.
  • the polypeptide comprises amino acids other than Ala, Ser and Pro, and the other amino acids are selected from the group consisting of Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Vai.
  • the other amino acids include one or more non-natural amino acids.
  • a random coil polypeptide domain comprises a plurality of “amino acid repeats”, i.e., the same amino acid sequence occurring two or more times in the domain, wherein the “amino acid repeats” consist of Ala, Ser, and Pro residues (depicted herein as “PAS”, or as “APS”).
  • PAS Ala, Ser, and Pro residues
  • no more than 6 consecutive amino acid residues are identical in the random coil polypeptide domain and the proline residues constitute more than about 4% and less than about 40% of the amino acids of the random coil polypeptide domain.
  • amino acid repeats consisting of Ala, Ser and Pro residues are provided herein; see, e g., SEQ ID NO: 835, SEQ ID NO: 837, SEQ ID NO: 839, SEQ ID NO: 841, SEQ ID NO: 843 and SEQ ID NO: 845 (Table 8). Fragments and/or multimers of these sequences are employed in some embodiments.
  • a “fragment” comprises at least 3 amino acids and comprises at least one Ala, one Ser and/or one Pro.
  • the aforementioned repeat sequences may be encoded by nucleic acid molecules having the sequences set forth in SEQ ID NO: 836, SEQ ID NO: 838, SEQ ID NO: 840, SEQ ID NO: 842, SEQ ID NO: 844 and/or SEQ ID NO: 846 (Table 8).
  • the amino acid repeat used in a random coil PAS polypeptide domain comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues, wherein the amino acid repeat comprises at least one Ala, Ser, and Pro residue. In one embodiment, the amino acid repeat does not comprise more than 100 amino acid residues.
  • the amino acid repeat in one embodiment, comprises at least about 4%, at least about 5%, at least about 6%, at least about 10%, at least about 15%, or at least about 20% Pro residues. In a further embodiment, the amino acid repeat comprises less than about 40%, e.g., less than about 35% Pro residues.
  • a random coil polypeptide domain comprises no more than 5 identical consecutive amino acid residues, e.g., no more than 4 identical consecutive amino acid residues, e.g., no more than 3 identical consecutive amino acid residues.
  • a random coil polypeptide domain comprises more than about 4% Ala residues but less than about 50% Ala residues, e.g., more than about 10% Ala residues but less than about 50% Ala residues, e.g., more than about 20% Ala residues but less than about 50% Ala residues.
  • a random coil polypeptide domain comprises more than about 4% Ser residues but less than about 50% Ser residues, e.g., more than about 10% Ser residues but less than about 50% Ser residues, e.g., more than about 20% Ser residues but less than about 50% Ser residues.
  • a random coil polypeptide domain comprises about 35% Pro residues, about 50% Ala residues and about 15% Ser residues.
  • a random coil polypeptide domain comprises about 35% Pro residues, about 15% Ala residues and about 50% Ser residues.
  • the IgG protease fusion protein comprises a PAS polypeptide in the random coil polypeptide domain encoded by the amino acid sequence set forth in SEQ ID NO: 831.
  • the IgG protease fusion protein comprises a PAS polypeptide in the random coil polypeptide domain encoded by the amino acid sequence set forth in SEQ ID NO: 832.
  • the IgG protease fusion protein comprises a PAS polypeptide in the random coil polypeptide domain encoded by the amino acid sequence set forth in SEQ ID NO: 833.
  • the present disclosure provides an isolated nucleic acid encoding an IgG protease polypeptide, an IgG protease variant polypeptide, or fusion protein of the invention.
  • the IgG protease is a deimmunized IgG protease.
  • the IgG protease in another embodiment, is present as a first domain within a fusion protein comprising at least a first domain and a second domain.
  • fusion protein is a recombinant IgG protease fusion protein.
  • An isolated nucleic acid is removed from its natural environment, and may additionally be in substantially pure, e.g., at least 90% pure, or in homogeneous form.
  • the isolated nucleic acid may be, for example, a synthetic DNA, a non-naturally occurring mRNA, or a cDNA.
  • Methods of producing the disclosed nucleic acids are well known to the one of skill in the art. See, e.g., Maniatis, T., 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., incorporated herein by reference in its entirety.
  • the disclosed nucleic acids may be produced by introducing one or more mutations into a related nucleic acid with a similar nucleic acid sequence using site-directed mutagenesis techniques known in the art, such as extension of overlapping gene segments by PCR, disclosed in Heckman and Pease (2007).
  • nucleic acids can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
  • the nucleic acid molecule which encodes a polypeptide (which can be a fusion protein) of the disclosure in one embodiment, is present in a nucleic acid vector.
  • the nucleic acid encoding an IgG protease or a fusion protein comprising the same is inserted into a vector, e.g., a plasmid, for multiplication of the nucleic acid.
  • the nucleic acid coding for an IgG protease or a recombinant IgG protease fusion protein comprising an IgG protease domain is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements, e.g., promoter sequences, terminator sequences, polyadenylation sequences, and enhancer sequences, for the transcription and translation of the inserted IgG protease-coding sequence.
  • Vectors may be plasmids, phage, phagemids, adenoviral, AAV, lentiviral, for example.
  • a variety of host- vector systems may be utilized to express the coding sequence of an IgG protease or a recombinant IgG protease fusion protein comprising an IgG protease domain.
  • Exemplary systems include mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.
  • the expression elements of these vectors vary in their strengths and specificities.
  • a bacterial expression vector is used.
  • the bacterial expression vector is for use with E. coli.
  • the promoter in the bacterial expression vector is a T5 promoter inducible by isopropyl 0-D-1 thiogalactopyranoside (IPTG) and repressible by glucose, a T7 promoter inducible by IPTG and repressible by glucose, a rhamnose (rham) promoter inducible by rhamnose and repressible by glucose, or an alkaline phosphatase (phoA) promoter inducible by phosphate starvation and repressible by the presence of phosphate.
  • IPTG isopropyl 0-D-1 thiogalactopyranoside
  • phoA alkaline phosphatase
  • any of the methods known for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a nucleic acid comprising one or more regulatory elements such as appropriate transcriptional/translational control signals and a coding sequence for an IgG protease or a recombinant IgG protease fusion protein operably linked thereto. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination).
