Ides variant proteins and methods of using the same

Abstract

This disclosure relates generally to variant IdeS polypeptides having lower immunogenicity and preserved or improved IdeS stability and IgG cleavage activity, and methods of using the same.

Description

Attorney Docket No.: TKT-002WO IDES VARIANT PROTEINS AND METHODS OF USING THE SAME CROSS-REFERENCE

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No.63 / 492,891, filed March 29, 2023, and U.S. Provisional Patent Application No.63 / 584,748, filed September 22, 2023, each of which is incorporated by reference in its entirety herein. FIELD OF THE DISCLOSURE

[0002] This disclosure relates to variant Ides polypeptides having lower immunogenicity and preserved or improved IdeS stability and IgG cleavage activity, and methods of using the same. BACKGROUND

[0003] IdeS (Immunoglobulin Degrading Enzyme from Streptococcus pyogenes) is a cysteine protease that catalyzes the rapid cleavage of all human IgG subclasses within the hinge region, severing the antigen binding domains of IgG from the Fc region that mediates immune effector functions. The decoupling of the antigen-binding Fab regions from the Fc moiety prevents Fc-mediated recruitment of immune effector functions to the antigen, including the recruitment of immune cells with killing capabilities (i.e. antibody-dependent cellular cytotoxicity or ADCC) and the deposition of activated complement (i.e. complement- dependent cytotoxicity or CDC).

[0004] 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.

[0005] However, IdeS is immunogenic and its administration elicits anti-IdeS antibodies, preventing possible uses where repeated long-term dosing is required. Furthermore, 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.

[0006] Described herein are 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. Such reduced immunogenic IdeS variants may be used to expand the reach of an IdeS-like drug into indications where repeated dosing is necessary. SUMMARY OF THE DISCLOSURE

[0007] Provided herein are 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.

[0008] Described herein, in certain embodiments, are 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. In some embodiments, 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. In some embodiments, the modification at position 277 is a substitution of leucine to cysteine. In some embodiments, 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 303 is a substitution of alanine to aspartate, a substitution of alanine to asparagine, or a substitution of alanine to glutamine. In some embodiments, the modification at position 306 is a substitution of valine to threonine. In some embodiments, the modification at position 318 is a substitution of isoleucine to lysine, aAttorney Docket No.: TKT-002WO substitution of isoleucine to aspartate, or a substitution of isoleucine to glycine. In some embodiments, the variant protein further comprises a modification at position 308. In some embodiments, the modification at position 308 is a substitution of isoleucine to leucine. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 positionAttorney Docket No.: TKT-002WO 308 to leucine. In some embodiments, 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: 85). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 toAttorney 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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, theAttorney Docket No.: TKT-002WO variant protein comprises a substitution of lysine at position 247 to asparagine. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0012] Described herein, in certain embodiments, are IdeS variant proteins comprising the amino acid sequence according to SEQ ID NO: 12.

[0013] Described herein, in certain embodiments, are IdeS variant proteins comprising the amino acid sequence according to SEQ ID NO: 13.

[0014] Described herein, in certain embodiments, are 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 QEEIAEGRRNPLRTAEWPMTKSTTDQ (SEQ ID NO: 85).

[0015] Described herein, in certain embodiments, are 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).

[0016] Described herein, in certain embodiments, are IdeS variant proteins comprising the amino acid sequence according to SEQ ID NOs: 86-94. In some embodiments, the variant protein has decreased presentation of epitopes on human leukocyte antigen (HLA) as compared to a wild-type IdeS protein. In some embodiments, 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.

[0018] Described herein, in certain embodiments, are 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. In some embodiments, the modification at position 31 is a substitution of serine to asparagine. In some embodiments, the modification at position 37 is a substitution of glutamate to asparagine. In some embodiments, the modification at position 39 is a substitution of arginine to threonine or asparagine. In some embodiments, the modification at position 42 is a substitution of glutamate to asparagine. In some embodiments, the modification at position 74 is a substitution of valine to asparagine. In some embodiments, the modification at position 76 is a substitution of asparagine to serine. In some embodiments, the modification at position 111 is a substitution of lysine to asparagine. In some embodiments, the modification at position 113 is a substitution of glutamine to threonine. In some embodiments, the modification at position 119 is a substitution of glutamate to asparagine. In some embodiments, the modification at position 121 is a substitution of histidine to threonine. In some embodiments, the modification atAttorney Docket No.: TKT-002WO position 130 is a substitution of asparagine to serine. In some embodiments, the modification at position 142 is a substitution of aspartate to asparagine. In some embodiments, the modification at position 144 is a substitution of lysine to threonine. In some embodiments, the modification at position 147 is a substitution of glutamine to serine. In some embodiments, the modification at position 148 is a substitution of leucine to asparagine. In some embodiments, the modification at position 198 is a substitution of glutamate to asparagine. In some embodiments, the modification at position 233 is a substitution of lysine to serine. In some embodiments, the modification at position 244 is a substitution of threonine to asparagine. In some embodiments, the modification at position 246 is a substitution of glycine to threonine. In some embodiments, the modification at position 311 is a substitution of lysine to asparagine. In some embodiments, the modification at position 313 is a substitution of isoleucine to threonine. In some embodiments, the modification at position 319 is a substitution of glycine to serine. In some embodiments, the variant protein comprises a substitution of glutamate at position 37 to asparagine and a substitution of arginine at position 39 to threonine. In some embodiments, 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. In some embodiments, 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.

[0019] Described herein, in certain embodiments, are 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. In some embodiments, the modification at position 47 is a substitution of threonine to asparagine. In some embodiments, the modification at position 49 is a substitution of glutamine to threonine or asparagine. In some embodiments, the modification at position 51 is a substitution of valine to threonine. In some embodiments, the modification at position 78 is a substitution of glycine to serine. In some embodiments, theAttorney Docket No.: TKT-002WO modification at position 111 is a substitution of lysine to asparagine. In some embodiments, the modification at position 113 is a substitution of glutamine to threonine. In some embodiments, the modification at position 123 is a substitution of glutamate to asparagine. In some embodiments, the modification at position 125 is a substitution of glutamine to threonine. In some embodiments, the modification at position 126 is a substitution of lysine to asparagine. In some embodiments, the modification at position 128 is a substitution of asparagine to serine. In some embodiments, the modification at position 142 is a substitution of aspartate to asparagine. In some embodiments, the modification at position 144 is a substitution of lysine to threonine. In some embodiments, the modification at position 148 is a substitution of leucine to asparagine. In some embodiments, the modification at position 198 is a substitution of glutamate to asparagine. In some embodiments, the modification at position 273 is a substitution of serine to asparagine. In some embodiments, the modification at position 275 is a substitution of glycine to serine. In some embodiments, the modification at position 278 is a substitution of lysine to serine. In some embodiments, 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. In some embodiments, 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. 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, 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. In some embodiments, 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. 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, 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. In some embodiments, 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. In some embodiments, 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. 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, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. 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, a substitution of glutamate at position 123 to asparagine, a substitution of glutamine at position 125 toAttorney Docket No.: TKT-002WO threonine, and a substitution of glutamate at position 198 to asparagine. 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, 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. 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, a substitution of leucine at position 148 to asparagine, and a substitution of glutamate at position 198 to asparagine.

[0020] 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. In some embodiments, the human serum albumin is conjugated to a N-terminal of the IdeS protein. In some embodiments, the human serum albumin is conjugated to a C-terminal of the IdeS protein. In some embodiments, the IdeS protein is conjugated to the human serum albumin using a linker. In some embodiments, 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. In some embodiments, the IdeS protein is fused to the human serum albumin.

[0021] Described herein, in certain embodiments, are 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; and b) a human serum albumin conjugated to the IdeS variant protein.

[0022] 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.

[0026] Described herein, in certain embodiments, are methods of treating a disease or disorder comprising administering an effective amount of the IdeS variant protein described herein or the pharmaceutical composition described herein. In some embodiments, the IdeS variant protein is co-administered with a gene therapy.

[0027] Described herein, in certain embodiments, are methods for reducing presentation on HLA-II of IdeS comprising administering an effective amount of the IdeS variant protein described herein, the polypeptide described herein, or the pharmaceutical composition described herein.

[0028] Described herein, in certain embodiments, are methods for prolonging serum half- life of IdeS comprising administering an effective amount of the IdeS variant protein described herein, the polypeptide described herein, or the pharmaceutical composition described herein.

[0029] Described herein, in certain embodiments, are methods for prolonging IgG depletion of IdeS comprising administering an effective amount of the IdeS variant protein described herein, the polypeptide described herein, or the pharmaceutical composition described herein.

[0030] Other embodiments and details of the disclosure are presented herein below. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] 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 µg) 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 withAttorney 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 µM).

[0032] 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). Residues that are highly represented in HLA-II presented peptides are in darker shading. Side chains of the catalytic triad are labeled and shown as black spheres (201). 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.

[0033] 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. Computationally predicted epitopes are shown (303), based on predicted binding to at least 4 HLA-II alleles in a test set of 14 alleles. From light to dark, the threshold for the rank percentage decreases from 20 to 15 to 10 to 5 % (i.e. the predictions increase in stringency). Experimentally identified HLA-II epitopes by MAPPs are shown (305), representing the number of unique peptides a residue is identified in, summed across 10 donors. In the bottom row, the sites of modifications to reduce immunogenicity of the protein are indicated (307). Lighter gray (301) indicates a modification was active in the screen.

[0034] FIG.4 depicts data from rapid screening of IdeS variants. To facilitate rapid screening of mutants, 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. Another set of example data from screening variants of IdeS directly from Expi293F expression medium. Upper panels show Coomassie-stained gels of degradation products when the expression medium is incubated with IVIG. The gel is run under non-reducing conditions. Lower panels show IdeS levels in the expression medium. Wt, wild type. C-94-S is a catalytic dead modification to the nucleophilic cysteine in the active site.

[0036] 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. The Coomassie-stained electrophoresis gel in the upper panel shows proteolytic products of IVIG after 1 h at 37 °C. The lower panel shows IdeS expression level in the medium. IdeS_D.3.3 was found to be as active as wild type IdeS.

[0037] 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. The proteins (20 nM) were incubated with 20 µM human monoclonal IgG1 (FIG.7A and FIG.7D), rabbit polyclonal IgG (FIG.7B and FIG.7E), or mouse polyclonal IgG (FIG.7C and FIG.7F) at 37 °C for the indicated times. Immunoglobulin degradation products were analyzed by Coomassie-stained SDS gels run under non-reducing conditions.

[0038] 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. Truncation variants IdeS_B.3.4 to IdeS_B.3.7 progressively shorten the N-terminus of the mature polypeptide by 5, 10, 16, and 19 residues, respectively. 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).

[0039] 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. Upper panels show Coomassie-stained electrophoresis gels following incubation of IdeS-transfected Expi293F expression medium with human IVIG. The gel was run under non-reducing conditions to observe the immunoglobulin degradation products. The lower Coomassie-stained gels show IdeS expression level in the medium. FIG.9B depicts a graph of melting temperatures of Variant 1 and its progeny as measured by DSF.

[0040] 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. Combining modifications from IdeS_D.3.3 (disulfide), Variant 94 (N-terminal chimera), Variant 77 (reduced immunogenic intermediate), plus others, yielded Variant 1 with reduced thermal stability. A small number of reversion and compensatory modifications led to Variant 6 (1001) and ultimately Variant 10 (1003).

[0041] 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. coli. L, load; FT, flow through. 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 µM). Degradation products were visualized on a Coomassie-stained polyacrylamide gel run under non-reducing conditions.

[0042] FIG.12 depicts graphs of binding of IdeS peptides to HLA-DR1. Wild-type (circles) and mutant (squares) peptides competed with a fluorescent reference peptide for binding to HLA-DRA1 / HLA-DRB1. Fluorescence polarization (FP) signal of the reference peptide is measured. Tighter binding peptides inhibit the FP signal at lower concentrations (i.e. curves of high affinity peptides are shifted to the left). Peptides that fail to bind with reasonable affinity to the HLA-II do not inhibit FP (e.g. peptide P6 at bottom-left).Attorney Docket No.: TKT-002WO

[0043] FIG.13 depicts graphs of binding of IdeS peptides to HLA-DR7. Wild-type (circles) and mutant (squares) peptides competed with a fluorescent reference peptide for binding to HLA-DRA1 / HLA-DRB7. FP of the reference peptide is plotted.

[0044] 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. Wild-type (circles) and mutant (squares) peptides competed with a fluorescent reference peptide for binding to HLA- DRA1 / HLA-DRB15. FP of the reference peptide is plotted.

[0046] 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.

[0047] 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.

[0048] 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). For orientation, 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. The IdeS proteinsAttorney Docket No.: TKT-002WO were purified and cleavage over 60 minutes of a monoclonal human IgG1 was monitored by SDS-PAGE. 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.

[0049] 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. Upper Coomassie-stained SDS electrophoretic gel (non-reducing conditions) shows cleavage of human IVIG incubated with expression medium. Lower gel shows expression of IdeS proteins.

[0050] 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.

[0051] 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.

[0052] 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 µM 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.

[0053] 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.

[0054] 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).

[0055] 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. At left, glycans are depicted using the symbol nomenclature for glycans (SNFG). At right, the van der Waals surfaces of the glycans are shown on top of the protein surface. FIG.22B depicts the protein surface of Variant 10.9 with amino acids that areAttorney 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.

[0056] FIG.23 depicts reduced anti-IdeS antibody recognition in ELISA when IdeS surface epitopes are shielded by the addition of glycans. Goat polyclonal anti-IdeS was used for capture and detection in ELISA of IdeS variants with increasing N-glycosylation sites. Error bars represent N=2 technical replicates.

[0057] 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.

[0058] FIG.24B depicts ELISA measurements of serum IgG in IdeS treated rabbits. Error bars represent N=2 technical replicates.

[0059] 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.

