EP4687953A2 - Ides-varianten-proteine und verfahren zur verwendung davon - Google Patents

Ides-varianten-proteine und verfahren zur verwendung davon

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Publication number
EP4687953A2
EP4687953A2 EP24782103.6A EP24782103A EP4687953A2 EP 4687953 A2 EP4687953 A2 EP 4687953A2 EP 24782103 A EP24782103 A EP 24782103A EP 4687953 A2 EP4687953 A2 EP 4687953A2
Authority
EP
European Patent Office
Prior art keywords
substitution
variant protein
ides
asparagine
threonine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24782103.6A
Other languages
English (en)
French (fr)
Inventor
Maximilian Sauer
Daniel Farrell
Kui CHAN
Benjamin DUTZAR
Emily Frazier
Lincoln LEWERKE
Gavin Robertson
Aaron AGUHOB
Jerry CHAN
David THIEKER
Grace DEVANE
Eric TARCHA
Yifan SONG
Erik PROCKO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cyrus Biotechnology Inc
Original Assignee
Cyrus Biotechnology Inc
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Filing date
Publication date
Application filed by Cyrus Biotechnology Inc filed Critical Cyrus Biotechnology Inc
Publication of EP4687953A2 publication Critical patent/EP4687953A2/de
Pending legal-status Critical Current

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    • 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)
    • 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)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • IdeS Immunoglobulin Degrading Enzyme from Streptococcus pyogenes
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement- dependent cytotoxicity
  • IdeS Due to the ability of IdeS to specifically target and cleave IgG, IdeS can significantly dampen antibody-mediated responses. This has been most comprehensively documented in the context of human kidney transplantation, where donor-specific antibodies (DSAs) in the transplant recipient recognize epitopes on the donor graft to drive transplant rejection. Patients with high levels of DSAs may be desensitized by administration of IdeS prior to transplantation, increasing success. Wild-type IdeS is used clinically for desensitization of kidney transplant recipients with high titers of anti-HLA donor-specific antibodies, and may have broader applications in autoimmunity, gene therapy with viral vectors where antibodies are prevalent, and to reduce antibody mediated rejection of xenografts.
  • DSAs donor-specific antibodies
  • IdeS is immunogenic and its administration elicits anti-IdeS antibodies, preventing possible uses where repeated long-term dosing is required.
  • S. Attorney Docket No.: TKT-002WO pyogenes is a ubiquitous pathogen and is the cause of strep throat infections. Many people have mounted antibody defenses to S. pyogenes and IdeS through prior exposure.
  • the presence and elicitation of anti-drug antibodies (ADAs) is of great concern when developing therapeutics, as ADAs have the potential to alter pharmacokinetics, drug activity, and bioavailability, or cause hypersensitivity reactions such as severe anaphylaxis.
  • IdeS variant peptides that have lower immunogenicity, including reduced presentation of peptides on HLA-II and masking of surface epitopes that would otherwise be available for B cell recognition, and preserved or improved IdeS stability and IgG cleavage activity.
  • reduced immunogenic IdeS variants may be used to expand the reach of an IdeS-like drug into indications where repeated dosing is necessary.
  • IdeS variant proteins comprising one or more than one amino acid modifications resulting in lower immunogenicity, measured as reduced presentation of peptides on HLA-II or masking of surface epitopes that are recognized by antibodies, and preserved or improved IdeS stability and IgG cleavage activity as compared to wild-type protein.
  • IdeS variant proteins comprising at least two modifications in positions selected from the group consisting of: 68, 75, 166, 187, 213, 236, 277, 302, 303, 306, and 318 of SEQ ID NO: 1.
  • the modification at position 68 is a substitution of valine to threonine. In some embodiments, the modification at position 75 is a substitution of alanine to proline. In some embodiments, the modification at position 166 is a substitution of threonine to arginine or a substitution of threonine to glycine. In some embodiments, the modification at position 187 is a substitution of serine to aspartate or a substitution of serine to glutamate. In some embodiments, the modification at position 213 is a substitution of threonine to glutamate. In some embodiments, the modification at position 236 is a substitution of serine to cysteine.
  • the modification at position 277 is a substitution of leucine to cysteine.
  • the modification at position 302 is a substitution of serine to aspartate, a substitution of serine to lysine, or a substitution of serine to glutamate.
  • the modification at position 303 is a substitution of alanine to aspartate, a substitution of alanine to asparagine, or a substitution of alanine to glutamine.
  • the modification at position 306 is a substitution of valine to threonine.
  • the modification at position 318 is a substitution of isoleucine to lysine, a Attorney Docket No.: TKT-002WO substitution of isoleucine to aspartate, or a substitution of isoleucine to glycine.
  • the variant protein further comprises a modification at position 308.
  • the modification at position 308 is a substitution of isoleucine to leucine.
  • the variant protein comprises a substitution of valine at position 68 to threonine, a substitution of alanine at position 75 to proline, a substitution of threonine at position 166 to arginine, a substitution of serine at position 187 to aspartate, a substitution of threonine at position 213 to glutamate, a substitution of serine at position 302 to lysine, and a substitution of alanine at position 303 to aspartate.
  • the variant protein comprises a substitution of valine at position 68 to threonine, a substitution of alanine at position 75 to proline, a substitution of threonine at position 166 to arginine, a substitution of serine at position 187 to aspartate, a substitution of threonine at position 213 to glutamate, a substitution of serine at position 302 to glutamate, and a substitution of alanine at position 303 to aspartate.
  • the variant protein comprises a substitution of valine at position 68 to threonine, a substitution of alanine at position 75 to proline, a substitution of threonine at position 166 to arginine, a substitution of serine at position 187 to aspartate, a substitution of threonine at position 213 to glutamate, a substitution of serine at position 302 to lysine, a substitution of alanine at position 303 to aspartate, and a substitution of valine at position 306 to threonine.
  • the variant protein comprises a substitution of valine at position 68 to threonine, a substitution of alanine at position 75 to proline, a substitution of threonine at position 166 to arginine, a substitution of serine at position 187 to aspartate, a substitution of threonine at position 213 to glutamate, a substitution of serine at position 302 to glutamate, a substitution of valine at position 306 to threonine, and a substitution of isoleucine at position 318 to lysine.
  • the variant protein comprises a substitution of valine at position 68 to threonine, a substitution of alanine at position 75 to proline, a substitution of threonine at position 166 to arginine, a substitution of serine at position 187 to aspartate, a substitution of threonine at position 213 to glutamate, a substitution at position 236 of serine to cysteine, a substitution of leucine at position 277 to cysteine, a substitution of serine at position 302 to glutamate, a substitution of alanine at position 303 to aspartate, and a substitution of valine at position 306 to threonine.
  • the variant protein comprises a substitution of valine at position 68 to threonine, a substitution of alanine at position 75 to proline, a substitution of threonine at position 166 to arginine, a substitution of serine at position 187 to aspartate, a substitution of threonine at position 213 to glutamate, a substitution at position 236 of serine to cysteine, a substitution of leucine at position 277 to cysteine, and a substitution of isoleucine at position Attorney Docket No.: TKT-002WO 308 to leucine.
  • the variant protein further comprises a substitution of DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2 with a sequence selected from the group consisting of: TTIQAETSKHTISKKDETLHQNQLSISKTAT (SEQ ID NO: 77), DDYQRNAMEAYAKEVPHQIT (SEQ ID NO: 78), DPNNENEVSNLEKIKKLYPKGFQYGN (SEQ ID NO: 79), DQKLKDYLKNDQLKGTELGKFLEEQGVTK (SEQ ID NO: 80), SAKVETGLPGELAPEEFSFPN (SEQ ID NO: 81), AQNKNPVTHYVNQFDGEEKEII (SEQ ID NO: 82), GSIGEKWDLLLDGIGLNSHRSS (SEQ ID NO: 83), AEPITLIWENYLSDSVSPDRDIR (SEQ ID NO: 84), and QEEIAEGRRNPLRTAEWPMTKSTTDQ (SEQ ID NO:
  • the variant protein further comprises a modification in positions selected from the group consisting of: 31, 32, 33, 38, 39, 43, 44, 45, 47, 54, 57, 60, 74, 77, 82, 85, 113, 115, 116, 127, 128, 129, 130, 133, 148, 153, 154, 159, 167, 168, 175, 188, 195, 197, 210, 218, 219, 220, 228, 233, 241, 244, 245, 247, 258, 273, 274, 278, 288, 289, 297, 299, 300, 307, 313, 314, 315, 316, 322, and 330 of SEQ ID NO: 1.
  • the variant protein comprises a substitution of serine at position 31 to aspartate or asparagine. In some embodiments, the variant protein comprises a substitution of phenylalanine at position 32 to lysine. In some embodiments, the variant protein comprises a substitution of serine at position 33 to glutamate. In some embodiments, the variant protein comprises a substitution of isoleucine at position 38 to valine. In some embodiments, the variant protein comprises a substitution of arginine at position 39 to asparagine or threonine. In some embodiments, the variant protein comprises a substitution of valine at position 43 to glutamate. In some embodiments, the variant protein comprises a substitution of threonine at position 44 to glutamate.
  • the variant protein comprises a substitution of proline at position 45 to glutamate. In some embodiments, the variant protein comprises a substitution of histidine at position 47 to lysine. In some embodiments, the variant protein comprises a substitution of lysine at position 54 to aspartate. In some embodiments, the variant protein comprises a substitution of threonine at position 57 to lysine or glutamine. In some embodiments, the variant protein comprises a substitution of alanine at position 60 to aspartate or glutamine. In some embodiments, the variant protein comprises a substitution of valine at position 74 to lysine. In some embodiments, the variant protein comprises a substitution of alanine at position 75 to asparagine or proline.
  • the variant protein comprises a substitution of glutamine at position 77 to glycine. In some embodiments, the variant protein comprises a substitution of isoleucine at position 82 to Attorney Docket No.: TKT-002WO methionine. In some embodiments, the variant protein comprises a substitution of threonine at position 85 to glutamine. In some embodiments, the variant protein comprises a substitution of glutamine at position 113 to aspartate. In some embodiments, the variant protein comprises a substitution of lysine at position 115 to histidine. In some embodiments, the variant protein comprises a substitution of arginine at position 116 to aspartate.
  • the variant protein comprises a substitution of isoleucine at position 127 to methionine. In some embodiments, the variant protein comprises a substitution of asparagine at position 128 to glycine. In some embodiments, the variant protein comprises a substitution of phenylalanine at position 129 to glutamate. In some embodiments, the variant protein comprises a substitution of asparagine at position 130 to glutamate. In some embodiments, the variant protein comprises a substitution of glutamine at position 133 to aspartate. In some embodiments, the variant protein comprises a substitution of leucine at position 148 to asparagine or arginine.
  • the variant protein comprises a substitution of phenylalanine at position 153 to methionine or tyrosine. In some embodiments, the variant protein comprises a substitution of glutamate at position 154 to aspartate. In some embodiments, the variant protein comprises a substitution of lysine at position 159 to asparagine. In some embodiments, the variant protein comprises a substitution of lysine at position 167 to proline. In some embodiments, the variant protein comprises a substitution of histidine at position 168 to aspartate or proline. In some embodiments, the variant protein comprises a substitution of histidine at position 175 to glutamine.
  • the variant protein comprises a substitution of leucine at position 188 to glycine or methionine. In some embodiments, the variant protein comprises a substitution of proline at position 195 to aspartate. In some embodiments, the variant protein comprises a substitution of lysine at position 197 to glutamate. In some embodiments, the variant protein comprises a substitution of alanine at position 210 to glycine. In some embodiments, the variant protein comprises a substitution of serine at position 218 to aspartate. In some embodiments, the variant protein comprises a substitution of lysine at position 219 to glycine. In some embodiments, the variant protein comprises a substitution of leucine at position 220 to glutamine.
  • the variant protein comprises a substitution of lysine at position 228 to glutamine. In some embodiments, the variant protein comprises a substitution of lysine at position 233 to serine. In some embodiments, the variant protein comprises a substitution of lysine at position 241 to aspartate. In some embodiments, the variant protein comprises a substitution of threonine at position 244 to lysine. In some embodiments, the variant protein comprises a substitution of glutamate at position 245 to aspartate. In some embodiments, the Attorney Docket No.: TKT-002WO variant protein comprises a substitution of lysine at position 247 to asparagine.
  • the variant protein comprises a substitution of valine at position 258 to aspartate. In some embodiments, the variant protein comprises a substitution of serine at position 273 to aspartate. In some embodiments, the variant protein comprises a substitution of asparagine at position 274 to glutamate. In some embodiments, the variant protein comprises a substitution of lysine at position 278 to aspartate or threonine. In some embodiments, the variant protein comprises a substitution of asparagine at position 288 to aspartate, glycine, or glutamine. In some embodiments, the variant protein comprises a substitution of alanine at position 289 to proline.
  • the variant protein comprises a substitution of phenylalanine at position 297 to aspartate. In some embodiments, the variant protein comprises a substitution of glycine at position 299 to aspartate. In some embodiments, the variant protein comprises a substitution of valine at position 300 to glutamate. In some embodiments, the variant protein comprises a substitution of alanine at position 307 to glycine or asparagine. In some embodiments, the variant protein comprises a substitution of isoleucine at position 313 to proline. In some embodiments, the variant protein comprises a substitution of lysine at position 314 to glycine. In some embodiments, the variant protein comprises a substitution of glutamate at position 315 to proline.
  • the variant protein comprises a substitution of aspartate at position 316 to proline. In some embodiments, the variant protein comprises a substitution of valine at position 322 to threonine. In some embodiments, the variant protein comprises a substitution of threonine at position 330 to aspartate. [0009] Described herein, in certain embodiments, are IdeS variant proteins comprising the amino acid sequence according to any one of SEQ ID NOs: 3-75. [0010] Described herein, in certain embodiments, are IdeS variant proteins comprising the amino acid sequence according to SEQ ID NO: 10. [0011] Described herein, in certain embodiments, are IdeS variant proteins comprising the amino acid sequence according to SEQ ID NO: 11.
  • IdeS variant proteins comprising the amino acid sequence according to SEQ ID NO: 12.
  • IdeS variant proteins comprising the amino acid sequence according to SEQ ID NO: 13.
  • IdeS variant proteins comprising a substitution of DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2 with a sequence at least 80% identical to the sequence selected from the group consisting of: Attorney Docket No.: TKT-002WO TTIQAETSKHTISKKDETLHQNQLSISKTAT (SEQ ID NO: 77), DPNNENEVSNLEKIKKLYPKGFQYGN (SEQ ID NO: 79), DQKLKDYLKNDQLKGTELGKFLEEQGVTK (SEQ ID NO: 80), SAKVETGLPGELAPEEFSFPN (SEQ ID NO: 81), AQNKNPVTHYVNQFDGEEKEII (SEQ ID NO: 82), GSIGEKWDLLLDGIGLNSHRSS (SEQ ID NO: 83), AEPITLIWENYLSDSVSPDRDIR (SEQ ID NO: 84), and QEE
  • IdeS variant proteins comprising a substitution of DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2 with the sequence DDYQRNAMEAYAKEVPHQIT (SEQ ID NO: 78).
  • IdeS variant proteins comprising the amino acid sequence according to SEQ ID NOs: 86-94.