  • nucleic acid sequence encoding an IgG protease or a recombinant IgG protease fusion protein comprising an IgG protease domain may be regulated by a second nucleic acid sequence so that the IgG protease or recombinant IgG protease fusion protein is expressed in a host transformed with the recombinant DNA molecule.
  • expression of an IgG protease or a recombinant IgG protease fusion protein comprising an IgG protease domain may be controlled by any promoter/enhancer element known in the art.
  • the nucleic acid comprises a nucleic acid sequence encoding the IgG protease or the recombinant IgG protease fusion protein operatively linked to a heterologous promoter.
  • exemplary promoters which may be used to control IgG protease or recombinant IgG protease fusion protein expression using a mammalian expression vector include the simian virus 40 (SV40) early promoter region, the promoter contained in the 3 ’ long terminal repeat of Rous sarcoma virus, the herpes thymidine kinase promoter, and the regulatory sequences of the metallothionine gene.
  • Exemplary promoters useful for prokaryotic expression vectors include the 0-lactamase promoter, the tac promoter, and the osmoregulated osmB promoter.
  • the present disclosure provides a host cell comprising a nucleic acid vector disclosed herein.
  • the host cell is capable of producing an IgG protease or fusion protein containing the same, disclosed herein.
  • the IgG protease is present as a fusion protein comprising (i) the IgG protease domain and a random coil domain or (ii) the IgG protease domain and an Fc domain.
  • the IgG protease fusion protein is a recombinant IgG protease fusion protein comprising on the IgG proteases disclosed herein.
  • Appropriate cell lines or host systems can be chosen based on the desired expression level and/or post-translational processing and modification (e.g., glycosylation, cleavage) of the IgG protease or recombinant IgG protease fusion protein.
  • Suitable host cells include bacteria (e.g., E. coli), mammalian cells, plant cells, insect cells, fungi, yeast and transgenic plants and animals.
  • Exemplary mammalian cell lines available in the art for expression of a heterologous protein include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, mouse melanoma cells, rat myeloma cells, human embryonic kidney cells, and human embryonic retina cells.
  • CHO Chinese hamster ovary
  • HeLa cells HeLa cells
  • baby hamster kidney cells mouse melanoma cells
  • rat myeloma cells human embryonic kidney cells
  • human embryonic retina cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, mouse melanoma cells, rat myeloma cells, human embryonic kidney cells, and human embryonic retina cells.
  • Introducing the vector into a host cell can be accomplished using techniques well known in the art.
  • suitable techniques may include calcium phosphate transfection, diethylaminoethyl (DEAE)-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, for example.
  • suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g., by culturing host cells under conditions for expression of the nucleic acid sequence encoding the IgG protease or recombinant IgG protease fusion protein, leading to respective protein production.
  • the nucleic acid encoding the IgG protease or IgG protease fusion protein is integrated into the genome, e.g., chromosome, of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.
  • the host cell is a mammalian cell, e.g., a Chinese hamster ovary (CHO) cell, or a human embryonic kidney cell.
  • the expression vector for use in the mammalian cell comprises the actin (e.g., chicken 0-actin), cytomegalovirus (CMV), CMV enhancer / elongation factor (CEF), CMV early enhancer/chicken 0-actin (CAG), hybrid CMV enhancer/chicken 0-actin (CBh), elongation factor-la (EFlalpha), glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), or simian virus 40 (SV40) promoter.
  • actin e.g., chicken 0-actin
  • CMV cytomegalovirus
  • CEF CMV enhancer / elongation factor
  • CAG CMV early enhancer/chicken 0-actin
  • CBh hybrid CMV enhancer/chicken 0-actin
  • the host cell is a yeast cell.
  • the expression vector for use in the yeast cell comprises the alcohol oxidase (AOX), glyceraldehyde-3 -phosphate dehydrogenase (GAP), alcohol dehydrogenase (ADH), or galactokinase 1 (GALI) promoter.
  • AOX alcohol oxidase
  • GAP glyceraldehyde-3 -phosphate dehydrogenase
  • ADH alcohol dehydrogenase
  • GALI galactokinase 1
  • the host cell is an insect cell infected with baculovirus.
  • the expression vector for use with the baculovirus comprises the polyhedrin gene promoter.
  • the host cell is bacteria, e.g., E. colt.
  • the expression vector for use in bacteria comprises the osmB promoter, the T7-lac promoter (see Shilling et al., Commun Biol 3, 214 (2020)), the pBAD promoter, the Tac promoter, a tet-inducible promoter, the cold-shock Protein A (cspA) promoter, or the inducible promoter of the alkaline phosphatase gene (phoA) derived from E. coli.
  • the present disclosure provides a method of recombinantly producing an IgG protease, an IgG protease variant, or a conjugate comprising the same (e.g., a recombinant IgG protease fusion protein) disclosed herein.
  • the method includes
  • culturing a host cell comprising a nucleic acid vector comprising a nucleic acid sequence encoding the IgG protease, the IgG protease variant, or the IgG protease conjugate (e.g., the recombinant IgG protease fusion protein) disclosed herein, wherein the nucleic acid sequence is operatively linked to a heterologous promoter under conditions to allow for expression of the nucleic acid sequence encoding the IgG protease, the IgG protease variant, or the IgG protease conjugate (e.g., the recombinant IgG protease fusion protein) and recombinant production of the IgG protease, the IgG protease variant, or the IgG protease conjugate by the host cell; and
  • the IgG proteases or IgG protease fusion proteins may be produced in any suitable cellculture system including prokaryotic cells, e.g., E. coli, BL21 or JM83, or eukaryotic cells, e.g., Pichia pastoris yeast strain X-33 or Chinese hamster ovary (CHO) cells. Further suitable cell lines known in the art are obtainable from cell line depositories, such as the American Type Culture Collection (ATCC).
  • the IgG proteases or IgG protease conjugates can be isolated from the growth medium, cellular lysates or cellular membrane fractions.
  • the isolation and purification of the recombinantly produced IgG proteases or conjugates comprising the same may be performed by any conventional means, including ammonium sulphate precipitation, affinity columns, column chromatography, gel electrophoresis and the like and may involve the use of monoclonal or polyclonal antibodies directed, e.g., against a tag fused with the biologically active protein of the invention.
  • the protein can be purified via the Strep-tag II using streptavidin affinity chromatography (Skerra and Schmidt (2000). Methods Enzymol 326, pp. 271-304).
  • the vector is under the control of an osmotic pressure sensitive promoter.
  • An osmotic pressure sensitive promoter initiates transcription as a result of increased osmotic pressure as sensed by the cell.