[0060] 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.

[0061] FIG.25C depicts purified Variant 10.14 with and without HSA fusion (20 nM) was incubated with human monoclonal IgG1 (20 µM) at 37 °C for 60 minutes. Cleavage products were separated by non-reducing SDS-PAGE.

[0062] FIG.25D depicts Coomassie-stained SDS gel of expression medium from Expi293F transfected with Variant 10.14 -HSA with different connecting linkers.

[0063] 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.

[0064] FIG.25F depicts ELISA using goat polyclonal anti-IdeS for capture and detection of Variant 10.14 -HSA linker variants.

[0065] FIG.26A depicts NZW rabbits (~4 kg) were IV administered 4 mg Variant 10.14 with (dotted line) and without (broken line) HSA fusion. Protein concentrations in serum were measured by ELISA. N=3 per group and serum collection was staggered between rabbits with N=1 per time point.

[0066] FIG.26B depicts serum IgG was measured by ELISA. Error bars represent N=2 technical replicates.

[0067] FIG.27 depicts goat polyclonal anti-IdeS was used for capture and detection in ELISA of glycosylated IdeS variants fused to HSA. Error bars represent N=2 technical replicates.Attorney Docket No.: TKT-002WO

[0068] FIG.28A depicts Coomassie-stained non-reducing SDS gel of human IgG1 (20 ^M) cleaved by IdeS-HSA variants (20 nM) at 37 ^C.

[0069] 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.

[0070] 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. pyogenes infection (FIG.29B), from a healthy donor (FIG.29C), or pooled IVIG (FIG.29D) were pre-incubated with increasing concentrations of Wt IdeS or variants as competitors. Blocked serum / IVIG was then added to the ELISA plates and antibody binding was measured.

[0071] 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.

[0072] FIG.30B depicts ELISA measurements of serum IgG in treated rabbits. Error bars represent N=2 technical replicates. Time points where serum samples were not collected are indicated as not determined / ND.

[0073] 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).

[0074] 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 WtAttorney Docket No.: TKT-002WO IdeS produced in Expi293F culture. IgG / scIgG levels (pale grey) were assayed using anti- rabbit F(ab')2for capture and anti-rabbit Fc for detection. IgG / scIgG / F(ab')2levels (dark grey) were assayed by directly coating ELISA plates with serum and detecting with anti- rabbit F(ab')2.

[0075] FIGs.32A-32B depict PK of wild type IdeS and variants in C57Bl / 6 mice that were administered IdeS proteins as a single dose of 1 mg / kg via the tail vein. Serum was collected and analyzed by quantitative ELISA to determine concentrations of IdeS until levels were below the detection limit. Data are mean ± SEM from N=3 male mice (6-8 weeks old) per time point. Variant 10.9 (solid line and triangles, FIG.32A) and Variant 10.2-HSA (solid line and diamonds, FIG.32B) are compared to 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.

[0077] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.

[0078] As used herein, 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.

[0079] As used herein, 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.

[0080] As used herein, the term “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. For examples of carriers, stabilizers and adjuvants, see e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .Attorney Docket No.: TKT-002WO

[0081] The terms “protein” or “polypeptide” are used interchangeably herein and refer to a polymer of repeating structural units connected by a peptide bond. Typically, 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. In certain aspects, proteins or polypeptides may be post-translationally modified (e.g. glycosylated, phosphorylated, lapidated, acetylated, or conjugation with a labeling component).

[0082] The term “heterologous” as used herein refers to a nucleic acid or polypeptide originating from a different genetic source or species.

[0083] The term “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.

[0084] 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. 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. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithmAttorney Docket No.: TKT-002WO involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, 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). For amino acid sequences, 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 BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, 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)).

[0087] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, 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.

[0088] As used herein, 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 “about,” as used herein when referring to a measurable value such as an amount of mass, weight, time, volume, concentration, or percentage, is meant to encompass variations of in certain aspects ±20%, in certain aspects ±10%, in certain aspects ±9%, in certain aspects ±8%, in certain aspects ±7%,Attorney Docket No.: TKT-002WO in certain aspects ±6%, in certain aspects ±5%, in certain aspects ±4%, in certain aspects ±3%, in certain aspects ±2%, in certain aspects ±1%, in certain aspects ±0.5%, and in certain aspects ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods and / or employ the disclosed compositions. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0089] As used herein, 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. For example, a functional fragment of a polypeptide or protein of the present application maintains its ability to perform its catalytic activity.

[0090] As used herein, 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.

[0091] 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. Substitution of one amino acid residue for another from a different class or group is referred to herein as a “non-conservative” substitution. In contrast, non-conservative amino acid substitutions tend to modify conformation and function of a protein. Example of Amino Acid Classification Small / Aliphatic residues: Gly, Ala, Val, Leu, IleCyclic Imino Acid: ProHydroxyl-containing Residues: Ser, Thr Acidic Residues: Asp, GluAttorney Docket No.: TKT-002WO Amide Residues: Asn, GlnBasic Residues: Lys, ArgImidazole Residue: HisAromatic Residues: Phe, Tyr, TrpSulfur-containing Residues: Met, Cys

[0092] In certain aspects, a conservative amino acid substitution includes substituting any of: glycine (G), alanine (A), isoleucine (I), valine (V), and leucine (L) for any other of these aliphatic amino acids; serine (S) for threonine (T) and vice versa; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; lysine (K) for arginine (R) and vice versa; phenylalanine (F), tyrosine (Y) and tryptophan (W) for any other of these aromatic amino acids; and methionine (M) for cysteine (C) and vice versa. Other 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. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pKs of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments (see, e.g., BIOCHEMISTRY at pp.13-15, 2nd ed. Lubert Stryer ed. (Stanford University); Henikoff et al., Proc. Nat'l Acad. Sci. USA (1992) 89:10915-10919; Lei et al., J. Biol. Chem. (1995) 270(20):11882-11886).

[0093] As used herein, the term “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).Attorney Docket No.: TKT-002WO

[0095] As used herein, “nucleic acid” or “oligonucleotide” or “polynucleotide” or grammatical equivalents used herein means at least two nucleotides covalently linked together. The term “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.

[0096] As used herein, 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. In certain aspects, the vector is a virus vector, such as a lentivirus vector.

[0097] As used herein, the term “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.

[0098] As used herein, the term “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.

[0099] As used herein, the term “sialylation” is the enzymatic addition of a neuraminic acid residue.

[0100] As used herein, the term “neuraminic acid” refers to a 9-carbon monosaccharide, a derivative of a ketonanose.

[0101] As used herein, the term “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: (Manα1-6[Manα1- 3)]Man(β1-4)GlcNAc(β1-4)GlcNAc(β1-Asn-X-Ser / Thr). The N-glycan core structure can be expanded through galactosylation, further GlcNAclyation, sialylation, fucosylation, or combinations thereof.

[0102] As used herein, the term “biantennary” refers to an N-linked glycan comprising the N-glycan core (Manα1-6[Manα1-3)]Man(β1-4)GlcNAc(β1-4)GlcNAc(β1-Asn-X-Ser / Thr) elongated with two GlcNAc residues linked to C-2 of the core mannose α1-3 and the mannose α1-6. This core structure can then be elongated or modified by various glycan structures.Attorney Docket No.: TKT-002WO

[0103] As used herein, the term “triantennary” refers to an N-linked glycan comprising an additional GlcNAc residue is added to either the C-4 of the core mannose α1-3 or the C-6 of the core mannose α1-6 of the biantennary core structure. This structure can then be elongated or modified by various glycan structures.

[0104] As used herein, the term “tetraantennary” refers to an N-linked glycan comprising two additional GlcNAc residues that are added to either the C-4 of the core mannose α1-3 or the C-6 of the core mannose α1-6 of the biantennary core structure. This core structure can then be elongated or modified by various glycan structures. I. Modified IdeS proteins

[0105] Described herein, in certain embodiments, are modified IdeS proteins comprising one or more modifications as compared to wild-type IdeS sequence. Wild-type IdeS with signal peptide (signal peptide underlined) MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGV TPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQN KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTK HLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRH DFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYV TDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO:1)

[0106] As used herein, references to amino acid residue positions are relative to SEQ ID NO: 1 (i.e. full-length wild-type IdeS sequence including signal peptide) unless stated otherwise. Wild-type IdeS (mature polypeptide) DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKT FNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKE AIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVK EGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTY ANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAK EIKEDNIGAQVLGLFTLSTGQDSWNQTN (SEQ ID NO: 2)

[0107] In some embodiments, the present disclosure provides an IdeS variant comprising one or more 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%,Attorney Docket No.: TKT-002WO 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. In some embodiments, 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. In some embodiments, the present disclosure provides an IdeS variant at least 95% identical to SEQ ID NO: 1 and having one or more modifications. In some embodiments, the present disclosure provides an IdeS variant at least 95% identical to SEQ ID NO: 1 and having at least two modifications. IdeS Variants

[0108] Described herein are modified IdeS proteins. In some embodiments, the IdeS protein is modified to have reduced presentation on HLA-II. In some embodiments, 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. In some embodiments, 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. In some embodiments, the IdeS protein is modified in multiple ways.

[0109] Described herein, in certain embodiments, are 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.

[0110] In some embodiments, the modification at position 68 is a substitution of valine to threonine.

[0111] In some embodiments, the modification at position 75 is a substitution of alanine to proline.

[0112] In some embodiments, the modification at position 166 is a substitution of threonine to arginine or a substitution of threonine to glycine.

[0113] 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 187 is a substitution of serine to aspartate. In some embodiments, the modification at position 187 is a substitution of serine to glutamate.

[0114] In some embodiments, the modification at position 213 is a substitution of threonine to glutamate.Attorney Docket No.: TKT-002WO

[0115] In some embodiments, the modification at position 236 is a substitution of serine to cysteine.

[0116] In some embodiments, the modification at position 277 is a substitution of leucine to cysteine.

[0117] In some embodiments, 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.

[0118] 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. In some embodiments, 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.

[0119] In some embodiments, the modification at position 306 is a substitution of valine to threonine.

[0120] 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. In some embodiments, 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.

[0121] 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.

[0122] In some embodiments, 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.

[0123] In some embodiments, 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, aAttorney 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.

[0124] In some embodiments, 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.

[0125] In some embodiments, 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.

[0126] In some embodiments, 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.

[0127] In some embodiments, 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.

[0128] Described herein, in certain embodiments, are 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), AEPITLIWENYLSDSVSPDRDIR (SEQ ID NO: 84), and QEEIAEGRRNPLRTAEWPMTKSTTDQ (SEQ ID NO: 85). In some embodiments, 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: 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). In some embodiments, 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). In some embodiments, 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).

[0129] Described herein, in certain embodiments, are 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 any one of SEQ ID NOs: 95-112. In some embodiments, the IdeS variant proteins comprise a substitution of DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2 with aAttorney 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. In some embodiments, 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.

[0130] Described herein, in certain embodiments, are 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.

[0131] In some embodiments, the IdeS variant protein comprises a substitution of serine at position 31 to aspartate or asparagine.

[0132] In some embodiments, the IdeS variant protein comprises a substitution of phenylalanine at position 32 to lysine.

[0133] In some embodiments, the IdeS variant protein comprises a substitution of serine at position 33 to glutamate.

[0134] In some embodiments, the IdeS variant protein comprises a substitution of isoleucine at position 38 to valine.

[0135] In some embodiments, the IdeS variant protein comprises a substitution of arginine at position 39 to asparagine or threonine.

[0136] In some embodiments, the IdeS variant protein comprises a substitution of valine at position 43 to glutamate.

[0137] In some embodiments, the IdeS variant protein comprises a substitution of threonine at position 44 to glutamate.

[0138] In some embodiments, the IdeS variant protein comprises a substitution of proline at position 45 to glutamate.

[0139] In some embodiments, the IdeS variant protein comprises a substitution of histidine at position 47 to lysine.

[0140] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 54 to aspartate.Attorney Docket No.: TKT-002WO

[0141] In some embodiments, the IdeS variant protein comprises a substitution of threonine at position 57 to lysine or glutamine.

[0142] In some embodiments, the IdeS variant protein comprises a substitution of alanine at position 60 to aspartate or glutamine.

[0143] In some embodiments, the IdeS variant protein comprises a substitution of valine at position 74 to lysine.

[0144] In some embodiments, the IdeS variant protein comprises a substitution of alanine at position 75 to asparagine or proline.

[0145] In some embodiments, the IdeS variant protein comprises a substitution of glutamine at position 77 to glycine.

[0146] In some embodiments, the IdeS variant protein comprises a substitution of isoleucine at position 82 to methionine.

[0147] In some embodiments, the IdeS variant protein comprises a substitution of threonine at position 85 to glutamine.

[0148] In some embodiments, the IdeS variant protein comprises a substitution of glutamine at position 113 to aspartate.

[0149] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 115 to histidine.

[0150] In some embodiments, the IdeS variant protein comprises a substitution of arginine at position 116 to aspartate.

[0151] In some embodiments, the IdeS variant protein comprises a substitution of isoleucine at position 127 to methionine.

[0152] In some embodiments, the IdeS variant protein comprises a substitution of asparagine at position 128 to glycine.

[0153] In some embodiments, the IdeS variant protein comprises a substitution of phenylalanine at position 129 to glutamate.

[0154] In some embodiments, the IdeS variant protein comprises a substitution of asparagine at position 130 to glutamate.

[0155] In some embodiments, the IdeS variant protein comprises a substitution of glutamine at position 133 to aspartate.

[0156] In some embodiments, 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

[0157] 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. In some embodiments, the IdeS variant protein comprises a substitution of phenylalanine at position 153 to tyrosine.

[0158] In some embodiments, the IdeS variant protein comprises a substitution of glutamate at position 154 to aspartate.

[0159] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 159 to asparagine.

[0160] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 167 to proline.