  • the variant protein has decreased presentation of epitopes on human leukocyte antigen (HLA) as compared to a wild-type IdeS protein.
  • HLA human leukocyte antigen
  • the variant protein has increased cleavage activity of immunoglobulins as compared to a wild-type IdeS protein. In some embodiments, the variant protein has a decrease in immunogenicity score more than 0 relative to SEQ ID NO: 1 or SEQ ID NO: 2. [0017] Described herein, in certain embodiments, are IdeS variant proteins comprising one or more glycosylation modifications as compared to wild-type.
  • IdeS variant proteins comprising at least one modification in positions selected from the group consisting of: 31, 37, 39, 42, 74, 76, 111, 113, 119, 121, 130, 142, 144, 147, 148, 198, 233, 244, 246, 311, 313, and 319 of SEQ ID NO: 1 to introduce a glycosylation site.
  • the modification at position 31 is a substitution of serine to asparagine.
  • the modification at position 37 is a substitution of glutamate to asparagine.
  • the modification at position 39 is a substitution of arginine to threonine or asparagine.
  • the modification at position 42 is a substitution of glutamate to asparagine.
  • the modification at position 74 is a substitution of valine to asparagine.
  • the modification at position 76 is a substitution of asparagine to serine.
  • the modification at position 111 is a substitution of lysine to asparagine.
  • the modification at position 113 is a substitution of glutamine to threonine.
  • the modification at position 119 is a substitution of glutamate to asparagine.
  • the modification at position 121 is a substitution of histidine to threonine.
  • the modification at Attorney Docket No.: TKT-002WO position 130 is a substitution of asparagine to serine.
  • the modification at position 142 is a substitution of aspartate to asparagine.
  • the modification at position 144 is a substitution of lysine to threonine.
  • the modification at position 147 is a substitution of glutamine to serine.
  • the modification at position 148 is a substitution of leucine to asparagine.
  • the modification at position 198 is a substitution of glutamate to asparagine.
  • the modification at position 233 is a substitution of lysine to serine.
  • the modification at position 244 is a substitution of threonine to asparagine.
  • the modification at position 246 is a substitution of glycine to threonine.
  • the modification at position 311 is a substitution of lysine to asparagine.
  • the modification at position 313 is a substitution of isoleucine to threonine.
  • the modification at position 319 is a substitution of glycine to serine.
  • the variant protein comprises a substitution of glutamate at position 37 to asparagine and a substitution of arginine at position 39 to threonine.
  • the variant protein comprises a substitution of valine at position 74 to asparagine and a substitution of asparagine at position 76 to serine. In some embodiments, the variant protein comprises a substitution of lysine at position 111 to asparagine and a substitution of glutamine at position 113 to threonine. In some embodiments, the variant protein comprises a substitution of glutamate at position 119 to asparagine and a substitution of histidine at position 121 to threonine. In some embodiments, the variant protein comprises a substitution of aspartate at position 142 to asparagine and a substitution of lysine at position 144 to threonine.
  • the variant protein comprises a substitution of threonine at position 244 to asparagine and a substitution of glycine at position 246 to threonine. In some embodiments, the variant protein comprises a substitution of lysine at position 311 to asparagine and a substitution of isoleucine at position 313 to threonine.
  • IdeS variant proteins comprising at least one modification in positions selected from the group consisting of: 47, 49, 51, 78, 111, 113, 123, 125, 126, 128, 142, 144, 148, 198, 273, 275, 278, 312, and 314 of SEQ ID NO: 12 to introduce a glycosylation site.
  • the modification at position 47 is a substitution of threonine to asparagine.
  • the modification at position 49 is a substitution of glutamine to threonine or asparagine.
  • the modification at position 51 is a substitution of valine to threonine.
  • the modification at position 78 is a substitution of glycine to serine.
  • the Attorney Docket No.: TKT-002WO modification at position 111 is a substitution of lysine to asparagine.
  • the modification at position 113 is a substitution of glutamine to threonine.
  • the modification at position 123 is a substitution of glutamate to asparagine.
  • the modification at position 125 is a substitution of glutamine to threonine.
  • the modification at position 126 is a substitution of lysine to asparagine.
  • the modification at position 128 is a substitution of asparagine to serine.
  • the modification at position 142 is a substitution of aspartate to asparagine.
  • the modification at position 144 is a substitution of lysine to threonine.
  • the modification at position 148 is a substitution of leucine to asparagine.
  • the modification at position 198 is a substitution of glutamate to asparagine.
  • the modification at position 273 is a substitution of serine to asparagine.
  • the modification at position 275 is a substitution of glycine to serine.
  • the modification at position 278 is a substitution of lysine to serine.
  • the modification at position 312 is a substitution of glutamate to asparagine. In some embodiments, the modification at position 314 is a substitution of lysine to serine. In some embodiments, the variant protein comprises a substitution of threonine at position 47 to asparagine and a substitution of glutamine at position 49 to threonine. In some embodiments, the variant protein comprises a substitution of glutamine at position 49 to asparagine and a substitution of valine at position 51 to threonine. In some embodiments, the variant protein comprises a substitution of glutamate at position 123 to asparagine and a substitution of glutamine at position 125 to threonine.
  • the variant protein comprises a substitution of lysine at position 126 to asparagine and a substitution of asparagine at position 128 to serine. In some embodiments, the variant protein comprises a substitution of serine at position 273 to asparagine and a substitution of glycine at position 275 to serine. In some embodiments, the variant protein comprises a substitution of glutamate at position 312 to asparagine and a substitution of lysine at position 314 to serine.
  • the variant protein comprises a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of aspartate at position 142 to asparagine, and a substitution of lysine at position 144 to threonine. In some embodiments, the variant protein comprises a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of aspartate at position 142 to asparagine, a substitution of lysine at position 144 to threonine, Attorney Docket No.: TKT-002WO and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of aspartate at position 142 to asparagine, a substitution of lysine at position 144 to threonine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of glutamine at position 49 to asparagine, a substitution of valine at position 51 to threonine, a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of lysine at position 126 to asparagine, a substitution of asparagine at position 128 to serine, a substitution of leucine at position 148 to asparagine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of glutamine at position 49 to asparagine, a substitution of valine at position 51 to threonine, a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of leucine at position 148 to asparagine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of lysine at position 126 to asparagine, a substitution of asparagine at position 128 to serine, a substitution of leucine at position 148 to asparagine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of glutamine at position 49 to asparagine, a substitution of valine at position 51 to threonine, a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of lysine at position 126 to asparagine, a substitution of asparagine at position 128 to serine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises, a substitution of threonine at position 47 to asparagine, a substitution of glutamine at position 49 to asparagine, a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of glutamine at position 49 to asparagine, a substitution of valine at position 51 to threonine, a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of glutamate at position 123 to asparagine, a substitution of glutamine at position 125 to Attorney Docket No.: TKT-002WO threonine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of lysine at position 126 to asparagine, a substitution of asparagine at position 128 to serine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of leucine at position 148 to asparagine, and a substitution of glutamate at position 198 to asparagine.
  • polypeptides comprising an IdeS protein comprising at least 90% sequence identity to any one of SEQ ID NOs: 1-75 conjugated to a human serum albumin.
  • the human serum albumin is conjugated to a N-terminal of the IdeS protein.
  • the human serum albumin is conjugated to a C-terminal of the IdeS protein.
  • the IdeS protein is conjugated to the human serum albumin using a linker.
  • the linker comprises GSGGGSG (SEQ ID NO: 113), GSGSGSGS (SEQ ID NO: 114), GSGGGSGGGSG (SEQ ID NO: 115), GSGSGSGSGSGS (SEQ ID NO: 116) or GS.
  • the IdeS protein is fused to the human serum albumin.
  • polypeptides polypeptide comprising: a) an IdeS variant protein comprising: i) at least two modifications in positions selected from the group consisting of: 68, 75, 166, 187, 213, 236, 277, 302, 303, 306, and 318 of SEQ ID NO: 1; and ii) at least one modification in positions selected from the group consisting of: 31, 37, 39, 42, 74, 76, 111, 113, 119, 121, 130, 142, 144, 147, 148, 198, 233, 244, 246, 311, 313, and 319 of SEQ ID NO: 1 or at least one modification in positions selected from the group consisting of: 47, 49, 51, 78, 111, 113, 123, 125, 126, 128, 142, 144, 148, 198, 273, 275, 278, 312, and 314 of SEQ ID NO: 12 to introduce a glycosylation site;
  • Described herein, in certain embodiments, are polynucleotides encoding the IdeS variant protein described herein. [0023] Described herein, in certain embodiments, are expression plasmids comprising the polynucleotide described herein and a promoter. [0024] Described herein, in certain embodiments, are cells comprising the polynucleotide described herein. Attorney Docket No.: TKT-002WO [0025] Described herein, in certain embodiments, are pharmaceutical compositions comprising the IdeS variant protein described herein and a pharmaceutically acceptable carrier.
  • the IdeS variant protein is co-administered with a gene therapy.
  • FIGs.1A-1G depict data showing IdeS expressed by mammalian cells is active and glycosylated.
  • FIG.1A depicts SDS-PAGE analysis of fractions from NiNTA affinity chromatography of his-tagged IdeS proteins.
  • L load; FT, flow through.
  • His-tagged IdeS proteins were expressed intracellularly in E. coli (EC) or secreted by Expi293F cells (XP). Proteins were eluted in PBS containing 250 mM imidazole. The calculated MW of tagged IdeS (excluding glycans) is 36 kD.
  • FIG.1B depicts eluates from NiNTA purification were concentrated and separated by SEC.
  • FIG.1C depicts IdeS (0.25 or 0.5 ⁇ g) purified from Expi293F expression medium was treated with PNGase F and analyzed by SDS-PAGE.
  • FIG.1D depicts IdeS-catalyzed cleavage of IgG produces fragments that are resolvable by non-reducing SDS-PAGE. The first cut in the hinge produces Fc/2 and single cut IgG (scIgG). The second cut in the neighboring hinge releases F(ab')2 and two Fc/2.
  • FIG.1E depicts expression medium from Expi293F cells transfected with IdeS was incubated with Attorney Docket No.: TKT-002WO IVIG and cleavage products were detected by SDS-PAGE (upper panels). Expression level of IdeS proteins was assessed by SDS-PAGE of the expression medium (lower panels). Asn residues in consensus N-glycosylation motifs (N-X-S/T) were mutated to Gln.
  • FIGs.1F-1G depict activity of purified IdeS (20 nM) from bacteria (FIG.1F) versus Expi293F culture (FIG.1G) was assessed based on proteolysis of a non-specific monoclonal IgG1 (20 ⁇ M).
  • FIGs.2A-2C depicts HLA-II epitopes in IdeS identified by MAPPs.
  • FIG.2A depicts monocyte-derived DCs from 10 donors were pulsed with IdeS. pMHC-II complexes were immunoprecipitated and bound IdeS peptides were identified by mass spectrometry. IdeS residues that are found in antigenic peptides with high frequency are in darker gray. The summed total of all peptide counts for each residue across the 10 donors is shown at the bottom.
  • FIG.2B depicts the summed peptide counts for each residue is mapped on to a model that was derived from the IdeS crystal structure (PDB 2AVW).
  • FIG.2C depicts epitope Clusters 1-8, which are labeled, on the IdeS structure. Side chains of the catalytic triad are shown as black spheres (203). Cluster 1 is only partially shown as it resides in the unstructured N-terminus that is missing in the model.
  • FIG.3 depicts a summary of predicted and experimentally identified HLA-II epitopes in IdeS and reduced immunogenic modifications. A schematic of the IdeS secondary structure is shown at the top.
  • FIG.4 depicts data from rapid screening of IdeS variants.
  • IdeS was expressed as a secreted protein in Expi293F cells and the expression medium was incubated directly with IVIG for 1 h at 37 ⁇ C. Upper panels show Coomassie-stained gels of IVIG degradation products under non-reducing conditions. Lower panels show IdeS levels in the expression medium (calculated MW of IdeS is 36 kD, Attorney Docket No.: TKT-002WO excluding glycans). This figure is representative of the screening process and shows results for a subset of the single point modifications that were evaluated. Wt, wild type. [0035] FIG.5 depicts data from rapid screening of IdeS variants.
  • FIG.6 depicts data from introduction of potential disulfide bonding cysteines into IdeS. Variants of IdeS with added cysteines, predicted to form disulfides, were expressed by Expi293F cells and the expression medium screened for proteolytic activity against IVIG.
  • FIGs.7A-7F depict data demonstrating a reduced immunogenic IdeS derivative is active and selective for human and rabbit IgG. Wild type (wt) IdeS (FIGs.7A-7C) and Variant 77 (FIGs.7D-7F) were purified from transfected Expi293F culture.
  • FIGs.8A-8B depict data from screening of IdeS truncation and chimera variants at the protein's N-terminus.
  • FIG.8A depicts Coomassie-stained SDS-gels to measure activity against human IVIG (upper gel image) and expression (lower gel image) of IdeS truncation variants and N-terminal chimeras.
  • Wt wild type.
  • FIG.8B depicts multiple sequence alignment of Wt IdeS with sequence and structural homologs used for creating N-terminal chimeras (SEQ ID NOS 196- 214, respectively, in order of appearance). Numbering at top is based on full-length IdeS (Wt).
  • FIGs.9A-9B depict data demonstrating combining multiple modifications for reducing immunogenicity risks destabilizing the protein fold.
  • FIG.9A depicts data of additional modifications for reducing immunogenicity and N-terminal chimerism were added to the Variant 77 intermediate to create derivatives IdeS Variants 100, 101, 102, 103, 1, 104, Attorney Docket No.: TKT-002WO 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, and 116. Based on its high expression and catalytic activity, Variant 1 was advanced. A small number of reversion and compensatory modifications (Variant 2 to Variant 7) were evaluated in an effort to increase thermal stability.
  • FIG.9B depicts a graph of melting temperatures of Variant 1 and its progeny as measured by DSF.
  • FIGs.10A-10B depict thermostability data of IdeS variants.
  • FIG.10A is a graph of DSF thermal melt curves of purified IdeS proteins. Wt, wild type.
  • FIG.10B is a graph of melting temperatures from DSF analysis.
  • FIGs.11A-11D depicts data demonstrating a reduced immunogenic IdeS protein has high catalytic activity in vitro.
  • FIG.11A depicts squared immunogenicity scores plotted across the sequences of wild type IdeS and Variant 10. Higher scores correspond to regions predicted to have high affinity to multiple HLA-II alleles.
  • FIG.11B depicts NiNTA affinity purification of Variant 10 expressed intracellularly in E.
  • FIG.11C depicts the NiNTA eluate was concentrated and separated by SEC. Peak fractions (numbered 11-13) are indicated by vertical broken lines. A Coomassie-stained electrophoretic gel of fractions 11-13 is shown at right. a.u., absorbance units.
  • FIG.11D depicts Purified Variant 10 (20 nM) is highly active for proteolyzing a human monoclonal IgG1 (20 ⁇ M). Degradation products were visualized on a Coomassie-stained polyacrylamide gel run under non-reducing conditions.
  • FIG.12 depicts graphs of binding of IdeS peptides to HLA-DR1.
