Novel IgG protease and method of use thereof

Abstract

This specification provides novel immunoglobulin G (IgG) proteases and fusion proteins containing them. The IgG proteases find utility in methods for treating diseases that are entirely or partially mediated by pathogenic immunoglobulin G (IgG) antibodies, including methods for preventing or treating antibody-mediated rejection (AMR) of organ allografts in organ transplant patients.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority from U.S. Provisional Application No. 63 / 517,080, filed on 1 August 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Reference to electronic sequence listings The contents of the electronic sequence listing (INMD_195_01WO_SeqList_ST26.xml, size: 3,051,481 bytes, created: July 29, 2024) are incorporated herein by reference in their entirety. [Background technology]

[0003] The immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) cleaves all subclasses of human IgG in the hinge region with high specificity. IdeS also catalyzes heavy chain cleavage of several subclasses of IgG in various animals. Pathogenic IgG antibodies contribute to the pathogenesis of many autoimmune conditions as well as acute and chronic transplant rejection. Furthermore, existing antibodies against gene therapy vectors can, in some cases, limit the ability of patients to receive such treatment. Therefore, the effective removal of such antibodies is a critical clinical challenge.

[0004] Streptococcus pyogenes (S. pyogenes) is a bacterial pathogen that causes common infections such as tonsillitis and streptococcal pharyngitis; therefore, most people have encountered IdeS and are likely to have anti-IdeS antibodies in their bloodstream. IdeS-specific antibodies have been detected in serum samples from random human subjects (likely due to previous streptococcal infections), as well as in intravenous immunoglobulin (IV-Ig) preparations, which are IgG preparations extracted from pooled serum of thousands of donors. Even if a subject does not have IdeS-specific antibodies before the first administration of IdeS, such antibodies are likely to be produced after administration. Therefore, because IdeS is an immunogenic protein, when it is used as a therapeutic agent, the immune system of a patient receiving IdeS often responds to it. The immune response may involve the production of IdeS-specific antibodies. In general, immune responses to IdeS, particularly the production of IdeS-specific anti-drug antibodies (ADAs), can lead to undesirable or even harmful complications, such as (i) reduced efficacy of IdeS due to ADA binding, and / or (ii) excessive inflammatory responses induced by immune complexes between ADA and IdeS.

[0005] This invention addresses the need for novel IgG proteases by providing novel proteases and methods for using them. [Overview of the Initiative]

[0006] In one embodiment, the present disclosure provides a variant of IdeS IgG protease ("IdeS variant"). IdeS IgG protease comprises the amino acid sequence described in SEQ ID NO: 2. The IdeS variant comprises (i) the following amino acid positions in SEQ ID NO: 2: S3, F4, A6, E9, I10, R11, Y12, Y18, V46, A47, N48, I54, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G (ii) an amino acid variant comprising one or more amino acid mutations in one or more of 222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, G296, and (ii) an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 98% identical to SEQ ID NO: 2. In a further embodiment, the IdeS variant has at least about 30% of the activity of IdeS in an IgG protease enzyme assay. In a further embodiment, the IdeS variant has at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% of the activity of IdeS in an IgG protease enzyme assay.

[0007] In one embodiment, one or more mutations in the IdeS variant include approximately 5 to 20, 10 to 20, 12 to 20, or 15 to 20 amino acid substitutions. In another embodiment, one or more mutations in the IdeS variant include approximately 10 to 35, or 24 to 29 amino acid substitutions.

[0008] In one embodiment, the IdeS variant includes one or more amino acid substitutions as listed in Table 1 disclosed herein. In a further embodiment, the IdeS variant does not have amino acid mutations at the following amino acid positions in SEQ ID NO: K56, C66, H234, D256, and D258. In other words, in this embodiment, the amino acids at positions 56, 66, 234, 256, and 258 are wild-type IdeS residues. In a further embodiment or another embodiment, the IdeS variant does not have amino acid mutations at one or more of the following amino acid positions in SEQ ID NO: A32, N33, T35, Q36, F41, D84, R88, E91, N102, M106, N117, H118, E170, S195, N197, K213, S245, K250, A261, K286, and S306. In other words, in this embodiment as well, the amino acids at the numbered positions mentioned above are wild-type IdeS residues.

[0009] In one embodiment, the IdeS variant is one of S3N, F4I, A6S, E9I, I10T, R11D and R11T, one of Y12N, Y18K, V46D, A47E, N48G, I54T, T57D, T57K, T57N, T57R, T57L and T57Q, one of N59D, G60S and G60T, one of K61R, E92R, H93Y, E104R, L120T, K123A, F125W, E126S, Y135H and Y135N, T138A, S15 The molecule contains one or more amino acid substitutions of Sequence ID No. 2, selected from the group consisting of 9K, T161W, T161G, and T161Y, one of N162D, T185G, D188T, F199L, K200R, E201N, N203T, G222A, L223I, one of V230A, and V230S, one of N233S, N246R, A251W, N273G, A275G, A275S, and A275W, D288G, Q293R, V294A, G296E, and combinations thereof.

[0010] In some embodiments, the IdeS variant includes a set of amino acid substitutions selected from one of the following for SEQ ID NO: (i) I10T, R11D, Y12N, Y18K and V46D, (ii) R11D, Y12N, Y18K, V46D and A47E, (iii) Y12N, Y18K, V46D, A47E and T57K, (iv) Y18K, V46D, A47E, T57K, H93Y, E104R and L120T, (v) I10T, R11D, Y12N, A275S, V294A and G296E, (vi) I10T, R11D, F125W, E126S, Y135H, A275W, V294A and G296E, (vii) I10T, R11D, Y12N, Y18K, K123A, F125W, E126S, V294A, and G296E, (viii) I10T, R11D, A275S, V294A, and G296E, (ix) I10T, R11D, V46D, A47E, A275S, V294A, and G296E, (x) Y12N, T57K, H93Y, E104R, L120T, and K123A, (xi) I10T, R11D, S159K, N162D, T185G, V230A, A251W, V294A, and G296E.

[0011] In some embodiments, the IdeS variant includes a set of amino acid substitutions for SEQ ID NO: 2, selected from one of the following:

[0012] (1) S3N, F4I, A6S, Y12N, Y18K, A47E, N48G, I54T, T57N, N59D, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0013] (2) Y12N, Y18K, A47E, N48G, T57K, N59D, G60S, K61R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, D188T, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0014] (3) Y12N, Y18K, A47E, N48G, T57K, N59D, G60S, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, L223I, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0015] (4) Y12N, Y18K, A47E, N48G, T57K, N59D, G60S, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0016] (5) Y12N, Y18K, A47E, N48G, T57K, N59D, G60S, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, N203T, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0017] (6) Y12N, Y18K, A47E, N48G, T57K, N59D, G60S, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, D188T, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0018] (7) Y12N, Y18K, A47E, N48G, T57K, N59D, G60S, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161Y, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0019] (8) Y12N, Y18K, A47E, N48G, T57K, N59D, G60S, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161Y, D188T, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0020] (9) Y12N, Y18K, A47E, N48G, T57Q, N59D, G60T, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0021] (10) Y12N, Y18K, A47E, N48G, T57R, N59D, G60T, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, L223I, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0022] (11) Y12N, Y18K, A47E, N48G, T57R, N59D, G60T, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, N203T, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0023] (12) Y12N, Y18K, A47E, N48G, T57R, N59D, G60T, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, D188T, F199L, K200R, E201N, L223I, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0024] (13) Y12N, Y18K, A47E, N48G, T57R, N59D, G60T, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161Y, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0025] (14) Y12N, Y18K, A47E, N48G, I54T, T57L, N59D, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0026] (15) Y12N, Y18K, A47E, N48G, I54T, T57L, N59D, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, L223I, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0027] (16) Y12N, Y18K, A47E, N48G, I54T, T57L, N59D, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161Y, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, Q293R.

[0028] (17) Y12N, Y18K, A47E, N48G, I54T, T57N, N59D, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0029] (18) Y12N, Y18K, A47E, N48G, I54T, T57N, N59D, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, D188T, F199L, K200R, E201N, L223I, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0030] (19) Y12N, Y18K, A47E, N48G, I54T, T57N, N59D, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161Y, G222A, L223I, V230S, N233S, N246R, N273G, A275S, and D288G.

[0031] (20) Y12N, Y18K, A47E, N48G, I54T, T57N, N59D, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161Y, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0032] (21) E9I, I10T, R11T, Y12N, Y18K, A47E, N48G, I54T, T57N, N59D, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

[0033] In yet another embodiment, the IdeS variant includes a set of amino acid substitutions for SEQ ID NO: 2, selected from one of the sets of amino acid substitutions listed in Tables 2, 3, and 4 disclosed herein. In one embodiment, the set of amino acid substitutions is listed in Table 2. In another embodiment, the set of amino acid substitutions is listed in Table 3. In yet another embodiment, the set of amino acid substitutions is listed in Table 4.

[0034] In some embodiments, the IdeS variant is selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, N102G, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, K213Q, N233G, S245A, N246E, K250E, A261P, K286E, and S306I, and does not have one or more amino acid substitutions of SEQ ID NO: 2.

[0035] In some embodiments, the IdeS variant includes an amino acid sequence selected from one of SEQ ID NOs: 1492, 1697, 1700, 1701, 1702, 1703, 1704, 1705, 1713, 1720, 1722, 1723, 1724, 1772, 1774, 1775, 1793, 1795, 1797, 1798, and 1949. In further embodiments, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the reference amino acid sequence described in one of SEQ ID NOs: 1492, 1697, 1700, 1701, 1702, 1703, 1704, 1705, 1713, 1720, 1722, 1723, 1724, 1772, 1774, 1775, 1793, 1795, 1797, 1798, and 1949, wherein the IdeS variant includes amino acid substitutions present in the reference amino acid sequence relative to SEQ ID NO: 2.

[0036] In some embodiments, the IdeS variant includes a first set of amino acid substitutions of SEQ ID NO: 2 and a second set of amino acid substitutions of SEQ ID NO: 2. The first set of amino acid substitutions of SEQ ID NO: 2 consists of Y12N, Y18K, A47E, H93Y, E104R, L120T, E126S, Y135N, S159K, V230S, and A275S. The second set of amino acid substitutions in Sequence ID No. 2 is a set of approximately 13 to 18 amino acid substitutions selected from the group consisting of S3N, F4I, A6S, E9I, I10T, R11T, N48G, I54T, T57K, T57N, T57R, T57L, and one of T57Q, one of N59D, G60S, and G60T, K61R, E92R, one of T138A, T161G, and T161Y, D188T, F199L, K200R, E201N, N203T, G222A, L223I, N233S, N246R, N273G, D288G, and Q293R. In one embodiment, an IdeS variant comprising a first set and a second set of amino acid substitutions of SEQ ID NO: 2 has an amino acid sequence that is at least about 88%, at least about 89%, at least about 90%, at least about 91%, or at least about 92% identical to SEQ ID NO: 2. In one embodiment, an IdeS variant comprising a first set and a second set of amino acid substitutions of SEQ ID NO: 2 does not have amino acid mutations at the following amino acid positions of SEQ ID NO: K56, C66, H234, D256, and D258. In another embodiment, an IdeS variant comprising a first set and a second set of amino acid substitutions of SEQ ID NO: 2 has no amino acid mutations at one or more of the following amino acid positions of SEQ ID NO: A32, N33, T35, Q36, F41, D84, R88, E91, N102, M106, N117, H118, E170, S195, N197, K213, S245, K250, A261, K286, S306.In further embodiments, the IdeS variant is selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, N102G, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, K213Q, N233G, S245A, N246E, K250E, A261P, K286E, and S306I, and does not have one or more amino acid substitutions of SEQ ID NO: 2.

[0037] In another aspect of the present disclosure, an IgG protease comprising an amino acid sequence selected from one of SEQ ID NOs: 27 to 302 is provided. In a further embodiment, an IgG protease variant of one of SEQ ID NOs: 27 to 302 is provided, the variant comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the amino acid sequence of each IgG protease from which the variant is derived. In one embodiment of the IgG protease, the IgG protease contains an amino acid sequence selected from one of the following: SEQ ID NOs: 36, 37, 39, 41, 50, 51, 54, 56, 60, 61, 65, 67, 137, 140, 144, 150, 159, 285, 286, 287, 288, 289, 292, 293, 294, 295, 296, 297, and 298. In another embodiment, a variant of an IgG protease is provided which includes one amino acid sequence from among SEQ ID NOs: 36, 37, 39, 41, 50, 51, 54, 56, 60, 61, 65, 67, 137, 140, 144, 150, 159, 285, 286, 287, 288, 289, 292, 293, 294, 295, 296, 297, and 298, wherein the variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the amino acid sequence of the respective IgG protease from which the variant is derived. In a further embodiment, an IgG protease is provided that contains an amino acid sequence selected from one of SEQ ID NOs. 54, 140, 293, 294, 295, 297, and 298.In yet another embodiment, a variant of an IgG protease is provided which includes one amino acid sequence from among SEQ ID NOs. 54, SEQ ID NOs. 140, SEQ ID NOs. 293, SEQ ID NOs. 294, SEQ ID NOs. 295, SEQ ID NOs. 297, and SEQ ID NOs. 298, wherein the variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the amino acid sequence of the respective IgG protease from which the variant is derived.

[0038] In another aspect of this disclosure, a variant of N142 IgG protease ("N142 variant") is provided. N142 IgG protease comprises the amino acid sequence of SEQ ID NO: 297. The N142 variant includes (i) one or more amino acid mutations at one or more of the following amino acid positions in SEQ ID NO: 297: I10, R11, Y12, V15, I35, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, K129, R157, S159, L160, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300, and (ii) an amino acid sequence that is at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 97%, or at least approximately 98% identical to SEQ ID NO: 297. In a further embodiment, the N142 variant has at least about 30% of the activity of IdeS in an IgG protease enzyme assay. In a further embodiment, the N142 variant has at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% of the activity of IdeS in an IgG protease enzyme assay.

[0039] In some embodiments, the N142 variant does not have an amino acid mutation at one or more of the following amino acid positions in SEQ ID NO: 297: A32, N33, Q36, F41, D84, R88, E91, E92, H93, M106, N117, H118, E126, E170, N197, N203, K213, L223, S245, K286, S306.

[0040] In one embodiment, one or more amino acid mutations in the N142 variant of SEQ ID NO: 297 are the following amino acid substitutions: one of I10A and I10Q, R11T, Y12K, one of V15H and V15K, I35T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G The variant includes one of L120T, Y127L, K129G, R157G, S159G, L160W, V230G, V230R, and V230S, one of N233S, K250S, A261K, F269N, one of S274D, A275F, A275S, and A275W, one of D288H and D288K, and one or more of Q293K, V294A, and L300T. One or more mutations in the N142 variant include, in one embodiment, about 5 to about 25, about 5 to about 20, about 10 to about 20, about 12 to about 20, about 15 to about 20, about 16 to about 25, or about 16 to about 17 amino acid substitutions.

[0041] In some embodiments, the N142 variant includes a set of amino acid substitutions selected from one of the following for SEQ ID NO: (i) I10A, V15H, I35T, and Y52F, (ii) I10A, V15H, I35T, and V46S, (iii) I10A, V15H, I35T, and V46D, (iv) I10A, V15H, I35T, V46D, and T57K, (v) V230S, A261K, S274D, A275F, and D288H, (vi) L120T, V230R, A261K, S274D, and A275F, (vii) V 230G, A261K, S274D, A275F, and D288H, (viii)I10A, V15H, V230S, A261K, and S274D, (ix)I10A, V15H, I35T, N48H, Y52S, T57K, N102G, L120T, S159G, V230R, K250S, A261K, S274D, A275F, D288H, and V294A, (x)I10A, V15H, I35T, N48H, T 57K, N102G, L120T, S159G, L160W, V230R, K250S, A261K, S274D, A275F, D288H, and L300T, (xi)I10Q, R11T, Y12K, V15K, N48G, Y52S, T57K, L120T, R157G, V230S, K250S, A261K, F269N, A275S, D288K, and Q293K, (xii)I10Q, R11T, Y12K, V15K, N48G, T57K, L120T, K129G, R157G, V230S, N233S, K250S, A261K, F269N, A275F, D288K, and Q293K, (xiii)I10Q, R11T, Y12K, V15K, N48G, T57K, L120T, K129G, R157G, V230S, N233S, K250S, A261K, F269N, A275W, D288K, and Q293K.

[0042] In one embodiment, the N142 variant comprises a set of amino acid mutations for SEQ ID NO: 297, selected from one of the sets of amino acid substitutions listed in Tables 6 and 7 disclosed herein.

[0043] In some embodiments, the N142 variant disclosed herein does not have one or more amino acid substitutions of SEQ ID NO: 297, selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, T57L, D84E, R88N, E91K, E92K, H93Y, M106L, N117D, H118S, E126D, N162D, E170R, N197Y, F199L, N203T, K213Q, L223I, N233G, S245A, A261P, K286E, and S306I.

[0044] In some embodiments, the N142 variant includes an amino acid sequence selected from one of SEQ ID NOs: 859, 868, 925, 936, and 942.

[0045] In some embodiments, the N142 variant includes an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 859, SEQ ID NO: 868, SEQ ID NO: 925, SEQ ID NO: 936, and SEQ ID NO: 942, and the N142 variant includes amino acid substitutions present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO: 297.

[0046] In one embodiment of an IgG protease variant, the variant is a variant of an IgG protease containing the amino acid sequence described in one of SEQ ID NOs. 27 to 302. In a further embodiment, the IgG protease variant is a variant of the amino acid sequence of SEQ ID NOs. 293 (the IgG protease containing the amino acid sequence of SEQ ID NOs. 293 is also referred to herein as N123). The N123 variant contains (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NOs. 293: Y12, L31, E34, F36, R37, Y38, N39, I44, A60, D133, N162, F274, A279, H280, V299, and (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NOs. 293. In a further embodiment, the N123 variant (iii) has at least about 25% of the activity of IdeS in an IgG protease enzyme assay. In further embodiments, the N123 variant exhibits at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% activity of IdeS in an IgG protease enzyme assay. In one embodiment of the N123 variant, the variant comprises an amino acid sequence that is at least about 90% or at least about 95% identical to SEQ ID NO: 293, and includes one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: Y12, L31, E34, F36, R37, Y38, N39, I44, A60, D133, N162, F274, A279, H280, V299.

[0047] In another embodiment, the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 298 (the IgG protease containing the amino acid sequence of SEQ ID NO: 298 is also referred to herein as N30). The N30 variant includes (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 298: I10, R11, Y12, V15, Y18, V46, N48, Y52, F101, N102, Q105, L120, F125, E126, Y127, F133, Y156, R157, S159, V230, R231, N233, D258, A261, V270, A275, F269, and (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 298. In a further embodiment, the N30 variant has at least about 25% of the activity of IdeS in an IgG protease enzyme assay. In a further embodiment, the N30 variant has at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% of the activity of IdeS in an IgG protease enzyme assay. In one embodiment, the N30 variant comprises an amino acid sequence that is at least about 90% or at least about 95% identical to SEQ ID NO: 298, and contains one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: I10, R11, Y12, V15, Y18, V46, N48, Y52, F101, N102, Q105, L120, F125, E126, Y127, F133, Y156, R157, S159, V230, R231, N233, D258, A261, V270, A275, F269.

[0048] In another embodiment, the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 294 (the IgG protease containing the amino acid sequence of SEQ ID NO: 294 is also referred to herein as N31). The N31 variant includes (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 294: I10, R11, Y12, E14, V15, Y18, V46, N48, Y52, D130, A132, Y157, N159, Y161, K162, V232, R233, N235, D260, A263, V272, S276, A277, and (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 294. In a further embodiment, the N31 variant (iii) has at least about 25% of the activity of IdeS in an IgG protease enzyme assay. In further embodiments, the N31 variant exhibits at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% of the activity of IdeS in an IgG protease enzyme assay. In one embodiment, the N31 variant comprises an amino acid sequence that is at least about 90% or at least about 95% identical to SEQ ID NO: 294, and includes one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: I10, R11, Y12, E14, V15, Y18, V46, N48, Y52, D130, A132, Y157, N159, Y161, K162, V232, R233, N235, D260, A263, V272, S276, A277.

[0049] In yet another embodiment, the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 54. The IgG protease variant includes (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: I10, I11, Y12, E14, Y18, H19V143, Y157, Y161, K162, and (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 54. In a further embodiment, the variant (iii) has at least about 25% of the activity of IdeS in an IgG protease enzyme assay. In a further embodiment, the variant has at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% of the activity of IdeS in an IgG protease enzyme assay. In one embodiment, the variant comprises an amino acid sequence that is at least about 90% or at least about 95% identical to SEQ ID NO: 54, and contains one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: I10, I11, Y12, E14, Y18, H19V143, Y157, Y161, K162.

[0050] In yet another embodiment, the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 140. The variant includes (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 140, M10, R11, Y12, V15, Y42, V46, A47, N48, Q49, N102, V104, I113, Y118, L120, D121, K123, Y127, Y156, R157, Y201, Q202, V230, F269, S274, A275, D288, G296, and (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 140. In yet another embodiment, the variant (iii) has at least about 25% of the activity of IdeS in an IgG protease enzyme assay. In further embodiments, the variant has at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% of the activity of IdeS in an IgG protease enzyme assay. In one embodiment, the variant comprises an amino acid sequence that is at least about 90% or at least about 95% identical to SEQ ID NO: 140, and contains one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: M10, R11, Y12, V15, Y42, V46, A47, N48, Q49, N102, V104, I113, Y118, L120, D121, K123, Y127, Y156, R157, Y201, Q202, V230, F269, S274, A275, D288, G296.

[0051] In another aspect, the Disclosure provides a variant of N144 IgG protease ("N144 Variant"). N144 IgG protease comprises the amino acid sequence described in SEQ ID NO: 295. The N144 Variant comprises (i) one or more amino acid positions of SEQ ID NO: 295: Y12, S18, I35, I46, A47, Y52, I54, T55, T57, N59, H93, F101, N102, E104, Q105, L120, F125, E126, Y127, T135, Y156, S159, L175, V183, R200, N201, N205, I207, V230, R231, N233, A24 1) One or more amino acid mutations in N244, E246, A261, Y269, V270, S274, A275, I280, S281, A282, I285, D288, V290, and (ii) an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 98% identical to SEQ ID NO: 295. In a further embodiment, the N144 variant has at least about 30% of the activity of IdeS in an IgG protease enzyme assay. In a further embodiment, the N144 variant has at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% of the activity of IdeS in an IgG protease enzyme assay.

[0052] In one embodiment, the N144 variant does not have an amino acid mutation at one or more of the following amino acid positions in SEQ ID NO: A32, N33, Q36, F41, D84, E91, M106, H118, E170, S195, N197, N203, S245, K286.

[0053] One or more mutations in the N144 variant, in one embodiment, include approximately 5 to 16, 10 to 16, 10 to 30, 12 to 16, 12 to 24, 12, 13, 14, 21, 22, 23, or 24 amino acid substitutions compared to SEQ ID NO: 295.

[0054] In one embodiment, the N144 variant is one of Y12N, S18K, I35E, I35K, and I35S, one of I46D, A47E, I54T, T55K, T55H, and T55R, one of T57L, N59D, H93Y, N102G, E104R, one of L120T, and L120S, one of F125W, T135H, S159K, L175A, L175N, and L175S, one of V183W, R200D, R200G, and R200T, one of N201E, N205D, I207T, one of V230H, and V230Q, R231W, The molecule contains one or more amino acid substitutions of sequence number 295, selected from the group consisting of N233G, A241G, E246G, and E246K; one of A261K, A261I, A261F, A261G, A261Q, and A261S; one of Y269D, Y269S, and Y269W; one of V270T, V272T, and V272Y; one of S274D and S274G; one of A275S and A275W; one of I280M, S281G, A282G, D288K, and D288T; V290F; and combinations thereof.In further embodiments, the N144 variant is one of Y12N, S18K, I35E, I35K, and I35S, one of I46D, A47E, I54T, T55K, T55H, and T55R, one of T57L, N59D, H93Y, N102G, E104R, one of L120T, and L120S, F125W, T135 H, S159K, L175A, L175N, and one of L175S, V183W, R200D, R200G, and one of R200T, N201E, N205D, I207T, one of V230H and V230Q, R231W, N233G, A241G, one of E246G and E246K, A261K, A2 One of 61I, A261F, A261G, A261Q, and A261S, one of Y269D, Y269S, and Y269W, one of V270T, V272T, and V272Y, one of S274D and S274G, one of A275S and A275W, I280M, S281G, A282G, D288K The amino acid substitutions of SEQ ID NO: 295 include approximately 5 to 16, 10 to 16, 10 to 30, 12 to 16, 12 to 24, 12, 13, 14, 21, 22, 23, or 24, selected from the group consisting of one of D288T, V290F, and combinations thereof.

[0055] In one embodiment, the N144 variant is selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, F199L, N203T, K213Q, L223I, S245A, N246E, K250E, K286E, and S306I, and does not have one or more amino acid substitutions of SEQ ID NO: 295.

[0056] In some embodiments, the N144 variant includes a set of amino acid substitutions for SEQ ID NO: 295, selected from one of the following: (1) Y12N, S18K, I46D, A47E, T55H, H93Y, E104R, L120T, F125W, T135H, S159K, and A275S, (2) Y12N, S18K, I46D, A47E, T55H, H93Y, E104R, L120T, F125W, T135H, S159K, A261I, and A275S, (3) Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261F, and A275S, (4) Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261G, and A275S, (5) Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261Q, and A275S, (6) Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261S, and A275S, (7) Y12N, S18K, I46D, A47E, T55R, H93Y, E104R, L120S, F125W, T135H, S159K, A261K, and A275S, (8) Y12N, S18K, I46D, A47E, T55R, H93Y, E104R, L120T, F125W, T135H, S159K, A261K, and A275S, (9) Y12N, S18K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, and A275S. (10) Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N201E, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F. (11) Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F. (12) Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F. (13) Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261Q, V270T, A275W, I280M, and V290F. (14) Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, A261S, Y269W, A275W, A282G, and V290F. (15) Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, A261S, Y269W, A275W, A282G, and D288T. (16) Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N201E, N233G, E246K, A261Q, Y269W, A275W, A282G, and D288T. (17) Y12N, S18K, I35E, I46D, A47E, T55R, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, A261K, Y269W, A275W, A282G, and D288T. (18) Y12N, S18K, I35E, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N201E, R231W, E246G, Y269W, A275W, A282G, and D288T. (19) Y12N, S18K, I35E, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, Y269W, A275W, A282G, and D288T. (20) Y12N, S18K, I35K, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F. (21) Y12N, S18K, I35K, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261S, V270T, A275W, I280M, and V290F. (22) Y12N, S18K, I35K, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F. (23) Y12N, S18K, I35K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246K, A261S, Y269W, S274G, A275S, A282G, and D288K. (24) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246G, V270T, A275W, I280M, and V290F. (25) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, R200G, R231W, E246K, A261K, V270T, A275W, I280M, and V290F. (26) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N201E, N233G, E246G, V270T, A275W, I280M, and V290F. (27) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N201E, R231W, E246K, A261K, V270T, A275W, I280M, and V290F. (28) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, V270T, A275W, I280M, and V290F. (29) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, V270T, A275W, I280M, and V290F. (30) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246G, A261K, V270T, A275W, I280M, and V290F. (31) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, V270T, A275W, I280M, and V290F. (32) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261K, V270T, A275W, I280M, and V290F. (33) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, R200T, R231W, E246K, A261K, V270T, A275W, I280M, and V290F. (34) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175S, N205D, N233G, E246G, V270T, A275W, I280M, and V290F. (35) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, V230H, E246K, Y269W, A275W, A282G, and D288T. (36) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F. (37) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, V183W, N205D, N233G, E246K, Y269W, S274D, A275S, A282G, and D288T. (38) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, V183W, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F. (39) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, V230H, E246K, A261K, Y269W, S274G, A275S, A282G, and D288T. (40) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, V230Q, E246G, A261K, Y269W, S274G, A275S, A282G, and D288T. (41) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246K, A261K, Y269W, S274G, A275S, A282G, and V290F. (42) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, Y269W, S274G, A275S, A282G, and V290F. (43) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, Y269W, A275W, A282G, and D288T. (44) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246G, Y269W, S274G, A275S, A282G, and V290F. (45) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, Y269W, S274G, A275S, A282G, and D288K. (46) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261K, Y269W, S274D, A275S, A282G, and V290F. (47) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261K, Y269W, S274D, A275S, A282G, and D288K. (48) Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175S, N205D, N233G, E246G, Y269W, A275W, A282G, and D288T. (49) Y12N, S18K, I35S, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F. (50) Y12N, S18K, I35S, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F. (51) Y12N, S18K, I35S, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261Q, V270T, A275W, I280M, and V290F. (52) Y12N, S18K, I35S, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, V230Q, A241G, Y269W, A275W, A282G, and V290F. (53) Y12N, S18K, I35S, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, Y269W, A275W, A282G, and V290F.

[0057] In one embodiment, the N144 variant is sequence numbers 1118, 1120, 1122, 1123, 1125, 1126, 1127, 1129, 1282, 1962, 1963, 1964, 1965, 1970, 1971, 1983, 2004, 2024, 2028, 2371, 2372, 2373, 2390, 2421, 2427, 2428, and 242 9, SEQ ID NOs: 2430, 2431, 2432, 2433, 2434, 2437, 2439, 2446, 2450, 2463, 2464, 2476, 2478, 2485, 2486, 2487, 2489, 2491, 2493, 2494, 2511, 2524, 2526, 2528, 2574, and 2575, each containing an amino acid sequence selected from one of these.

[0058] In another embodiment, the N144 variant is sequence numbers 1118, 1120, 1122, 1123, 1125, 1126, 1127, 1129, 1282, 1962, 1963, 1964, 1965, 1970, 1971, 1983, 2004, 2024, 2028, 2371, 2372, 2373, 2390, 2421, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2437, 243 9. The N144 variant includes an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 2446, SEQ ID NO: 2450, SEQ ID NO: 2463, SEQ ID NO: 2464, SEQ ID NO: 2476, SEQ ID NO: 2478, SEQ ID NO: 2485, SEQ ID NO: 2487, SEQ ID NO: 2489, SEQ ID NO: 2491, SEQ ID NO: 2493, SEQ ID NO: 2494, SEQ ID NO: 2511, SEQ ID NO: 2524, SEQ ID NO: 2526, SEQ ID NO: 2528, SEQ ID NO: 2574, and SEQ ID NO: 2575, wherein the N144 variant includes amino acid substitutions present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO: 295.

