IdeS variant protein and method of using it

Abstract

This disclosure relates, as a whole, to variant IdeS polypeptides that have lower immunogenicity and maintain or improve IdeS stability and IgG cleavage activity, as well as to methods for using these variant IdeS polypeptides.

Description

[Technical Field]

[0001] cross reference

[0001] This application claims benefit and priority to U.S. Provisional Patent Application No. 63 / 492,891 filed on 29 March 2023 and U.S. Provisional Patent Application No. 63 / 584,748 filed on 22 September 2023, which are incorporated herein by reference in their entirety.

[0002] Areas of this disclosure

[0002] This disclosure relates to variant Ides polypeptides having lower immunogenicity and maintaining or improving IdeS stability and IgG cleavage activity, as well as methods for using these variant Ides polypeptides. [Background technology]

[0003] background

[0003] IdeS (an immunoglobulin-degrading enzyme derived from Streptococcus pyogenes) is a cysteine ​​protease that catalyzes the rapid cleavage of all human IgG subclasses within the hinge region, cleaving the antigen-binding domain of IgG from the Fc region which mediates immune effector function. Separation of the antigen-binding Fab region from the Fc portion prevents Fc-mediated recruitment of immune effector functions to antigens, such as the recruitment of lethal immune cells (i.e., antibody-dependent cell-mediated cytotoxicity or ADCC), and the deposition of activated complement (i.e., complement-dependent cell-mediated cytotoxicity or CDC).

[0004]

[0004] Due to IdeS's ability to specifically target and cleave IgG, IdeS can significantly reduce antibody-mediated reactions. This has been most comprehensively documented in the context of human kidney transplantation, where donor-specific antibodies (DSAs) in transplant recipients recognize epitopes on the donor graft and trigger transplant rejection. Patients with higher levels of DSA can be desensitized by administering IdeS pre-transplant, thereby increasing the success rate. Wild-type IdeS has been clinically used to desensitize kidney transplant recipients with higher titers of anti-HLA donor-specific antibodies and may have broader applications in autoimmunity, gene therapy with antibody-rich viral vectors, and to reduce antibody-mediated rejection of xenografts. [Overview of the project] [Problems that the invention aims to solve]

[0005]

[0005] However, IdeS is immunogenic, and its administration induces anti-IdeS antibodies, which hinders its use when long-term repeated administration is required. Furthermore, S. pyogenes is a widely distributed pathogen and a cause of streptococcal pharyngitis infection. Many people have developed antibody protection against S. pyogenes and IdeS through previous exposure. The presence and induction of anti-drug antibodies (ADAs) are a major concern in the development of therapeutics because ADAs have the potential to alter pharmacokinetics, drug activity, and bioavailability, or to cause hypersensitivity reactions such as severe anaphylaxis.

[0006]

[0006] Described herein are IdeS variant peptides that have lower immunogenicity, such as reduced peptide presentation on HLA-II and masking of surface epitopes that can otherwise be used for B cell recognition, while maintaining or improving IdeS stability and IgG cleavage activity. Using such immunogenic IdeS variants, the application of IdeS-like drugs to indications requiring repeated administration can be expanded. [Means for solving the problem]

[0007] Summary of this disclosure

[0007] Provided herein are IdeS variant proteins comprising one or more amino acid modifications that result in reduced peptide presentation on HLA-II or reduced immunogenicity as measured by masking of surface epitopes recognized by antibodies, as well as maintenance or improvement of IdeS stability and IgG cleavage activity compared to wild-type proteins.

[0008]

[0008] Described herein are IdeS variant proteins that, in certain embodiments, include at least two modifications at 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 threonine to glycine. In some embodiments, the modification at position 187 is a substitution of serine to aspartic acid or serine to glutamic acid. In some embodiments, the modification at position 213 is a substitution of threonine to glutamic acid. 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 aspartic acid, serine to lysine, or serine to glutamic acid. In some embodiments, the modification at position 303 is a substitution of alanine to aspartic acid, alanine to asparagine, or 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, isoleucine to aspartic acid, or isoleucine to glycine. In some embodiments, the variant protein further includes 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 includes substitutions of valine to threonine at position 68, alanine to proline at position 75, threonine to arginine at position 166, serine to aspartic acid at position 187, threonine to glutamic acid at position 213, serine to lysine at position 302, and alanine to aspartic acid at position 303.In some embodiments, the variant protein includes a valine to threonine substitution at position 68, alanine to proline substitution at position 75, a threonine to arginine substitution at position 166, a serine to aspartic acid substitution at position 187, a threonine to glutamic acid substitution at position 213, a serine to glutamic acid substitution at position 302, and alanine to aspartic acid substitution at position 303. In some embodiments, the variant protein includes a valine to threonine substitution at position 68, alanine to proline substitution at position 75, a threonine to arginine substitution at position 166, a serine to aspartic acid substitution at position 187, a threonine to glutamic acid substitution at position 213, a serine to lysine substitution at position 302, alanine to aspartic acid substitution at position 303, and a valine to threonine substitution at position 306. In some embodiments, the variant protein includes substitutions of valine to threonine at position 68, alanine to proline at position 75, threonine to arginine at position 166, serine to aspartic acid at position 187, threonine to glutamic acid at position 213, serine to glutamic acid at position 302, valine to threonine at position 306, and isoleucine to lysine at position 318. In some embodiments, the variant protein includes a valine-to-threonine substitution at position 68, an alanine-to-proline substitution at position 75, a threonine-to-arginine substitution at position 166, a serine-to-aspartic acid substitution at position 187, a threonine-to-glutamic acid substitution at position 213, a serine-to-cysteine ​​substitution at position 236, a leucine-to-cysteine ​​substitution at position 277, a serine-to-glutamic acid substitution at position 302, alanine-to-aspartic acid substitution at position 303, and a valine-to-threonine substitution at position 306.In some embodiments, the variant protein includes substitutions of valine to threonine at position 68, alanine to proline at position 75, threonine to arginine at position 166, serine to aspartic acid at position 187, threonine to glutamic acid at position 213, serine to cysteine ​​at position 236, leucine to cysteine ​​at position 277, and isoleucine to leucine at position 308. In some embodiments, the variant protein further includes substitutions of DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2 with sequences 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 includes modifications at 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 includes a substitution of serine to aspartic acid or asparagine at position 31. In some embodiments, the variant protein includes a substitution of phenylalanine to lysine at position 32.In some embodiments, the variant protein includes a substitution of serine to glutamate at position 33. In some embodiments, the variant protein includes a substitution of isoleucine to valine at position 38. In some embodiments, the variant protein includes a substitution of arginine to asparagine or threonine at position 39. In some embodiments, the variant protein includes a substitution of valine to glutamate at position 43. In some embodiments, the variant protein includes a substitution of threonine to glutamate at position 44. In some embodiments, the variant protein includes a substitution of proline to glutamate at position 45. In some embodiments, the variant protein includes a substitution of histidine to lysine at position 47. In some embodiments, the variant protein includes a substitution of lysine to aspartate at position 54. In some embodiments, the variant protein includes a substitution of threonine to lysine or glutamine at position 57. In some embodiments, the variant protein includes a substitution of alanine to aspartate or glutamine at position 60. In some embodiments, the variant protein includes a substitution of valine to lysine at position 74. In some embodiments, the variant protein includes a substitution of alanine to asparagine or proline at position 75. In some embodiments, the variant protein includes a substitution of glutamine to glycine at position 77. In some embodiments, the variant protein includes a substitution of isoleucine to methionine at position 82. In some embodiments, the variant protein includes a substitution of threonine to glutamine at position 85. In some embodiments, the variant protein includes a substitution of glutamine to aspartic acid at position 113. In some embodiments, the variant protein includes a substitution of lysine to histidine at position 115. In some embodiments, the variant protein includes a substitution of arginine to aspartic acid at position 116. In some embodiments, the variant protein includes a substitution of isoleucine to methionine at position 127.In some embodiments, the variant protein includes a substitution of asparagine to glycine at position 128. In some embodiments, the variant protein includes a substitution of phenylalanine to glutamic acid at position 129. In some embodiments, the variant protein includes a substitution of asparagine to glutamic acid at position 130. In some embodiments, the variant protein includes a substitution of glutamine to aspartic acid at position 133. In some embodiments, the variant protein includes a substitution of leucine to asparagine or arginine at position 148. In some embodiments, the variant protein includes a substitution of phenylalanine to methionine or tyrosine at position 153. In some embodiments, the variant protein includes a substitution of glutamic acid to aspartic acid at position 154. In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 159. In some embodiments, the variant protein includes a substitution of lysine to proline at position 167. In some embodiments, the variant protein includes a histidine to aspartic acid or proline substitution at position 168. In some embodiments, the variant protein includes a histidine to glutamine substitution at position 175. In some embodiments, the variant protein includes a leucine to glycine or methionine substitution at position 188. In some embodiments, the variant protein includes a proline to aspartic acid substitution at position 195. In some embodiments, the variant protein includes a lysine to glutamic acid substitution at position 197. In some embodiments, the variant protein includes an alanine to glycine substitution at position 210. In some embodiments, the variant protein includes a serine to aspartic acid substitution at position 218. In some embodiments, the variant protein includes a lysine to glycine substitution at position 219. In some embodiments, the variant protein includes a leucine to glutamine substitution at position 220. In some embodiments, the variant protein includes a lysine to glutamine substitution at position 228.In some embodiments, the variant protein includes a substitution of lysine to serine at position 233. In some embodiments, the variant protein includes a substitution of lysine to aspartic acid at position 241. In some embodiments, the variant protein includes a substitution of threonine to lysine at position 244. In some embodiments, the variant protein includes a substitution of glutamic acid to aspartic acid at position 245. In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 247. In some embodiments, the variant protein includes a substitution of valine to aspartic acid at position 258. In some embodiments, the variant protein includes a substitution of serine to aspartic acid at position 273. In some embodiments, the variant protein includes asparagine at position 274. This includes substitution from to glutamic acid. In some embodiments, the variant protein includes substitution from lysine to aspartic acid or threonine at position 278. In some embodiments, the variant protein includes substitution from asparagine to aspartic acid, glycine, or glutamine at position 288. In some embodiments, the variant protein includes substitution from alanine to proline at position 289. In some embodiments, the variant protein includes substitution from phenylalanine to aspartic acid at position 297. In some embodiments, the variant protein includes substitution from glycine to aspartic acid at position 299. In some embodiments, the variant protein includes substitution from valine to glutamic acid at position 300. In some embodiments, the variant protein includes substitution from alanine to glycine or asparagine at position 307. In some embodiments, the variant protein includes substitution from isoleucine to proline at position 313. In some embodiments, the variant protein includes substitution from lysine to glycine at position 314. In some embodiments, the variant protein includes a glutamate-to-proline substitution at position 315. In some embodiments, the variant protein includes an aspartate-to-proline substitution at position 316. In some embodiments, the variant protein includes a valine-to-threonine substitution at position 322. In some embodiments, the variant protein includes a threonine-to-aspartate substitution at position 330.

[0009]

[0009] In certain embodiments, the IdeS variant protein described herein is an IdeS variant protein comprising the amino acid sequence described in any one of SEQ ID NOs: 3 to 75.

[0010]

[0010] In certain embodiments, the IdeS variant protein described herein is an IdeS variant protein comprising the amino acid sequence described in SEQ ID NO: 10.

[0011]

[0011] In certain embodiments, the IdeS variant protein described herein is an IdeS variant protein comprising the amino acid sequence described in SEQ ID NO: 11.

[0012]

[0012] In certain embodiments, the IdeS variant protein described herein is an IdeS variant protein comprising the amino acid sequence described in SEQ ID NO: 12.

[0013]

[0013] In certain embodiments, the IdeS variant protein described herein is an IdeS variant protein comprising the amino acid sequence described in SEQ ID NO: 13.

[0014]

[0014] In certain embodiments described herein, an IdeS variant protein is provided which DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2, comprising a substitution with a sequence that is at least 80% identical to a 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).

[0015]

[0015] In certain embodiments, the IdeS variant protein described herein is one which includes a substitution of the sequence DDYQRNAMEAYAKEVPHQIT (Sequence ID 78) for DSFSANQEIRYSEVTPYHVT (Sequence ID 76) of SEQ ID NO: 2.

[0016]

[0016] Described herein are IdeS variant proteins that, in certain embodiments, contain the amino acids listed in SEQ ID NOs: 86-94. In some embodiments, the variant protein exhibits reduced epitope presentation on human leukocyte antigens (HLA) compared to the wild-type IdS protein. In some embodiments, the variant protein exhibits increased immunoglobulin cleavage activity compared to the wild-type IdeS protein. In some embodiments, the variant protein exhibits a reduced immunogenicity score greater than 0 compared to SEQ ID NOs: 1 or SEQ ID NOs: 2.

[0017]

[0017] In certain embodiments, the IdeS variant proteins described herein include one or more glycosylation modifications compared to the wild type.

[0018]

[0018] Described herein are IdeS variant proteins that, in certain embodiments, include at least one modification at a position 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 in order 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 glutamic acid 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 glutamic acid 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 glutamic acid to asparagine. In some embodiments, the modification at position 121 is a substitution of histidine to threonine. In some embodiments, the modification at position 130 is a substitution of asparagine to serine. In some embodiments, the modification at position 142 is a substitution of aspartic acid 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 glutamic acid 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 includes a substitution of glutamic acid to asparagine at position 37 and a substitution of arginine to threonine at position 39. In some embodiments, the variant protein includes a substitution of valine to asparagine at position 74 and a substitution of asparagine to serine at position 76. In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 111 and a substitution of glutamine to threonine at position 113. In some embodiments, the variant protein includes a substitution of glutamic acid to asparagine at position 119 and a substitution of histidine to threonine at position 121. In some embodiments, the variant protein includes a substitution of aspartic acid to asparagine at position 142 and a substitution of lysine to threonine at position 144. In some embodiments, the variant protein includes a substitution of threonine to asparagine at position 244 and a substitution of glycine to threonine at position 246. In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 311 and a substitution of isoleucine to threonine at position 313.

[0019]

[0019] Described herein is an IdeS variant protein that, in certain embodiments, includes at least one modification at a position 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 in order to introduce a glycosylation site. In some embodiments, the modification at position 47 is the substitution of threonine with asparagine. In some embodiments, the modification at position 49 is the substitution of glutamine with threonine or asparagine. In some embodiments, the modification at position 51 is the substitution of valine with threonine. In some embodiments, the modification at position 78 is the substitution of glycine with serine. In some embodiments, the modification at position 111 is the substitution of lysine with 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 glutamic acid 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 aspartic acid 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 glutamic acid 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 glutamic acid to asparagine. In some embodiments, the modification at position 314 is a substitution of lysine to serine.In some embodiments, the variant protein includes a substitution of threonine to asparagine at position 47 and a substitution of glutamine to threonine at position 49. In some embodiments, the variant protein includes a substitution of glutamine to asparagine at position 49 and a substitution of valine to threonine at position 51. In some embodiments, the variant protein includes a substitution of glutamic acid to asparagine at position 123 and a substitution of glutamine to threonine at position 125. In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 126 and a substitution of asparagine to serine at position 128. In some embodiments, the variant protein includes a substitution of serine to asparagine at position 273 and a substitution of glycine to serine at position 275. In some embodiments, the variant protein includes a substitution of glutamic acid to asparagine at position 312 and a substitution of lysine to serine at position 314. In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of aspartic acid to asparagine at position 142, and a substitution of lysine to threonine at position 144. In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, and a substitution of glutamic acid to asparagine at position 198. In some embodiments, the variant protein includes a substitution of aspartic acid to asparagine at position 142, a substitution of lysine to threonine at position 144, and a substitution of glutamic acid to asparagine at position 198. In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of aspartic acid to asparagine at position 142, a substitution of lysine to threonine at position 144, and a substitution of glutamic acid to asparagine at position 198.In some embodiments, the variant protein includes a substitution of glutamine to asparagine at position 49, a substitution of valine to threonine at position 51, a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of lysine to asparagine at position 126, a substitution of asparagine to serine at position 128, a substitution of leucine to asparagine at position 148, and a substitution of glutamic acid to asparagine at position 198. In some embodiments, the variant protein includes a substitution of glutamine to asparagine at position 49, a substitution of valine to threonine at position 51, a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of leucine to asparagine at position 148, and a substitution of glutamic acid to asparagine at position 198. In some embodiments, the variant protein includes substitutions of lysine to asparagine at position 111, glutamine to threonine at position 113, lysine to asparagine at position 126, asparagine to serine at position 128, leucine to asparagine at position 148, and glutamic acid to asparagine at position 198. In some embodiments, the variant protein includes substitutions of glutamine to asparagine at position 49, valine to threonine at position 51, lysine to asparagine at position 111, glutamine to threonine at position 113, lysine to asparagine at position 126, asparagine to serine at position 128, and glutamic acid to asparagine at position 198. In some embodiments, the variant protein includes substitutions of threonine to asparagine at position 47, glutamine to asparagine at position 49, lysine to asparagine at position 111, glutamine to threonine at position 113, and glutamic acid to asparagine at position 198.In some embodiments, the variant protein comprises a substitution of glutamine to asparagine at position 49, a substitution of valine to threonine at position 51, a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, and a substitution of glutamic acid to asparagine at position 198. In some embodiments, the variant protein comprises a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of glutamic acid to asparagine at position 123, a substitution of glutamine to threonine at position 125, and a substitution of glutamic acid to asparagine at position 198. In some embodiments, the variant protein comprises a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of lysine to asparagine at position 126, a substitution of asparagine to serine at position 128, and a substitution of glutamic acid to asparagine at position 198. In some embodiments, the variant protein comprises a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of leucine to asparagine at position 148, and a substitution of glutamic acid to asparagine at position 198.

[0020]

[0020] In certain embodiments, as described herein, a polypeptide comprising an IdeS protein having at least 90% sequence identity to any one of SEQ ID NOs: 1-75, conjugated to human serum albumin. In some embodiments, the human serum albumin is conjugated to the N-terminus of the IdeS protein. In some embodiments, the human serum albumin is conjugated to the C-terminus of the IdeS protein. In some embodiments, the IdeS protein is conjugated to 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 human serum albumin.

[0021]

[0021] In certain embodiments described herein, a polypeptide comprising: a) an IdeS variant protein, i) at least two modifications at 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 at a position 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 for introducing a glycosylation site, or at least one modification at a position 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; and b) human serum albumin conjugated to the IdeS variant protein.

[0022]

[0022] In certain embodiments described herein, a polynucleotide encoding an IdeS variant protein described herein.

[0023]

[0023] In certain embodiments described herein, an expression plasmid comprising a polynucleotide described herein and a promoter.

[0024]

[0024] In certain embodiments described herein, a cell comprising a polynucleotide described herein.

[0025]

[0025] In certain embodiments described herein, a pharmaceutical composition comprising an IdeS variant protein described herein and a pharmaceutically acceptable carrier.

[0026]

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

[0027]

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

[0028]

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

[0029]

[0029] In certain embodiments described herein are methods for prolonging IgS depletion of IdeS, comprising administering an effective amount of an IdeS variant protein, a polypeptide, or a pharmaceutical composition as described herein.