  • FIG.13 depicts graphs of binding of IdeS peptides to HLA-DR7.
  • FIG.14 depicts binding of IdeS peptides to HLA-DR11. Wild-type (circles) and mutant (squares) peptides competed with a fluorescent reference peptide for binding to HLA- DRA1/HLA-DRB11. FP of the reference peptide is plotted. [0045] FIG.15 depicts binding of IdeS peptides to HLA-DR15.
  • FIGs.16A-16B depict data from reduced immunogenic IdeS variants administered intravenously showing rapid degradation of IgG in rabbits.
  • FIG.16A depicts data from two rabbits (indicated by light and dark grey circles beneath the sample lanes) that were administered Variant 10 (4 mg dose) and blood was collected at the indicated times via ear vein. Serum was analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions.
  • FIG.16B depicts serum IgG levels measured by ELISA following intravenous injection of 4 mg of wild type (Wt) IdeS or Variant 10 purified from E. coli.
  • FIG.17 depicts graphs of levels of anti-IdeS antibodies evaluated by ELISA in serum from 10 healthy males (upper left), 10 healthy females (upper right), and 9 donors convalescent for recent S. pyogenes infection (lower left). Plates were coated with wild-type IdeS. For reference, IVIG and polyclonal goat anti-IdeS are also shown and a high titer healthy donor serum sample (donor HMN799367; yellow) is highlighted.
  • donor HMN799367 high titer healthy donor serum sample
  • FIGs.18A-18F depict results of IdeS variants with custom N-glycosylation sites.
  • FIG.18A depicts single N-glycosylation motifs were added to wild-type IdeS and combinations of motifs Variants 10.1, 10.2, 10.3, and 10.4 were added to Variant 10. The proteins were expressed and secreted by Expi293F cells.
  • Upper Coomassie-stained electrophoresis gels show human IVIG degradation products following incubation with expression medium. The gel was run under non-reducing conditions. The lower gels were run under reducing conditions and show IdeS expression in the medium and shifts to higher MW for some of the mutants.
  • FIG.18B depicts structural model of IdeS (labeled in figure) bound to cleaved product IgG Fc (labeled in figure) with positions 111, 148, and 198 that were sites of custom N-glycosylation labeled and shown as spheres.
  • Wild-type residue N61 (labeled and shown as spheres) is also glycosylated (see FIG.1E).
  • catalytic residue C94 is labeled and shown as spheres.
  • FIG.18C depicts variants 10.1, 10.2, 10.3, and 10.4 combine custom glycosylation sites at positions 111, 148, and 198.
  • FIG.18D depicts DSF analysis of purified IdeS hyperglycosylation mutants.
  • FIG.18E depicts melting temperatures of hyperglycosylated IdeS variants.
  • FIG.18F depicts purified IdeS variants were analyzed on a Coomassie-stained electrophoresis gel.
  • FIG.19A depicts custom N-glycosylation sites that were introduced into Variant 10 and the variant proteins were secreted into the expression medium of transfected Expi293F cells.
  • FIGs.19B-19C depict purified IdeS variants containing (FIG.19B) 5-6 or (FIG. 19C) 4 N-glycosylation sites that were incubated with human monoclonal IgG1 and cleavage products were analyzed by non-reducing SDS-PAGE.
  • FIG.19D depicts thermal stability by conventional DSF of purified IdeS variants carrying, at left, 5-6 N-glycosylation sites, or at right, 4 N-glycosylation sites. Control samples are buffer solution only.
  • FIGs.20A-20C depict properties of glycosylated IdeS variants.
  • FIG.20A depicts melting temperatures of IdeS proteins measured by conventional DSF.
  • FIG.20B depicts purified IdeS proteins that were separated by SDS-PAGE before (at left) and after (at right) treatment with PNGase F. The theoretical MW of wild-type IdeS without any added glycans is 36 kD.
  • FIG.20C depicts 20 ⁇ M IgG from different species that was incubated with 20 nM Variant 10.9 at 37 °C for up to 60 minutes. Cleavage products were separated by non- reducing SDS-PAGE.
  • FIG.21A depicts His-tagged Variant 10.9 expressed by Expi293F cells that was purified by NiNTA affinity chromatography. M, marker; FT, flow through. The eluted protein has an apparent MW of ⁇ 50 kD versus aglycosylated IdeS has a theoretical MW of 36 kD.
  • FIG.21B depicts SEC separation on a Superdex 200 increase 10/300 GL column of aglycosylated Variant 10.14 (broken line) and hyperglycosylated Variant 10.9 (solid line).
  • FIG.22A-22B depict occlusion of the accessible surface on a hyperglycosylated IdeS variant by glycans.
  • FIG.22A shows a surface representation of Variant 10.9 modeled as homogenously glycosylated with tetraantennary glycans at the 4 N-glycosylation sites labeled in boxes.
  • glycans are depicted using the symbol nomenclature for glycans (SNFG).
  • SNFG symbol nomenclature for glycans
  • FIG.22B depicts the protein surface of Variant 10.9 with amino acids that are Attorney Docket No.: TKT-002WO occluded by glycans shaded in darker grey.
  • the contact surface for Fc substrate/product is based on PDB 8A47 and is outlined in a broken line and hatched.
  • FIG.24A depicts IdeS concentrations as measured by ELISA in serum collected from NZW rabbits intravenously administered a 4 mg dose ( ⁇ 1 mg/kg). IdeS levels in rabbit serum are below the limit of detection for all variants after 24 h.
  • FIG.24B depicts ELISA measurements of serum IgG in IdeS treated rabbits.
  • FIG.25A depicts structural representation for size comparison showing HSA (surface) at the C-terminus of IdeS (ribbon). Nucleophile C94 is labeled and shown as spheres and glycosylated asparagines of Variant 10.1 are labeled and shown as spheres.
  • FIG.25B depicts Coomassie-stained SDS gel of Expi293F expression medium showing secreted Variant 10.14 without (left lane) and with (right lane) fusion to HSA.
  • FIG.25C depicts purified Variant 10.14 with and without HSA fusion (20 nM) was incubated with human monoclonal IgG1 (20 ⁇ M) at 37 °C for 60 minutes. Cleavage products were separated by non-reducing SDS-PAGE.
  • FIG.25D depicts Coomassie-stained SDS gel of expression medium from Expi293F transfected with Variant 10.14 -HSA with different connecting linkers.
  • FIG.25E depicts cleavage products of monoclonal human IgG1 separated by non- reducing SDS-PAGE after incubation with linker variants of Variant 10.14 -HSA.
  • FIG.25F depicts ELISA using goat polyclonal anti-IdeS for capture and detection of Variant 10.14 -HSA linker variants.
  • FIG.28A depicts Coomassie-stained non-reducing SDS gel of human IgG1 (20 ⁇ M) cleaved by IdeS-HSA variants (20 nM) at 37 ⁇ C.
  • FIG.28B depicts Nano DSF measurements of change in intrinsic tryptophan fluorescence of IdeS variants as they are heated. HSA has a single tryptophan that has minor contribution to the fluorescence signal.
  • FIGs.29A-29D show reduced reactivity of IdeS variants towards human serum.
  • FIG.29A depicts a plot of EC50 values (measured as a dilution factor) from ELISA experiments in which plates were directly coated with equal concentrations of Wt IdeS (E. coli produced, black), Variant 10.2-HSA (dark grey), and Variant 10.9 (pale grey). Reactivity towards pooled IVIG or human serum from 5 donors was assayed and error bars show 95% confidence intervals.
  • FIGs.29B-29D depict competition ELISA analysis. ELISA plates were coated with Wt IdeS (E. coli produced) and high titer serum from a donor recently recovered from S.
  • FIG.30A depicts IdeS-HSA concentrations as measured by ELISA in serum collected from NZW rabbits that were intravenously administered Variant 10.2-HSA at 0.3 mg/kg (open squares and black line) or 1 mg/kg (filled triangles and grey line). For comparison, data from rabbits administered Variant 10.14-HSA (an aglycosylated variant, filled triangles and broken black line) are also shown.
  • FIG.30C depicts quantitative IgG levels calculated as a percent of baseline for each individual rabbit following administration ( ⁇ 1 mg/kg) with Wt IdeS (E. coli produced, black line and diamonds), Variant 10.2-HSA (grey line and triangles), or Variant 10.9 (open squares and broken line).
  • FIGs.31A-31D show that antigen-binding fragments are cleared in 24 hours following IdeS-catalyzed IgG cleavage in rabbits.
  • FIG.31A-31C show anti-rabbit Fab western blots of serum samples collected from rabbits treated with 1 mg/kg of Variant 10.9 (FIG.31A), Variant 10.2-HSA (FIG.31B), or Wt IdeS produced in Expi293F culture (FIG. 31C).
  • FIG.31D depicts ELISA analysis of serum samples from rabbits administered Wt Attorney Docket No.: TKT-002WO IdeS produced in Expi293F culture. IgG/scIgG levels (pale grey) were assayed using anti- rabbit F(ab') 2 for capture and anti-rabbit Fc for detection.
  • Variant 10.9 solid line and triangles, FIG.32A
  • Variant 10.2-HSA solid line and diamonds, FIG.32B
  • Wt Ides solid line and squares, E. coli produced
  • Proteins treated with Arthrobacter ureafaciens neuraminidase (NA) are shown in broken lines.
  • DETAILED DESCRIPTION [0076] The present application provides for a modified IdeS protein comprising lower immunogenicity, measured as reduced presentation of peptides on HLA-II or shielding of surface epitopes that are recognized by antibodies, and preserved or improved IdeS stability and IgG cleavage activity. To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.
  • the terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.
  • the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably include humans.
  • the term “pharmaceutical formulation” refers to the combination of an active agent (e.g., an IdeS variant) with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.
  • an active agent e.g., an IdeS variant
  • a carrier inert or active
  • pharmaceutically acceptable carrier refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • proteins or polypeptide are used interchangeably herein and refer to a polymer of repeating structural units connected by a peptide bond.
  • the repeating structural units of the peptide are amino acids including naturally occurring amino acids, non- naturally occurring amino acids, analogues of amino acids or any combination of these.
  • proteins or polypeptides may be post-translationally modified (e.g.
  • sequence identity means the proportion of amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 85%, the percentage denotes the fraction of matches over the length of sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; for example, wherein gap lengths of 5 amino acids or less, optionally 3 amino acids or less, are usually used.
  • Percent sequence identity can be any integer from 60% to 100%. Exemplary embodiments include at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. [0085] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared.
  • sequence comparison algorithm When using a sequence comparison algorithm, 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. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. [0086] 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 et al. (1990) J. Mol. Biol.215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively.
  • TKT-002WO high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always >0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are 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, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
  • 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)).
  • BLAST algorithm 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.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10 -5 , and most preferably less than about 10 -20 .
  • the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, “about” means within a standard deviation using measurements generally acceptable in the art.
  • the term “functional fragment thereof” refers to a portion of a protein or polypeptide that maintains the ability to perform a biological function of the whole protein or polypeptide.
  • a functional fragment of a polypeptide or protein of the present application maintains its ability to perform its catalytic activity.
  • the terms “mutation,” “modification,” and “substitution” are used interchangeably and refer to the alteration of an amino acid, in the context of a reference amino acid sequence, to another amino acid.
  • an alteration of an amino acid in a reference amino acid sequence can occur at the N-terminal or C-terminal position or anywhere between those terminal positions. Modifications may be interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a substitution can be, but need not necessarily be, a conservative substitution. Twenty amino acids are commonly found in proteins. Those amino acids can be grouped into nine classes or groups based on the chemical properties of their side chains. Substitution of one amino acid residue for another within the same class or group is referred to herein as a “conservative” substitution. Conservative amino acid substitutions can frequently be made in a protein without significantly altering the conformation or function of the protein.
  • a conservative amino acid substitution includes substituting any of: glycine (G), alanine (A), isoleucine (I), valine (V
  • substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein.
  • G glycine
  • A alanine
  • V valine
  • M Methionine
  • L Lysine
  • R arginine
  • deletion refers to the removal of one or more than one amino acid residue in the context of a reference amino acid sequence. A deletion can occur at the N-terminal or C-terminal position or anywhere between those terminal positions.
  • Deletions may be interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence. When occurring at the N-terminal or C-terminal positions, a deletion of one or more than one contiguous amino acids can also be referred to as a “truncation.” [0094] As used herein, the terms “mutant” and “variant” are used interchangeably and refer to a protein, or enzyme having one or more than one mutation and/or deletion in the context of a reference sequence (e.g., a wild-type sequence).
  • nucleic acid or “oligonucleotide” or “polynucleotide” or grammatical equivalents used herein means at least two nucleotides covalently linked together.
  • nucleic acid includes single-, double-, or multiple-stranded DNA, RNA and analogs (derivatives) thereof.
  • Oligonucleotides can be from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 45 or more nucleotides in length, up to about 100 nucleotides in length.
  • Nucleic acids and polynucleotides are polymers of any length, including longer lengths, e.g., 200, 300, 440, 1000, 2000, 3000, 4400, 7000, 10,000, etc. nucleotides in length.
  • the term “vector” refers to a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • the vector is a virus vector, such as a lentivirus vector.
  • N-glycosylation refers to the attachment of a sugar molecule oligosaccharide known as glycan to a nitrogen atom of an amino acid in a protein.
  • O-glycosylation refers to the attachment of a sugar molecule oligosaccharide (i.e., a glycan) to an oxygen atom of an amino acid in a protein.
  • sialylation is the enzymatic addition of a neuraminic acid residue.
  • N-glycan refers to a core structure generally comprising two N-Acetyl-D-glucosamine (GlcNAc) and three mannose molecules. N-glycans can be added to an asparagine (Asn) side chain via N-linked glycosylation initiated by oligosaccharyltransferase complex in the endoplasmic reticulum membrane.
  • N-glycans are attached to Asn located in a sequence of Asn-X-Serine/Threonine, whereby X can be any amino acid apart from proline.
  • N-glycans may be expressed as: (Man ⁇ 1-6[Man ⁇ 1- 3)]Man( ⁇ 1-4)GlcNAc( ⁇ 1-4)GlcNAc( ⁇ 1-Asn-X-Ser/Thr).
  • the N-glycan core structure can be expanded through galactosylation, further GlcNAclyation, sialylation, fucosylation, or combinations thereof.
  • the term “biantennary” refers to an N-linked glycan comprising the N-glycan core (Man ⁇ 1-6[Man ⁇ 1-3)]Man( ⁇ 1-4)GlcNAc( ⁇ 1-4)GlcNAc( ⁇ 1-Asn-X-Ser/Thr) elongated with two GlcNAc residues linked to C-2 of the core mannose ⁇ 1-3 and the mannose ⁇ 1-6.
  • This core structure can then be elongated or modified by various glycan structures.
  • triantennary refers to an N-linked glycan comprising an additional GlcNAc residue is added to either the C-4 of the core mannose ⁇ 1-3 or the C-6 of the core mannose ⁇ 1-6 of the biantennary core structure. This structure can then be elongated or modified by various glycan structures.
  • tetraantennary refers to an N-linked glycan comprising two additional GlcNAc residues that are added to either the C-4 of the core mannose ⁇ 1-3 or the C-6 of the core mannose ⁇ 1-6 of the biantennary core structure.