[0161] In some embodiments, the IdeS variant protein comprises a substitution of histidine at position 168 to aspartate or proline. In some embodiments, 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.

[0162] In some embodiments, the IdeS variant protein comprises a substitution of histidine at position 175 to glutamine.

[0163] 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.

[0164] In some embodiments, the IdeS variant protein comprises a substitution of proline at position 195 to aspartate.

[0165] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 197 to glutamate.

[0166] In some embodiments, the IdeS variant protein comprises a substitution of alanine at position 210 to glycine.

[0167] In some embodiments, the IdeS variant protein comprises a substitution of serine at position 218 to aspartate.

[0168] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 219 to glycine.

[0169] In some embodiments, the IdeS variant protein comprises a substitution of leucine at position 220 to glutamine.Attorney Docket No.: TKT-002WO

[0170] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 228 to glutamine.

[0171] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 233 to serine.

[0172] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 241 to aspartate.

[0173] In some embodiments, the IdeS variant protein comprises a substitution of threonine at position 244 to lysine.

[0174] In some embodiments, the IdeS variant protein comprises a substitution of glutamate at position 245 to aspartate.

[0175] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 247 to asparagine.

[0176] In some embodiments, the IdeS variant protein comprises a substitution of valine at position 258 to aspartate.

[0177] In some embodiments, the IdeS variant protein comprises a substitution of serine at position 273 to aspartate.

[0178] In some embodiments, the IdeS variant protein comprises a substitution of asparagine at position 274 to glutamate.

[0179] In some embodiments, 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.

[0180] 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. In some embodiments, 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.

[0181] In some embodiments, the IdeS variant protein comprises a substitution of alanine at position 289 to proline.

[0182] In some embodiments, the IdeS variant protein comprises a substitution of phenylalanine at position 297 to aspartate.

[0183] In some embodiments, the IdeS variant protein comprises a substitution of glycine at position 299 to aspartate.Attorney Docket No.: TKT-002WO

[0184] In some embodiments, the IdeS variant protein comprises a substitution of valine at position 300 to glutamate.

[0185] In some embodiments, the IdeS variant protein comprises a substitution of alanine at position 307 to glycine or asparagine. In some embodiments, the IdeS variant protein comprises a substitution of alanine at position 307 to glycine. In some embodiments, the IdeS variant protein comprises a substitution of alanine at position 307 to asparagine.

[0186] In some embodiments, the IdeS variant protein comprises a substitution of isoleucine at position 313 to proline.

[0187] In some embodiments, the IdeS variant protein comprises a substitution of lysine at position 314 to glycine.

[0188] In some embodiments, the IdeS variant protein comprises a substitution of glutamate at position 315 to proline.

[0189] In some embodiments, the IdeS variant protein comprises a substitution of aspartate at position 316 to proline.

[0190] In some embodiments, the IdeS variant protein comprises a substitution of valine at position 322 to threonine.

[0191] In some embodiments, the IdeS variant protein comprises a substitution of threonine at position 330 to aspartate.

[0192] In some embodiments, provided herein are 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. Table 1A – IdeS Variant Sequences SEQ Description Sequence ID NO T I Y T I Y S T I Y T I Y T I Y T I Y TAttorney Docket No.: TKT-002WO QIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEK AFPYLSRKHLGVFPDHVIDMFINGYRLDLTNHGPTPVKEGSKDPRGGI FDAVFERGDQSKLLTSRHDFKEKNLKEICDLIKKELTEGKALGLSHTY T I Y E T I Y T I Y T I Y N H L Q L I N H L Q L I N H L Q L IAttorney Docket No.: TKT-002WO Q-133-D DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVAN Variant 15 QGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEH PEKQKINFNGEDMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHL Q L I N H D N N H L Q L I N H D N N H L Q L I N H L Q N H L Q L I N H L Q LAttorney Docket No.: TKT-002WO WGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEI KEDNIGAQVLGLFTLSTGQDSWNQTN S-273-D DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVAN H L Q L EI N H L Q L I N H L Q L I N H L Q L I N H L Q L EI N H L Q L I N H L Q L I N H LAttorney Docket No.: TKT-002WO GVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQ SKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINL WGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSQGKVAISAKEI N H L Q L I N H L Q L I N H L Q L I E G VI N H L Q L I N H L Q L I N H L Q L IAttorney Docket No.: TKT-002WO T-44-E DSFSANQEIRYSEVEPYHVTSVWTKGVTPPANFTQGEDVFHAPYVAN Variant 38 QGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEH PEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHL Q L I N H L Q L I N H L Q L I N H L Q L I N H L Q L I N H L Q L I N H L Q L I N H L Q LAttorney Docket No.: TKT-002WO WGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEI KEDNIGAQVLGLFTLSTGQDSWNQTN G-299-D DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVAN H L Q L I N H L Q L N H L Q L I N H L Q L I N H L Q L I N H L Q L I N H L Q L I S H LAttorney Docket No.: TKT-002WO GVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQ SKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINL WGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEI N H L Q L I N H L Q L I N H D N N H L Q L I N H L Q L I N H L Q L E N H L Q L IAttorney Docket No.: TKT-002WO N-61-Q DSFSANQEIRYSEVTPYHVTSVWTKGVTPPAQFTQGEDVFHAPYVAN Variant 61 QGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEH PEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHL Q L I N H L Q L EI N H L Q L I N H L D I A N H L N H D I N H L Q NAttorney Docket No.: TKT-002WO HLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRG DQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVI NLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISA N H H D N N H L Q L I N H L Q L I T I Y T I Y N YAttorney Docket No.: TKT-002WO FKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDP RGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLS HTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVG V E K G VI V I Y S Q Y P S G V E K G VI Y L T R H S Y L T R H S Y LAttorney Docket No.: TKT-002WO EEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLST KHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTR GDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINH S V I Y S

[0193] Described herein, in some embodiments, are IdeS variant proteins comprising the amino acid sequence according to any one of SEQ ID NOs: 3-75 and 86-94. In some embodiments, 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. In some embodiments, 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.

[0194] Described herein, in some embodiments, are IdeS variant proteins comprising the amino acid sequence according to SEQ ID NO: 10. In some embodiments, 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. In some embodiments, 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.

[0195] Described herein, in some embodiments, are IdeS variant proteins comprising the amino acid sequence according to SEQ ID NO: 11. In some embodiments, the IdeS variant protein comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at leastAttorney 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. In some embodiments, 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.

[0196] Described herein, in some embodiments, are IdeS variant proteins comprising the amino acid sequence according to SEQ ID NO: 12. In some embodiments, 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. In some embodiments, 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.

[0197] Described herein, in some embodiments, are IdeS variant proteins comprising the amino acid sequence according to SEQ ID NO: 13. In some embodiments, 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. In some embodiments, 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.

[0198] Further described herein, in some embodiments, are IdeS variant proteins comprising one or more glycosylation modifications. In some embodiments, provided herein are 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. In some embodiments, the IdeS variant protein comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 117-153. In some embodiments, 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 aminoAttorney 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. In some embodiments, 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. Table 1B – IdeS Glycosylation Variants SEQ Name Mutation(s) Sequence ID added to E G K A E G K A N L P R Q G I N LAttorney Docket No.: TKT-002WO LCGAATAGNMLHWWFDQNKDQIKRYLEEHP EKQKINFNGEQMFDVKEAIDTKNHQLDSKLF EYFKEKAFPYLSTKHLGVFPDHVIDMFINGYR Q G I N L P R Q G I N L P R Q G I N L P R Q G I N L P E L S I N LAttorney Docket No.: TKT-002WO LCGAATAGNMLHWWFDQNKDQIKRYLEEHP EKQKINFNGEQMFDVKEAINTTNHQLDSKLFE YFKEKAFPYLSTKHLGVFPDHVIDMFINGYRL S I N L P E L S I N L P F R Q G I N L P R Q G I N L P R Q G I N LAttorney Docket No.: TKT-002WO LCGAATAGNMLHWWFDQNKDQIKRYLEEHP EKQKINFNGEQMFDVKEAIDTKNHQLDSKLF EYFKEKAFPYLSTKHLGVFPDHVIDMFINGYR Q I K N L P R Q G N L P R Q G I K I D D K I DAttorney Docket No.: TKT-002WO SNGNLKAIYVTDSDSNASIGMKKYFVGVNKD GKVAISAKEIKEDNIGAQVLGLFTLSTGQDSW N TN K F D A L K Q K I D D K I D D K I D D KAttorney Docket No.: TKT-002WO KRYLEEHPEKQKINFNGEQMFDVKEAIDTKN HQLDSKLFEYFKEKAFPYLSRKHLGVFPDHVI DMFINGYRLDLTNHGPTPVKEGSKDPRGGIFD K I D D K K D A L K Q K K D A L K Q K I D DAttorney Docket No.: TKT-002WO GKVAISAKEIKEDNIGAQVLGLFTLSTGQDSW NQTN Vi 104 K111N 1EEIAEGRRNPLRTAEWPMTKSTTD SVWTK K D A L K Q K K Q M V T G V T K K D A L K Q K K Q M V T G V T K K QAttorney Docket No.: TKT-002WO 6N_N128S_ LDSKLFEYFKEKAFPYLSRKHLGVFPDHVIDM E198N FINGYRLDLTNHGPTPVKNGSKDPRGGIFDAV FERGD SKLLTSRHDFKEKNLKEISDLIKKELT G V T K K D A L K Q K K D A L K Q K K D A L K Q K K Q M V T G VAttorney Docket No.: TKT-002WO AISAKEIKEDNIGAQVLGLFTLSTGQDSWNQT N 153V i 1013 K111N 1EEIAEGRRNPLRTAEWPMTKSTTD SVWTK K D A L K Qcomprising 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. In some embodiments, 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

[0200] In some embodiments, the modification at position 31 is a substitution of serine to asparagine. In some embodiments, the modification at position 37 is a substitution of glutamate to asparagine.

[0201] In some embodiments, the modification at position 39 is a substitution of arginine to threonine or asparagine.

[0202] In some embodiments, the modification at position 42 is a substitution of glutamate to asparagine.

[0203] In some embodiments, the modification at position 74 is a substitution of valine to asparagine.

[0204] In some embodiments, the modification at position 76 is a substitution of asparagine to serine.

[0205] In some embodiments, the modification at position 111 is a substitution of lysine to asparagine.

[0206] In some embodiments, the modification at position 113 is a substitution of glutamine to threonine.Attorney Docket No.: TKT-002WO

[0207] In some embodiments, the modification at position 119 is a substitution of glutamate to asparagine.

[0208] In some embodiments, the modification at position 121 is a substitution of histidine to threonine.

[0209] In some embodiments, the modification at position 130 is a substitution of asparagine to serine.

[0210] In some embodiments, the modification at position 142 is a substitution of aspartate to asparagine.

[0211] In some embodiments, the modification at position 144 is a substitution of lysine to threonine.

[0212] In some embodiments, the modification at position 147 is a substitution of glutamine to serine.

[0213] In some embodiments, the modification at position 148 is a substitution of leucine to asparagine.

[0214] In some embodiments, the modification at position 198 is a substitution of glutamate to asparagine.

[0215] In some embodiments, the modification at position 233 is a substitution of lysine to serine.

[0216] In some embodiments, the modification at position 244 is a substitution of threonine to asparagine.

[0217] In some embodiments, the modification at position 246 is a substitution of glycine to threonine.

[0218] In some embodiments, the modification at position 311 is a substitution of lysine to asparagine.

[0219] In some embodiments, the modification at position 313 is a substitution of isoleucine to threonine.

[0220] In some embodiments, the modification at position 319 is a substitution of glycine to serine.

[0221] In some embodiments, the variant protein comprises a substitution of glutamate at position 37 to asparagine and a substitution of arginine at position 39 to threonine.

[0222] In some embodiments, the variant protein comprises a substitution of valine at position 74 to asparagine and a substitution of asparagine at position 76 to serine.

[0223] 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.Attorney Docket No.: TKT-002WO

[0224] 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.

[0225] 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.

[0226] In some embodiments, the variant protein comprises a substitution of threonine at position 244 to asparagine and a substitution of glycine at position 246 to threonine.

[0227] 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.

[0228] Further described herein, in some embodiments, are 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. In some embodiments, 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.

[0229] In some embodiments, the modification at position 47 is a substitution of threonine to asparagine.

[0230] In some embodiments, the modification at position 49 is a substitution of glutamine to threonine or asparagine.

[0231] In some embodiments, the modification at position 51 is a substitution of valine to threonine.

[0232] In some embodiments, the modification at position 78 is a substitution of glycine to serine.

[0233] In some embodiments, the modification at position 111 is a substitution of lysine to asparagine.

[0234] In some embodiments, the modification at position 113 is a substitution of glutamine to threonine.

[0235] In some embodiments, the modification at position 123 is a substitution of glutamate to asparagine.

[0236] In some embodiments, the modification at position 125 is a substitution of glutamine to threonine.

[0237] In some embodiments, the modification at position 126 is a substitution of lysine to asparagine.

[0238] In some embodiments, the modification at position 128 is a substitution of asparagine to serine.Attorney Docket No.: TKT-002WO

[0239] In some embodiments, the modification at position 142 is a substitution of aspartate to asparagine.

[0240] In some embodiments, the modification at position 144 is a substitution of lysine to threonine.

[0241] In some embodiments, the modification at position 148 is a substitution of leucine to asparagine.

[0242] In some embodiments, the modification at position 198 is a substitution of glutamate to asparagine.

[0243] In some embodiments, the modification at position 273 is a substitution of serine to asparagine.

[0244] In some embodiments, the modification at position 275 is a substitution of glycine to serine.

[0245] In some embodiments, the modification at position 278 is a substitution of lysine to serine.

[0246] In some embodiments, the modification at position 312 is a substitution of glutamate to asparagine.

[0247] In some embodiments, the modification at position 314 is a substitution of lysine to serine.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] In some embodiments, the variant protein comprises a substitution of lysine at position 126 to asparagine and a substitution of asparagine at position 128 to serine.