[0030]

[0030] Other embodiments and details of the present disclosure are shown below in this specification. [Brief explanation of the drawing]

[0031] Brief explanation of the drawing [Figure 1A]

[0031] Figures 1A-1G show data indicating that IdeS expressed by mammalian cells is active and glycosylated. Figure 1A shows SDSA-PAGE analysis of fractions from NiNTA affinity chromatography of his-tagged IdeS protein. L, load; FT, flow-through. His-tagged IdeS protein was expressed in Escherichia coli (E. coli) cells (EC) or secreted by Expi293F cells (XP). The protein was eluted with PBS containing 250 mM imidazole. The calculated MW of tagged IdeS (excluding glycan) is 36 kD. [Figure 1B]

[0031] Figure 1B shows that the eluate from NiNTA purification was concentrated and separated by SEC. [Figure 1C]

[0031] Figure 1C shows that IdeS (0.25 or 0.5 μg) purified from Expi293F expression medium was treated with PNGase F and analyzed by SDS-PAGE. [Figure 1D]

[0031] Figure 1D shows that cleavage of IgG catalyzed by IdeS generates fragments that can be degraded by non-reducing SDS-PAGE. The first cleavage of the hinge generates Fc / 2 and single-cut IgG (scIgG). The second cleavage of the adjacent hinge releases F(ab')2 and two Fc / 2 molecules. [Figure 1E]

[0031] Figure 1E shows that the expression medium from Expi293F cells transfected with IdeS was incubated with IVIG, and the cleavage products were detected by SDS-PAGE (upper panel). The expression level of the IdeS protein was evaluated by SDS-PAGE of the expression medium (lower panel). The Asn residue in the consensus N-glycosylation motif (NXS / T) was mutated to Gln. [Figure 1F]

[0031] Figure 1F shows that the activity of purified IdeS (20 nM) derived from bacteria was evaluated based on the proteolysis of nonspecific monoclonal IgG1 (20 μM). [Figure 1G]

[0031] Figure 1G shows that the activity of purified IdeS (20 nM) from the Expi293F culture was evaluated based on the proteolysis of nonspecific monoclonal IgG1 (20 μM). [Figure 2A]

[0032] Figures 2A-2C show HLA-II epitopes in IdeS identified by MAPP. Figure 2A shows pulsed treatment of monocyte-derived dendritic cells (DCs) from 10 donors with IdeS. The pMHC-II complex was immunoprecipitated, and the bound IdeS peptide was identified by mass spectrometry. IdeS residues frequently found in antigen peptides are shown in darker gray. The total number of peptides for each residue across the 10 donors is shown at the bottom. [Figure 2B]

[0032] Figure 2B shows the mapping of the total peptide number of each residue to a model derived from the IdeS crystal structure (PDB 2AVW). Residues that are expressed more frequently in the HLA-II-presenting peptide are shown with darker shading. The side chains of the three catalytic residues are labeled and shown as black spheres (201). [Figure 2C]

[0032] Figure 2C shows epitope clusters 1-8 (labeled) on the IdeS structure. The side chains of the three catalytic residues are shown as black spheres (203). Cluster 1 is only partially shown because it is located in the unstructured N-terminus, which is not present in this model. [Figure 3]

[0033] Figure 3 shows an overview of predicted and experimentally identified HLA-II epitopes in IdeS and modifications that reduced immunogenicity. A schematic of the IdeS secondary structure is shown at the top. Calculation-predicted epitopes are shown based on predicted binding to at least four HLA-II alleles in a test set of 14 alleles (303). From light to dark colors, the rank percentage threshold decreases from 20% to 15%, 10%, and 5% (i.e., the prediction becomes more precise). Experimentally identified HLA-II epitopes by MAPP are shown (305), representing the total number of unique peptide residues identified across 10 donors. The bottom row shows modification sites to reduce the immunogenicity of the protein (307). Light gray (301) indicates that the modification was active during screening. [Figure 4]

[0034] Figure 4 shows data from rapid screening of IdeS variants. To facilitate rapid screening of variants, IdeS was expressed as a secreted protein in Expi293F cells, and the expression medium was directly incubated with IVIG at 37°C for 1 hour. The upper panel shows the Coomassie-stained gel of IVIG degradation products under non-reducing conditions. The lower panel shows the IdeS levels in the expression medium (excluding glycans, the calculated MW of IdeS is 36 kD). This figure is representative of the screening process and shows the results of a subset of the single-point variants evaluated. Wt, wild type. [Figure 5]

[0035] Figure 5 shows data from rapid screening of IdeS variants. Another exemplary set of data from screening IdeS variants directly from Expi293F expression medium. The top panel shows Coomassie-stained gels of degradation products when the expression medium is incubated with IVIG. These gels are run under non-reducing conditions. The bottom panel shows IdeS levels in the expression medium. Wt, wild type. C-94-S is a catalytic inactivation modification of the nucleophilic cysteine ​​in the active site. [Figure 6]

[0036] Figure 6 shows data from the introduction of a potential disulfide-bonded cysteine ​​into IdeS. A variant of IdeS with cysteine ​​(predicted to form a disulfide) was expressed in Expi293F cells, and the expression medium was screened for proteolytic activity against IVIG. The Coomassie-stained electrophoresis gel in the upper panel shows the IVIG proteolytic products after 1 hour at 37°C. The lower panel shows the IdeS expression levels in the culture medium. IdeS_D.3.3 was found to be as active as wild-type IdeS. [Figure 7A]

[0037] Figures 7A–7F show data demonstrating that immunogenic IdeS derivatives are active and selective for human and rabbit IgG. Wild-type (wt) IdeS was purified from transfected Expi293F cultures. The protein (20 nM) was incubated with 20 μM human monoclonal IgG1 at 37°C for the indicated time. Immunoglobulin degradation products were analyzed by Coomassie-stained SDS gel electrophoresis under non-reducing conditions. [Figure 7B]

[0037] Wild-type (wt) IdeS was purified from a culture of transfected Expi293F. The protein (20 nM) was incubated with 20 μM rabbit polyclonal IgG at 37°C for the indicated time. Immunoglobulin degradation products were analyzed by Coomassie-stained SDS gel electrophoresis under non-reducing conditions. [Figure 7C]

[0037] Wild-type (wt) IdeS was purified from a culture of transfected Expi293F. The protein (20 nM) was incubated with 20 μM mouse polyclonal IgG at 37°C for the indicated time. Immunoglobulin degradation products were analyzed by Coomassie-stained SDS gel electrophoresis under non-reducing conditions. [Figure 7D]

[0037] Variant 77 was purified from a culture of transfected Expi293F. The protein (20 nM) was incubated with 20 μM human monoclonal IgG1 at 37°C for the indicated time. Immunoglobulin degradation products were analyzed by Coomassie-stained SDS gel electrophoresis under non-reducing conditions. [Figure 7E]

[0037] Variant 77 was purified from a culture of transfected Expi293F. The protein (20 nM) was incubated with 20 μM rabbit polyclonal IgG at 37°C for the indicated time. Immunoglobulin degradation products were analyzed by Coomassie-stained SDS gel electrophoresis under non-reducing conditions. [Figure 7F]

[0037] Variant 77 was purified from a culture of transfected Expi293F. The protein (20 nM) was incubated with 20 μM mouse polyclonal IgG at 37°C for the indicated time. Immunoglobulin degradation products were analyzed by Coomassie-stained SDS gel electrophoresis under non-reducing conditions. [Figure 8A]

[0038] Figures 8A-8B show data from screening for IdeS truncation and chimeric variants at the N-terminus of the protein. Figure 8A shows Coomassie-stained SDS gels for measuring activity against human IVIG (upper gel image) and expression of IdeS truncation variants and N-terminal chimeras (lower gel image). Wt, wild type. Truncation variants IdeS_B.3.4-IdeS_B.3.7 are characterized by gradual shortening of the N-terminus of the mature polypeptide by 5, 10, 16, and 19 residues, respectively. [Figure 8B]

[0038] Figure 8B shows the multiple sequence alignment of Wt IdeS and the sequence and structural homologues used to create the N-terminal chimera (sequence numbers 196-214, in order of appearance). The numbering at the top is based on the full-length IdeS(Wt). [Figure 9A]

[0039] Figures 9A–9B show data demonstrating the combination of multiple modifications to reduce the immunogenicity risk that destabilizes the protein fold. Figure 9A shows data for additional modifications to reduce immunogenicity, where N-terminal chimerization was added to variant 77 intermediate to create derivative IdeS variants 100, 101, 102, 103, 1, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, and 116. Based on high expression and catalytic activity, variant 1 was used. A small number of revertant and compensatory modifications (variants 2–7) were evaluated to enhance thermal stability. The top panel shows Coomassie-stained electrophoresis gels after incubation of IdeS-transfected Expi293F expression medium with human IVIG. These gels were run under non-reducing conditions to observe immunoglobulin degradation products. The Coomassie-stained gel at the bottom shows the IdeS expression level in the culture medium. [Figure 9B]

[0039] Figure 9B shows a graph of the melting temperatures of variant 1 and its descendants when measured by DSF. [Figure 10A]

[0040] Figures 10A-10B show the thermal stability data of IdeS variants. Figure 10A is a graph of the DSF thermal fusion curve of purified IdeS protein. Wt, wild type. [Figure 10B]

[0040] Figure 10B is a graph of melting temperatures from DSF analysis. By combining IdeS_D.3.3 (disulfide), variant 94 (N-terminal chimera), variant 77 (intermediate with reduced immunogenicity), and modifications from other sources, variant 1 with reduced thermal stability was obtained. With a few reverting and compensatory modifications, variant 6 (1001) was obtained, and finally variant 10 (1003) was obtained. [Figure 11A]

[0041] Figures 11A–11D show data demonstrating that the immunogenic IdeS protein exhibits high catalytic activity in vitro. Figure 11A shows the squared immunogenicity scores plotted across the sequences of wild-type IdeS and variant 10. Higher scores correspond to regions predicted to have high affinity for multiple HLA-II alleles. [Figure 11B]

[0041] Figure 11B shows the NiNTA affinity purification of variant 10 expressed in cells of Escherichia coli (E. coli). L, load; FT, flow-through. [Figure 11C]

[0041] Figure 11C shows the NiNTA eluate concentrated and separated by SEC. Peak fractions (numbered 11-13) are shown by vertical dashed lines. Coomassie-stained electrophoresis gels of fractions 11-13 are shown on the right. au, absorbance units. [Figure 11D]

[0041] Figure 11D shows that purified variant 10 (20 nM) has high activity against the proteolysis of human monoclonal IgG1 (20 μM). The degradation products were visualized on a Coomassie-stained polyacrylamide gel run under non-reducing conditions. [Figure 12]

[0042] Figure 12 shows a graph of IdeS peptide binding to HLA-DR1. Wild-type (circular) and mutant (square) peptides competed with the fluorescent reference peptide for binding to HLA-DRA1 / HLA-DRB1. The fluorescence polarization (FP) signal of the reference peptide was measured. Peptides that bind more strongly inhibit the FP signal at lower concentrations (i.e., the curve for high-affinity peptides shifts to the left). Peptides that cannot bind to HLA-II with adequate affinity do not inhibit FP (e.g., peptide P6 in the lower left). [Figure 13]

[0043] Figure 13 shows a graph of IdeS peptide binding to HLA-DR7. The wild-type (circular) and mutant (square) peptides competed with the fluorescent reference peptide for binding to HLA-DRA1 / HLA-DRB7. The FP of the reference peptide is plotted. [Figure 14]

[0044] Figure 14 shows the binding of the IdeS peptide to HLA-DR11. The wild-type (circular) and mutant (square) peptides competed with the fluorescent reference peptide for binding to HLA-DRA1 / HLA-DRB11. The FP of the reference peptide is plotted. [Figure 15]

[0045] Figure 15 shows the binding of the IdeS peptide to HLA-DR15. The wild-type (circular) and mutant (square) peptides competed with the fluorescent reference peptide for binding to HLA-DRA1 / HLA-DRB15. The FP of the reference peptide is plotted. [Figure 16A]

[0046] Figures 16A–16B show data from intravenously administered, immunogenic IdeS variants demonstrating rapid IgG degradation in rabbits. Figure 16A shows data from two rabbits (indicated by light gray and dark gray circles below the sample lane) administered variant 10 (4 mg dose) and collected via ear vein at the indicated time. Serum was analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. [Figure 16B]

[0046] Figure 16B shows the serum IgG levels measured by ELISA after intravenous injection of 10 4 mg of wild-type (Wt) IdeS or a variant purified from Escherichia coli (E. coli). [Figure 17]

[0047] Figure 17 shows graphs of ELISA-evaluated levels of anti-IdeS antibodies in serum from 10 healthy males (top left), 10 healthy females (top right), and 9 donors (bottom left) recovering from recent S. pyogenes infections. The plates were coated with wild-type IdeS. For reference, IVIG and polyclonal goat anti-IdeS are also shown, with high titer healthy donor serum samples (donor HMN799367; yellow) highlighted. [Figure 18A]

[0048] Figures 18A–18F show the results for IdeS variants with custom N-glycosylation sites. Figure 18A shows the addition of a single N-glycosylation motif to wild-type IdeS, and the addition of combinations of motif variants 10.1, 10.2, 10.3, and 10.4 to variant 10. The proteins were expressed and secreted by Expi293F cells. The upper Coomassie-stained electrophoresis gel shows human IVIG degradation products after incubation with expression medium. The gel was run under non-reducing conditions. The lower gel was run under reducing conditions and shows IdeS expression in the medium and a shift to higher MW for some of the variants. [Figure 18B]

[0048] Figure 18B shows a structural model of IdeS (labeled in the figure) bound to the cleavage product IgG Fc (labeled in the figure), with the custom N-glycosylation sites at positions 111, 148, and 198 labeled and shown as spheres. The wild-type residue N61 (labeled and shown as a sphere) is also glycosylated (see Figure 1E). For orientation purposes, the catalytic residue C94 is labeled and shown as a sphere. [Figure 18C]

[0048] Figure 18C shows that variants 10.1, 10.2, 10.3, and 10.4 combine custom glycosylation sites at positions 111, 148, and 198. The IdeS protein was purified and cleavage of monoclonal human IgG1 was monitored by SDS-PAGE for 60 minutes. [Figure 18D]

[0048] Figure 18D shows the DSF analysis of the purified IdeS hyperglycosylated mutant. [Figure 18E]

[0048] Figure 18E shows the melting temperature of the highly glycosylated IdeS variant. [Figure 18F]

[0048] Figure 18F shows the analysis of the purified IdeS variant using Coomassie-stained electrophoresis gel. [Figure 19A]

[0049] Figure 19A shows the custom N-glycosylation site introduced into variant 10, and this variant protein was secreted into the expression medium of transfected Expi293F cells. The upper Coomassie-stained SDS electrophoresis gel (non-reducing conditions) shows cleavage of human IVIG incubated with expression medium. The lower gel shows expression of IdeS protein. [Figure 19B]

[0050] Figure 19B shows purified IdeS variants containing 5-6 N-glycosylation sites incubated with human monoclonal IgG1, and the cleavage products were analyzed by non-reducing SDS-PAGE. [Figure 19C]

[0050] Figure 19C shows a purified IdeS variant containing four N-glycosylation sites incubated with human monoclonal IgG1, and the cleavage products were analyzed by non-reducing SDS-PAGE. [Figure 19D]

[0051] Figure 19D shows the conventional thermal stability of purified IdeS variants with 5-6 N-glycosylation sites on the left, or purified IdeS variants with 4 N-glycosylation sites on the right, using DSF. The control sample is buffer solution only. [Figure 20A]

[0052] Figures 20A-20C show the characteristics of glycosylated IdeS variants. Figure 20A shows the melting temperature of IdeS protein as measured by conventional DSF. [Figure 20B]

[0052] Figure 20B shows purified IdeS protein isolated by SDS-PAGE before (left) and after (right) treatment with PNGase F. The theoretical MW of wild-type IdeS without any glycans attached is 36 kD. [Figure 20C]

[0052] Figure 20C shows 20 μM IgG from various species incubated with 20 nM variant 10.9 at 37°C for up to 60 minutes. The cleavage products were separated by non-reducing SDS-PAGE. [Figure 21A]

[0053] Figure 21A shows the His-tagged variant 10.9 expressed by Expi293F cells and purified by NiNTA affinity chromatography. M, marker; FT, flow-through. The eluted protein has an apparent MW of approximately 50 kD, while the non-glycosylated IdeS has a theoretical MW of 36 kD. [Figure 21B]

[0054] Figure 21B shows the SEC separation of the non-glycosylated variant 10.14 (dashed line) and the highly glycosylated variant 10.9 (solid line) using a Superdex 200 increase 10 / 300 GL column. [Figure 22A]

[0055] Figures 22A–22B show the occlusion of accessible surfaces on a highly glycosylated IdeS variant by glycans. Figure 22A shows the surface representation of variant 10.9 modeled as homogeneous glycosylation by a ternary glycan at four N-glycosylation sites labeled in a box. On the left, the glycan is shown using the Symbolic Nomenclature for Glycans (SNFG). On the right, the van der Waals surface of the glycan is shown on top of the protein surface. [Figure 22B]

[0055] Figure 22B shows the protein surface of variant 10.9, with glycan-occluded amino acids shaded in darker gray. The Fc substrate / product contact surface is based on PDB 8A47 and is enclosed in dashed lines and hatched. [Figure 23]

[0056] Figure 23 shows the decreased recognition of anti-IdeS antibody in ELISA when the IdeS surface epitope is shielded by glycan addition. Goat polyclonal anti-IdeS was used for the capture and detection of IdeS variants with increased N-glycosylation sites in ELISA. Error bars represent technical repeats of N=2. [Figure 24A]

[0057] Figure 24A shows the IdeS concentrations measured by ELISA in serum collected from NZW rabbits administered a 4 mg dose (approximately 1 mg / kg) intravenously. IdeS levels in rabbit serum were below the detection limit for all variants after 24 hours. [Figure 24B]

[0058] Figure 24B shows ELISA measurements of serum IgG in IdeS-treated rabbits. Error bars represent technical repeats of N=2. [Figure 25A]

[0059] Figure 25A shows a structural representation for size comparison of the HSA (surface) at the C-terminus of IdeS (ribbon). The nucleophile C94 is labeled and shown as a sphere, and the glycosylated asparagine of variant 10.1 is labeled and shown as a sphere. [Figure 25B]

[0060] Figure 25B shows Coomassie-stained SDS gels of Expi293F expression medium showing secretory variant 10.14 with and without fusion with HSA (left lane) and with fusion with HSA (right lane). [Figure 25C]

[0061] Figure 25C shows purified variant 10.14 with and without fusion with HSA, incubated with human monoclonal IgG1 (20 μM) for 60 minutes at 37°C. The cleavage products were separated by non-reducing SDS-PAGE. [Figure 25D]

[0062] Figure 25D shows Coomassie-stained SDS gels of expression medium from Expi293F transfected with variant 10.14-HSA having various linking linkers. [Figure 25E]

[0063] Figure 25E shows the cleavage products of monoclonal human IgG1 isolated by non-reducing SDS-PAGE after incubation with the linker variant of variant 10.14-HSA. [Figure 25F]

[0064] Figure 25F shows an ELISA using goat polyclonal anti-IdeS for the capture and detection of variant 10.14-HSA linker variant. [Figure 26A]

[0065] Figure 26A shows the results of intravenous administration of 10.14 4 mg of variant 10.14 with HSA fusion (dotted line) and without HSA fusion (dashed line) to NZW rabbits (approximately 4 kg). Serum protein concentrations were measured by ELISA. N=3 per group, and serum collection was staggered for each rabbit, with N=1 per time point. [Figure 26B]

[0066] Figure 26B shows the measurement of serum IgG by ELISA. Error bars represent technical repeats of N=2. [Figure 27]

[0067] Figure 27 shows the use of goat polyclonal anti-IdeS for the capture and detection of glycosylated IdeS variants fused with HSA in ELISA. Error bars represent technical repeats of N=2. [Figure 28A]

[0068] Figure 28A shows a Coomassie-stained, unreduced SDS gel of human IgG1 (20 μM) cleaved with the IdeS-HSA variant (20 nM) at 37°C. [Figure 28B]