  • This core structure can then be elongated or modified by various glycan structures.
  • I. Modified IdeS proteins [00105] Described herein, in certain embodiments, are modified IdeS proteins comprising one or more modifications as compared to wild-type IdeS sequence.
  • the present disclosure provides an IdeS variant comprising at least two modifications as compared to wild-type IdeS sequence, and having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO:1.
  • the present disclosure provides an IdeS variant at least 95% identical to SEQ ID NO: 1 and having one or more modifications.
  • the present disclosure provides an IdeS variant at least 95% identical to SEQ ID NO: 1 and having at least two modifications.
  • IdeS Variants [00108] Described herein are modified IdeS proteins.
  • the IdeS protein is modified to have reduced presentation on HLA-II.
  • the IdeS protein is modified (e.g., by introduction of one or more glycosylation sites, or by fusion to serum albumin) to have reduced recognition by anti-IdeS antibodies.
  • the IdeS protein is modified (e.g., by fusion to serum albumin) to prolong half-life of the modified IdeS protein and/or IgG depletion.
  • the IdeS protein is modified in multiple ways.
  • IdeS variant proteins comprising at least two modifications in positions selected from the group consisting of: 68, 75, 166, 187, 213, 236, 277, 302, 303, 306, and 318 of SEQ ID NO: 1.
  • the modification at position 68 is a substitution of valine to threonine.
  • the modification at position 75 is a substitution of alanine to proline.
  • the modification at position 166 is a substitution of threonine to arginine or a substitution of threonine to glycine.
  • the modification at position 187 is a substitution of serine to aspartate or a substitution of serine to glutamate. In some embodiments, the modification at position 187 is a substitution of serine to aspartate. In some embodiments, the modification at position 187 is a substitution of serine to glutamate. [00114] In some embodiments, the modification at position 213 is a substitution of threonine to glutamate. Attorney Docket No.: TKT-002WO [00115] In some embodiments, the modification at position 236 is a substitution of serine to cysteine. [00116] In some embodiments, the modification at position 277 is a substitution of leucine to cysteine.
  • the modification at position 302 is a substitution of serine to aspartate, a substitution of serine to lysine, or a substitution of serine to glutamate. In some embodiments, the modification at position 302 is a substitution of serine to aspartate. In some embodiments, the modification at position 302 is a substitution of serine to lysine. In some embodiments, the modification at position 302 is a substitution of serine to glutamate. [00118] In some embodiments, the modification at position 303 is a substitution of alanine to aspartate, a substitution of alanine to asparagine, or a substitution of alanine to glutamine.
  • the modification at position 303 is a substitution of alanine to aspartate. In some embodiments, the modification at position 303 is a substitution of alanine to asparagine. In some embodiments, the modification at position 303 is a substitution of alanine to glutamine. [00119] In some embodiments, the modification at position 306 is a substitution of valine to threonine. [00120] In some embodiments, the modification at position 318 is a substitution of isoleucine to lysine, a substitution of isoleucine to aspartate, or a substitution of isoleucine to glycine. In some embodiments, the modification at position 318 is a substitution of isoleucine to lysine.
  • the modification at position 318 is a substitution of isoleucine to aspartate. In some embodiments, the modification at position 318 is a substitution of isoleucine to glycine. [00121] Described herein, in certain embodiments, are IdeS variant proteins, wherein the IdeS variant proteins further comprise a modification at position 308. In some embodiments, the modification at position 308 is a substitution of isoleucine to leucine.
  • the IdeS variant protein comprises a substitution of valine at position 68 to threonine, a substitution of alanine at position 75 to proline, a substitution of threonine at position 166 to arginine, a substitution of serine at position 187 to aspartate, a substitution of threonine at position 213 to glutamate, a substitution of serine at position 302 to lysine, and a substitution of alanine at position 303 to aspartate.
  • the IdeS variant protein comprises a substitution of valine at position 68 to threonine, a substitution of alanine at position 75 to proline, a substitution of threonine at position 166 to arginine, a substitution of serine at position 187 to aspartate, a Attorney Docket No.: TKT-002WO substitution of threonine at position 213 to glutamate, a substitution of serine at position 302 to glutamate, and a substitution of alanine at position 303 to aspartate.
  • the IdeS variant protein comprises a substitution of valine at position 68 to threonine, a substitution of alanine at position 75 to proline, a substitution of threonine at position 166 to arginine, a substitution of serine at position 187 to aspartate, a substitution of threonine at position 213 to glutamate, a substitution of serine at position 302 to lysine, a substitution of alanine at position 303 to aspartate, and a substitution of valine at position 306 to threonine.
  • the IdeS variant protein comprises a substitution of valine at position 68 to threonine, a substitution of alanine at position 75 to proline, a substitution of threonine at position 166 to arginine, a substitution of serine at position 187 to aspartate, a substitution of threonine at position 213 to glutamate, a substitution of serine at position 302 to glutamate, a substitution of valine at position 306 to threonine, and a substitution of isoleucine at position 318 to lysine.
  • the IdeS variant protein comprises a substitution of valine at position 68 to threonine, a substitution of alanine at position 75 to proline, a substitution of threonine at position 166 to arginine, a substitution of serine at position 187 to aspartate, a substitution of threonine at position 213 to glutamate, a substitution at position 236 of serine to cysteine, a substitution of leucine at position 277 to cysteine, a substitution of serine at position 302 to glutamate, a substitution of alanine at position 303 to aspartate, and a substitution of valine at position 306 to threonine.
  • the IdeS variant protein comprises a substitution of valine at position 68 to threonine, a substitution of alanine at position 75 to proline, a substitution of threonine at position 166 to arginine, a substitution of serine at position 187 to aspartate, a substitution of threonine at position 213 to glutamate, a substitution at position 236 of serine to cysteine, a substitution of leucine at position 277 to cysteine, and a substitution of isoleucine at position 308 to leucine.
  • IdeS variant proteins wherein the IdeS variant proteins comprise a substitution of DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2 with a sequence selected from the group consisting of: TTIQAETSKHTISKKDETLHQNQLSISKTAT (SEQ ID NO: 77), DDYQRNAMEAYAKEVPHQIT (SEQ ID NO: 78), DPNNENEVSNLEKIKKLYPKGFQYGN (SEQ ID NO: 79), DQKLKDYLKNDQLKGTELGKFLEEQGVTK (SEQ ID NO: 80), Attorney Docket No.: TKT-002WO SAKVETGLPGELAPEEFSFPN (SEQ ID NO: 81), AQNKNPVTHYVNQFDGEEKEII (SEQ ID NO: 82), GSIGEKWDLLLDGIGLNSHRSS (SEQ ID NO: 83), AEPITLIWE
  • the IdeS variant proteins comprise a substitution of DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2 with a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical to the sequence selected from the group consisting of: TTIQAETSKHTISKKDETLHQNQLSISKTAT (SEQ ID NO: 77), DDYQRNAMEAYAKEVPHQIT (SEQ ID NO: 78), DPNNENEVSNLEKIKKLYPKGFQYGN (SEQ ID NO: 79), DQKLKDYLKNDQLKGTELGKFLEEQGVTK (SEQ ID NO:
  • the IdeS variant proteins comprise a substitution of DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2 with a sequence at least 80% identical to the sequence selected from the group consisting of: TTIQAETSKHTISKKDETLHQNQLSISKTAT (SEQ ID NO: 77), DPNNENEVSNLEKIKKLYPKGFQYGN (SEQ ID NO: 79), DQKLKDYLKNDQLKGTELGKFLEEQGVTK (SEQ ID NO: 80), SAKVETGLPGELAPEEFSFPN (SEQ ID NO: 81), AQNKNPVTHYVNQFDGEEKEII (SEQ ID NO: 82), GSIGEKWDLLLDGIGLNSHRSS (SEQ ID NO: 83), AEPITLIWENYLSDSVSPDRDIR (SEQ ID NO: 84), and QEEIAEGRRNPLRTAEWPMTKSTTDQ (SEQ ID NO: 85).
  • the IdeS variant proteins comprise a substitution of DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2 with the sequence DDYQRNAMEAYAKEVPHQIT (SEQ ID NO: 78).
  • the IdeS variant proteins comprise a substitution of DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2 with a Attorney Docket No.: TKT-002WO sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical to the sequence selected from the group consisting of any one of SEQ ID NOs: 95-112.
  • the IdeS variant proteins comprise a substitution of DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2 with a sequence at least 80% identical to the sequence selected from the group consisting of any one of SEQ ID NOs: 95-112.
  • IdeS variant proteins wherein the IdeS variant proteins comprises a modification in positions selected from the group consisting of: 31, 32, 33, 38, 39, 43, 44, 45, 47, 54, 57, 60, 74, 77, 82, 85, 113, 115, 116, 127, 128, 129, 130, 133, 148, 153, 154, 159, 167, 168, 175, 188, 195, 197, 210, 218, 219, 220, 228, 233, 241, 244, 245, 247, 258, 273, 274, 278, 288, 289, 297, 299, 300, 307, 313, 314, 315, 316, 322, and 330 of SEQ ID NO: 1.
  • the IdeS variant protein comprises a substitution of serine at position 31 to aspartate or asparagine. [00132] In some embodiments, the IdeS variant protein comprises a substitution of phenylalanine at position 32 to lysine. [00133] In some embodiments, the IdeS variant protein comprises a substitution of serine at position 33 to glutamate. [00134] In some embodiments, the IdeS variant protein comprises a substitution of isoleucine at position 38 to valine. [00135] In some embodiments, the IdeS variant protein comprises a substitution of arginine at position 39 to asparagine or threonine.
  • the IdeS variant protein comprises a substitution of valine at position 43 to glutamate. [00137] In some embodiments, the IdeS variant protein comprises a substitution of threonine at position 44 to glutamate. [00138] In some embodiments, the IdeS variant protein comprises a substitution of proline at position 45 to glutamate. [00139] In some embodiments, the IdeS variant protein comprises a substitution of histidine at position 47 to lysine. [00140] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 54 to aspartate.
  • the IdeS variant protein comprises a substitution of threonine at position 57 to lysine or glutamine. [00142] In some embodiments, the IdeS variant protein comprises a substitution of alanine at position 60 to aspartate or glutamine. [00143] In some embodiments, the IdeS variant protein comprises a substitution of valine at position 74 to lysine. [00144] In some embodiments, the IdeS variant protein comprises a substitution of alanine at position 75 to asparagine or proline. [00145] In some embodiments, the IdeS variant protein comprises a substitution of glutamine at position 77 to glycine.
  • the IdeS variant protein comprises a substitution of isoleucine at position 82 to methionine. [00147] In some embodiments, the IdeS variant protein comprises a substitution of threonine at position 85 to glutamine. [00148] In some embodiments, the IdeS variant protein comprises a substitution of glutamine at position 113 to aspartate. [00149] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 115 to histidine. [00150] In some embodiments, the IdeS variant protein comprises a substitution of arginine at position 116 to aspartate.
  • the IdeS variant protein comprises a substitution of isoleucine at position 127 to methionine. [00152] In some embodiments, the IdeS variant protein comprises a substitution of asparagine at position 128 to glycine. [00153] In some embodiments, the IdeS variant protein comprises a substitution of phenylalanine at position 129 to glutamate. [00154] In some embodiments, the IdeS variant protein comprises a substitution of asparagine at position 130 to glutamate. [00155] In some embodiments, the IdeS variant protein comprises a substitution of glutamine at position 133 to aspartate.
  • the IdeS variant protein comprises a substitution of leucine at position 148 to asparagine or arginine. In some embodiments, the IdeS variant protein comprises a substitution of leucine at position 148 to asparagine. In some embodiments, the IdeS variant protein comprises a substitution of leucine at position 148 to arginine. Attorney Docket No.: TKT-002WO [00157] In some embodiments, the IdeS variant protein comprises a substitution of phenylalanine at position 153 to methionine or tyrosine. In some embodiments, the IdeS variant protein comprises a substitution of phenylalanine at position 153 to methionine.
  • the IdeS variant protein comprises a substitution of phenylalanine at position 153 to tyrosine. [00158] In some embodiments, the IdeS variant protein comprises a substitution of glutamate at position 154 to aspartate. [00159] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 159 to asparagine. [00160] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 167 to proline. [00161] In some embodiments, the IdeS variant protein comprises a substitution of histidine at position 168 to aspartate or proline.
  • the IdeS variant protein comprises a substitution of histidine at position 168 to aspartate. In some embodiments, the IdeS variant protein comprises a substitution of histidine at position 168 to proline. [00162] In some embodiments, the IdeS variant protein comprises a substitution of histidine at position 175 to glutamine. [00163] In some embodiments, the IdeS variant protein comprises a substitution of leucine at position 188 to glycine or methionine. In some embodiments, the IdeS variant protein comprises a substitution of leucine at position 188 to glycine. In some embodiments, the IdeS variant protein comprises a substitution of leucine at position 188 to methionine.
  • the IdeS variant protein comprises a substitution of proline at position 195 to aspartate. [00165] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 197 to glutamate. [00166] In some embodiments, the IdeS variant protein comprises a substitution of alanine at position 210 to glycine. [00167] In some embodiments, the IdeS variant protein comprises a substitution of serine at position 218 to aspartate. [00168] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 219 to glycine.
  • the IdeS variant protein comprises a substitution of leucine at position 220 to glutamine. Attorney Docket No.: TKT-002WO [00170] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 228 to glutamine. [00171] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 233 to serine. [00172] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 241 to aspartate. [00173] In some embodiments, the IdeS variant protein comprises a substitution of threonine at position 244 to lysine.
  • the IdeS variant protein comprises a substitution of glutamate at position 245 to aspartate. [00175] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 247 to asparagine. [00176] In some embodiments, the IdeS variant protein comprises a substitution of valine at position 258 to aspartate. [00177] In some embodiments, the IdeS variant protein comprises a substitution of serine at position 273 to aspartate. [00178] In some embodiments, the IdeS variant protein comprises a substitution of asparagine at position 274 to glutamate.
  • the IdeS variant protein comprises a substitution of lysine at position 278 to aspartate or threonine. In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 278 to aspartate. In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 278 to threonine. [00180] In some embodiments, the IdeS variant protein comprises a substitution of asparagine at position 288 to aspartate, glycine, or glutamine. In some embodiments, the IdeS variant protein comprises a substitution of asparagine at position 288 to aspartate.
  • the IdeS variant protein comprises a substitution of asparagine at position 288 to glycine. In some embodiments, the IdeS variant protein comprises a substitution of asparagine at position 288 to glutamine. [00181] In some embodiments, the IdeS variant protein comprises a substitution of alanine at position 289 to proline. [00182] In some embodiments, the IdeS variant protein comprises a substitution of phenylalanine at position 297 to aspartate. [00183] In some embodiments, the IdeS variant protein comprises a substitution of glycine at position 299 to aspartate.
  • the IdeS variant protein comprises a substitution of valine at position 300 to glutamate.
  • the IdeS variant protein comprises a substitution of alanine at position 307 to glycine or asparagine.
  • the IdeS variant protein comprises a substitution of alanine at position 307 to glycine.
  • the IdeS variant protein comprises a substitution of alanine at position 307 to asparagine.