  • the host cell is A. coli
  • the promoter is the osmB promoter, the T7-lac promoter, the pBAD promoter, the Tac promoter, a tet-inducible promoter, the cold-shock Protein A (cspA) promoter, or the inducible promoter of the alkaline phosphatase gene (phoA) derived from E. coli.
  • the IgG protease, the IgG protease variant, or a conjugate comprising the same is isolated using a cationic surfactant, for example, cetyl pyridinium chloride (CPC).
  • CPC cetyl pyridinium chloride
  • the method further comprises purifying the recombinantly produced IgG protease or conjugate thereof using, for example, affinity chromatography, ammonium sulfate fractionation, or sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis.
  • the IgG protease or conjugate thereof can be made according to the methods described in International Patent Application Publication No.
  • the IgG protease or conjugate thereof is produced in bacteria and, following isolation, subjected to endotoxin mitigation using methods known in the art, such as one disclosed in the examples of the present application.
  • the present disclosure provides a pharmaceutical composition comprising an IgG protease, an IgG protease variant, or a fusion protein comprising the same and a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials known to those skilled in the art for formulating protein therapeutics.
  • a pharmaceutically acceptable moiety e.g., a salt, dosage form, or excipient
  • a pharmaceutically acceptable moiety has one or more benefits that outweigh any deleterious effect that the moiety may have. Deleterious effects may include, for example, excessive toxicity, irritation, allergic response, and other problems and complications.
  • the pharmaceutical composition may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art for formulating protein therapeutics. Such materials are non-toxic and do not interfere with the efficacy of the variants described herein.
  • Such materials may include, for example, solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents.
  • pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, and ethanol, as well as combinations thereof.
  • the pharmaceutical composition includes an isotonic agent, for example, a sugar, and/or a polyalcohol, such as mannitol or sorbitol, or sodium chloride.
  • additional examples of pharmaceutically acceptable substances are wetting agents or auxiliary substances, such as emulsifying agents, preservatives or buffers, which increase the shelf life or effectiveness.
  • the pharmaceutical composition comprising the IgG protease, the IgG protease variant, or a fusion protein comprising the same may be formulated in liquid, semi-solid or solid forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, powders, liposomes, and suppositories.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions e.g., injectable and infusible solutions
  • powders e.g., liposomes, and suppositories.
  • Formulations may include excipients, or combinations of excipients, for example: sugars, amino acids and surfactants.
  • Liquid formulations may include a wide range of IgG protease concentrations and pH. Solid formulations may be produced by lyophilization, spray drying, or drying by supercritical fluid technology, for example.
  • the active ingredient may be in a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pK, isotonicity, and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pK, isotonicity, and stability.
  • isotonic vehicles such as sodium chloride solution, Ringer’s solution, and a lactated Ringer’s solution.
  • Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included.
  • the pharmaceutical composition is formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to contain a high IgG protease concentration.
  • Sterile injectable solutions can be prepared by incorporating an IgG protease in an appropriate solvent with one or a combination of ingredients enumerated above, followed by filtered sterilization.
  • dispersions are prepared by incorporating the IgG protease into a sterile vehicle that contains a dispersion medium and other ingredients from those enumerated above.
  • the methods of preparation include vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by using a coating such as lecithin, by maintaining the particle size of a dispersion, or by using surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • the pharmaceutical composition may be prepared with a carrier that protects the IgG protease against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a carrier that protects the IgG protease against rapid release
  • a controlled release formulation including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • the pharmaceutical composition is a solution of an IgG protease or an IgG protease variant disclosed herein, or a fusion protein comprising the same.
  • the solution in one embodiment, is a phosphate buffered saline solution containing the IgG protease.
  • the solution is sterile and suitable for injection, e.g., intravenous injection or subcutaneous injection.
  • the polypeptides, fusion proteins and compositions of the present invention are used in vitro, ex vivo or in vivo to cleave IgG into F(ab’)2 and Fc-fragments.
  • the method comprises, in one embodiment, contacting IgG with a compound of the invention, or a composition comprising the same.
  • the method in a further embodiment, is carried out ex vivo.
  • the present invention provides a method of treating or preventing a disease mediated in whole or in part by pathogenic immunoglobulin G (IgG) antibodies.
  • the method comprises, administering to the patient in need of treatment a therapeutically or prophylactically effective amount of a polypeptide or fusion protein of the invention, or a composition comprising the same.
  • the method comprises multiple administrations of the polypeptide or fusion protein of the invention, or composition comprising the same, to the patient in need of treatment.
  • the method of treating is an ex vivo method. That is, blood taken from the patient having a disease or condition mediated in whole or part by IgG antibodies, and the method comprises contacting the blood with a polypeptide of the invention, or fusion protein comprising the same.
  • treatment or “treating,” or “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit.
  • Therapeutic benefit refers to any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.
  • treating in one embodiment, includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in the patient that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition (e.g., arresting, reducing or delaying the development of the disease, or a relapse thereof in case of maintenance treatment, of at least one clinical or subclinical symptom thereof); (3) relieving the condition (for example, by causing regression, or reducing the severity of the state, disorder or condition or at least one of its clinical or subclinical symptoms).
  • polypeptides e.g., IgG proteases or fusion proteins comprising the same
  • compositions comprising the same are administered to a subject already suffering from a disorder or condition, in an amount effective to treat (e.g., cure, alleviate or partially arrest the condition or one or more of its symptoms).
  • Such therapeutic treatment may result in a decrease in severity of disease symptoms, or an increase in frequency or duration of symptom-free periods.
  • polypeptides or compositions comprising the same are administered to a subject not yet exhibiting symptoms of a disorder or condition, or at risk for developing the disease or condition, in an amount sufficient to prevent or delay the development of symptoms and/or onset of disease.
  • the term “effective amount” or “therapeutically effective amount” refers to the amount of an agent that is sufficient to achieve an outcome, for example, to effect beneficial or desired results.
  • the therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like.
  • the amount of agent administered can be referred to as a “prophy lactically effective amount”.
  • the subject may have been identified as being at risk of developing the disease or condition by any suitable means.
  • the terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, such as a mammal.
  • the mammal may be, for example, a mouse, a rat, a rabbit, a cat, a dog, a pig, a sheep, a horse, a non-human primate (e.g., cynomolgus monkey, chimpanzee), or a human.