[0069] Figure 28B shows Nano DSF measurements of the change in intrinsic tryptophan fluorescence of IdeS variants upon heating. HSA has a single tryptophan that contributes only slightly to the fluorescence signal. [Figure 29A]

[0070] Figures 29A–29D show the reduced reactivity of IdeS variants to human serum. Figure 29A shows a plot of EC50 values ​​(measured as dilution factors) from ELISA experiments in which plates were directly coated with isoconcentrations of Wt IdeS (produced by Escherichia coli, black), variant 10.2-HSA (dark gray), and variant 10.9 (light gray). Reactivity to pooled IVIG or human serum from five donors was assayed, and error bars indicate 95% confidence intervals. [Figure 29B]

[0070] Figures 29B-29D show competitive ELISA analysis. ELISA plates were coated with Wt IdeS (produced by Escherichia coli), and high-titer serum from donors who had recently recovered from S. pyogenes infection was pre-incubated with Wt IdeS or a variant as a competitor, increasing in concentration. Blocked serum / IVIG was then added to the ELISA plates, and antibody binding was measured. [Figure 29C]

[0070] ELISA plates were coated with Wt IdeS (produced by Escherichia coli), and high-titer serum from healthy donors was pre-incubated with Wt IdeS or a variant as a competitor, increasing in concentration. Blocked serum / IVIG was then added to the ELISA plates, and antibody binding was measured. [Figure 29D]

[0070] ELISA plates were coated with Wt IdeS (produced by Escherichia coli), and pooled IVIG was incubated with Wt IdeS or a variant as a competitor, with increasing concentrations. Blocked serum / IVIG was then added to the ELISA plates, and antibody binding was measured. [Figure 30A]

[0071] Figure 30A shows the IdeS-HSA concentrations measured by ELISA in serum collected from NZW rabbits administered variant 10.2-HSA intravenously at 0.3 mg / kg (outlined square and black line) or 1 mg / kg (filled triangle and gray line). For comparison, data from rabbits administered variant 10.14-HSA (non-glycosylated variant, filled black triangle and black dashed line) are also shown. [Figure 30B]

[0072] Figure 30B shows ELISA measurements of serum IgG in treated rabbits. Error bars represent technical repeats of N=2. Points in time when serum samples were not collected are indicated as undecided / ND. [Figure 30C]

[0073] Figure 30C shows the quantitative IgG levels, calculated as a percentage of baseline for each individual rabbit after administration (approximately 1 mg / kg) of Wt IdeS (produced by Escherichia coli, black line and diamond), variant 10.2-HSA (gray line and triangle), or variant 10.9 (white square and dashed line). [Figure 31A]

[0074] Figures 31A–31D show that antigen-binding fragments are removed within 24 hours after IdeS-catalyzed IgG cleavage in rabbits. Figure 31A shows anti-rabbit Fab Western blots of serum samples taken from rabbits treated with 1 mg / kg variant 10.9. [Figure 31B]

[0074] Figure 31B shows anti-rabbit Fab Western blots of serum samples taken from rabbits treated with 1 mg / kg variant 10.2-HSA. [Figure 31C]

[0074] Figure 31C shows an anti-rabbit Fab Western blot of serum samples taken from rabbits treated with 1 mg / kg of Wt IdeS produced in Expi293F culture. [Figure 31D]

[0074] Figure 31D shows the ELISA analysis of serum samples from rabbits administered with Wt IdeS produced in Expi293F culture. IgG / scIgG levels (light gray) were assayed using anti-rabbit F(ab')2 for capture and anti-rabbit Fc for detection. IgG / scIgG / F(ab')2 levels (dark gray) were assayed by directly coating ELISA plates with serum and detecting them with anti-rabbit F(ab')2. [Figure 32A]

[0075] Figures 32A-32B show the PK of wild-type IdeS and variants in C57Bl / 6 mice administered a single dose of 1 mg / kg of IdeS protein via the tail vein. Serum was collected and analyzed by quantitative ELISA to determine the concentration of IdeS until the level was below the detection limit. Data are mean ± SEM for male mice (6-8 weeks old) with N=3 per time point. Variant 10.9 (solid line and triangle) is compared to Wt Ides (solid line and square, produced by Escherichia coli). Protein treated with Arthrobacter ureafaciens neuraminidase (NA) is shown by the dashed line. [Figure 32B]

[0075] Variant 10.2-HSA (solid line and diamond) is compared with Wt Ides (solid line and square, produced by Escherichia coli (E. coli)). Proteins treated with Arthrobacter ureafaciens neuraminidase (NA) are shown by dashed lines. [Modes for carrying out the invention]

[0032] Detailed explanation

[0076] This application provides a modified IdeS protein that exhibits lower immunogenicity (measured as reduced peptide presentation on HLA-II or shielding of surface epitopes recognized by antibodies), while maintaining or improving IdeS stability and IgG cleavage activity. To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0033]

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

[0034]

[0078] As used herein, the terms “subject” and “patient” refer to an organism treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., mice, monkeys, horses, cattle, pigs, dogs, cats, etc.), and more preferably, humans.

[0035]

[0079] As used herein, the term “pharmaceutical composition” refers to a combination of an active agent (e.g., an IdeS variant) and an inert or active carrier that makes the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use.

[0036]

[0080] As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutically acceptable carriers, such as phosphate-buffered saline, water, emulsions (e.g., oil / water emulsions or water / oil emulsions), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .

[0037]

[0081] The terms “protein” and “polypeptide” are used herein as synonymous and refer to polymers of repeating structural units linked by peptide bonds. Typically, the repeating structural units of a peptide are amino acids, such as native amino acids, non-native amino acids, amino acid analogs, or any combination thereof. In certain embodiments, proteins or polypeptides may undergo post-translational modifications (e.g., glycosylation, phosphorylation, lapidation, acetylation, or conjugation with labeling components).

[0038]

[0082] As used herein, the term "heterogeneous" refers to nucleic acids or polypeptides derived from different genetic sources or species.

[0039]

[0083] The term "sequence identity" refers to the degree of amino acid agreement between two amino acid sequences. When sequence homology is expressed as a percentage (e.g., 85%), this percentage represents the degree of agreement in sequence length compared to several other sequences. To maximize matching, gaps (present in either of the two sequences) are permitted; for example, gap lengths of five or fewer amino acids, or optionally three or fewer amino acids, are commonly used.

[0040]

[0084] The percentage of sequence identity can be any integer between 60% and 100%. Exemplary embodiments, using the program described herein and preferably using BLAST with standard parameters as described below, include at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% compared to a reference sequence. Those skilled in the art will recognize that these values ​​can be appropriately adjusted, taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc., to determine the corresponding identity of proteins encoded by two nucleotide sequences.

[0041]

[0085] For sequence comparison, typically one sequence acts as a reference sequence, against which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into the computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are specified. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence to the reference sequence based on the program parameters.

[0042]

[0086] Suitable algorithms for determining the percentage of 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 analysis is available from the National Center for Biotechnology Information (NCBI) website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W within the query sequence, which, when aligned with words of the same length in the database sequence, match or satisfy a certain positive threshold score T. T is referred to as the neighbor word score threshold (see Altschul et al., above). These first neighbor word hits serve as a seed to initiate a search for longer HSPs containing them. Next, word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. The cumulative score is calculated for nucleotide sequences using parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for mismatched residues; always < 0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of word hits in each direction is stopped when the cumulative alignment score decreases by an amount X from its maximum value, when the cumulative score becomes 0 or less due to the accumulation of one or more negative scoring residue alignments, or when 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, by default, a word size (W) of 28, an expected value (E) of 10, M=1, N=-2, and comparison of both strands.Regarding amino acid sequences, the BLASTP program uses a word size (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix by default (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0043]

[0087] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indicator of the probability that the match between two nucleotide or amino acid sequences will occur by chance. For example, the minimum sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.01, and more preferably about 10. -5 It is less than, and most preferably about 10 -20 If the value is less than the reference sequence, the nucleic acid is considered similar to the reference sequence.

[0044]

[0088] Where used herein, the term “about” means a range of values ​​including a specified value, which a person skilled in the art would reasonably consider to be similar to the specified value. In embodiments, “about” means within a standard deviation using measurements generally accepted in the art. Where used herein, the term “about” refers to a measurable value such as a mass, weight, time, volume, concentration, or percentage, and means to include variations from a specified amount of ±20%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, and ±0.1%, which are suitable for carrying out the methods of the disclosure and / or for using the compositions of the disclosure. Therefore, unless otherwise indicated, the numerical parameters described herein and in the appended claims are approximations that may vary depending on the desired characteristics to be obtained by the subject matter of this disclosure.

[0045]

[0089] As used herein, the term “functional fragment” refers to a portion of a protein or polypeptide that maintains its ability to perform the overall biological function of the protein or polypeptide. For example, the functional fragment of the polypeptide or protein of this application maintains its ability to exert catalytic activity.

[0046]

[0090] As used herein, the terms “mutation,” “modification,” and “substitution” are used synonymously and refer to the change of one amino acid to another in the context of a reference amino acid sequence. Amino acid changes in a reference amino acid sequence can occur at the N-terminal or C-terminal position, or anywhere between these terminal positions. Modifications may be scattered individually among residues in the reference sequence, or they may be scattered within one or more consecutive groups within the reference sequence.

[0047]

[0091] Substitutions can be conservative, but are not necessarily so. Twenty amino acids are commonly found in proteins. These amino acids can be classified into nine classes or groups based on the chemical identification of their side chains. Substitution of one amino acid residue within the same class or group for another is referred to herein as a “conservative” substitution. Conservative amino acid substitutions can frequently occur within proteins without significantly altering the protein’s structure or function. Substitution of one amino acid residue for another amino acid residue in a different class or group is referred to herein as a “non-conservative” substitution. In contrast, non-conservative amino acid substitutions tend to alter the structure and function of proteins.

[0048] [Table 1]

[0049]

[0092] In certain embodiments, conserved amino acid substitutions include any of the following substitutions: glycine (G), alanine (A), isoleucine (I), valine (V), and leucine (L) to any other of these aliphatic amino acids; serine (S) to threonine (T), and vice versa; aspartic acid (D) to glutamic acid (E), and vice versa; glutamine (Q) to asparagine (N), and vice versa; lysine (K) to arginine (R), and vice versa; phenylalanine (F), tyrosine (Y), and tryptophan (W) to any other of these aromatic amino acids; and methionine (M) to cysteine ​​(C), and vice versa. Depending on the environment of a particular amino acid and its role in the three-dimensional structure of a protein, other substitutions may also be considered conserved. For example, glycine (G) and alanine (A) may be as frequently interchangeable as alanine (A) and valine (V). Relatively hydrophobic methionine (M) can frequently be exchanged with leucine and isoleucine, and sometimes even with valine. Lysine (K) and arginine (R) are frequently interchangeable when the charge of the amino acid residue is a key characteristic and the different pK values ​​of these two amino acid residues are not important. Further changes may be considered "conservative" under certain circumstances (see, for example, 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).

[0050]

[0093] As used herein, the term “deletion” refers to the removal of one or more amino acid residues in the context of a reference amino acid sequence. Deletions can occur at the N-terminus, the C-terminus, or anywhere between these terminal locations. Deletions may be scattered individually among residues in the reference sequence, or they may be scattered within one or more consecutive groups in the reference sequence. When occurring at the N-terminus or C-terminus, the deletion of one or more consecutive amino acid residues may also be referred to as a “truncation.”

[0051]

[0094] As used herein, the terms “mutant” and “variant” are used synonymously and refer to a protein or enzyme having one or more mutations and / or deletions in the context of a reference sequence (e.g., a wild-type sequence).

[0052]

[0095] As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide,” or their grammatical equivalents as used herein, mean at least two nucleotides covalently linked to one another. The term “nucleic acid” includes single-stranded, double-stranded, or multi-stranded DNA, RNA, and their analogues (derivatives). Oligonucleotides can be about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 45, or more nucleotides, 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.).

[0053]

[0096] As used herein, the term “vector” refers to a nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector may contain a nucleic acid sequence that enables replication within the host cell, such as an origin of replication. A vector may also contain one or more select marker genes and other genetic elements known in the art. In certain embodiments, a vector is a viral vector, such as a lentiviral vector.

[0054]

[0097] As used herein, the term "N-glycosylation" refers to the attachment of oligosaccharides, sugar molecules known as glycans, to the nitrogen atom of an amino acid in a protein.

[0055]

[0098] As used herein, the term "O-glycosylation" refers to the attachment of a sugar molecule, an oligosaccharide (i.e., a glycan), to the oxygen atom of an amino acid in a protein.

[0056]

[0099] As used herein, the term "sialylation" refers to the enzymatic addition of a neuraminic acid residue.

[0057]

[0100] As used herein, the term "neuraminic acid" refers to a nine-carbon monosaccharide that is a derivative of ketonanose.

[0058]

[0101] As used herein, the term “N-glycan” generally refers to a core structure comprising two N-acetyl-D-glucosamine (GlcNAc) molecules and three mannose molecules. N-glycans can be attached to asparagine (Asn) side chains via N-linked glycosylation initiated by an oligosaccharide transferase complex in the endoplasmic reticulum membrane. The N-glycan is attached to the Asn in the Asn-X-serine / threonine sequence, where X can be any amino acid other than proline. An N-glycan may be represented as (Manα1-6[Manα1-3)]Man(β1-4)GlcNAc(β1-4)GlcNAc(β1-Asn-X-Ser / Thr). N-glycan core structures can be extended by galactosylation, further GlcNAcation, sialylation, fucosylation, or a combination thereof.

[0059]

[0102] As used herein, the term “bibranched” refers to an N-linked glycan containing an N-glycan core (Manα1-6[Manα1-3)]Man(β1-4)GlcNAc(β1-4)GlcNAc(β1-Asn-X-Ser / Thr) extended by two GlcNAc residues linked to C-2 of coremannose α1-3 and mannose α1-6. This core structure can then be extended or modified by various glycan structures.

[0060]

[0103] As used herein, the term “tribranched” refers to an N-linked glycan containing an additional GlcNAc residue attached to either C-4 of coremannose α1-3 or C-6 of coremannose α1-6 of a bibranched core structure. This core structure can then be extended or modified by various glycan structures.

[0061]

[0104] As used herein, the term “quadrilateral” refers to an N-linked glycan containing two additional GlcNAc residues attached to either C-4 of coremannose α1-3 or C-6 of coremannose α1-6 of a bilateral core structure. This core structure can then be extended or modified by various glycan structures.

[0062] I. Modified IdeS protein

[0105] Described herein are, in certain embodiments, modified IdeS proteins that include one or more modifications compared to the wild-type IdeS sequence. Wild-type IdeS with a signal peptide (signal peptide is underlined)

[0063] [ka]

[0064]

[0106] Where used herein, references to amino acid residue positions refer to SEQ ID NO: 1 (i.e., the full-length wild-type IdeS sequence containing the signal peptide) unless otherwise specified. Wild-type IdeS (mature polypeptide)

[0065] [ka]

[0066]

[0107] In some embodiments, the Disclosure provides IdeS variants comprising one or more modifications compared to a wild-type IdeS sequence and being 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 disclosure provides an IdeS variant comprising at least two modifications compared to a wild-type IdeS sequence and being 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 disclosure provides an IdeS variant that is at least 95% identical to SEQ ID NO: 1 and has one or more modifications. In some embodiments, the disclosure provides an IdeS variant that is at least 95% identical to SEQ ID NO: 1 and has at least two modifications.

[0067] IdeS variant

[0108] This specification describes modified IdeS proteins. In some embodiments, the IdeS protein is modified to reduce its presentation on HLA-II. In some embodiments, the IdeS protein is modified (e.g., by introducing one or more glycosylation sites or by fusing to serum albumin) to reduce its recognition by anti-IdeS antibodies. In some embodiments, the IdeS protein is modified (e.g., by fusing to serum albumin) to extend the half-life and / or IgG depletion of the modified IdeS protein. In some embodiments, the IdeS protein is modified in multiple ways.

[0068]

[0109] Described herein is an IdeS variant protein that, in a particular embodiment, includes at least two modifications at positions selected from the group consisting of 68, 75, 166, 187, 213, 236, 277, 302, 303, 306, and 318 of SEQ ID NO: 1.

[0069]

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

[0070]

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

[0071]

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

[0072]

[0113] In some embodiments, the modification at position 187 is a substitution of serine to aspartic acid, or a substitution of serine to glutamic acid.

[0073]

[0114] In some embodiments, the modification at position 213 is the substitution of threonine to glutamic acid.

[0074]

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

[0075]

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

[0076]

[0117] In some embodiments, the modification at position 302 is a substitution of serine to aspartic acid, a substitution of serine to lysine, or a substitution of serine to glutamic acid.

[0077]

[0118] In some embodiments, the modification at position 303 is a substitution of alanine to aspartic acid, a substitution of alanine to asparagine, or a substitution of alanine to glutamine.

[0078]

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

[0079]

[0120] In some embodiments, the modification at position 318 is a substitution of isoleucine with lysine, a substitution of isoleucine with aspartic acid, or a substitution of isoleucine with glycine. In some embodiments, the modification at position 318 is a substitution of isoleucine with lysine. In some embodiments, the modification at position 318 is a substitution of isoleucine with aspartic acid. In some embodiments, the modification at position 318 is a substitution of isoleucine with glycine.

[0080]

[0121] Described herein are, in certain embodiments, IdeS variant proteins that further include a modification at position 308. In some embodiments, the modification at position 308 is an isoleucine to leucine substitution.

[0081]

[0122] In some embodiments, the IdeS variant protein includes substitutions of valine to threonine at position 68, alanine to proline at position 75, threonine to arginine at position 166, serine to aspartic acid at position 187, threonine to glutamic acid at position 213, serine to lysine at position 302, and alanine to aspartic acid at position 303.

[0082]

[0123] In some embodiments, the IdeS variant protein includes a valine-to-threonine substitution at position 68, an alanine-to-proline substitution at position 75, a threonine-to-arginine substitution at position 166, a serine-to-aspartic acid substitution at position 187, a threonine-to-glutamic acid substitution at position 213, a serine-to-glutamic acid substitution at position 302, and an alanine-to-aspartic acid substitution at position 303.

[0083]

[0124] In some embodiments, the IdeS variant protein includes a substitution of valine to threonine at position 68, alanine to proline at position 75, threonine to arginine at position 166, serine to aspartic acid at position 187, threonine to glutamic acid at position 213, serine to lysine at position 302, alanine to aspartic acid at position 303, and valine to threonine at position 306.

[0084]

[0125] In some embodiments, the IdeS variant protein includes a substitution of valine to threonine at position 68, alanine to proline at position 75, threonine to arginine at position 166, serine to aspartic acid at position 187, threonine to glutamic acid at position 213, serine to glutamic acid at position 302, valine to threonine at position 306, and isoleucine to lysine at position 318.

[0085]

[0126] In some embodiments, the IdeS variant protein includes a valine-to-threonine substitution at position 68, an alanine-to-proline substitution at position 75, a threonine-to-arginine substitution at position 166, a serine-to-aspartic acid substitution at position 187, a threonine-to-glutamic acid substitution at position 213, a serine-to-cysteine ​​substitution at position 236, a leucine-to-cysteine ​​substitution at position 277, a serine-to-glutamic acid substitution at position 302, alanine-to-aspartic acid substitution at position 303, and a valine-to-threonine substitution at position 306.