  • the IdeS variant protein comprises a substitution of isoleucine at position 313 to proline.
  • the IdeS variant protein comprises a substitution of lysine at position 314 to glycine. [00188] In some embodiments, the IdeS variant protein comprises a substitution of glutamate at position 315 to proline. [00189] In some embodiments, the IdeS variant protein comprises a substitution of aspartate at position 316 to proline. [00190] In some embodiments, the IdeS variant protein comprises a substitution of valine at position 322 to threonine. [00191] In some embodiments, the IdeS variant protein comprises a substitution of threonine at position 330 to aspartate.
  • IdeS variants having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity to any one of the IdeS variant sequences listed in Table 1A.
  • the IdeS variant protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 3-75 and 86-94.
  • the IdeS variant protein comprises an amino acid sequence at least 90% identical any one of SEQ ID NOs: 3- 75 and 86-94. In some embodiments, the IdeS variant protein comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 3-75 and 86-94. In some embodiments, the IdeS variant protein comprises an amino acid sequence at least 96% identical to any one of SEQ ID NOs: 3-75 and 86-94. In some embodiments, the IdeS variant protein comprises an amino acid sequence at least 97% identical to any one of SEQ ID NOs: 3-75 and 86-94.
  • the IdeS variant protein comprises an amino acid sequence at least 98% identical to any one of SEQ ID NOs: 3-75 and 86-94. In some embodiments, the IdeS variant protein comprises an amino acid sequence at least 99% Attorney Docket No.: TKT-002WO identical to any one of SEQ ID NOs: 3-75 and 86-94. In some embodiments, the IdeS variant protein comprises an amino acid sequence 100% identical to any one of SEQ ID NOs: 3-75 and 86-94.
  • the IdeS variant protein comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence according to any one of SEQ ID NOs: 3-75 and 86-94.
  • the IdeS variant protein comprises a sequence at least 95% identical to the amino acid sequence according to any one of SEQ ID NOs: 3-75 and 86-94 and having one or more than one modifications to these enumerated amino acid sequences. [00194] Described herein, in some embodiments, are IdeS variant proteins comprising the amino acid sequence according to SEQ ID NO: 10.
  • the IdeS variant protein comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence according to SEQ ID NO: 10.
  • the IdeS variant protein comprises a sequence at least 95% identical to the amino acid sequence according to SEQ ID NO: 10 and having one or more than one modifications.
  • IdeS variant proteins comprising the amino acid sequence according to SEQ ID NO: 11.
  • the IdeS variant protein comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least Attorney Docket No.: TKT-002WO 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence according to SEQ ID NO: 11.
  • the IdeS variant protein comprises a sequence at least 95% identical to the amino acid sequence according to SEQ ID NO: 11 and having one or more than one modifications. [00196] Described herein, in some embodiments, are IdeS variant proteins comprising the amino acid sequence according to SEQ ID NO: 12.
  • the IdeS variant protein comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence according to SEQ ID NO: 12.
  • the IdeS variant protein comprises a sequence at least 95% identical to the amino acid sequence according to SEQ ID NO: 12 and having one or more than one modifications.
  • IdeS variant proteins comprising the amino acid sequence according to SEQ ID NO: 13.
  • the IdeS variant protein comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence according to SEQ ID NO: 13.
  • the IdeS variant protein comprises a sequence at least 95% identical to the amino acid sequence according to SEQ ID NO: 13 and having one or more than one modifications. [00198] Further described herein, in some embodiments, are IdeS variant proteins comprising one or more glycosylation modifications.
  • IdeS variants having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity to any one of the IdeS variant sequences listed in Table 1B.
  • the IdeS variant protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 117-153.
  • the IdeS variant protein comprises an amino acid sequence at least 90% identical any one of SEQ ID NOs: 117-153. In some embodiments, the IdeS variant protein comprises an amino Attorney Docket No.: TKT-002WO acid sequence at least 95% identical to any one of SEQ ID NOs: 117-153. In some embodiments, the IdeS variant protein comprises an amino acid sequence at least 96% identical to any one of SEQ ID NOs: 117-153. In some embodiments, the IdeS variant protein comprises an amino acid sequence at least 97% identical to any one of SEQ ID NOs: 117- 153. In some embodiments, the IdeS variant protein comprises an amino acid sequence at least 98% identical to any one of SEQ ID NOs: 117-153.
  • the IdeS variant protein comprises an amino acid sequence at least 99% identical to any one of SEQ ID NOs: 117-153. In some embodiments, the IdeS variant protein comprises an amino acid sequence 100% identical to any one of SEQ ID NOs: 117-153.
  • the IdeS variant proteins comprising at least one modification in positions selected from the group consisting of: 37, 39, 74, 76, 111, 113, 119, 121, 142, 144, 147, 148, 198, 244, and 246 of SEQ ID NO: 1 to introduce a glycosylation site
  • the modification at position 31 is a substitution of serine to asparagine.
  • the modification at position 37 is a substitution of glutamate to asparagine.
  • the modification at position 39 is a substitution of arginine to threonine or asparagine.
  • the modification at position 42 is a substitution of glutamate to asparagine.
  • the modification at position 74 is a substitution of valine to asparagine.
  • the modification at position 76 is a substitution of asparagine to serine.
  • the modification at position 111 is a substitution of lysine to asparagine.
  • the modification at position 113 is a substitution of glutamine to threonine. Attorney Docket No.: TKT-002WO [00207]
  • the modification at position 119 is a substitution of glutamate to asparagine.
  • the modification at position 121 is a substitution of histidine to threonine.
  • the modification at position 130 is a substitution of asparagine to serine.
  • the modification at position 142 is a substitution of aspartate to asparagine.
  • the modification at position 144 is a substitution of lysine to threonine.
  • the modification at position 147 is a substitution of glutamine to serine.
  • the modification at position 148 is a substitution of leucine to asparagine.
  • the modification at position 198 is a substitution of glutamate to asparagine.
  • the modification at position 233 is a substitution of lysine to serine.
  • the modification at position 244 is a substitution of threonine to asparagine.
  • the modification at position 246 is a substitution of glycine to threonine.
  • the modification at position 311 is a substitution of lysine to asparagine.
  • the modification at position 313 is a substitution of isoleucine to threonine.
  • the modification at position 319 is a substitution of glycine to serine.
  • the variant protein comprises a substitution of glutamate at position 37 to asparagine and a substitution of arginine at position 39 to threonine.
  • the variant protein comprises a substitution of valine at position 74 to asparagine and a substitution of asparagine at position 76 to serine.
  • the variant protein comprises a substitution of lysine at position 111 to asparagine and a substitution of glutamine at position 113 to threonine.
  • the variant protein comprises a substitution of glutamate at position 119 to asparagine and a substitution of histidine at position 121 to threonine.
  • the variant protein comprises a substitution of aspartate at position 142 to asparagine and a substitution of lysine at position 144 to threonine.
  • the variant protein comprises a substitution of threonine at position 244 to asparagine and a substitution of glycine at position 246 to threonine.
  • the variant protein comprises a substitution of lysine at position 311 to asparagine and a substitution of isoleucine at position 313 to threonine.
  • IdeS variant proteins comprising at least one modification in positions selected from the group consisting of: 47, 49, 51, 78, 111, 113, 123, 125, 126, 128, 142, 144, 148, 198, 273, 275, 278, 312, and 314 of SEQ ID NO: 12 to introduce a glycosylation site.
  • the IdeS variant proteins comprises at least one modification in positions selected from the group consisting of: 47, 49, 51, 123, 125, 126, and 128 of SEQ ID NO: 12 to introduce a glycosylation site.
  • the modification at position 47 is a substitution of threonine to asparagine.
  • the modification at position 49 is a substitution of glutamine to threonine or asparagine.
  • the modification at position 51 is a substitution of valine to threonine.
  • the modification at position 78 is a substitution of glycine to serine.
  • the modification at position 111 is a substitution of lysine to asparagine.
  • the modification at position 113 is a substitution of glutamine to threonine.
  • the modification at position 123 is a substitution of glutamate to asparagine.
  • the modification at position 125 is a substitution of glutamine to threonine.
  • the modification at position 126 is a substitution of lysine to asparagine.
  • the modification at position 128 is a substitution of asparagine to serine.
  • the modification at position 142 is a substitution of aspartate to asparagine.
  • the modification at position 144 is a substitution of lysine to threonine.
  • the modification at position 148 is a substitution of leucine to asparagine.
  • the modification at position 198 is a substitution of glutamate to asparagine.
  • the modification at position 273 is a substitution of serine to asparagine.
  • the modification at position 275 is a substitution of glycine to serine.
  • the modification at position 278 is a substitution of lysine to serine.
  • the modification at position 312 is a substitution of glutamate to asparagine.
  • the modification at position 314 is a substitution of lysine to serine.
  • the variant protein comprises a substitution of threonine at position 47 to asparagine and a substitution of glutamine at position 49 to threonine.
  • the variant protein comprises a substitution of glutamine at position 49 to asparagine and a substitution of valine at position 51 to threonine.
  • the variant protein comprises a substitution of glutamate at position 123 to asparagine and a substitution of glutamine at position 125 to threonine.
  • the variant protein comprises a substitution of lysine at position 126 to asparagine and a substitution of asparagine at position 128 to serine.
  • the variant protein comprises a substitution of serine at position 273 to asparagine and a substitution of glycine at position 275 to serine.
  • the variant protein comprises a substitution of glutamate at position 312 to asparagine and a substitution of lysine at position 314 to serine.
  • the variant protein comprises a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of aspartate at position 142 to asparagine, and a substitution of lysine at position 144 to threonine.
  • Attorney Docket No.: TKT-002WO the variant protein comprises a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of aspartate at position 142 to asparagine, a substitution of lysine at position 144 to threonine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of aspartate at position 142 to asparagine, a substitution of lysine at position 144 to threonine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of glutamine at position 49 to asparagine, a substitution of valine at position 51 to threonine, a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of lysine at position 126 to asparagine, a substitution of asparagine at position 128 to serine, a substitution of leucine at position 148 to asparagine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of glutamine at position 49 to asparagine, a substitution of valine at position 51 to threonine, a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of leucine at position 148 to asparagine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of lysine at position 126 to asparagine, a substitution of asparagine at position 128 to serine, a substitution of leucine at position 148 to asparagine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of glutamine at position 49 to asparagine, a substitution of valine at position 51 to threonine, a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of lysine at position 126 to asparagine, a substitution of asparagine at position 128 to serine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises, a substitution of threonine at position 47 to asparagine, a substitution of glutamine at position 49 to asparagine, a Attorney Docket No.: TKT-002WO substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of glutamine at position 49 to asparagine, a substitution of valine at position 51 to threonine, a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of glutamate at position 123 to asparagine, a substitution of glutamine at position 125 to threonine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of lysine at position 126 to asparagine, a substitution of asparagine at position 128 to serine, and a substitution of glutamate at position 198 to asparagine.
  • the variant protein comprises a substitution of lysine at position 111 to asparagine, a substitution of glutamine at position 113 to threonine, a substitution of leucine at position 148 to asparagine, and a substitution of glutamate at position 198 to asparagine.
  • IdeS variant proteins comprising one or more glycans.
  • the one or more glycans are glycans of a mammalian glycoprotein.
  • the one or more glycans are N-linked glycans (i.e., N-glycans) or O-linked glycans (i.e., O-glycans) of a mammalian glycoprotein.
  • the one or more glycans are N-linked glycans (i.e., N-glycans).
  • the one or more glycans comprise an N-glycan core structure.
  • the one or more glycans comprise an N-glycan core structure of: (Man ⁇ 1- 6[Man ⁇ 1-3)]Man( ⁇ 1-4)GlcNAc( ⁇ 1-4)GlcNAc( ⁇ 1-Asn-X-Ser/Thr).
  • the one or more glycans are selected from the group consisting of: a biantennary glycan, a triantennary glycan, and a tetraantennary glycan.
  • the one or more glycans terminate with a galactose, an N- acetylgalactosamine, a glucose, an N-acetylglucosamine, a fucose, an N-acetylneuraminic acid, a mannose or a sialic acid. In some embodiments, the one or more glycans terminate with a galactose. In some embodiments, the one or more glycans terminate with an N- acetylgalactosamine. In some embodiments, the one or more glycans terminate with a glucose.
  • the one or more glycans terminate with an N- Attorney Docket No.: TKT-002WO acetylglucosamine. In some embodiments, the one or more glycans terminate with a fucose. In some embodiments, the one or more glycans terminate with an N-acetylneuraminic acid. In some embodiments, the one or more glycans terminate with a mannose. In some embodiments, the one or more glycans terminate with a sialic acid.
  • the one or more glycans is selected from the group consisting of: a biantennary glycan that terminates with mannose, a biantennary glycan that terminates with sialic acid, a triantennary glycan that terminates with a mannose, a triantennary glycan that terminates with a sialic acid, a tetraantennary glycan that terminates with mannose, and a tetraantennary glycan that terminates with sialic acid.
  • the one or more glycans is a biantennary glycan that terminates with a galactose.
  • the one or more glycans is a biantennary glycan that terminates with an N-acetylgalactosamine. In some embodiments, the one or more glycans is a biantennary glycan that terminates with a glucose. In some embodiments, the one or more glycans is a biantennary glycan that terminates with an N-acetylglucosamine. In some embodiments, the one or more glycans is a biantennary glycan that terminates with a fucose.
  • the one or more glycans is a biantennary glycan that terminates with an N-acetylneuraminic acid. In some embodiments, the one or more glycans is a biantennary glycan that terminates with mannose. In some embodiments, the one or more glycans is a biantennary glycan that terminates with sialic acid. In some embodiments, the one or more glycans is a triantennary glycan that terminates with a galactose. In some embodiments, the one or more glycans is a triantennary glycan that terminates with an N-acetylgalactosamine.
  • the one or more glycans is a triantennary glycan that terminates with a glucose. In some embodiments, the one or more glycans is a triantennary glycan that terminates with an N-acetylglucosamine. In some embodiments, the one or more glycans is a triantennary glycan that terminates with a fucose. In some embodiments, the one or more glycans is a triantennary glycan that terminates with an N-acetylneuraminic acid. In some embodiments, the one or more glycans is a triantennary glycan that terminates with a mannose.
  • the one or more glycans is a triantennary glycan that terminates with a sialic acid. In some embodiments, the one or more glycans is a tetraantennary glycan that terminates with a galactose. In some embodiments, the one or more glycans is a tetraantennary glycan that terminates with an N-acetylgalactosamine. In some embodiments, the one or more glycans is a tetraantennary glycan that terminates with a glucose.
  • the one or more glycans is a tetraantennary glycan that terminates with an N-acetylglucosamine. In some embodiments, the one or more glycans is a Attorney Docket No.: TKT-002WO tetraantennary glycan that terminates with a fucose. In some embodiments, the one or more glycans is a tetraantennary glycan that terminates with an N-acetylneuraminic acid.
  • the one or more glycans is a tetraantennary glycan that terminates with mannose, and a tetraantennary glycan that terminates with sialic acid. In some embodiments, the one or more glycans is a tetraantennary glycan that terminates with sialic acid. [00270] In some embodiments, the glycan is selected from the glycans listed in Table 10.