[0059] In some embodiments, the N144 variant includes a first set of amino acid substitutions of SEQ ID NO: 295 and a second set of amino acid substitutions of SEQ ID NO: 295. The first set of amino acid substitutions of SEQ ID NO: 295 consists of Y12N, S18K, I46D, A47E, H93Y, E104R, F125W, T135H, and S159K. The second set of amino acid substitutions for Sequence ID No. 295 is one of I35K, I35E, and I35S, one of I54T, T55K, T55R, and T55H, one of T57L, N59D, N102G, one of L120T and L120S, one of L175N, L175A, and L175S, one of V183W, one of R200G and R200T, one of N201E, N205D, one of V230H and V230Q, R231W, N It is a set of about 3 to about 15 amino acid substitutions selected from the group consisting of 233G, A241G, E246K and one of E246G, A261K, A261Q, A261S, A261I, A261F and one of A261G, Y269W, V270T, one of S274G and S274D, one of A275W and A275S, I280M, A282G, D288T and D288K and V290F. In a further embodiment, the N144 variant has an amino acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or at least about 96% identical to SEQ ID NO: 295. In one embodiment, the N144 variant, which includes a first set and a second set of amino acid substitutions of SEQ ID NO: 295, does not have an amino acid mutation at one or more of the following amino acid positions of SEQ ID NO: A32, N33, Q36, F41, D84, E91, M106, H118, E170, S195, N197, N203, S245, K286.In further embodiments, the N144 variant does not have one or more amino acid substitutions of SEQ ID NO: 295, selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, F199L, N203T, K213Q, L223I, S245A, N246E, K250E, K286E, and S306I.

[0060] In one embodiment of the polypeptide variants provided herein, the polypeptide variant is depleted of T cell epitopes compared to the protein from which the variant's amino acid sequence is derived.

[0061] Another aspect of this disclosure provides a fusion protein comprising (i) a first domain comprising one of the polypeptides or variants thereof described herein, and (ii) a second domain. In a further embodiment, the fusion protein comprises a third domain.

[0062] In one embodiment of the fusion protein, the second domain comprises a random coil polypeptide. In another embodiment, the second domain comprises a human immunoglobulin class G (IgG) immunoglobulin fragment crystallizable (Fc) domain. In yet another embodiment, the second domain comprises an albumin protein.

[0063] In yet another aspect of this disclosure, a method is provided for treating a patient for a disease that is mediated whole or partially by an immunoglobulin G (IgG) antibody. The method comprises administering to a patient in need of treatment one of the polypeptides or variants thereof described herein, or one of the fusion proteins. In one embodiment, the disease is chronic inflammatory demyelinating polyneuropathy (CIDP). In another embodiment, the disease is acute inflammatory demyelinating polyneuropathy (AIDP).

[0064] In yet another aspect of this disclosure, a method is provided for preventing or treating antibody-mediated rejection (AMR) of an organ allograft in an organ transplant patient requiring treatment. This method comprises administering to the patient one of the polypeptides or variants thereof, or one of the fusion proteins described herein. In one embodiment, the administration is performed before the transplantation of the organ allograft, for example, about 3 weeks to about 1 hour before the transplantation of the organ allograft. In one embodiment, the organ is a solid organ, such as a kidney, small intestine, pancreas, heart, lung, or liver. [Brief explanation of the drawing]

[0065] [Figure 1] This is a schematic diagram of a PBMC (peripheral blood mononuclear cell) assay used to measure CD4+ T cell activation and epitopes for N142 and wild-type IdeS, as well as selected variants of each. [Figure 2] This graph shows the complete CD4+ T cell epitope mapping of N142 using a PBMC assay. [Figure 3]This graph shows the targeted CD4+ T cell epitope mapping of selected N142 variants compared to parental N142, as determined by a PBMC assay. [Figure 4] This graph shows the complete CD4+ T cell epitope mapping of wild-type IdeS cells using a PBMC assay. [Figure 5] This graph shows the targeted CD4+ T cell epitope mapping of selected IdeS variants compared to wild-type IdeS, as determined by a PBMC assay. [Modes for carrying out the invention]

[0066] Unless otherwise indicated, all figures used herein and in the claims, representing quantities of ingredients, reaction conditions, etc., should be understood in all cases to be modified by the term “approximately.” Therefore, unless otherwise indicated, the numerical parameters described herein and in the appended claims are approximations that may vary depending on the desired properties to be obtained by this application. Generally, as used herein, with respect to measurable values ​​such as weight, time, and dose, the term “approximately” means that the value is within a degree of variation acceptable in the art. In some embodiments, the degree of variation is based on FDA guidelines.

[0067] Furthermore, as used herein, "and / or" means and encompasses any and all possible combinations of one or more of the related enumerated items, as well as the absence of any combination when interpreted as an alternative ("or").

[0068] When used in the context of comparing the thermal stability of an IgG protease variant to the thermal stability of a reference IgG protease, such as the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2, or the IdeZ enzyme containing the amino acid sequence described in SEQ ID NO: 830, the term "substantially identical thermal stability" refers to a measure of thermal stability of the IgG protease variant, such as melting temperature in Celsius, that is identical to or differs by less than 10%, less than 5%, less than 3%, or less than 1% from the same measure of thermal stability of the reference IgG protease.

[0069] For sequence comparison and to determine the "percentage of identity" between two peptide, polypeptide, or nucleic acid sequences, typically one sequence serves as the reference sequence against which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, and if necessary, subsequence coordinates and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity for the test sequence relative to the reference sequence based on the program parameters.

[0070] Suitable algorithms for determining sequence identity percentages and sequence similarity percentages are the BLAST and BLAST2.0 algorithms, described in Altschul et al. (1990) J.Mol.Biol.215:403-410 and Altschul et al. (1997). Nucleic Acids Res.25, pp.3389-3402, respectively. Software for performing BLAST analysis is publicly available through 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 in the query sequence that, 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 (Altschul et al. (1997). Nucleic Acids Res. 25, pp. 3389-3402). These initial neighbor word hits act as seeds to initiate a search for longer HSPs containing them. Word hits are then extended in both directions along each sequence as long as the cumulative alignment score can increase. For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of word hits in each direction stops when the cumulative alignment score falls by an amount X from its maximum achieved value, when the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment.For amino acid sequences, the BLASTP program uses a word size (W) of 3, an estimated value (E) of 10, and the BLOSUM62 scoring matrix by default (see Henikoff & Henikoff. (1992). Proc. Natl. Acad. Sci. USA 89, pp. 10915-10919).

[0071] The BLAST algorithm also performs 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 occurs by chance.

[0072] A “T cell epitope” refers to a peptide epitope that binds to an MHCII molecule and subsequently forms a ternary complex with a congener T cell receptor (TCR). In aspects of this disclosure, recombinant IgG proteases or polynucleotides containing them are provided that are “T cell epitope depleted” compared to a corresponding IgG protease protein. A protein is “T cell epitope depleted” if one or more amino acid mutations are present in one or more T cell epitopes, resulting in the T cell epitopes in the IgG protease being depleted compared to a corresponding IgG protease protein, e.g., wild-type (WT) IgG protease protein. In one embodiment, if a peptide derived from an IgG protease variant cannot bind to an MHCII molecule, or if the peptide binds to an MHCII molecule but does not subsequently bind to a TCR, for example, due to a lack of affinity for the MHCII molecule, then one or more T cell epitopes are “depleted” from the IgG protease variant. Such IgG protease variants are, in some cases, referred to herein as “immune-deimmunized IgG protease variants” or “T-cell epitope depletion” IgG protease variants. The use of such variants and methods for producing them are also described herein.

[0073] The terms “variant protein,” “protein variant,” “polypeptide variant,” and “variant polypeptide” are terms of the art and are used interchangeably herein. Each refers to a protein that is distinguished from a corresponding protein, such as the WT form of the protein, based on the presence of one or more amino acid modifications, such as one or more amino acid substitutions, insertions, deletions, or combinations thereof. In this disclosure, the terms “variant protein,” “protein variant,” “polypeptide variant,” and “variant polypeptide” encompass the full-length and cleaved forms of IgG protease variants, such as catalytically active fragments of IgG protease variants. The term “mutant gene” is a term of the art and refers to a gene that is distinguished from the WT form of the gene based on the presence of one or more nucleic acid modifications, such as one or more nucleic acid substitutions, insertions, deletions, or combinations thereof. In some embodiments, a mutant gene encodes a variant protein. However, mutations in a gene may also be silent mutations, i.e., mutations that do not affect the amino acid sequence in the protein encoded by the gene. Mutants / variants may be naturally occurring or engineered.

[0074] When it is disclosed that one IgG protease variant is several percent identical to another IgG protease variant (reference variant), the IgG protease variant includes amino acid mutations present in the reference variant. For example, if IgG protease variant A' is 90% identical to (reference) IgG protease variant A, then IgG protease variant A' also includes amino acid mutations present in IgG protease variant A.

[0075] As used herein, the term “wild type” (abbreviated as “WT”) means, unless otherwise specified, the most common form of an organism, strain, gene, protein, or characteristic that is naturally occurring and / or distinguishable from any variant or mutated form.

[0076] The full-length amino acid sequence of IdeS is publicly available as NCBI reference sequence number WP010922160.1 and is provided herein as Sequence ID No. 1. The full-length sequence includes an N-terminal methionine followed by a secretion signal sequence of 28 amino acids. The N-terminal methionine and signal sequence (a total of 29 amino acids at the N-terminus) are typically removed to form the mature IdeS protein, whose sequence is publicly available as Genbank accession number ADF13949.1 and is provided herein, together with the N-terminal methionine residue, as Sequence ID No. 2. The IgG protease variants described herein include (i) polypeptides containing an N-terminal methionine and secretion signal sequence, and (ii) polypeptides that do not contain an N-terminal methionine and secretion signal sequence.

[0077] Unless otherwise specified, all references to amino acid position numbering in the IdeS variant polypeptides disclosed herein are based on the numbering of the corresponding positions in SEQ ID NO: 2, starting from the N-terminal methionine residue. Thus, SEQ ID NO: 2 is otherwise identical to SEQ ID NO: 1, except that it lacks the 28-amino acid signal sequence.

[0078] If an N-terminal methionine residue is absent in a polypeptide disclosed herein, an alternative embodiment includes an IgG protease variant of the same sequence that includes an N-terminal methionine residue. Similarly, if an N-terminal methionine residue is present in an IgG protease variant, an alternative embodiment includes an IgG protease variant of the same sequence that does not include an N-terminal methionine residue. Similarly, if a signal sequence (e.g., a secretion signal sequence) is absent in a polypeptide disclosed herein, an alternative embodiment includes an IgG protease variant of the same sequence that includes a signal sequence. Alternatively, if a signal sequence is present in an IgG protease variant, an alternative embodiment includes an IgG protease variant of the same sequence that does not include a signal sequence and does not include an N-terminal methionine residue.

[0079] In one embodiment, the present disclosure provides a variant of IdeS IgG protease ("IdeS variant"). The IdeS IgG protease provided above comprises the amino acid sequence described in SEQ ID NO: 2. The IdeS variant has (i) the following amino acid positions in SEQ ID NO: 2: S3, F4, A6, E9, I10, R11, Y12, Y18, V46, A47, N48, I54, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, (ii) Contains one or more amino acid mutations in one or more of the following: T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, G296, and (ii) an amino acid sequence that is at least approximately 75% identical to SEQ ID NO: 2.

[0080] In one embodiment, the IdeS variant does not have amino acid mutations at the following amino acid positions in SEQ ID NO: K56, C66, H234, D256, and D258. In a further embodiment or another embodiment, the IdeS variant does not have amino acid mutations at one or more of the following amino acid positions in SEQ ID NO: A32, N33, T35, Q36, F41, D84, R88, E91, N102, M106, N117, H118, E170, S195, N197, K213, S245, K250, A261, K286, and S306.

[0081] In one embodiment of the IdeS variant, mutations in the IdeS variant at one or more of the aforementioned amino acid positions eliminate one or more dominant immunogenic T cell epitopes present in the WT IdeS enzyme, resulting in the IdeS variant being T cell epitope depleted compared to WT IdeS (i.e., polypeptide of SEQ ID NO: 2).

[0082] The amino acid mutations of this disclosure include, but are not limited to, amino acid exchanges, insertions, deletions, additions, substitutions, inversions, and / or duplications. These mutations / modifications also include conservative and / or homologous amino acid exchanges. In preferred embodiments of the present invention, one or more mutations in the IdeS variant are one or more amino acid substitutions.

[0083] In one embodiment, the IdeS variant contains 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more amino acid mutations compared to the wild-type IdeS enzyme of SEQ ID NO: 2, where the mutations are at the following amino acid positions of SEQ ID NO: S3, F4, A6, E9, I10, R11, Y12, Y18, V46, A47, N48, I The mutations (e.g., substitutions) are selected from 54, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, and G296. In a further embodiment, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid mutations are two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid substitutions. In a further embodiment, the amino acid substitution is selected from the amino acid substitutions of Sequence ID No. 2 listed in Table 1.

[0084] In one embodiment, the IdeS variant is WT of SEQ ID NO: 2 The IdeS enzyme contains approximately 5 to 23 amino acid mutations, selected from the following amino acid positions in Sequence ID No. 2: S3, F4, A6, E9, I10, R11, Y12, Y18, V46, A47, N48, I54, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, and G296. In further embodiments, approximately 5 to 23 amino acid mutations are approximately 5 to 23 amino acid substitutions. In further embodiments, approximately 5 to 23 amino acid substitutions are selected from the amino acid substitutions of Sequence ID No. 2 listed in Table 1.

[0085] In another embodiment, the IdeS variant has approximately 5 to 20 amino acid mutations, approximately 10 to 20 amino acid mutations, approximately 10 to 18 amino acid mutations, approximately 10 to 16 amino acid mutations, approximately 10 to 14 amino acid mutations, approximately 10 to 13 amino acid mutations, approximately 10 to 12 amino acid mutations, approximately 11 to 20 amino acid mutations, approximately 11 to 13 amino acid mutations, approximately 12 to 20 amino acid mutations, approximately 12 to 14 amino acid mutations, approximately 13 to 20 amino acid mutations, approximately 14 to 20 amino acid mutations, approximately 15 to 20 amino acid mutations, approximately 10 to 35 amino acid mutations, approximately 15 to 30 amino acid mutations, and approximately 20 to 30 amino acid mutations compared to the WT IdeS enzyme of SEQ ID NO: 2 It contains approximately 24 to 35 amino acid mutations, approximately 24 to 32 amino acid mutations, or approximately 24 to 29 amino acid mutations, and the mutations are at the following amino acid positions in SEQ ID NO: S3, F4, A6, E9, I10, R11, Y12, Y18, V46, A47, N48, I54, T57, N59, G60, K61, E92, H93, E104 The mutations are selected from L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, and G296. In a further embodiment, each of the amino acid mutations is an amino acid substitution. In a further embodiment, the amino acid substitution is selected from the amino acid substitutions of Sequence ID No. 2 listed in Table 1.

[0086] In one embodiment, the IdeS variant is WT of SEQ ID NO: 2 The IdeS enzyme contains approximately 5 to 20 amino acid mutations, selected from the following amino acid residues in Sequence ID No. 2: S3, F4, A6, E9, I10, R11, Y12, Y18, V46, A47, N48, I54, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, and G296. In further embodiments, each amino acid mutation is an amino acid substitution. In further embodiments, approximately 5 to 20 amino acid substitutions are selected from the amino acid substitutions of Sequence ID No. 2 listed in Table 1.

[0087] In one embodiment, the IdeS variant is WT of SEQ ID NO: 2 The IdeS enzyme contains approximately 10 to 20 amino acid mutations, selected from the following amino acid residues of SEQ ID NO: S3, F4, A6, E9, I10, R11, Y12, Y18, V46, A47, N48, I54, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, G296. In further embodiments, each amino acid mutation is an amino acid substitution. In further embodiments, about 10 to 20 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO: 2 listed in Table 1.

[0088] In one embodiment, the IdeS variant is WT of SEQ ID NO: 2 The IdeS enzyme contains approximately 12 to 20 amino acid mutations, selected from the following amino acid residues of SEQ ID NO: S3, F4, A6, E9, I10, R11, Y12, Y18, V46, A47, N48, I54, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, G296. In further embodiments, each amino acid mutation is an amino acid substitution. In further embodiments, about 12 to about 20 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO: 2 listed in Table 1.

[0089] In one embodiment, the IdeS variant is WT of SEQ ID NO: 2 The IdeS enzyme contains approximately 15 to 20 amino acid mutations, selected from the following amino acid residues of SEQ ID NO: S3, F4, A6, E9, I10, R11, Y12, Y18, V46, A47, N48, I54, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159, T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, G296. In further embodiments, each amino acid mutation is an amino acid substitution. In further embodiments, about 15 to 20 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO: 2 listed in Table 1.

[0090] In another embodiment, the IdeS variant includes one of the sets of amino acid substitutions of SEQ ID NO: 2 listed in Table 2 of this specification.

[0091] In yet another embodiment, the IdeS variant includes one of the sets of amino acid substitutions of SEQ ID NO: 2 listed in Table 3 of this specification.

[0092] In yet another embodiment, the IdeS variant includes one of the sets of amino acid substitutions of SEQ ID NO: 2 listed in Table 4 of this specification.

[0093] [Table 1]

[0094] [Table 2]

[0095] [Table 3]

[0096] [Table 4-1]

[0097] [Table 4-2]

[0098] [Table 4-3]

[0099] [Table 4-4]

[0100] [Table 4-5]

[0101] [Table 4-6]

[0102] Table 4-7

[0103] Table 4-8

[0104] Table 4-9

[0105] Table 4-10

[0106] Table 4-11

[0107] Table 4-12

[0108] Table 4-13

[0109] Table 4-14

[0110] Table 4-15

[0111] Table 4-16

[0112] [Table 4-17]

[0113] [Table 4-18]

[0114] [Table 4-19]

[0115] In some embodiments, the IdeS variant is selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, N102G, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, K213Q, N233G, S245A, N246E, K250E, A261P, K286E, and S306I, and does not have one or more amino acid substitutions of SEQ ID NO: 2.

[0116] In some embodiments, the IdeS variant has an amino acid sequence that is at least about 75% identical to SEQ ID NO: 2. In some embodiments, the IdeS variant has an amino acid sequence that is at least about 80% identical to SEQ ID NO: 2. In some embodiments, the IdeS variant has an amino acid sequence that is at least about 85% identical to SEQ ID NO: 2. In some embodiments, the IdeS variant has an amino acid sequence that is at least about 90% identical to SEQ ID NO: 2. In some embodiments, the IdeS variant has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 2. In some embodiments, the IdeS variant has an amino acid sequence that is at least about 97% identical to SEQ ID NO: 2. In some embodiments, the IdeS variant has an amino acid sequence that is at least about 98% identical to SEQ ID NO: 2.

[0117] In some embodiments, the IdeS variant includes an amino acid sequence described in one of SEQ ID NOs: 3 to 26. In further embodiments, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence described in one of SEQ ID NOs: 3 to 26, and the IdeS variant includes amino acid substitutions present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO: 2.

[0118] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 3. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 3, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 3 relative to SEQ ID NO: 2.

[0119] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 4. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 4, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 4 relative to SEQ ID NO: 2.

[0120] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 5. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 5, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 5 relative to SEQ ID NO: 2.

[0121] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 6. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 6, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 6 with respect to SEQ ID NO: 2.

[0122] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 7. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 7, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 7 with respect to SEQ ID NO: 2.

[0123] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 8. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 8, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 8 with respect to SEQ ID NO: 2.

[0124] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 9. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 9, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 9 with respect to SEQ ID NO: 2.

[0125] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 10. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 10, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 10 with respect to SEQ ID NO: 2.

[0126] In one embodiment, the IdeS variant comprises the amino acid sequence described in SEQ ID NO: 11. In a further embodiment, the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 11, and the IdeS variant comprises amino acid substitutions present in the amino acid sequence of SEQ ID NO: 11 with respect to SEQ ID NO: 2.

[0127] In one embodiment, the IdeS variant comprises the amino acid sequence described in SEQ ID NO: 12. In a further embodiment, the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 12, and the IdeS variant comprises amino acid substitutions present in the amino acid sequence of SEQ ID NO: 12 relative to SEQ ID NO: 2.

[0128] In one embodiment, the IdeS variant comprises the amino acid sequence described in SEQ ID NO: 13. In a further embodiment, the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 13, and the IdeS variant comprises amino acid substitutions present in the amino acid sequence of SEQ ID NO: 13 relative to SEQ ID NO: 2.

[0129] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 14. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 14, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 14 with respect to SEQ ID NO: 2.

[0130] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 15. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 15, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 15 relative to SEQ ID NO: 2.

[0131] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 16. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 16, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 16 with respect to SEQ ID NO: 2.

[0132] In one embodiment, the IdeS variant comprises the amino acid sequence described in SEQ ID NO: 17. In a further embodiment, the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 17, and the IdeS variant comprises amino acid substitutions present in the amino acid sequence of SEQ ID NO: 17 relative to SEQ ID NO: 2.

[0133] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 18. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 18, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 18 with respect to SEQ ID NO: 2.

[0134] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 19. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 19, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 19 with respect to SEQ ID NO: 2.

[0135] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 20. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 20, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 20 with respect to SEQ ID NO: 2.

[0136] In one embodiment, the IdeS variant comprises the amino acid sequence described in SEQ ID NO: 21. In a further embodiment, the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 21, and the IdeS variant comprises amino acid substitutions present in the amino acid sequence of SEQ ID NO: 21 with respect to SEQ ID NO: 2.

[0137] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 22. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 22, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 22 with respect to SEQ ID NO: 2.

[0138] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 23. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 23, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 23 relative to SEQ ID NO: 2.

[0139] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 24. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 24, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 24 with respect to SEQ ID NO: 2.

[0140] In one embodiment, the IdeS variant includes the amino acid sequence described in SEQ ID NO: 25. In a further embodiment, the IdeS variant includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 25, and the IdeS variant includes amino acid substitutions present in the amino acid sequence of SEQ ID NO: 25 relative to SEQ ID NO: 2.

[0141] In one embodiment, the IdeS variant comprises the amino acid sequence described in SEQ ID NO: 26. In a further embodiment, the IdeS variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 26, and the IdeS variant comprises amino acid substitutions present in the amino acid sequence of SEQ ID NO: 26 relative to SEQ ID NO: 2.

[0142] In some embodiments, the IdeS variant includes an amino acid sequence described in one of SEQ ID NOs: 1491 to 1951. In further embodiments, the IdeS variant of the present disclosure includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence described in one of SEQ ID NOs: 1491 to 1951, and the IdeS variant includes an amino acid substitution present in the reference amino acid sequence with respect to SEQ ID NO: 2. For example, an IdeS variant that is 85% identical to the variant of SEQ ID NO: 1491 includes an amino acid substitution present in SEQ ID NO: 1491 (a substitution for SEQ ID NO: 2).

[0143] In some embodiments, the IdeS variant includes an amino acid sequence selected from one of SEQ ID NOs: 1492, 1697, 1700, 1701, 1702, 1703, 1704, 1705, 1713, 1720, 1722, 1723, 1724, 1772, 1774, 1775, 1793, 1795, 1797, 1798, and 1949. In further embodiments, the IdeS variant of the present disclosure includes an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the reference amino acid sequence described in one of SEQ ID NOs: 1492, 1697, 1700, 1701, 1702, 1703, 1704, 1705, 1713, 1720, 1722, 1723, 1724, 1772, 1774, 1775, 1793, 1795, 1797, 1798, and 1949, wherein the IdeS variant includes, with respect to SEQ ID NO: 2, an amino acid substitution present in the reference amino acid sequence.

[0144] In some embodiments, the IdeS variant includes a first set of amino acid substitutions of SEQ ID NO: 2 and a second set of amino acid substitutions of SEQ ID NO: 2. The first set of amino acid substitutions of SEQ ID NO: 2 consists of Y12N, Y18K, A47E, H93Y, E104R, L120T, E126S, Y135N, S159K, V230S, and A275S. The second set of amino acid substitutions in Sequence ID No. 2 is a set of approximately 13 to 18 amino acid substitutions selected from the group consisting of S3N, F4I, A6S, E9I, I10T, R11T, N48G, I54T, T57K, T57N, T57R, T57L, and one of T57Q, one of N59D, G60S, and G60T, K61R, E92R, one of T138A, T161G, and T161Y, D188T, F199L, K200R, E201N, N203T, G222A, L223I, N233S, N246R, N273G, D288G, and Q293R. In one embodiment, an IdeS variant containing a first set and a second set of amino acid substitutions of SEQ ID NO: 2 has an amino acid sequence that is at least about 88% identical to SEQ ID NO: 2. In another embodiment, an IdeS variant has an amino acid sequence that is at least about 89%, at least about 90%, at least about 91%, or at least about 92% identical to SEQ ID NO: 2. In yet another embodiment, an IdeS variant has an amino acid sequence that is about 88% to about 92%, about 89% to about 92%, or about 90% to about 92% identical to SEQ ID NO: 2. In one embodiment, an IdeS variant containing a first set and a second set of amino acid substitutions of SEQ ID NO: 2 does not have amino acid mutations at the following amino acid positions of SEQ ID NO: K56, C66, H234, D256, and D258. In another embodiment, the IdeS variant does not have an amino acid mutation at one or more of the following amino acid positions in SEQ ID NO: A32, N33, T35, Q36, F41, D84, R88, E91, N102, M106, N117, H118, E170, S195, N197, K213, S245, K250, A261, K286, S306.In further embodiments, the IdeS variant is selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, N102G, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, K213Q, N233G, S245A, N246E, K250E, A261P, K286E, and S306I, and does not have one or more amino acid substitutions of SEQ ID NO: 2.

[0145] In one embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of approximately 13 to 17 amino acid substitutions. In another embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of approximately 13 to 16 amino acid substitutions. In yet another embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of approximately 13 to 15 amino acid substitutions. In yet another embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of 13, 14, 15, 16, 17, or 18 amino acid substitutions.

[0146] In some embodiments, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 18 amino acid substitutions selected from the group consisting of N48G, I54T, T57K, T57N, T57R, and T57L, N59D, G60S, and G60T, K61R, E92R, T138A, T161G, and T161Y, D188T, F199L, K200R, E201N, N203T, L223I, N233S, N246R, N273G, D288G, and Q293R. In one embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 17 amino acid substitutions. In another embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 16 amino acid substitutions. In another embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of approximately 13 to approximately 15 amino acid substitutions. In yet another embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of 13, 14, 15, 16, 17, or 18 amino acid substitutions.

[0147] In some embodiments, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 18 amino acid substitutions selected from the group consisting of N48G, I54T, T57K and T57N, N59D, G60S and G60T, K61R, E92R, T138A, T161G and T161Y, D188T, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R. In one embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 17 amino acid substitutions. In another embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 16 amino acid substitutions. In yet another embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 15 amino acid substitutions. In another embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of 13, 14, 15, 16, 17, or 18 amino acid substitutions.

[0148] In some embodiments, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 18 amino acid substitutions selected from the group consisting of N48G, I54T, T57K and T57N, N59D, G60S, K61R, E92R, T138A, T161G and T161Y, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R. In one embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 17 amino acid substitutions. In another embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 16 amino acid substitutions. In yet another embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 15 amino acid substitutions. In another embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of 13, 14, 15, 16, 17, or 18 amino acid substitutions.

[0149] In some embodiments, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 18 amino acid substitutions selected from the group consisting of N48G, I54T, T57K and T57N, N59D, G60T, K61R, E92R, T138A, T161G and T161Y, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R. In one embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 17 amino acid substitutions. In another embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 16 amino acid substitutions. In yet another embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 15 amino acid substitutions. In another embodiment, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of 13, 14, 15, 16, 17, or 18 amino acid substitutions.

[0150] In some embodiments, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of about 13 to about 17 amino acid substitutions selected from the group consisting of N48G, I54T, T57K, N59D, G60S, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

[0151] In some embodiments, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 15 amino acid substitutions selected from the group consisting of N48G, I54T, N59D, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

[0152] In some embodiments, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 14 amino acid substitutions selected from the group consisting of N48G, N59D, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

[0153] In some embodiments, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of 13 amino acid substitutions consisting of N48G, N59D, K61R, E92R, T138A, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

[0154] In some embodiments, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of 13 amino acid substitutions consisting of S3N, F4I, A6S, N48G, I54T, T57N, N59D, T138A, N233S, N246R, N273G, D288G, and Q293R.

[0155] In some embodiments, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of 15 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, T138A, D188T, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

[0156] In some embodiments, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of 17 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161G, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.

[0157] In some embodiments, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of 16 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

[0158] In some embodiments, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of 17 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N203T, N233S, N246R, N273G, D288G, and Q293R.

[0159] In some embodiments, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of 17 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161G, D188T, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

[0160] In some embodiments, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of 16 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161Y, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

[0161] In some embodiments, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of 17 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161Y, D188T, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

[0162] In some embodiments, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of 16 amino acid substitutions consisting of N48G, T57Q, N59D, G60T, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

[0163] In some embodiments, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of 17 amino acid substitutions consisting of N48G, T57R, N59D, G60T, K61R, E92R, T138A, T161G, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.

[0164] In some embodiments, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of 17 amino acid substitutions consisting of N48G, T57R, N59D, G60T, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N203T, N233S, N246R, N273G, D288G, and Q293R.

[0165] In some embodiments, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of 18 amino acid substitutions consisting of N48G, T57R, N59D, G60T, K61R, E92R, T138A, T161G, D188T, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.

[0166] In some embodiments, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of 16 amino acid substitutions consisting of N48G, T57R, N59D, G60T, K61R, E92R, T138A, T161Y, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

[0167] In some embodiments, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of 13 amino acid substitutions consisting of N48G, I54T, T57L, N59D, T138A, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

[0168] In some embodiments, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of 16 amino acid substitutions consisting of N48G, I54T, T57L, N59D, E92R, T138A, T161G, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.

[0169] In some embodiments, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of 15 amino acid substitutions consisting of N48G, I54T, T57L, N59D, E92R, T138A, T161Y, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

[0170] In some embodiments, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of 15 amino acid substitutions consisting of N48G, I54T, T57N, N59D, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

[0171] In some embodiments, the second set of amino acid substitutions of SEQ ID NO: 2 is a set of 17 amino acid substitutions consisting of N48G, I54T, T57N, N59D, E92R, T138A, T161G, D188T, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.

[0172] In some embodiments, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of 13 amino acid substitutions consisting of N48G, I54T, T57N, N59D, E92R, T138A, T161Y, G222A, L223I, N233S, N246R, N273G, and D288G.

[0173] In some embodiments, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of 15 amino acid substitutions consisting of N48G, I54T, T57N, N59D, E92R, T138A, T161Y, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

[0174] In some embodiments, the second set of amino acid substitutions in SEQ ID NO: 2 is a set of 13 amino acid substitutions consisting of E9I, I10T, R11T, N48G, I54T, T57N, N59D, T138A, N233S, N246R, N273G, D288G, and Q293R.

[0175] In some embodiments, the IdeS variants disclosed herein have at least 25% of the activity of the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2 in an IgG protease enzyme assay. In some embodiments, the IdeS variants have at least 30% of the activity of the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2 in an IgG protease enzyme assay. In some embodiments, the IdeS variants have at least about 40% of the activity of the IdeS enzyme containing the amino acid sequence of SEQ ID NO: 2, at least about 50% of the activity, at least about 60% of the activity, at least about 70% of the activity, at least about 75% of the activity, at least about 80% of the activity, at least about 90% of the activity, at least about 100% of the activity, at least about 110% of the activity, and at least about 120% of the activity of the IdeS enzyme containing the amino acid sequence of SEQ ID NO: 2 in an IgG protease enzyme assay. In some embodiments, the IdeS variant exhibits at least about 25% activity, at least about 40% activity, at least about 50% activity, at least about 60% activity, at least about 70% activity, at least about 80% activity, at least about 90% activity, at least about 100% activity, at least about 110% activity, and at least about 120% activity of the IdeZ enzyme containing the amino acid sequence of SEQ ID NO: 830 in an IgG protease enzyme assay. In some embodiments, the IgG protease activity in an IgG protease enzyme assay is measured by an enzyme-linked immunosorbent assay that includes digestion of an immobilized antibody substrate.