[0086]

[0127] In some embodiments, the IdeS variant protein includes a valine-to-threonine substitution at position 68, an alanine-to-proline substitution at position 75, a threonine-to-arginine substitution at position 166, a serine-to-aspartic acid substitution at position 187, a threonine-to-glutamic acid substitution at position 213, a serine-to-cysteine ​​substitution at position 236, a leucine-to-cysteine ​​substitution at position 277, and an isoleucine-to-leucine substitution at position 308.

[0087]

[0128] Described herein is an IdeS variant protein, in certain embodiments, comprising a substitution of the sequence 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, this IdeS variant protein is DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2, 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: 8 3) The substitution includes a sequence that is 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 a sequence selected from the group consisting of AEPITLIWENYLSDSVSPDRDIR (SEQ ID NO: 84) and QEEIAEGRRNPLRTAEWPMTKSTTDQ (SEQ ID NO: 85). In some embodiments, the IdeS variant protein includes substitutions of sequence DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2 with a 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), which are at least 80% identical.In some embodiments, this IdeS variant protein includes a substitution of the sequence DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) in SEQ ID NO: 2 with the sequence DDYQRNAMEAYAKEVPHQIT (SEQ ID NO: 78).

[0088]

[0129] Described herein is an IdeS variant protein comprising a substitution of sequence in SEQ ID NO: 2 DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) with a sequence selected from the group consisting of SEQ ID NOs. 95-112. In some embodiments, this IdeS variant protein comprises a substitution of sequence in SEQ ID NO: 2 DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) with a sequence selected from the group consisting of SEQ ID NOs. 95-112 that is 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 in SEQ ID NO: 2 DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) with a sequence selected from the group consisting of SEQ ID NOs. 95-112. In some embodiments, the IdeS variant protein includes a substitution of sequence that is at least 80% identical to the sequence of DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2, selected from the group consisting of any one of SEQ ID NOs: 95-112.

[0089]

[0130] Described herein, in certain embodiments, is an IdeS variant protein, which is sequence numbers 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, 17 of SEQ ID NO: 1. This is an IdeS variant protein that includes modifications at positions selected from the group consisting of 5, 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.

[0090]

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

[0091]

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

[0092]

[0133] In some embodiments, the IdeS variant protein includes a serine-to-glutamic acid substitution at position 33.

[0093]

[0134] In some embodiments, the IdeS variant protein includes an isoleucine-to-valine substitution at position 38.

[0094]

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

[0095]

[0136] In some embodiments, the IdeS variant protein includes a valine-to-glutamic acid substitution at position 43.

[0096]

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

[0097]

[0138] In some embodiments, the IdeS variant protein includes a proline-to-glutamic acid substitution at position 45.

[0098]

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

[0099]

[0140] In some embodiments, the IdeS variant protein includes a substitution from lysine to aspartic acid at position 54.

[0100]

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

[0101]

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

[0102]

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

[0103]

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

[0104]

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

[0105]

[0146] In some embodiments, the IdeS variant protein includes an isoleucine-to-methionine substitution at position 82.

[0106]

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

[0107]

[0148] In some embodiments, the IdeS variant protein includes a substitution from glutamine to aspartic acid at position 113.

[0108]

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

[0109]

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

[0110]

[0151] In some embodiments, the IdeS variant protein includes an isoleucine-to-methionine substitution at position 127.

[0111]

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

[0112]

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

[0113]

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

[0114]

[0155] In some embodiments, the IdeS variant protein includes a substitution from glutamine to aspartic acid at position 133.

[0115]

[0156] In some embodiments, the IdeS variant protein includes a substitution of leucine to asparagine or arginine at position 148. In some embodiments, the IdeS variant protein includes a substitution of leucine to asparagine at position 148. In some embodiments, the IdeS variant protein includes a substitution of leucine to arginine at position 148.

[0116]

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

[0117]

[0158] In some embodiments, the IdeS variant protein includes a substitution of glutamic acid to aspartic acid at position 154.

[0118]

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

[0119]

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

[0120]

[0161] In some embodiments, the IdeS variant protein includes a histidine to aspartic acid or proline substitution at position 168. In some embodiments, the IdeS variant protein includes a histidine to aspartic acid substitution at position 168. In some embodiments, the IdeS variant protein includes a histidine to proline substitution at position 168.

[0121]

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

[0122]

[0163] In some embodiments, the IdeS variant protein includes a leucine-to-glycine or methionine substitution at position 188. In some embodiments, the IdeS variant protein includes a leucine-to-glycine substitution at position 188. In some embodiments, the IdeS variant protein includes a leucine-to-methionine substitution at position 188.

[0123]

[0164] In some embodiments, the IdeS variant protein includes a proline-to-aspartic acid substitution at position 195.

[0124]

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

[0125]

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

[0126]

[0167] In some embodiments, the IdeS variant protein includes a serine-to-aspartic acid substitution at position 218.

[0127]

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

[0128]

[0169] In some embodiments, the IdeS variant protein includes a leucine-to-glutamine substitution at position 220.

[0129]

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

[0130]

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

[0131]

[0172] In some embodiments, the IdeS variant protein includes a substitution from lysine to aspartic acid at position 241.

[0132]

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

[0133]

[0174] In some embodiments, the IdeS variant protein includes a substitution of glutamic acid to aspartic acid at position 245.

[0134]

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

[0135]

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

[0136]

[0177] In some embodiments, the IdeS variant protein includes a serine-to-aspartic acid substitution at position 273.

[0137]

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

[0138]

[0179] In some embodiments, the IdeS variant protein includes a substitution of lysine to aspartic acid or threonine at position 278. In some embodiments, the IdeS variant protein includes a substitution of lysine to aspartic acid at position 278. In some embodiments, the IdeS variant protein includes a substitution of lysine to threonine at position 278.

[0139]

[0180] In some embodiments, the IdeS variant protein includes a substitution of asparagine to aspartic acid, glycine, or glutamine at position 288. In some embodiments, the IdeS variant protein includes a substitution of asparagine to aspartic acid at position 288. In some embodiments, the IdeS variant protein includes a substitution of asparagine to glycine at position 288. In some embodiments, the IdeS variant protein includes a substitution of asparagine to glutamine at position 288.

[0140]

[0181] In some embodiments, the IdeS variant protein includes an alanine-to-proline substitution at position 289.

[0141]

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

[0142]

[0183] In some embodiments, the IdeS variant protein includes a substitution of glycine to aspartic acid at position 299.

[0143]

[0184] In some embodiments, the IdeS variant protein includes a valine-to-glutamic acid substitution at position 300.

[0144]

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

[0145]

[0186] In some embodiments, the IdeS variant protein includes an isoleucine-to-proline substitution at position 313.

[0146]

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

[0147]

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

[0148]

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

[0149]

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

[0150]

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

[0151]

[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 proteins listed in Table 1A. In some embodiments, this IdeS variant protein contains an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 3-75 and 86-94. In some embodiments, this IdeS variant protein contains an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs. 3-75 and 86-94. In some embodiments, this IdeS variant protein contains an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 3-75 and 86-94. In some embodiments, this IdeS variant protein contains an amino acid sequence that is at least 96% identical to one of SEQ ID NOs: 3-75 and 86-94. In some embodiments, this IdeS variant protein contains an amino acid sequence that is at least 97% identical to one of SEQ ID NOs: 3-75 and 86-94. In some embodiments, this IdeS variant protein contains an amino acid sequence that is at least 98% identical to one of SEQ ID NOs: 3-75 and 86-94. In some embodiments, this IdeS variant protein contains an amino acid sequence that is at least 99% identical to one of SEQ ID NOs: 3-75 and 86-94. In some embodiments, this IdeS variant protein contains an amino acid sequence that is 100% identical to one of SEQ ID NOs: 3-75 and 86-94.

[0152] [Table 2]

[0153] Table 3

[0154] Table 4

[0155] Table 5

[0156] Table 6

[0157] Table 7

[0158] Table 8

[0159] Table 9

[0160] Table 10

[0161] Table 11

[0162] Table 12

[0163] Table 13

[0164]

[0193] Described herein are, in some embodiments, IdeS variant proteins comprising the amino acid sequences described in any one of SEQ ID NOs: 3-75 and 86-94. In some embodiments, this IdeS variant protein comprises sequences that are 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 sequences described in any one of SEQ ID NOs: 3-75 and 86-94. In some embodiments, this IdeS variant protein comprises sequences that are at least 95% identical to the amino acid sequences described in any one of SEQ ID NOs: 3-75 and 86-94, and have one or more modifications to these enumerated amino acid sequences.

[0165]

[0194] Described herein are, in some embodiments, IdeS variant proteins comprising the amino acid sequence described in SEQ ID NO: 10. In some embodiments, this IdeS variant protein comprises a sequence that is 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 described in SEQ ID NO: 10. In some embodiments, this IdeS variant protein comprises a sequence that is at least 95% identical to the amino acid sequence described in SEQ ID NO: 10 and has one or more modifications.

[0166]

[0195] Described herein are, in some embodiments, IdeS variant proteins comprising the amino acid sequence described in SEQ ID NO: 11. In some embodiments, this IdeS variant protein comprises a sequence that is 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 described in SEQ ID NO: 11. In some embodiments, this IdeS variant protein comprises a sequence that is at least 95% identical to the amino acid sequence described in SEQ ID NO: 11 and has one or more modifications.

[0167]

[0196] Described herein are, in some embodiments, IdeS variant proteins comprising the amino acid sequence described in SEQ ID NO: 12. In some embodiments, this IdeS variant protein comprises a sequence that is 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 described in SEQ ID NO: 12. In some embodiments, this IdeS variant protein comprises a sequence that is at least 95% identical to the amino acid sequence described in SEQ ID NO: 12 and has one or more modifications.

[0168]

[0197] Described herein are, in some embodiments, IdeS variant proteins comprising the amino acid sequence described in SEQ ID NO: 13. In some embodiments, this IdeS variant protein comprises a sequence that is 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 described in SEQ ID NO: 13. In some embodiments, this IdeS variant protein comprises a sequence that is at least 95% identical to the amino acid sequence described in SEQ ID NO: 13 and has one or more modifications.

[0169]

[0198] Further described herein are IdeS variant proteins comprising one or more glycosylation modifications in some embodiments. 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, this IdeS variant protein contains an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 117-153. In some embodiments, this IdeS variant protein contains an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs. 117-153. In some embodiments, this IdeS variant protein contains an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs. 117-153. In some embodiments, this IdeS variant protein contains an amino acid sequence that is at least 96% identical to any one of SEQ ID NOs. 117-153. In some embodiments, this IdeS variant protein contains an amino acid sequence that is at least 97% identical to any one of SEQ ID NOs. 117-153. In some embodiments, this IdeS variant protein contains an amino acid sequence that is at least 98% identical to any one of SEQ ID NOs. 117-153. In some embodiments, this IdeS variant protein contains an amino acid sequence that is at least 99% identical to any one of SEQ ID NOs. 117-153. In some embodiments, this IdeS variant protein contains an amino acid sequence that is 100% identical to any one of SEQ ID NOs. 117-153.

[0170] [Table 14]

[0171] Table 15

[0172] Table 16

[0173] Table 17

[0174] Table 18

[0175] Table 19

[0176] Table 20

[0177] Table 21

[0178] Table 22

[0179]

[0199] Further described herein are IdeS variant proteins comprising, in some embodiments, at least one modification at a position 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, for introducing a glycosylation site.

[0180]

[0200] In some embodiments, the modification at position 31 is the substitution of serine with asparagine. In some embodiments, the modification at position 37 is the substitution of glutamic acid with asparagine.

[0181]

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

[0182]

[0202] In some embodiments, the modification at position 42 is the substitution of glutamic acid with asparagine.

[0183]

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

[0184]

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

[0185]

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

[0186]

[0206] In some embodiments, the modification at position 113 is the substitution of glutamine with threonine.

[0187]

[0207] In some embodiments, the modification at position 119 is a substitution from glutamic acid to asparagine.

[0188]

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

[0189]

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

[0190]

[0210] In some embodiments, the modification at position 142 is a substitution from aspartic acid to asparagine.

[0191]

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

[0192]

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

[0193]

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

[0194]

[0214] In some embodiments, the modification at position 198 is a substitution from glutamic acid to asparagine.

[0195]

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

[0196]

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

[0197]

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

[0198]

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

[0199]

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

[0200]

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

[0201]

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

[0202]

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

[0203]

[0223] In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 111 and a substitution of glutamine to threonine at position 113.

[0204]

[0224] In some embodiments, the variant protein includes a substitution of glutamic acid to asparagine at position 119 and a substitution of histidine to threonine at position 121.

[0205]

[0225] In some embodiments, the variant protein includes a substitution of aspartic acid to asparagine at position 142 and a substitution of lysine to threonine at position 144.

[0206]

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

[0207]

[0227] In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 311 and a substitution of isoleucine to threonine at position 313.

[0208]

[0228] Further described herein are IdeS variant proteins that, in some embodiments, include at least one modification at a position 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 in order to introduce a glycosylation site.

[0209]

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

[0210]

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

[0211]

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

[0212]

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

[0213]

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

[0214]

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

[0215]

[0235] In some embodiments, the modification at position 123 is a substitution from glutamic acid to asparagine.

[0216]

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

[0217]

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

[0218]

[0238] In some embodiments, the modification at position 128 is a substitution from asparagine to serine.

[0219] <00,00976> In some embodiments, the modification at position 142 is a substitution from aspartic acid to asparagine.

[0220]

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

[0221]

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

[0222]

[0242] In some embodiments, the modification at position 198 is a substitution from glutamic acid to asparagine.

[0223]

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

[0224]

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

[0225]

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

[0226]

[0246] In some embodiments, the modification at position 312 is the substitution of glutamic acid with asparagine.

[0227]

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

[0228]

[0248] In some embodiments, the variant protein includes a substitution of threonine to asparagine at position 47 and a substitution of glutamine to threonine at position 49.

[0229]

[0249] In some embodiments, the variant protein includes a substitution of glutamine to asparagine at position 49 and a substitution of valine to threonine at position 51.

[0230]

[0250] In some embodiments, the variant protein includes a substitution of glutamic acid to asparagine at position 123 and a substitution of glutamine to threonine at position 125.

[0231]

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

[0232]

[0252] In some embodiments, the variant protein includes a serine-to-asparagine substitution at position 273 and a glycine-to-serine substitution at position 275.

[0233]

[0253] In some embodiments, the variant protein includes a substitution of glutamic acid to asparagine at position 312 and a substitution of lysine to serine at position 314.

[0234]

[0254] In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of aspartic acid to asparagine at position 142, and a substitution of lysine to threonine at position 144.

[0235]

[0255] In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, and a substitution of glutamic acid to asparagine at position 198.

[0236]

[0256] In some embodiments, the variant protein includes a substitution of aspartic acid to asparagine at position 142, a substitution of lysine to threonine at position 144, and a substitution of glutamic acid to asparagine at position 198.

[0237]

[0257] In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of aspartic acid to asparagine at position 142, a substitution of lysine to threonine at position 144, and a substitution of glutamic acid to asparagine at position 198.

[0238]

[0258] In some embodiments, the variant protein includes substitutions of glutamine to asparagine at position 49, valine to threonine at position 51, lysine to asparagine at position 111, glutamine to threonine at position 113, lysine to asparagine at position 126, asparagine to serine at position 128, leucine to asparagine at position 148, and glutamic acid to asparagine at position 198.

[0239]

[0259] In some embodiments, the variant protein includes a substitution of glutamine to asparagine at position 49, a substitution of valine to threonine at position 51, a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of leucine to asparagine at position 148, and a substitution of glutamic acid to asparagine at position 198.

[0240]

[0260] In some embodiments, the variant protein includes substitutions of lysine to asparagine at position 111, glutamine to threonine at position 113, lysine to asparagine at position 126, asparagine to serine at position 128, leucine to asparagine at position 148, and glutamic acid to asparagine at position 198.

[0241]

[0261] In some embodiments, the variant protein includes substitutions of glutamine to asparagine at position 49, valine to threonine at position 51, lysine to asparagine at position 111, glutamine to threonine at position 113, lysine to asparagine at position 126, asparagine to serine at position 128, and glutamic acid to asparagine at position 198.

[0242]

[0262] In some embodiments, the variant protein includes substitutions of threonine to asparagine at position 47, glutamine to asparagine at position 49, lysine to asparagine at position 111, glutamine to threonine at position 113, and glutamic acid to asparagine at position 198.

[0243]

[0263] In some embodiments, the variant protein includes a substitution of glutamine to asparagine at position 49, a substitution of valine to threonine at position 51, a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, and a substitution of glutamic acid to asparagine at position 198.

[0244]

[0264] In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of glutamic acid to asparagine at position 123, a substitution of glutamine to threonine at position 125, and a substitution of glutamic acid to asparagine at position 198.

[0245]

[0265] In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of lysine to asparagine at position 126, a substitution of asparagine to serine at position 128, and a substitution of glutamic acid to asparagine at position 198.

[0246]

[0266] In some embodiments, the variant protein includes a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of leucine to asparagine at position 148, and a substitution of glutamic acid to asparagine at position 198.

[0247]

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

[0248]

[0268] In some embodiments, one or more glycans are terminated with galactose, N-acetylgalactosamine, glucose, N-acetylglucosamine, fucose, N-acetylneuraminic acid, mannose, or sialic acid. In some embodiments, this one or more glycans are terminated with galactose. In some embodiments, one or more glycans are terminated with N-acetylgalactosamine. In some embodiments, one or more glycans are terminated with glucose. In some embodiments, one or more glycans are terminated with N-acetylglucosamine. In some embodiments, one or more glycans are terminated with fucose. In some embodiments, one or more glycans are terminated with N-acetylneuraminic acid. In some embodiments, one or more glycans are terminated with mannose. In some embodiments, one or more glycans are terminated with sialic acid.

[0249]

[0269] In some embodiments, one or more glycans are selected from the group consisting of bibranched glycans ending with mannose, bibranched glycans ending with sialic acid, tribranched glycans ending with mannose, tribranched glycans ending with sialic acid, tetrabranched glycans ending with mannose, and tetrabranched glycans ending with sialic acid. In some embodiments, this one or more glycans is a bibranched glycan ending with galactose. In some embodiments, this one or more glycans is a bibranched glycan ending with N-acetylgalactosamine. In some embodiments, this one or more glycans is a bibranched glycan ending with glucose. In some embodiments, this one or more glycans is a bibranched glycan ending with N-acetylglucosamine. In some embodiments, this one or more glycans is a bibranched glycan ending with fucose. In some embodiments, this one or more glycans is a bibranched glycan ending with N-acetylneuraminic acid. In some embodiments, one or more glycans are bibranched glycans terminated with mannose. In some embodiments, one or more glycans are bibranched glycans terminated with sialic acid. In some embodiments, one or more glycans are tribranched glycans terminated with galactose. In some embodiments, one or more glycans are tribranched glycans terminated with N-acetylgalactosamine. In some embodiments, one or more glycans are tribranched glycans terminated with glucose. In some embodiments, one or more glycans are tribranched glycans terminated with N-acetylglucosamine. In some embodiments, one or more glycans are tribranched glycans terminated with fucose. In some embodiments, one or more glycans are tribranched glycans terminated with N-acetylneuraminic acid. In some embodiments, one or more glycans are tribranched glycans terminated with mannose. In some embodiments, one or more of these glycans are tribranched glycans terminated with sialic acid.In some embodiments, one or more glycans are tetrabranched glycans terminated with galactose. In some embodiments, one or more glycans are tetrabranched glycans terminated with N-acetylgalactosamine. In some embodiments, one or more glycans are tetrabranched glycans terminated with glucose. In some embodiments, one or more glycans are tetrabranched glycans terminated with N-acetylglucosamine. In some embodiments, one or more glycans are tetrabranched glycans terminated with fucose. In some embodiments, one or more glycans are tetrabranched glycans terminated with N-acetylneuraminic acid. In some embodiments, one or more glycans are tetrabranched glycans terminated with mannose and tetrabranched glycans terminated with sialic acid. In some embodiments, one or more glycans are tetrabranched glycans terminated with sialic acid.