  • the glycan is selected from the group consisting of Man( ⁇ 1-6)[Man( ⁇ 1- 3)]Man( ⁇ 1-4)GlcNAc( ⁇ 1-4)GlcNAc( ⁇ 1-ASN) (i.e., Glycan 1), Neu5Ac( ⁇ 2-6)Gal( ⁇ 1- 4)GlcNAc( ⁇ 1-2)Man( ⁇ 1-6)[Neu5Ac( ⁇ 2-6)Gal( ⁇ 1-4)GlcNAc( ⁇ 1-2)Man( ⁇ 1-3)]Man( ⁇ 1- 4)GlcNAc( ⁇ 1-4)GlcNAc( ⁇ 1-ASN) (i.e., Glycan 2), and Neu5Ac( ⁇ 2-6)Gal( ⁇ 1-4)GlcNAc( ⁇ 1- 6) [Neu5Ac( ⁇ 2-6)Gal( ⁇ 1-4)GlcNAc( ⁇ 1-2)]Man( ⁇ 1-6)[Neu5Ac( ⁇ 2-6)Gal( ⁇ 1-4)GlcNAc( ⁇ 1- 2)[Neu5Ac( ⁇ 2-6)G
  • the one or more glycans terminate with an N- acetylglucosamine (i.e. GlcNAc).
  • GlcNAc N- acetylglucosamine
  • the one or more glycans shield the surface area of the variant protein. In some embodiments, the one or more glycans shield at least 10-64% of the total accessible surface area of the variant protein. In some embodiments, the one or more glycans shield at least 20-50% of the total accessible surface area of the variant protein. In some embodiments, the one or more glycans shield at least 25-45% of the total accessible surface area of the variant protein.
  • the one or more glycans shield at least 20-35% of the total accessible surface area of the variant protein. In some embodiments, the one or more glycans shield at least 25% of the total accessible surface area of the variant protein. In some embodiments, the one or more glycans shield at least 30% of the total accessible surface area of the variant protein. In some embodiments, the one or more glycans shield at least 35% of the total accessible surface area of the variant protein. In some embodiments, the one or more glycans shield at least 40% of the total accessible surface area of the variant protein. In some embodiments, the one or more glycans shield at least 45% of the total accessible surface area of the variant protein.
  • the IdeS variant proteins comprising one or more glycans have reduced immunogenicity compared to aglycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS. In some embodiments, the IdeS variant proteins comprising one or more glycans have increased serum stability compared to aglycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS. In some Attorney Docket No.: TKT-002WO embodiments, the IdeS variant proteins comprising one or more glycans persist longer in serum than aglycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS.
  • the IdeS variant proteins comprising one or more glycans have a serum half-life approximately two times longer than the serum half-life of aglycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS. In some embodiments, the IdeS variant proteins comprising one or more glycans have increased size (i.e., Molecular Weight (MW)) compared to aglycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS.
  • MW Molecular Weight
  • the IdeS variant proteins comprising one or more glycans have reduced renal elimination compared to aglycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS.
  • reduced renal elimination is due to an increase in variant protein’s size (i.e., MW) as compared to aglycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS.
  • the IdeS variant proteins comprising one or more glycans have a longer duration of IgG depletion compared to aglycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS.
  • the longer duration of IgG depletion is correlated with a greater extent of IdeS variant protein glycosylation.
  • IdeS variant proteins fused to Human Serum Albumin HSA
  • the variant proteins fused to HSA comprise one or more glycans.
  • the variant proteins fused to HSA have a reduced EC50 value for polyclonal anti-IdeS reactivity compared to the EC50 value of aglycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS.
  • the EC50 value of variant proteins fused to HSA is approximately half the EC50 value of aglycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS.
  • the IdeS variant proteins fused to HSA have a longer duration of IgG depletion compared to aglycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS.
  • the IdeS variant proteins fused to HSA have a serum half-life approximately two, three, four, five, six, or seven times longer than the serum half-life of aglycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS.
  • IdeS Polypeptides [00275] Described herein, in certain embodiments, are polypeptides comprising an IdeS protein comprising at least 90% sequence identity to any one of SEQ ID NOs: 1-75 conjugated to a human serum albumin.
  • the human serum albumin is Attorney Docket No.: TKT-002WO conjugated to a N-terminal of the IdeS protein.
  • the human serum albumin is conjugated to a C-terminal of the IdeS protein.
  • IdeS polypeptides having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity to any one of the IdeS polypeptide sequences listed in Table 1C.
  • the IdeS polypeptide comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 154-161.
  • the IdeS polypeptide comprises an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 154-161. In some embodiments, the IdeS polypeptide comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 154-161. In some embodiments, the IdeS polypeptide comprises an amino acid sequence at least 96% identical to any one of SEQ ID NOs: 154-161. In some embodiments, the IdeS polypeptide comprises an amino acid sequence at least 97% identical to any one of SEQ ID NOs: 154-161. In some embodiments, the IdeS polypeptide comprises an amino acid sequence at least 98% identical to any one of SEQ ID NOs: 154-161.
  • the IdeS polypeptide comprises an amino acid sequence at least 99% identical to any one of SEQ ID NOs: 154-161. In some embodiments, the IdeS polypeptide comprises an amino acid sequence 100% identical to any one of SEQ ID NOs: 154-161.
  • the linker is a peptide linker. In some embodiments, the peptide linker comprises a flexible peptide linker. In some embodiments, the peptide linker comprises a rigid peptide linker. In some embodiments, the peptide linker comprises a cleavable peptide linker. [00278] In some embodiments, the linker comprises at least 2 to about 30 amino acids.
  • the linker comprises about 2 to about 35 amino acids, about 2 to about 40 amino acids, about 2 to about 35 amino acids, about 2 to about 35 amino acids, about 2 to about 30 amino acids, about 2 to about 25 amino acids, about 2 to about 20 amino acids, about 2 to about 15 amino acids, about 2 to about 10 amino acids, about 5 to about 30 amino Attorney Docket No.: TKT-002WO acids, about 5 to about 30 amino acids, about 5 to about 25 amino acids, about 5 to about 20 amino acids, about 5 to about 15 amino acids, or about 5 to about 10 amino acids.
  • the linker comprises a sequence selected from the group consisting of (GS)n (SEQ ID NO: 162), (G2S)n (SEQ ID NO: 163), (G3S)n (SEQ ID NO: 164), (G4S)n (SEQ ID NO: 165), and (G)n (SEQ ID NO: 166), and wherein n is an integer from 1 to 20.
  • n is an integer from 1 to 18, from 1 to 16, from 1 to 14, from 1 to 12, from 1 to 10, from 1 to 8, from 1 to 6, from 1 to 4, from 4 to 20, from 6 to 20, from 8 to 20, from 10 to 20, from 12 to 20, from 14 to 20, from 16 to 20, or from 18 to 20.
  • the linker comprises a sequence selected from the group consisting of (GGSGGD)n (SEQ ID NO: 167) or (GGSGGE)n (SEQ ID NO: 168), and wherein n is an integer from 1 to 6.
  • the linker comprises a sequence selected from the group consisting of (GGGSGSGGGGS)n (SEQ ID NO: 169) and (GGGGGPGGGGP)n (SEQ ID NO: 170), and wherein n is an integer from 1 to 3.
  • the linker comprises a sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n (SEQ ID NO: 171), and (GzX)n, wherein z is between 1 and 20.
  • z is between 1 and 18, 1 and 16, 1 and 14, 1 and 12, 1 and 10, 1 and 8, 1 and 6, 1 and 4, 4 and 20, 6 and 20, 8 and 20, 10 and 20, 12 and 20, 14 and 20, 16 and 20, or 18 and 20.
  • X is serine, aspartic acid, glutamic acid, threonine, or proline.
  • the linker comprises GSGGGSG (SEQ ID NO: 113), GSGSGSGS (SEQ ID NO: 114), GSGGGSGGGSG (SEQ ID NO: 115), or GSGSGSGSGSGSGS (SEQ ID NO: 116). In some embodiments, the linker comprises GS.
  • the IdeS protein is fused to the human serum albumin. [00285] Further described herein, in certain embodiments, are polypeptides comprising an IdeS variant protein described herein conjugated to a human serum albumin.
  • the IdeS variant protein comprises at least two modifications in positions selected from the group consisting of: 68, 75, 166, 187, 213, 236, 277, 302, 303, 306, and 318 of SEQ ID NO: 1 and at least one modification in positions selected from the group consisting of: 37, 39, 74, 76, 111, 113, 119, 121, 142, 144, 147, 148, 198, 244, and 246 of SEQ ID NO: 1 or at least one modification in positions selected from the group consisting of: 47, 49, 51, 123, 125, 126, and 128 of SEQ ID NO: 12 to introduce a glycosylation site.
  • the IdeS variant protein comprises at least two modifications in positions selected from the group consisting of: 68, 75, 166, 187, 213, 236, 277, 302, 303, 306, and 318 of SEQ ID NO: 1 and at least one modification in positions selected from the group consisting of: 31, 37, 39, 42, 74, 76, 111, 113, 119, 121, 130, 142, 144, 147, 148, 198, 233, 244, 246, 311, 313, and 319 of SEQ ID NO: 1 or at least one modification in positions selected from the group consisting of: 47, 49, 51, 78, 111, 113, 123, 125, 126, 128, 142, 144, 148, 198, 273, 275, 278, 312,
  • the IdeS variant protein or IdeS polypeptide described herein comprises increased level of expression when expressed by a cell as compared to the level of expression of wild-type IdeS by the same cell.
  • the IdeS variant protein or IdeS polypeptide comprises an increase of at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more than 500% in the level of expression as compared to the level of expression of wild-type IdeS by the same cell.
  • the IdeS variant protein or IdeS polypeptide comprises an increase in a range of about 5-95%, 10-90%, 20-80%, 30-70%, 40-60%, 50-95%, 65-85%, 75-95%, 10-200%, 20-200%, 30-200%, 40-200%, 50-200%, 75-200%, 100-200%, 150- 200%, 10-300%, 20-300%, 30-300%, 40-300%, 50-300%, 75-300%, 100-300%, 150-300%, 10-400%, 20-400%, 30-400%, 40-400%, 50-400%, 75-400%, 100-400%, 150-400%, 10- 500%, 20-500%, 30-500%, 40-500%, 50-500%, 75-500%, 100-500%, or 150-500% in the level of expression when expressed by a cell as compared to the level of expression of wild- type IdeS.
  • the IdeS variant protein or IdeS polypeptide described herein comprises levels of expression in a cell ⁇ about 1-20%, ⁇ about 2-20%, ⁇ about 4- 20%, ⁇ about 6-20%, ⁇ about 8-20%, ⁇ about 10-20%, ⁇ about 12-20%, ⁇ about 14-20%, ⁇ about 16-20%, ⁇ about 18-20%, about 1-18%, ⁇ about 2-18%, ⁇ about 4-18%, ⁇ about 6- 18%, ⁇ about 8-18%, ⁇ about 10-18%, ⁇ about 12-18%, ⁇ about 14-18%, ⁇ about 16-18%, 1- 16%, ⁇ about 2-16%, ⁇ about 4-16%, ⁇ about 6-16%, ⁇ about 8-16%, ⁇ about 10-16%, ⁇ about 12-16%, ⁇ about 14-16%, about 1-14%, ⁇ about 2-14%, ⁇ about 4-14%, ⁇ about 6- 14%, ⁇ about 8-14%, ⁇ about 10-14%, ⁇ about 10-14%,
  • the IdeS variant protein or IdeS polypeptide described herein comprises increased stability of the IdeS variant protein as compared to the stability of wild-type IdeS.
  • the IdeS variant protein or IdeS polypeptide comprises an increase of at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more than 500% in the stability as compared to the stability of wild-type IdeS.
  • the IdeS variant protein or IdeS polypeptide comprises an increase in a range of about 5-95%, 10-90%, 20-80%, 30-70%, 40- 60%, 50-95%, 65-85%, 75-95%, 10-200%, 20-200%, 30-200%, 40-200%, 50-200%, 75- 200%, 100-200%, 150-200%, 10-300%, 20-300%, 30-300%, 40-300%, 50-300%, 75-300%, 100-300%, 150-300%, 10-400%, 20-400%, 30-400%, 40-400%, 50-400%, 75-400%, 100- 400%, 150-400%, 10-500%, 20-500%, 30-500%, 40-500%, 50-500%, 75-500%, 100-500%, or 150-500% increase in the stability as compared to the stability of wild-type IdeS.
  • the IdeS variant protein or IdeS polypeptide described herein comprises an increased enzymatic activity as compared to the activity of wild-type IdeS.
  • the enzymatic activity is cleavage (e.g., proteolytic) activity of immunoglobulins.
  • the IdeS variant protein or IdeS polypeptide comprises an increase of at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more than 500% in the enzymatic activity as compared to the activity of wild-type IdeS.
  • the IdeS variant protein or IdeS polypeptide comprises an increase in a range of about 5-95%, 10-90%, 20-80%, 30-70%, 40- 60%, 50-95%, 65-85%, 75-95%, 10-200%, 20-200%, 30-200%, 40-200%, 50-200%, 75- 200%, 100-200%, 150-200%, 10-300%, 20-300%, 30-300%, 40-300%, 50-300%, 75-300%, 100-300%, 150-300%, 10-400%, 20-400%, 30-400%, 40-400%, 50-400%, 75-400%, 100- 400%, 150-400%, 10-500%, 20-500%, 30-500%, 40-500%, 50-500%, 75-500%, 100-500%, or 150-500% in the enzymatic activity as compared to the activity to wild-type IdeS.
  • the IdeS variant protein or IdeS polypeptide described herein comprises decreased presentation of epitopes on human leukocyte antigen (HLA) as compared to a wild-type IdeS protein.
  • the IdeS variant protein or IdeS polypeptide comprises a decrease of at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more than 500% in the presentation of epitopes on human leukocyte antigen (HLA) as compared to a wild-type IdeS protein.
  • the IdeS variant protein or IdeS polypeptide comprises a decrease in a range of about 5-95%, 10-90%, 20-80%, 30-70%, 40-60%, 50-95%, 65-85%, 75-95%, 10- 200%, 20-200%, 30-200%, 40-200%, 50-200%, 75-200%, 100-200%, 150-200%, 10-300%, 20-300%, 30-300%, 40-300%, 50-300%, 75-300%, 100-300%, 150-300%, 10-400%, 20- 400%, 30-400%, 40-400%, 50-400%, 75-400%, 100-400%, 150-400%, 10-500%, 20-500%, 30-500%, 40-500%, 50-500%, 75-500%, 100-500%, or 150-500% in the presentation of epitopes on human leukocyte antigen (HLA) as compared to a wild-type IdeS protein.
  • HLA human leukocyte antigen
  • the HLA is an HLA class II allele.
  • the HLA class II allele is selected from the group consisting of: HLA-DRB1*01:01, HLA-DRB1*03:01, HLA-DRB1*04:01, HLA-DRB1*07:01, HLA-DRB1*08:02, HLA-DRB1*11:01, HLA- DRB1*13:02, HLA-DRB1*15:01, HLA-DRB1*09:01, HLA-DRB3*01:01, HLA- DRB4*01:01, HLA-DRB5*01:01, HLA-DQA1*05:01-DQB1*03:01, and HLA- DQA1*03:01-DQB1*03:02.