[0252] 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.

[0253] 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.

[0254] 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, a substitution of aspartate at position 142 to asparagine, and a substitution of lysine at position 144 to threonine.Attorney Docket No.: TKT-002WO

[0255] 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.

[0256] In some embodiments, 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.

[0257] 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, 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.

[0258] In some embodiments, 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.

[0259] In some embodiments, 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.

[0260] 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, 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.

[0261] In some embodiments, 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.

[0262] In some embodiments, the variant protein comprises, a substitution of threonine at position 47 to asparagine, a substitution of glutamine at position 49 to asparagine, aAttorney 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.

[0263] In some embodiments, 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.

[0264] 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, 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.

[0265] 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, 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.

[0266] 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, a substitution of leucine at position 148 to asparagine, and a substitution of glutamate at position 198 to asparagine.

[0267] Further described herein, in some embodiments, are IdeS variant proteins comprising one or more glycans. In some embodiments, the one or more glycans are glycans of a mammalian glycoprotein. In some embodiments, 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. In some embodiments, the one or more glycans are N-linked glycans (i.e., N-glycans). In some embodiments the one or more glycans comprise an N-glycan core structure. In some embodiments the one or more glycans comprise an N-glycan core structure of: (Manα1- 6[Manα1-3)]Man(β1-4)GlcNAc(β1-4)GlcNAc(β1-Asn-X-Ser / Thr). In some embodiments, the one or more glycans are selected from the group consisting of: a biantennary glycan, a triantennary glycan, and a tetraantennary glycan.

[0268] In some embodiments, 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. In some embodiments, 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.

[0269] In some embodiments, 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. In some embodiments, the one or more glycans is a biantennary glycan that terminates with a galactose. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the one or more glycans is a tetraantennary glycan that terminates with an N-acetylglucosamine. In some embodiments, the one or more glycans is aAttorney 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. In some embodiments, 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.

[0270] In some embodiments, the glycan is selected from the glycans listed in Table 10. In some embodiments, the glycan is selected from the group consisting of Man(α1-6)[Man(α1- 3)]Man(β1-4)GlcNAc(β1-4)GlcNAc(β1-ASN) (i.e., Glycan 1), Neu5Ac(α2-6)Gal(β1- 4)GlcNAc(β1-2)Man(α1-6)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-2)Man(α1-3)]Man(β1- 4)GlcNAc(β1-4)GlcNAc(β1-ASN) (i.e., Glycan 2), and Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1- 6) [Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-2)]Man(α1-6)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1- 2)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-4)]Man(α1-3)]Man(β1-4)GlcNAc(β1-4)GlcNAc(β1- ASN) (i.e., Glycan 3).

[0271] In some embodiments the one or more glycans terminate with an N- acetylglucosamine (i.e. GlcNAc).

[0272] In some embodiments, 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. In some embodiments, 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.

[0273] In some embodiments, 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 someAttorney 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the longer duration of IgG depletion is correlated with a greater extent of IdeS variant protein glycosylation.

[0274] Further described herein, in some embodiments, are IdeS variant proteins fused to Human Serum Albumin (HSA). In some embodiments, the variant proteins fused to HSA comprise one or more glycans. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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

[0275] 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. In some embodiments, the human serum albumin isAttorney Docket No.: TKT-002WO conjugated to a N-terminal of the IdeS protein. In some embodiments, the human serum albumin is conjugated to a C-terminal of the IdeS protein.

[0276] In some embodiments, provided herein are 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. In some embodiments, the IdeS polypeptide comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 154-161. In some embodiments, 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. In some embodiments, 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. Table 1C – IdeS-HSA polypeptides SEQ Name Sequence ID A V A L S L A A P EAttorney Docket No.: TKT-002WO AKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLE KCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE LGEYKF NALLVRYTKKVP VSTPTLVEVSRNLGKV S F V A V A L S L C C F S E T Q R E E A V A L S L G C C F S EAttorney Docket No.: TKT-002WO CKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKT YETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQ NCELFE LGEYKF NALLVRYTKKVP VSTPTLVEVSR E E A V A L S L F F M L S R N C P K A V A L S L F F M L SAttorney Docket No.: TKT-002WO SKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLR LAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQN LIK NCELFE LGEYKF NALLVRYTKKVP VSTPTLV C P K A V A L S L A A P E E S F V A V A D G S E P AAttorney Docket No.: TKT-002WO EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTL EKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELF E LGEYKF NALLVRYTKKVP VSTPTLVEVSRNLGK V T A A V A D G S E P A F V T A

[0277] In some embodiments, the IdeS protein is conjugated to the human serum albumin using a linker. In some embodiments, 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.

[0278] In some embodiments, the linker comprises at least 2 to about 30 amino acids. In some embodiments, 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 aminoAttorney 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.

[0279] In some embodiments, 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. In some embodiments, 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.

[0280] In some embodiments, 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.

[0281] In some embodiments, 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.

[0282] In some embodiments, 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. In some embodiments, 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. In some embodiments, X is serine, aspartic acid, glutamic acid, threonine, or proline.

[0283] In some embodiments, the linker comprises GSGGGSG (SEQ ID NO: 113), GSGSGSGS (SEQ ID NO: 114), GSGGGSGGGSG (SEQ ID NO: 115), or GSGSGSGSGSGS (SEQ ID NO: 116). In some embodiments, the linker comprises GS.

[0284] In some embodiments, the IdeS protein is fused to the human serum albumin.

[0285] Further described herein, in certain embodiments, are polypeptides comprising an IdeS variant protein described herein conjugated to a human serum albumin. In some embodiments, 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.

[0286] Further described herein, in certain embodiments, are polypeptides comprising an IdeS variant protein described herein conjugated to a human serum albumin. In someAttorney Docket No.: TKT-002WO embodiments, 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, and 314 of SEQ ID NO: 12 to introduce a glycosylation site.

[0287] In some embodiments, 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. In some embodiments 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. In some embodiments, 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.

[0288] In some embodiments, 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 12-14%, ± about 1-12%, ± about 2-12%, ± about 4-12%, ± about 6-12%, ± about 8-12%, ± about 10-12%, ± about 1-10%, ± about 2- 10%, ± about 4-10%, ± about 6-10%, ± about 8-10%, ± about 1-8%, ± about 2-8%, ± about 4-8%, ± about 6-8%, ± about 1-6%, ± about 2-6%, ± about 4-6%, ± about 1-4%, ± about 2- 4%, or about 1-2% the levels of expression of wild-type IdeS in the same cell.Attorney Docket No.: TKT-002WO

[0289] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0290] In some embodiments, the IdeS variant protein or IdeS polypeptide described herein comprises an increased enzymatic activity as compared to the activity of wild-type IdeS. In some embodiments, the enzymatic activity is cleavage (e.g., proteolytic) activity of immunoglobulins. In some embodiments, 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. In some embodiments, 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.

[0291] In some embodiments, 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. In some embodiments, 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. InAttorney Docket No.: TKT-002WO some embodiments, 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. In some embodiments, the HLA is an HLA class II allele. In some embodiments, 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.

[0292] In some embodiments, the IdeS variant protein’s or IdeS polypeptide’s binding to the HLA-II allele is measured. In some embodiments, 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.

[0293] In some embodiments, the IdeS variant protein or IdeS polypeptide described herein comprises reduced immunogenicity as compared to a wild-type IdeS protein. In some embodiments, 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. In some embodiments, 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.

[0294] In some embodiments, 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. In other words, 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 theAttorney Docket No.: TKT-002WO peptide is expected to bind. In some embodiments, 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. In some embodiments, 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. In some embodiments, the IdeS variant protein has a decrease in immunogenicity score more than 0 relative to SEQ ID NO: 1 or SEQ ID NO: 2.

[0295] In some embodiments, the IdeS variant protein or IdeS polypeptide described herein comprises reduced antibody binding as compared to a wild-type IdeS protein. In some embodiments, 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. In some embodiments, the IdeS variant protein or IdeS polypeptide binding to the antibody is determined using ELISA.

[0296] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. II. Pharmaceutical compositions

[0297] IdeS variant proteins of the present disclosure can be used in the manufacture of pharmaceutical compositions. In certain aspects, pharmaceutical compositions disclosed herein include IdeS variant proteins of the present disclosure and a pharmaceuticallyAttorney Docket No.: TKT-002WO acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. By “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.

[0298] In certain aspects, 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. Pharmaceutical 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. Examples of 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. In certain aspects pharmaceutical 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.

[0299] 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.

[0300] In certain aspects, pharmaceutical compositions disclosed herein can be formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal administration. For example, a pharmaceutical composition of the present disclosure is administered intravenously to a subject in need thereof. III. Nucleic Acids and Vectors

[0301] 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. For example, in certain aspects, a nucleic acid molecule encoding the IdeS variant protein of the present disclosure can be incorporated into a vector and introduced into a cell. In certain aspects, a cell has one or more than one nucleic acid encoding the IdeS variant protein described herein.Attorney Docket No.: TKT-002WO

[0302] In some embodiments, the nucleic acid is a DNA, for example a linear DNA, a plasmid DNA, or a minicircle DNA. In some embodiments, the nucleic acid is an RNA, for example a mRNA. In some embodiments, the nucleic acid is provided in a vector. In some embodiments, 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. In some embodiments, 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-OXB20, pSF-Tac, pRI 101-AN DNA, pCambia2301,pTYB21, pKLAC2, pAc5.1 / V5-His A, and pDEST8.

[0303] In some embodiments, the nucleic acid comprises a promoter. In some embodiments, the promoter is selected from the group consisting of a mini promoter, an inducible promoter, a constitutive promoter, and derivatives thereof. In some embodiments, 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.

[0304] In some embodiments, the nucleic acid is provided in a virus. In some embodiments, 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. In some embodiments, the virus is an alphavirus. In some embodiments, the virus is a parvovirus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is an AAV. In some embodiments, the virus is a baculovirus. In some embodiments, the virus is a Dengue virus. In some embodiments, the virus is a lentivirus. In some embodiments, 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.

[0305] In some embodiments, 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-HSC10, AAV-HSC11, AAV- HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, or a derivative thereof. In some embodiments, the herpesvirus is HSV type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.

[0306] In some embodiments, 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. In some embodiments, the nucleic acid is provided in a lipid nanoparticle (LNP).

[0307] 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. Examples of 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)).

[0308] 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 promoterAttorney 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. Examples of a suitable marker are a gene that encodes green fluorescent protein or a gene that encodes resistance to an antibiotic. III. Methods of Use

[0309] Described herein, in some embodiments, are methods of treating a disease or disorder comprising administering an effective amount of IdeS variant proteins described herein or pharmaceutical compositions comprising the IdeS variant proteins described herein. In some embodiments, methods described herein improve the efficiency of gene editing with viral vectors. In some embodiments, the IdeS variant protein is co-administered with a gene therapy. In some embodiments, the IdeS variant protein is provided as a gene therapy.

[0310] Methods for gene editing, in some embodiments, comprises a nuclease for targeting of a particular target nucleic acid sequence. Types of 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. In some instances, the nuclease is wild-type, genetically modified, or recombinant. In some embodiments, the gene editing system comprises CRISPR / Cas9.

[0311] Described herein, in some embodiments, are methods of treating an autoantibody- mediated autoimmune disease comprising administering an effective amount of the IdeS variant protein described herein, the polypeptide described herein, or the pharmaceutical described herein. EXAMPLES

[0312] 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.Attorney Docket No.: TKT-002WO EXAMPLE 1: Reduced Immunogenic Bacterial Proteases for Degrading Immunoglobulin G

[0313] This Example describes generation of reduced immunogenic IdeS variant proteins for degrading immunoglobulin G (IgG).

[0314] Screen for Expression and Activity of IdeS Variants

[0315] To rapidly screen IdeS variants without purification, IdeS was expressed with a strong signal peptide in Expi293F cells, a suspension culture derivative of HEK293 that gives high yield of secreted protein into medium. Using this format, the proteolytic activity of IdeS variants could be rapidly screened directly from the expression medium without purification. IdeS produced in bacteria was non-glycosylated and ran as a sharp peak by size exclusion chromatography (SEC) as seen in FIGs.1A-1B. By comparison, 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). Based on changes in electrophoretic mobility following mutations to the potential N-glycosylation sites, 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.

[0316] Identification of HLA-II epitopes in IdeS by MAPPs

[0317] Monocyte-derived DCs cultured from 10 healthy donors were pulsed with purified IdeS, and peptide-HLA-II complexes were immunoprecipitated using a pan-HLA-DR antibody 24 hours later. Peptides were analyzed by LC-MS / MS and the data were processed to identify peptides derived from IdeS processing. A high number of peptides bound HLA-II alleles across multiple donors (FIG.2A). In all, there were 8 significant clusters of antigenic peptides across the IdeS sequence, found at residues ~30-50 (bound HLA-II alleles in 5 of the 10 donors), residues ~50-90 (bound HLA-II alleles in 3 donors), residues ~110-130 (2 donors), residues ~150-170 (7 donors), residues ~180-200 (4 donors), residues ~210-220 (4 donors), residues ~250-270 (6 donors), and residues ~280-320 (8 donors). These are referred to as Clusters 1 to 8, respectively. The eighth cluster may represent two or more individualAttorney Docket No.: TKT-002WO epitopes that are overlapping or adjacent to each other. The donors represented a broad set of different HLA-II alleles (Table 2). Table 2. 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 6seen 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. Furthermore, residues in Clusters 2, 4, 6, 7 and 8 contribute to the binding interface with IgG substrates based on modeling using the crystal structure of IgG1 Fc bound to S. pyogenes Mac-2. Modifications that reduce immunogenicity of the protein must carefully consider the structural context of each epitope to minimize deleterious perturbations, while epistatic effects are expected to be especially prominent when modifications are combined between clusters that form close atomic interactions.