[0176] In some embodiments, the IdeS variant is depleted of one or more T cell epitopes compared to the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2, for example, two or more, three or more, or four or more T cell epitopes.

[0177] In some embodiments, the IdeS variant has substantially the same thermal stability as the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2. In other embodiments, the IdeS variant has higher thermal stability than the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2. In some embodiments, thermal stability is measured by differential scanning fluorescence quantification.

[0178] In some embodiments, the IdeS variant has substantially the same thermal stability as the IdeZ enzyme containing the amino acid sequence described in SEQ ID NO: 830. In other embodiments, the IdeS variant is more thermally stable than the IdeZ enzyme containing the amino acid sequence described in SEQ ID NO: 830. In some embodiments, thermal stability is measured by differential scanning fluorescence quantification.

[0179] In another aspect of the present disclosure, an IgG protease comprising an amino acid sequence selected from one of SEQ ID NOs: 27 to 302 is provided. In another embodiment, an IgG protease variant is provided which is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the amino acid sequence of each IgG protease from which the variant is derived.

[0180] In one embodiment, an IgG protease is provided comprising an amino acid sequence selected from one of SEQ ID NOs: 36, 37, 39, 41, 50, 51, 54, 56, 60, 61, 65, 67, 137, 140, 144, 150, 159, 285, 286, 287, 288, 289, 292, 293, 294, 295, 296, 297, and 298. In another embodiment, an IgG protease variant is provided, comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the amino acid sequence of each IgG protease from which the variant is derived.

[0181] In further embodiments, an IgG protease is provided comprising an amino acid sequence selected from one of SEQ ID NOs. 54, 140, 293, 294, 295, 297, and 298. In another embodiment, an IgG protease variant is provided, comprising an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to the amino acid sequence of each IgG protease from which the variant is derived.

[0182] In one embodiment of the IgG protease, the IgG protease comprises the amino acid sequence described in SEQ ID NO: 54. In another embodiment, an IgG protease variant is provided, which comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to SEQ ID NO: 54.

[0183] In one embodiment of the IgG protease, the IgG protease comprises the amino acid sequence described in SEQ ID NO: 140. In another embodiment, an IgG protease variant is provided, which comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to SEQ ID NO: 140.

[0184] In one embodiment of the IgG protease, the IgG protease comprises the amino acid sequence described in SEQ ID NO: 293. In another embodiment, an IgG protease variant is provided, which comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to SEQ ID NO: 293.

[0185] In one embodiment of the IgG protease, the IgG protease comprises the amino acid sequence described in SEQ ID NO: 294. In another embodiment, an IgG protease variant is provided, which comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to SEQ ID NO: 294.

[0186] In one embodiment of the IgG protease, the IgG protease comprises the amino acid sequence described in SEQ ID NO: 295. In another embodiment, an IgG protease variant is provided, which comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to SEQ ID NO: 295.

[0187] In one embodiment of the IgG protease, the IgG protease comprises the amino acid sequence described in SEQ ID NO: 297. In another embodiment, an IgG protease variant is provided, which comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to SEQ ID NO: 297.

[0188] In one embodiment of the IgG protease, the IgG protease comprises the amino acid sequence described in SEQ ID NO: 298. In another embodiment, an IgG protease variant is provided, which comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, or at least about 95% identical to SEQ ID NO: 298.

[0189] Unless otherwise specified, all references to the numbering of amino acid positions in the N142 variants disclosed herein are based on the numbering of the corresponding positions in SEQ ID NO: 297, starting with the N-terminal methionine residue (i.e., residue 1 in SEQ ID NO: 297). As provided herein, the variants of this disclosure include both (i) polypeptides containing an N-terminal methionine and (ii) polypeptides not containing an N-terminal methionine.

[0190] In one aspect of the present disclosure, a variant of a predicted N142 IgG protease is provided. The N142 IgG protease provided above comprises the amino acid sequence described in SEQ ID NO: 297. In one embodiment, the N142 variant comprises (i) one or more amino acid mutations at the following amino acid positions of SEQ ID NO: 297: I10, R11, Y12, V15, I35, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, K129, R157, S159, L160, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300, and (ii) an amino acid sequence that is at least about 75% identical to SEQ ID NO: 297.

[0191] In one embodiment, an N142 variant having one or more mutations in one or more of the aforementioned amino acid positions is T cell epitope depleted compared to the WT N142 enzyme containing the amino acid sequence of SEQ ID NO: 297. In other words, in some embodiments, the N142 variant is depleted of one or more dominant immunogenic T cell epitopes compared to the immunogenic T cell epitopes present in the WT N142 enzyme containing the amino acid sequence of SEQ ID NO: 297. Amino acid mutations used herein include, but are not limited to, amino acid exchanges, insertions, deletions, additions, substitutions, inversions, and / or duplications. These mutations / modifications also include conservative and / or homologous amino acid exchanges. In preferred embodiments of the present invention, one or more mutations are one or more amino acid substitutions.

[0192] In one embodiment, the N142 variant contains one or more amino acid mutations at the following amino acid positions in SEQ ID NO: 297: I10, R11, Y12, V15, I35, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, L300.

[0193] In some embodiments, the N142 variant does not have an amino acid mutation at one or more of the following amino acid positions in SEQ ID NO: 297: A32, N33, Q36, F41, D84, R88, E91, E92, H93, M106, N117, H118, E126, E170, N197, N203, K213, L223, S245, K286, S306.

[0194] In one embodiment, the N142 variant is a WT containing the amino acid sequence of SEQ ID NO: 297. The N142 enzyme contains 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more amino acid mutations, and the mutations are selected from the following amino acid positions of SEQ ID NO: 297: I10, R11, Y12, V15, I35, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, K129, R157, S159, L160, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300. In further embodiments, the mutations are selected from the following amino acid positions of SEQ ID NO: 297: I10, R11, Y12, V15, I35, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, L300. In further embodiments, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid mutations are two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid substitutions. In further embodiments, the amino acid substitution is selected from the amino acid substitutions of Sequence ID No. 297 listed in Table 5.In further embodiments, the amino acid substitutions of SEQ ID NO: 297 include one of I10A and I10Q, one of R11T, Y12K, one of V15H and V15K, one of I35T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G, L120T, Y127L, K129G, and R15 The following are selected from the group consisting of 7G, S159G, L160W, V230G, V230R, and V230S, one of N233S, K250S, A261K, F269N, S274D, A275F, A275S, and A275W, one of D288H and D288K, Q293K, V294A, L300T, and combinations thereof.

[0195] In one embodiment, the N142 variant has approximately 5 to 25 amino acid mutations, approximately 5 to 20 amino acid mutations, approximately 5 to 15 amino acid mutations, approximately 8 to 20 amino acid mutations, approximately 10 to 20 amino acid mutations, approximately 10 to 18 amino acid mutations, approximately 10 to 16 amino acid mutations, approximately 10 to 14 amino acid mutations, approximately 10 to 13 amino acid mutations, approximately 10 to 12 amino acid mutations, approximately 11 to 20 amino acid mutations, approximately 11 to 13 amino acid mutations, approximately 12 to 14 amino acid mutations, approximately 13 to 20 amino acid mutations, approximately 14 to 20 amino acid mutations, and approximately 15 to 2 The amino acid mutations include 0 amino acid mutations, approximately 16 to 20 amino acid mutations, approximately 16 to 25 amino acid mutations, or approximately 16 to 17 amino acid mutations, and the amino acid mutations include at least 5 or at least 10 amino acid mutations at the following amino acid positions of SEQ ID NO: 297: I10, R11, Y12, V15, I35, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, K129, R157, S159, L160, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300. In further embodiments, the amino acid mutations include at least five or at least ten amino acid mutations at the following amino acid positions in SEQ ID NO: 297: I10, R11, Y12, V15, I35, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, L300. In further embodiments, each of the amino acid mutations is an amino acid substitution. In further embodiments, the at least five or at least ten amino acid substitutions at the above amino acid positions are selected from the at least five or at least ten amino acid substitutions of SEQ ID NO: 297 listed in Table 5.In further embodiments, at least five or at least ten amino acid substitutions in SEQ ID NO: 297 are one of I10A and I10Q, one of R11T, Y12K, one of V15H and V15K, one of I35T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G, L120T, Y127L, The following are selected from the group consisting of K129G, R157G, S159G, L160W, V230G, V230R and V230S, one of N233S, K250S, A261K, F269N, S274D, A275F, A275S and A275W, one of D288H and D288K, Q293K, V294A, L300T, and combinations thereof.

[0196] In one embodiment, the N142 variant contains approximately 5 to 20 amino acid mutations compared to the WT N142 enzyme of SEQ ID NO: 297. In a further embodiment, approximately 5 to 20 amino acid mutations are approximately 5 to 20 amino acid substitutions compared to the wild-type N142 enzyme of SEQ ID NO: 297. In one embodiment, the N142 variant contains approximately 5 to 20 amino acid mutations compared to the WT N142 enzyme of SEQ ID NO: 297, and the amino acid mutations include approximately 5 to 20 amino acid mutations in the following amino acid residues of SEQ ID NO: 297: I10, R11, Y12, V15, I35, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, K129, R157, S159, L160, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300. In further embodiments, the amino acid mutations include approximately 5 to 20 amino acid mutations in the following amino acid residues of SEQ ID NO: 297: I10, R11, Y12, V15, I35, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, and L300. In further embodiments, each of the amino acid mutations is an amino acid substitution. In further embodiments, approximately 5 to 20 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO: 297 listed in Table 5. In further embodiments, the approximately 5 to 20 amino acid substitutions in SEQ ID NO: 297 are one of I10A and I10Q, R11T, Y12K, one of V15H and V15K, I35T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G, L120T, Y127L, K129 The following are selected from the group consisting of G, R157G, S159G, L160W, V230G, V230R, and V230S, one of N233S, K250S, A261K, F269N, S274D, A275F, A275S, and A275W, one of D288H and D288K, Q293K, V294A, L300T, and combinations thereof.

[0197] In one embodiment, the N142 variant contains approximately 5 to 16 amino acid mutations compared to the wild-type N142 enzyme of SEQ ID NO: 297. In a further embodiment, the approximately 5 to 16 amino acid mutations are approximately 5 to 16 amino acid substitutions compared to the WT N142 enzyme of SEQ ID NO: 297. In one embodiment, the N142 variant contains approximately 5 to 16 amino acid mutations compared to the WT N142 enzyme of SEQ ID NO: 297, and the amino acid mutations include approximately 5 to 16 amino acid mutations in the following amino acid residues of SEQ ID NO: 297: I10, R11, Y12, V15, I35, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, K129, R157, S159, L160, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300. In further embodiments, the amino acid mutations include approximately 5 to 16 amino acid mutations in the following amino acid residues of SEQ ID NO: 297: I10, R11, Y12, V15, I35, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, and L300. In further embodiments, each of the amino acid mutations is an amino acid substitution. In further embodiments, approximately 5 to 16 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO: 297 listed in Table 5. In further embodiments, approximately 5 to 16 amino acid substitutions in Sequence ID No. 297 are one of I10A and I10Q, R11T, Y12K, one of V15H and V15K, I35T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G, L120T, Y127L, K129 The following are selected from the group consisting of G, R157G, S159G, L160W, V230G, V230R, and V230S, one of N233S, K250S, A261K, F269N, S274D, A275F, A275S, and A275W, one of D288H and D288K, Q293K, V294A, L300T, and combinations thereof.

[0198] In one embodiment, the N142 variant contains approximately 10 to 20 amino acid mutations compared to the WT N142 enzyme containing the amino acid sequence of SEQ ID NO: 297. In a further embodiment, the approximately 10 to 20 amino acid mutations are approximately 10 to 20 amino acid substitutions compared to the WT N142 enzyme of SEQ ID NO: 297. In one embodiment, the N142 variant contains approximately 10 to 20 amino acid mutations compared to the WT N142 enzyme of SEQ ID NO: 297, and the amino acid mutations include approximately 10 to 20 amino acid mutations in the following amino acid residues of SEQ ID NO: 297: I10, R11, Y12, V15, I35, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, K129, R157, S159, L160, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300. In further embodiments, the amino acid mutations include approximately 10 to 20 amino acid mutations in the following amino acid residues of SEQ ID NO: 297: I10, R11, Y12, V15, I35, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, and L300. In further embodiments, each of the amino acid mutations is an amino acid substitution. In further embodiments, the approximately 10 to 20 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO: 297 listed in Table 5. In a further embodiment, the approximately 10 to 20 amino acid substitutions in SEQ ID NO: 297 are one of I10A and I10Q, R11T, Y12K, one of V15H and V15K, I35T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G, L120T, Y127L, K129 The following are selected from the group consisting of G, R157G, S159G, L160W, V230G, V230R, and V230S, one of N233S, K250S, A261K, F269N, S274D, A275F, A275S, and A275W, one of D288H and D288K, Q293K, V294A, L300T, and combinations thereof.

[0199] In one embodiment, the N142 variant contains approximately 12 to 18 amino acid mutations compared to the WT N142 enzyme of SEQ ID NO: 297. In a further embodiment, the approximately 12 to 18 amino acid mutations are approximately 12 to 18 amino acid substitutions compared to the WT N142 enzyme of SEQ ID NO: 297. In one embodiment, the N142 variant contains approximately 12 to 18 amino acid mutations in each of the following amino acid residues of SEQ ID NO: 297: I10, R11, Y12, V15, I35, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, K129, R157, S159, L160, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300. In further embodiments, the amino acid mutations include approximately 12 to 18 amino acid mutations in the following amino acid residues of SEQ ID NO: 297: I10, R11, Y12, V15, I35, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, L300. In further embodiments, each of the amino acid mutations is an amino acid substitution. In further embodiments, approximately 12 to 18 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO: 297 listed in Table 5. In further embodiments, approximately 12 to 18 amino acid substitutions in SEQ ID NO: 297 are one of I10A and I10Q, R11T, Y12K, one of V15H and V15K, I35T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G, L120T, Y127L, K129 The following are selected from the group consisting of G, R157G, S159G, L160W, V230G, V230R, and V230S, one of N233S, K250S, A261K, F269N, S274D, A275F, A275S, and A275W, one of D288H and D288K, Q293K, V294A, L300T, and combinations thereof.

[0200] In one embodiment, the N142 variant contains approximately 15 to 18 amino acid mutations compared to the WT N142 enzyme of SEQ ID NO: 297. In a further embodiment, the approximately 15 to 18 amino acid mutations are approximately 15 to 18 amino acid substitutions compared to the WT N142 enzyme of SEQ ID NO: 297. In one embodiment, the N142 variant contains approximately 15 to 18 amino acid mutations in the following amino acid residues of SEQ ID NO: 297: I10, R11, Y12, V15, I35, Y45, V46, N48, Y52, T57, N102, L120, E126, Y127, K129, R157, S159, L160, V230, R231, N233, K250, A261, F269, V272, S274, A275, D288, Q293, V294, L300. In further embodiments, the amino acid mutations include approximately 15 to 18 amino acid mutations in the following amino acid residues of SEQ ID NO: 297: I10, R11, Y12, V15, I35, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, and L300. In further embodiments, each of the amino acid mutations is an amino acid substitution. In further embodiments, the approximately 15 to 18 amino acid substitutions are selected from the amino acid substitutions of SEQ ID NO: 297 listed in Table 5. In a further embodiment, approximately 15 to 18 amino acid substitutions in SEQ ID NO: 297 are one of I10A and I10Q, R11T, Y12K, one of V15H and V15K, I35T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, T57K, N102G, L120T, Y127L, K129 The following are selected from the group consisting of G, R157G, S159G, L160W, V230G, V230R, and V230S, one of N233S, K250S, A261K, F269N, S274D, A275F, A275S, and A275W, one of D288H and D288K, Q293K, V294A, L300T, and combinations thereof.

[0201] In some embodiments, the N142 variant disclosed herein does not have one or more amino acid substitutions of SEQ ID NO: 297, selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, T57L, D84E, R88N, E91K, E92K, H93Y, M106L, N117D, H118S, E126D, N162D, E170R, N197Y, F199L, N203T, K213Q, L223I, N233G, S245A, A261P, K286E, and S306I.

[0202] In one embodiment, the N142 variant includes one or more amino acid substitutions of SEQ ID NO: 297, selected from the group consisting of I10A, V15H, I35T, V46D, V46S, N48H, Y52F, T57K, N102G, L120T, Y127L, V230G, V230R, V230S, K250S, A261K, S274D, A275F, D288H, and combinations thereof. In further embodiments, the one or more amino acid substitutions in the N142 variant include approximately 5 to 16, approximately 8 to 16, approximately 10 to 16, or approximately 12 to 16 of the above-mentioned amino acid substitutions. In further embodiments, the N142 variant includes one of the group of mutations listed in Table 6.

[0203] In one embodiment, the N142 variant includes one or more amino acid substitutions of SEQ ID NO: 297, selected from the group consisting of I10A, V15H, I35T, V46D, V46S, N48H, Y52F, T57K, N102G, L120T, Y127L, V230G, V230R, V230S, K250S, A261K, S274D, A275F, D288H, and combinations thereof. In one embodiment, the one or more amino acid substitutions in the N142 variant include approximately 5 to 20, approximately 10 to 20, approximately 12 to 20, or approximately 15 to 20 amino acid substitutions at one or more amino acid positions. In further embodiments, the N142 variant includes one of the group of mutations listed in Table 7.

[0204] [Table 5]

[0205] [Table 6-1]

[0206] [Table 6-2]

[0207] [Table 6-3]

[0208] [Table 6-4]

[0209] [Table 6-5]

[0210] [Table 7]

[0211] In one embodiment, the N142 variant includes one of the sets of mutations listed in Table 6.

[0212] In one embodiment, the N142 variant includes one of the sets of mutations listed in Table 7.

[0213] In one embodiment, the N142 variant includes the following amino acid mutations in SEQ ID NO: 297: I10A, V15H, I35T, Y52F (Table 7, N1).

[0214] In one embodiment, the N142 variant includes the following amino acid mutations in SEQ ID NO: 297: I10A, V15H, I35T, V46S (Table 7, N2).

[0215] In one embodiment, the N142 variant includes the following amino acid mutations in SEQ ID NO: 297: I10A, V15H, I35T, V46D (Table 7, N3).

[0216] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: I10A, V15H, I35T, V46D, and T57K (Table 7, N4).

[0217] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: I10A, V15H, I35T, Y52F, and T57K (Table 7, N5).

[0218] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: I10A, V15H, I35T, Y52F, and N102G (Table 7, N6).

[0219] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: L120T, V230R, A261K, S274D, and A275F (Table 7, N7).

[0220] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: V230S, A261K, S274D, A275F, and D288H (Table 7, N8).

[0221] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: V230G, A261K, S274D, A275F, and D288H (Table 7, N9).

[0222] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: I10A, V15H, V230S, A261K, S274D (Table 7, N10).

[0223] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: I10A, V15H, I35T, V46D, T57K, L120T, V230R, A261K, S274D, A275F, and D288H (Table 7, N11).

[0224] In one embodiment, an N142 variant is provided comprising (i) the amino acid sequence described in SEQ ID NO: 303, or (ii) an amino acid sequence that is at least about 90% or 95% identical to SEQ ID NO: 303. In a further embodiment, the N142 variant comprises the amino acid sequence described in SEQ ID NO: 303.

[0225] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: I10A, V15H, I35T, V46S, T57K, L120T, V230R, A261K, S274D, A275F, and D288H (Table 7, N12).

[0226] In one embodiment, an N142 variant is provided comprising (i) the amino acid sequence described in SEQ ID NO: 304, or (ii) an amino acid sequence that is at least about 90% or 95% identical to SEQ ID NO: 304. In a further embodiment, the N142 variant comprises the amino acid sequence described in SEQ ID NO: 304.

[0227] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: I10A, V15H, I35T, N48H, Y52F, T57K, L120T, V230R, A261K, S274D, A275F, and D288H (Table 7, N13).

[0228] In one embodiment, an N142 variant is provided comprising (i) the amino acid sequence described in SEQ ID NO: 305, or (ii) an amino acid sequence that is at least about 90% or 95% identical to SEQ ID NO: 305. In a further embodiment, the N142 variant comprises the amino acid sequence described in SEQ ID NO: 305.

[0229] In one embodiment, the N142 variant comprises the following amino acid mutations in SEQ ID NO: 297: I10A, V15H, I35T, Y52F, T57K, L120T, V230R, A261K, S274D, A275F, and D288H (Table 7, N14).

[0230] In one embodiment, there is provided an N142 variant comprising (i) the amino acid sequence set forth in SEQ ID NO: 306, or (ii) an amino acid sequence that is at least about 90% identical or 95% identical to SEQ ID NO: 306. In a further embodiment, the N142 variant comprises the amino acid sequence set forth in SEQ ID NO: 306.

[0231] In one embodiment, the N142 variant comprises the following amino acid mutations in SEQ ID NO: 297: I10A, V15H, I35T, Y52F, T57K, L120T, V230R, K250S, A261K, S274D, A275F, and D288H (Table 7, N15).

[0232] In one embodiment, there is provided an N142 variant comprising (i) the amino acid sequence set forth in SEQ ID NO: 307, or (ii) an amino acid sequence that is at least about 90% identical or 95% identical to SEQ ID NO: 307. In a further embodiment, the N142 variant comprises the amino acid sequence set forth in SEQ ID NO: 307.

[0233] In one embodiment, the N142 variant comprises the following amino acid mutations in SEQ ID NO: 297: I10A, V15H, I35T, Y52F, T57K, L120T, V230S, A261K, S274D, A275F, and D288H (Table 7, N16).

[0234] In one embodiment, there is provided an N142 variant comprising (i) the amino acid sequence set forth in SEQ ID NO: 308, or (ii) an amino acid sequence that is at least about 90% identical or 95% identical to SEQ ID NO: 308. In a further embodiment, the N142 variant comprises the amino acid sequence set forth in SEQ ID NO: 308.

[0235] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: I10A, V15H, I35T, Y52F, N102G, L120T, V230G, A261K, S274D, A275F, and D288H (Table 7, N17).

[0236] In one embodiment, an N142 variant is provided comprising (i) the amino acid sequence described in SEQ ID NO: 309, or (ii) an amino acid sequence that is at least about 90% or 95% identical to SEQ ID NO: 309. In a further embodiment, the N142 variant comprises the amino acid sequence described in SEQ ID NO: 309.

[0237] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: I10A, V15H, I35T, Y52F, Y127L, V230G, A261K, S274D, A275F, and D288H (Table 7, N18).

[0238] In one embodiment, an N142 variant is provided comprising (i) the amino acid sequence described in SEQ ID NO: 310, or (ii) an amino acid sequence that is at least about 90% or 95% identical to SEQ ID NO: 310. In a further embodiment, the N142 variant comprises the amino acid sequence described in SEQ ID NO: 310.

[0239] In one embodiment, an N142 variant is provided which includes an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs: 303 to 310. In a further embodiment, the N142 variant includes amino acid substitutions present in the reference amino acid sequence to the amino acid sequence of SEQ ID NO: 297.

[0240] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: I10A, V15H, I35T, N48H, Y52S, T57K, N102G, L120T, S159G, V230R, K250S, A261K, S274D, A275F, D288H, and V294A.

[0241] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: I10A, V15H, I35T, N48H, T57K, N102G, L120T, S159G, L160W, V230R, K250S, A261K, S274D, A275F, D288H, and L300T.

[0242] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: I10Q, R11T, Y12K, V15K, N48G, Y52S, T57K, L120T, R157G, V230S, K250S, A261K, F269N, A275S, D288K, and Q293K.

[0243] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: I10Q, R11T, Y12K, V15K, N48G, T57K, L120T, K129G, R157G, V230S, N233S, K250S, A261K, F269N, A275F, D288K, and Q293K.

[0244] In one embodiment, the N142 variant includes the following amino acid mutations of SEQ ID NO: 297: I10Q, R11T, Y12K, V15K, N48G, T57K, L120T, K129G, R157G, V230S, N233S, K250S, A261K, F269N, A275W, D288K, and Q293K.

[0245] In some embodiments, the N142 variant includes the amino acid sequence described in one of SEQ ID NOs. 358 to 430.

[0246] In some embodiments, the N142 variant includes the amino acid sequence described in one of SEQ ID NOs: 431 to 448.

[0247] In some embodiments, the N142 variant includes the amino acid sequence described in one of SEQ ID NOs: 449 to 485.

[0248] In some embodiments, the N142 variant includes the amino acid sequence described in one of SEQ ID NOs: 486 to 489.

[0249] In some embodiments, the N142 variant includes the amino acid sequence described in one of SEQ ID NOs: 490 to 596.

[0250] In some embodiments, the N142 variant includes an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 358 to SEQ ID NOs. 596, and the N142 variant includes amino acid substitutions present in the reference amino acid sequence relative to the amino acid sequence of SEQ ID NO. 297.

[0251] In some embodiments, the N142 variant includes the amino acid sequence described in one of SEQ ID NOs: 847 to 1005.

[0252] In some embodiments, the N142 variant includes an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 847 to SEQ ID NOs. 1005, and the N142 variant includes amino acid substitutions present in the reference amino acid sequence relative to the amino acid sequence of SEQ ID NOs. 297.

[0253] In some embodiments, the N142 variant includes an amino acid sequence selected from one of SEQ ID NOs: 859, 868, 925, 936, and 942.

[0254] In some embodiments, the N142 variant includes an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 859, SEQ ID NO: 868, SEQ ID NO: 925, SEQ ID NO: 936, and SEQ ID NO: 942, and the N142 variant includes amino acid substitutions present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO: 297.

[0255] In some embodiments, the N142 variant has at least 25% of the activity of an IdeS enzyme comprising the amino acid sequence set forth in SEQ ID NO: 2 in an IgG protease enzyme assay. In some embodiments, the N142 variant has at least 30% of the activity of an IdeS enzyme comprising the amino acid sequence set forth in SEQ ID NO: 2 in an IgG protease enzyme assay. In some embodiments, the N142 variant has at least about 40% of the activity, at least about 50% of the activity, at least about 60% of the activity, at least about 70% of the activity, at least about 75% of the activity, at least about 80% of the activity, at least about 90% of the activity, at least about 100% of the activity, at least about 110% of the activity, or at least about 120% of the activity of an IdeS enzyme comprising the amino acid sequence of SEQ ID NO: 2 in an IgG protease enzyme assay. In some embodiments, the N142 variant has at least about 40% of the activity, at least about 50% of the activity, at least about 60% of the activity, at least about 70% of the activity, at least about 80% of the activity, at least about 90% of the activity, at least about 100% of the activity, at least about 110% of the activity, or at least about 120% of the activity of an IdeZ enzyme comprising the amino acid sequence of SEQ ID NO: 830 in an IgG protease enzyme assay. In some embodiments, IgG protease activity in an IgG protease enzyme assay is measured by an enzyme-linked immunosorbent assay comprising digestion of an immobilized antibody substrate.

[0256] In some embodiments, the N142 variant is depleted of one or more T-cell epitopes compared to a polypeptide having the amino acid sequence of SEQ ID NO: 297, for example, the N142 variant is depleted of two or more, three or more, or four or more T-cell epitopes.

[0257] In some embodiments, the N142 variant has substantially the same thermostability as an IdeS enzyme comprising the amino acid sequence set forth in SEQ ID NO: 2. In other embodiments, the N142 variant has higher thermostability than an IdeS enzyme comprising the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, thermostability is measured by differential scanning fluorimetry.

[0258] In some embodiments, the N142 variant has substantially the same thermal stability as the IdeZ enzyme containing the amino acid sequence described in SEQ ID NO: 830. In other embodiments, the N142 variant has higher thermal stability than the IdeZ enzyme containing the amino acid sequence described in SEQ ID NO: 830. In some embodiments, thermal stability is measured by differential scanning fluorescence quantification.

[0259] In some embodiments, the N142 variant provided herein has an amino acid sequence that is at least about 75% identical to SEQ ID NO: 297. In some embodiments, the N142 variant has an amino acid sequence that is at least about 80% identical to SEQ ID NO: 297. In some embodiments, the N142 variant has an amino acid sequence that is at least about 85% identical to SEQ ID NO: 297. In some embodiments, the N142 variant has an amino acid sequence that is at least about 90% identical to SEQ ID NO: 297. In some embodiments, the N142 variant has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 297. In some embodiments, the N142 variant has an amino acid sequence that is at least about 97% identical to SEQ ID NO: 297. In some embodiments, the N142 variant has an amino acid sequence that is at least about 98% identical to SEQ ID NO: 297.

[0260] In yet another embodiment, the disclosure provides an IgG protease variant from among SEQ ID NOs: 27 to 302. In one embodiment, the IgG protease variant has an amino acid sequence that is at least about 75% identical to the amino acid sequence of the corresponding WT enzyme selected from one of SEQ ID NOs: 27 to 302 from which the IgG protease variant is derived. In a further embodiment, the amino acid sequence of the IgG protease variant is at least about 80% identical to the amino acids of the corresponding WT enzyme, or at least about 85% identical to the amino acids of the corresponding WT enzyme, or at least about 90% identical to the amino acids of the corresponding WT enzyme, or at least about 95% identical to the amino acids of the corresponding WT enzyme, or at least about 97% identical to the amino acids of the corresponding WT enzyme, or at least about 98% identical to the amino acids of the corresponding WT enzyme.

[0261] In one embodiment, the IgG protease variant is depleted of T cell epitopes compared to the corresponding WT enzyme. In other words, in embodiments of the variants provided herein, the variant is depleted of one or more dominant immunogenic T cell epitopes compared to the immunogenic T cell epitopes present in the corresponding WT enzyme.

[0262] In a further embodiment, the IgG protease variant is an enzyme variant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 39, 41, 50, 51, 54, 56, 60, 61, 65, 67, 137, 140, 144, 150, 159, 285, 286, 287, 288, 289, 292, 293, 294, 295, 296, 297, and 298. In a further embodiment, the IgG protease variant is an amino acid sequence variant selected from the group consisting of SEQ ID NOs: 54, 140, 293, 294, 295, 297, and 298.

[0263] In yet another embodiment, the IgG protease variant comprises the amino acid sequence described in one of SEQ ID NOs: 318 to 829. In yet another embodiment, the IgG protease variant comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the reference amino acid sequence described in one of SEQ ID NOs: 318 to 829, wherein the IgG protease variant comprises amino acid substitutions present in the reference amino acid sequence with respect to the corresponding enzyme of the reference enzyme.

[0264] In a further embodiment, the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 293[N123]. The IgG protease variant includes (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 293: Y12, L31, E34, F36, R37, Y38, N39, I44, A60, D133, N162, F274, A279, H280, V299, and (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 293. In a further embodiment, the IgG protease variant has (iii) at least about 25% of the activity of IdeS in an IgG protease enzyme assay. In a further embodiment, the IgG protease variant has at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% of the activity of IdeS in an IgG protease enzyme assay. In further embodiments, the variant has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, or at least about 98% identical to SEQ ID NO: 293.