  • the IdeS variant protein’s or IdeS polypeptide’s binding to the HLA-II allele is measured.
  • the IdeS variant protein’s or IdeS polypeptide’s binding to the HLA-II allele is determined as a probability ranking against a reference set (e.g., of 100,000 peptides). This is referred to as the rank-percentage. For example, a rank-percentage of 5% indicates a peptide is predicted to bind a particular HLA with more confidence than 95% of the peptides in the reference set.
  • the IdeS variant protein or IdeS polypeptide described herein comprises reduced immunogenicity as compared to a wild-type IdeS protein.
  • the IdeS variant protein or IdeS polypeptide comprises reduced immunogenicity of at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, as compared to a wild-type IdeS protein.
  • the IdeS variant protein or IdeS polypeptide comprises reduced immunogenicity in a range of about 5-95%, 10-90%, 20-80%, 30-70%, 40-60%, 50- 95%, 65-85%, or 75-95% as compared to a wild-type IdeS protein.
  • immunogenicity of the IdeS variant protein or IdeS polypeptide is calculated based on the number of HLA-II alleles that present peptide epitopes. This can be referred to as an immunogenicity score. For example, peptides in IdeS are predicted to be presented on a HLA-II allele if they are within the 10% highest affinity sequences.
  • a rank-percentage of 10% means the peptide is predicted to fall within the 10% of peptides of highest affinity from a reference set of 100,000.
  • the immunogenicity score for a peptide will then be the number of HLA-II alleles to which the Attorney Docket No.: TKT-002WO peptide is expected to bind.
  • a number of HLA-II alleles that present a peptide from an IdeS variant protein is reduced as compared to a number of HLA-II alleles that present an equivalent peptide from an IdeS protein comprising SEQ ID NO: 1 or SEQ ID NO: 2.
  • a number of HLA-II alleles that present a peptide from an IdeS variant protein is reduced as compared to a number of HLA-II alleles that present an equivalent peptide from an IdeS protein comprising to wild-type IdeS.
  • the IdeS variant protein has a decrease in immunogenicity score more than 0 relative to SEQ ID NO: 1 or SEQ ID NO: 2.
  • the IdeS variant protein or IdeS polypeptide described herein comprises reduced antibody binding as compared to a wild-type IdeS protein.
  • the IdeS variant protein or IdeS polypeptide comprises reduced antibody binding of at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, as compared to a wild-type IdeS protein. In some embodiments, the IdeS variant protein or IdeS polypeptide comprises reduced antibody binding in a range of about 5-95%, 10-90%, 20-80%, 30-70%, 40-60%, 50- 95%, 65-85%, or 75-95% as compared to a wild-type IdeS protein.
  • the IdeS variant protein or IdeS polypeptide binding to the antibody is determined using ELISA.
  • the IdeS variant protein or IdeS polypeptide described herein comprises one or more glycosylation (e.g., N-glycosylation) modifications or is fused to albumin resulting in reduced antibody binding as compared to a wild-type IdeS protein.
  • the IdeS variant protein or IdeS polypeptide comprises one or more glycosylation (e.g., N-glycosylation) modifications or is fused to albumin resulting in reduced antibody binding of at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, as compared to a wild-type IdeS protein.
  • glycosylation e.g., N-glycosylation
  • the IdeS variant protein or IdeS polypeptide comprises one or more glycosylation (e.g., N-glycosylation) modifications or is fused to albumin resulting in reduced antibody binding in a range of about 5-95%, 10-90%, 20-80%, 30-70%, 40-60%, 50-95%, 65-85%, or 75-95% as compared to a wild-type IdeS protein.
  • glycosylation e.g., N-glycosylation
  • compositions disclosed herein include IdeS variant proteins of the present disclosure and a pharmaceutically Attorney Docket No.: TKT-002WO acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc.
  • pharmaceutically acceptable it is meant a material that is not toxic or otherwise undesirable, i.e. the material may be administered to a subject without causing any undesirable biological effects.
  • pharmaceutical compositions can comprise sterile aqueous and non-aqueous injection solutions, which are optionally isotonic with the blood of the subject to whom the pharmaceutical composition is to be delivered.
  • compositions can contain anti-oxidants, buffers, bacteriostats and solutes, which render the composition isotonic with the blood of the intended subject to be administered.
  • Aqueous and non-aqueous sterile suspensions, solutions and emulsions can include suspending agents and thickening agents.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • compositions comprise pharmaceutically acceptable vehicles and can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. [00299] In certain aspects, pharmaceutical compositions can be presented in unit/dose or multi-dose containers, for example, in sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use.
  • sterile liquid carrier for example, saline or water-for-injection immediately prior to use.
  • compositions disclosed herein can be formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal administration.
  • a pharmaceutical composition of the present disclosure is administered intravenously to a subject in need thereof.
  • Effective concentrations of IdeS variant proteins of the present disclosure can be achieved via the transient or stable expression of a nucleic acid molecule encoding the IdeS variant proteins.
  • a nucleic acid molecule encoding the IdeS variant protein of the present disclosure can be incorporated into a vector and introduced into a cell.
  • a cell has one or more than one nucleic acid encoding the IdeS variant protein described herein.
  • the nucleic acid is a DNA, for example a linear DNA, a plasmid DNA, or a minicircle DNA.
  • the nucleic acid is an RNA, for example a mRNA.
  • the nucleic acid is provided in a vector.
  • the nucleic acid is provided in a plasmid (e.g., circular DNA molecules that can autonomously replicate inside a cell), cosmid (e.g., pWE or sCos vectors), artificial chromosome, human artificial chromosome (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosome (BAC), P1-derived artificial chromosomes (PAC), phagemid, phage derivative, bacmid, or virus.
  • cosmid e.g., pWE or sCos vectors
  • HAC human artificial chromosome
  • YAC yeast artificial chromosomes
  • BAC bacterial artificial chromosome
  • PAC P1-derived artificial chromosomes
  • the nucleic acid is provided in a vector selected from the list consisting of: pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV- NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV- FLAG(R)-6His, pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEF1a-mCherry-N1 vector, pEF1a-tdTomato vector, pSF-CMV-FMDV-Hygro, pSF-CMV- PGK-Puro, pMCP-tag(m), pSF-CMV-PURO-NH2-CMYC, pSF-OXB20-BetaGal,pSF- OXB20-Fluc, pSF-O
  • the nucleic acid comprises a promoter.
  • the promoter is selected from the group consisting of a mini promoter, an inducible promoter, a constitutive promoter, and derivatives thereof.
  • the promoter is selected from the group consisting of CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p19, p40, Synapsin, CaMKII, GRK1, and derivatives thereof.
  • the nucleic acid is provided in a virus.
  • the virus is an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, or a retrovirus.
  • the virus is an alphavirus.
  • the virus is a parvovirus.
  • the virus is an adenovirus.
  • the virus is an AAV.
  • the virus is a baculovirus.
  • the virus is a Dengue virus.
  • the virus is a lentivirus.
  • the virus is a herpesvirus. In some embodiments, the virus is a poxvirus. In some embodiments, the virus is an anellovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a vaccinia virus. In some embodiments, the virus is or a retrovirus.
  • the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, Attorney Docket No.: TKT-002WO AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV- Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC
  • the herpesvirus is HSV type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.
  • the nucleic acid is provided in a non-viral delivery system. In some embodiments, the nucleic acid is comprised in a liposome. In some embodiments, the nucleic acid is associated with a lipid.
  • the nucleic acid associated with a lipid in some embodiments, is encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the nucleic acid, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • the nucleic acid is provided in a lipid nanoparticle (LNP).
  • Vectors can be introduced into a cell by a variety of methods, including transformation, transfection, direct uptake, projectile bombardment, and by encapsulation of the vector in a liposome or nanoparticle.
  • suitable methods of transfecting or transforming cells are calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. Such methods are described in more detail, for example, in Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition (Cold Spring Harbor University Press, New York (2014)); and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York (2015)).
  • a variety of vectors for the delivery and expression of polynucleotides encoding exogenous polypeptides in a mammalian cell have been developed. Examples of expression vectors are disclosed in, e.g., WO 1994/011026 and are incorporated herein by reference. Expression vectors for use in the compositions and methods described herein contain a polynucleotide sequence that encodes an IdeS variant protein of the present disclosure as well as, e.g., additional sequence elements used for the expression of the polypeptide and/or the integration of the polynucleotide sequence into the genome of a mammalian cell.
  • Certain vectors that can be used include plasmids that contain regulatory sequences, such as promoter Attorney Docket No.: TKT-002WO and enhancer regions, which direct gene transcription.
  • Other useful vectors contain polynucleotide sequences that enhance the rate of translation or improve the stability or nuclear export of mRNA. These sequence elements include, e.g., 5’ and 3’ UTR regions, an internal ribosomal entry site (IRES), and polyA in order to direct efficient transcription of the gene carried on the expression vector.
  • the expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector.
  • a suitable marker are a gene that encodes green fluorescent protein or a gene that encodes resistance to an antibiotic.
  • nucleases include, but are not limited to, Transcription Activator-Like Effector Nuclease (TALEN), zinc finger nuclease (ZFN), meganuclease, Argonaute, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein.
  • TALEN Transcription Activator-Like Effector Nuclease
  • ZFN zinc finger nuclease
  • meganuclease Argonaute
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas Clustered Regularly Interspaced Short Palindromic Repeats
  • the nuclease is wild-type, genetically modified, or recombinant.
  • the gene editing system comprises CRISPR/Cas9.
  • EXAMPLES [00312] The disclosure now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the scope of the disclosure in any way.
  • IdeS produced in bacteria was non-glycosylated and ran as a sharp peak by size exclusion chromatography (SEC) as seen in FIGs.1A-1B.
  • SEC size exclusion chromatography
  • IdeS purified from Expi293F culture ran by SEC as a broad peak at higher molecular weight (MW) and its electrophoretic mobility increased following PNGase F treatment as seen in FIGs.1A-1C, consistent with the protein being glycosylated.
  • the IdeS sequence has three consensus motifs for N-glycosylation in mammalian cells at positions 61, 288, and 336 (a fourth potential N- glycosylation site is added by the C-terminal linker and purification tag).
  • N61 was identified as the site of glycosylation (FIG.1E).
  • Protein purified from Expi293F culture had similar catalytic activity to non-glycosylated IdeS from bacteria (FIGs.1F-1G). From the results, it was concluded that the mammalian expression system, despite producing IdeS with non-native glycosylation at N61, is suitable for rapidly determining effects of modifications.
  • the eighth cluster may represent two or more individual Attorney Docket No.: TKT-002WO epitopes that are overlapping or adjacent to each other.
  • HLA haplotype of donors for MAPPs Donor HLA Haplotype DRB111 DRB115 DRB302 DRB501 DQA10 DQA10 DQB103 DQB106 1 01 01 02 01 102 505 01 02 6 6 2 6 seen in FIGs.2B-2C. Most of the residues in Cluster 1 were not resolved in the electron density and are presumably disordered. Clusters 2-8 cover the protein surface and hydrophobic interior. There were frequent residue interactions between epitope clusters, and residues within Clusters 2, 7 and 8 shape the active site.
  • the IdeS sequence was scanned for 15-mer peptides, and their predicted immunogenicity score is based on calculated HLA-II affinity (e.g. a rank-percentage of 10% means the peptide is predicted to fall within the 10% of peptides of highest affinity from a reference set of 100,000) and the number of HLA-II alleles to which the peptide is expected to bind.
  • HLA-II affinity e.g. a rank-percentage of 10% means the peptide is predicted to fall within the 10% of peptides of highest affinity from a reference set of 100,000
  • the number of HLA-II alleles to which the peptide is expected to bind As the Attorney Docket No.: TKT-002WO threshold for the rank-percentage is loosened, more of the IdeS sequence is predicted to be immunogenic, whereas stringent thresholds are expected to predict the most likely HLA-II epitopes.
  • IVIG intravenous immune globulin
  • FIG.4 and FIG.5 The expression medium was then screened for proteolytic activity against intravenous immune globulin (IVIG; polyclonal immunoglobulin pooled from many donors) (FIG.4 and FIG.5). Since IdeS activity in this screen is directly tested from expression medium without purification, proteolytic cleavage of IVIG is a function of both IdeS variant expression level and intrinsic catalytic activity. Indeed, activity often correlated with the expression level of IdeS based on Coomassie- stained SDS-PAGE analysis of the medium (FIG.4 and FIG.5). 260 single amino acid substitutions were screened at 141 positions and were found to have varying levels of expression and catalytic activity (FIGs.3-5 and Table 3).
  • cysteine substitutions predicted to form disulfides were strategically introduced into IdeS to potentially stabilize the protein fold (FIG.6 and Table 4); of 3 cysteine pairs screened, 1 yielded soluble protein in the expression medium with activity on par with wild type IdeS. Table 3. Screening of IdeS mutants with substitutions in single epitope clusters M odification(s) ⁇ Immuno. Rel. Modification(s) Rel. 2 ⁇ Immuno.
  • the introduced cysteine pair predicted to form a disulfide was determined to reduce the Tm of IdeS by 2 oC (FIGs.10A- 10B) and it was thus removed from the reduced immunogenic IdeS derivatives.
  • One of the reduced immunogenic IdeS proteins from bacteria was expressed and purified: Variant 10, with a chimeric N-terminus and 7 substitutions, predicted to reduce HLA-II presentation of epitopes in Clusters 1, 2, 4, 5, 6, and 8 (FIGs.11A-11B).
  • peptides were synthesized spanning regions of Variant 10 that were mutated (Table 7). Increasing concentrations of wild type and mutant IdeS peptides were incubated with HLA-II (purified with low affinity placeholder peptides to maintain stability) in the presence of a constant concentration of a fluorescent reference peptide.
  • HLA-II-specific Attorney Docket No.: TKT-002WO chaperone and peptide exchange catalyst HLA-DM the placeholder peptide is replaced in an exchange reaction that mimics the biological process of HLA-II antigen presentation.
  • fluorescence polarization signal increases. IdeS peptides compete with the reference peptide for HLA-II binding and inhibition of fluorescence polarization acts as an indirect readout of relative peptide affinity.
  • N-glycosylation motifs were introduced into wild-type IdeS. Following expression by Expi293F cells, 8 of the IdeS variants were both active and had apparent increases in MW by electrophoresis, consistent with the attachment of additional glycan groups (Table 8 and FIG.18A). 4 combinations of N-glycosylation motifs in the reduced immunogenic Variant 10 at positions 111, 142, and 198 were evaluated, which Attorney Docket No.: TKT-002WO structural analysis suggested could be combined without negative interference (FIG.18B and Table 9).
  • FIGs.18A and 18C Two were found to have high catalytic activity (FIGs.18A and 18C) with cooperative unfolding, in which the melting temperatures were slightly reduced compared to parental Variant 10 (FIGs.18D and 18E).
  • the other 2 combinations put together custom N- glycosylation motifs at positions 111 and 142, and these produced proteins with low activity that correlated with steady and poorly cooperative unfolding that began at low temperatures.