[0250]

[0270] In some embodiments, the glycan is selected from the glycans listed in Table 10. In some embodiments, this glycan is 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). The following are selected from the group consisting of , 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).

[0251]

[0271] In some embodiments, one or more glycans are terminated with N-acetylglucosamine (i.e., GlcNAc).

[0252]

[0272] In some embodiments, one or more glycans shield the surface area of ​​the variant protein. In some embodiments, one or more glycans shield at least 10–64% of the total accessible surface area of ​​the variant protein. In some embodiments, one or more glycans shield at least 20–50% of the total accessible surface area of ​​the variant protein. In some embodiments, one or more glycans shield at least 25–45% of the total accessible surface area of ​​the variant protein. In some embodiments, one or more glycans shield at least 20–35% of the total accessible surface area of ​​the variant protein. In some embodiments, one or more glycans shield at least 25% of the total accessible surface area of ​​the variant protein. In some embodiments, one or more glycans shield at least 30% of the total accessible surface area of ​​the variant protein. In some embodiments, one or more glycans shield at least 35% of the total accessible surface area of ​​the variant protein. In some embodiments, one or more glycans shield at least 40% of the total accessible surface area of ​​the variant protein. In some embodiments, one or more glycans shield at least 45% of the total accessible surface area of ​​the variant protein.

[0253]

[0273] In some embodiments, IdeS variant proteins containing one or more glycans exhibit reduced immunogenicity compared to non-glycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS. In some embodiments, IdeS variant proteins containing one or more glycans exhibit increased serum stability compared to non-glycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS. In some embodiments, IdeS variant proteins containing one or more glycans persist longer in serum compared to non-glycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS. In some embodiments, IdeS variant proteins containing one or more glycans have approximately twice the serum half-life compared to non-glycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS. In some embodiments, IdeS variant proteins containing one or more glycans exhibit increased size (i.e., molecular weight (MW)) compared to non-glycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS. In some embodiments, IdeS variant proteins containing one or more glycans exhibit reduced renal excretion compared to non-glycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS. In some embodiments, the reduced renal excretion is attributed to the increased size (i.e., MW) of the variant protein compared to non-glycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS. In some embodiments, IdeS variant proteins containing one or more glycans exhibit a longer duration of IgS depletion compared to non-glycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS. In some embodiments, this longer duration of IgS depletion correlates with a greater degree of IdeS variant protein glycosylation.

[0254]

[0274] Further described herein are, in some embodiments, IdeS variant proteins fused with human serum albumin (HSA). In some embodiments, the HSA-fused variant protein contains one or more glycans. In some embodiments, the HSA-fused variant protein has a reduced EC50 value compared to the polyclonal anti-IdeS reactivity EC50 values ​​of non-glycosylated variant proteins, variant proteins with fewer glycans, or wild-type IdeS. In some embodiments, the EC50 value of the HSA-fused variant protein is about half that of the non-glycosylated variant protein, the variant protein with fewer glycans, or wild-type IdeS. In some embodiments, the HSA-fused IdeS variant protein exhibits a longer duration of IgS depletion compared to the non-glycosylated variant protein, the variant protein with fewer glycans, or wild-type IdeS. In some embodiments, the IdeS variant protein fused with HSA has a serum half-life approximately 2, 3, 4, 5, 6, or 7 times longer than that of the non-glycosylated variant protein, the variant protein with fewer glycans, or the wild-type IdeS.

[0255] IdeS polypeptide

[0275] Described herein are polypeptides comprising, in certain embodiments, an IdeS protein conjugated to human serum albumin and having at least 90% sequence identity to any one of SEQ ID NOs: 1-75. In some embodiments, this human serum albumin is conjugated to the N-terminus of the IdeS protein. In some embodiments, this human serum albumin is conjugated to the C-terminus of the IdeS protein.

[0256]

[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 contains an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs. 154-161. In some embodiments, the IdeS polypeptide contains an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs. 154-161. In some embodiments, the IdeS polypeptide contains an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs. 154-161. In some embodiments, the IdeS polypeptide contains an amino acid sequence that is at least 96% identical to any one of SEQ ID NOs. 154-161. In some embodiments, the IdeS polypeptide contains an amino acid sequence that is at least 97% identical to any one of SEQ ID NOs. 154-161. In some embodiments, the IdeS polypeptide contains an amino acid sequence that is at least 98% identical to any one of SEQ ID NOs. 154-161. In some embodiments, the IdeS polypeptide contains an amino acid sequence that is at least 99% identical to any one of SEQ ID NOs. 154-161. In some embodiments, the IdeS polypeptide contains an amino acid sequence that is 100% identical to any one of SEQ ID NOs. 154-161.

[0257] [Table 23]

[0258] [Table 24]

[0259] [Table 25]

[0260] [Table 26]

[0261] [Table 27]

[0262]

[0277] In some embodiments, the IdeS protein is coupled to human serum albumin using a linker. In some embodiments, this linker is a peptide linker. In some embodiments, this peptide linker includes a flexible peptide linker. In some embodiments, this peptide linker includes a rigid peptide linker. In some embodiments, this peptide linker includes a cleavable peptide linker.

[0263]

[0278] In some embodiments, the linker contains at least 2 to about 30 amino acids. In some embodiments, the linker contains about 2 to about 35 amino acids, about 2 to about 40 amino acids, about 2 to about 35 amino acids, about 2 to about 35 amino acids, about 2 to about 30 amino acids, about 2 to about 25 amino acids, about 2 to about 20 amino acids, about 2 to about 15 amino acids, about 2 to about 10 amino acids, about 5 to about 30 amino 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.

[0264]

[0279] In some embodiments, the linker includes an array selected from the group consisting of (GS)n (sequence number 162), (G2S)n (sequence number 163), (G3S)n (sequence number 164), (G4S)n (sequence number 165), and (G)n (sequence number 166), where n is an integer from 1 to 20. In some embodiments, n is an integer from 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 4 to 20, 6 to 20, 8 to 20, 10 to 20, 12 to 20, 14 to 20, 16 to 20, or 18 to 20.

[0265]

[0280] In some embodiments, the linker includes an array selected from the group consisting of (GGSGGD)n (sequence number 167) or (GGSGGE)n (sequence number 168), where n is an integer from 1 to 6.

[0266]

[0281] In some embodiments, the linker includes an array selected from the group consisting of (GGGSGSGGGGS)n (sequence number 169) and (GGGGGPGGGGP)n (sequence number 170), where n is an integer from 1 to 3.

[0267]

[0282] In some embodiments, the linker includes a sequence selected from the group consisting of (GX)n, (GGX)n, (GGGX)n, (GGGGX)n (SEQ ID NO: 171), and (GzX)n, where z is 1 to 20. In some embodiments, z is 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 4 to 20, 6 to 20, 8 to 20, 10 to 20, 12 to 20, 14 to 20, 16 to 20, or 18 to 20. In some embodiments, X is serine, aspartic acid, glutamic acid, threonine, or proline.

[0268]

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

[0269]

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

[0270]

[0285] Further described herein are polypeptides comprising the IdeS variant protein described herein, conjugated to human serum albumin, in certain embodiments. In some embodiments, the IdeS variant protein includes at least two modifications at 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 at 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 at positions selected from the group consisting of 47, 49, 51, 123, 125, 126, and 128 of SEQ ID NO: 12, in order to introduce glycosylation sites.

[0271]

[0286] Further described herein are polypeptides comprising the IdeS variant protein described herein, conjugated to human serum albumin, in certain embodiments. In some embodiments, this IdeS variant protein is modified to introduce glycosylation sites by at least two modifications at positions selected from the group consisting of 68, 75, 166, 187, 213, 236, 277, 302, 303, 306, and 318 of SEQ ID NO: 1, as well as 31, 37, 39, 42, 74, 76, 111, 113, 119, 121, 130, 142, and 144 of SEQ ID NO: 1. , including at least one modification at a position selected from the group consisting of 147, 148, 198, 233, 244, 246, 311, 313, and 319, or at least one modification at a position 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 Sequence ID No. 12.

[0272]

[0287] In some embodiments, the IdeS variant protein or IdeS polypeptide described herein, when expressed by cells, exhibits elevated expression levels compared to the expression level of wild-type IdeS in the same cells. In some embodiments, the expression level of this IdeS variant protein or IdeS polypeptide is 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% higher compared to the expression level of wild-type IdeS in the same cells. In some embodiments, when this IdeS variant protein or IdeS polypeptide is expressed by cells, the expression level is approximately 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%, 1 High in the ranges of 0-300%, 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%.

[0273]

[0288] In some embodiments, the expression level of the IdeS variant protein or IdeS polypeptide described herein in a cell is approximately ±1-20%, ±2-20%, ±4-20%, ±6-20%, ±8-20%, ±10-20%, ±12-20%, ±14-20%, ±16-20%, ±18-20%, ±18-20%, ±18-18%, ±2-18%, ±4-18%, ±6-18%, ±8-18%, ±10-18%, ±12-18%, ±14-18%, ±16-18%, ±16-18%, ±16-16%, ±2-16%, ±4- 16%, ±6-16%, ±8-16%, ±10-16%, ±12-16%, ±14-16%, ±1-14%, ±2-14%, ±4-14%, ±6-14%, ±8-14%, ±10-14%, ±12-14%, ±1-12%, ±2-12%, ±4-12%, ±6-1 The percentages are 2%, ±8-12%, ±10-12%, ±1-10%, ±2-10%, ±4-10%, ±6-10%, ±8-10%, ±1-8%, ±2-8%, ±4-8%, ±6-8%, ±1-6%, ±2-6%, ±4-6%, ±1-4%, ±2-4%, or approximately 1-2%.

[0274]

[0289] In some embodiments, the IdeS variant protein or IdeS polypeptide described herein exhibits increased stability compared to the stability of wild-type IdeS. In some embodiments, this IdeS variant protein or IdeS polypeptide is 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% higher than the stability of wild-type IdeS. In some embodiments, the stability of this IdeS variant protein or IdeS polypeptide is approximately 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%, and 20-300% compared to the stability of wild-type IdeS. The increase is high in the range of 0%, 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%.

[0275]

[0290] In some embodiments, the IdeS variant protein or IdeS polypeptide described herein exhibits higher enzymatic activity compared to that of wild-type IdeS. In some embodiments, this enzymatic activity is immunoglobulin cleavage (e.g., proteolytic activity). In some embodiments, this IdeS variant protein or IdeS polypeptide exhibits 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% higher enzymatic activity compared to that of wild-type IdeS. In some embodiments, the IdeS protein or IdeS polypeptide exhibits enzymatic activity of approximately 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%, and 20-300% compared to the enzymatic activity of wild-type IdeS. High in the range of 00%, 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%.

[0276]

[0291] In some embodiments, the IdeS variant protein or IdeS polypeptide described herein exhibits reduced presentation of epitopes on human leukocyte antigens (HLA) compared to the wild-type IdeS protein. In some embodiments, the presentation of epitopes on human leukocyte antigens (HLA) is reduced by 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% compared to the wild-type IdeS protein. In some embodiments, the IdeS variant protein or IdeS polypeptide exhibits epitope presentation on human leukocyte antigens (HLA) at approximately 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%, and 10- The reductions are in the range of 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 some embodiments, 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.

[0277]

[0292] In some embodiments, the binding of IdeS variant proteins or IdeS polypeptides to HLA-II alleles is measured. In some embodiments, the binding of IdeS variant proteins or IdeS polypeptides to HLA-II alleles is determined as a probability ranking against a reference set (e.g., 100,000 peptides). This is referred to as the rank-percentage. For example, a rank-percentage of 5% indicates that the peptide is predicted to bind to a particular HLA with a higher confidence than 95% of the peptides in the reference set.

[0278]

[0293] In some embodiments, the IdeS variant protein or IdeS polypeptide described herein has reduced immunogenicity compared to wild-type IdeS protein. In some embodiments, the immunogenicity of this IdeS variant protein or IdeS polypeptide is reduced by 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% compared to wild-type IdeS protein. In some embodiments, the immunogenicity of this IdeS variant protein or IdeS polypeptide is reduced by about 5–95%, 10–90%, 20–80%, 30–70%, 40–60%, 50–95%, 65–85%, or 75–95% compared to wild-type IdeS protein.

[0279]

[0294] In some embodiments, the immunogenicity of an IdeS variant protein or IdeS polypeptide is calculated based on the number of HLA-II alleles presenting the peptide epitope. This may be referred to as the immunogenicity score. For example, a peptide in IdeS is predicted to be presented on an HLA-II allele if it is located within the 10% of sequences with the highest affinity. In other words, a rank percentage of 10% means that the peptide is predicted to fall within the 10% of the highest affinity peptides from a reference set of 100,000. The immunogenicity score of the peptide is then the number of HLA-II alleles to which the peptide is expected to bind. In some embodiments, the number of HLA-II alleles presenting the peptide derived from the IdeS variant protein is reduced compared to the number of HLA-II alleles presenting the equivalent peptide derived from the IdeS protein, including SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the number of HLA-II alleles presenting peptides derived from the IdeS variant protein is reduced compared to the number of HLA-II alleles presenting equivalent peptides derived from the IdeS protein, including wild-type IdeS. In some embodiments, the IdeS variant protein has a reduced immunogenicity score of more than 0 compared to SEQ ID NO: 1 or SEQ ID NO: 2.

[0280]

[0295] In some embodiments, the IdeS variant protein or IdeS polypeptide described herein has reduced antibody binding compared to the wild-type IdeS protein. In some embodiments, the antibody binding of this IdeS variant protein or IdeS polypeptide is reduced by 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% compared to the wild-type IdeS protein. In some embodiments, the antibody binding of this IdeS variant protein or IdeS polypeptide is reduced by about 5–95%, 10–90%, 20–80%, 30–70%, 40–60%, 50–95%, 65–85%, or 75–95% compared to the wild-type IdeS protein. In some embodiments, the binding affinity of this IdeS variant protein or IdeS polypeptide to an antibody is determined using ELISA.

[0281]

[0296] In some embodiments, the IdeS variant protein or IdeS polypeptide described herein contains one or more glycosylation (e.g., N-glycosylation) modifications or is fused with albumin, resulting in reduced antibody binding compared to wild-type IdeS protein. In some embodiments, this IdeS variant protein or IdeS polypeptide contains one or more glycosylation (e.g., N-glycosylation) modifications or is fused with albumin, resulting in reduced antibody binding by 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% compared to wild-type IdeS protein. In some embodiments, the IdeS variant protein or IdeS polypeptide contains one or more glycosylation (e.g., N-glycosylation) modifications or is fused with albumin, resulting in a reduction in antibody binding compared to wild-type IdeS protein by approximately 5–95%, 10–90%, 20–80%, 30–70%, 40–60%, 50–95%, 65–85%, or 75–95%.

[0282] II. Pharmaceutical Compositions

[0297] The IdeS variant protein of this disclosure may be used in the manufacture of pharmaceutical compositions. In certain embodiments, the pharmaceutical compositions disclosed herein include the IdeS variant protein of this disclosure, a pharmaceutically acceptable carrier, and optionally other agents, pharmaceuticals, stabilizers, buffers, carriers, adjuvants, diluents, etc. "pharmaceutically acceptable" means a substance that is not toxic or otherwise undesirable, i.e., the substance can be administered to a subject without causing any undesirable biological effects.

[0283]

[0298] In certain embodiments, the pharmaceutical composition may include sterile aqueous and non-aqueous injectable solutions, which are optionally isotonic with the blood of the subject to which the pharmaceutical composition is delivered. The pharmaceutical composition may also include antioxidants, buffers, bacteriostatic agents, and solutes, thereby making the composition isotonic with the blood of the subject to which it is intended to be administered. Sterile aqueous and non-aqueous suspensions, solutions, and emulsions may include suspending agents and thickeners. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, e.g., olive oil, and organic esters for injection, e.g., ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions (including physiological saline and buffering media). In certain embodiments, the pharmaceutical composition may include pharmaceutically acceptable excipients, which may include sodium chloride solution, ringer's dextrose, dextrose, and sodium chloride, Ringer's lactate solution, or fixing oils. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0284]

[0299] In certain embodiments, pharmaceutical compositions may be provided in unit dose or multi-dose containers (e.g., sealed ampoules and vials) and may be stored under freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier (e.g., physiological saline or water for injection) immediately before use.

[0285]

[0300] In certain embodiments, the pharmaceutical compositions disclosed herein may be formulated for intravenous, intramuscular, subcutaneous, or intraperitoneal administration. For example, the pharmaceutical compositions disclosed herein may be administered intravenously to the target subject.

[0286] III. Nucleic acids and vectors

[0301] Effective concentrations of the IdeS variant proteins of this disclosure can be achieved by transient or stable expression of the nucleic acid molecule encoding the IdeS variant protein. For example, in certain embodiments, the nucleic acid molecule encoding the IdeS variant protein of this disclosure can be incorporated into a vector and introduced into a cell. In certain embodiments, the cell has one or more nucleic acids encoding the IdeS variant proteins described herein.

[0287]

[0302] In some embodiments, the nucleic acid is DNA, such as linear DNA, plasmid DNA, or minicircle DNA. In some embodiments, the nucleic acid is RNA, such as mRNA. In some embodiments, the nucleic acid is provided as a vector. In some embodiments, the nucleic acid is provided as a plasmid (e.g., a circular DNA molecule capable of autonomous replication within a cell), a cosmid (e.g., a pWE or sCos vector), an artificial chromosome, a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a P1-derived artificial chromosome (PAC), a phagemide, a phage derivative, a bacmid, or a virus. In some embodiments, this nucleic acid is 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 Provided in the form of a vector selected from a list consisting of DNA, pCambia2301, pTYB21, pKLAC2, pAc5.1 / V5-His A, and pDEST8.

[0288]

[0303] In some embodiments, the nucleic acid includes a promoter. In some embodiments, the promoter is selected from the group consisting of minipromoters, inductive promoters, constitutive promoters, 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.

[0289]

[0304] In some embodiments, the nucleic acid is provided by a virus. In some embodiments, this virus is an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anerovirus, bocavirus, vacciniavirus, or retrovirus. In some embodiments, this virus is an alphavirus. In some embodiments, this virus is a parvovirus. In some embodiments, this virus is an adenovirus. In some embodiments, this virus is an AAV. In some embodiments, this virus is a baculovirus. In some embodiments, this virus is a dengue virus. In some embodiments, this virus is a lentivirus. In some embodiments, this virus is a herpesvirus. In some embodiments, this virus is a poxvirus. In some embodiments, this virus is an anerovirus. In some embodiments, this virus is a bocavirus. In some embodiments, this virus is a vacciniavirus. In some embodiments, this virus is a retrovirus.

[0290]

[0305] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh1 0, 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, A The herpesviruses are AV-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 derivatives thereof. In some embodiments, the herpesviruses are HSV1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.

[0291]

[0306] In some embodiments, the nucleic acid is provided in a non-viral delivery system. In some embodiments, the nucleic acid is contained within a liposome. In some embodiments, the nucleic acid is associated with a lipid. In some embodiments, the lipid-associated nucleic acid is encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule associated with both the liposome and the nucleic acid, captured within a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in or complexed with a micelle, or otherwise associated with a lipid. In some embodiments, the nucleic acid is provided as lipid nanoparticles (LNPs).