[0319] To complement the MAPPs results, epitopes using the methodology from King et al.13for a representative set of HLA-II alleles were computationally predicted. 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. As theAttorney 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. Using a threshold of 10%, the in-silico predictions qualitatively agree with the experimental MAPPs data, with some differences (FIG.3). The immunogenicity score is thus sufficiently predictive for identifying mutations that might decrease peptide presentation on HLA-II.

[0320] Removal of HLA-II Epitopes

[0321] Single modifications computationally predicted to decrease immunogenicity, and located within the epitope clusters identified by MAPPs, were introduced into IdeS and expressed by Expi293F cells. Modifications were avoided within the active site and were predicted to be minimally destabilizing based on modeling. 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). Furthermore, 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 Modification(s)^Immuno.Rel.Modification(s)Rel. 2^Immuno.Score1Activity Score ActivityAttorney Docket No.: TKT-002WO T44E 0 ++ Y163M 0 + P45E 0 ++ Y163N -2 + H47A 0 ++ Y163P -2 +Attorney Docket No.: TKT-002WO Y117H 0 + T244N_G246T 0 ++ Y117W 0 + E245D -2 ++ L118T 0 + K247N -2 ++Attorney Docket No.: TKT-002WO Y155N -2 - I318D 0 ++ Y155P -4 + I318G 0 ++ Y155Q -2 - I318K 0 ++Mutation(s) ^Immuno. Rel. Score1Activity2q y reduced immunogenicity multiple HLA-II epitopes within a single IdeS polypeptide (Table 5). This was accomplished in stages by successively adding sets of modifications. A reduced immunogenic IdeS derivative, Variant 77, carrying 6 modifications 'hitting' 5 epitope clusters plus a putative disulfide-bonding pair of cysteines was purified and characterized. Variant 77 was highly active for the first cut of a non-specific human IgG1 monoclonal, but slower than wild type IdeS for making the second cut that releases the F(ab')2fragment (FIGs.7A-7F). Variant 77 and wild type IdeS were both selective for human and rabbit IgG versus mouse IgG (FIGs.Attorney Docket No.: TKT-002WO 7A-7F). However, Variant 77 had reduced thermal stability (melting temperature Tm = 46 ºC versus 53 ºC for wild type IdeS) (FIG.10B). Variant 77 was used as an intermediate for final rounds of reduction of immunogenicity and stabilization. Table 5. Screening of IdeS variants that combine mutations across multiple epitope clusters. Mutation(s)^ImmRel. Score1uno.Activity2S31D A 1 T166R D316P 0Attorney Docket No.: TKT-002WO V43E_V48N_A71N_L188G_V258P_A303Q -9 ++ V43E_A60Q_F69P_S187D_K241D_V258D_A303D -11 ++ V43E A60Q L188G K241D V258PA303Q -10 ++Attorney Docket No.: TKT-002WO V68T_A75P_T166R_N190D_T213E_S236C_L277C_I308L -3 - V68T_A75P_L169E_S187D_T213E_S236C_L277C_I308L -8 ++ V68T A75P H168D N190D T213E S236C L277CI308L -4 -Attorney Docket No.: TKT-002WO del(30_49)_49ins(QEEIAEGRRNPLRTAEWPMTKSTTDQ (SEQ ID NO: -21 - 110))_M134W_Y163P_S187D_L188G_S218D_K241D_V258D_F297D del(30 49) 49ins(QEEIAEGRRNPLRTAEWPMTKSTTDQ (SEQ ID NO: -25 -Attorney Docket No.: TKT-002WO del(30_49)_49ins(QEEIAEGRRNPLRTAEWPMTKSTTDQ (SEQ ID NO: ++ 110))_V68T_A75P_T166R_S187D_T213E_S302K_A303D (Variant 10) del(30 49) 49ins(QEEIAEGRRNPLRTAEWPMTKSTTDQ (SEQ ID NO: ++unstructured region that extends N-terminally from the protease domain. Deletions of the N- terminal extension were evaluated to remove the Cluster 1 epitope but IdeS expression decreased as the length of the deleted segment increased (FIG.8A and Table 3). Instead of deleting the IdeS N-terminus, this region was replaced with the equivalent N-terminal flanking sequences from homologous proteases or from proteins of unknown function that are structurally similar to IdeS, creating chimeras (FIG.8B and Table 6). Chimeric constructs were screened with diverse N-terminal sequences and found to have high proteolytic activity (FIG.8A). The N-terminal extension transplanted from a protein within a metagenomics database was chosen for incorporating into Variant 77 based on its favorable immunogenicity score. Table 6. Alternative sequences for replacing the IdeS N-terminus (a.a. D30-T49) SEQ Construct Source (Accession Sequence ID NO Identifier No.)1K T1All references to accession numbers throughout the application refer to the versions that were current in the database as of the filing date of the application.Attorney Docket No.: TKT-002WO 105 Variant 90 GMGC10.182_811_4 YYTFNLDVPQTNESSPRIQW 24.UNKNOWN 106 Variant 91 GMGC10.000 551 9 TLLISKIEVKGEREKITLNTTNE EVariant 77 together with N-terminal chimerism to create combination variants IdeS Variants 100, 101, 102, 103, 1, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, and 116. Following screening of the variants in Expi293F expression medium (FIG.9A), Variant 1 was selected based on its high expression, high proteolytic activity against IVIG, and reduced immunogenicity score. Variant 1 has a chimeric N-terminus, 8 substitutions to remove HLA- II epitopes, and an introduced pair of disulfide-bonding cysteines. However, the thermal stability of this derivative was even further diminished (Tm = 43 ºC, FIG.9B). A subset of modified residues were reverted back to their wild type identities and / or compensatory modifications (designed proteins Variant 2 to Variant 7) were added to partially rescue stability (Tm values between 42 ºC and 50 ºC, FIG.9B). 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). The protein eluted as a sharp peak by SEC (FIG.11C), and the Tm (53 ºC, FIG.10B) and proteolytic activity against a human monoclonal IgG1 (FIG.11D) were indistinguishable from wild type IdeS.

[0325] To validate reduction in antigenic peptide presentation on HLA-II, 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. In the presence of the HLA-II-specificAttorney 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. When the reference peptide is bound by HLA-II, 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. Using this assay to examine peptide binding to HLA-DR alleles with beta chains HLA-DRB1*01:01 (FIG.12), DRB1*07:01 (FIG.13), DRB1*11:01 (FIG.14), and HLA-DRB1*15:01 (FIG. 15), some peptides were observed to strongly compete with the reference peptide or not at all for distinct HLA-DRB alleles. In nearly all instances, mutant IdeS peptides either had no change in competition compared to the wild type IdeS peptide or competed weaker. Mutations in Variant 10 were found to reduce HLA-II binding affinity to some HLA-II alleles as designed. Table 7. Peptide Sequences Paired peptides from wild-type IdeS and Variant 10  Peptide Residues of Sequence1SEQAttorney Docket No.: TKT-002WO Variant 10- KKYFVGVNKDGKVAI 189 P7b K294-I308 R f P tid

[0326] Derivative Variant 10 was purified from bacteria and its pharmacokinetics (PK) and pharmacodynamics (PD) were evaluated and compared to wild type IdeS, also purified from bacteria and therefore without glycosylation. The proteins were administered as a single intravenous (IV) dose (1 mg / kg) to New Zealand white rabbits. Blood was collected at time intervals via ear vein and enzymatic activity was immediately inhibited with iodoacetic acid to prevent continued proteolytic turnover of rabbit IgG. Serum levels of IdeS proteins and the corresponding reduction and recovery of the IgG pool were measured (FIGs.16A- 16B). Reduced immunogenic Variant 10 was as effective as wild type IdeS in rapidly reducing serum IgG within 3 minutes. IgG levels began to recover at 24-48 h and took ~7 days to return to initial levels. The modified changes in the IdeS sequence to remove HLA-II epitopes thus have not compromised in vivo activity.

[0327] Shielding B Cell Epitopes Exposed on the IdeS Surface using N-Glycosylation

[0328] It is expected that many individuals will have anti-IdeS memory B cells due to prior exposure to S. pyogenes, which may be a source of ADAs even if activation of CD4+ T cells is reduced. This expectation is supported by low titers of anti-IdeS antibodies present in healthy human serum as seen in FIG.17 as well as reported by others, which increases 10- 100-fold two weeks following IdeS administration. To minimize exposure of potential B cell epitopes, the IdeS surface was partially shielded by the addition of custom N-glycosylation sites.

[0329] Using rational design and comparisons to homologous sequences to find suitable sites for modification, 16 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, whichAttorney Docket No.: TKT-002WO structural analysis suggested could be combined without negative interference (FIG.18B and Table 9). 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. Based on its superior activity and thermal melt properties, 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 Activity1Glycosylation Mobility ShiftWt; -, inactive.Attorney Docket No.: TKT-002WO Table 9. Hyperglycosylated derivatives of Variant 10 Name Custom N- Mutations Activity2Glycosylation Sites1

[0330] 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).

[0331] 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). TheAttorney 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). The 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). Despite large changes on the protein surface to block potential B cell epitopes, hyperglycosylated Variant 10.9 retained preferential specificity for human and rabbit IgG, with slower cleavage of IgG from cynomolgus monkey and rat (FIG.20C).

[0332] To quantify the surface area of IdeS that is shielded by glycosylation, 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. Based on the crystal structure of IdeS bound to a cleaved IgG Fc product (PDB 8A47), 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. Of the remaining IdeS surface, the 4 glycans of Variant 10.9 are primarily localized to one side of the protein (FIGs.22A-22B). Using a sphere of 10 Å radius to approximate antibody accessibility, 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 Å 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. Accessible Surface Area of Variant 10.9 that is Shielded by Glycosylation Spherical Probe (Representation andAttorney Docket No.: TKT-002WO Glycan 2: Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1- 2)Man(α1-6)[Neu5Ac(α2-6)Gal(β1- 4 Gl NA 12 M 13 M 1 4% 28% 36%IdeS polyclonal antibody, consistent with surface epitopes being shielded (FIG.23). For a set of IdeS variants that share the same primary sequence and differ only by the number of N- glycosylation sites, the sequential addition of glycans going from 0 (Variant 10.14, which is equivalent to Variant 10 with mutation N61Q to remove the glycosylation site of wild type IdeS) to 1 (Variant 10) to 3 (Variant 10.2) to 4 glycans (Variant 10.9) is associated with decreasing anti-IdeS reactivity.

[0334] 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. Notably, 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. Curiously, 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. Overall, hyperglycosylationAttorney Docket No.: TKT-002WO succeeded not only in shielding potential B cell epitopes on the IdeS surface but also extended PK and PD duration.

[0335] Shielding B Cell Epitopes Exposed on the IdeS Surface via Fusion with Human Serum Albumin (HSA)

[0336] The effects of fusion of HSA on shielding B cell epitopes was determined. See FIG.25A. First, the C-terminus of aglycosylated Variant 10.14 was fused to the N-terminus of mature HSA. The fusion protein was secreted into the medium of transfected Expi293F cells at high levels (FIG.25B) and once purified was active for the cleavage of a human IgG1 monoclonal, albeit at reduced levels (FIG.25C). The length of the glycine / serine-rich linker connecting IdeS to HSA (Table 11 and FIG.25D) 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. Linker sequences connecting C-terminus of IdeS with N-terminus of HSA Name of Linker Sequence SEQ ID NO: No Linker GS (cloning scar)HSA had increased serum half-life compared to Variant 10.14 without HSA fusion (t1 / 2= 29 versus 1.2 h) and increased exposure (AUC0-t= 3040 versus 24 μg / ml ^h) (FIG.26A). Rabbit IgG levels remained low for the entire duration of the study (8 days; FIG.26B). HSA fusion therefore accomplished epitope shielding while substantially extending PK and PD.

[0338] IdeS Variants that Combine HLA-II Epitope Reduction, Glycan Shielding, and HSA Fusion

[0339] IdeS variants with HLA-II Epitope Reduction, glycan shielding, and HSA fusion were generated. 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. Based on ELISA using polyclonal goat anti-IdeS, there is a sequential reduction of surface epitopes on the HSA-fused proteins as N-glycan number is increased (FIG.27). However,Attorney Docket No.: TKT-002WO catalytic activity of the most heavily glycosylated HSA-fusion variant (Variant 10.9-HSA) was abrogated (FIG.28A). Thermostability without dye using nano DSF was determined. Loss of catalytic activity in the Variant 10.9-HSA construct correlated with steady unfolding over a wide temperature range beginning at ~30 ^C, whereas other variants displayed cooperative unfolding at higher temperatures (FIG.28B). The addition of custom N- glycosylation sites and HSA fusion are therefore not necessarily independent of each other and in one of the constructs interacted negatively.

[0340] Variant 10.9, with 4 N-glycans and no HSA fusion, and Variant 10.2-HSA, with 3 N-glycans and fused at the C-terminus to HSA, were chosen for further characterization. The reactivity of the proteins towards polyclonal anti-IdeS in serum from individual donors or from pooled donors was evaluated by direct ELISA (FIG.29A). The EC50 values for reactivity of the two variants are approximately half those of wild type IdeS. Unlike reactivity measured against goat polyclonal anti-IdeS (FIG.27), 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. In the competition ELISA (FIG.29B), wild type IdeS as the competitor completely blocked anti- IdeS in human serum, whereas Variant 10.9 and Variant 10.2-HSA only blocked approximately half the reactivity of the human serum samples at the highest concentrations tested. ELISA results are consistent with modeling of glycan shielding (described above), which estimated that a third of the IdeS surface is occluded by hyperglycosylation.