  • These low stability IdeS variants separated by electrophoresis as broad heterogenous mixtures (FIG.18F) suggestive that their partial unfolding during biogenesis was exposing additional sites for glycosylation, presumably native positions N288 and N336.
  • Variant 10.2 was further modified with 3 N- glycosylation sites in total: at wild-type position 61 and at custom positions 111 and 198.
  • Table 8. Screening the addition of single N-glycosylation motifs Name Custom N- Mutation(s) Electrophoretic Activity 1 Glycosylation Mobility Shift Wt; -, inactive. Attorney Docket No.: TKT-002WO Table 9. Hyperglycosylated derivatives of Variant 10 Name Custom N- Mutations Activity 2 Glycosylation Sites 1 [00330] Glycan shielding was maximized by screening mutations at 8 new sites for creating additional N-glycosylation motifs.
  • the IdeS variants were screened in Expi293F expression medium (Table 8); 4 of the variants were both active for cleavage of IVIG and had reduced electrophoretic mobility consistent with the addition of glycans (FIG.19A). Additional N- glycosylation motifs were combined into Variant 10.2 to create variants with 4 to 6 N- glycans in total (Table 9). While IdeS variants with 5 or 6 N-glycans had varying levels of catalytic activity (FIG.19B), they were unstable and exposed hydrophobic core residues even at ambient temperature (FIG.19D). The IdeS variants with 4 N-glycans were all highly active and had cooperative unfolding by DSF (FIGs.19C and 19D).
  • Variant 10.9 was found to be hyperglycosylated with N-glycans at positions 47, 61, 111 and 198, folded at physiological temperature, and did not bind any of the hydrophobic DSF dye until it began to cooperatively unfold between ⁇ 45-60 ⁇ C (FIG.19D).
  • the Attorney Docket No.: TKT-002WO thermostability of Variant 10.9 was comparable to its 3 N-glycan and 1 N-glycan parental sequences, Variant 10.2 and Variant 10, respectively, as well as wild-type IdeS purified from Expi293F cells with a single N-glycan (FIGs.20A-20C).
  • N-glycan groups added substantial size to Variant 10.9 compared to aglycosylated protein based on SEC (FIGs.21A- 21B) and the added MW was lost following enzymatic release on N-glycans with PNGase F (FIG.20B).
  • hyperglycosylated Variant 10.9 retained preferential specificity for human and rabbit IgG, with slower cleavage of IgG from cynomolgus monkey and rat (FIG.20C).
  • Variant 10.9 was modeled with 3 common glycans of mammalian glycoproteins: a biantennary glycan that terminates with mannose, a biantennary glycan that terminates in sialic acid, and a tetraantennary glycan that terminates with sialic acid (Table 10). N-terminal residues up to amino acid 39, which are predicted to be unstructured, were excluded from analysis.
  • the footprint of the substrate is 36% of the IdeS surface and possibly larger if the IgG Fab domains also contribute to the IdeS-substrate interface. At least 36% of the IdeS surface is therefore unavailable for shielding without adversely impacting substrate accessibility and catalytic activity.
  • the 4 glycans of Variant 10.9 are primarily localized to one side of the protein (FIGs.22A-22B).
  • the glycans shield 25-45% of the total accessible surface area on IdeS (Table 10), corresponding to 39-70% of the IdeS surface that does not contribute to substrate recognition. Accessibility of a sphere with radius 7.2 ⁇ was analyzed, which mimics the size of a single CDR hypervariable loop and thus approximates the smallest antibody paratopes. Using this very conservative computational model of antibody accessibility, the glycans of Variant 10.9 were calculated at minimum to shield 20-35% of the total protein surface (Table 10), corresponding to 31-55% of the IdeS surface that does not contribute to substrate recognition. Table 10.
  • Wild type IdeS (1 glycan), Variant 10.2 (3 glycans), Variant 10.9 (4 glycans), and Variant 10.14 (an aglycosylated variant) were purified from Expi293F culture and administered IV to NZW rabbits (1 mg/kg dose). Hyperglycosylated Variant 10.2 and Variant 10.9 persisted longer in serum than aglycosylated Variant 10.14; while Variant 10.14 in rabbit serum was below the limit of detection after 8 h post-administration, the glycosylated variants were detected for 24 h (FIG.24A). Even wild type IdeS produced by Expi293F cells with a single glycan had significantly extended PK.
  • aglycosylated IdeS has a theoretical MW of ⁇ 35 kD and proteins less than 45 kD are rapidly filtered by the kidneys, suggesting added glycans may significantly reduce renal elimination through increasing the protein's size.
  • initial clearance of the hyperglycosylated Variant 10.2 and Variant 10.9 variants was greater than for Expi293F-produced wild type IdeS, which we hypothesize is due to fractions of the hyperglycosylated proteins having low sialylated glycoforms that are rapidly cleared via asialoglycoprotein receptors in the liver.
  • Extended PK of the glycosylated IdeS variants translated into a longer duration of IgG depletion, with IgG levels beginning to increase back towards baseline by 48 h in Variant 10.14 treated animals but not until 72 h, 120 h, and 144 h following wild type IdeS, Variant 10.2, and Variant 10.9 treatment, respectively (FIG.24B).
  • the trend for duration of IgG depletion is therefore correlated with the extent of IdeS glycosylation.
  • hyperglycosylation Attorney Docket No.: TKT-002WO succeeded not only in shielding potential B cell epitopes on the IdeS surface but also extended PK and PD duration.
  • the length of the glycine/serine-rich linker connecting IdeS to HSA was increased but found no differences in catalytic activity (FIG.25E).
  • the HSA fusion proteins were found to have reduced reactivity towards goat polyclonal anti-IdeS by ELISA, indicating surface epitopes exposed to B cells and their antibodies were indeed being hidden (FIG.25F).
  • the length of the connecting linker had no effect on masking of surface epitopes, suggesting the HSA fusion partner may lie on the IdeS surface in a preferred conformation. Table 11.
  • IdeS Variants that Combine HLA-II Epitope Reduction, Glycan Shielding, and HSA Fusion
  • HSA was fused to the C-termini of: (i) Variant 10, with partial removal of HLA-II epitopes and 1 N-glycan at native position N61; (ii) Variant 10.2, a derivative of Variant 10 with 3 N-glycans; and (iii) Variant 10.9, another derivative with 4 N-glycans.
  • the EC50 values for reactivity of the two variants are approximately half those of wild type IdeS.
  • Variant 10.9 had consistently lower reactivity than Variant 10.2-HSA, suggesting the addition of the fourth glycan at N47 is shielding a more dominant epitope targeted by human anti-IdeS antibodies than HSA fusion at the C-terminus.
  • Epitope shielding was further confirmed by competition ELISA, in which human serum or IVIG was pre-incubated with competing IdeS variants before testing reactivity against wild type IdeS coated on the ELISA plate surface.
  • Variant 10.2-HSA Elimination of Variant 10.2-HSA from rabbit serum followed the same trajectory as aglycosylated Variant 10.14-HSA (FIG.30A), with the exception of an initial phase of rapid clearance that has similarities to initial clearance in rabbits of hyperglycosylated IdeS variants without HSA.
  • This initial phase of rapid clearance of glycosylated IdeS variants is due to a fraction of protein having poorly sialylated glycoforms, leading to targeted removal by the liver.
  • the degree to which glycosylated IdeS variants are initially cleared may therefore be modulated through optimized production methods focused on glycan composition.
  • the long PK of Variant 10.2-HSA was associated with extended duration of IgG depletion for approximately 2 weeks (FIG.30B).
  • ELISA analysis of IgG depletion in rabbits measures full-length IgG or single cut IgG (scIgG), in which the Fab and Fc regions remain together. Clearance of antigen-binding F(ab')2 fragments, a product of IdeS-catalyzed IgG cleavage, was qualitatively assessed by immunoblot analysis of serum from rabbits treated with wild type and variant IdeS proteins (FIGs.31A-31C). We consistently observed that F(ab')2 cleavage products are cleared in rabbits by 24 hours post IdeS administration.
  • IdeS variants with extended PK provide a substantially longer window of time in which antigen-binding IgG and F(ab')2 products are depleted. This offers significant benefits for applications in AAV-mediated gene therapy and IgG-mediated autoimmunity, due to extended removal of neutralizing anti-AAV and pathogenic autoantigen-reactive antibodies and antibody fragments, respectively.
  • PK of Variant 10.9 and Variant 10.2-HSA were also evaluated in inbred C57Bl/6 mice with increased animal number for more accurate determination of PK properties. Hyperglycosylation was found to increase half-life from 2.6 h (bacteria-produced wild type IdeS) to 5.1 h (Variant 10.9), with a further extension to 17.8 h (Variant 10.2-HSA) following HSA fusion (FIGs.32A-32B and Table 12).
  • HSA-fused IdeS will have substantially longer duration of action in human patients than observed in rabbit or mouse models.
  • Variant 10.9 and Variant 10.2-HSA with neuraminidase caused a massive reduction in half-life and exposure, consistent with sialylation of glycans being necessary for optimum PK.
  • Table 12. PK properties of wild type IdeS and variants in C57Bl/6 mice AUC0-inf (3) IdeS variant (1) t1 2 ⁇ (2) (h) MRT0 i f (3) (h) Attorney Docket No.: TKT-002WO Wt (E. coli produced) 2.6 7.3 0.7 Variant 10.9 5.1 9.0 5.0 i 1 2 H A 1 1 1 1 ea a - e s e e e o - , - a a .
  • the signal peptide of influenza A hemagglutinnin (KTIIALSYIFCLVFA (SEQ ID NO: 193)) was fused to the N-terminus of mature IdeS (a.a.30-339) and the IdeS C-terminus was fused to a GSG linker and 8xHis affinity tag (SEQ ID NO: 194).
  • the gene was cloned into the NheI-XhoI sites of pcDNA3.1(+) with a strong Kozak sequence (GCCACCATG, where ATG is the start methionine) at the 5’ end. Single substitution modifications were made using overlap extension PCR, while constructs with multiple modifications were synthesized as DNA fragments.
  • MAPPs uses dendritic cells (DCs) cultured from multiple donors with mass spectrometry to identity peptides that are presented on MHC class II molecules that decorate the DC surface. Briefly, buffy coats from 10 healthy donors were collected and used as a source for culturing monocyte-derived DCs. DCs were proliferated and on day 7 were pulsed with test construct (IdeS).
  • DCs dendritic cells
  • the DCs were harvested and lysed in a hypotonic buffer.
  • pMHC-II molecules were captured via immunoprecipitation using a pan- HLA-DR antibody.
  • Peptides bound to the MHC-II were then analyzed using nano-LC- MS/MS, and includes peptides derived from processing of the test construct together with peptides from other proteins available in the culture.
  • the nano-LC-MS/MS data was analyzed to find peptides with sequences matching the test construct.
  • MHC haplotypes of donors were also determined to match peptide presentation to specific MHC-II alleles (Table 2).
  • IdeS crystal structure (PDB 2AVW) was prepared by first modeling residues not resolved in the electron density and to revert the catalytic C94A modification back to cysteine. This model was then further refined.
  • PDB 2AVW The IdeS crystal structure
  • To prepare a model of the IdeS- IgG1-Fc complex the crystal structure of IgG1-Fc bound to Mac-2 (PDB 8A47) was modified and used as. Using PyMOL, a 50 residue GS linker was added between the chains of the Fc (chains A and B). The linker was not sampled and existed only as 'padding' for the machine learning algorithm.
  • IdeS sequences that are less immunogenic were generated using computational methods, in which point variants were evaluated and scored based on their rotamer compatibility within an 8 ⁇ sphere and on their predicted immunogenicity. To expedite the reduction of immunogenicity process, only single positions were allowed to modify at any given time. Modifications were focused to regions highlighted by MAPPs and where epitope prediction found the position was part of an epitope/s that was predicted to bind at least 4 HLA-II alleles with a rank-percentage of less than 10.0.
  • DDG folding free energies
  • Disulfide Bond Engineering Two methods were used to find disulfides within IdeS that might stabilize the protein while simultaneously reducing epitope presentation. The first method was used to design cysteine modifications that would be likely to yield disulfide bonds based on the IdeS crystal structure. Briefly, the crystal structure of IdeS (PDB 2AVW) was cartesian relaxed 18 and this relaxed structure was used as an input. All residue-residue pairs with C ⁇ atoms less than or equal to 5 ⁇ from each other were evaluated.
  • the C1 ⁇ -C1 ⁇ -C2 ⁇ -C2 ⁇ dihedral angles of these residue pairs are then checked for compatibility with representatives from all possible ideal-disulfide bond backbone configurations.
  • the residue pairs that pass that filter are then ranked based on their reduction in unfolded state entropy.
  • the method predicted 13 disulfides possible within the IdeS structure but only the designed disulfide between residues 62 and 283 had a dslf_fa13 score ⁇ 0 and therefore was the only one chosen for experimental testing.
  • structural similarity searches were used to find proteins with similar topologies to IdeS, and then these structures were mined for their disulfides.
  • the culture was induced with 0.2 mM isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG) and the temperature was lowered to 30 °C.
  • IPTG isopropyl ⁇ -D-1-thiogalactopyranoside
  • Cells were harvested after ⁇ 12 h by centrifugation (10 minutes, 4000 ⁇ g, 4 °C). Cells were resuspended in phosphate-buffered saline (PBS) and sonicated. The lysate was cleared by centrifugation (15,000 ⁇ g, 4 °C, 10 minutes). The supernatant was incubated with Ni-NTA resin while rotating for 1 hour at 4 °C.
  • PBS phosphate-buffered saline
  • Resin was collected by passing through a gravity column, washed with 8 column volumes (CV) PBS, and washed with 8 CV PBS containing 20 mM imidazole pH 7.8. Protein was eluted with 250 mM imidazole in PBS. The eluate was concentrated using a centrifugal filtration device with a 10,000 molecular weight cut off (MWCO). The protein was then further purified using size exclusion chromatography on a Superdex 75 (S75) 10-300 GL increase column with PBS as the running buffer.
  • CV column volumes
  • S75 Superdex 75
  • Expi293F cells were cultured at 37 °C, 125 rpm, 8% CO2, maintaining a density of ⁇ 0.25-4 ⁇ ⁇ 10 6 cells/ml. Cells were transfected at a density of 2 ⁇ 10 6 cells/ml using ExpiFectamine according to the manufacturer’s directions, using 1 mg pcDNA3-IdeS-8h variant per ml culture. ExpiFectamine Transfection Enhancers 1 and 2 were added 18-22 h post-transfection. Expression medium was harvested 4-7 days post-transfection.
  • IdeS constructs cloned in pcDNA3 were transfected into Expi293F cells as described above. Expression medium was harvested 4-5 days post-transfection. Proteolytic activity was measured against clinical-grade human IVIG or purified human polyclonal IgG (MP) with sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE).
  • Rabbit 2 1, 2, 8, 24, 72, and 168 h. Pre-bleeds were taken at -120 h. Rabbits were treated with 0.5 ml acepromazine injected subcutaneously prior to ear vein sampling. Enzymatic activity in the blood draws was immediately inhibited by addition of iodoacetic acid (1-2 mM final) and the blood was allowed to clot. Post centrifugation, serum samples were stored at - 20 ⁇ C.

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