[0292]

[0307] Vectors can be introduced into cells by various methods, including transformation, transfection, direct uptake, projectile bombardment, and encapsulation of the vector in liposomes or nanoparticles. Examples of preferred methods for transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. Such methods are described in more detail, for example, 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)).

[0293]

[0308] Various vectors have been developed for the delivery and expression of polynucleotides encoding exogenous polypeptides in mammalian cells. Examples of expression vectors are disclosed, for example, in International Publication No. 1994 / 011026, which is incorporated herein by reference. Expression vectors used in the compositions and methods described herein include a polynucleotide sequence encoding the IdeS variant protein of this disclosure, as well as additional sequence elements used, for example, for polypeptide expression and / or integration of the polynucleotide sequence into the genome of a mammalian cell. Certain vectors that may be used include plasmids containing regulatory sequences (e.g., promoter and enhancer regions) that induce gene transcription. Other useful vectors include polynucleotide sequences that increase translation rate or improve mRNA stability or nuclear export. Examples of these sequence elements include 5'UTR and 3'UTR regions, internal ribosome entry sites (IRES), and poly(A) for inducing efficient transcription of the gene carried by the expression vector. Expression vectors suitable for use in the compositions and methods described herein may also include polynucleotides encoding markers for selecting cells containing such vectors. Suitable markers include genes encoding green fluorescent protein or genes encoding antibiotic resistance.

[0294] III.How to use

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

[0295]

[0310] In some embodiments, gene editing methods include a nuclease for targeting a specific target nucleic acid sequence. Examples of nuclease types include, but are not limited to, activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), meganucleases, Argonauts, and clustered regularly interspaced short palindromic repeats (CRISPR)-related (Cas) proteins. In some cases, the nuclease may be wild-type, genetically modified, or recombinant. In some embodiments, the gene editing system includes CRISPR / Cas9.

[0296]

[0311] In some embodiments, the methods described herein involve administering an effective amount of an IdeS variant protein, a polypeptide, or a pharmaceutical product as described herein, for treating autoantibody-mediated autoimmune diseases. [Examples]

[0297]

[0312] The generally described disclosure will be more readily understood by referring to the following examples, which are included solely to illustrate certain aspects and embodiments of the disclosure and are not intended to limit the scope of the disclosure in any way.

[0298] Example 1: Bacterial protease with reduced immunogenicity for degrading immunoglobulin G

[0313] This example describes the generation of immunogenic IdeS variant proteins for the degradation of immunoglobulin G (IgG).

[0299]

[0314] Screening for IdeS variant expression and activity

[0315] To rapidly screen IdeS variants without purification, IdeS was expressed in Expi293F cells, a suspension medium derivative of HEL293 that yields high-yielding secreted protein to the culture medium, along with a potent signal peptide. Using this format, the proteolytic activity of IdeS variants can be rapidly screened directly from the expression medium without purification. IdeS produced in bacteria is not glycosylated and is shown as a sharp peak by size exclusion chromatography (SEC), as shown in Figures 1A-1B. In comparison, IdeS purified from Expi293F cultures is shown as a broader peak with a higher molecular weight (MW) by SEC, and its electrophoretic mobility increases after PNGase F treatment, as shown in Figures 1A-1C, which is consistent with the protein being glycosylated. The IdeS sequence possesses three consensus motifs for N-glycosylation in mammalian cells at positions 61, 288, and 336 (a fourth potential N-glycosylation site is added via a C-terminal linker and purification tag). Based on changes in electrophoretic mobility after mutation to the potential N-glycosylation site, N61 was identified as the glycosylation site (Figure 1E). The protein purified from Expi293F culture exhibited catalytic activity similar to that of non-glycosylated IdeS derived from bacteria (Figures 1F-1G). From these results, it was concluded that the mammalian expression system is suitable for rapidly determining the effects of modifications, despite producing IdeS with non-natural glycosylation at N61.

[0300]

[0316] Identification of HAL-II epitopes in IdeS using MPP

[0317] Monocyte-derived dendritic cells (DCs) cultured from 10 healthy donors were pulsed with purified IdeS, and after 24 hours, peptide-HLA-II complexes were immunoprecipitated using a pan-HLA-DR antibody. Peptides were analyzed by LC-MS / MS, and the data were processed to identify peptides obtained from IdeS treatment. Numerous peptides bound to HLA-II alleles across multiple donors (Figure 2A). Overall, prominent clusters of eight antigenic peptides were found throughout the IdeS sequence, as follows: approximately residues 30–50 (bound to HLA-II alleles of 5 out of 10 donors), approximately residues 50–90 (bound to HLA-II alleles of 3 donors), approximately residues 110–130 (2 donors), approximately residues 150–170 (7 donors), approximately residues 180–200 (4 donors), approximately residues 210–220 (4 donors), approximately residues 250–270 (6 donors), and approximately residues 280–320 (8 donors). These are referred to as clusters 1–8, respectively. Cluster 8 may represent two or more distinct epitopes that overlap or are adjacent to each other. The donors represented a broad set of various HLA-II alleles (Table 2).

[0301] [Table 28]

[0302]

[0318] The positions of the antigen peptide were mapped to the structure of IdeS, as shown in Figures 2B-2C. Most of the residues in cluster 1 are not degraded by electron density and are likely disordered. Clusters 2-8 cover the protein surface and hydrophobic interior. Residue interactions occur frequently between epitope clusters, with residues in clusters 2, 7, and 8 forming active sites. Furthermore, residues in clusters 2, 4, 6, 7, and 8 contribute to the binding interface with the IgG substrate, based on modeling using the crystal structure of IgG1 bound to S. pyogenes Mac-2. Modifications to reduce the immunogenicity of a protein must carefully consider the structural status of each epitope to minimize harmful perturbations, but epistasis effects are expected to be particularly pronounced when modifications are combined between clusters that form close atomic interactions.

[0303]

[0319] To complement the MAPP results, King et al. discussed a representative set of HLA-II alleles. 13 Epitopes were computationally predicted using the following methodology. IdeS sequences were scanned for 15-mer peptides, and their predicted immunogenicity scores were based on the calculated HLA-II affinity (e.g., a rank percentage of 10% means the peptide is predicted to fall within the top 10% of the most affinity peptides in a reference set of 100,000) and the number of HLA-II alleles to which the peptide is predicted to bind. A looser rank percentage threshold predicts more IdeS sequences as immunogenic, while a stricter threshold predicts the most likely HLA-II epitopes. Using a 10% threshold, the in silico predictions qualitatively agree with experimental MAPP data, although there are some differences (Figure 3). Therefore, the immunogenicity scores have sufficient predictive power to identify mutations that may reduce peptide presentation on HLA-II.

[0304]

[0320] Removal of HLA-II epitopes

[0321] A single modification located within an epitope cluster identified by MAPP, which was computationally predicted to reduce immunogenicity, was introduced into IdeS and expressed in Expi293F cells. Modification within the active site was avoided, and based on modeling, it was predicted that destabilization would be minimized. The expression medium was then screened for proteolytic activity against intravenous immunoglobulin (IVIG; polyclonal immunoglobulin pooled from multiple donors) (Figures 4 and 5). Since IdeS activity in this screening is tested directly from the expression medium without purification, IVIG proteolytic cleavage is a function of both the IdeS variant expression level and its intrinsic catalytic activity. Indeed, activity often correlates with IdeS expression levels based on Coomassie-stained SDS-PAGE analysis of the medium (Figures 4 and 5). 260 single amino acid substitutions were screened at 141 locations and found to have varying levels of expression and catalytic activity (Figures 3-5 and Table 3). Furthermore, to potentially stabilize the protein fold, cysteine ​​substitutions predicted to form disulfides were strategically introduced into IdeS (Figure 6 and Table 4); of the three screened cysteine ​​pairs, one produced a soluble protein in expression medium with activity equivalent to that of wild-type IdeS.

[0305] [Table 29]

[0306] [Table 30]

[0307] [Table 31]

[0308] [Table 32]

[0309] [Table 33]

[0310]

[0322] Subsequently, the modifications were combined to reduce the immunogenicity of multiple HLA-II epitopes within a single IdeS polypeptide (Table 5). This was achieved stepwise by sequentially adding a series of modifications. A reduced-immunogenic IdeS derivative (Variant 77) with six modifications "hitting" five epitope clusters and a putative cysteine ​​disulfide bond pair was purified and characterized. Variant 77 was more active for the initial cleavage of nonspecific human IgG1 monoclonal but slower than wild-type IdeS in forming the second cleavage releasing the F(ab')2 fragment (Figures 7A-7F). Both Variant 77 and wild-type IdeS were selective for human and rabbit IgG over mouse IgG (Figures 7A-7F). However, Variant 77 had reduced thermal stability (melting temperature Tm = 46°C, compared to 53°C for wild-type IdeS) (Figure 10B). Variant 77 was used as an intermediate in the final stage of immunogenicity reduction and stabilization.

[0311] [Table 34]

[0312] [Table 35]

[0313] [Table 36]

[0314] [Table 37]

[0315] [Table 38]

[0316] [Table 39]

[0317] [Table 40]

[0318] [Table 41]

[0319]

[0323] Modifications could not be modeled within epitope cluster 1, which is located in the unstructured region extending from the protease domain to the N-terminus. Deletion of the N-terminal extension was evaluated to remove the cluster 1 epitope, but IdeS expression decreased as the length of the deletion segment increased (Figure 8A and Table 3). Instead of the IdeS N-terminal deletion, chimeras were created by replacing this region with an equivalent N-terminal flanking sequence from a homologous protease or a functionally unknown protein structurally similar to IdeS (Figure 8B and Table 6). Chimeric constructs were screened with diverse N-terminal sequences and found to possess high proteolytic activity (Figure 8A). N-terminal extensions transplanted from proteins in a metagenomics database were selected for incorporation into variant 77 based on favorable immunogenicity scores.

[0320] [Table 42]

[0321]

[0324] The final set of modified variants with reduced immunogenicity was added to variant 77 simultaneously with N-terminal chimerization to create the combined variants IdeS variants 100, 101, 102, 103, 1, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, and 116. After screening the variants in Expi293F expression medium (Figure 9A), variant 1 was selected based on high expression, high proteolytic activity against IVIG, and reduced immunogenicity score. Variant 1 has a chimeric N-terminus, eight substitutions to remove the HLA-II epitope, and introduced pairs of disulfide-bonded cysteine. However, the thermal stability of this derivative was further reduced (Tm=43°C, Figure 9B). A subset of the modified residues was restored to the wild-type identity, and / or compensatory modifications (designed protein variants 2-7) were added to partially restore stability (Tm values ​​at 42°C-50°C, Figure 9B). The introduced cysteine ​​pair, which was predicted to form a disulfide, was found to decrease the Tm of IdeS by 2°C (Figures 10A-10B), and therefore this cysteine ​​pair was removed from the immunogenic IdeS derivative. Variant 10 (with a chimeric N-terminus and 7 substitutions), which is one of the immunogenic IdeS proteins of bacterial origin and is predicted to reduce HLA-II presentation of epitopes in clusters 1, 2, 4, 5, 6, and 8, was expressed and purified (Figures 11A-11B). The protein was eluted as a sharp peak by SEC (Figure 11C), and its Tm (53°C, Figure 10B) and proteolytic activity against human monoclonal IgG1 (Figure 11D) were indistinguishable from wild-type IdeS.

[0322]

[0325] To verify the reduction of antigen peptide presentation on HLA-II, peptides were synthesized across the region of mutated variant 10 (Table 7). Wild-type and mutant IdeS peptides, increasing in concentration, were incubated with HLA-II (purified with a low-affinity placeholder peptide to maintain stability) in the presence of a constant concentration of a fluorescent reference peptide. In the presence of an HLA-II-specific chaperone and the peptide exchange catalyst HLA-DM, this placeholder peptide was replaced in an exchange reaction that mimicked the biological process of HLA-II antigen presentation. When HLA-II binds to the reference peptide, the fluorescence polarization signal increases. The IdeS peptide competes with the reference peptide for HLA-II binding, and the inhibition of fluorescence polarization functions as an indirect reading of relative peptide affinity. Using this assay, the binding of peptides to HLA-DR alleles containing beta-chain HLA-DRB1*01:01 (Figure 12), DRB1*07:01 (Figure 13), DRB1*11:01 (Figure 14), and HLA-DRB1*15:01 (Figure 15) was examined, and it was observed that some peptides strongly competed with the reference peptide or did not compete at all with respect to different HLA-DRB alleles. In almost all cases, the mutant IdeS peptide showed no change in competition or weak competition compared to the wild-type IdeS peptide. The mutation in variant 10 was found to reduce the HLA-II binding affinity to some HLA-II alleles as designed.

[0323] [Table 43]

[0324]

[0326] Derivative variant 10 was purified from bacteria, and its pharmacokinetics (PK) and pharmacodynamics (PD) were evaluated and compared with wild-type IdeS (and therefore unglycosylated) similarly purified from bacteria. The protein was administered to New Zealand white rabbits as a single intravenous (IV) dose (1 mg / kg). Blood was collected from the ear vein at regular intervals, and enzyme activity was immediately inhibited with iodoacetic acid to prevent the continuation of rabbit IgG proteolytic turnover. Serum levels of IdeS protein, as well as the reduction and recovery of the corresponding IgG pool, were measured (Figures 16A-16B). Variant 10, with reduced immunogenicity, showed comparable efficacy to wild-type IdeS, with a rapid reduction of serum IgG within 3 minutes. IgG levels began to recover within 24-48 hours and took approximately 7 days to return to their initial levels. Therefore, modification of the IdeS sequence to remove the HLA-II epitope does not impair in vivo activity.

[0325]

[0327] Shielding of B cell epitopes exposed on the IdeS surface using N-glycosylation

[0328] Many individuals possess anti-IdeS memory B cells due to past exposure to S. pyogenes, which suggests they may be a source of ADA even with reduced CD4+ T cell activation. This prediction is supported by the low titers of anti-IdeS antibodies present in the serum of healthy individuals, as seen in Figure 17 and as reported elsewhere, which increase 10- to 100-fold two weeks after IdeS administration. To minimize exposure of potential B cell epitopes, the IdeS surface was partially shielded by the addition of custom N-glycosylation sites.

[0326]

[0329] Using rational design and comparison with homologous sequences, suitable sites for modification were identified, and 16 N-glycosylation motifs were introduced into wild-type IdeS. After expression in Expi293F cells, eight IdeS variants were active and showed a clear increase in MW by electrophoresis, consistent with the attachment of additional glycan groups (Table 8 and Figure 18A). Four combinations of N-glycosylation motifs in variant 10, which had reduced immunogenicity at positions 111, 142, and 198, were evaluated, and structural analysis suggested that they could be combined without negative interference (Figure 18B and Table 9). Two were found to have high catalytic activity with cooperative development (Figures 18A and 18C), and the melting temperature was slightly lower compared to the parent variant 10 (Figures 18D and 18E). The other two combinations combined custom N-glycosylation motifs at positions 111 and 142, producing a low-activity protein correlated with stable, less cooperative development starting at low temperatures. These less stable IdeS variants were separated as a broad heterogeneous mixture by electrophoresis (Figure 18F), suggesting that partial development during biosynthesis exposed additional glycosylation sites, likely at native positions N288 and N336. Based on their superior activity and thermal melting properties, variant 10.2 was further modified with a total of three N-glycosylation sites: the wild-type position 61, and custom positions 111 and 198.

[0327] [Table 44]

[0328] [Table 45]

[0329]

[0330] Glycan shielding was maximized by screening mutations at eight novel sites to create additional N-glycosylation motifs. IdeS variants were screened in Expi293F expression medium (Table 8); four of the variants were active for IVIG cleavage and exhibited reduced electrophoretic mobility consistent with glycan addition (Figure 19A). Additional N-glycosylation motifs were combined with variant 10.2 to create variants with a total of 4–6 N-glycans (Table 9). IdeS variants with 5 or 6 N-glycans exhibited varying levels of catalytic activity (Figure 19B), but were unstable, with hydrophobic core residues exposed even at ambient temperature (Figure 19D). All IdeS variants with 4 N-glycans were highly active and cooperatively deployed by DSF (Figures 19C and 19D).

[0330]

[0331] Variant 10.9 was found to be highly glycosylated by N-glycans at positions 47, 61, 111, and 198, folded at physiological temperatures, and not bind to any hydrophobic DSF dyes until it began to unfold cooperatively at approximately 45–60°C (Figure 19D). The thermal stability of variant 10.9 was comparable to that of variants 10.2 and 10, which have parent sequences with three N-glycans and one N-glycan, respectively, as well as wild-type IdeS purified from Expi293F cells, which has a single N-glycan (Figures 20A–20C). The N-glycan group added a considerable size to variant 10.9 compared to SEC-based non-glycosylated proteins (Figures 21A–21B), and the added MW was lost after enzymatic release of the N-glycan by PNGase F (Figure 20B). Despite significant changes to the protein surface to block potential B cell epitopes, the highly glycosylated variant 10.9 retained preferential specificity for human and rabbit IgG, and slowed cleavage of IgG from cynomolgus monkeys and rats (Figure 20C).

[0331]

[0332] To quantify the surface area of ​​IdeS shielded by glycosylation, variant 10.9 was modeled with the following three common mammalian glycoprotein glycans: a mannose-terminating bibranched glycan, a sialic acid-terminating bibranched glycan, and a sialic acid-terminating tetrabranched glycan (Table 10). N-terminal residues up to 39 amino acids, which were predicted to be unstructured, were excluded from the analysis. Based on the crystal structure of IdeS bound to the cleaved IgG Fc product (PDB 8A47), the substrate footprint is 36% of the IdeS surface, and may be even larger if the IgG Fab domain also contributes to the IdeS substrate interface. Therefore, at least 36% of the IdeS surface cannot be used for shielding without adversely affecting substrate accessibility and catalytic activity. Of the remaining IdeS surface, the four glycans of variant 10.9 are mainly localized on one side of the protein (Figures 22A-22B). Using a sphere with a radius of 10 Å to approximate antibody accessibility, the glycan shields 25–45% of the total accessible surface area on IdeS (Table 10), which corresponds to 39–70% of the IdeS surface that does not contribute to substrate recognition. We analyzed the accessibility of a sphere with a radius of 7.2 Å, which mimics the size of a single CDR hypervariable loop and thus approximates the smallest antibody paratope. Using this very conservative antibody accessibility calculation model, we calculated that the glycan variant 10.9 shields at least 20–35% of the total protein surface (Table 10), which corresponds to 31–55% of the IdeS surface that does not contribute to substrate recognition.

[0332] [Table 46]

[0333]

[0333] Highly glycosylated IdeS variants showed lower reactivity to goat anti-IdeS polyclonal antibodies in ELISA, consistent with shielding of surface epitopes (Figure 23). In the case of a set of IdeS variants that share the same primary sequence and differ only in the number of N-glycosylation sites, sequential glycan addition from 0 (variant 10.14, which corresponds to variant 10 having mutation N61Q to remove the glycosylation site of wild-type IdeS) to 1 (variant 10), 3 (variant 10.2), and 4 (variant 10.9) was associated with reduced anti-IdeS reactivity.