[0265] In another embodiment, the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 298[N30]. The variant includes (i) one or more amino acid mutations at one or more amino acid positions in SEQ ID NO: 298: I10, R11, Y12, V15, Y18, V46, Y52, F101, N102, Q105, L120, F125, E126, Y127, Y156, R157, S159, V230, R231, N233, A261, F269, V270, A275, F269, and (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 298. In a further embodiment, the IgG protease variant (iii) has at least about 25% of the activity of IdeS in an IgG protease enzyme assay. In further embodiments, the IgG protease variant exhibits at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% of the activity of IdeS in an IgG protease enzyme assay. In further embodiments, the variant has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, or at least about 98% identical to SEQ ID NO: 298.

[0266] In another embodiment, the IgG protease variant is [N31], a variant of the amino acid sequence of SEQ ID NO: 294. The IgG protease variant includes (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 294: I10, R11, Y12, E14, V15, Y18, V46, N48, Y52, D130, A132, Y157, N159, Y161, K162, V232, R233, N235, D260, A263, V272, S276, A277, and (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 294. In a further embodiment, the IgG protease variant (iii) has at least about 25% of the activity of IdeS in an IgG protease enzyme assay. In further embodiments, the IgG protease variant exhibits at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% of the activity of IdeS in an IgG protease enzyme assay. In further embodiments, the variant has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, or at least about 98% identical to SEQ ID NO: 294.

[0267] In yet another embodiment, the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 54. The IgG protease variant includes (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: I10, I11, Y12, E14, Y18, H19, V143, Y157, Y161, K162, and (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 54. In yet another embodiment, the IgG protease variant has (iii) at least about 25% of the activity of IdeS in an IgG protease enzyme assay. In yet another embodiment, the IgG protease variant has at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% of the activity of IdeS in an IgG protease enzyme assay. In further embodiments, the variant has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, or at least about 98% identical to SEQ ID NO: 54.

[0268] In yet another embodiment, the IgG protease variant is a variant of the amino acid sequence of SEQ ID NO: 140. The IgG protease variant includes (i) one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: 140: M10, R11, Y12, V15, Y42, V46, A47, N48, Q49, N102, V104, I113, Y118, L120, D121, K123, Y127, Y156, R157, Y201, Q202, V230, F269, S274, A275, D288, G296, and (ii) an amino acid sequence that is at least about 80% identical to SEQ ID NO: 140. In yet another embodiment, the IgG protease variant (iii) has at least about 25% of the activity of IdeS in an IgG protease enzyme assay. In further embodiments, the IgG protease variant exhibits at least about 50%, at least about 75%, at least about 90%, at least about 100%, or at least about 110% of the activity of IdeS in an IgG protease enzyme assay. In further embodiments, the IgG protease variant has an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, or at least about 98% identical to SEQ ID NO: 140.

[0269] In one embodiment, the present disclosure provides a variant of N144 IgG protease. The N144 IgG protease provided above comprises the amino acid sequence described in SEQ ID NO: 295. The N144 variant comprises (i) one or more amino acid positions of SEQ ID NO: 295: Y12, S18, I35, I46, A47, Y52, I54, T55, T57, N59, H93, F101, N102, E104, Q105, L120, F125, E126, Y127, T135, Y156, S159, L175, V183, R200, N2 (ii) The variant includes one or more amino acid mutations in 01, N205, I207, V230, R231, N233, A241, N244, E246, A261, Y269, V270, S274, A275, I280, S281, A282, I285, D288, and V290, and (ii) an amino acid sequence that is at least about 75% identical to SEQ ID NO: 295. In further embodiments, the variant has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 98% identical to SEQ ID NO: 295.

[0270] In one embodiment, the N144 variant does not have an amino acid mutation at one or more of the following amino acid positions in SEQ ID NO: A32, N33, Q36, F41, D84, E91, M106, H118, E170, S195, N197, N203, S245, K286.

[0271] In one embodiment of the N144 variant of this disclosure, a mutation in the N144 variant at one or more of the aforementioned amino acid positions eliminates one or more dominant immunogenic T cell epitopes present in wild-type N144, resulting in the N144 variant being depleted of T cell epitopes compared to wild-type N144.

[0272] The amino acid mutations of this disclosure include, but are not limited to, amino acid exchanges, insertions, deletions, additions, substitutions, inversions, and / or duplications. These mutations / modifications also include conservative and / or homologous amino acid exchanges. In preferred embodiments of the present invention, one or more mutations in the N144 variant are one or more amino acid substitutions. In further embodiments, one or more amino acid substitutions in SEQ ID NO: 295 in the N144 variant are one of Y12N, S18K, I35E, I35K, and I35S, one of I46D, A47E, I54T, T55K, T55H, and T55R, one of T57L, N59D, H93Y, N102G, E104R, one of L120T, and L120S, one of F125W, T135H, S159K, one of L175A, L175N, and L175S, one of V183W, R200D, R200G, and R200T, N201E, N205D, I207T, V The following are selected from the group consisting of one of 230H and V230Q, one of R231W, N233G, A241G, E246G and E246K, one of A261K, A261I, A261F, A261G, A261Q and A261S, one of Y269D, Y269S and Y269W, one of V270T, V272T and V272Y, one of S274D and S274G, one of A275S and A275W, one of I280M, S281G, A282G, D288K and D288T, V290F, and combinations thereof.

[0273] In one embodiment, the N144 variant contains 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more amino acid mutations compared to WT N144 containing the amino acid sequence of SEQ ID NO: 295, where the mutations are at the following amino acid positions: Y12, S18, I35, I46, A47, Y52, I54, T55, T57, N59, H93, F101, N102, The mutations are selected from E104, Q105, L120, F125, E126, Y127, T135, Y156, S159, L175, V183, R200, N201, N205, I207, V230, R231, N233, A241, N244, E246, A261, Y269, V270, S274, A275, I280, S281, A282, I285, D288, and V290. A further embodiment is that two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid mutations are two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more amino acid substitutions.In further embodiments, the amino acid substitutions of SEQ ID NO: 295 include one of Y12N, S18K, I35E, I35K, and I35S, one of I46D, A47E, I54T, T55K, T55H, and T55R, one of T57L, N59D, H93Y, N102G, E104R, one of L120T, and L120S, one of F125W, T135H, S159K, one of L175A, L175N, and L175S, one of V183W, R200D, R200G, and R200T, N201E, N205D, I207T, V230H, and V23 The following are selected from the group consisting of one of 0Q, one of R231W, N233G, A241G, one of E246G and E246K, one of A261K, A261I, A261F, A261G, A261Q and A261S, one of Y269D, Y269S and Y269W, one of V270T, V272T and V272Y, one of S274D and S274G, one of A275S and A275W, one of I280M, S281G, A282G, D288K and D288T, V290F, and combinations thereof.

[0274] In another embodiment, the N144 variant includes approximately 10 to 30 amino acid mutations, approximately 10 to 25 amino acid mutations, approximately 12 to 24 amino acid mutations, approximately 12 to 20 amino acid mutations, approximately 12 to 16 amino acid mutations, approximately 21 to 24 amino acid mutations, or 12, 13, 14, 21, 22, 23, or 24 amino acid mutations relative to WT N144 containing the amino acid sequence of SEQ ID NO: 295, Y12, S18, I35, I46, A47, Y52, I The mutations are selected from 54, T55, T57, N59, H93, F101, N102, E104, Q105, L120, F125, E126, Y127, T135, Y156, S159, L175, V183, R200, N201, N205, I207, V230, R231, N233, A241, N244, E246, A261, Y269, V270, S274, A275, I280, S281, A282, I285, D288, and V290. In further embodiments, each amino acid mutation is an amino acid substitution. In further embodiments, the amino acid substitutions of SEQ ID NO: 295 include one of Y12N, S18K, I35E, I35K, and I35S, one of I46D, A47E, I54T, T55K, T55H, and T55R, one of T57L, N59D, H93Y, N102G, E104R, one of L120T, and L120S, one of F125W, T135H, S159K, one of L175A, L175N, and L175S, one of V183W, R200D, R200G, and R200T, N201E, N205D, I207T, V230H, and V23 The following are selected from the group consisting of one of 0Q, one of R231W, N233G, A241G, one of E246G and E246K, one of A261K, A261I, A261F, A261G, A261Q and A261S, one of Y269D, Y269S and Y269W, one of V270T, V272T and V272Y, one of S274D and S274G, one of A275S and A275W, one of I280M, S281G, A282G, D288K and D288T, V290F, and combinations thereof.

[0275] In one embodiment, the N144 variant is selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, F199L, N203T, K213Q, L223I, S245A, N246E, K250E, K286E, and S306I, and does not have one or more amino acid substitutions of SEQ ID NO: 295.

[0276] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I46D, A47E, T55H, H93Y, E104R, L120T, F125W, T135H, S159K, and A275S.

[0277] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I46D, A47E, T55H, H93Y, E104R, L120T, F125W, T135H, S159K, A261I, and A275S.

[0278] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261F, and A275S.

[0279] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261G, and A275S.

[0280] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261Q, and A275S.

[0281] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261S, and A275S.

[0282] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I46D, A47E, T55R, H93Y, E104R, L120S, F125W, T135H, S159K, A261K, and A275S.

[0283] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I46D, A47E, T55R, H93Y, E104R, L120T, F125W, T135H, S159K, A261K, and A275S.

[0284] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, and A275S.

[0285] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N201E, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

[0286] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

[0287] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.

[0288] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261Q, V270T, A275W, I280M, and V290F.

[0289] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, A261S, Y269W, A275W, A282G, and V290F.

[0290] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, A261S, Y269W, A275W, A282G, and D288T.

[0291] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N201E, N233G, E246K, A261Q, Y269W, A275W, A282G, and D288T.

[0292] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35E, I46D, A47E, T55R, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, A261K, Y269W, A275W, A282G, and D288T.

[0293] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35E, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N201E, R231W, E246G, Y269W, A275W, A282G, and D288T.

[0294] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35E, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, Y269W, A275W, A282G, and D288T.

[0295] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

[0296] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261S, V270T, A275W, I280M, and V290F.

[0297] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.

[0298] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246K, A261S, Y269W, S274G, A275S, A282G, and D288K.

[0299] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.

[0300] In one embodiment, the N144 variant includes the following amino acid substitutions of SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, R200G, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

[0301] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N201E, N233G, E246G, V270T, A275W, I280M, and V290F.

[0302] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N201E, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

[0303] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.

[0304] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, V270T, A275W, I280M, and V290F.

[0305] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246G, A261K, V270T, A275W, I280M, and V290F.

[0306] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, V270T, A275W, I280M, and V290F.

[0307] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

[0308] In one embodiment, the N144 variant includes the following amino acid substitutions of SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, R200T, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

[0309] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175S, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.

[0310] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, V230H, E246K, Y269W, A275W, A282G, and D288T.

[0311] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F.

[0312] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, V183W, N205D, N233G, E246K, Y269W, S274D, A275S, A282G, and D288T.

[0313] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, V183W, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F.

[0314] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, V230H, E246K, A261K, Y269W, S274G, A275S, A282G, and D288T.

[0315] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, V230Q, E246G, A261K, Y269W, S274G, A275S, A282G, and D288T.

[0316] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246K, A261K, Y269W, S274G, A275S, A282G, and V290F.

[0317] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, Y269W, S274G, A275S, A282G, and V290F.

[0318] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, Y269W, A275W, A282G, and D288T.

[0319] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246G, Y269W, S274G, A275S, A282G, and V290F.

[0320] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, Y269W, S274G, A275S, A282G, and D288K.

[0321] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261K, Y269W, S274D, A275S, A282G, and V290F.

[0322] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261K, Y269W, S274D, A275S, A282G, and D288K.

[0323] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175S, N205D, N233G, E246G, Y269W, A275W, A282G, and D288T.

[0324] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35S, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

[0325] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35S, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.

[0326] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35S, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261Q, V270T, A275W, I280M, and V290F.

[0327] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35S, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, V230Q, A241G, Y269W, A275W, A282G, and V290F.

[0328] In one embodiment, the N144 variant includes the following amino acid substitutions in SEQ ID NO: 295: Y12N, S18K, I35S, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, Y269W, A275W, A282G, and V290F.

[0329] In one embodiment, the N144 variant includes the amino acid sequence described in one of SEQ ID NOs. 597 to 657. In another embodiment, the N144 variant includes an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 597 to 657, and the N144 variant includes amino acid substitutions present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NOs. 295.

[0330] In one embodiment, the N144 variant includes an amino acid sequence described in one of SEQ ID NOs: 1006 to 1490 and SEQ ID NOs: 1952 to 2590. In another embodiment, the N144 variant includes an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs: 1006 to 1490 and SEQ ID NOs: 1952 to 2590, and the N144 variant includes an amino acid substitution present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO: 295.

[0331] In one embodiment, the N144 variant is sequence numbers 1118, 1120, 1122, 1123, 1125, 1126, 1127, 1129, 1282, 1962, 1963, 1964, 1965, 1970, 1971, 1983, 2004, 2024, 2028, 2371, 2372, 2373, 2390, 2421, 2427, 2428, and 242 9, SEQ ID NOs: 2430, 2431, 2432, 2433, 2434, 2437, 2439, 2446, 2450, 2463, 2464, 2476, 2478, 2485, 2486, 2487, 2489, 2491, 2493, 2494, 2511, 2524, 2526, 2528, 2574, and 2575, each containing an amino acid sequence selected from one of these.In another embodiment, the N144 variant is sequence numbers 1118, 1120, 1122, 1123, 1125, 1126, 1127, 1129, 1282, 1962, 1963, 1964, 1965, 1970, 1971, 1983, 2004, 2024, 2028, 2371, 2372, 2373, 2390, 2421, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2437, 243 9. The N144 variant includes an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 2446, SEQ ID NO: 2450, SEQ ID NO: 2463, SEQ ID NO: 2464, SEQ ID NO: 2476, SEQ ID NO: 2478, SEQ ID NO: 2485, SEQ ID NO: 2487, SEQ ID NO: 2489, SEQ ID NO: 2491, SEQ ID NO: 2493, SEQ ID NO: 2494, SEQ ID NO: 2511, SEQ ID NO: 2524, SEQ ID NO: 2526, SEQ ID NO: 2528, SEQ ID NO: 2574, and SEQ ID NO: 2575, wherein the N144 variant includes amino acid substitutions present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO: 295.

[0332] In one embodiment, the N144 variant includes an amino acid sequence selected from one of the following: SEQ ID NOs: 1118, 1120, 1122, 1123, 1125, 1126, 1127, 1129, 1282, 1970, 1971, 1983, 2004, 2024, 2028, 2390, 2421, 2446, 2450, 2463, 2464, 2476, 2478, 2485, 2486, 2487, 2489, 2491, 2493, 2494, 2511, 2574, and 2575. In another embodiment, the N144 variant is sequence numbers 1118, 1120, 1122, 1123, 1125, 1126, 1127, 1129, 1282, 1970, 1971, 1983, 2004, 2024, 2028, 2390, 2421, 2446, 2450, 2463, 2464, 2476, 2478, and 248. 5. The N144 variant includes an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs: 2486, 2487, 2489, 2491, 2493, 2494, 2511, 2574, and 2575, wherein the N144 variant includes amino acid substitutions present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO: 295.

[0333] In some embodiments, the N144 variant includes a first set of amino acid substitutions of SEQ ID NO: 295 and a second set of amino acid substitutions of SEQ ID NO: 295. The first set of amino acid substitutions of SEQ ID NO: 295 consists of Y12N, S18K, I46D, A47E, H93Y, E104R, F125W, T135H, and S159K. The second set of amino acid substitutions for Sequence ID No. 295 is one of I35K, I35E, and I35S, one of I54T, T55K, T55R, and T55H, one of T57L, N59D, N102G, one of L120T and L120S, one of L175N, L175A, and L175S, one of V183W, one of R200G and R200T, one of N201E, N205D, one of V230H and V230Q, R231W, N This is a set of approximately 3 to 15 amino acid substitutions selected from the group consisting of 233G, A241G, E246K and one of E246G, one of A261K, A261Q, A261S, A261I, A261F and one of A261G, one of Y269W, V270T, S274G and S274D, one of A275W and A275S, one of I280M, A282G, D288T and D288K, and V290F. In a further embodiment, the N144 variant, which includes a first set and a second set of amino acid substitutions of SEQ ID NO: 295, does not have an amino acid mutation at one or more of the following amino acid positions of SEQ ID NO: A32, N33, Q36, F41, D84, E91, M106, H118, E170, S195, N197, N203, S245, K286. In further embodiments, the N144 variant does not have one or more amino acid substitutions of SEQ ID NO: 295, selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, F199L, N203T, K213Q, L223I, S245A, N246E, K250E, K286E, and S306I.

[0334] In one embodiment, the N144 variant, comprising a first set and a second set of amino acid substitutions of SEQ ID NO: 295, has an amino acid sequence that is at least about 90% identical to SEQ ID NO: 295. In another embodiment, the N144 variant has an amino acid sequence that is at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, or at least about 96% identical to SEQ ID NO: 295. In yet another embodiment, the N144 variant has an amino acid sequence that is about 90% to about 96%, or about 92% to about 96%, identical to SEQ ID NO: 295. In one embodiment, the second set of amino acid substitutions of SEQ ID NO: 295 is a set of about 3 to about 5 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of SEQ ID NO: 295 is a set of about 12 to about 15 amino acid substitutions. In yet another embodiment, the second set of amino acid substitutions of SEQ ID NO: 295 is a set of about 5 to about 12 amino acid substitutions. In another embodiment, the second set of amino acid substitutions in SEQ ID NO: 295 is a set of approximately 5 to 15 amino acid substitutions.

[0335] In some embodiments, the second set of amino acid substitutions for SEQ ID NO: 295 is a set of approximately 3 to 15 amino acid substitutions selected from the group consisting of one of I35K and I35E, one of I54T, T55K, T57L, N59D, N102G, L120T, L175N, N205D, R231W, N233G, one of E246K and E246G, one of A261K and A261Q, one of Y269W, V270T, one of A275W and A275S, I280M, A282G, D288T, and V290F. In one embodiment, the second set of amino acid substitutions for SEQ ID NO: 295 is a set of approximately 3 to 5 amino acid substitutions. In another embodiment, the second set of amino acid substitutions for SEQ ID NO: 295 is a set of approximately 12 to 15 amino acid substitutions. In another embodiment, the second set of amino acid substitutions in SEQ ID NO: 295 is a set of approximately 5 to 12 amino acid substitutions. In yet another embodiment, the second set of amino acid substitutions in SEQ ID NO: 295 is a set of approximately 5 to 15 amino acid substitutions.

[0336] In some embodiments, the second set of amino acid substitutions for SEQ ID NO: 295 is a set of approximately 3 to 15 amino acid substitutions selected from the group consisting of I35K, I54T, T55K, T57L, N59D, N102G, L120T, L175N, N205D, N233G, E246K, Y269W, V270T, one of A275W and A275S, I280M, A282G, and V290F. In one embodiment, the second set of amino acid substitutions for SEQ ID NO: 295 is a set of approximately 3 to 5 amino acid substitutions. In another embodiment, the second set of amino acid substitutions for SEQ ID NO: 295 is a set of approximately 12 to 15 amino acid substitutions. In yet another embodiment, the second set of amino acid substitutions for SEQ ID NO: 295 is a set of approximately 5 to 12 amino acid substitutions. In another embodiment, the second set of amino acid substitutions in SEQ ID NO: 295 is a set of approximately 5 to 15 amino acid substitutions.

[0337] In some embodiments, the second set of amino acid substitutions in SEQ ID NO: 295 is a set of approximately 3 to approximately 13 amino acid substitutions selected from the group consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F. In one embodiment, the second set of amino acid substitutions in SEQ ID NO: 295 is a set of approximately 3 to approximately 5 amino acid substitutions. In another embodiment, the second set of amino acid substitutions in SEQ ID NO: 295 is a set of approximately 12 to approximately 13 amino acid substitutions. In yet another embodiment, the second set of amino acid substitutions in SEQ ID NO: 295 is a set of approximately 5 to approximately 13 amino acid substitutions.

[0338] In one embodiment, the second set of amino acid substitutions in SEQ ID NO: 295 is a set of three amino acid substitutions consisting of T55H, L120T, and A275S.

[0339] In one embodiment, the second set of amino acid substitutions in SEQ ID NO: 295 is a set of four amino acid substitutions consisting of T55H, L120T, A261I, and A275S.

[0340] In one embodiment, the second set of amino acid substitutions in Sequence ID No. 295 is a set of four amino acid substitutions consisting of T55K, L120T, A261F, and A275S.

[0341] In one embodiment, the second set of amino acid substitutions in Sequence ID No. 295 is a set of four amino acid substitutions consisting of T55K, L120T, A261G, and A275S.

[0342] In one embodiment, the second set of amino acid substitutions in SEQ ID NO: 295 is a set of four amino acid substitutions consisting of T55K, L120T, A261Q, and A275S.

[0343] In one embodiment, the second set of amino acid substitutions in SEQ ID NO: 295 is a set of four amino acid substitutions consisting of T55K, L120T, A261S, and A275S.

[0344] In one embodiment, the second set of amino acid substitutions in SEQ ID NO: 295 is a set of four amino acid substitutions consisting of T55R, L120S, A261K, and A275S.

[0345] In one embodiment, the second set of amino acid substitutions in SEQ ID NO: 295 is a set of four amino acid substitutions consisting of T55R, L120T, A261K, and A275S.

[0346] In one embodiment, the second set of amino acid substitutions in SEQ ID NO: 295 is a set of five amino acid substitutions consisting of I54T, T57L, N59D, L120T, and A275S.

[0347] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35E, T55K, N102G, L120T, L175N, N201E, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

[0348] In one embodiment, the second set of amino acid substitutions in Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35E, T55K, N102G, L120T, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

[0349] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35E, T55K, N102G, L120T, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.

[0350] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35E, T55K, N102G, L120T, L175N, N205D, R231W, E246K, A261Q, V270T, A275W, I280M, and V290F.

[0351] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 12 amino acid substitutions consisting of I35E, T55K, L120T, L175A, N205D, N233G, E246K, A261S, Y269W, A275W, A282G, and V290F.

[0352] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 12 amino acid substitutions consisting of I35E, T55K, L120T, L175A, N205D, N233G, E246K, A261S, Y269W, A275W, A282G, and D288T.

[0353] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 12 amino acid substitutions consisting of I35E, T55K, L120T, L175N, N201E, N233G, E246K, A261Q, Y269W, A275W, A282G, and D288T.

[0354] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 12 amino acid substitutions consisting of I35E, T55R, L120T, L175A, N205D, N233G, E246K, A261K, Y269W, A275W, A282G, and D288T.

[0355] In one embodiment, the second set of amino acid substitutions in Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35E, I54T, T57L, N59D, L120T, L175A, N201E, R231W, E246G, Y269W, A275W, A282G, and D288T.

[0356] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35E, I54T, T57L, N59D, L120T, L175A, N205D, N233G, E246K, Y269W, A275W, A282G, and D288T.

[0357] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, T55K, N102G, L120T, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

[0358] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, T55K, N102G, L120T, L175N, N205D, N233G, E246G, A261S, V270T, A275W, I280M, and V290F.

[0359] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, T55K, N102G, L120T, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.

[0360] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, T55K, L120T, L175N, N205D, N233G, E246K, A261S, Y269W, S274G, A275S, A282G, and D288K.

[0361] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175A, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.

[0362] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, R200G, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

[0363] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N201E, N233G, E246G, V270T, A275W, I280M, and V290F.

[0364] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N201E, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

[0365] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.

[0366] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N205D, N233G, A241G, V270T, A275W, I280M, and V290F.

[0367] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N205D, R231W, E246G, A261K, V270T, A275W, I280M, and V290F.

[0368] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N205D, R231W, E246K, V270T, A275W, I280M, and V290F.

[0369] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N205D, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

[0370] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, R200T, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

[0371] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175S, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.

[0372] In one embodiment, the second set of amino acid substitutions in Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175A, N205D, V230H, E246K, Y269W, A275W, A282G, and D288T.

[0373] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175A, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F.

[0374] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, V183W, N205D, N233G, E246K, Y269W, S274D, A275S, A282G, and D288T.

[0375] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, V183W, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F.

[0376] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, V230H, E246K, A261K, Y269W, S274G, A275S, A282G, and D288T.

[0377] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, V230Q, E246G, A261K, Y269W, S274G, A275S, A282G, and D288T.

[0378] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, N233G, E246K, A261K, Y269W, S274G, A275S, A282G, and V290F.

[0379] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, N233G, A241G, Y269W, S274G, A275S, A282G, and V290F.

[0380] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, N233G, A241G, Y269W, A275W, A282G, and D288T.

[0381] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, R231W, E246G, Y269W, S274G, A275S, A282G, and V290F.

[0382] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, R231W, E246K, Y269W, S274G, A275S, A282G, and D288K.

[0383] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, R231W, E246K, A261K, Y269W, S274D, A275S, A282G, and V290F.

[0384] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, R231W, E246K, A261K, Y269W, S274D, A275S, A282G, and D288K.

[0385] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175S, N205D, N233G, E246G, Y269W, A275W, A282G, and D288T.

[0386] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35S, T55K, N102G, L120T, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

[0387] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35S, T55K, N102G, L120T, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.

[0388] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35S, T55K, N102G, L120T, L175N, N205D, R231W, E246K, A261Q, V270T, A275W, I280M, and V290F.

[0389] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35S, I54T, T57L, N59D, L120T, L175N, N205D, V230Q, A241G, Y269W, A275W, A282G, and V290F.

[0390] In one embodiment, the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35S, I54T, T57L, N59D, L120T, L175N, N205D, R231W, E246K, Y269W, A275W, A282G, and V290F.

[0391] In some embodiments, the IgG protease variant of the present disclosure, for example, the N144 variant of the present disclosure, has at least 25% activity of the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2 in an IgG protease enzyme assay. In some embodiments, the IgG protease variant of the present disclosure, for example, the N144 variant of the present disclosure, has at least 30% activity of the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2 in an IgG protease enzyme assay. In some embodiments, the IgG protease variant of the present disclosure, for example, the N144 variant of the present disclosure, has at least about 40% activity, at least about 50% activity, at least about 60% activity, at least about 70% activity, at least about 75% activity, at least about 80% activity, at least about 90% activity, at least about 100% activity, at least about 110% activity, and at least about 120% activity of the IdeS enzyme containing the amino acid sequence of SEQ ID NO: 2 in an IgG protease enzyme assay. In some embodiments, the IgG protease variant of the present disclosure, for example, the N144 variant of the present disclosure, has at least about 25% activity, at least about 40% activity, at least about 50% activity, at least about 60% activity, at least about 70% activity, at least about 80% activity, at least about 90% activity, at least about 100% activity, at least about 110% activity, and at least about 120% activity of the IdeZ enzyme comprising the amino acid sequence of SEQ ID NO: 830 in an IgG protease enzyme assay. In some embodiments, the IgG protease activity in an IgG protease enzyme assay is measured by an enzyme-linked immunosorbent assay that includes digestion of an immobilized antibody substrate.

[0392] In some embodiments, the N144 variant is depleted of one or more T cell epitopes compared to the polypeptide of the amino acid sequence of SEQ ID NO: 295, for example, two or more, three or more, or four or more T cell epitopes.

[0393] In some embodiments, the IgG protease variant of the present disclosure, for example, the N144 variant of the present disclosure, has substantially the same thermal stability as the IdeS enzyme comprising the amino acid sequence described in SEQ ID NO: 2. In other embodiments, the IgG protease variant of the present disclosure, for example, the N144 variant of the present disclosure, has higher thermal stability than the IdeS enzyme comprising the amino acid sequence described in SEQ ID NO: 2. In some embodiments, thermal stability is measured by differential scanning fluorescence quantification.

[0394] In some embodiments, the IgG protease variant of the Disclosure, for example, the N144 variant of the Disclosure, has substantially the same thermal stability as the IdeZ enzyme comprising the amino acid sequence described in SEQ ID NO: 830. In other embodiments, the IgG protease variant of the Disclosure, for example, the N144 variant of the Disclosure, has higher thermal stability than the IdeZ enzyme comprising the amino acid sequence described in SEQ ID NO: 830. In some embodiments, thermal stability is measured by differential scanning fluorescence quantification.

[0395] The melting temperatures of the polypeptides described herein can be determined by methods known to those skilled in the art. For example, in one embodiment, the melting temperature (Tm) of the purified polypeptide is measured using a thermal shift assay (also known as differential scanning fluorimetry, DSF). In one embodiment of the thermal shift assay for determining the Tm of the polypeptide of the present invention, a solution containing the protein is heated in a quantitative polymerase chain reaction (qPCR) instrument in combination with a dye, such as SYPRO® Orange. By systematically increasing the temperature and simultaneously monitoring the SYPRO Orange fluorescence emission, it is possible to monitor the thermal denaturation of the protein.

[0396] The activity of IgG proteases, such as the IgG protease variants provided herein, or fusion proteins containing them, can be determined by methods known to those skilled in the art. For example, an enzyme-linked immunosorbent assay (ELISA) may be used. In one embodiment, individual purified IgG proteases (or combinations thereof) of the present invention are added individually to the wells of an assay plate, for example, a 96-well plate, and anti-human IgG(Fc)-HRP (with horseradish peroxidase added) is added. Then, 3,3',5,5'-tetramethylbenzidine (TMB) is added to the wells as a substrate for HRP, and the wells are incubated. In one embodiment, the absorbance is read at 450 nm and corrected for background absorbance by subtracting the absorbance at a reference wavelength of 620 nm.

[0397] In one embodiment of the IgG protease or IgG protease variant, or fusion protein containing the same, disclosed herein, the polypeptide is not PEGylated. In another embodiment, the polypeptide is PEGylated, i.e., the polypeptide is covalently conjugated to polyethylene glycol (PEG) at, for example, one or more lysine residues. The IgG protease, IgG protease variant, or fusion protein containing the same may be conjugated to any desired number, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 PEG or monomethoxypoly(ethylene glycol) (mPEG) molecules. In one embodiment, the IgG protease, IgG protease variant, or fusion protein containing the same is conjugated to about 8 to about 11 PEG or mPEG molecules, about 9 PEG or mPEG molecules, or about 10 PEG or mPEG molecules. In one embodiment, the average total molecular weight of the PEG portion of the PEGylated IgG protease, IgG protease variant, or fusion protein is approximately 10 kDa to 150 kDa, approximately 30 kDa to 120 kDa, or approximately 50 kDa to 100 kDa.