  • a subject’s tissues, cells, or derivatives thereof, obtained in vivo or cultured in vitro are also encompassed.
  • a human subject may be an adult, a teenager, a child (2 years to 14 years of age), an infant (1 month to 24 months), or a neonate (up to 1 month).
  • the adults are seniors about 65 years or older, or about 60 years or older.
  • the subject is a pregnant woman or a woman intending to become pregnant. In one embodiment, the subject is >18 to ⁇ 85 years of age.
  • the polypeptide may be co-administered with an immune-suppressive agent.
  • polypeptide or composition comprising the same in one embodiment, is administered parenterally.
  • administration is by intravenous infusion.
  • administration is intradermal, subcutaneous, percutaneous, intramuscular, intraarterial, intraperitoneal, intraarticular or intraosseous administration.
  • the polypeptides of the invention may be administered to treat or prevent a disease or condition mediated in whole or part by pathogenic IgG antibodies.
  • the invention provides a polypeptide of the invention for use in the treatment or prevention of a disease or condition mediated by pathogenic IgG antibodies.
  • the invention also provides a method of treating or preventing a disease or condition mediated by pathogenic IgG antibodies comprising administering to an individual a polypeptide of the invention. The method may comprise repeat administration of the said polypeptide.
  • the invention also provides a polypeptide of the invention for use in the manufacture of a medicament for the treatment or prevention of a disease or condition mediated in whole or part by pathogenic IgG antibodies.
  • the pathogenic antibodies may typically be specific for an antigen which is targeted in an autoimmune disease or other condition mediated wholly or in part by antibodies.
  • a polypeptide of the invention may be used to treat any of these diseases or conditions.
  • the polypeptide is particularly effective for the treatment or prevention of autoimmune disease which is mediated in whole or in part by pathogenic IgG antibodies.
  • the disease associated with IgG antibodies is selected from Addision’s disease, anti-glomerular basement membrane (anti-GBM) glomerulonephritis, Goodpasteur syndrome, anti-neutrophil cytoplasmic antibody-associated vasculidity (ANCA associated vasculitis), Churg-Strauss syndrome, microscopic polyangiitis, anti-N-methyl-D-aspartate receptor (anti -NMD AR) encephalitis, anti-phospholipid antibody syndrome (APS) (e.g., catastrophic APS), autoimmune bullous skin disease, pemphigus (e.g., pemphigus foliaceus, fogo selvagem, pemphigus vulgaris), autoimmune hemolytic anemia (AIHA), autoimmune hepatitis (AIH), autoimmune neutropenia (AIN), bullous pemphigoid (BP), celiac disease, chronic utricaria, complete congenital heart block (CCHB), diabetes type 1 A, epider
  • the disease associated with IgG antibodies and treated via a method provided herein is chronic inflammatory demyelinating polyneuropathy (CIDP).
  • CIDP chronic inflammatory demyelinating polyneuropathy
  • the disease associated with IgG antibodies and treated via a method provided herein is acute inflammatory demyelinating polyneuropathy (AIDP).
  • AIDP acute inflammatory demyelinating polyneuropathy
  • a method for preventing and/or treating antibody-mediated rejection (AMR) of an organ allograft in a patient in need of treatment comprises, in one embodiment, administering a polypeptide, fusion protein or composition of the invention to a patient in need thereof.
  • DSAs donor specific antibodies
  • a polypeptide or fusion protein of the invention, or composition comprising the same can be administered to the patient prior to organ transplant, during organ transplant, or subsequent to organ transplant.
  • DSAs donor specific antibodies
  • AMR acute antibody-mediated rejection
  • the method of the present invention allows the removal of DSAs in a potential transplant recipient.
  • Administering a polypeptide or fusion protein of the invention, or composition comprising the same, prior to transplantation has the capacity to effectively desensitize a sensitized patient, thereby allowing transplantation and avoiding acute antibody-mediated rejection.
  • the method of the present invention when used in organ transplant patients, either prior, during or subsequent to transplant, may allow for the removal of DSAs in a potential transplant recipient.
  • the organ in one embodiment, is a solid organ. In a further embodiment, the organ is a kidney, small intestine, pancreas, heart, lung or liver. In even a further embodiment, the organ is a kidney.
  • the patient to be treated in one embodiment is sensitized. By “sensitized” it is meant that the patient has developed antibodies to human major histocompatibility (MHC) antigens (also referred to as human leukocyte antigens (HLA)).
  • MHC major histocompatibility
  • HLA human leukocyte antigens
  • the anti-HLA antibodies originate from allogenically sensitized B-cells and can be present in patients that have previously been sensitized by blood transfusion, prior transplantation or pregnancy.
  • Whether or not a potential transplant recipient is sensitized may be determined by any suitable method.
  • a Panel Reactive Antibody (PRA) test may be used to determine if a recipient is sensitized.
  • a PRA score >30% is typically taken to mean that the patient is “high immunologic risk” or “sensitized”.
  • a cross match test may be conducted, in which a sample of the potential transplant donor’s blood is mixed with that of the intended recipient.
  • a positive cross-match means that the recipient has antibodies which react to the donor sample, indicating that the recipient is sensitized.
  • Cross-match tests are typically conducted as a final check immediately prior to transplantation.
  • IVIG intravenous immunoglobulin
  • the subject is administered the pharmaceutical composition chronically. That is, the subject is administered the composition for their entire life, once treatment for the particular disease/syndrome is initiated.
  • 24- well deep well plates were prefilled with 3 mL of Terrific Broth supplemented with appropriate selective antibiotic.
  • Pre-filled 24-well plates were inoculated with a 1:20 dilution of the overnight cultures, and the plates were sealed with gas-permeable membranes and incubated at 37 °C with 300 rpm of rotation.
  • HisPurTM Cobalt Spin plates (Thermo #90095) or HisPurTM Ni-NTA Spin Plates (Thermo #88230) were equilibrated following manufacturer instructions with Wash Buffer (25 mM Tris HC1 pH 7.5, 500 mM NaCl, 10 mM imidazole). Clarified lysate was applied to the plate. The plate was incubated for 15 min. on wet ice for each application of clarified lysate. The plate assembly was then centrifuged in the following sequence: 10 x g for 3 min., 25 x g for 3 min., and 500 x g for 3 min. for each application of clarified lysate, and the flow through discarded after each application. The plate was then washed with wash buffer following the manufacturer’s instructions.