[0341] Variant 10.2-HSA was IV administered to NZW rabbits at 0.3 and 1 mg / kg doses. 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. We again speculate that 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). To better understand howAttorney Docket No.: TKT-002WO long IgG levels are depleted, IgG in rabbit serum was rigorously quantified by ELISA measurements at multiple sample dilutions that were compared to standards. We found that whereas IgG recovers to baseline by Day 7 in rabbits treated with wild type IdeS from bacteria, IgG levels remain >80% reduced up to Day 15 in rabbits treated with Variant 10.2- HSA (FIG.30C). Notably, both 0.3 and 1 mg / kg doses of Variant 10.2-HSA caused equivalent reductions in IgG over the 15 day study, suggesting the doses are saturating and can be decreased. Lower dose is anticipated in-and-of-itself to reduce immunogenicity further.

[0342] 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. This was confirmed by anti-Fab ELISA of serum from rabbits treated with wild type IdeS; whereas full-length IgG is destroyed in minutes, clearance of F(ab')2 products occurs over 24 hours (FIG.31D). Compared to wild type enzyme, 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.

[0343] The 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). We note that the half-life of human serum albumin in humans is markedly longer (19 days) than in rabbits (2 days) or mice (1 day), and it is thus likely that HSA-fused IdeS will have substantially longer duration of action in human patients than observed in rabbit or mouse models. Desialylation of 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 1ea a - e s e e e o - , - a a . , o - a a 10.2-HSA). (3) Abbreviations: AUC, area under curve; MRT, mean residence time.

[0344] METHODS

[0345] Sequences and Plasmids

[0346] The numbering for S. pyogenes IdeS is based on the NCBI Reference Sequence WP_010922160.1, identical to UniProt ID Q7DAM2. Residues 1-29 form a Sec signal peptide. Residues 30-339 form the mature polypeptide. For expression in Expi293F cells, 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. The inserts of all plasmids were verified by Sanger sequencing. For expression in E. coli, the mature polypeptide of IdeS (a.a.30-339) was cloned with a N- terminal 10xHis tag (SEQ ID NO: 195) and TEV cleavage site upstream of the IdeS starting residue (D30) into the NdeI-BamHI sites of pET-9a.

[0347] MHC Class II Epitope Prediction

[0348] Potential CD4+ T cell activating regions of wild-type IdeS were identified. Briefly, for every possible overlapping 15-mer peptide sequence in the IdeS polypeptide, and for each of the following human leukocyte antigen (HLA) class II alleles (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, HLA-DQA1*03:01-DQB1*03:02), the peptide’s binding to the HLA-II alleles and as a probability ranking against a reference set of 100,000 peptides from the human genome were predicted. This is referred to as the rank-percentage. 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.Attorney Docket No.: TKT-002WO

[0349] MHC Class II Associated Peptide Proteomics (MAPPs)

[0350] 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). Once matured (~24 h), 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).

[0351] Preparation of IdeS Models

[0352] The 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.

[0353] 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.

[0354] Computational HLA-II Epitope Removal

[0355] 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 Å 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. The positions selected were 10-21, 52-83, 91-120, 127-137, 146-196, 200-220, 232-268, 271-289, 293-331, and for each position the protocol was run 40 times and all returned variants were taken to the next step. After reduced immunogenicity prediction, the structures were evaluated, selecting the model of lowest score / energy out of 20 minimization runs. Next, the protocol was run, and the calculated changes in folding free energies (DDG) were collated along with epitope scoresAttorney Docket No.: TKT-002WO into a database for analysis. In total, 18,498 constructs were generated with this methodology. To filter the results down to a set for experimental testing, modifications with high ΔΔG (>= 4.5 Energy Units or EU) were discarded. Models of variant IdeS were then manually inspected for final selection.

[0356] Disulfide Bond Engineering

[0357] 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 relaxed18and this relaxed structure was used as an input. All residue-residue pairs with Cβatoms less than or equal to 5 Å from each other were evaluated. The C1ɑ-C1β-C2β-C2ɑ 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.

[0358] In the second method, structural similarity searches were used to find proteins with similar topologies to IdeS, and then these structures were mined for their disulfides. Structures were aligned to the 2AVW chain A crystal structure with TMAlign21and all structures were manually evaluated for potential disulfides based on intra-chain cysteine proximity. Disulfides were then manually mapped onto the IdeS crystal structure. This methodology yielded two additional disulfide variants at IdeS residues 83-148 and 236-277 for experimental testing.

[0359] Engineering of N-Glycosylation Sites

[0360] Two computational protocols were used to generate IdeS variants with custom N- glycosylation sites.

[0361] In the first protocol, the set of structures sharing the IdeS topology that were curated from the foldseek server and rebuilt using PULCHERA (see description above in the subsection on Disulfide Bond Engineering), were searched for the presence of NX(T / S) motifs, where X is any amino acid except proline. Motifs identified by this method were transplanted based on structural alignment using TMAlign to their corresponding positions in IdeS. These positions were then manually inspected to ensure that they were surface exposedAttorney Docket No.: TKT-002WO and the sequences computationally evaluated as described above to ensure predicted immunogenicity didn't increase.

[0362] In the second protocol, N-glycosylation motifs were manually introduced into the IdeS structure based on surface exposure using structure visualization software. The sequence changes were validated to not increase predicted immunogenicity.

[0363] Bacterial Expression and Purification of IdeS

[0364] An overnight starter culture (LB medium containing 50 mg / ml kanamycin, 37 °C, 250 rpm) of E. coli (BL21) transformed with pET9a-10his-TEV-IdeS was used to inoculate larger flasks. Once the OD at 600 nm reached 0.5-0.8, the culture was induced with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and the temperature was lowered to 30 °C. 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. 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.

[0365] Mammalian Cell Expression and Purification of IdeS

[0366] Expi293F cells were cultured at 37 °C, 125 rpm, 8% CO2, maintaining a density of ~0.25-4 ^ ^ 106cells / ml. Cells were transfected at a density of 2 ^ 106cells / 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. Cells were removed by centrifugation at 800 ^ g, 4 °C, 10 minutes, and debris was removed by centrifugation at 15,000 ^ g, 4 °C, 20 minutes. Supernatant was incubated with HisPur Ni- NTA resin for 2 h at 4 °C with rotation. Resin was collected by passing over a column and washed with ~20 CV PBS and ~5 CV PBS containing 20 mM imidazole. Protein was eluted with ~8 CV PBS containing 250 mM imidazole. The protein was concentrated with a 10,000 MWCO filtration device and then separated on a S7510-300 GL increase column equilibrated in PBS. Fractions containing monomeric IdeS were pooled, concentrated, and aliquots were snap frozen in liquid nitrogen and stored at -80 °C.Attorney Docket No.: TKT-002WO

[0367] High Throughput Activity Screen

[0368] 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). In this assay, 20 µM IVIG is mixed with 1 / 12.5 dilution of expression medium in 20 mM sodium phosphate, pH 6.5, 150 mM NaCl, 1.25 µM bovine serum albumin (assay buffer) for 1 h at 37 °C. Samples were then combined with non-reducing load dye, heated to 95 °C for 5 minutes, and analyzed by SDS-PAGE.

[0369] IgG Proteolysis Assay

[0370] Kinetics of proteolytic IdeS activity for monoclonal IgGs and IgGs from selected species were determined by SDS-PAGE. A mixture of 20 µM IgG and 20 nM IdeS in assay buffer was incubated at 37 ^C. The enzymatic reaction was stopped at different time points (0- 18h) with 2 mM iodoacetic acid. Samples were then combined with non-reducing sample buffer, heated to 95 °C for 5 minutes, and analyzed by SDS-PAGE.

[0371] Differential Scanning Fluorimetry (DSF)

[0372] The thermal stability of IdeS variants was measured by differential scanning fluorimetry (DSF) on a Quantstudio3 device. The experiment was performed using the Protein Thermal Shift Dye Kit and according to manufacturer's standard instructions. In brief, a constant concentration of 0.4 mg / ml IdeS variant in PBS was mixed with thermal shift dye and reaction buffer in a 96-well plate. The plate was sealed with adhesive film and the sample fluorescence signal monitored from 25 to 95 ^C in 0.05 ^C per s increments. The data was analyzed and thermal melt (Tm) values determined using a Boltzmann fitting model.

[0373] In Vivo Activity

[0374] NZW rabbits were housed outside at the R&R Research facility (Stanwood, Washington). Rabbits 3.77-3.96 kg were injected in the marginal ear vein with 4 mg of IdeS proteins (prepared at 5 mg / ml in PBS). Each test group had two rabbits that were bled (1-2 ml via ear vein) at alternating time points. Rabbit 1: 0.05, 0.17, 0.5, 4, 48, 96, 192, and 240 h. 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.Attorney Docket No.: TKT-002WO

[0375] Rabbit IgG ELISA

[0376] Nunc MaxiSorp plates were coated 2 h at 37 ^C with F(ab')2fragment goat anti- rabbit IgG specific for the F(ab')2fragment diluted in PBS to 0.1ug / ml. The remaining steps were at room temperature. Plates were washed four times with PBS containing 0.05% Tween 20 (PBS-T) and blocked with SuperBlock blocking buffer for 1 h. Plates were washed four times with PBS-T and incubated for 1 h with a serial dilution (1:102to 1:107) of serum in PBS containing 1% bovine serum albumin. Plates were washed four times with PBS-T and incubated for 1 h with peroxidase-conjugated goat anti-rabbit IgG specific for the Fc fragment diluted 1:25,000 in PBS. Plates were washed four times with PBS-T and developed with 1-Step TMB substrate for 20 minutes prior to addition of ELISA Stop. Absorbance at 450 nm was read.

[0377] This Example shows generation of IdeS variants that reduce presentation of peptides on HLA-II while preserving IdeS stability and IgG cleavage activity References 1. Vidarsson, G., Dekkers, G. & Rispens, T. IgG Subclasses and Allotypes: From Structure to Effector Functions. Front. Immunol.5, 520 (2014). 2. Hansa Biopharma AB. An Open-label, Controlled, Randomized Phase 3 Trial Evaluating 12-month Kidney Function in Highly Sensitized (cPRA ≥99.9%) Kidney Tx Patients With Positive XM Against a Deceased Donor, Comparing Desensitization Using Imlifidase With SoC. https: / / clinicaltrials.gov / ct2 / show / NCT04935177 (2022). 3. Agniswamy, J. et al. Crystal Structure of Group A Streptococcus Mac-1: Insight into Dimer-Mediated Specificity for Recognition of Human IgG. Structure 14, 225–235 (2006). 4. Vultaggio, A. et al. How to Prevent and Mitigate Hypersensitivity Reactions to Biologicals Induced by Anti-Drug Antibodies? Front. Immunol.12, (2021). 5. Schellekens, H. Immunogenicity of therapeutic proteins: Clinical implications and future prospects. Clin. Ther.24, 1720–1740 (2002). 6. Rosenberg, A. Immunogenicity of biological therapeutics: a hierarchy of concerns. Dev. Biol.112, 15–21 (2003). 7. Zhao, L., Ren, T. & Wang, D. D. Clinical pharmacology considerations in biologics development. Acta Pharmacol. Sin.33, 1339–1347 (2012).Attorney Docket No.: TKT-002WO 8. Zinsli, L. V., Stierlin, N., Loessner, M. J. & Schmelcher, M. Deimmunization of protein therapeutics – Recent advances in experimental and computational epitope prediction and deletion. Comput. Struct. Biotechnol. J.19, 315–329 (2021). 9. Yao, B., Zheng, D., Liang, S. & Zhang, C. Conformational B-Cell Epitope Prediction on Antigen Protein Structures: A Review of Current Algorithms and Comparison with Common Binding Site Prediction Methods. PLOS ONE 8, e62249 (2013). 10. Kringelum, J. V., Lundegaard, C., Lund, O. & Nielsen, M. Reliable B Cell Epitope Predictions: Impacts of Method Development and Improved Benchmarking. PLOS Comput. Biol.8, e1002829 (2012). 11. Nagata, S. & Pastan, I. Removal of B cell epitopes as a practical approach for reducing the immunogenicity of foreign protein-based therapeutics☆. Adv. Drug Deliv. Rev.61, 977– 985 (2009). 12. Yachnin, B. J., Mulligan, V. K., Khare, S. D. & Bailey-Kellogg, C. MHCEpitopeEnergy, a Flexible Rosetta-Based Biotherapeutic Deimmunization Platform. J. Chem. Inf. Model.61, 2368–2382 (2021). 13. King, C. et al. Removing T-cell epitopes with computational protein design. Proc. Natl. Acad. Sci.111, 8577–8582 (2014). 14. Fleishman, S. J. et al. RosettaScripts: A Scripting Language Interface to the Rosetta Macromolecular Modeling Suite. PLoS ONE 6, e20161 (2011). 15. Frenz, B. et al. Prediction of Protein Mutational Free Energy: Benchmark and Sampling Improvements Increase Classification Accuracy. Front. Bioeng. Biotechnol.8, 558247 (2020). 16. Park, H. et al. Simultaneous Optimization of Biomolecular Energy Functions on Features from Small Molecules and Macromolecules. J. Chem. Theory Comput.12, 6201–6212 (2016). 17. Bhardwaj, G. et al. Accurate de novo design of hyperstable constrained peptides. Nature 538, 329–335 (2016). 18. Conway, P., Tyka, M. D., DiMaio, F., Konerding, D. E. & Baker, D. Relaxation of backbone bond geometry improves protein energy landscape modeling: Relaxation of Backbone Bond Geometry. Protein Sci.23, 47–55 (2014). 19. Kempen, M. van et al. Foldseek: fast and accurate protein structure search. 2022.02.07.479398 Preprint at https: / / doi.org / 10.1101 / 2022.02.07.479398 (2022).Attorney Docket No.: TKT-002WO 20. Rotkiewicz, P. & Skolnick, J. Fast procedure for reconstruction of full-atom protein models from reduced representations. J. Comput. Chem.29, 1460–1465 (2008). 21. Zhang, Y. & Skolnick, J. TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic Acids Res.33, 2302–2309 (2005). 22. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012). INCORPORATION BY REFERENCE