[0398] In one embodiment, an IgG protease or IgG protease variant, or a fusion protein containing the same, disclosed herein, is covalently bonded to PEG, such as mPEG, via a biocompatible linking group using methods known in the art, such as those described in, for example, Park et al., Anticancer Res., 1981, 1:373-376, and Zaplipsky and Lee, Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, edited by J.M. Harris, 1992, Plenum Press, New York, Chapter 21 (each of these disclosures is incorporated herein by reference in whole). The biocompatible linking group is nontoxic and can be used in vitro or in vivo without causing injury, disease, illness, or death. In some embodiments, PEG may be bonded to the linking group via, for example, an ether linkage, an ester linkage, a thiol linkage, or an amide linkage. Suitable biocompatible linking groups include, for example, ester groups, amide groups, imide groups, carbamate groups, carboxyl groups, hydroxyl groups, carbohydrates, succinimide groups, epoxide groups, oxycarbonylimidazole groups, nitrophenyl groups, trisilicic acid groups, aldehyde groups, isocyanate groups, vinylsulfone groups, tyrosine groups, cysteine ​​groups, histidine groups, or primary amines.

[0399] In another embodiment, an IgG protease or IgG protease variant disclosed herein, or a fusion protein containing the same, is conjugated to a PEG without a linking group, such as mPEG, via an amino group, sulfhydryl group, hydroxyl group, or carboxyl group of the IgG protease, IgG protease variant, or fusion protein. In one embodiment, PEG is conjugated to one or more lysine residues on the IgG protease, IgG protease variant, or fusion protein. In one embodiment, PEG is conjugated to one or more cysteine ​​residues on the IgG protease, IgG protease variant, or fusion protein. In one embodiment, PEG is conjugated to one or more serine residues on the IgG protease, IgG protease variant, or fusion protein. In yet another embodiment, PEG is conjugated to one or more lysine residues, one or more cysteine ​​residues, one or more serine residues, or any combination thereof.

[0400] In one aspect of the present invention, a fusion protein comprising two or more domains and / or a polynucleotide encoding it is provided. One of the domains is an IgG protease domain, i.e., a domain comprising one of the IgG protease polypeptides or IgG protease variant polypeptides described herein. In one embodiment, the second domain is a domain that increases the half-life of the IgG protease, for example, an immunoglobulin fragment crystallizable (Fc) domain and / or a random coil polypeptide domain.

[0401] A "fusion protein" refers to a protein composed of multiple polypeptide components (or polypeptide "domains") that are typically not linked in their native state, but whose respective N-terminuses and C-terminuses are linked via peptide bonds to form a single continuous polypeptide. In the embodiments described herein, the fusion protein is produced via recombinant expression of a single DNA sequence containing the fusion protein domain.

[0402] As used herein, the term “domain” refers to any region / part of an amino acid sequence that can autonomously adopt a particular structure and / or function. In the context of the present invention, “domain” may represent a functional domain or a structural domain. As described herein, the fusion protein of the present invention comprises at least one IgG protease domain and (i) at least one domain that forms a random coil conformation (e.g., a PAS polypeptide domain), and (ii) a domain that increases the half-life of the IgG protease, e.g., an Fc domain or an albumin domain. In one embodiment, the IgG protease fusion protein of the present invention comprises two domains.

[0403] In some embodiments, the fusion protein of the present disclosure comprises three or more domains. In one embodiment, the three or more domains comprise (i) an IgG protease domain, (ii) a domain that increases the half-life of the IgG protease, and (iii) a random coil domain. In another embodiment, the three or more domains comprise an IgG protease domain and two domains that increase the half-life of the IgG protease, for example, the domains that increase the half-life of the IgG protease are the N-terminus and the C-terminus of the IgG protease. In yet another embodiment, the three or more domains comprise an IgG protease domain and two random coil domains, for example, the random coil domains are the N-terminus and the C-terminus of the IgG protease.

[0404] The domains of the fusion proteins provided herein may be linked directly or via amino acid or peptide linkers. In one embodiment, the amino acid spacer linker is 1 amino acid long, 2 amino acid long, 3 amino acid long, 4 amino acid long, or 5 amino acid long. In another embodiment, the amino acid spacer sequence is about 2 amino acid long to about 5 amino acid long. In yet another embodiment, the amino acid spacer sequence is about 2 amino acid long to about 4 amino acid long. In a further embodiment, the amino acid spacer sequence is 2 amino acid long. In a further embodiment, the spacer sequence is Gly-Ser.

[0405] In one preferred embodiment, the IgG protease polypeptide domain is conjugated to a second domain at the DNA level by operably linking the IgG protease DNA sequence to a DNA sequence encoding the second domain such that the DNA sequence is located within a single open reading frame, followed by recombinant expression of the sequential DNA sequences encoding the first and second domains. The linking of the two domains at the DNA level eliminates the need for in vitro coupling or modification steps to achieve conjugate synthesis, such steps are required, for example, for the coupling of polyethylene glycol (PEG) and IgG protease in other embodiments described herein.

[0406] IgG protease fusion proteins may be combinations of two, three, or four or more different domains. IgG protease fusion proteins may also include fusions with heterologous and homologous leader sequences, with or without an N-terminal methionine residue, as well as fusion proteins containing additional sequences (e.g., polyhistidine tags) for the purification of the fusion protein.

[0407] In one embodiment, the IgG protease fusion protein includes additional effector domains such as a protease-sensitive cleavage site, an affinity tag such as a polyhistidine tag or Strep tag, a signal peptide, a retention peptide, a targeted peptide such as a membrane-transfer peptide, or an antibody fragment for tumor targeting associated with an enzyme for activation of an antitumor toxin or prodrug.

[0408] In one embodiment, the fusion protein provided herein comprises a first domain containing an IgG protease or IgG protease variant provided herein, and a second domain containing an immunoglobulin fragment crystallizable (Fc) domain. In this embodiment, the Fc domain gene sequence is fused to the IgG protease gene sequence, and the fusion protein is produced by standard molecular biology techniques. The Fc domain extends the serum half-life of the protein. While we do not wish to be bound by theory, the extension of the half-life is thought to be due to pH-dependent binding of the neonatal Fc receptor (FcRn). Such binding recovers the protein from degradation in endosomes. Furthermore, renal clearance of the fusion protein is limited due to increased mass resulting from the homodimer nature of the Fc fusion domain. Often, the Fc region improves the biophysical properties of its fusion partner, such as protein solubility and stability. In addition, high expression of the Fc fusion protein, secretion into tissue culture media, and protein A affinity purification simplify downstream manufacturing processes.

[0409] The Fc domain of the fusion protein disclosed herein may include a natural (or naturally occurring or wild-type) Fc polypeptide, or a variant Fc polypeptide (e.g., a non-natural Fc polypeptide) containing, for example, one, two, three, four, five, one to five, one to ten, or five to ten or more amino acid mutations, such as substitutions, additions, or deletions. For example, a variant Fc polypeptide may contain an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the wild-type Fc polypeptide. In some embodiments, Fc polypeptides or variant Fc polypeptides suitable for use in the Fc domain of the fusion protein provided herein are disclosed in U.S. Patent Application Publication No. 2024 / 0229002 (which is incorporated herein by reference in its entirety for all purposes). Exemplary Fc polypeptides and variant Fc polypeptides include those containing amino acid sequences selected from one of SEQ ID NOs. 2591 to 2606 (Table A). Further variants of the aforementioned exemplary Fc polypeptides or variant Fc polypeptides may also be used, containing amino acid sequences having at least about 50%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with their counterpart exemplary Fc polypeptides or variant Fc polypeptides.

[0410] [Table 8-1]

[0411] [Table 8-2]

[0412] In one embodiment, the Fc domain of the fusion protein provided herein dimerizes with a second Fc domain, thus providing a dimerized protein. In one embodiment, the dimerized protein is a homodimer. In another embodiment, the dimerized protein is a heterodimer.

[0413] In one embodiment, the Fc domain is at the N-terminus of the IgG protease domain. In another embodiment, the Fc domain is at the C-terminus of the IgG protease domain. In yet another embodiment, the fusion protein includes an Fc domain at the N-terminus of the IgG protease domain and a second Fc domain at the C-terminus of the IgG protease domain.

[0414] In one embodiment, the Fc domain is the IgG1 Fc domain. In another embodiment, the Fc domain is the IgG2 Fc domain. In another embodiment, the Fc domain is the IgG3 Fc domain. In another embodiment, the Fc domain is the IgG4 Fc domain.

[0415] In one embodiment, the IgG protease or IgG protease variant provided herein is part of a fusion protein with an albumin protein domain. In this embodiment, the albumin gene sequence is fused to the IgG protease gene sequence, and the fusion protein is produced via standard molecular biology techniques. The albumin protein has a cyclic half-life of 19 days, and therefore, the fusion protein provided herein, comprising the albumin domain, has an extended half-life compared to the half-life of the IgG protease when it exists as a single-domain protein.

[0416] Furthermore, the renal clearance of the fusion protein is limited due to the increased mass resulting from the size of the albumin domain. In one embodiment, the albumin domain improves the biophysical properties of the IgG protease, such as protein solubility and stability.

[0417] In one embodiment, the albumin domain is the N-terminus of the IgG protease domain. In another embodiment, the albumin domain is the C-terminus of the IgG protease domain.

[0418] In another embodiment of the fusion protein, the fusion protein comprises at least two domains: (i) a first domain (IgG protease domain) comprising one of the IgG proteases or IgG protease variants disclosed herein; and (ii) a second domain comprising a first random coil polypeptide (referred to as the “first random coil polypeptide domain” in some embodiments), wherein the first domain is conjugated to the second domain.

[0419] In another embodiment, the IgG protease fusion protein comprises (i) a first domain comprising an IgG protease or IgG protease variant disclosed herein, and (ii) a second domain comprising a random coil polypeptide domain which is the C-terminus of the IgG protease domain. In yet another embodiment of the IgG protease fusion protein, the fusion protein comprises (i) an IgG protease or IgG protease variant disclosed herein, and (ii) a random coil polypeptide domain which is the N-terminus of the IgG protease domain. In yet another embodiment of the IgG protease fusion protein, the fusion protein comprises (i) an IgG protease or IgG protease variant disclosed herein, (ii) a random coil polypeptide domain which is the C-terminus of the IgG protease domain, and (iii) a random coil polypeptide domain which is the N-terminus of the IgG protease domain. In a further embodiment, the random coil polypeptide domain comprises a PAS polypeptide.

[0420] As used herein, the term “random coil” refers to a conformation of a polymer molecule, including an amino acid polymer, in which the individual monomer elements forming the polymer structure are oriented essentially randomly relative to adjacent monomer elements, but are still chemically bonded to those adjacent monomer elements. In particular, polypeptides or amino acid polymers employing / having / forming a “random coil” conformation substantially lack defined secondary and tertiary structures. The properties of polypeptide random coils and methods for their experimental identification are known to those skilled in the art. A random coil polypeptide suitable for use in an IgG protease fusion protein disclosed herein is described in International Patent Application Publication 2024 / 124142, which is incorporated herein by reference in its entirety.

[0421] The IgG protease fusion proteins provided herein are, in one embodiment, recombinant IgG protease fusion proteins. In the embodiments described herein, the first domain may be the C-terminus or N-terminus of the second domain. In some embodiments, a linker peptide, such as Gly-Ser(GS), is located between the IgG protease domain and a C-terminal random coil PAS polypeptide domain (e.g., PAS10, PAS20, or PAS30) or a C-terminal random coil XTEN polypeptide domain.

[0422] In one embodiment, the IgG protease fusion protein includes a first random coil polypeptide domain. The first random coil polypeptide domain contains at least about 100 amino acid residues. In some embodiments, the first random coil polypeptide domain is a PA polypeptide, a PAS polypeptide, or an XTEN polypeptide. While we do not wish to be bound by theory, the random coil conformation mediates an increase in the in vivo and / or in vitro stability of the IgG protease. Furthermore, while we do not wish to be bound by theory, since the random coil polypeptide domain is not thought to adopt a stable structure or function on its own, the biological activity of the IgG protease to which it is conjugated is essentially conserved.

[0423] In one embodiment of the random coil polypeptide domain, the random coil polypeptide domain comprises two amino acids, proline (Pro) and alanine (Ala). In a further embodiment, the random coil polypeptide consists of two amino acids, proline (Pro) and alanine (Ala). When all amino acid residues in the polypeptide are Pro and Ala, or substantially all amino acids in the polypeptide are Pro and Ala, such polypeptide is referred to herein as a "PA polypeptide". In one embodiment, the IgG protease comprises a PA polypeptide domain. In further embodiments, the PA polypeptide is at least about 100 amino acid lengths, at least about 150 amino acid lengths, at least about 200 amino acid lengths, at least about 250 amino acid lengths, at least about 300 amino acid lengths, at least about 350 amino acid lengths, at least about 400 amino acid lengths, at least about 450 amino acid lengths, at least about 500 amino acid lengths, or at least about 550 amino acid lengths. For example, the PA polypeptide is about 200 amino acid lengths, about 300 amino acid lengths, about 400 amino acid lengths, about 500 amino acid lengths, or about 600 amino acid lengths.

[0424] In one embodiment of an IgG protease polypeptide, the polypeptide comprises two domains: (i) IgG protease and (ii) a first random coil polypeptide domain. In a further embodiment, the random coil polypeptide domain comprises three amino acids: proline (Pro), alanine (Ala), and serine (Ser). In a further embodiment, the random coil polypeptide consists of three amino acids: proline (Pro), alanine (Ala), and serine (Ser). If all amino acid residues in the polypeptide are Pro, Ala, and Ser, or substantially all amino acids in the polypeptide are Pro, Ala, and Ser, such a polypeptide is referred to herein as a "PAS polypeptide." A PAS polypeptide may also be referred to as a PAS domain if the PAS polypeptide is present within a fusion protein. In one embodiment, the IgG protease fusion protein comprises a PAS polypeptide, the PAS polypeptide comprising about 10 to about 30 tandem copies of a PAS sequence containing Pro, Ala, and Ser, for example, PAS10 having 10 tandem copies and a total of about 200 amino acids, PAS20 having 20 tandem copies and a total of about 400 amino acids, and PAS30 having 30 tandem copies and a total of about 600 amino acids. Exemplary amino acid sequences of PAS10, PAS20, and PAS30 are shown in SEQ ID NOs. 831, 832, and 833, respectively.

[0425] In yet another embodiment, the PAS amino acid sequence for use in the random coil domain includes one of the amino acid sequences listed in Table 8.

[0426] [Table 9]

[0427] In another embodiment, the random coil polypeptide is an elongated recombinant (XTEN) polypeptide. The XTEN polypeptide contains six amino acids, A, E, G, S, P, and T, in a variable proportion to form a long, non-structural hydrophilic amino acid sequence (Pasut, Polymers 2014, 6, 160-178). In one embodiment, the XTEN polypeptide is one of the polypeptides disclosed in U.S. Patent Application Publication 2015 / 0037359, the contents of which are incorporated by reference in whole for any purpose. In one embodiment, the XTEN polypeptide is at least about 800 amino acids long, consisting of six hydrophilic, chemically stable amino acids Ala, Asp, Gly, Pro, Ser, and Thr in a non-repeating manner. In one embodiment, the XTEN polypeptide is 864 residues long. In another embodiment, the XTEN polypeptide is an 864aa fragment of the XTEN polypeptide. In one embodiment, the XTEN polypeptide contains the amino acid sequence described in Sequence ID No. 834. The XTEN polypeptide may be conjugated to a first domain containing an IgG protease described herein via chemical conjugation, or it may be produced as a fusion protein with an IgG protease.

[0428] In some embodiments, the second domain of the IgG protease fusion protein comprises a PAS polypeptide and an amino acid sequence containing at least about 100 amino acid residues that form a random coil conformation. The at least about 100 amino acid residues that form the random coil include the amino acids proline (Pro), alanine (Ala), and serine (Ser). In the PAS domain, all or substantially all of the amino acids are Pro, Ala, and Ser. While we do not wish to be bound by theory, the random coil conformation mediates an increase in the in vivo and / or in vitro stability of the IgG protease enzyme. Details relating to various types of PAS polypeptides and the nucleic acids encoding them for use in the present invention can be found in International Publication Application 2008 / 155134, the contents of which are incorporated in whole by reference for all purposes.

[0429] Random coils of IgG protease fusion proteins are formed under physiological conditions. For example, in one embodiment, the physiological conditions are parameters that are typically effective for higher organisms, particularly mammals, most preferably humans. Thus, the physiological conditions may be those normally found in the body fluids of mammals. The physiological conditions may relate to corresponding parameters found in a healthy body, as well as parameters found in a diseased mammal or human patient. For example, a diseased mammal or human patient may have higher but physiological body temperature conditions if the mammal or human is suffering from a fever.

[0430] Several buffers in the experimental setup (e.g., for use in determining protein structure, particularly circular dichroism (CD) measurement, and other methods for determining the structural properties of protein / amino acid stretches), solvents and / or excipients for pharmaceutical compositions are considered to represent physiological solutions and / or physiological conditions in vitro. Examples of such buffers include, for example, phosphate-buffered saline, Tris buffer, acetate buffer, citrate buffer, or similar buffers. Generally, the pH of buffers representing physiological solution conditions is in the range of 6.5 to 8.5, e.g., 7.0 to 8.0, e.g., 7.2 to 7.7, and the osmotic pressure may be in the range of 10 to 1000 mmol / kg of H2O, more specifically in the range of 50 to 500 mmol / kg of H2O, e.g., 200 to 350 mmol / kg of H2O.

[0431] Methods for determining whether an amino acid polymer forms / adopts a random coil conformation are known in the art. Such methods include CD spectroscopy, which represents optical absorption spectroscopy that measures the difference in absorbance of right-circularly polarized and left-circularly polarized light by a substance. The secondary structure of a protein can be determined by CD spectroscopy using far-ultraviolet spectra with wavelengths of approximately 190–250 nm. At these wavelengths, α-helices, parallel and antiparallel β-sheets, and random coil conformations each produce characteristic shapes and sizes of CD spectra, so that different secondary structures commonly found in polypeptides can be analyzed. Therefore, by using CD spectroscopy, those skilled in the art can easily determine whether an amino acid polymer forms / adopts a random coil conformation under physiological conditions. Other established biophysical methods include nuclear magnetic resonance (NMR) spectroscopy, absorption spectroscopy, infrared and Raman spectroscopy, hydrodynamic volume measurement by size exclusion chromatography, analytical ultracentrifugation or dynamic / static light scattering, and measurement of friction coefficient or intrinsic viscosity.

[0432] In one embodiment, the random coil polypeptide domain contains at least about 100 amino acid residues, at least about 150 amino acid residues, at least about 200 amino acid residues, at least about 250 amino acid residues, at least about 300 amino acid residues, at least about 350 amino acid residues, or at least about 400 amino acid residues. In another embodiment, the random coil polypeptide domain contains up to about 1000 amino acid residues, up to about 900 amino acid residues, up to about 800 amino acid residues, up to about 700 amino acid residues, or up to about 600 amino acid residues. In one embodiment, the random coil polypeptide domain contains up to about 500 amino acid residues or up to about 450 amino acid residues.

[0433] In one embodiment, the random coil polypeptide domain contains about 100 to about 3000 amino acid residues. In a further embodiment, the random coil polypeptide domain contains about 100 to about 1000 amino acid residues. In some embodiments, the random coil polypeptide domain contains about 100 to about 800, about 100 to about 700, about 100 to about 600, about 100 to about 500, about 100 to about 400, or about 100 to about 300 amino acid residues.

[0434] In one embodiment, the random coil polypeptide domain includes an amino acid sequence in which proline residues account for approximately 4% to 40% of the random coil polypeptide domain. In further embodiments, alanine and serine residues constitute the remaining approximately 60% to 96% of the random coil polypeptide domain. In some embodiments, the random coil polypeptide domain includes additional amino acids different from Ala, Ser, and Pro as trace components. As used in this context, the term “trace components” means that up to 10% of the amino acids in the random coil polypeptide domain are different from alanine, serine, and proline; for example, up to 8% of the amino acids in the random coil polypeptide domain; for example, up to 6% of the amino acids in the random coil polypeptide domain; for example, up to 5% of the amino acids in the random coil polypeptide domain; for example, up to 4% of the amino acids in the random coil polypeptide domain; for example, up to 3% of the amino acids in the random coil polypeptide domain; for example, up to 2% of the amino acids in the random coil polypeptide domain; and up to 1% of the amino acids in the random coil polypeptide domain are different from Ala, Ser, and Pro. In one embodiment of the random coil polypeptide domain, the polypeptide comprises amino acids other than Ala, Ser, and Pro, the other amino acids being selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. In another embodiment, the other amino acids comprise one or more non-natural amino acids.

[0435] In another embodiment, the random coil polypeptide domain includes multiple “amino acid repeats,” i.e., the same amino acid sequence appearing two or more times within the domain, where each “amino acid repeat” consists of Ala, Ser, and Pro residues (referred to herein as “PAS” or “APS”). In a further embodiment, six or fewer consecutive amino acid residues are identical in the random coil polypeptide domain, and proline residues constitute more than approximately 4% but less than approximately 40% of the amino acids in the random coil polypeptide domain. Non-limiting examples of “amino acid repeats” consisting of Ala, Ser, and Pro residues are provided herein; see, for example, SEQ ID NOs. 835, 837, 839, 841, 843, and 845 (Table 8). Fragments and / or polymers of these sequences are used in several embodiments. A “fragment” contains at least three amino acids, including at least one Ala, one Ser, and / or one Pro.

[0436] The aforementioned repeating sequences can be encoded by nucleic acid molecules having the sequences described in SEQ ID NO: 836, SEQ ID NO: 838, SEQ ID NO: 840, SEQ ID NO: 842, SEQ ID NO: 844 and / or SEQ ID NO: 846 (Table 8).

[0437] In one embodiment, the amino acid repeats used in the random coil PAS polypeptide domain include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues, and the amino acid repeat includes at least one Ala, Ser, and Pro residue. In one embodiment, the amino acid repeat does not include more than 100 amino acid residues. In one embodiment, the amino acid repeat includes at least about 4%, at least about 5%, at least about 6%, at least about 10%, at least about 15%, or at least about 20% Pro residues. In a further embodiment, the amino acid repeat includes less than about 40%, for example, less than about 35% Pro residues.

[0438] In one embodiment, the random coil polypeptide domain includes five or fewer identical consecutive amino acid residues, for example, four or fewer identical consecutive amino acid residues, for example, three or fewer identical consecutive amino acid residues.

[0439] In one embodiment, the random coil polypeptide domain contains more than 4% Ala residues but less than 50% Ala residues, for example, more than 10% Ala residues but less than 50% Ala residues, for example, more than 20% Ala residues but less than 50% Ala residues.

[0440] In another embodiment, the random coil polypeptide domain contains more than 4% but less than 50% of Ser residues, for example, more than 10% but less than 50% of Ser residues, for example, more than 20% but less than 50% of Ser residues.

[0441] In one embodiment, the random coil polypeptide domain comprises approximately 35% Pro residues, approximately 50% Ala residues, and approximately 15% Ser residues. Alternatively, the random coil polypeptide domain comprises approximately 35% Pro residues, approximately 15% Ala residues, and approximately 50% Ser residues.

[0442] In one embodiment of the present invention, the IgG protease fusion protein contains a PAS polypeptide in a random coil polypeptide domain encoded by the amino acid sequence described in SEQ ID NO: 831.

[0443] In one embodiment of the present invention, the IgG protease fusion protein contains a PAS polypeptide in a random coil polypeptide domain encoded by the amino acid sequence described in SEQ ID NO: 832.

[0444] In one embodiment of the present invention, the IgG protease fusion protein contains a PAS polypeptide in a random coil polypeptide domain encoded by the amino acid sequence described in SEQ ID NO: 833.

[0445] In other embodiments, the present disclosure provides isolated nucleic acids encoding IgG protease polypeptides, IgG protease variant polypeptides, or fusion proteins of the present invention. In one embodiment, the IgG protease is a deimmunized IgG protease. In another embodiment, the IgG protease exists as a first domain in a fusion protein comprising at least a first domain and a second domain. In a further embodiment, the fusion protein is a recombinant IgG protease fusion protein. The isolated nucleic acids are taken out of their natural environment and may be substantially pure, for example, at least 90% pure, or in a homogeneous form. The isolated nucleic acids may be, for example, synthetic DNA, mRNA that does not exist in nature, or cDNA. Methods for producing the disclosed nucleic acids are well known to those skilled in the art. See, for example, Maniatis, T., 1990, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, which is incorporated herein by reference in its entirety. Furthermore, or alternatively, the disclosed nucleic acids may be generated by introducing one or more mutations into a related nucleic acid having a similar nucleic acid sequence using site-directed mutagenesis techniques known in the art, such as the PCR-based extension of duplicated gene segments disclosed in Heckman and Pease (2007). Nat Protoc. 2, pp. 924-932, which is incorporated herein by reference in its entirety. The disclosed nucleic acids may be cleaved at appropriate sites using restriction endonucleases, followed by, if desired, further enzymatic modification, isolation, and in vitro ligation.

[0446] In one embodiment, the nucleic acid molecule encoding the polypeptide (which may be a fusion protein) of this disclosure is present in a nucleic acid vector. In one embodiment, the nucleic acid encoding an IgG protease or a fusion protein containing the IgG protease is inserted into a vector, such as a plasmid, for nucleic acid replication. In another embodiment, the nucleic acid encoding an IgG protease or a recombinant IgG protease fusion protein containing an IgG protease domain is inserted into a suitable expression vector, i.e., a vector containing elements necessary for the transcription and translation of the inserted IgG protease coding sequence, such as a promoter sequence, a terminator sequence, a polyadenylation sequence, and an enhancer sequence. The vector may be, for example, a plasmid, a phage, a phagemid, an adenovirus, an AAV, or a lentivirus. Various host-vector systems can be used to express the coding sequence of an IgG protease or a recombinant IgG protease fusion protein containing an IgG protease domain. Exemplary systems include mammalian cell lines infected with viruses (e.g., vaccinia virus, adenovirus, etc.); insect cell lines infected with viruses (e.g., baculovirus); and microorganisms such as yeast containing a yeast vector, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA. The expression elements of these vectors differ in their intensity and specificity. In one embodiment, a bacterial expression vector is used. In a further embodiment, the bacterial expression vector is intended for use with E. coli. In one embodiment, the promoter in the bacterial expression vector is a T5 promoter that is induceable by isopropyl β-D-1 thiogalactopyranoside (IPTG) and repressible by glucose, a T7 promoter that is induceable by IPTG and repressible by glucose, a rhamnose (rham) promoter that is induceable by rhamnose and repressible by glucose, or an alkaline phosphatase (phoA) promoter that is induceable by phosphate starvation and repressible in the presence of phosphate.

[0447] Expression vectors can be constructed using any known method for inserting DNA fragments into a vector, containing nucleic acids comprising one or more regulatory elements, such as appropriate transcription / translation control signals, and a coding sequence for an IgG protease or recombinant IgG protease fusion protein operably linked thereto. These methods may include in vitro recombinant DNA and synthetic techniques, as well as in vivo recombination (genetic recombination). The expression of a nucleic acid sequence encoding an IgG protease or a recombinant IgG protease fusion protein comprising an IgG protease domain may be regulated by a second nucleic acid sequence so that the IgG protease or recombinant IgG protease fusion protein is expressed in a host transformed with a recombinant DNA molecule. For example, the expression of an IgG protease or a recombinant IgG protease fusion protein comprising an IgG protease domain may be regulated by any promoter / enhancer element known in the art. In some embodiments, the nucleic acid comprises a nucleic acid sequence encoding an IgG protease or recombinant IgG protease fusion protein operably linked to a heterologous promoter. Exemplary promoters that can be used to control the expression of IgG proteases or recombinant IgG protease fusion proteins using mammalian expression vectors include the Simian virus 40 (SV40) initial promoter region, the promoter contained in the 3' long-term repeat of Roussarcoma virus, the herpesthymidine kinase promoter, and the regulatory sequence of the metallothionein gene. Exemplary promoters useful for prokaryotic expression vectors include the β-lactamase promoter, the tac promoter, and the osmotically regulated osmB promoter.

[0448] In another embodiment, the disclosure provides a host cell comprising a nucleic acid vector disclosed herein. In a further embodiment, the host cell can produce an IgG protease or a fusion protein containing the IgG protease disclosed herein. In one embodiment, the IgG protease exists as a fusion protein comprising (i) an IgG protease domain and a random coil domain, or (ii) an IgG protease domain and an Fc domain. In a further embodiment, the IgG protease fusion protein is a recombinant IgG protease fusion protein comprising an IgG protease disclosed herein. A suitable cell line or host system may be selected based on the desired expression level of the IgG protease or recombinant IgG protease fusion protein and / or post-translational processing and modification (e.g., glycosylation, cleavage). Suitable host cells include bacteria (e.g., E. coli), mammalian cells, plant cells, insect cells, fungi, yeasts, and transgenic plants and animals. Exemplary mammalian cell lines available in the art for the expression of heterologous proteins, such as IgG proteases or recombinant IgG protease fusion proteins containing the IgG protease domain disclosed herein, include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, mouse melanoma cells, rat myeloma cells, human embryonic kidney cells, and human embryonic retinal cells. The introduction of the vector into host cells can be achieved using techniques well known in the art. For eukaryotic cells, preferred techniques include, for example, calcium phosphate transfection, diethylaminoethyl (DEAE)-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses. For bacterial cells, preferred techniques include calcium chloride transformation, electroporation, and transfection using bacteriophages.Following introduction, expression from the nucleic acid can be induced or enabled, for example, by culturing host cells under conditions for the expression of nucleic acid sequences encoding IgG protease or recombinant IgG protease fusion proteins, leading to the production of the respective proteins. In one embodiment, the nucleic acid encoding IgG protease or IgG protease fusion protein is integrated into the genome of the host cell, e.g., chromosomes. Integration can be facilitated by including sequences that promote recombination with the genome, according to standard techniques.

[0449] In one embodiment, the host cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell or a human fetal kidney cell. In a further embodiment, the expression vector for use in mammalian cells comprises actin (e.g., chicken β-actin), cytomegalovirus (CMV), CMV enhancer / elongation factor (CEF), CMV early enhancer / chicken β-actin (CAG), hybrid CMV enhancer / chicken β-actin (CBh), elongation factor-1α (EF1 alpha), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or Simian virus 40 (SV40) promoter.

[0450] In another embodiment, the host cell is a yeast cell. In a further embodiment, the expression vector for use in yeast cells comprises an alcohol oxidase (AOX), glyceraldehyde-3-phosphate dehydrogenase (GAP), alcohol dehydrogenase (ADH), or galactokinase 1 (GAL1) promoter.

[0451] In another embodiment, the host cell is an insect cell infected with a baculovirus. In a further embodiment, the expression vector for use with the baculovirus includes a polyhedrin gene promoter.

[0452] In another embodiment, the host cell is a bacterium, for example, E. coli. In a further embodiment, the expression vector for use in bacteria includes an osmB promoter, a T7-lac promoter (see Shilling et al., Commun Biol 3, 214 (2020)), a pBAD promoter, a Tac promoter, a tet-inducible promoter, a cold shock protein A (cspA) promoter, or an inducible promoter of the alkaline phosphatase gene (phoA) derived from E. coli.

[0453] In another aspect, the Disclosure provides a method for recombinantly producing IgG proteases, IgG protease variants, or conjugates containing them as disclosed herein (e.g., recombinant IgG protease fusion proteins).