  • the purification plate was then placed on a new collection plate and 220 pL of elution buffer (25 mM Tris HC1 pH 7.5, 500 mM NaCl, 500 mM imidazole) was added to each well, and incubated for 15 min. on wet ice.
  • the plate assembly was then centrifuged in the following sequence: 10 x g for 3 min., 25 x g for 3 min., and 500 x g for 3 min., and elution collected.
  • High binding capacity plates were coated with 50-100 pL /well of human TNF-a (stock solution 0.1 mg/ml) at a 1:800 dilution or various non-cleavable capture antibodies at varying dilutions in carbonate/bicarbonate buffer.
  • the plate was sealed and incubated 16-48 hr. at 4 °C or up to 8 hr. at 37 °C.
  • the plate was washed 3-5 x with plate washer and phosphate buffered saline with Tween 20 (PBS-T) wash buffer and tapped dry.
  • PBS-T phosphate buffered saline with Tween 20
  • Wells were incubated with 75-100 pL of purified library variants or controls prepared and normalized separately in PBS for varying times, at 37 °C. Plates were washed 3-5x with plate washer and PBS-T wash buffer and tapped dry. 75-100 pL of anti-human, horseradish peroxidase (HRP) conjugated secondary antibody was added to each well at a dilution of 1 :5, 000-1 :30,000 in LowCross buffer. Plates were incubated at 37 °C for 20-40 min. Plates were washed 3-5 x with plate washer and PBS-T wash buffer and tapped dry.
  • HRP horseradish peroxidase
  • TMB 3,3',5,5'-tetramethylbenzidine
  • Example 1 Identification of Polypeptides with IgG Protease Activity via Ancestral Reconstruction.
  • PSIBlast (v2.12.0) was used to search the National Center for Biotechnology Information’s (NCBI’s) non-redundant (nr) database with an e-value threshold 0.005 (see, e.g., Altschul et al. (1997). Nucleic Acids Research 25, pp. 3389-3402, the contents of which are incorporated by reference herein in their entirety).
  • Predicted proteolytic domains were isolated using InterProScan (see, e.g., Blum et al. (2021). Nucleic Acids Research 49, Database issue doi:
  • Sequences were selected from the PSIBlast search that had at least 40% identity to IdeS (SEQ ID NO:2) or IdeZ (SEQ ID NO:830), with or without the N-terminal signal sequence.
  • MSA multiple sequence alignment
  • a phylogenetic tree was constructed using the MSA with RaxML (v8.2.12) using options “-m PROTCATJTT -f a -x 42 -N autoMRE”. It is noted that the “-x” option turns on rapid bootstrapping, (cme.h-its.org/exelixis/web/software/raxml/. Stamatakis (2014). Bioinformatics 30(9), pp. 1312-1313, doi: 10.1093/bioinformatics/btu033, incorporated by reference herein in its entirety).
  • Ancestral sequences were generated using the multiple sequence alignment (MSA) and the phylogenetic tree with FastML (v3.11.0) using options “ — seqType aa”. See, e.g., Moshe and Pupko (2019). Bioinformatics 35(11), pp. 2562-2568, incorporated by reference in its entirety. (http://fastml.tau.ac.il/credits.php).
  • IgG Protease predicted ancestral protein sequences are provided in SEQ ID NOS:27-302.
  • Consensus sequences were generated according to the methodology set forth in Biswas et al. (2021). Nat Methods 18, 389-396, the disclosure of which is incorporated by reference herein in its entirety.
  • a metropolis-hastings sampling algorithm was used. The algorithm was modified from the code provided at github.com/churchlab/low-N-protein-engineering/blob/master/analysis/ A009_chip_l_GFP_follow_up_designs/002_consensus_ sequence design, ipynb.
  • Additional consensus sequences were generated from an MSA generated as above from sequences with at least 40% sequence identity to IdeS or IdeZ.
  • the seed sequence for these consensus designs was the highest likelihood sequence from the position-specific scoring matrix (PSSM).
  • PSSM position-specific scoring matrix
  • Libraries were generated in a sequential process using standard molecular biology techniques, iteratively increasing the mutational load based on predicted epitope deletion and predicted fitness. Individual gene inserts were synthesized.
  • N142 SEO ID NO:297
  • N144 SEO ID NO:295
  • This example describes additional engineering of IgG protease variants of N142 (SEQ ID NO:297) and N144 (SEQ ID NO:295) identified in Example 2.
  • the additional engineering was carried out by continuing to iteratively design, construct, express, and screen libraries of N142 and N144 IgG protease variants based on predicted T epitope depletion, thermostability measured by melting temperature, and functional fitness measured by IgG protease activity.
  • the melting temperatures and IgG protease activities of the variants as well as wild type IdeS for comparison were determined by using the methods described in the “Methods” section.
  • Table 13A shows 159 exemplary functional IgG protease variants of N142 with at least 30% of the IgG protease activity of wild type IdeS measured at the 4 pg/mL concentration and 1 hour reaction time.
  • variant concentrations and reaction time used for measuring the IgG protease activity of each variant with the IgG protease activity of a variant presented both as percent reduction in corrected absorbance at 450 nm of the variant relative to PBS (negative control) and as percentage of IgG protease activity of wild type IdeS, as described in the “Methods.”
  • IgG protease activity of a variant presented both as percent reduction in corrected absorbance at 450 nm of the variant relative to PBS (negative control) and as percentage of IgG protease activity of wild type IdeS, as described in the “Methods.”
  • All of the high performing N142 variants exhibited about 50-70% activity of wild type IdeS measured at 4 pg/mL with 1 h reaction time, with N142 variants of SEQ ID NOs:925, 936, and 942 having melting temperatures similar to or better than the melting temperature of about 50°C of wild type IdeS.
  • Table 14A shows 1124 exemplary functional IgG protease variants of N144 with at least 30% of the IgG protease activity of wild type IdeS measured at the 4 pg/mL concentration and 1 hour reaction time.
  • variant concentrations and reaction time used for measuring the IgG protease activity of each variant are also indicated in the table.
  • the IgG protease activity of a variant presented both as percent reduction in corrected absorbance at 450 nm of the variant relative to PBS (negative control) and as percentage of IgG protease activity of wild type IdeS, as described in the “Methods.”
  • the exemplary IgG protease variants of N144 listed in Table 14A 53 high performing N144 variants with the best overall activity, thermostability, and predicted T epitope depletion profile were identified (Table 14B).