[0378] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes. EQUIVALENTS

[0379] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the disclosure described herein. Various structural elements of the different embodiments and various disclosed method steps may be utilized in various combinations and permutations, and all such variants are to be considered forms of the disclosure. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.Attorney Docket No.: TKT-002WO SEQUENCE LISTING SEQ Description Sequence ID NO L K L E V T N H L Q L I H K A V K H K A V K H K A V K H K A V K H K A VAttorney Docket No.: TKT-002WO RINHVINLWGADFDSNGNCKAIYVTDSDSNASIGMKKYFVGVNKDG KTAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN Variant 6 QEEIAEGRRNPLRTAEWPMTKSTTDQSVWTKGVTPPANFTQGEDTFH K A V H K A V K H K A V K H K A V H K A V H K A V K N H L Q L I N H LAttorney Docket No.: TKT-002WO GVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQ SKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINL WGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEI N H L Q L I N H L Q L I N H D N N H L Q L I N H D N N H L Q L I N H L QAttorney Docket No.: TKT-002WO E-245-D DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVAN Variant 21 QGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEH PEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHL Q L I N H L Q L I N H L Q L EI N H L Q L I N H L Q L I N H L Q L I N H L Q L EI N H L Q LAttorney Docket No.: TKT-002WO WGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSDGKVAISAKEI KEDNIGAQVLGLFTLSTGQDSWNQTN A-303-N DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVAN H L Q L I N H L Q L I N H L Q L I N H L Q L I N H L Q L I E G VI N H L Q L I N H LAttorney Docket No.: TKT-002WO GVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQ SKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINL WGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEI N H L Q L I N H L Q L I N H L Q L I N H L Q L I N H L Q L I N H L Q L I N H L Q L IAttorney Docket No.: TKT-002WO A-210-G DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVAN Variant 44 QGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEH PEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHL Q L I N H L Q L I N H L Q L I N H L Q L P N H L Q L I N H L Q L I N H L Q L I N H L Q LAttorney Docket No.: TKT-002WO WGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEI KEDNIGAQVLGLFTLSTGQDSWNQTN E-42-N DSFSANQEIRYSNVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVAN H L Q L I S H L Q L I N H L Q L I N H L Q L I N H D N N H L Q L I N H L Q L I N H LAttorney Docket No.: TKT-002WO GVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQ SKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINL WGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISANE N H L Q L I N H L Q L I N H L Q L I N H L Q L I N H L D N K N H L N Q P L Q EAttorney Docket No.: TKT-002WO S-33-E_Q-77- DSFEANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVAN G_S-187- GGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEH D S-236- PEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHL Q L E N G VI N H H D N N H L Q L I N H L Q L I H K A V K H K AAttorney Docket No.: TKT-002WO DQ (SEQ ID VFERGDQSKLLTSRHDFKEKNLKEICDLIKKELTEGKALGLSHTYANV NO: RINHVINLWGADFDSNGNCKAIYVTDSDSNASIGMKKYDVGVNSAG 110)) V68T KVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN G T A E G VI H K A V K EAttorney Docket No.: TKT-002WO KDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFK EKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRG GIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHT E G VI V E K G VI V E K G VI V E K G VI H K A V KAttorney Docket No.: TKT-002WO Variant 86 DQKLKDYLKNDQLKGTELGKFLEEQGVTK Variant 87 EDDFFQNHPAFIKEIPGVKY N H L Q L I N H L Q L I N H L Q L I N H L Q L I S H L QAttorney Docket No.: TKT-002WO SKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINL WGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEI KEDNIGAQVLGLFTLSTGQDSWNQTN N H L Q L I N T L Q L I N H L Q L I N H L Q L I N H L Q L I N H D N N H L Q L I N HAttorney Docket No.: TKT-002WO PEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHL GVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQ SKLLTSRHDFKEKNLSEISDLIKKELTEGKALGLSHTYANVRINHVINL I N H L Q L I N H L Q L N H L Q L I H K A V H K A V H K A V H K P A VAttorney Docket No.: TKT-002WO Variant 10.19 QEEIAEGRRNPLRTAEWPMTKSTTDQSVWTKGVTPPANFTQGEDTFH - APYVPNQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIK K126N N128 RYLEEHPEKQKISFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPY V R H K A V H K A V K H K A V H K A V H K A V H K A V H K A VAttorney Docket No.: TKT-002WO RINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNKDG KVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN Variant 10.5 - QEEIAEGRRNPLRTAEWPMTKSTTDNSTWTKGVTPPANFTQGEDTFH K A V H K P A V H K A V H K Y V R H K A V H K A V H K P A V H K YAttorney Docket No.: TKT-002WO T_K126N_N1 LSRKHLGVFPDHVIDMFINGYRLDLTNHGPTPVKNGSKDPRGGIFDAV 28S_E198N FERGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVR INHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNKDGK H K P A V H K A V R E Q A L C V P N S L L H K A V S T R S T S L R T I H KAttorney Docket No.: TKT-002WO YLSRKHLGVFPDHVIDMFINGYRLDLTNHGPTPVKEGSKDPRGGIFDA VFERGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANV RINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNKDG K K R A L K E L R T I H K A V T Y L V P T A H K A V S T Y L V P TAttorney Docket No.: TKT-002WO LSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA DDKETCFAEEGKKLVAASQAALGL Variant 10- QEEIAEGRRNPLRTAEWPMTKSTTDQSVWTKGVTPPANFTQGEDTFH K A V R E Q A L C V P N S L L H K A V R E Q A L C V P N S L L H K A V R E Q A L CAttorney Docket No.: TKT-002WO CHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEV ENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHP DYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQN S L L

Claims

Attorney Docket No.: TKT-002WO CLAIMS WHAT IS CLAIMED IS:

1. An IdeS variant protein 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.

2. The IdeS variant protein of claim 1, wherein the modification at position 68 is a substitution of valine to threonine.

3. The IdeS variant protein of any one of claims 1-2, wherein the modification at position 75 is a substitution of alanine to proline.

4. The IdeS variant protein of any one of claims 1-3, wherein the modification at position 166 is a substitution of threonine to arginine or a substitution of threonine to glycine.

5. The IdeS variant protein of any one of claims 1-4, wherein the modification at position 187 is a substitution of serine to aspartate or a substitution of serine to glutamate.

6. The IdeS variant protein of any one of claims 1-5, wherein the modification at position 213 is a substitution of threonine to glutamate.

7. The IdeS variant protein of any one of claims 1-6, wherein the modification at position 236 is a substitution of serine to cysteine.

8. The IdeS variant protein of any one of claims 1-7, wherein the modification at position 277 is a substitution of leucine to cysteine.

9. The IdeS variant protein of any one of claims 1-8, wherein 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.

10. The IdeS variant protein of any one of claims 1-9, wherein 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.

11. The IdeS variant protein of any one of claims 1-10, wherein the modification at position 306 is a substitution of valine to threonine.

12. The IdeS variant protein of any one of claims 1-11, wherein 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.

13. The IdeS variant protein of any one of claims 1-12, wherein the variant protein further comprises a modification at position 308.

14. The IdeS variant protein of claim 13, wherein the modification at position 308 is a substitution of isoleucine to leucine.Attorney Docket No.: TKT-002WO 15. The IdeS variant protein of any one of claims 1-14, wherein 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.

16. The IdeS variant protein of any one of claims 1-14, wherein 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.

17. The IdeS variant protein of any one of claims 1-14, wherein 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.

18. The IdeS variant protein of any one of claims 1-14, wherein 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.

19. The IdeS variant protein of any one of claims 1-14, wherein 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.

20. The IdeS variant protein of claim 14, wherein 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 substitutionAttorney Docket No.: TKT-002WO 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.

21. The IdeS variant protein of any one of claims 1-20, wherein 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: 85).

22. The IdeS variant protein of any one of claims 1-21, wherein 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.

23. The IdeS variant protein of claim 22, wherein the variant protein comprises a substitution of serine at position 31 to aspartate or asparagine.

24. The IdeS variant protein of any one of claims 22-23, wherein the variant protein comprises a substitution of phenylalanine at position 32 to lysine.

25. The IdeS variant protein of any one of claims 22-24, wherein the variant protein comprises a substitution of serine at position 33 to glutamate.

26. The IdeS variant protein of any one of claims 22-25, wherein the variant protein comprises a substitution of isoleucine at position 38 to valine.

27. The IdeS variant protein of any one of claims 22-26, wherein the variant protein comprises a substitution of arginine at position 39 to asparagine or threonine.

28. The IdeS variant protein of any one of claims 22-27, wherein the variant protein comprises a substitution of valine at position 43 to glutamate.

29. The IdeS variant protein of any one of claims 22-28, wherein the variant protein comprises a substitution of threonine at position 44 to glutamate.

30. The IdeS variant protein of any one of claims 22-29, wherein the variant protein comprises a substitution of proline at position 45 to glutamate.Attorney Docket No.: TKT-002WO 31. The IdeS variant protein of any one of claims 22-30, wherein the variant protein comprises a substitution of histidine at position 47 to lysine.

32. The IdeS variant protein of any one of claims 22-31, wherein the variant protein comprises a substitution of lysine at position 54 to aspartate.

33. The IdeS variant protein of any one of claims 22-32, wherein the variant protein comprises a substitution of threonine at position 57 to lysine or glutamine.

34. The IdeS variant protein of any one of claims 22-33, wherein the variant protein comprises a substitution of alanine at position 60 to aspartate or glutamine.

35. The IdeS variant protein of any one of claims 22-34, wherein the variant protein comprises a substitution of valine at position 74 to lysine.

36. The IdeS variant protein of any one of claims 22-35, wherein the variant protein comprises a substitution of alanine at position 75 to asparagine or proline.

37. The IdeS variant protein of any one of claims 22-36, wherein the variant protein comprises a substitution of glutamine at position 77 to glycine.

38. The IdeS variant protein of any one of claims 22-37, wherein the variant protein comprises a substitution of isoleucine at position 82 to methionine.

39. The IdeS variant protein of any one of claims 22-38, wherein the variant protein comprises a substitution of threonine at position 85 to glutamine.

40. The IdeS variant protein of any one of claims 22-39, wherein the variant protein comprises a substitution of glutamine at position 113 to aspartate.

41. The IdeS variant protein of any one of claims 22-40, wherein the variant protein comprises a substitution of lysine at position 115 to histidine.

42. The IdeS variant protein of any one of claims 22-41, wherein the variant protein comprises a substitution of arginine at position 116 to aspartate.

43. The IdeS variant protein of any one of claims 22-42, wherein the variant protein comprises a substitution of isoleucine at position 127 to methionine.

44. The IdeS variant protein of any one of claims 22-43, wherein the variant protein comprises a substitution of asparagine at position 128 to glycine.

45. The IdeS variant protein of any one of claims 22-44, wherein the variant protein comprises a substitution of phenylalanine at position 129 to glutamate.

46. The IdeS variant protein of any one of claims 22-45, wherein the variant protein comprises a substitution of asparagine at position 130 to glutamate.

47. The IdeS variant protein of any one of claims 22-46, wherein the variant protein comprises a substitution of glutamine at position 133 to aspartate.Attorney Docket No.: TKT-002WO 48. The IdeS variant protein of any one of claims 22-47, wherein the variant protein comprises a substitution of leucine at position 148 to asparagine or arginine.

49. The IdeS variant protein of any one of claims 22-48, wherein the variant protein comprises a substitution of phenylalanine at position 153 to methionine or tyrosine.

50. The IdeS variant protein of any one of claims 22-49, wherein the variant protein comprises a substitution of glutamate at position 154 to aspartate.

51. The IdeS variant protein of any one of claims 22-50, wherein the variant protein comprises a substitution of lysine at position 159 to asparagine.

52. The IdeS variant protein of any one of claims 22-51, wherein the variant protein comprises a substitution of lysine at position 167 to proline.

53. The IdeS variant protein of any one of claims 22-52, wherein the variant protein comprises a substitution of histidine at position 168 to aspartate or proline.

54. The IdeS variant protein of any one of claims 22-53, wherein the variant protein comprises a substitution of histidine at position 175 to glutamine.

55. The IdeS variant protein of any one of claims 22-54, wherein the variant protein comprises a substitution of leucine at position 188 to glycine or methionine.

56. The IdeS variant protein of any one of claims 22-55, wherein the variant protein comprises a substitution of proline at position 195 to aspartate.

57. The IdeS variant protein of any one of claims 22-56, wherein the variant protein comprises a substitution of lysine at position 197 to glutamate.

58. The IdeS variant protein of any one of claims 22-57, wherein the variant protein comprises a substitution of alanine at position 210 to glycine.

59. The IdeS variant protein of any one of claims 22-58, wherein the variant protein comprises a substitution of serine at position 218 to aspartate.

60. The IdeS variant protein of any one of claims 22-59, wherein the variant protein comprises a substitution of lysine at position 219 to glycine.

61. The IdeS variant protein of any one of claims 22-60, wherein the variant protein comprises a substitution of leucine at position 220 to glutamine.

62. The IdeS variant protein of any one of claims 22-61, wherein the variant protein comprises a substitution of lysine at position 228 to glutamine.

63. The IdeS variant protein of any one of claims 22-62, wherein the variant protein comprises a substitution of lysine at position 233 to serine.

64. The IdeS variant protein of any one of claims 22-63, wherein the variant protein comprises a substitution of lysine at position 241 to aspartate.Attorney Docket No.: TKT-002WO 65. The IdeS variant protein of any one of claims 22-64, wherein the variant protein comprises a substitution of threonine at position 244 to lysine.

66. The IdeS variant protein of any one of claims 22-65, wherein the variant protein comprises a substitution of glutamate at position 245 to aspartate.

67. The IdeS variant protein of any one of claims 22-66, wherein the variant protein comprises a substitution of lysine at position 247 to asparagine.