[0334]

[0334] Wild-type IdeS (1 glycan), variant 10.2 (3 glycans), variant 10.9 (4 glycans), and variant 10.14 (non-glycosylated variant) were purified from Expi293F cultures and administered intravenously to NZW rabbits (1 mg / kg dose). Highly glycosylated variants 10.2 and 10.9 persisted longer in serum than the non-glycosylated variant 10.14. Variant 10.14 in rabbit serum was below the detection limit 8 hours after administration, while the glycosylated variants were detected for 24 hours (Figure 24A). Even wild-type IdeS produced by Expi293F cells with a single glycan showed a significant prolongation of pharmacokinetic activity (PK). In particular, non-glycosylated IdeS has a theoretical MW of approximately 35 kD, and proteins less than 45 kD are rapidly filtered by the kidney, suggesting that the added glycans can significantly reduce renal excretion by increasing the size of the protein. Curiously, the initial clearance of the highly glycosylated variant 10.2 and variant 10.9 was higher than that of wild-type IdeS produced by Expi293F, which we hypothesize is due to a fraction of highly glycosylated proteins with a low sialylated glycosylation that is rapidly cleared in the liver via the asialoclycoprotein receptor. The prolonged PK of the glycosylated IdeS variants resulted in a longer duration of IgG depletion; in animals treated with variant 10.14, IgG levels began to increase toward baseline at 48 hours, but did not increase until 72, 120, and 144 hours, respectively, after treatment with wild-type IdeS, variant 10.2, and variant 10.9 (Figure 24B). Therefore, the trend in the duration of IgG depletion correlated with the degree of IdeS glycosylation. Overall, high glycosylation was successful not only in shielding potential B cell epitopes on the IdeS surface but also in prolonging PK and PD durations.

[0335]

[0335] Shielding of B cell epitopes exposed on the IdeS surface by fusion with human serum albumin (HSA)

[0336] The fusion effect of HSA on shielding B cell epitopes was determined. See Figure 25A. First, the C-terminus of the non-glycosylated variant 10.14 was fused with the N-terminus of a mature HSA. This fusion protein was secreted at high levels into the culture medium of transfected Expi293F cells (Figure 25B), and although the levels decreased after purification, it remained active in cleaving human IgG1 monoclonal (Figure 25C). The length of the glycine / serine-rich linker connecting IdeS to HSA increased (Table 11 and Figure 25D), but there was no difference in catalytic activity (Figure 25E). ELISA showed that the HSA fusion protein exhibited reduced reactivity to goat polyclonal anti-IdeS, indicating that surface epitopes exposed to B cells and their antibodies were indeed hidden (Figure 25F). The length of the connecting linker did not affect the masking of surface epitopes, suggesting that HSA fusion partners may be present on the IdeS surface in a favorable structure.

[0336] [Table 47]

[0337]

[0337] When administered to NZW rabbits (1 mg / kg IV dose), variant 10.14-HSA had an increased serum half-life compared to variant 10.14 without HSA fusion (t 1 / 2 (=29 vs 1.2 hours), and exposure was increasing (AUC 0-t =3040 vs 24 μg / ml × h) (Figure 26A). Rabbit IgG levels remained low throughout the entire duration of this study (8 days; Figure 26B). Therefore, HSA fusion achieved epitope shielding while significantly extending PK and PD.

[0338]

[0338] IdeS variant combining HLA-II epitope reduction, glycan shielding, and HSA fusion

[0339] IdeS variants with reduced HLA-II epitopes, glycan shielding, and HSA fusion were generated. HSA was fused to the C-terminus of (i) variant 10, in which the HLA-II epitope and one N-glycan at the native position N61 were partially removed; (ii) variant 10.2, a derivative of variant 10 with three N-glycans; and (iii) variant 10.9, another derivative with four N-glycans. Based on ELISA using polyclonal goat anti-IdeS, the surface epitope on the HSA fusion protein was sequentially reduced as the number of N-glycans increased (Figure 27). However, the catalytic activity of the most highly glycosylated HSA fusion variant (variant 10.9-HSA) was suppressed (Figure 28A). Dye-free thermal stability was determined using nanoDSF. The loss of catalytic activity in variant 10.9-HSA construct correlated with stable development over a broad temperature range starting at approximately 30°C, while other variants exhibited cooperative development at higher temperatures (Figure 28B). Therefore, the addition of the custom N-glycosylation site and HSA fusion were not necessarily independent of each other and negatively interacted in one of the constructs.

[0339]

[0340] For further characterization, we selected variant 10.9, which has four N-glycans and no HSA fusion, and variant 10.2-HSA, which has three N-glycans and is fused to HSA at the C-terminus. The reactivity of the proteins to polyclonal anti-IdeS in serum from individual or pooled donors was evaluated by direct ELISA (Figure 29A). The EC50 values ​​of the reactivity of these two variants were approximately half that of wild-type IdeS. Unlike the reactivity measured against goat polyclonal anti-IdeS (Figure 27), variant 10.9 consistently showed lower reactivity compared to variant 10.2-HSA, suggesting that the addition of the fourth glycan at N47 shields a more dominant epitope targeted by human anti-IdeS antibodies compared to HSA fusion at the C-terminus. Epitope shielding was further confirmed by competitive ELISA, where human serum or IVIG was pre-incubated with competing IdeS variants, and then tested for reactivity to wild-type IdeS coated on the surface of ELISA plates. In the competitive ELISA (Figure 29B), wild-type IdeS as the competitor completely blocked anti-IdeS in human serum, whereas variants 10.9 and 10.2-HSA blocked only about half of the reactivity of human serum samples at the highest concentrations tested. The ELISA results are consistent with the glycan shielding model (above), and it is estimated that one-third of the IdeS surface is occluded by hyperglycosylation.

[0340]

[0341] Variant 10.2-HSA was administered intravenously to NZW rabbits at doses of 0.3 and 1 mg / kg. The elimination of variant 10.2-HSA from rabbit serum followed the same trajectory as the non-glycosylated variant 10.14-HSA, except for an initial stage of rapid clearance similar to the initial clearance of the HSA-less highly glycosylated IdeS variant in rabbits (Figure 30A). We again hypothesize that this initial stage of rapid clearance of the glycosylated IdeS variant is due to a portion of the protein having an insufficiently sialylated sugar structure, leading to targeted elimination by the liver. Therefore, the degree to which the glycosylated IdeS variant is initially eliminated can be controlled through optimized manufacturing methods that focus on glycan composition. The long PK of variant 10.2-HSA was associated with an extended duration of IgG depletion, lasting approximately two weeks (Figure 30B). To better understand the duration of IgG depletion, IgG in rabbit serum was rigorously quantified by ELISA measurement at multiple sample dilutions compared to a standard. We found that in rabbits treated with bacterial wild-type IdeS, IgG recovered to baseline levels by day 7, whereas in rabbits treated with variant 10.2-HSA, IgG levels remained reduced by more than 80% until day 15 (Figure 30C). In particular, doses of 0.3 and 1 mg / kg of variant 10.2-HSA resulted in similar reductions of IgG over the 15-day study, suggesting that the dose is saturated and can be reduced further. Lower doses are predicted to further reduce immunogenicity.

[0341]

[0342] ELISA analysis of IgG depletion in rabbits was used to measure full-length IgG or single-cleavage IgG (scIgG) in which both the Fab and Fc regions remain. The clearance of antigen-binding F(ab')2 fragments, products of IdeS-catalyzed IgG cleavage, was qualitatively evaluated by immunoblotting of rabbit serum treated with wild-type and variant IdeS proteins (Figures 31A-31C). The inventors consistently observed that in rabbits, the F(ab')2 cleavage product was removed within 24 hours after IdeS administration. This was confirmed by anti-Fab ELISA of rabbit serum treated with wild-type IdeS, where full-length IgG was destroyed within minutes, but the clearance of the F(ab')2 product took 24 hours (Figure 31D). Compared with the wild-type enzyme, the IdeS variant with an extended PK significantly extended the time frame for the depletion of antigen-binding IgG and F(ab')2 product. This brings significant benefits to applications in AAV-mediated gene therapy and IgG-mediated autoimmunity, due to the long-term removal of neutralizing anti-AAV, as well as pathogenic autoantigen-reactive antibodies and antibody fragments.

[0342]

[0343] The pharmacokinetics (PKs) of variant 10.9 and variant 10.2-HSA were also evaluated in inbred C57Bl / 6 mice with an increased number of animals to more accurately determine the PK properties. It was found that hyperglycosylation increased the half-life from 2.6 hours (bacterial-produced wild-type IdeS) to 5.1 hours (variant 10.9), and after HSA fusion, it was further extended to 17.8 hours (variant 10.2-HSA) (Figures 32A-32B and Table 12). The inventors note that the half-life of human serum albumin is significantly longer in humans (19 days) compared to rabbits (2 days) or mice (1 day), and therefore, HSA-fused IdeS is likely to exhibit a significantly longer duration of action in human patients compared to observations in rabbit or mouse models. Desialization of variants 10.9 and 10.2-HSA by neuraminidase significantly reduced half-life and exposure, consistent with the need for glycan sialization for optimal PK.

[0343] [Table 48]

[0344]

[0344] Method

[0345] Sequences and plasmids

[0346] The numbering of S. pyogenes IdeS is based on the NCBI reference sequence WP_010922160.1 and is identical to UniProt ID Q7DAM2. Residues 1-29 form the Sec signal peptide. Residues 30-339 form the mature polypeptide. For expression in Expi293F cells, the influenza A hemagglutinin signal peptide (KTIIALSYIFCLVFA (SEQ ID NO: 193)) was fused to the N-terminus (aa30-339) of the mature IdeS, and the C-terminus of IdeS was fused to the GSG linker and 8xHis affinity tag (SEQ ID NO: 194). This gene was cloned into the NheI-XhoI site of pcDNA3.1(+) which has a strong Kozak sequence (GCCACCATG, where ATG is the initiation methionine) at the 5' end. Single substitution modifications were performed using overlap extension PCR, and constructs containing multiple modifications were synthesized as DNA fragments. Insertions in the entire plasmid were validated by Sanger sequencing. For expression in Escherichia coli (E. coli), the mature polypeptide of IdeS (aa30-339), along with the N-terminal 10xHis tag (SEQ ID NO: 195) and the TEV cleavage site upstream of the IdeS start residue (D30), was cloned into the NdeI-BamHI site of pET-9a.

[0345]

[0347] Prediction of MHC Class II epitopes

[0348] Potential CD4+ T cell activation regions of wild-type IdeS were identified. In short, all potentially redundant 15-mer peptide sequences in the IdeS polypeptide, as well as 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: For each of the HLA-II alleles (HLA-DQA1*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), we predicted the binding of the peptide to the HLA-II allele and its probability ranking relative to a reference set of 100,000 peptides from the human genome. This is referred to as the rank percentage. A rank percentage of 5% indicates that the peptide is predicted to bind to a particular HLA with higher confidence than 95% of the peptides in the reference set.

[0346]

[0349] MHC class II-related peptide proteomics (MAPP)

[0350] MAPP uses mass spectrometry to identify peptides presented on MHC class II molecules that adorn the surface of dendritic cells (DCs) cultured from multiple donors. Briefly, pia mater was collected from 10 healthy donors and used as a source for culturing monocyte-derived DCs. The DCs were grown and pulsed with a test construct (IdeS) on day 7. Upon maturation (approximately 24 hours), the DCs were harvested and lysed in hypotonic buffer. pMHC-II molecules were captured by immunoprecipitation using a pan-HLA-DR antibody. MHC-II-bound peptides were then analyzed using nano-LC-MS / MS, which included peptides obtained from the test construct treatment along with peptides from other proteins available in the culture. Analysis of nano-LC-MS / MS data identified peptides with sequences matching the test construct. Donor MHC haplotypes were also determined to match peptide presentation to specific MHC-II alleles (Table 2).

[0347]

[0351] Preparation of the IdeS model

[0352] The IdeS crystal structure (PDB2AVW) was initially modeled by first identifying residues that were not degraded by electron density, and then prepared by reverting the catalyst C94A modification back to cysteine. Subsequently, this model was further improved.

[0348]

[0353] To prepare a model of the IdeS-IgG1-Fc complex, we used a modified crystal structure of Mac-2-bound IgG1-Fc (PDB 8A47). Using PyMOL, a 50-residue GS linker was added between the Fc chains (chains A and B). This linker was not sampled and existed only as "padding" for the machine learning algorithm.

[0349]

[0354] Computational removal of HLA-II epitopes

[0355] An IdeS sequence with low immunogenicity was generated using a computational approach, and point variants were evaluated and scored based on their rotational isomeric compatibility within an 8 Å sphere and predicted immunogenicity. To facilitate the immunogenicity reduction process, only a single position was modified at any given time. The modifications were concentrated in regions highlighted by MAPP, and epitope prediction revealed that this position is part of an epitope predicted to bind to at least four HLA-II alleles with a rank percentage of less than 10.0. The selected positions were 10 - 21, 52 - 83, 91 - 120, 127 - 137, 146 - 196, 200 - 220, 232 - 268, 271 - 289, 293 - 331. For each position, the protocol was run 40 times and all returned variants were advanced to the next step. After predicting immunogenicity reduction, the structure was evaluated and the model with the lowest score / energy from 20 minimization runs was selected. Next, the protocol was run and the change in the calculated unfolding free energy (DDG) was collated and analyzed against the epitope score in a database. In total, 18,498 constructs were generated by this methodology. To narrow down the results to a set for experimental testing, modifications with a high ΔΔG (≥ 4.5 energy units or EU) were discarded. Then, the models of the variant IdeS were manually inspected to make the final selection.

[0350]

[0356] Manipulation of disulfide bonds

[0357] Two methods were used to find disulfides within IdeS that could potentially stabilize the protein while simultaneously reducing epitope presentation. Using the first method, based on the IdeS crystal structure, cysteine modifications that were likely to form disulfide bonds were designed. Briefly, the crystal structure of IdeS (PDB No. 2AVW) was relaxed in Cartesian coordinates 18 and this relaxed structure was used as the input. All residue-residue pairs with C β atoms within 5 Å of each other were evaluated. Then, the C1 α -C1 β -C2 β -C2 αThe dihedral angles are examined in relation to representative values ​​for all possible ideal disulfide bond backbone structures. Then, residue pairs that pass this filter are ranked based on the reduction of unfolded state entropy. This method predicted that 13 disulfides are possible within the IdeS structure, but only the disulfide designed between residues 62 and 283 had a dslf_fa13 score < 0, and therefore was the only one selected for experimental testing.

[0351]

[0358] In the second method, structural similarity searches were used to identify proteins with a topology similar to IdeS, and then disulfides were obtained from these structures. 21 The 2AVW chain A crystal structure was aligned using [a specific method], and all structures were manually evaluated for potential disulfides based on the proximity of intrachain cysteine. The 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.

[0352]

[0359] Manipulation of N-glycosylation sites

[0360] We generated IdeS variants with custom N-glycosylation sites using two different computational protocols.

[0353]

[0361] In the first protocol, a set of structures sharing the IdeS topology, curated from the foldseek server and reconstructed using PULCHERA (see the description in the subsection on disulfide bond manipulation above), were searched for the presence of NX(T / S) motifs, where X is any amino acid other than proline. Motifs identified in this way were transplanted to their corresponding positions in IdeS based on structural alignment using TMAlign. These positions were then manually inspected to confirm surface exposure, and the sequences were computationally evaluated as described above to ensure no increased immunogenicity was expected.

[0354]

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

[0355]

[0363] Bacterial expression and purification of IdeS

[0364] E. coli (BL21) transformed with pET9a-10his-TEV-IdeS was inoculated into a larger flask using an overnight starter culture (LB medium containing 50 mg / ml kanamycin, 37°C, 250 rpm). When 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 reduced to 30°C. After approximately 12 hours, cells were collected by centrifugation (10 minutes, 4000 × g, 4°C). The cells were resuspended in phosphate-buffered saline (PBS) and sonicated. The lysate was clarified by centrifugation (15,000 × g, 4°C, 10 minutes). The supernatant was incubated with Ni-NTA resin at 4°C for 1 hour with rotation. The resin was collected by passing it through a gravity column, washed with 8 CV of PBS, and then washed again with 8 CV of PBS (pH 7.8) containing 20 mM imidazole. The protein was eluted with 250 mM imidazole in PBS. The eluate was concentrated using a centrifugal filtration device with a molecular weight cutoff (MWCO). The protein was then further purified by size exclusion chromatography using a Superdex 75 (S75) 10-300GL increasing column with PBS as the running buffer.

[0356]

[0365] Mammalian cell expression and purification of IdeS

[0366] Expi293F cells were cultured at 37°C, 125 rpm, and 8% CO2, resulting in approximately 0.25-4 × 10⁶ cells. 6A cell density of 10 cells / ml was maintained. Cells were cultured 2 × 10⁶ times using ExpiFectamine, according to the manufacturer's instructions, with 1 mg of pcDNA3-IdeS-8h variant per 1 ml of culture. 6 Transfected at a density of 10 cells / ml. ExpiFectamine Transfection Enhancers 1 and 2 were added 18–22 hours after transfection. Expression medium was collected 4–7 days after transfection. Cells were removed by centrifugation at 800×g, 4°C, 10 minutes, and the residue was removed by centrifugation at 15,000×g, 4°C, 20 minutes. The supernatant was incubated with HisPur Ni-NTA resin at 4°C for 2 hours with rotation. The resin was collected through a column and washed with approximately 20 CV of PBS and approximately 5 CV of PBS containing 20 mM imidazole. Proteins were eluted with approximately 8 CV of PBS containing 250 mM imidazole. Proteins were concentrated using a 10,000 MWCO filtration device and then separated using an S75 10-300 GL increasing column equilibrated with PBS. The fractions containing monomer IdeS were pooled, concentrated, and aliquots were rapidly frozen with liquid nitrogen and stored at -80°C.

[0357]

[0367] High-throughput active screen

[0368] The IdeS construct cloned into pcDNA3 was transfected into Expi293F cells as described above. The expression medium was collected 4-5 days after transfection. Proteolytic activity against clinical-grade human IVIG or purified human polyclonal IgG (MP) was measured by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). In this assay, 20 μM IVIG was mixed with a 1 / 12.5 dilution of the expression medium in 20 mM sodium phosphate (pH 6.5), 150 mM NaCl, and 1.25 μM bovine serum albumin (assay buffer) at 37°C for 1 hour. The sample was then combined with a non-reducing load dye, heated to 95°C for 5 minutes, and analyzed by SDS-PAGE.

[0358]

[0369] IgG proteolysis assay

[0370] The kinetics of proteolytic IdeS activity against monoclonal IgG and IgG 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 various time points (0–18 hours) with 2 mM iodoacetic acid. The samples were then combined with non-reducing sample buffer, heated to 95°C for 5 minutes, and analyzed by SDS-PAGE.

[0359]

[0371] Differential scanning fluorescence (DSF)

[0372] The thermal stability of the IdeS variant was measured by differential scanning fluorescence (DSF) using a Quantstudio3 device. This experiment was conducted using the Protein Thermal Shift Dye Kit, following the manufacturer's standard instructions. Briefly, a constant concentration of 0.4 mg / ml IdeS variant in PBS was mixed with the thermal shift dye and reaction buffer in a 96-well plate. The plate was sealed with adhesive film, and the fluorescence signal of the sample was monitored at 25–95°C increments of 0.05°C / s. The data were analyzed, and the thermal fusion (Tm) value was determined using a Boltzmann fitting model.

[0360]

[0373] In vivo activity

[0374] New Zealand rabbits were raised outdoors at the R&R Research Facility (Stanwood, Washington). 4 mg of IdeS protein (prepared to 5 mg / ml in PBS) was injected into the ear vein of rabbits weighing 3.77–3.96 kg. Each test group consisted of two rabbits, and blood samples were collected alternately at the following time points (1–2 ml from the ear vein): Rabbit 1: 0.05, 0.17, 0.5, 4, 48, 96, 192, and 240 hours; Rabbit 2: 1, 2, 8, 24, 72, and 168 hours. Pre-collection was performed at -120 hours. Rabbits were subcutaneously injected with 0.5 ml of acepromazine before ear vein sampling. Enzyme activity in the collected blood was immediately inhibited by adding iodoacetic acid (final 1–2 mM), causing blood coagulation. After centrifugation, serum samples were stored at -20°C.