  • All of the high performing N144 variants exhibited about 53-114% activity of wild type IdeS measured at 4 pg/mL with 1 h reaction time, with 33 of the 53 high performing N144 variants of SEQ ID NOs:1118, 1120, 1122, 1123, 1125, 1126, 1127, 1129, 1282, 1970, 1971, 1983, 2004, 2024, 2028, 2390, 2421, 2446, 2450, 2463, 2464, 2476, 2478, 2485, 2486, 2487, 2489, 2491, 2493, 2494, 2511, 2574, and 2575 having melting temperatures similar to or better than the melting temperature of about 50°C of wild type IdeS.
  • All of the high performing N144 variants are also predicted to be significantly deimmunized as compared to the N144 parent, because, as discussed in detail below, each of the high performing N144 variants by design had a number of T cell epitope targeting amino acid substitutions corresponding to those present in the IdeS variant of SEQ ID NO: 13, as well as additional T cell epitope targeting amino acid substitutions.
  • the IdeS variant of SEQ ID NO: 13 is shown in Example 3.1 to have markedly reduced T-cell responses by PBMC assay as compared to its parent, i.e., wild type IdeS.
  • the mutation load of the 53 high performing IgG protease variants of N144 ranged from 12 to 24 amino acid substitutions per variant relative to the parent N144.
  • Table 14C summarizes the amino acid substitutions of SEQ ID NO:295 identified among the 53 high performing N144 variants. Additionally, individual amino acid substitutions are ranked in descending order of occurrence frequency among the 53 high performing N144 variants (see the “Occurrence frequency ranking” column in Table 14C, with the ranking of 1 signifying the highest frequency).
  • each of the 53 high performing N144 variants harbored two sets of amino acid substitutions.
  • the first set of amino acid substitutions was present in all of the 53 high performing N144 variants and included 9 amino acid substitutions, i.e., Y12N, S18K, I46D, A47E, H93Y, E104R, F125W, T135H, and S159K.
  • amino acid substitutions of Y12N, S18K, A47E, H93Y, E104R, L120T, T135H, and S 159K relative to the parent N144 occurred most frequently in the high performing N144 variants with the rankings of 1 or 2.
  • Those amino acid substitutions correspond to the amino acid substitutions of Y12N, Y18K, A47E, H93Y, E104R, L120T, Y135N, and S159K, respectively, in the IdeS variant of SEQ ID NO: 13 relative to wild type IdeS, which together made up 8 of the 11 total amino acid substitutions in the IdeS variant of SEQ ID NO: 13.
  • Example 3.1 As the IdeS variant of SEQ ID NO: 13 is shown in Example 3.1 to be markedly deimmunized by PBMC assay as compared to wild type IdeS, the high performing N144 variants identified in this example are expected to have favorable deimmunization profiles as compared to the N144 parent.
  • Example 2.2 Immunogenicity analyses of and mapping of T-cell epitopes in N142 and select variants of N142 by ex vivo cellular immunoassay using human peripheral blood mononuclear cells (PBMCs)
  • PBMCs peripheral blood mononuclear cells
  • This example describes immunogenicity analyses of and mapping of T-cell epitopes in N142 comprising the amino acid sequence of SEQ ID NO:297 and select variants of N142 comprising an amino acid sequence selected from one of SEQ ID NOS: 859, 868, and 925, identified as high performing N142 variants in Example 2.1 (see Table 13B), by ex vivo cellular immunoassay using human peripheral blood mononuclear cells (PBMCs), also referred to as the “PBMC assay.”
  • PBMCs peripheral blood mononuclear cells
  • the study results of this example demonstrate that all of the select variants of N142 had reductions in T cell responses, with the N142 variant comprising the amino acid sequence of SEQ ID NO:925 exhibiting the least overall residual immunogenicity risk, as compared with the parent N142.
  • PBMC assay human peripheral blood mononuclear cells
  • PBMCs from healthy human donors were expanded with IL-2 in the presence of N142 or one of its variant proteins for 13 days, followed by restimulation with peptides corresponding to the protein used for initial stimulation. Surface and intracellular staining for flow cytometry was then used to evaluate CD4 T cell cytokine production.
  • PBMCs were seeded at 6.25* 10 6 cell/mL RPMI media with 5% human serum into appropriate culture vessels.
  • Antigen stimulation or vehicle control was added at the time of plating.
  • IL-2 was added on day 4 and maintained thereafter with media changes every 2-3 days.
  • cells from each stimulation condition were counted and redistributed into 96 well plates.
  • Restimulation with peptides or pools of peptides was at a final concentration of 2 pM and all samples were treated with brefeldin A (to block cytokine secretion) overnight.
  • Flow cytometric staining was then performed with the following surface stains: Zombie AquaTM, and anti-CD3, anti-CD4 and anti-CD8 antibodies.
  • Intracellular staining for IFN-y, TNF-a and IL-2 was performed using BD Cytofix/CytopermTM Fixation/Permeabilization staining kit as per the manufacturer's instructions. Following the last step, samples were resuspended in PBS and acquired on a Cytek® Northern LightsTM full spectrum flow cytometer.
  • Peptides used for restimulation of cells expanded in the presence of N142 for the purpose of full CD4+ T cell epitope mapping of N142 are listed in Table 15.
  • the peptides were 18 or 20 amino acids (aa) in length with an offset of 2-4 aa.
  • epitope mapping the peptides during restimulation were used in pools of two sequential peptides walking down the length of the protein as indicated in Table 15.
  • FIG. 1 is a schematic of the PBMC assay measuring T-cell activation, with the details of the assay described in the “Methods” section.
  • immune cells from individual human donors were incubated with N142 or a select N142 variant over a 14-day expansion period.
  • T cells were then evaluated by flow cytometry for cytokine (i.e., IL-2, IFN-y, and TNF-a) production as an indicator of activation.
  • cytokine i.e., IL-2, IFN-y, and TNF-a
  • the sum of the % CD4+ T cells positive for each of the cytokines is termed the “summed cytokine” value.
  • the donors used to evaluate T cell responses represented all 7 of the HLA II supertypes with coverage of 27 alleles contributing to the supertypes (see Greenbaum el al., Immunogenetics. 2011 Jun;63(6):325-35, incorporated herein by reference in its entirety). As such, those donors were a good representation of possible responses.
  • the HLA II alleles covered for each supertype are shown in Table 17.