[0454] The method is, (i) Culturing a host cell comprising a nucleic acid vector comprising a nucleic acid sequence encoding an IgG protease, IgG protease variant, or IgG protease conjugate (e.g., recombinant IgG protease fusion protein) disclosed herein, wherein the nucleic acid sequence is operably linked to a heterologous promoter under conditions that enable expression of the nucleic acid sequence encoding an IgG protease, IgG protease variant, or IgG protease conjugate (e.g., recombinant IgG protease fusion protein) and recombinant production of the IgG protease, IgG protease variant, or IgG protease conjugate by the host cell, (ii) Isolating an IgG protease, an IgG protease variant, or an IgG protease conjugate produced by recombinant DNA.

[0455] IgG proteases or IgG protease fusion proteins may be produced in any suitable cell culture system, including prokaryotic cells, e.g., E. coli, BL21, or JM83, or eukaryotic cells, e.g., Pichia pastris yeast strain X-33 or Chinese hamster ovary (CHO) cells. Further suitable cell lines known in the art are available from cell line depositories, such as the American Type Culture Collection (ATCC). IgG proteases or IgG protease conjugates can be isolated from growth media, cell lysates, or cell membrane fractions. Isolation and purification of recombinantly produced IgG proteases or conjugates containing them may be carried out by any conventional means, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, etc., and may involve, for example, the use of monoclonal or polyclonal antibodies against the tag fused with the biologically active protein of the present invention. For example, proteins can be purified via Strep-tag II using streptavidin affinity chromatography (Skerra and Schmidt (2000). Methods Enzymol 326, pp. 271-304).

[0456] In one embodiment, the vector is under the control of an osmotically sensitive promoter. The osmotically sensitive promoter initiates transcription as a result of an increase in osmotic pressure, as sensed by the cell. In one embodiment, the host cell is E. coli, and the promoter is the osmB promoter, T7-lac promoter, pBAD promoter, Tac promoter, tet-inducible promoter, cold shock protein A (cspA) promoter, or an inducible promoter of the alkaline phosphatase gene (phoA) derived from E. coli.

[0457] In one embodiment, an IgG protease, an IgG protease variant, or a conjugate containing the same is isolated using a cationic surfactant, such as cetylpyridinium chloride (CPC). In one embodiment, this method further comprises purifying the recombinantly produced IgG protease or its conjugate using, for example, affinity chromatography, ammonium sulfate fractionation, or sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. For example, an IgG protease or its conjugate may be prepared according to the method described in International Patent Application Publication No. 2000 / 008196 (which is incorporated herein by reference in its entirety). In one embodiment, the IgG protease or its conjugate is produced in bacteria and, after isolation, subjected to endotoxin reduction using methods known in the art, such as those disclosed in the examples of this application.

[0458] In another embodiment, the Disclosure provides a pharmaceutical composition comprising an IgG protease, an IgG protease variant, or a fusion protein containing the same, and a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other material known to those skilled in the art for formulating protein therapeutics.

[0459] Unless otherwise specified, the term “pharmaceutically acceptable” is used to characterize a part (e.g., a salt, dosage form, or excipient) as appropriate for use in accordance with sound medical judgment. Generally, a pharmaceutically acceptable part has one or more benefits that outweigh any adverse effects it may have. Adverse effects may include, for example, excessive toxicity, irritation, allergic reactions, and other problems and complications.

[0460] The pharmaceutical composition may contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art for formulating protein therapeutics. Such materials are non-toxic and do not impair the efficacy of the variants described herein. Examples of such materials include solvents, dispersions, antimicrobial and antifungal agents, isotonic agents, and absorption retarders. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate-buffered saline, dextrose, glycerol, and ethanol, and combinations thereof. In one embodiment, the pharmaceutical composition contains an isotonic agent, such as a sugar, and / or a polyhydric alcohol such as mannitol or sorbitol, or sodium chloride. Further examples of pharmaceutically acceptable substances are wetting agents or auxiliary substances, such as emulsifiers, preservatives, or buffers, which increase shelf life or efficacy.

[0461] In one embodiment, a pharmaceutical composition comprising an IgG protease, an IgG protease variant, or a fusion protein containing the same may be formulated in liquid, semi-solid, or solid form, such as a liquid solution (e.g., injectable and injectable solutions), a dispersion or suspension, a powder, liposomes, and suppositories. The preferred form depends on the intended mode of administration, the therapeutic application, the physicochemical properties of the IgG protease, and the delivery route. The formulation may contain excipients, such as sugars, amino acids, and surfactants, or combinations of excipients. Liquid formulations may encompass a wide range of IgG protease concentrations and pH. Solid formulations can be produced, for example, by freeze-drying, spray-drying, or drying using supercritical fluid technology.

[0462] For intravenous or intragraft injection, the active ingredient may be in the form of a parenterally acceptable aqueous solution that is pyrogenic and has a suitable pK, isotonicity, and stability. Those skilled in the art can easily prepare suitable solutions using isotonic vehicles such as sodium chloride solution, Ringer's solution, and lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included.

[0463] In some embodiments, pharmaceutical compositions are formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for containing high concentrations of IgG protease. Sterile injectable solutions can be prepared by incorporating IgG protease with one or a combination of the components listed above into a suitable solvent, followed by filtration sterilization. Generally, dispersions are prepared by incorporating IgG protease into a sterile vehicle containing a dispersion medium and other components listed above. For sterile powders for the preparation of sterile injectable solutions, preparation methods include vacuum drying and freeze-drying, from a pre-sterile filtered solution to obtain a powder of the active ingredient and any additional desired components. Appropriate fluidity of the solution can be maintained, for example, by using a coating such as lecithin, by maintaining the particle size of the dispersion, or by using a surfactant. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as monostearate and gelatin, in the composition.

[0464] In some embodiments, the pharmaceutical composition may be prepared with a carrier that protects the IgG protease from rapid release, such as an implant, a transdermal patch, and a sustained-release formulation including a microencapsulation delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used.

[0465] In one embodiment, the pharmaceutical composition is a solution of an IgG protease or IgG protease variant disclosed herein, or a fusion protein containing the same. In one embodiment, the solution is phosphate-buffered saline containing IgG protease. In further embodiments, the solution is sterile and suitable for injection, for example, intravenous or subcutaneous injection.

[0466] In one embodiment, the polypeptides, fusion proteins, and compositions of the present invention are used in vitro, ex vivo, or in vivo to cleave IgG into F(ab')2 and Fc fragments. In one embodiment, this method involves contacting IgG with the compound of the present invention or a composition containing it. In a further embodiment, this method is carried out ex vivo.

[0467] In another embodiment, the present invention provides a method for treating or preventing diseases that are mediated whole or partially by pathogenic immunoglobulin G (IgG) antibodies. This method comprises administering to a patient in need of treatment a therapeutically or prophylactically effective amount of the polypeptide or fusion protein of the present invention, or a composition containing the same. In one embodiment, this method comprises administering to a patient in need of treatment a plurality of times the polypeptide or fusion protein of the present invention, or a composition containing the same.

[0468] In one embodiment of this method, the treatment method is an ex vivo method. That is, blood is collected from a patient having a disease or condition that is mediated whole or partially by an IgG antibody, and this method involves contacting this blood with the polypeptide of the present invention or a fusion protein containing the same.

[0469] As used herein, “treat,” “treat,” and “improve” are interchangeable. These terms refer to an approach to obtain beneficial or desirable outcomes, including but not limited to therapeutic and / or preventive benefits. Therapeutic benefit refers to any therapeutically related improvement or effect on one or more diseases, conditions, or symptoms under treatment. In one embodiment, the term “treat” includes (1) preventing or delaying the onset of clinical symptoms of a condition, disorder, or pathology in a patient who is suffering from or may be susceptible to the condition, disorder, or pathology but has not yet experienced or shown any clinical or subclinical symptoms of the condition, disorder, or pathology; (2) inhibiting the condition, disorder, or pathology (e.g., stopping, reducing, or delaying the onset of at least one clinical or subclinical symptom of the disease, or, in the case of maintenance treatment, its recurrence); and (3) alleviating the condition (e.g., by causing a regression or reduction in the severity of at least one of the conditions, disorder, or pathology, or its clinical or subclinical symptoms).

[0470] The methods provided herein may be therapeutic or prophylactic. In therapeutic use, a polypeptide (e.g., an IgG protease or a fusion protein containing the same), or a composition containing the same, is administered to a subject already suffering from a disorder or condition in an amount effective to treat it (e.g., cure, alleviate or partially cessate one or more of the conditions or symptoms thereof). Such therapeutic treatment may result in a reduction in the severity of disease symptoms or an increase in the frequency or duration of asymptomatic periods. In prophylactic use, a polypeptide or a composition containing the same is administered to a subject who is not yet showing symptoms of a disorder or condition, or who is at risk of developing the disease or condition, in an amount sufficient to prevent or delay the onset of symptoms and / or the onset of the disease.

[0471] The term “effective dose” or “therapeutic effective dose” refers to the amount of drug sufficient to achieve a certain outcome, for example, to produce a beneficial or desirable result. The therapeutic effective dose may vary depending on one or more of the following: the subject being treated and the disease state, the subject’s weight and age, the severity of the disease state, the mode of administration, etc. In some embodiments, when the polypeptide of the present invention is administered to a subject before the onset of disease or pathology, the amount of drug administered may be referred to as the “preventive effective dose.” The subject may be identified by any preferred means as being at risk of developing disease or pathology.

[0472] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to vertebrates such as mammals. Mammals may be, for example, mice, rats, rabbits, cats, dogs, pigs, sheep, horses, non-human primates (e.g., cynomolgus macaques, chimpanzees), or humans. Subject tissues, cells, or derivatives thereof obtained in vivo or cultured in vitro are also included. Human subjects may be adults, teenagers, children (2 to 14 years), infants (1 to 24 months), or neonates (up to 1 month). In some embodiments, adults are about 65 years of age or older, or elderly people about 60 years of age or older. In some embodiments, subjects are pregnant women or women intending to become pregnant. In one embodiment, subjects are ≥18 to ≤85 years of age.

[0473] In the embodiments described herein, the polypeptide may be administered concurrently with the immunosuppressant.

[0474] In one embodiment, the polypeptide or a composition containing the same is administered parenterally. In a further embodiment, administration is by intravenous infusion. In yet another embodiment, administration is by intradermal, subcutaneous, transdermal, intramuscular, intra-arterial, intraperitoneal, intra-articular, or intraosseous administration.

[0475] In therapeutic methods provided herein, the polypeptides of the present invention may be administered to treat or prevent diseases or conditions that are entirely or partially mediated by pathogenic IgG antibodies. Accordingly, the present invention provides polypeptides of the present invention for use in the treatment or prevention of diseases or conditions that are mediated by pathogenic IgG antibodies. The present invention also provides a method for treating or preventing diseases or conditions that are mediated by pathogenic IgG antibodies, comprising administering the polypeptides of the present invention to an individual. This method may include repeated administration of the polypeptides. The present invention also provides polypeptides of the present invention for use in the manufacture of pharmaceuticals for the treatment or prevention of diseases or conditions that are entirely or partially mediated by pathogenic IgG antibodies.

[0476] Pathogenic antibodies are typically specific to antigens targeted in autoimmune diseases or other conditions that are entirely or partially mediated by antibodies. The polypeptides of the present invention can be used to treat any of these diseases or conditions. The polypeptides are particularly effective in treating or preventing autoimmune diseases that are entirely or partially mediated by pathogenic IgG antibodies.

[0477] In one embodiment, diseases associated with IgG antibodies include Addison's disease, anti-glomerular basement membrane (anti-GBM) glomerulonephritis, Goodpasture syndrome, anti-neutrophil cytoplasmic antibody (ANCA-associated vasculitis), Churg-Strauss syndrome, microscopic polyangiitis, anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis, anti-phospholipid antibody syndrome (APS) (e.g., catastrophic APS), autoimmune bullous skin diseases, pemphigus (e.g., pemphigus foliaceus, endemic pemphigus, pemphigus vulgaris), autoimmune hemolytic anemia (AIHA), autoimmune hepatitis (AIH), and autoimmune neutropenia (autoimmune Neutropenia (AIN), bullous pemphigoid (BP), celiac disease, chronic urticaria, complete congenital heart block (CCHB), type 1A diabetes, epidermolysis bullosa, essential mixed cryoglobulinemia, Graves' disease, Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), acute inflammatory demyelinating polyneuropathy (AIDP), acute motor axonal neuropathy (AMAN), acquired factor VIII deficiency, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), Lambert-Eaton myasthenic syndrome myasthenic syndrome (LEMS), mixed connective tissue disease (mixed connective tissue disease)The following conditions are selected: multiple myeloma, myasthenia gravis, myasthenic crisis, myocarditis, dilated cardiomyopathy (DCM), congestive cardiomyopathy, neuromyelitis optica (NMD), primary biliary cirrhosis (PBC), primary progressive multiple sclerosis (PPMS), systemic lupus erythematosus (SLE) (e.g., lupus nephritis), stiff-person syndrome (SPS), Sjögren's syndrome (SS), systemic sclerosis (scleroderma), rheumatoid arthritis (RA), rheumatic heart disease (RHD), serum sickness syndrome, or type III immune complex hypersensitivity.

[0478] In one embodiment, the disease treated by the method provided herein, in relation to IgG antibodies, is chronic inflammatory demyelinating polyneuropathy (CIDP).

[0479] In another embodiment, the disease associated with IgG antibodies and treated by the method provided herein is acute inflammatory demyelinating polyneuropathy (AIDP).

[0480] In yet another aspect of the present invention, a method is provided for preventing and / or treating antibody-mediated rejection (AMR) of an organ allograft in a patient requiring treatment. In one embodiment, this method comprises administering a polypeptide, fusion protein, or composition of the present invention to a patient in need. Depending on when donor-specific antibodies (DSAs) are identified, the polypeptide or fusion protein of the present invention, or a composition containing the same, may be administered to the patient before, during, or after organ transplantation.

[0481] In transplant patients, the presence of high-titer antibodies against the potential donor's MHC antigen (known as "donor-specific antibodies" or "DSA") is a direct contraindication to transplantation due to the risk of acute antibody-mediated rejection (AMR) of the transplanted organ. Many patients awaiting transplantation are sensitized, and often highly sensitized, to the donor MHC antigen, and an accumulation of patients who cannot proceed with transplantation has been identified.

[0482] One approved strategy to overcome the DSA barrier is to reduce DSA levels to a level where transplantation can be considered, often by applying plasmapheresis or immunoadsorption in combination with intravenous gamma globulin (IVIG) or rituximab. However, plasmapheresis, immunoadsorption, and IVIG therapy have the disadvantages of being inefficient and requiring rigorous planning due to the need for repeated treatments over a long period.

[0483] In contrast, the method of the present invention enables the removal of DSA in potential transplant recipients. Administration of the polypeptide or fusion protein of the present invention, or a composition containing the same, prior to transplantation has the ability to effectively desensitize sensitized patients, thereby enabling transplantation and avoiding acute antibody-mediated rejection.

[0484] While we do not wish to be bound by theory, when used in organ transplant patients, either pre-transplant, during transplantation, or post-transplantation, the method of the present invention may enable the removal of DSA in potential transplant recipients.

[0485] In one embodiment, the organ is a solid organ. In a further embodiment, the organ is a kidney, small intestine, pancreas, heart, lung, or liver. In yet another embodiment, the organ is a kidney.

[0486] In one embodiment, the patient being treated is sensitized. "Sensitized" means that the patient has developed antibodies against major histocompatibility (MHC) antigens (also known as human leukocyte antigens, HLA). Anti-HLA antibodies originate from allosensitized B cells and may be present in patients who have been previously sensitized by blood transfusion prior to transplantation or pregnancy.

[0487] Whether a potential transplant recipient is sensitized can be determined by any preferred method. For example, a panel-reactive antibody (PRA) test can be used to determine if a recipient is sensitized. A PRA score > 30% is typically interpreted as meaning the patient is at "high immunological risk" or "sensitized." Alternatively, a cross-matching test may be performed, in which a blood sample from the potential transplant donor is mixed with a blood sample from the intended recipient. A positive cross-match means the recipient has antibodies that react to the donor sample, indicating that the recipient is sensitized. Cross-matching tests are typically performed as a final check immediately before transplantation.

[0488] In one embodiment of a method for treating AMR in patients requiring treatment for AMR, the patient is further administered intravenous immunoglobulin (IVIG).

[0489] In one embodiment, the subject is administered the pharmaceutical composition chronically; that is, after treatment for a specific disease / syndrome is initiated, the subject is administered the composition throughout their lifetime. [Examples]

[0490] The present invention will be further described with reference to the following embodiments. However, it should be noted that these embodiments are illustrative, as are the embodiments described above, and should not be construed as limiting the scope of the present invention.

[0491] method IgG protease library construction NEBuilder HiFi DNA Assembly Cloning The amplified, purified, validated, and linearized plasmid backbone and individual protease gene fragments were equally divided into 96-well PCR plates, each containing a combined total volume of 5 μL. The plates were placed on moist ice, and 5 μL of 2×NEBuilder HiFi DNA Assembly Master Mix (NEB#E2621L) was equally divided into each well. The wells were sealed and incubated in a preheated thermocycle at 50°C for 15–30 minutes. The plates were placed on ice, and each well was diluted with 30 μL of molecular-grade water.

[0492] Transformation 5 μL of commercially available chemically competent E. coli cell line was placed in each well of a 96-well PCR plate maintained on moist ice. 1 μL of diluted NEBuilder reaction solution was added to each well, and the plate was sealed. The cells were modified and transformed using 100 μL of SOC (Super Optimal broth with Catabolite repression) medium for growth, according to the manufacturer's instructions.

[0493] 100 μL of SOC medium was added to each well, and the wells were gently mixed with a pipette. The entire contents of the wells were transferred to a 96-well deep-well plate, the plate was sealed, and incubated at 37°C for 1 hour at 220 rpm. A new 96-well deep-well plate was pre-filled with 1 mL of Terrific Broth medium supplemented with the appropriate antibiotic. The entire volume of deep wells filled with SOC was transferred to the new plate, sealed with a gas-permeable membrane, and incubated at 37°C overnight at 220 rpm.

[0494] Expression 24-well deep-well plates were pre-filled with 3 mL of Terrific Broth medium supplemented with appropriate selective antibiotics. A 1:20 dilution of the culture was inoculated into the pre-filled 24-well plates overnight, the plates were sealed with a gas-permeable membrane, and incubated at 37°C at 300 rpm.

[0495] When the consensus OD600 value reached >0.8 (approximately 2 hours), expression was induced with L-arabinose, IPTG, or other suitable inducers, and the protein was expressed at 37°C for 2–4 hours or at 16–30°C for 18–24 hours at 300 rpm. The cell pellet was collected by centrifugation of the plate at 3,200 × g for 10 minutes. The supernatant was discarded, and the plate was sealed with foil seal. The foil-sealed plate was stored at -80°C before lysis.

[0496] Dissolution Before cell lysis, a foil-sealed 24-well plate was removed from -80°C and allowed to thaw completely at room temperature. 400–800 μL of B-Per (Thermo#78248), B-Per (Thermo#78248) containing benzonase (MilliporeSigma#706643), or B-Per Complete (Thermo#89821) was added to each well, and the wells were mixed by pipetting. The wells were incubated at room temperature for 15 minutes. The plate was centrifuged at 3,200 × g for 15 minutes. The clarified lysates containing the expressed proteins were pipetted from the wells and transferred to a 96-well deep-well plate, and stored on moist ice until purification.

[0497] HisPur® NiNTA or HisPur® Cobalt Refining HisPur® Cobalt Spin Plates (Thermo#90095) or HisPur® Ni-NTA Spin Plates (Thermo#88230) were equilibrated with washing buffer (25 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10 mM imidazole) according to the manufacturer's instructions. The clarified lysates were applied to the plates. The plates were incubated on wet ice for 15 minutes after each application of the clarified lysate. The plate assemblies were then centrifuged at 10 × g for 3 minutes, 25 × g for 3 minutes, and 500 × g for 3 minutes, in that order, after each application of the clarified lysate, discarding the flow-through after each application. The plates were then washed with washing buffer according to the manufacturer's instructions.

[0498] Next, the purified plate was placed on a new collection plate, and 220 μL of elution buffer (25 mM Tris-HCl (pH 7.5), 500 mM NaCl, 500 mM imidazole) was added to each well and incubated on wet ice for 15 minutes. The plate assembly was then centrifuged at 10 × g for 3 minutes, 25 × g for 3 minutes, and 500 × g for 3 minutes in that order, and the eluted material was collected.

[0499] Desalting For each sample, two Zeba spin desalting plates (Thermo#PI8980) were used (110 μL per plate). The plates were equilibrated with final buffer (25 mM Tris HCl (pH 7.5), 150 mM NaCl) according to the manufacturer's instructions. The Zeba plates were placed on a new collection plate, and 110 μL of eluate was applied to the center of the resin bed. The plate assembly was centrifuged at 1,000 × g for 2 minutes to collect the buffer-exchanged proteins. The two desalted samples were combined into a single new plate and quantified by bicinchoninic acid (BCA) protein assay (Thermo#23225) according to the manufacturer's protocol.

[0500] Differential scanning fluorescence (SCFL) for determining the melting temperature of proteins In a 96-well PCR plate, 22.5 μL of protein and 2.5 μL of 50×SYPRO Orange (Invitrogen #S6650) were added to each well. The plate was sealed with polymerase chain reaction (PCR) optical film. Using a BioRad CFX96 Touch real-time PCR detection system, the program was run for 10 seconds at 0.5°C intervals within a temperature range of 10°C to 95°C.

[0501] Activity assessment using a custom ELISA-based assay High-binding capacity plates were coated with 50–100 μL / well of human TNF-α (stock solution 0.1 mg / mL) at a 1:800 dilution or with various non-cleavable capture antibodies at various dilutions in carbonate / bicarbonate buffer. The plates were sealed and incubated at 4°C for 16–48 hours or at 37°C for up to 8 hours. The plates were washed 3–5 times in a plate washer and with phosphate-buffered saline (PBS-T) wash buffer containing Tween 20, then gently tapped and dried.

[0502] The plates were blocked with 175–200 μL of 1× Superblock Buffer (Thermo#37515) at 22–37°C for 15–30 minutes. The blocking buffer was removed, and the plates were gently tapped to dry. Next, 75–100 μL of adalimumab was added in LowCross Buffer (Boca#NC9048027) at dilutions of 1:2,500–1:10,000, or various other antibody substrates at various dilutions, and the plates were sealed. The plates were then incubated at 37°C for 1–4 hours. Finally, the plates were washed 3–5 times in a plate washer with PBS-T wash buffer, and gently tapped to dry.

[0503] Each well was prepared separately in PBS and incubated with 75–100 μL of normalized purified library variant or control at 37°C for varying durations. The plate was washed 3–5 times in a plate washer and PBS-T wash buffer, then tapped and dried. 75–100 μL of secondary antibody conjugated with anti-human horseradish peroxidase (HRP) was added to each well at a dilution of 1:5,000–1:30,000 in LowCross buffer. The plate was incubated at 37°C for 20–40 minutes. The plate was washed 3–5 times in a plate washer and PBS-T wash buffer, then tapped and dried.

[0504] 75–100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added to each well. The plate was incubated at room temperature (RT) with intermittent shaking until sufficient color development occurred. Next, 75–100 μL of stop solution (Invitrogen #SS04) was added per well, and the plate was incubated at 550 rpm for 1 minute. Absorbance was read at 450 nm and corrected for background absorbance by subtracting the absorbance at a reference wavelength of 620 nm. The IgG protease activity of the variant was presented as a percentage of the IgG protease activity of its parent enzyme or wild-type IdeS. In several studies, the IgG protease activity of the variant has also been presented as the percentage decrease in corrected absorbance at 450 nm compared to the corrected absorbance at 450 nm using PBS (negative control), i.e., the percentage decrease in corrected absorbance at 450 nm using the variant compared to PBS = (1 - corrected absorbance at 450 nm using the variant / corrected absorbance at 450 nm using PBS). * 100

[0505] Example 1 - Identification of polypeptides with IgG protease activity via ancestral reconstruction. method search Using PSIBlast (v2.12.0), a non-redundant (nr) database of the National Center for Biotechnology Information (NCBI) was searched with an e-value threshold of 0.005 (see, e.g., Altschul et al. (1997). Nucleic Acids Research 25, pp. 3389-3402, the entire contents of which are incorporated herein by reference). Predicted proteolytic domains were isolated using InterProScan (see, e.g., Blum et al. (2021). Nucleic Acids Research 49, Database issue doi:10.1093 / nar / gkaa977, the entire contents of which are incorporated herein by reference). Where overlapping domains existed in the sequence, they were combined into a single sequence (blast.ncbi.nlm.nih.gov / doc / blast-help / references.html#references).

[0506] Ancestor Sequence Reconstruction Sequences having at least 40% identity with IdeS (SEQ ID NO: 2) or IdeZ (SEQ ID NO: 830), whether or not they have an N-terminal signal sequence, were selected from the PSIBlast search.

[0507] A total of 180 array elements were selected.

[0508] Multiple sequence alignments (MSAs) were constructed from a pool of selected sequences using ClustalOmega (v1.2.4) with the option "-iterations 3-full-full-iter". (www.clustal.org / omega / , Sievers et al. (2011). Molecular Systems Biology 7:539 doi:10.1038 / msb.2011.75, the entire paper is incorporated herein by reference).

[0509] Using RaxML (v8.2.12), a phylogenetic tree was constructed using MSA with the option "-m PROTCATJTT-f ax 42-N autoMRE". Note that the "-x" option enables fast bootstrapping. (cme.h-its.org / exelixis / web / software / raxml / ,Stamatakis(2014).Bioinformatics 30(9),pp.1312-1313,doi:10.1093 / bioinformatics / btu033, the entire text is incorporated herein by reference).

[0510] The ancestral sequences were generated using FastML (v3.11.0) with the option "-seqType aa" using multiple sequence alignment (MSA) and phylogenetic trees. See, for example, Moshe and Pupko (2019). Bioinformatics 35(11), pp. 2562–2568. The entire work is incorporated herein by reference (http: / / fastml.tau.ac.il / credits.php).

[0511] Predicted ancestral protein sequences for IgG protease are provided in SEQ ID NOs: 27 to 302.

[0512] Generation of Concomitant Sequences The consensus sequence was generated according to the method described in Biswas et al. (2021). Nat Methods 18, 389-396 (its disclosure is incorporated herein by reference in its entirety). The Metropolis-Hastings sampling algorithm was used to sample the highest-probability sequence from the multiple sequence alignment. The algorithm was modified from the code provided at github.com / churchlab / low-N-protein-engineering / blob / master / analysis / A009_chip_1_GFP_follow_up_designs / 002_consensus_sequence_design.ipynb.

[0513] For IdeS and IdeZ, MSAs were generated using ClustalOmega (Sievers et al. (2011). Molecular Systems Biology 7:539 doi:10.1038 / msb.2011.75, the entire report of which is incorporated herein by reference) from sequences having at least 40% sequence identity with each protein (i.e., IdeS or IdeZ). Subsequently, Metropolis-Hastings sampling was performed, starting from each protein (IdeS or IdeZ), using the position-specific scoring matrix (PSSM) generated from the MSAs as a probability distribution.

[0514] Further consensus sequences were generated from MSAs produced as described above, using sequences with at least 40% sequence identity to IdeS or IdeZ. The seed sequences for these consensus designs were the most likely sequences from the position-specific scoring matrix (PSSM).

[0515] Measurement of IgG protease activity and melting temperature Procedures for evaluating melting temperature and IgG protease activity are provided above. Results for specific IgG proteases in this disclosure are provided in Table 9 below. IgG protease activity is presented as a percentage of the IgG protease activity of wild-type IdeS.

[0516] [Table 10]

[0517] Example 2 - Modified variant of ancestral IgG protease polypeptide Fishing protease variant Seven candidates were selected from the top 29 proteases from Example 1 for further manipulation. The candidates were selected based on activity, thermal stability, and IdeS, IdeZ, and identity compared to one another. Initial libraries were generated for each of the seven candidates based on predicted epitope depletion and predicted fitness. 34–47 point mutations were tested for each candidate.

[0518] Further point mutation and multiple mutation designs were tested for N142, and further multiple mutation designs were tested for N144.

[0519] All other IDs are NCBI accession numbers, where everything after the pipe "|" character indicates the range of positions used in the array. For example, WP_165623594.1|5-383 is the array from position 5 to 383 of NCBI accession number WP_165623594.1.

[0520] Procedures for evaluating melting temperature and IgG protease activity are provided above. Results for specific IgG protease variants of SEQ ID NO: 297 are provided in Tables 10 and 11. The activity in Table 10 is presented as a percentage of N142 (SEQ ID NO: 297) IgG protease activity. The activity in Table 11 is presented as a percentage of the corresponding wild-type enzyme (parent enzyme) IgG protease activity.

[0521] [Table 11]

[0522] [Table 12-1]

[0523] [Table 12-2]

[0524] [Table 12-3]

[0525] Table 12-4

[0526] Table 12-5

[0527] Table 12-6

[0528] Table 12-7

[0529] Table 12-8

[0530] Table 12-9

[0531] Table 12-10

[0532] Table 12-11

[0533] Table 12-12

[0534] Table 12-13

[0535] Table 12-14

[0536] Table 12-15

[0537] Table 12-16

[0538] Table 12-17

[0539] Table 12-18

[0540] Table 12-19

[0541] Table 12-20

[0542] Table 12-21

[0543] Table 12-22

[0544] Table 12-23

[0545] Table 12-24

[0546] Table 12-25

[0547] Table 12-26

[0548] Table 12-27

[0549] Table 12-28

[0550] Table 12-29

[0551] Table 12-30

[0552] Table 12-31

[0553] Table 12-32

[0554] Table 12-33

[0555] Table 12-34

[0556] Table 12-35

[0557] Table 12-36

[0558] Table 12-37

[0559] Table 12-38

[0560] Table 12-39

[0561] Table 12-40

[0562] Table 12-41

[0563] Table 12-42

[0564] Table 12-43

[0565] [Table 12-44]

[0566] [Table 12-45]

[0567] [Table 12-46]

[0568] [Table 12-47]

[0569] Example 3 - Manipulated variant of IdeS (SEQ ID NO: 2) The library was generated using standard molecular biology techniques in a sequential process that iteratively increased mutation weights based on predicted epitope deletions and predicted fitness. Individual gene insertions were then synthesized.

[0570] Procedures for evaluating melting temperature and IgG protease activity are provided above. Results for specific IgG protease variants of Sequence ID No. 2 are provided in Table 12. Activity is presented as a percentage of the IgG protease activity of IdeS.

[0571] [Table 13-1]

[0572] [Table 13-2]

[0573] Example 2.1 - Further manipulation and screening of functional IgG protease variants N142 (SEQ ID NO: 297) and N144 (SEQ ID NO: 295) that are predicted to have T cell epitope deletions. This example describes further manipulation of the IgG protease variants N142 (SEQ ID NO: 297) and N144 (SEQ ID NO: 295) identified in Example 2. Further manipulation was carried out by iteratively designing, constructing, expressing, and screening libraries of the IgG protease variants N142 and N144 based on predicted T epitope depletion, thermal stability measured by melting temperature, and functional fitness measured by IgG protease activity. The melting temperature and IgG protease activity of variants and wild-type IdeS for comparison were determined using the methods described in the "Methods" section.