68. The IdeS variant protein of any one of claims 22-67, wherein the variant protein comprises a substitution of valine at position 258 to aspartate.

69. The IdeS variant protein of any one of claims 22-68, wherein the variant protein comprises a substitution of serine at position 273 to aspartate.

70. The IdeS variant protein of any one of claims 22-69, wherein the variant protein comprises a substitution of asparagine at position 274 to glutamate.

71. The IdeS variant protein of any one of claims 22-70, wherein the variant protein comprises a substitution of lysine at position 278 to aspartate or threonine.

72. The IdeS variant protein of any one of claims 22-71, wherein the variant protein comprises a substitution of asparagine at position 288 to aspartate, glycine, or glutamine.

73. The IdeS variant protein of any one of claims 22-72, wherein the variant protein comprises a substitution of alanine at position 289 to proline.

74. The IdeS variant protein of any one of claims 22-73, wherein the variant protein comprises a substitution of phenylalanine at position 297 to aspartate.

75. The IdeS variant protein of any one of claims 22-74, wherein the variant protein comprises a substitution of glycine at position 299 to aspartate.

76. The IdeS variant protein of any one of claims 22-75, wherein the variant protein comprises a substitution of valine at position 300 to glutamate.

77. The IdeS variant protein of any one of claims 22-76, wherein the variant protein comprises a substitution of alanine at position 307 to glycine or asparagine.

78. The IdeS variant protein of any one of claims 22-77, wherein the variant protein comprises a substitution of isoleucine at position 313 to proline.

79. The IdeS variant protein of any one of claims 22-78, wherein the variant protein comprises a substitution of lysine at position 314 to glycine.

80. The IdeS variant protein of any one of claims 22-79, wherein the variant protein comprises a substitution of glutamate at position 315 to proline.

81. The IdeS variant protein of any one of claims 22-80, wherein the variant protein comprises a substitution of aspartate at position 316 to proline.Attorney Docket No.: TKT-002WO 82. The IdeS variant protein of any one of claims 22-81, wherein the variant protein comprises a substitution of valine at position 322 to threonine.

83. The IdeS variant protein of any one of claims 22-82, wherein the variant protein comprises a substitution of threonine at position 330 to aspartate.

84. An IdeS variant protein comprising the amino acid sequence according to any one of SEQ ID NOs: 3-75.

85. An IdeS variant protein comprising the amino acid sequence according to SEQ ID NO:

10.

86. An IdeS variant protein comprising the amino acid sequence according to SEQ ID NO:

11.

87. An IdeS variant protein comprising the amino acid sequence according to SEQ ID NO:

12.

88. An IdeS variant protein comprising the amino acid sequence according to SEQ ID NO:

13.

89. An IdeS variant protein 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: 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).

90. An IdeS variant protein comprising a substitution of DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2 with the sequence DDYQRNAMEAYAKEVPHQIT (SEQ ID NO: 78).

91. An IdeS variant protein comprising the amino acid sequence according to SEQ ID NOs: 86-94.

92. The IdeS variant protein of any one of claims 1-91, wherein the variant protein has decreased presentation of epitopes on human leukocyte antigen (HLA) as compared to a wild- type IdeS protein.

93. The IdeS variant protein of any one of claims 1-92, wherein the variant protein has increased cleavage activity of immunoglobulins as compared to a wild-type IdeS protein.

94. The IdeS variant protein of any one of claims 1-93, wherein the variant protein has a decrease in immunogenicity score more than 0 relative to SEQ ID NO: 1 or SEQ ID NO: 2.Attorney Docket No.: TKT-002WO 95. An IdeS variant protein comprises one or more glycosylation modifications as compared to wild-type.

96. An IdeS variant protein 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.

97. The IdeS variant protein of claim 96, wherein the modification at position 31 is a substitution of serine to asparagine.

98. The IdeS variant protein of any one of claims 96-97, wherein the modification at position 37 is a substitution of glutamate to asparagine.

99. The IdeS variant protein of any one of claims 96-98, wherein the modification at position 39 is a substitution of arginine to threonine or asparagine.

100. The IdeS variant protein of any one of claims 96-99, wherein the modification at position 42 is a substitution of glutamate to asparagine.

101. The IdeS variant protein of any one of claims 96-100, wherein the modification at position 74 is a substitution of valine to asparagine.

102. The IdeS variant protein of any one of claims 96-101, wherein the modification at position 76 is a substitution of asparagine to serine.

103. The IdeS variant protein of any one of claims 96-102, wherein the modification at position 111 is a substitution of lysine to asparagine.

104. The IdeS variant protein of any one of claims 96-103, wherein the modification at position 113 is a substitution of glutamine to threonine.

105. The IdeS variant protein of any one of claims 96-104, wherein the modification at position 119 is a substitution of glutamate to asparagine.

106. The IdeS variant protein of any one of claims 96-105, wherein the modification at position 121 is a substitution of histidine to threonine.

107. The IdeS variant protein of any one of claims 96-106, wherein the modification at position 130 is a substitution of asparagine to serine.

108. The IdeS variant protein of any one of claims 96-107, wherein the modification at position 142 is a substitution of aspartate to asparagine.

109. The IdeS variant protein of any one of claims 96-108, wherein the modification at position 144 is a substitution of lysine to threonine.

110. The IdeS variant protein of any one of claims 96-109, wherein the modification at position 147 is a substitution of glutamine to serine.

111. The IdeS variant protein of any one of claims 96-110, wherein the modification at position 148 is a substitution of leucine to asparagine.Attorney Docket No.: TKT-002WO 112. The IdeS variant protein of any one of claims 96-111, wherein the modification at position 198 is a substitution of glutamate to asparagine.

113. The IdeS variant protein of any one of claims 96-112, wherein the modification at position 233 is a substitution of lysine to serine.

114. The IdeS variant protein of any one of claims 96-113, wherein the modification at position 244 is a substitution of threonine to asparagine.

115. The IdeS variant protein of any one of claims 96-114, wherein the modification at position 246 is a substitution of glycine to threonine.

116. The IdeS variant protein of any one of claims 96-115, wherein the modification at position 311 is a substitution of lysine to asparagine.

117. The IdeS variant protein of any one of claims 96-116, wherein the modification at position 313 is a substitution of isoleucine to threonine.

118. The IdeS variant protein of any one of claims 96-117, wherein the modification at position 319 is a substitution of glycine to serine.

119. The IdeS variant protein of claim 96, wherein the variant protein comprises a substitution of glutamate at position 37 to asparagine and a substitution of arginine at position 39 to threonine.

120. The IdeS variant protein of claim 96, wherein the variant protein comprises a substitution of valine at position 74 to asparagine and a substitution of asparagine at position 76 to serine.

121. The IdeS variant protein of claim 96, wherein the variant protein comprises a substitution of lysine at position 111 to asparagine and a substitution of glutamine at position 113 to threonine.

122. The IdeS variant protein of claim 96, wherein the variant protein comprises a substitution of glutamate at position 119 to asparagine and a substitution of histidine at position 121 to threonine.

123. The IdeS variant protein of claim 96, wherein the variant protein comprises a substitution of aspartate at position 142 to asparagine and a substitution of lysine at position 144 to threonine.

124. The IdeS variant protein of claim 96, wherein the variant protein comprises a substitution of threonine at position 244 to asparagine and a substitution of glycine at position 246 to threonine.

125. The IdeS variant protein of claim 96, wherein the variant protein comprises a substitution of lysine at position 311 to asparagine and a substitution of isoleucine at position 313 to threonine.Attorney Docket No.: TKT-002WO 126. An IdeS variant protein 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.

127. The IdeS variant protein of claim 126, wherein the modification at position 47 is a substitution of threonine to asparagine.

128. The IdeS variant protein of any one of claims 126-127, wherein the modification at position 49 is a substitution of glutamine to threonine or asparagine.

129. The IdeS variant protein of any one of claims 126-128, wherein the modification at position 51 is a substitution of valine to threonine.

130. The IdeS variant protein of any one of claims 126-129, wherein the modification at position 78 is a substitution of glycine to serine.

131. The IdeS variant protein of any one of claims 126-130, wherein the modification at position 111 is a substitution of lysine to asparagine.

132. The IdeS variant protein of any one of claims 126-131, wherein the modification at position 113 is a substitution of glutamine to threonine.

133. The IdeS variant protein of any one of claims 126-132, wherein the modification at position 123 is a substitution of glutamate to asparagine.

134. The IdeS variant protein of any one of claims 126-133, wherein the modification at position 125 is a substitution of glutamine to threonine.

135. The IdeS variant protein of any one of claims 126-134, wherein the modification at position 126 is a substitution of lysine to asparagine.

136. The IdeS variant protein of any one of claims 126-135, wherein the modification at position 128 is a substitution of asparagine to serine.

137. The IdeS variant protein of any one of claims 126-136, wherein the modification at position 142 is a substitution of aspartate to asparagine.

138. The IdeS variant protein of any one of claims 126-137, wherein the modification at position 144 is a substitution of lysine to threonine.

139. The IdeS variant protein of any one of claims 126-138, wherein the modification at position 148 is a substitution of leucine to asparagine.

140. The IdeS variant protein of any one of claims 126-139, wherein the modification at position 198 is a substitution of glutamate to asparagine.

141. The IdeS variant protein of any one of claims 126-140, wherein the modification at position 273 is a substitution of serine to asparagine.

142. The IdeS variant protein of any one of claims 126-141, wherein the modification at position 275 is a substitution of glycine to serine.Attorney Docket No.: TKT-002WO 143. The IdeS variant protein of any one of claims 126-142, wherein the modification at position 278 is a substitution of lysine to serine.

144. The IdeS variant protein of any one of claims 126-143, wherein the modification at position 312 is a substitution of glutamate to asparagine.

145. The IdeS variant protein of any one of claims 126-144, wherein the modification at position 314 is a substitution of lysine to serine.

146. The IdeS variant protein of claim 126, wherein the variant protein comprises a substitution of threonine at position 47 to asparagine and a substitution of glutamine at position 49 to threonine.

147. The IdeS variant protein of claim 126, wherein the variant protein comprises a substitution of glutamine at position 49 to asparagine and a substitution of valine at position 51 to threonine.

148. The IdeS variant protein of claim 126, wherein the variant protein comprises a substitution of glutamate at position 123 to asparagine and a substitution of glutamine at position 125 to threonine.

149. The IdeS variant protein of claim 126, wherein the variant protein comprises a substitution of lysine at position 126 to asparagine and a substitution of asparagine at position 128 to serine.

150. The IdeS variant protein of claim 126, wherein the variant protein comprises a substitution of serine at position 273 to asparagine and a substitution of glycine at position 275 to serine.

151. The IdeS variant protein of claim 126, wherein the variant protein comprises a substitution of glutamate at position 312 to asparagine and a substitution of lysine at position 314 to serine.

152. The IdeS variant protein of claim 126, wherein 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.

153. The IdeS variant protein of claim 126, wherein 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.

154. The IdeS variant protein of claim 126, wherein 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.Attorney Docket No.: TKT-002WO 155. The IdeS variant protein of claim 126, wherein 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.

156. The IdeS variant protein of claim 126, wherein 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.

157. The IdeS variant protein of claim 126, wherein 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.

158. The IdeS variant protein of claim 126, wherein 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.

159. The IdeS variant protein of claim 126, wherein 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.

160. The IdeS variant protein of claim 126, wherein 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.

161. The IdeS variant protein of claim 126, wherein 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.Attorney Docket No.: TKT-002WO 162. The IdeS variant protein of claim 126, wherein 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.

163. The IdeS variant protein of claim 126, wherein 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.

164. The IdeS variant protein of claim 126, wherein 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.

165. A polypeptide 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.

166. The polypeptide of claim 165, wherein the human serum albumin is conjugated to a N- terminal of the IdeS protein.

167. The polypeptide of claim 165, wherein the human serum albumin is conjugated to a C- terminal of the IdeS protein.

168. The polypeptide of any one of claims 165-167, wherein the IdeS protein is conjugated to the human serum albumin using a linker.

169. The polypeptide of claim 168, wherein 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.

170. The polypeptide of any one of claims 165-167, wherein the IdeS protein is fused to the human serum albumin.

171. A 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,Attorney Docket No.: TKT-002WO 148, 198, 273, 275, 278, 312, and 314 of SEQ ID NO: 12 to introduce a glycosylation site; and b) a human serum albumin conjugated to the IdeS variant protein.

172. A polynucleotide encoding the IdeS variant protein of any one of claims 1-171.

173. An expression plasmid comprising the polynucleotide as in claim 172 and a promoter.

174. A cell comprising the polynucleotide as in claim 172.

175. A pharmaceutical composition comprising the IdeS variant protein of any one of claims 1-171, and a pharmaceutically acceptable carrier.

176. A method of treating a disease or disorder comprising administering an effective amount of the IdeS variant protein of any one of claims 1-164, the polypeptide of any one of claims 165-171, or the pharmaceutical composition of claim 175.

177. The method of claim 176, wherein the IdeS variant protein is co-administered with a gene therapy.

178. A method for reducing presentation on HLA-II of IdeS comprising administering an effective amount of the IdeS variant protein of any one of claims 1-164, the polypeptide of any one of claims 165-171, or the pharmaceutical composition of claim 175.

179. A method for prolonging serum half-life of IdeS comprising administering an effective amount of the IdeS variant protein of any one of claims 1-164, the polypeptide of any one of claims 165-171, or the pharmaceutical composition of claim 175.

180. A method for prolonging IgG depletion of IdeS comprising administering an effective amount of the IdeS variant protein of any one of claims 1-164, the polypeptide of any one of claims 165-171, or the pharmaceutical composition of claim 175.

181. A method of treating an autoantibody-mediated autoimmune disease comprising administering an effective amount of the IdeS variant protein of any one of claims 1-164, the polypeptide of any one of claims 165-171, or the pharmaceutical composition of claim 175.

Images