  • FIG. 2 shows the full CD4+ T cell epitope mapping results of N142 using the PBMC assay, in which healthy human peripheral blood mononuclear cells (PBMCs) from each donor were expanded in the presence of N142 for 14 days, restimulated with synthetic peptides covering the length of the protein (see Table 15) and then evaluated for T cell activation as measured by intracellular cytokine staining and flow cytometry analysis.
  • PBMCs peripheral blood mononuclear cells
  • the summed cytokine value (% IL-2+, INF-y+, and TNF-a+ populations of CD4+ T cells) with background subtraction was plotted as a heatmap, with the cytokine value for each amino acid (AA) position in the protein being the highest cytokine value of the overlapping peptides covering the position.
  • the top bar labeled “Aggregate,” is the max of the summed cytokine values across all donors.
  • the full CD4+ T cell epitope mapping results of FIG. 2 identified the most highly immunogenic regions in N142 that needed to be addressed in deimmunized N142 variant candidates.
  • FIG. 3 shows the targeted CD4+ T cell epitope mapping results of select N142 variants comprising an amino acid sequence selected from one of SEQ ID NOS:859, 868, and 925, as compared to the parent N142 using the PBMC assay, in which healthy human PBMCs from each donor were expanded in the presence of N142 or a select N142 variant for 14 days, restimulated with synthetic peptide corresponding to the protein used for initial stimulation (see Table 16) covering the regions indicated by black bars above the heatmaps and then evaluated for T cell activation as measured by intracellular cytokine staining and flow cytometry analysis.
  • N142 variants displayed markedly reduced T cell responses as compared with N 142.
  • the N142 variant comprising the amino acid sequence of SEQ ID NO: 925 exhibited the least overall residual immunogenicity risk, which was mainly present in two regions approximately corresponding to amino acid (AA) positions 220-240 and AA positions 260-290, respectively.
  • the two other high performing N142 variants comprising the amino acid sequence of SEQ ID NO: 936 or 942 identified in Example 2.1 each had an extra T-cell epitope targeting mutation of N233S in the AA 220-240 region, but otherwise had similar mutations as compared to the select N142 variant comprising the amino acid sequence of SEQ ID NO:925, the former two high performing N142 variants are expected to have similar or better deimmunization profiles as compared to the latter variant.
  • Example 3.1 Immunogenicity analyses of and mapping of T-cell epitopes in wild type IdeS and select variants of IdeS by ex vivo cellular immunoassay using human peripheral blood mononuclear cells (PBMCs)
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • PBMCs from healthy human donors were expanded with IL-2 in the presence of WT IdeS or one of its variant proteins for 13 days, followed by restimulation with peptides corresponding to the protein used for initial stimulation. Surface and intracellular staining for flow cytometry was then used to evaluate CD4 T cell cytokine production.
  • PBMCs were seeded at 6.25* 10 6 cell/mL RPMI media with 5% human serum into appropriate culture vessels.
  • Antigen stimulation or vehicle control was added at the time of plating.
  • IL-2 was added on day 4 and maintained thereafter with media changes every 2-3 days.
  • cells from each stimulation condition were counted and redistributed into 96 well plates.
  • Restimulation with peptides or pools of peptides was at a final concentration of 2 pM and all samples were treated with brefeldin A (to block cytokine secretion) overnight.
  • Flow cytometric staining was then performed with the following surface stains: Zombie AquaTM, and anti-CD3, anti-CD4 and anti-CD8 antibodies.
  • Intracellular staining for IFN-y, TNF-a and IL-2 was performed using BD Cytofix/CytopermTM Fixation/Permeabilization staining kit as per the manufacturer's instructions. Following the last step, samples were resuspended in PBS and acquired on a Cytek® Northern LightsTM full spectrum flow cytometer.
  • Peptides used for restimulation of cells expanded in the presence of WT IdeS for the purpose of full CD4+ T cell epitope mapping of WT IdeS are listed in Table 18.
  • the peptides were 18 or 20 amino acids (aa) in length with an offset of 2-4 aa.
  • epitope mapping the peptides during restimulation were used in pools of two or four sequential peptides walking down the length of the protein as indicated in Table 18.
  • FIG. 1 is a schematic of the PBMC assay measuring T-cell activation, with the details of the assay described in the “Methods” section.
  • immune cells from individual human donors were incubated with WT IdeS or a select IdeS variant over a 14-day expansion period.
  • T cells were then evaluated by flow cytometry for cytokine (i.e., IL-2, IFN-y, and TNF-a) production as an indicator of activation.
  • cytokine i.e., IL-2, IFN-y, and TNF-a
  • the sum of the % CD4+ T cells positive for each of the cytokines is termed the “summed cytokine” value.
  • the donors used to evaluate T cell responses represented all 7 of the HLA II supertypes with coverage of 27 alleles contributing to the supertypes (see Greenbaum el al., Immunogenetics. 2011 Jun;63(6):325-35, incorporated herein by reference in its entirety). As such, those donors were a good representation of possible responses.
  • the HLA II alleles covered for each supertype are shown in Table 20.
  • FIG. 4 shows the full CD4+ T cell epitope mapping results of WT IdeS using the PBMC assay, in which healthy human peripheral blood mononuclear cells (PBMCs) from each donor were expanded in the presence of WT IdeS for 14 days, restimulated with synthetic peptides covering the length of the protein (see Table 18) and then evaluated for T cell activation as measured by intracellular cytokine staining and flow cytometry analysis.
  • PBMCs peripheral blood mononuclear cells
  • the summed cytokine value (% IL-2+, INF-y+, and TNF-a+ populations of CD4+ T cells) with background subtraction was plotted as a heatmap, with the cytokine value for each amino acid (AA) position in the protein being the highest cytokine value of the overlapping peptides covering the position.
  • the top bar labeled “Aggregate,” is the max of the summed cytokine values across all donors.
  • the full CD4+ T cell epitope mapping results of FIG. 4 identified the most highly immunogenic regions in WT IdeS that needed to be addressed in deimmunized IdeS variant candidates.
  • FIG. 5 shows the targeted CD4+ T cell epitope mapping results of select IdeS variants comprising an amino acid sequence selected from one of SEQ ID NOS: 4, 6, 8, 9, 10, 13, 14, and 22, as compared to WT IdeS using the PBMC assay, in which healthy human PBMCs from each donor were expanded in the presence of WT IdeS or a select IdeS variant for 14 days, restimulated with synthetic peptide corresponding to the protein used for initial stimulation (see Table 19) covering the regions indicated by black bars above the heatmaps and then evaluated for T cell activation as measured by intracellular cytokine staining and flow cytometry analysis.