[0574] Table 13A shows 159 exemplary functional IgG protease variants of N142 that have at least 30% of the IgG protease activity of wild-type IdeS, measured at a concentration of 4 μg / mL and a reaction time of 1 hour. The table includes the sequence number for the amino acid sequence of each variant, the mutation of sequence number 297 from the parent N142 amino acid sequence, and the IgG protease activity and melting temperature (T) of each variant. m The results are shown in the table. The table also shows the variant concentrations and reaction times used to measure the IgG protease activity of each variant. The IgG protease activity of each variant is presented both as a percentage decrease in corrected absorbance at 450 nm of the variant compared to PBS (negative control), as described in the "Methods," and as a percentage of the IgG protease activity of wild-type IdeS.

[0575] [Table 14-1]

[0576] [Table 14-2]

[0577] Table 14-3

[0578] Table 14-4

[0579] Table 14-5

[0580] Table 14-6

[0581] Table 14-7

[0582] Table 14-8

[0583] Table 14-9

[0584] Table 14-10

[0585] Table 14-11

[0586] Table 14-12

[0587] [Table 14-13]

[0588] [Table 14-14]

[0589] [Table 14-15]

[0590] [Table 14-16]

[0591] [Table 14-17]

[0592] From the exemplary IgG protease variants of N142 listed in Table 13A, five high-performance variants with the best overall activity, thermal stability, and predicted T-epitope depletion profile were identified (Table 13B). All high-performance N142 variants showed approximately 50–70% activity of wild-type IdeS measured at 4 μg / mL with a reaction time of 1 hour, while the N142 variants of SEQ ID NOs. 925, 936, and 942 showed approximately 50% activity of wild-type IdeS. o It had a melting temperature similar to or better than that of C.

[0593] [Table 15]

[0594] Table 14A shows 1124 exemplary functional IgG protease variants of N144 that have at least 30% of the IgG protease activity of wild-type IdeS, measured at a concentration of 4 μg / mL and a reaction time of 1 hour. The table includes the sequence number for the amino acid sequence of each variant, the mutation of sequence number 295 from the parent N144 amino acid sequence, and the IgG protease activity and melting temperature (T) of each variant. m The results are shown in the table. The table also shows the variant concentrations and reaction times used to measure the IgG protease activity of each variant. The IgG protease activity of each variant is presented both as a percentage decrease in corrected absorbance at 450 nm of the variant compared to PBS (negative control), as described in the "Methods," and as a percentage of the IgG protease activity of wild-type IdeS.

[0595] [Table 16-1]

[0596] [Table 16-2]

[0597] [Table 16-3]

[0598] [Table 16-4]

[0599] [Table 16-5]

[0600] [Table 16-6]

[0601] Table 16-7

[0602] Table 16-8

[0603] Table 16-9

[0604] Table 16-10

[0605] Table 16-11

[0606] Table 16-12

[0607] Table 16-13

[0608] Table 16-14

[0609] Table 16-15

[0610] Table 16-16

[0611] Table 16-17

[0612] Table 16-18

[0613] Table 16-19

[0614] Table 16-20

[0615] Table 16-21

[0616] Table 16-22

[0617] Table 16-23

[0618] Table 16-24

[0619] Table 16-25

[0620] Table 16-26

[0621] Table 16-27

[0622] Table 16-28

[0623] Table 16-29

[0624] Table 16-30

[0625] Table 16-31

[0626] Table 16-32

[0627] Table 16-33

[0628] Table 16-34

[0629] Table 16-35

[0630] Table 16-36

[0631] Table 16-37

[0632] Table 16-38

[0633] Table 16-39

[0634] Table 16-40

[0635] Table 16-41

[0636] Table 16-42

[0637] Table 16-43

[0638] Table 16-44

[0639] Table 16-45

[0640] Table 16-46

[0641] Table 16-47

[0642] Table 16-48

[0643] Table 16-49

[0644] Table 16-50

[0645] Table 16-51

[0646] Table 16-52

[0647] Table 16-53

[0648] Table 16-54

[0649] Table 16-55

[0650] Table 16-56

[0651] Table 16-57

[0652] Table 16-58

[0653] Table 16-59

[0654] Table 16-60

[0655] Table 16-61

[0656] Table 16-62

[0657] Table 16-63

[0658] Table 16-64

[0659] Table 16-65

[0660] Table 16-66

[0661] Table 16-67

[0662] Table 16-68

[0663] Table 16-69

[0664] Table 16-70

[0665] Table 16-71

[0666] Table 16-72

[0667] Table 16-73

[0668] Table 16-74

[0669] Table 16-75

[0670] Table 16-76

[0671] Table 16-77

[0672] Table 16-78

[0673] Table 16-79

[0674] Table 16-80

[0675] Table 16-81

[0676] Table 16-82

[0677] Table 16-83

[0678] Table 16-84

[0679] Table 16-85

[0680] Table 16-86

[0681] Table 16-87

[0682] Table 16-88

[0683] Table 16-89

[0684] Table 16-90

[0685] Table 16-91

[0686] Table 16-92

[0687] Table 16-93

[0688] Table 16-94

[0689] Table 16-95

[0690] Table 16-96

[0691] Table 16-97

[0692] Table 16-98

[0693] Table 16-99

[0694] Table 16-100

[0695] Table 16-101

[0696] Table 16-102

[0697] Table 16-103

[0698] Table 16-104

[0699] Table 16-105

[0700] Table 16-106

[0701] Table 16-107

[0702] Table 16-108

[0703] Table 16-109

[0704]

Table 16-110

[0705] Table 16-111

[0706] Table 16-112

[0707] Table 16-113

[0708] Table 16-114

[0709] Table 16-115

[0710] Table 16-116

[0711] Table 16-117

[0712] Table 16-118

[0713] Table 16-119

[0714] Table 16-120

[0715] Table 16-121

[0716] Table 16-122

[0717] Table 16-123

[0718] From the exemplary IgG protease variants of N144 listed in Table 14A, 53 high-performance N144 variants with the best overall activity, thermal stability, and predicted T-epitope depletion profile were identified (Table 14B). All high-performance N144 variants showed approximately 53–114% of the activity of wild-type IdeS measured at 4 μg / mL with a reaction time of 1 hour, and include SEQ ID NOs: 1118, 1120, 1122, 1123, 1125, 1126, 1127, 1129, 1282, 1970, 1971, 1983, 2004, 2024, 2028, and sequence numbers. Of the 53 high-performance N144 variants, number 2390, SEQ ID NO: 2421, SEQ ID NO: 2446, SEQ ID NO: 2450, SEQ ID NO: 2463, SEQ ID NO: 2464, SEQ ID NO: 2476, SEQ ID NO: 2478, SEQ ID NO: 2485, SEQ ID NO: 2486, SEQ ID NO: 2487, SEQ ID NO: 2489, SEQ ID NO: 2491, SEQ ID NO: 2493, SEQ ID NO: 2494, SEQ ID NO: 2511, SEQ ID NO: 2574, and SEQ ID NO: 2575, 33 of them are approximately 50% of wild-type IdeS. ℃ It had a melting temperature similar to or better than that of the original. As will be discussed in detail below, each of the high-performance N144 variants, by design, had several T cell epitope-targeting amino acid substitutions corresponding to those present in the IdeS variant of SEQ ID NO: 13, as well as additional T cell epitope-targeting amino acid substitutions; therefore, all of the high-performance N144 variants are also predicted to be significantly deimmunized compared to the N144 parent. The IdeS variant of SEQ ID NO: 13 has a significantly reduced T cell response by PBMC assay compared to its parent, i.e., wild-type IdeS, as shown in Example 3.1.

[0719] [Table 17-1]

[0720] [Table 17-2]

[0721] Table 17-3

[0722] Table 17-4

[0723] Table 17-5

[0724] Table 17-6

[0725] Table 17-7

[0726] Table 17-8

[0727] Table 17-9

[0728] Table 17-10

[0729] Table 17-11

[0730] The mutational weights of the 53 high-performance IgG protease variants of N144 ranged from 12 to 24 amino acid substitutions per variant compared to the parent N144. Table 14C summarizes the amino acid substitutions of SEQ ID NO: 295 identified among the 53 high-performance N144 variants. Furthermore, each amino acid substitution is ranked in descending order of frequency among the 53 high-performance N144 variants (see the "Frequency Ranking" column in Table 14C; Rank 1 indicates the highest frequency).

[0731] [Table 18]

[0732] As shown in Table 14C, each of the 53 high-performance N144 variants had two sets of amino acid substitutions. The first set of amino acid substitutions was present in all 53 high-performance N144 variants and included nine amino acid substitutions: Y12N, S18K, I46D, A47E, H93Y, E104R, F125W, T135H, and S159K. The second set of amino acid substitutions is one of I35K, I35E, and I35S, one of I54T, T55K, T55R, and T55H, one of T57L, N59D, N102G, one of L120T and L120S, one of L175N, L175A, and L175S, one of V183W, one of R200G and R200T, one of N201E, N205D, one of V230H and V230Q, R231W, N233 The variants contained 3 to 15 additional amino acid substitutions selected from the group consisting of G, A241G, E246K and one of E246G, A261K, A261Q, A261S, A261I, A261F and one of A261G, Y269W, V270T, one of S274G and S274D, one of A275W and A275S, one of I280M, A282G, D288T and D288K, and V290F. Note that the amino acid substitutions of Y12N, S18K, A47E, H93Y, E104R, L120T, T135H and S159K relative to the parent N144 occurred most frequently in high-performance N144 variants ranked 1 or 2. These amino acid substitutions correspond to the Y12N, Y18K, A47E, H93Y, E104R, L120T, Y135N, and S159K amino acid substitutions in the IdeS variant of SEQ ID NO: 13 relative to wild-type IdeS, and together they constitute eight of the eleven total amino acid substitutions in the IdeS variant of SEQ ID NO: 13. Since the IdeS variant of SEQ ID NO: 13 was shown to be significantly deimmunized by the PBMC assay compared to wild-type IdeS in Example 3.1, the high-performance N144 variant identified in this example is expected to have a favorable deimmunization profile compared to the N144 parent.

[0733] Example 2.2 - Immunogenicity analysis and mapping of T cell epitopes in N142 and selected variants of N142 by ex vivo cell immunoassay using human peripheral blood mononuclear cells (PBMCs). This example describes the immunogenicity analysis and mapping of T cell epitopes in N142 containing the amino acid sequence of SEQ ID NO: 297, and in selected variants of N142 containing an amino acid sequence selected from one of the high-performance N142 variants identified in Example 2.1 (see Table 13B), namely SEQ ID NO: 859, SEQ ID NO: 868, and SEQ ID NO: 925, using an ex vivo cell immunoassay using human peripheral blood mononuclear cells (PBMCs), also known as the "PBMC assay." The results of this example demonstrate that T cell response was reduced in all selected variants of N142, and that the N142 variant containing the amino acid sequence of SEQ ID NO: 925 showed the lowest overall residual immunogenicity risk compared to the parent N142.

[0734] method Immunogenicity analysis of N142 and its variants using ex vivo cell immunoassay (PBMC assay) with human peripheral blood mononuclear cells (PBMCs). PBMCs from healthy human donors were grown for 13 days using IL-2 in the presence of N142 or one of its variant proteins, and then restimulated with a peptide corresponding to the protein used for the initial stimulation. CD4 T cell cytokine production was then evaluated using surface and intracellular staining for flow cytometry.

[0735] Simply put, PBMCs are divided into 6.25 × 10⁶ units per 1 mL of RPMI medium containing 5% human serum. 6Cells were seeded in appropriate culture vessels. Antigen stimulation or vehicle control was added at the time of plating. IL-2 was added on day 4, and the culture medium was changed every 2-3 days thereafter. After 13 days of growth, cells from each stimulation condition were counted and redistributed into 96-well plates. Restimulation with peptides or a peptide pool was performed at a final concentration of 2 μM, and all samples were treated overnight with brefeldin A (to block cytokine secretion). Next, flow cytometry staining was performed using the following surface stain: Zombie Aqua®, and anti-CD3, anti-CD4, and anti-CD8 antibodies. Intracellular staining for IFN-γ, TNF-α, and IL-2 was performed using the BD Cytofix / Cytoperm® fixation / permeabilization staining kit according to the manufacturer's instructions. After the final step, the samples were resuspended in PBS and measured using a Cytek® Northern Lights® full-spectrum flow cytometer.

[0736] Table 15 lists the peptides used to restimulate cells grown in the presence of N142 for the purpose of complete CD4+ T cell epitope mapping of N142. The peptides were 18 or 20 amino acid lengths with 2-4 amino acid (aa) offsets. For epitope mapping, the peptides used during restimulation were in a pool of two consecutive peptides covering the full length of the protein, as shown in Table 15.

[0737] [Table 19-1]

[0738] [Table 19-2]

[0739] To compare the CD4+ T cell response to different epitopes in selected N142 variants with that in parental N142, a subset of regions in each variant was selected and evaluated. Regions were selected based on predicted immunogenicity, prioritizing those containing epitopes with high predicted immunogenicity and covering the mutation sites in the selected N142 variants. To cover these regions, peptides were selected for each variant to cover the core epitopes within the selected regions. Peptides with start positions matching the selected variant peptides were evaluated for parental N142 for comparison. During restimulation, each peptide corresponding to the sequence of the protein used for proliferation was used, as shown in Table 16.

[0740] [Table 20-1]

[0741] [Table 20-2]

[0742] The immunogenicity of N142 and its variants was reported as the sum of the percentages of CD4+ T cells positive for each cytokine (IFN-γ, TNF-α, and IL-2) from each donor (total cytokine). For graphing the heatmap, the maximum total cytokine value of any peptide covering its amino acid (AA) is shown. Background signal was subtracted for each condition.

[0743] result The immunogenicity of selected N142 variants containing an amino acid sequence chosen from one of the high-performance N142 variants identified in Example 2.1—SEQ ID NO: 859, SEQ ID NO: 868, and SEQ ID NO: 925—was evaluated by ex vivo cell immunoassay (also referred to as the “PBMC assay”) using human peripheral blood mononuclear cells (PBMCs), compared to their parent N142. Figure 1 is a schematic diagram of the PBMC assay for measuring T cell activation; details of the assay are described in the “Methods” section. In the PBMC assay of this example, immune cells derived from individual human donors were incubated with N142 or the selected N142 variant for a 14-day proliferation period. T cells were then evaluated by flow cytometry for cytokine (i.e., IL-2, IFN-γ, and TNF-α) production as an indicator of activation. The sum of the percentage of CD4+ T cells positive for each cytokine is referred to as the “total cytokine” value. The donors used to evaluate the T cell response represented all seven HLA II supertypes and covered all 27 alleles contributing to the supertypes (see Greenbaum et al., Immunogenetics. 2011 Jun;63(6):325-35, the entire report of which is incorporated herein by reference). Therefore, these donors represented the potential response well. Table 17 shows the HLA II alleles covering each supertype.

[0744] [Table 21]

[0745] 1. Immunogenicity analysis of N142 Figure 2 shows the complete CD4+ T cell epitope mapping results for N142 using a PBMC assay. Here, healthy human peripheral blood mononuclear cells (PBMCs) from each donor were grown in the presence of N142 for 14 days, restimulated with synthetic peptides covering the length of the protein (see Table 15), and then evaluated for T cell activation as measured by intracellular cytokine staining and flow cytometry. For each evaluated donor, the total cytokine values ​​(%IL-2+, INF-γ+, and TNF-α+ populations of CD4+ T cells) with background subtraction were plotted as a heatmap, and the cytokine value at each amino acid (AA) position in the protein was the highest cytokine value of the overlapping peptide covering that position. In Figure 2, the top bar labeled "Overall" represents the maximum total cytokine value across all donors. The grayscale shows the total cytokine value represented by the range of gray shades. Values ​​exceeding the indicated range are presented in black (maximum cytokine value = 10). The complete CD4+ T cell epitope mapping results in Figure 2 identified the most immunogenic regions within N142 that need to be addressed in candidate deimmunized N142 variants.

[0746] 2. Comparison of immunogenicity of N142 and selected N142 variants Figure 3 shows the results of targeted CD4+ T cell epitope mapping of selected N142 variants containing amino acid sequences selected from one of SEQ ID NOs: 859, 868, and 925, compared to parental N142 using a PBMC assay. Here, healthy human PBMCs from each donor were grown for 14 days in the presence of N142 or the selected N142 variant, restimulated with synthetic peptides corresponding to the proteins used for initial stimulation (see Table 16) covering the region indicated by the black bars on the heatmap, and then evaluated for T cell activation as measured by intracellular cytokine staining and flow cytometry analysis. The heatmap in Figure 3 shows the maximum total cytokine values ​​(%CD4+ T cells positive for IL-2, INF-γ, and TNF-α) with background subtracted for all donors for N142 or its selected variant, and the cytokine value for each amino acid (AA) position in the protein is the highest cytokine value of the duplicate peptide covering that position. For comparison, the complete epitope mapping of N142 shown in Figure 2 is shown at the top of Figure 3. *The marks indicate the location of the mutation in each selected N142 variant. The grayscale key shows various total cytokine values ​​represented by the range of gray shades. Values ​​exceeding the indicated range are presented in black (maximum cytokine value = 10). The data in Figure 3 show that the most immunogenic region of N142 is covered by the region selected for evaluation of the selected variants. The data also show the effect of the mutation in the selected N142 variants on the T cell response in those regions. That is, all selected N142 variants showed a significantly reduced T cell response compared to N142. The N142 variant containing the amino acid sequence of SEQ ID NO: 925 showed the lowest overall residual immunogenicity risk, which was mainly located in two regions roughly corresponding to amino acid (AA) positions 220-240 and 260-290, respectively. Two other high-performance N142 variants containing the amino acid sequences of SEQ ID NO: 936 or 942, identified in Example 2.1, each had an additional T cell epitope targeting mutation of N233S in the AA220-240 region, but otherwise had similar mutations compared to the selected N142 variant containing the amino acid sequence of SEQ ID NO: 925. Therefore, the former two high-performance N142 variants are expected to have similar or better deimmunization profiles compared to the latter variant.

[0747] Example 3.1 - Immunogenicity analysis and mapping of T cell epitopes in wild-type IdeS and selected variants of IdeS by ex vivo cell immunoassay using human peripheral blood mononuclear cells (PBMCs). This example describes the immunogenicity analysis and mapping of T cell epitopes in wild-type IdeS containing the amino acid sequence of SEQ ID NO: 2, and in selected variants of IdeS containing amino acid sequences selected from one of SEQ ID NOs: 4, 6, 8, 9, 10, 13, 14, and 22, which were identified in Example 3 (see Table 12), using an ex vivo cell immunoassay, also known as the "PBMC assay," which utilizes human peripheral blood mononuclear cells (PBMCs). The results of this example demonstrate that all selected variants of IdeS showed a reduced T cell response compared to wild-type IdeS, with the variant containing the amino acid sequence of SEQ ID NO: 13 showing the best overall reduction.

[0748] method Immunogenicity analysis of wild-type (WT) IdeS and its variants using ex vivo cell immunoassay (PBMC assay) with human peripheral blood mononuclear cells (PBMCs). PBMCs derived from healthy human donors were grown for 13 days using IL-2 in the presence of WT IdeS or one of its variant proteins, and then restimulated with peptides corresponding to the protein used for initial stimulation. CD4 T cell cytokine production was then evaluated using surface and intracellular staining for flow cytometry.

[0749] Simply put, PBMCs are used in RPMI medium containing 5% human serum at a rate of 6.25 × 10⁶ per 1 mL. 6Cells were seeded in appropriate culture vessels. Antigen stimulation or vehicle control was added at the time of plating. IL-2 was added on day 4, and the culture medium was changed every 2-3 days thereafter. After 13 days of growth, cells from each stimulation condition were counted and redistributed into 96-well plates. Restimulation with peptides or a peptide pool was performed at a final concentration of 2 μM, and all samples were treated overnight with brefeldin A (to block cytokine secretion). Next, flow cytometry staining was performed using the following surface stain: Zombie Aqua®, and anti-CD3, anti-CD4, and anti-CD8 antibodies. Intracellular staining for IFN-γ, TNF-α, and IL-2 was performed using the BD Cytofix / Cytoperm® fixation / permeabilization staining kit according to the manufacturer's instructions. After the final step, the samples were resuspended in PBS and measured using a Cytek® Northern Lights® full-spectrum flow cytometer.

[0750] Table 18 lists the peptides used to restimulate cells grown in the presence of WT IdeS for the purpose of complete CD4+ T cell epitope mapping of WT IdeS. The peptides were 18 or 20 amino acid lengths with 2-4 amino acid (aa) offsets. For epitope mapping, the peptides used during restimulation were pooled in pairs of two or four consecutive peptides covering the protein length, as shown in Table 18.

[0751] [Table 22-1]

[0752] [Table 22-2]

[0753] To compare the CD4+ T cell response to different epitopes in selected IdeS variants with that in WT IdeS, a subset of regions in each variant was selected and evaluated. Regions were selected based on predicted immunogenicity, prioritizing those containing epitopes with high predicted immunogenicity and covering the mutation sites in the selected IdeS variants. To cover these regions, peptides were selected for each variant to cover the core epitopes within the selected regions. Peptides with start sites matching the selected variant peptides were evaluated for WT IdeS for comparison. During restimulation, each peptide corresponding to the sequence of the protein used for proliferation was used, as shown in Table 19.

[0754] [Table 23-1]

[0755] [Table 23-2]

[0756] [Table 23-3]

[0757] The immunogenicity of WT IdeS and its variants was reported as the sum of the percentages of CD4+ T cells positive for each cytokine (IFN-γ, TNF-α, and IL-2) from each donor (total cytokine). For graphing the heatmap, the maximum total cytokine value of any peptide covering its amino acid (AA) is shown. Background signal was subtracted for each condition.

[0758] result The immunogenicity of selected variants of IdeS containing an amino acid sequence selected from one of SEQ ID NOs: 4, 6, 8, 9, 10, 13, 14, and 22, relative to the WT IdeS identified in Example 3, was evaluated by an ex vivo cell immunoassay using human peripheral blood mononuclear cells (PBMCs), also known as the "PBMC assay." Figure 1 is a schematic diagram of the PBMC assay for measuring T cell activation; details of the assay are described in the "Methods" section. In this example's PBMC assay, immune cells derived from individual human donors were incubated with WT IdeS or the selected IdeS variant for a 14-day proliferation period. T cells were then evaluated by flow cytometry for cytokine (i.e., IL-2, IFN-γ, and TNF-α) production as an indicator of activation. The sum of the percentage of CD4+ T cells positive for each cytokine is called the "total cytokine" value. The donors used to evaluate the T cell response represented all seven HLA II supertypes and covered all 27 alleles contributing to the supertypes (see Greenbaum et al., Immunogenetics. 2011 Jun;63(6):325-35, the entire report of which is incorporated herein by reference). Therefore, these donors represented the potential response well. Table 20 shows the HLA II alleles covering each supertype.

[0759] [Table 24-1]

[0760] [Table 24-2]

[0761] 1. Immunogenicity analysis of WT IdeS Figure 4 shows the complete CD4+ T cell epitope mapping results for WT IdeS using a PBMC assay. Here, healthy human peripheral blood mononuclear cells (PBMCs) from each donor were grown for 14 days in the presence of WT IdeS, restimulated with synthetic peptides covering the length of the protein (see Table 18), and then evaluated for T cell activation as measured by intracellular cytokine staining and flow cytometry. For each evaluated donor, the total cytokine values ​​(%IL-2+, INF-γ+, and TNF-α+ populations of CD4+ T cells) with background subtraction were plotted as a heatmap, and the cytokine value at each amino acid (AA) position in the protein was the highest cytokine value of the overlapping peptide covering that position. In Figure 4, the top bar labeled "Overall" represents the maximum total cytokine value across all donors. The grayscale shows the total cytokine value represented by the range of gray shades. Values ​​exceeding the indicated range are presented in black (maximum cytokine value = 10). The complete CD4+ T cell epitope mapping results in Figure 4 identified the most immunogenic regions in WT IdeS that needed to be addressed in candidate deimmunized IdeS variants.

[0762] 2. Comparison of immunogenicity of WT IdeS and selected IdeS variants Figure 5 shows the targeted CD4+ T cell epitope mapping results of selected IdeS variants comprising an amino acid sequence selected from one of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 22, compared to wild-type IdeS using a PBMC assay, wherein normal human PBMCs from each donor were expanded for 14 days in the presence of wild-type IdeS or the selected IdeS variant, restimulated with a synthetic peptide corresponding to the protein (see Table 19) used for initial stimulation covering the region indicated by the black bar above the heatmap, and then assessed for T cell activation as measured by intracellular cytokine staining and flow cytometry analysis. The heatmap of Figure 5 shows, for wild-type IdeS and the selected variants thereof, the background-subtracted maximum total cytokine values (% CD4+ T cells positive for IL-2, INF-γ, and TNF-α) for all donors, wherein the cytokine value at each amino acid (AA) position in the protein is the highest cytokine value of overlapping peptides covering that position. For comparison, the complete epitope mapping of wild-type IdeS shown in Figure 4 is presented at the top of Figure 5. In Figure 5 * marks represent the positions of mutations in each selected IdeS variant. The grayscale key indicates the different total cytokine values represented by ranges of gray shades. Values exceeding the indicated range are presented as black (maximum cytokine value = 10). The data in Figure 5 shows that the highest immunogenic region of wild-type IdeS is covered by the regions selected for evaluation of the selected variants. The data also shows the effect of mutations in the selected IdeS variants on T cell responses in those regions. Namely, all of the selected IdeS variants showed a marked reduction in T cell response compared to wild-type IdeS, and the IdeS variant comprising the amino acid sequence of SEQ ID NO: 13 showed the best overall reduction.

[0763] Example 3.2 - Further engineering and screening of functional IgG protease variants of IdeS (SEQ ID NO: 2) predicted to have T cell epitope deletions This example describes further manipulation of the IgG protease variant of IdeS (SEQ ID NO: 2) identified in Example 3. Further manipulation was carried out by iteratively designing, constructing, expressing, and screening libraries of IgG protease variants of IdeS based on predicted T-epitope depletion, thermal stability measured by melting temperature, and functional fitness measured by IgG protease activity. The melting temperatures and IgG protease activity of IdeS variants and wild-type IdeS were determined using the methods described in the "Methods" section.

[0764] Table 21A shows 461 exemplary functional IgG protease variants of IdeS that have at least 30% of the IgG protease activity of wild-type IdeS, measured at a concentration of 4 μg / mL and a reaction time of 1 hour. The table includes the amino acid sequence number of each variant, the mutation of the parent IdeS amino acid sequence in SEQ ID NO: 2, and the IgG protease activity and melting temperature (T) of each variant. m The results are shown in the table. The table also shows the variant concentrations and reaction times used to measure the IgG protease activity of each variant. The IgG protease activity of each variant is presented both as a percentage reduction in the corrected absorbance of the variant at 450 nm relative to PBS (negative control) and as a percentage of the IgG protease activity of (parental) wild-type IdeS, as described in "Methods".

[0765] [Table 25-1]

[0766] [Table 25-2]

[0767] [Table 25-3]

[0768] Table 25-4

[0769] Table 25-5

[0770] Table 25-6

[0771] Table 25-7

[0772] Table 25-8

[0773] Table 25-9

[0774] Table 25-10

[0775] Table 25-11

[0776] Table 25-12

[0777] Table 25-13

[0778] Table 25-14

[0779] Table 25-15

[0780] Table 25-16

[0781] Table 25-17

[0782] Table 25-18

[0783] Table 25-19

[0784] Table 25-20

[0785] Table 25-21

[0786] Table 25-22

[0787] Table 25-23

[0788] Table 25-24

[0789] Table 25-25

[0790] Table 25-26

[0791] Table 25-27

[0792] Table 25-28

[0793] Table 25-29

[0794] Table 25-30

[0795] Table 25-31

[0796] Table 25-32

[0797] Table 25-33

[0798] Table 25-34

[0799] Tables 25-35

[0800] Table 25-36

[0801] Table 25-37

[0802] Table 25-38

[0803] Table 25-39

[0804] Table 25-40

[0805] Table 25-41

[0806] Table 25-42

[0807] Table 25-43

[0808] Table 25-44

[0809] Table 25-45

[0810] Table 25-46

[0811] Table 25-47

[0812] Table 25-48

[0813] Table 25-49

[0814] Table 25-50

[0815] From the exemplary IgG protease variants of IdeS listed in Table 21A, 21 high-performance variants with the best overall activity, thermal stability, and predicted T-epitope depletion profile were identified (Table 21B). These high-performance variants were designed using the IdeS variant of SEQ ID NO: 13 (with 11 amino acid substitutions compared to wild-type IdeS; see Table 12 in Example 3) as a scaffold, to which 13–18 additional amino acid substitutions were introduced. The IdeS variant of SEQ ID NO: 13 was selected as the scaffold because it was determined to have the most desirable deimmunization profile in Example 3.1. The high-performance IdeS variants retained approximately 39–77% of the activity of wild-type IdeS measured at 4 μg / mL after a reaction time of 1 hour, and all but four variants (SEQ ID NO: 1492, 1795, 1797, and 1949) retained approximately 50% of the activity of wild-type IdeS. ℃ The high-performance IdeS variants had melting temperatures similar to or better than those of the original IdeS variant. Furthermore, each high-performance IdeS variant had 13 to 18 additional amino acid substitutions targeting immunogenic T cell epitopes, particularly T cell epitopes within the residual immunogenicity risk regions around amino acid positions 40-60, 220-240, and 260-290 of the scaffold IdeS variant of SEQ ID NO: 13, and was therefore predicted to be more deimmunized than the scaffold IdeS variant of SEQ ID NO: 13 (see Figure 5). Despite the fact that some of the targeted T cell epitopes in the high-performance IdeS variants extended to the active site of WT IdeS, the high-performance IdeS variants unexpectedly retained satisfactory activity and thermal stability.

[0816] [Table 26-1]

[0817] [Table 26-2]

[0818] [Table 26-3]

[0819] [Table 26-4]

[0820] [Table 26-5]

[0821] In accordance with the variant design described above, each of the 21 high-performance IgG protease variants of IdeS had two sets of amino acid substitutions. Overall, the first set of amino acid substitutions for the 21 high-performance IgG protease variants of IdeS included the 11 amino acid substitutions present in the scaffold variant of SEQ ID NO: 13, Y12N, Y18K, A47E, H93Y, E104R, L120T, E126S, Y135N, S159K, V230S, and A275S. The second set of amino acid substitutions for the 21 high-performance IgG protease variants of IdeS included 13 to 18 additional amino acid substitutions designed to enhance T cell epitope targeting in order to further reduce immunogenicity (see the "Variations in the Amino Acid Sequence of SEQ ID NO: 13 against the Scaffold IdeS Variant" column in Table 21B). Table 21C summarizes a second set of amino acid substitutions identified among the 21 high-performance IgG protease variants of IdeS. Furthermore, each amino acid substitution in this second set is ranked in descending order of frequency among the 21 high-performance IgG protease variants of IdeS (see the “Frequency Ranking” column in Table 21C; Rank 1 indicates the highest frequency).