[0361]

[0375] Rabbit IgG ELISA

[0376] Nunc MaxiSorp plates were coated with F(ab')2 fragment-specific goat anti-rabbit IgG diluted to 0.1 ug / ml in PBS at 37°C for 2 hours. The remaining steps were performed at room temperature. The plates were washed four times with PBS (PBS-T) containing 0.05% Tween 20 and blocked with SuperBlock blocking buffer for 1 hour. The plates were washed four times with PBS-T and serially diluted serum (1:10) in PBS containing 1% bovine serum albumin. 2 ~1:10 7 The plates were incubated with the following for 1 hour: The plates were washed four times with PBS-T and incubated with peroxidase-conjugated goat anti-rabbit IgG specific to the Fc fragment, diluted 1:25,000 in PBS, for 1 hour. The plates were washed four times with PBS-T and chromogenically treated with a one-step TMB substrate for 20 minutes, after which ELISA Stop was added. The absorbance at 450 nm was read.

[0362]

[0377] This example demonstrates the generation of an IdeS variant that reduces peptide presentation on HLA-II while maintaining IdeS stability and IgG cleavage activity.

[0363] 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). 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). 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, CA, Rasband, WS & Eliceiri, KWNIH Image to ImageJ:25 years of image analysis.Nat.Methods 9,671-675(2012).

[0364] Built-in by reference

[0378] The full disclosures of each patent document and scientific paper referred to herein are incorporated by reference for all purposes.

[0365] Equal portions

[0379] This disclosure can be embodied in other specific forms without departing from its spirit or essential features. Therefore, the embodiments described herein should be considered illustrative in all respects, rather than limiting the disclosure as described herein. Various structural elements of various embodiments and steps of various methods of this disclosure may be used in various combinations and permutations, and all such variations should be considered forms of this disclosure. Accordingly, the scope of this disclosure is indicated not by the foregoing description but by the appended claims, and all modifications that fall within the meaning and scope of the equivalents of the claims are intended to be encompassed therein.

[0366] [Table 49]

[0367] [Table 50]

[0368] [Table 51]

[0369] [Table 52]

[0370] [Table 53]

[0371] Table 54

[0372] Table 55

[0373] Table 56

[0374] Table 57

[0375] Table 58

[0376] Table 59

[0377] Table 60

[0378] Table 61

[0379] Table 62

[0380] Table 63

[0381] Table 64

[0382] Table 65

[0383] Table 66

[0384] Table 67

[0385] Table 68

[0386] Table 69

Claims

1. An IdeS variant protein comprising at least two modifications at 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 according to claim 1, wherein the modification at position 68 is a substitution of valine to threonine.

3. The IdeS variant protein according to claim 1 or 2, wherein the modification at position 75 is a substitution of alanine to proline.

4. The IdeS variant protein according to any one of claims 1 to 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 according to any one of claims 1 to 4, wherein the modification at position 187 is a substitution of serine to aspartic acid, or a substitution of serine to glutamic acid.

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

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

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

9. The IdeS variant protein according to any one of claims 1 to 8, wherein the modification at position 302 is a substitution of serine to aspartic acid, a substitution of serine to lysine, or a substitution of serine to glutamic acid.

10. The IdeS variant protein according to any one of claims 1 to 9, wherein the modification at position 303 is a substitution of alanine to aspartic acid, a substitution of alanine to asparagine, or a substitution of alanine to glutamine.

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

12. The IdeS variant protein according to any one of claims 1 to 11, wherein the modification at position 318 is a substitution of isoleucine to lysine, a substitution of isoleucine to aspartic acid, or a substitution of isoleucine to glycine.

13. The IdeS variant protein according to any one of claims 1 to 12, further comprising a modification at position 308.

14. The IdeS variant protein according to claim 13, wherein the modification at position 308 is a substitution of isoleucine to leucine.

15. The IdeS variant protein according to any one of claims 1 to 14, wherein the variant protein comprises a substitution of valine to threonine at position 68, a substitution of alanine to proline at position 75, a substitution of threonine to arginine at position 166, a substitution of serine to aspartic acid at position 187, a substitution of threonine to glutamic acid at position 213, a substitution of serine to lysine at position 302, and a substitution of alanine to aspartic acid at position 303.

16. The IdeS variant protein according to any one of claims 1 to 14, wherein the variant protein comprises a substitution of valine to threonine at position 68, a substitution of alanine to proline at position 75, a substitution of threonine to arginine at position 166, a substitution of serine to aspartic acid at position 187, a substitution of threonine to glutamic acid at position 213, a substitution of serine to glutamic acid at position 302, and a substitution of alanine to aspartic acid at position 303.

17. The IdeS variant protein according to any one of claims 1 to 14, wherein the variant protein comprises a substitution of valine to threonine at position 68, a substitution of alanine to proline at position 75, a substitution of threonine to arginine at position 166, a substitution of serine to aspartic acid at position 187, a substitution of threonine to glutamic acid at position 213, a substitution of serine to lysine at position 302, a substitution of alanine to aspartic acid at position 303, and a substitution of valine to threonine at position 306.

18. The IdeS variant protein according to any one of claims 1 to 14, wherein the variant protein comprises a substitution of valine to threonine at position 68, a substitution of alanine to proline at position 75, a substitution of threonine to arginine at position 166, a substitution of serine to aspartic acid at position 187, a substitution of threonine to glutamic acid at position 213, a substitution of serine to glutamic acid at position 302, a substitution of valine to threonine at position 306, and a substitution of isoleucine to lysine at position 318.

19. The IdeS variant protein according to any one of claims 1 to 14, wherein the variant protein comprises a substitution of valine to threonine at position 68, a substitution of alanine to proline at position 75, a substitution of threonine to arginine at position 166, a substitution of serine to aspartic acid at position 187, a substitution of threonine to glutamic acid at position 213, a substitution of serine to cysteine ​​at position 236, a substitution of leucine to cysteine ​​at position 277, a substitution of serine to glutamic acid at position 302, a substitution of alanine to aspartic acid at position 303, and a substitution of valine to threonine at position 306.

20. The IdeS variant protein according to claim 14, wherein the variant protein comprises a substitution of valine to threonine at position 68, a substitution of alanine to proline at position 75, a substitution of threonine to arginine at position 166, a substitution of serine to aspartic acid at position 187, a substitution of threonine to glutamic acid at position 213, a substitution of serine to cysteine ​​at position 236, a substitution of leucine to cysteine ​​at position 277, and a substitution of isoleucine to leucine at position 308.

21. The variant proteins are DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76) of SEQ ID NO: 2, TTIQAETSKHTISKKDETLHQNQLSISKTAT (SEQ ID NO: 77), DDYQRNAMEAYAKEVPHQIT (SEQ ID NO: 78), DPNNENEVSNLEKIKKLYPKGFQYGN (SEQ ID NO: 79), DQKLKDYLKNDQLKGTELGKFLEEEQGVTK (SEQ ID NO: 80), and SAKVETGLPGELA The IdeS variant protein according to any one of claims 1 to 20, further comprising substitution with a sequence selected from the group consisting of PEEFSFPN (SEQ ID NO: 81), AQNKNPVTHYVNQFDGEEKEII (SEQ ID NO: 82), GSIGEKWDLLLDGIGLNSHRSS (SEQ ID NO: 83), AEPITLIWENYLSDSVSPDRDIR (SEQ ID NO: 84), and QEEIAEGRRRNPLRTAEWPMTKSTTDQ (SEQ ID NO: 85).

22. The variant proteins are 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 of SEQ ID NO: 1 The IdeS variant protein according to any one of claims 1 to 21, further comprising modification at a position selected from the group consisting of 220, 228, 233, 241, 244, 245, 247, 258, 273, 274, 278, 288, 289, 297, 299, 300, 307, 313, 314, 315, 316, 322, and 330.

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

24. The IdeS variant protein according to claim 22 or 23, wherein the variant protein comprises a substitution from phenylalanine to lysine at position 32.

25. The IdeS variant protein according to any one of claims 22 to 24, wherein the variant protein comprises a substitution from serine to glutamic acid at position 33.

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

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

28. The IdeS variant protein according to any one of claims 22 to 27, wherein the variant protein comprises a substitution from valine to glutamic acid at position 43.

29. The IdeS variant protein according to any one of claims 22 to 28, wherein the variant protein comprises a substitution from threonine to glutamic acid at position 44.

30. The IdeS variant protein according to any one of claims 22 to 29, wherein the variant protein comprises a substitution of proline to glutamic acid at position 45.

31. The IdeS variant protein according to any one of claims 22 to 30, wherein the variant protein comprises a histidine to lysine substitution at position 47.

32. The IdeS variant protein according to any one of claims 22 to 31, wherein the variant protein comprises a substitution from lysine to aspartic acid at position 54.

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

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

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

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

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

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

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

40. The IdeS variant protein according to any one of claims 22 to 39, wherein the variant protein comprises a substitution from glutamine to aspartic acid at position 113.

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

42. The IdeS variant protein according to any one of claims 22 to 41, wherein the variant protein comprises a substitution from arginine to aspartic acid at position 116.

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

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

45. The IdeS variant protein according to any one of claims 22 to 44, wherein the variant protein comprises a substitution from phenylalanine to glutamic acid at position 129.

46. The IdeS variant protein according to any one of claims 22 to 45, wherein the variant protein comprises a substitution from asparagine to glutamic acid at position 130.

47. The IdeS variant protein according to any one of claims 22 to 46, wherein the variant protein comprises a substitution from glutamine to aspartic acid at position 133.

48. The IdeS variant protein according to any one of claims 22 to 47, wherein the variant protein comprises a substitution of leucine to asparagine or arginine at position 148.

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

50. The IdeS variant protein according to any one of claims 22 to 49, wherein the variant protein comprises a substitution from glutamic acid to aspartic acid at position 154.

51. The variant protein is the IdeS variant protein according to any one of claims 22 to 50, comprising a substitution of lysine to asparagine at position 159.

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

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

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

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

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

57. The IdeS variant protein according to any one of claims 22 to 56, wherein the variant protein comprises a substitution from lysine to glutamic acid at position 197.

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

59. The IdeS variant protein according to any one of claims 22 to 58, wherein the variant protein comprises a substitution from serine to aspartic acid at position 218.

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

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

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

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

64. The IdeS variant protein according to any one of claims 22 to 63, wherein the variant protein comprises a substitution from lysine to aspartic acid at position 241.

65. The IdeS variant protein according to any one of claims 22 to 64, wherein the variant protein comprises a substitution from threonine to lysine at position 244.

66. The IdeS variant protein according to any one of claims 22 to 65, wherein the variant protein comprises a substitution from glutamic acid to aspartic acid at position 245.

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

68. The IdeS variant protein according to any one of claims 22 to 67, wherein the variant protein comprises a substitution from valine to aspartic acid at position 258.

69. The IdeS variant protein according to any one of claims 22 to 68, wherein the variant protein comprises a substitution from serine to aspartic acid at position 273.

70. The IdeS variant protein according to any one of claims 22 to 69, wherein the variant protein comprises a substitution from asparagine to glutamic acid at position 274.

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

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

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

74. The IdeS variant protein according to any one of claims 22 to 73, wherein the variant protein comprises a substitution from phenylalanine to aspartic acid at position 297.

75. The IdeS variant protein according to any one of claims 22 to 74, wherein the variant protein comprises a substitution from glycine to aspartic acid at position 299.

76. The IdeS variant protein according to any one of claims 22 to 75, wherein the variant protein comprises a substitution from valine to glutamic acid at position 300.

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

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

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

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

81. The variant protein is the IdeS variant protein according to any one of claims 22 to 80, comprising a substitution of aspartic acid to proline at position 316.

82. The IdeS variant protein according to any one of claims 22 to 81, wherein the variant protein comprises a substitution from valine to threonine at position 322.

83. The IdeS variant protein according to any one of claims 22 to 82, wherein the variant protein comprises a substitution from threonine to aspartic acid at position 330.

84. An IdeS variant protein containing the amino acid sequence described in any one of SEQ ID NOs: 3 to 75.

85. An IdeS variant protein containing the amino acid sequence described in SEQ ID NO:

10.

86. An IdeS variant protein containing the amino acid sequence described in SEQ ID NO:

11.

87. An IdeS variant protein containing the amino acid sequence described in SEQ ID NO:

12.

88. An IdeS variant protein containing the amino acid sequence described in SEQ ID NO:

13.

89. DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 76), SEQ ID NO: 2, TTIQAETSKHTISKKDETLHQNQLSISKTAT (SEQ ID NO: 77), DPNNENEVSNLEKIKKLYPKGFQYGN (SEQ ID NO: 79), DQKLKDYLKNDQLKGTELGKFLEEQGVTK (SEQ ID NO: 80), SAKVETGLPGELAPEEFSFPN (SEQ ID NO: 81), A An IdeS variant protein comprising a substitution with a sequence that is at least 80% identical to a sequence selected from the group consisting of QNKNPVTHYVNQFDGEEKEII (SEQ ID NO: 82), GSIGEKWDLLLDGIGLNSHRSS (SEQ ID NO: 83), AEPITLIWENYLSDSVSPDRDIR (SEQ ID NO: 84), and QEEIAEGRRRNPLRTAEWPMTKSTTDQ (SEQ ID NO: 85).

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

2.

91. IdeS variant proteins containing the amino acids listed in SEQ ID NOs. 86-94.

92. The IdeS variant protein according to any one of claims 1 to 91, wherein the variant protein exhibits reduced presentation of epitopes on human leukocyte antigens (HLA) compared to the wild-type IdS protein.

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

94. The IdeS variant protein according to any one of claims 1 to 93, wherein the variant protein has an immunogenicity score reduced to more than 0 compared to SEQ ID NO: 1 or SEQ ID NO:

2.

95. IdeS variant proteins containing one or more glycosylation modifications compared to the wild type.

96. An IdeS variant protein comprising at least one modification at a position 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, in order to introduce a glycosylation site.

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

98. The IdeS variant protein according to claim 96 or 97, wherein the modification at position 37 is a substitution of glutamic acid with asparagine.

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

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

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

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

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

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

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

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

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

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

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

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

111. The IdeS variant protein according to any one of claims 96 to 110, wherein the modification at position 148 is a substitution of leucine to asparagine.

112. The IdeS variant protein according to any one of claims 96 to 111, wherein the modification at position 198 is a substitution of glutamic acid with asparagine.

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

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

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

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

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

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

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

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

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

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

123. The IdeS variant protein according to claim 96, wherein the variant protein comprises a substitution from aspartic acid to asparagine at position 142 and a substitution from lysine to threonine at position 144.

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

125. The IdeS variant protein according to claim 96, wherein the variant protein comprises a substitution of lysine to asparagine at position 311 and a substitution of isoleucine to threonine at position 313.

126. An IdeS variant protein comprising at least one modification at a position 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, in order to introduce a glycosylation site.

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

128. The IdeS variant protein according to claim 126 or 127, wherein the modification at position 49 is a substitution of glutamine with threonine or asparagine.

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

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

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

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

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

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

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

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

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

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

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

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

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

142. The IdeS variant protein according to any one of claims 126 to 141, wherein the modification at position 275 is a substitution of glycine to serine.

143. The IdeS variant protein according to any one of claims 126 to 142, wherein the modification at position 278 is a substitution of lysine to serine.

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

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

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

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

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

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

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

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

152. The IdeS variant protein according to claim 126, wherein the variant protein comprises a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of aspartic acid to asparagine at position 142, and a substitution of lysine to threonine at position 144.

153. The IdeS variant protein according to claim 126, wherein the variant protein comprises a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, and a substitution of glutamic acid to asparagine at position 198.

154. The IdeS variant protein according to claim 126, wherein the variant protein comprises a substitution of aspartic acid to asparagine at position 142, a substitution of lysine to threonine at position 144, and a substitution of glutamic acid to asparagine at position 198.

155. The IdeS variant protein according to claim 126, wherein the variant protein comprises a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of aspartic acid to asparagine at position 142, a substitution of lysine to threonine at position 144, and a substitution of glutamic acid to asparagine at position 198.

156. The IdeS variant protein according to claim 126, wherein the variant protein comprises a substitution of glutamine to asparagine at position 49, a substitution of valine to threonine at position 51, a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of lysine to asparagine at position 126, a substitution of asparagine to serine at position 128, a substitution of leucine to asparagine at position 148, and a substitution of glutamic acid to asparagine at position 198.

157. The IdeS variant protein according to claim 126, wherein the variant protein comprises a substitution of glutamine to asparagine at position 49, a substitution of valine to threonine at position 51, a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of leucine to asparagine at position 148, and a substitution of glutamic acid to asparagine at position 198.

158. The IdeS variant protein according to claim 126, wherein the variant protein comprises a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of lysine to asparagine at position 126, a substitution of asparagine to serine at position 128, a substitution of leucine to asparagine at position 148, and a substitution of glutamic acid to asparagine at position 198.

159. The IdeS variant protein according to claim 126, wherein the variant protein comprises a substitution of glutamine to asparagine at position 49, a substitution of valine to threonine at position 51, a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of lysine to asparagine at position 126, a substitution of asparagine to serine at position 128, and a substitution of glutamic acid to asparagine at position 198.

160. The IdeS variant protein according to claim 126, wherein the variant protein comprises a substitution of threonine to asparagine at position 47, a substitution of glutamine to asparagine at position 49, a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, and a substitution of glutamic acid to asparagine at position 198.

161. The IdeS variant protein according to claim 126, wherein the variant protein comprises a substitution of glutamine to asparagine at position 49, a substitution of valine to threonine at position 51, a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, and a substitution of glutamic acid to asparagine at position 198.

162. The IdeS variant protein according to claim 126, wherein the variant protein comprises a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of glutamic acid to asparagine at position 123, a substitution of glutamine to threonine at position 125, and a substitution of glutamic acid to asparagine at position 198.

163. The IdeS variant protein according to claim 126, wherein the variant protein comprises a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of lysine to asparagine at position 126, a substitution of asparagine to serine at position 128, and a substitution of glutamic acid to asparagine at position 198.

164. The IdeS variant protein according to claim 126, wherein the variant protein comprises a substitution of lysine to asparagine at position 111, a substitution of glutamine to threonine at position 113, a substitution of leucine to asparagine at position 148, and a substitution of glutamic acid to asparagine at position 198.

165. A polypeptide comprising an IdeS protein conjugated to human serum albumin, exhibiting at least 90% sequence identity to one of sequence numbers 1-75.

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

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

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

169. The polypeptide according to claim 168, wherein the linker comprises GSGGGGSG (SEQ ID NO: 113), GSGSGSGS (SEQ ID NO: 114), GSGGGGGGGGSG (SEQ ID NO: 115), GSGSGSGSGSGS (SEQ ID NO: 116), or GS.

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

171. It is a polypeptide, a) It is an IdeS variant protein, i) at least two modifications of Sequence ID No. 1 at positions selected from the group consisting of 68, 75, 166, 187, 213, 236, 277, 302, 303, 306, and 318; and ii) At least one modification of Sequence ID No. 1 at a position 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, for introducing a glycosylation site, or at least one modification of Sequence ID No. 12 at a position 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. IdeS variant proteins containing, b) Human serum albumin coupled to the IdeS variant protein and Polypeptides containing this material.

172. A polynucleotide encoding an IdeS variant protein according to any one of claims 1 to 171.

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

174. A cell containing polynucleotides according to claim 172.

175. A pharmaceutical composition comprising an IdeS variant protein according to any one of claims 1 to 171 and a pharmaceutically acceptable carrier.

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

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

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

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

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

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

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