  • FIG. 5 show the max summed cytokine value (% CD4+ T cells positive for IL-2, INF- y, and TNF-a) with background subtraction for all donors for WT IdeS and its select variants, with the cytokine value for each amino acid (AA) position in the protein being the highest cytokine value of the overlapping peptides covering that position.
  • AA amino acid
  • FIG. 5 shows the full epitope mapping of WT IdeS shown in FIG. 4 at the top of FIG. 5.
  • the * symbol in FIG. 5 represents the positions of mutations in each select IdeS variant.
  • the data of FIG. 5 indicate that the most highly immunogenic regions of WT IdeS are covered by the regions selected for evaluation of the select variants.
  • the data also indicate the impact of the mutations in the select IdeS variants on T cell responses in those regions. Namely, all of the select IdeS variants displayed markedly reduced T cell responses as compared with WT IdeS, with the IdeS variant comprising the amino acid sequence of SEQ ID NO: 13 exhibiting the best overall reduction.
  • This example describes additional engineering of IgG protease variants of IdeS (SEQ ID NO:2) identified in Example 3.
  • the additional engineering was carried out by continuing to iteratively design, construct, express, and screen libraries of IgG protease variants of IdeS based on predicted T epitope depletion, thermostability measured by melting temperature, and functional fitness measured by IgG protease activity.
  • the melting temperatures and IgG protease activities of the IdeS variants as well as wild type IdeS were determined by using the methods described in the “Methods” section.
  • Table 21 A shows 461 exemplary functional IgG protease variants of IdeS with at least 30% of the IgG protease activity of wild type IdeS measured at the 4 pg/mL concentration and 1 hour reaction time.
  • variant concentrations and reaction time used for measuring the IgG protease activity of each variant are also indicated in the table.
  • the IgG protease activity of a variant presented both as percent reduction in corrected absorbance at 450 nm of the variant relative to PBS (negative control) and as percentage of IgG protease activity of (parent) wild type IdeS, as described in the “Methods.
  • IgG protease variants of IdeS listed in Table 21A 21 high performing variants with the best overall activity, thermostability, and predicted T epitope depletion profile were identified (Table 21B). Those high performing variants were designed using the IdeS variant of SEQ ID NO: 13 (with 11 amino acid substitutions relative to wild type IdeS, see Table 12 in Example 3) as the scaffold, to which from 13 to 18 additional amino acid substitutions were introduced. The IdeS variant of SEQ ID NO: 13 was selected as the scaffold because it was determined to have a most desirable deimmunization profile in Example 3.1.
  • the high performing IdeS variants retained about 39-77% activity of wild type IdeS measured at 4 pg/mL with 1 h reaction time, with all but four variants of SEQ ID NOS: 1492, 1795, 1797, and 1949 having melting temperatures similar to or better than the melting temperature of about 50°C of wild type IdeS.
  • the high performing IdeS variants are predicted to be even more deimmunized than the scaffold IdeS variant of SEQ ID NO: 13, as each of the high performing variants had from 13 to 18 additional amino acid substitutions targeting immunogenic T cell epitopes, particularly the T cell epitopes in the residual immunogenicity risk regions around amino acid positions 40-60, 220-240, and 260-290 of the scaffold IdeS variant of SEQ ID NO: 13 (see FIG. 5).
  • the high performing IdeS variants unexpectedly retained satisfactory activity and thermostability. Table 21B.
  • each of the 21 high performing IgG protease variants of IdeS harbored two sets of amino acid substitutions.
  • the first set of amino acid substitutions of the 21 high performing IgG protease variants of IdeS included 11 amino acid substitutions present in the scaffold variant of SEQ ID NO: 13, i.e., Y12N, Y18K, A47E, H93Y, E104R, L120T, E126S, Y135N, S159K, V230S, and A275S.
  • the second set of amino acid substitutions of the 21 high performing IgG protease variants of IdeS included from 13 to 18 additional amino acid substitutions that were designed for enhanced targeting of T cell epitopes to further reduce immunogenicity (see the “Mutations to scaffold IdeS variant of amino acid sequence of SEQ ID NO: 13” column of Table 21B).
  • Table 21C summarizes the second set of amino acid substitutions identified among the 21 high performing IgG protease variants of IdeS. Additionally, individual amino acid substitutions in the second set are ranked in descending order of occurrence frequency among the 21 high performing IgG protease variants of IdeS (see the “Occurrence frequency ranking” column in Table 21 C, with the ranking of 1 signifying the highest frequency).
  • the second set of amino acid substitutions present in the 21 high performing IgG protease variants of IdeS included from 13 to 18 amino acid substitutions selected from the group consisting of S3N, F4I, A6S, E9I, HOT, RUT, N48G, I54T, one of T57K, T57N, T57R, T57L, and T57Q, N59D, one of G60S and G60T, K61R, E92R, T138A, one of T161G and T161 Y, D188T, F199L, K200R, E201N, N203T, G222A, L223I, N233S, N246R, N273G, D288G, and Q293R.
  • N48G, N59D, N233S, N246R, N273G, D288G, and Q293R which were present in the high performing IdeS variants most frequently with the rankings of 1 or 2, were intended to target the residual immunogenicity risk regions around amino acid positions 40-60, 220-240, and 260-290 of the scaffold IdeS variant of SEQ ID NO: 13.
  • the high performing IdeS variants are expected to have better deimmunization profiles than the scaffold IdeS variant of SEQ ID NO: 13.
  • Patents, patent applications, patent application publications, journal articles and protocols referenced herein are incorporated by reference in their entireties, for all purposes.

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Abstract

L'invention concerne de nouvelles protéases d'immunoglobuline G (IgG) et des protéines de fusion les comprenant. Les protéases d'IgG trouvent une utilité dans des méthodes de traitement de maladies médiées en totalité ou en partie par des anticorps d'immunoglobuline G (IgG) pathogènes, y compris des méthodes de prévention ou de traitement d'un rejet à médiation par anticorps (AMR) d'une allogreffe d'organe chez un patient ayant subi une greffe d'organe.
PCT/US2024/040614 2023-08-01 2024-08-01 Nouvelles protéases d'igg et leurs procédés d'utilisation Pending WO2025030053A1 (fr)

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