[0822] [Table 27]

[0823] As shown in Table 21C, the second set of amino acid substitutions present in the 21 high-performance IgG protease variants of IdeS included 13 to 18 amino acid substitutions selected from the group consisting of one of S3N, F4I, A6S, E9I, I10T, R11T, N48G, I54T, T57K, T57N, T57R, T57L, and T57Q, one of N59D, G60S, and G60T, one of K61R, E92R, T138A, T161G, and T161Y, D188T, F199L, K200R, E201N, N203T, G222A, L223I, N233S, N246R, N273G, D288G, and Q293R. As discussed above, among the second set of amino acid substitutions, N48G, N59D, N233S, N246R, N273G, D288G, and Q293R, which were most frequently present in the high-performance IdeS variants ranked 1 or 2, were intended to target residual immunogenicity risk regions around amino acid positions 40-60, 220-240, and 260-290 of the scaffold IdeS variant of SEQ ID NO: 13. As a result, the high-performance IdeS variants are expected to have a better deimmunization profile than the scaffold IdeS variant of SEQ ID NO: 13. *******

[0824] While the described invention is described with reference to its specific embodiments, it should be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. Furthermore, many modifications may be made to adapt specific circumstances, materials, substance compositions, processes, or one or more process steps to the spirit and scope of the described invention. All such modifications are intended to fall within the claims appended herein.

[0825] The patents, patent applications, patent publications, academic papers, and protocols referenced herein are incorporated in their entirety for all purposes.

Claims

1. A polypeptide variant of the amino acid sequence of SEQ ID NO: 2 [IdeS], wherein the polypeptide variant has one or more amino acid positions of SEQ ID NO: S3, F4, A6, E9, I10, R11, Y12, Y18, V46, A47, N48, I54, T57, N59, G60, K61, E92, H93, E104, L120, K123, F125, E126, Y135, T138, S159 A polypeptide variant comprising one or more amino acid mutations in T161, N162, T185, D188, F199, K200, E201, N203, G222, L223, V230, N233, N246, A251, N273, A275, D288, Q293, V294, G296, wherein the polypeptide variant comprises an amino acid sequence that is at least about 75% identical to SEQ ID NO:

2.

2. The polypeptide variant according to claim 1, wherein the polypeptide variant does not have amino acid mutations at the following amino acid positions of SEQ ID NO: K56, C66, H234, D256, and D258 of SEQ ID NO:

2.

3. The polypeptide variant according to claim 1 or 2, wherein the polypeptide variant does not have an amino acid mutation in one or more of the following amino acid positions of SEQ ID NO: A32, N33, T35, Q36, F41, D84, R88, E91, N102, M106, N117, H118, E170, S195, N197, K213, S245, K250, A261, K286, S306.

4. A polypeptide variant according to claim 1 or 2, comprising an amino acid sequence that is at least about 80% identical to SEQ ID NO:

2.

5. A polypeptide variant according to claim 1 or 2, comprising an amino acid sequence that is at least about 85% identical to SEQ ID NO:

2.

6. A polypeptide variant according to claim 1 or 2, comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO:

2.

7. A polypeptide variant according to claim 1 or 2, comprising an amino acid sequence that is at least about 95% identical to SEQ ID NO:

2.

8. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations are one or more amino acid substitutions.

9. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations are two or more amino acid mutations.

10. The polypeptide variant according to claim 9, wherein the two or more amino acid mutations are two or more amino acid substitutions.

11. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations are three or more amino acid mutations.

12. The polypeptide variant according to claim 11, wherein the three or more amino acid mutations are three or more amino acid substitutions.

13. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations are four or more amino acid mutations.

14. The polypeptide variant according to claim 13, wherein the four or more amino acid mutations are four or more amino acid substitutions.

15. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations are five or more amino acid mutations.

16. The polypeptide variant according to claim 15, wherein the five or more amino acid mutations are five or more amino acid substitutions.

17. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations are six or more amino acid mutations.

18. The polypeptide variant according to claim 17, wherein the six or more amino acid mutations are six or more amino acid substitutions.

19. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations are seven or more amino acid mutations.

20. The polypeptide variant according to claim 19, wherein the seven or more amino acid mutations are seven or more amino acid substitutions.

21. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations are eight or more amino acid mutations.

22. The polypeptide variant according to claim 21, wherein the eight or more amino acid mutations are eight or more amino acid substitutions.

23. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations are nine or more amino acid mutations.

24. The polypeptide variant according to claim 23, wherein the nine or more amino acid mutations are nine or more amino acid substitutions.

25. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations are ten or more amino acid mutations.

26. The polypeptide variant according to claim 25, wherein the 10 or more amino acid mutations are 10 or more amino acid substitutions.

27. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations are 11 or more amino acid mutations.

28. The polypeptide variant according to claim 27, wherein the 11 or more amino acid mutations are 11 or more amino acid substitutions.

29. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations consist of approximately 5 to approximately 23 amino acid mutations.

30. The polypeptide variant according to claim 29, wherein the aforementioned approximately 5 to approximately 23 amino acid mutations are approximately 5 to approximately 23 amino acid substitutions.

31. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations consist of approximately 5 to approximately 20 amino acid mutations.

32. The polypeptide variant according to claim 31, wherein the aforementioned approximately 5 to approximately 20 amino acid mutations are approximately 5 to approximately 20 amino acid substitutions.

33. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations consist of approximately 10 to approximately 20 amino acid mutations.

34. The polypeptide variant according to claim 33, wherein the approximately 10 to approximately 20 amino acid mutations are approximately 10 to approximately 20 amino acid substitutions.

35. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations consist of approximately 11 to approximately 20 amino acid mutations.

36. The polypeptide variant according to claim 35, wherein the aforementioned approximately 11 to approximately 20 amino acid mutations are approximately 11 to approximately 20 amino acid substitutions.

37. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations consist of approximately 12 to approximately 20 amino acid mutations.

38. The polypeptide variant according to claim 37, wherein the aforementioned approximately 12 to approximately 20 amino acid mutations are approximately 12 to approximately 20 amino acid substitutions.

39. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations consist of approximately 15 to approximately 20 amino acid mutations.

40. The polypeptide variant according to claim 39, wherein the aforementioned approximately 15 to approximately 20 amino acid mutations are approximately 15 to approximately 20 amino acid substitutions.

41. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations consist of approximately 10 to approximately 35 amino acid mutations.

42. The polypeptide variant according to claim 41, wherein the aforementioned approximately 10 to approximately 35 amino acid mutations are approximately 10 to approximately 35 amino acid substitutions.

43. The polypeptide variant according to any one of claims 1 to 7, wherein the one or more amino acid mutations consist of approximately 15 to approximately 30 amino acid mutations.

44. The polypeptide variant according to claim 43, wherein the approximately 15 to approximately 30 amino acid mutations are approximately 15 to approximately 30 amino acid substitutions.

45. The polypeptide variant according to any one of claims 1 to 6, wherein the one or more amino acid mutations consist of approximately 20 to approximately 30 amino acid mutations.

46. The polypeptide variant according to claim 45, wherein the approximately 20 to approximately 30 amino acid mutations are approximately 20 to approximately 30 amino acid substitutions.

47. The polypeptide variant according to any one of claims 1 to 6, wherein the one or more amino acid mutations consist of approximately 24 to approximately 35 amino acid mutations.

48. The polypeptide variant according to claim 47, wherein the approximately 24 to approximately 35 amino acid mutations are approximately 24 to approximately 35 amino acid substitutions.

49. The polypeptide variant according to any one of claims 1 to 6, wherein the one or more amino acid mutations consist of approximately 24 to approximately 32 amino acid mutations.

50. The polypeptide variant according to claim 49, wherein the aforementioned approximately 24 to approximately 32 amino acid mutations are approximately 24 to approximately 32 amino acid substitutions.

51. The polypeptide variant according to any one of claims 1 to 6, wherein the one or more amino acid mutations consist of approximately 24 to approximately 29 amino acid mutations.

52. The polypeptide variant according to claim 51, wherein the approximately 24 to approximately 29 amino acid mutations are approximately 24 to approximately 29 amino acid substitutions.

53. One of S3N, F4I, A6S, E9I, I10T, R11D and R11T, one of Y12N, Y18K, V46D, A47E, N48G, I54T, T57D, T57K, T57N, T57R, T57L and T57Q, one of N59D, G60S and G60T, one of K61R, E92R, H93Y, E104R, L120T, K123A, F125W, E126S, Y135H and Y135N, one of T138A, S159K, T161W, T161G and T161Y A polypeptide variant according to any one of claims 1 to 52, comprising one amino acid substitution of Sequence ID No. 2, selected from the group consisting of one of N162D, T185G, D188T, F199L, K200R, E201N, N203T, G222A, L223I, V230A and V230S, one of N233S, N246R, A251W, N273G, A275G, A275S and A275W, D288G, Q293R, V294A, G296E, and combinations thereof.

54. A polypeptide variant according to any one of claims 1 to 53, selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, N102G, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, K213Q, N233G, S245A, N246E, K250E, A261P, K286E, and S306I, which does not have one or more amino acid substitutions of SEQ ID NO:

2.

55. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, R11D, Y12N, Y18K, and V46D.

56. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include R11D, Y12N, Y18K, V46D, and A47E.

57. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, V46D, A47E, and T57K.

58. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y18K, V46D, A47E, T57K, H93Y, E104R, and L120T.

59. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, R11D, Y12N, A275S, V294A, and G296E.

60. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, R11D, F125W, E126S, Y135H, A275W, V294A, and G296E.

61. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, R11D, Y12N, Y18K, K123A, F125W, E126S, V294A, and G296E.

62. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, R11D, A275S, V294A, and G296E.

63. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, R11D, V46D, A47E, A275S, V294A, and G296E.

64. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, T57K, H93Y, E104R, L120T, and K123A.

65. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, R11D, S159K, N162D, T185G, V230A, A251W, V294A, and G296E.

66. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, Y12N, V46D, H93Y, F125W, E126S, N162D, V294A, and G296E.

67. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in Sequence ID No. 2 include one of I10T, R11D, Y12N, Y18K, V46D, A47E, T57D and T57K, one of H93Y, E104R, L120T, K123A, F125W, E126S, Y135H and Y135N, one of S159K, T161W and T161Y, one of N162D, T185G, one of V230A and V230S, one of A251W, A275G, A275S and A275W, V294A, and G296E.

68. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in Sequence ID No. 2 include one of I10T, R11D, Y12N, Y18K, V46D, A47E, T57D and T57K, one of H93Y, E104R, L120T, K123A, F125W, E126S, Y135H and Y135N, one of S159K, T161W and T161Y, one of N162D, T185G, one of V230A and V230S, one of A251W, A275G, A275S and A275W, V294A, and G296E.

69. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, R11D, Y12N, L120T, K123A, S159K, V230S, V294A, and G296E.

70. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include R11D, Y18K, T57K, H93Y, E104R, L120T, Y135N, S159K, T161W, T185G, V230A, and A275G.

71. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include R11D, Y18K, T57K, H93Y, E104R, L120T, Y135N, S159K, T161W, V230A, and A275W.

72. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include R11D, Y18K, T57K, H93Y, E104R, L120T, E126S, Y135N, S159K, T161Y, V230A, and A275W.

73. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include R11D, Y18K, T57K, H93Y, E104R, L120T, F125W, Y135H, S159K, T161Y, N162D, T185G, V230S, and A275S.

74. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include R11D, Y18K, T57K, H93Y, E104R, L120T, F125W, Y135H, S159K, T161Y, T185G, V230S, and A275S.

75. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include R11D, Y18K, T57K, H93Y, E104R, L120T, F125W, Y135H, S159K, T161Y, T185G, A275S, and G296E.

76. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, V46D, A47E, H93Y, E104R, L120T, K123A, E126S, Y135N, S159K, and A275S.

77. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, T57D, H93Y, E104R, L120T, E126S, Y135N, S159K, V230S, and A275S.

78. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, T57K, H93Y, E104R, L120T, E126S, Y135N, S159K, V230S, A275S, and V294A.

79. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, T57K, H93Y, E104R, L120T, E126S, Y135N, S159K, V230S, A275S, and G296E.

80. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, H93Y, E104R, L120T, E126S, Y135N, S159K, V230S, and A275S.

81. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, H93Y, E104R, L120T, E126S, Y135N, S159K, A275S, and V294A.

82. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, Y12N, Y18K, V46D, A47E, T57D, H93Y, E104R, L120T, K123A, F125W, E126S, Y135N, S159K, T161W, T185G, V230S, A275W, and V294A.

83. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, Y12N, Y18K, V46D, A47E, T57K, H93Y, E104R, L120T, K123A, E126S, Y135N, S159K, T161W, T185G, V230A, A275W, and V294A.

84. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, Y12N, Y18K, V46D, A47E, T57K, H93Y, E104R, L120T, K123A, E126S, Y135N, S159K, T161W, T185G, V230A, A251W, and A275S.

85. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, Y12N, Y18K, V46D, A47E, T57K, H93Y, E104R, L120T, K123A, E126S, Y135N, S159K, T161W, T185G, V230S, and A275S.

86. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, Y12N, Y18K, V46D, A47E, T57K, H93Y, E104R, L120T, K123A, E126S, Y135N, S159K, T161W, T185G, V230S, A251W, and A275S.

87. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, Y12N, Y18K, V46D, A47E, T57K, H93Y, E104R, L120T, K123A, F125W, E126S, Y135N, S159K, T161W, T185G, V230A, A275S, and V294A.

88. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, Y12N, Y18K, V46D, A47E, T57K, H93Y, E104R, L120T, K123A, F125W, E126S, Y135N, S159K, T161W, T185G, V230A, and A275S.

89. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, Y12N, Y18K, V46D, A47E, T57K, H93Y, E104R, L120T, K123A, F125W, E126S, Y135N, S159K, T161W, T185G, V230A, A251W, and A275S.

90. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, Y12N, Y18K, V46D, A47E, T57K, H93Y, E104R, L120T, K123A, F125W, E126S, Y135N, S159K, T161W, T185G, V230S, and A275S.

91. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, Y12N, Y18K, V46D, A47E, T57K, H93Y, E104R, L120T, K123A, F125W, E126S, Y135N, S159K, T161W, T185G, V230S, A275W, and V294A.

92. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, Y12N, Y18K, V46D, A47E, T57K, H93Y, E104R, L120T, K123A, F125W, E126S, Y135N, S159K, T161W, T185G, V230S, A251W, and A275S.

93. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include I10T, Y12N, Y18K, V46D, A47E, H93Y, E104R, L120T, F125W, Y135H, S159K, and A275S.

94. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include S3N, F4I, A6S, Y12N, Y18K, A47E, N48G, I54T, T57N, N59D, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

95. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, T57K, N59D, G60S, K61R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, D188T, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

96. A polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, T57K, N59D, G60S, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, L223I, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

97. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, T57K, N59D, G60S, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

98. A polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, T57K, N59D, G60S, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, N203T, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

99. A polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, T57K, N59D, G60S, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, D188T, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

100. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, T57K, N59D, G60S, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161Y, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

101. A polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, T57K, N59D, G60S, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161Y, D188T, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

102. A polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, T57Q, N59D, G60T, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

103. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, T57R, N59D, G60T, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, L223I, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

104. A polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, T57R, N59D, G60T, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, N203T, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

105. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, T57R, N59D, G60T, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, D188T, F199L, K200R, E201N, L223I, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

106. A polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, T57R, N59D, G60T, K61R, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161Y, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

107. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, I54T, T57L, N59D, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

108. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, I54T, T57L, N59D, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, L223I, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

109. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, I54T, T57L, N59D, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161Y, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

110. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, I54T, T57N, N59D, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

111. A polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, I54T, T57N, N59D, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161G, D188T, F199L, K200R, E201N, L223I, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

112. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, I54T, T57N, N59D, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161Y, G222A, L223I, V230S, N233S, N246R, N273G, A275S, and D288G.

113. A polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include Y12N, Y18K, A47E, N48G, I54T, T57N, N59D, E92R, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, T161Y, F199L, K200R, E201N, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

114. The polypeptide variant according to claim 53 or 54, wherein one or more amino acid substitutions in SEQ ID NO: 2 include E9I, I10T, R11T, Y12N, Y18K, A47E, N48G, I54T, T57N, N59D, H93Y, E104R, L120T, E126S, Y135N, T138A, S159K, V230S, N233S, N246R, N273G, A275S, D288G, and Q293R.

115. A polypeptide variant according to claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 26.

116. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

3.

117. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

4.

118. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

5.

119. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

6.

120. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

7.

121. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

8.

122. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

9.

123. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

10.

124. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

11.

125. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

12.

126. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

13.

127. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

14.

128. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

15.

129. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

16.

130. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

17.

131. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

18.

132. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

19.

133. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

20.

134. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

21.

135. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

22.

136. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

23.

137. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

24.

138. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

25.

139. A polypeptide variant according to claim 115, comprising the amino acid sequence described in SEQ ID NO:

26.

140. The polypeptide variant according to claim 1, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to SEQ ID NOs: 26, wherein the polypeptide variant comprises amino acid mutations present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO:

2.

141. A polypeptide variant according to claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1491 to 1951.

142. The polypeptide variant according to claim 1, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs: 1491 to SEQ ID NOs: 1951, wherein the polypeptide variant comprises amino acid mutations present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO:

2.

143. A polypeptide variant according to claim 1, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 1492, SEQ ID NO: 1697, SEQ ID NO: 1700, SEQ ID NO: 1701, SEQ ID NO: 1702, SEQ ID NO: 1703, SEQ ID NO: 1704, SEQ ID NO: 1705, SEQ ID NO: 1713, SEQ ID NO: 1720, SEQ ID NO: 1722, SEQ ID NO: 1723, SEQ ID NO: 1724, SEQ ID NO: 1772, SEQ ID NO: 1774, SEQ ID NO: 1775, SEQ ID NO: 1793, SEQ ID NO: 1795, SEQ ID NO: 1797, SEQ ID NO: 1798, and SEQ ID NO: 1949, wherein the polypeptide variant comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 1492, SEQ ID NO: 1697, SEQ ID NO: 1700, SEQ ID NO: 1701, SEQ ID NO: 1701, SEQ ID NO: 1702, SEQ ID NO: 1703, SEQ ID NO: 1704, SEQ ID NO: 1705, SEQ ID NO: 1713, SEQ ID NO: 1720, SEQ ID NO: 1722, SEQ ID NO: 17

144. A polypeptide variant of the amino acid sequence of SEQ ID NO: 2 [IdeS], wherein the polypeptide variant comprises a first set of amino acid substitutions of SEQ ID NO: 2 and a second set of amino acid substitutions of SEQ ID NO:

2. The first set of amino acid substitutions in Sequence ID No. 2 consists of Y12N, Y18K, A47E, H93Y, E104R, L120T, E126S, Y135N, S159K, V230S, and A275S, and the second set of amino acid substitutions in Sequence ID No. 2 consists of S3N, F4I, A6S, E9I, I10T, R11T, N48G, I54T, T57K, T57N, T57R, T57L, and T57 A set of approximately 13 to 18 amino acid substitutions selected from the group consisting of one of Q, one of N59D, one of G60S and G60T, one of K61R, E92R, one of T138A, one of T161G and T161Y, D188T, F199L, K200R, E201N, N203T, G222A, L223I, N233S, N246R, N273G, D288G, and Q293R. The polypeptide variant is a polypeptide variant that includes an amino acid sequence that is at least about 88% identical to SEQ ID NO:

2.

145. The polypeptide variant according to claim 144, wherein the polypeptide variant does not have amino acid mutations at the following amino acid positions of SEQ ID NO: K56, C66, H234, D256, and D258.

146. The polypeptide variant according to claim 144 or 145, wherein the polypeptide variant does not have an amino acid mutation in one or more of the following amino acid positions of SEQ ID NO: A32, N33, T35, Q36, F41, D84, R88, E91, N102, M106, N117, H118, E170, S195, N197, K213, S245, K250, A261, K286, S306.

147. The polypeptide variant according to any one of claims 144 to 146, wherein the polypeptide variant is selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, N102G, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, K213Q, N233G, S245A, N246E, K250E, A261P, K286E, and S306I, and does not have one or more amino acid substitutions of SEQ ID NO:

2.

148. A polypeptide variant according to any one of claims 144 to 147, comprising an amino acid sequence that is at least about 89% identical to SEQ ID NO:

2.

149. A polypeptide variant according to any one of claims 144 to 147, comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO:

2.

150. A polypeptide variant according to any one of claims 144 to 147, comprising an amino acid sequence that is at least about 91% identical to SEQ ID NO:

2.

151. A polypeptide variant according to any one of claims 144 to 147, comprising an amino acid sequence that is at least about 92% identical to SEQ ID NO:

2.

152. A polypeptide variant according to any one of claims 144 to 147, comprising an amino acid sequence that is approximately 88% to approximately 92% identical to SEQ ID NO:

2.

153. A polypeptide variant according to any one of claims 144 to 147, comprising an amino acid sequence that is approximately 89% to approximately 92% identical to SEQ ID NO:

2.

154. A polypeptide variant according to any one of claims 144 to 147, comprising an amino acid sequence that is approximately 90% to approximately 92% identical to SEQ ID NO:

2.

155. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of about 13 to about 18 amino acid substitutions selected from the group consisting of N48G, I54T, T57K, T57N, T57R, and T57L, N59D, G60S, and G60T, K61R, E92R, T138A, T161G, and T161Y, D188T, F199L, K200R, E201N, N203T, L223I, N233S, N246R, N273G, D288G, and Q293R.

156. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of about 13 to about 18 amino acid substitutions selected from the group consisting of N48G, I54T, T57K and T57N, N59D, G60S and G60T, K61R, E92R, T138A, T161G and T161Y, D188T, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.

157. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of about 13 to about 18 amino acid substitutions selected from the group consisting of one of N48G, I54T, T57K and T57N, one of N59D, G60S, K61R, E92R, T138A, T161G and T161Y, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.

158. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of about 13 to about 18 amino acid substitutions selected from the group consisting of one of N48G, I54T, T57K and T57N, one of N59D, G60T, K61R, E92R, T138A, T161G and T161Y, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.

159. The polypeptide variant according to any one of claims 144 to 158, wherein the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 17 amino acid substitutions.

160. The polypeptide variant according to any one of claims 144 to 158, wherein the second set of amino acid substitutions in SEQ ID NO: 2 is a set of about 13 to about 16 amino acid substitutions.

161. The polypeptide variant according to any one of claims 144 to 158, wherein the second set of amino acid substitutions in Sequence ID No. 2 is a set of about 13 to about 15 amino acid substitutions.

162. The polypeptide variant according to any one of claims 144 to 158, wherein the second set of amino acid substitutions in Sequence ID No. 2 is a set of 13, 14, 15, 16, 17, or 18 amino acid substitutions.

163. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of about 13 to about 17 amino acid substitutions selected from the group consisting of N48G, I54T, T57K, N59D, G60S, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

164. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of about 13 to about 15 amino acid substitutions selected from the group consisting of N48G, I54T, N59D, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

165. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of about 13 to about 14 amino acid substitutions selected from the group consisting of N48G, N59D, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

166. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 13 amino acid substitutions consisting of N48G, N59D, K61R, E92R, T138A, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

167. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 13 amino acid substitutions consisting of S3N, F4I, A6S, N48G, I54T, T57N, N59D, T138A, N233S, N246R, N273G, D288G, and Q293R.

168. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 15 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, T138A, D188T, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

169. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 17 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161G, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.

170. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 16 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

171. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 17 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N203T, N233S, N246R, N273G, D288G, and Q293R.

172. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 17 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161G, D188T, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

173. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 16 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161Y, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

174. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 17 amino acid substitutions consisting of N48G, T57K, N59D, G60S, K61R, E92R, T138A, T161Y, D188T, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

175. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 16 amino acid substitutions consisting of N48G, T57Q, N59D, G60T, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

176. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 17 amino acid substitutions consisting of N48G, T57R, N59D, G60T, K61R, E92R, T138A, T161G, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.

177. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 17 amino acid substitutions consisting of N48G, T57R, N59D, G60T, K61R, E92R, T138A, T161G, F199L, K200R, E201N, N203T, N233S, N246R, N273G, D288G, and Q293R.

178. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 18 amino acid substitutions consisting of N48G, T57R, N59D, G60T, K61R, E92R, T138A, T161G, D188T, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.

179. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 16 amino acid substitutions consisting of N48G, T57R, N59D, G60T, K61R, E92R, T138A, T161Y, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

180. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 13 amino acid substitutions consisting of N48G, I54T, T57L, N59D, T138A, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

181. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 16 amino acid substitutions consisting of N48G, I54T, T57L, N59D, E92R, T138A, T161G, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.

182. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 15 amino acid substitutions consisting of N48G, I54T, T57L, N59D, E92R, T138A, T161Y, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

183. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 15 amino acid substitutions consisting of N48G, I54T, T57N, N59D, E92R, T138A, T161G, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

184. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 17 amino acid substitutions consisting of N48G, I54T, T57N, N59D, E92R, T138A, T161G, D188T, F199L, K200R, E201N, L223I, N233S, N246R, N273G, D288G, and Q293R.

185. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 13 amino acid substitutions consisting of N48G, I54T, T57N, N59D, E92R, T138A, T161Y, G222A, L223I, N233S, N246R, N273G, and D288G.

186. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 15 amino acid substitutions consisting of N48G, I54T, T57N, N59D, E92R, T138A, T161Y, F199L, K200R, E201N, N233S, N246R, N273G, D288G, and Q293R.

187. The polypeptide variant according to any one of claims 144 to 154, wherein the second set of amino acid substitutions of Sequence ID No. 2 is a set of 13 amino acid substitutions consisting of E9I, I10T, R11T, N48G, I54T, T57N, N59D, T138A, N233S, N246R, N273G, D288G, and Q293R.

188. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1492.

189. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1697.

190. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1700.

191. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1701.

192. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1702.

193. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1703.

194. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1704.

195. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1705.

196. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1713.

197. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1720.

198. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1722.

199. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1723.

200. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1724.

201. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1772.

202. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1774.

203. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1775.

204. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1793.

205. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1795.

206. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1797.

207. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1798.

208. A polypeptide variant according to claim 144, comprising the amino acid sequence of SEQ ID NO: 1949.

209. The polypeptide variant according to any one of claims 1 to 208, wherein the polypeptide variant has at least about 25% of the activity of the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2 in an IgG protease enzyme assay.

210. The polypeptide variant according to any one of claims 1 to 208, wherein the polypeptide variant has at least about 30% of the activity of the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2 in an IgG protease enzyme assay.

211. The polypeptide variant according to any one of claims 1 to 208, wherein the polypeptide variant has at least about 40% activity, at least about 50% activity, at least about 60% activity, at least about 70% activity, at least about 75% activity, at least about 80% activity, at least about 90% activity, at least about 100% activity, at least about 110% activity, and at least about 120% activity of the IdeS enzyme containing the amino acid sequence of SEQ ID NO: 2 in an IgG protease enzyme assay.

212. The polypeptide variant according to any one of claims 1 to 211, wherein the polypeptide variant has at least about 25% activity, at least about 40% activity, at least about 50% activity, at least about 60% activity, at least about 70% activity, at least about 80% activity, at least about 90% activity, at least about 100% activity, at least about 110% activity, and at least about 120% activity of the IdeZ enzyme containing the amino acid sequence of SEQ ID NO: 830 in an IgG protease enzyme assay.

213. The polypeptide variant according to any one of claims 209 to 212, wherein the IgG protease activity in the IgG protease enzyme assay is measured by an enzyme-linked immunosorbent assay including digestion of an immobilized antibody substrate.

214. The polypeptide variant according to any one of claims 1 to 213, wherein the polypeptide variant has one or more T cell epitopes depleted compared to the IdeS enzyme containing the amino acid sequence described in SEQ ID NO:

2.

215. The polypeptide variant according to any one of claims 1 to 214, wherein the polypeptide variant has substantially the same thermal stability as the IdeS enzyme comprising the amino acid sequence described in SEQ ID NO:

2.

216. The polypeptide variant according to any one of claims 1 to 215, wherein the polypeptide has substantially the same thermal stability as the IdeZ enzyme comprising the amino acid sequence described in SEQ ID NO:

830.

217. The polypeptide variant according to any one of claims 1 to 214, wherein the polypeptide variant has higher thermal stability than the IdeS enzyme containing the amino acid sequence described in SEQ ID NO:

2.

218. The polypeptide variant according to any one of claims 1 to 215 and 217, wherein the polypeptide variant has higher thermal stability than the IdeZ enzyme containing the amino acid sequence described in SEQ ID NO:

830.

219. A polypeptide variant according to any one of claims 215 to 218, wherein the thermal stability is measured by differential scanning fluorescence quantification.

220. A polypeptide variant of the amino acid sequence of SEQ ID NO: 297 [N142], wherein the polypeptide variant contains one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: I10, R11, Y12, V15, I35, V46, N48, Y52, T57, N102, L120, Y127, K129, R157, S159, L160, V230, N233, K250, A261, F269, S274, A275, D288, Q293, V294, L300, and the polypeptide variant contains an amino acid sequence that is at least about 75% identical to SEQ ID NO:

297.

221. The polypeptide variant according to claim 220, wherein the polypeptide variant does not have an amino acid mutation at one or more of the following amino acid positions of SEQ ID NO: A32, N33, Q36, F41, D84, R88, E91, E92, H93, M106, N117, H118, E126, E170, N197, N203, K213, L223, S245, K286, S306.

222. A polypeptide variant according to claim 220 or 221, comprising an amino acid sequence that is at least about 80% or at least about 85% identical to SEQ ID NO:

297.

223. A polypeptide variant according to claim 220 or 221, comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO:

297.

224. A polypeptide variant according to claim 220 or 221, comprising an amino acid sequence that is at least about 95% identical to SEQ ID NO:

297.

225. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations are one or more amino acid substitutions.

226. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations are two or more amino acid mutations.

227. The polypeptide variant according to claim 226, wherein the two or more amino acid mutations are two or more amino acid substitutions.

228. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations are three or more amino acid mutations.

229. The polypeptide variant according to claim 228, wherein the three or more amino acid mutations are three or more amino acid substitutions.

230. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations are four or more amino acid mutations.

231. The polypeptide variant according to claim 230, wherein the four or more amino acid mutations are four or more amino acid substitutions.

232. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations are five or more amino acid mutations.

233. The polypeptide variant according to claim 232, wherein the five or more amino acid mutations are five or more amino acid substitutions.

234. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations are six or more amino acid mutations.

235. The polypeptide variant according to claim 234, wherein the six or more amino acid mutations are six or more amino acid substitutions.

236. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations are seven or more amino acid mutations.

237. The polypeptide variant according to claim 236, wherein the seven or more amino acid mutations are seven or more amino acid substitutions.

238. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations are eight or more amino acid mutations.

239. The polypeptide variant according to claim 238, wherein the eight or more amino acid mutations are eight or more amino acid substitutions.

240. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations are nine or more amino acid mutations.

241. The polypeptide variant according to claim 240, wherein the nine or more amino acid mutations are nine or more amino acid substitutions.

242. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations are ten or more amino acid mutations.

243. The polypeptide variant according to claim 242, wherein the 10 or more amino acid mutations are 10 or more amino acid substitutions.

244. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations are eleven or more amino acid mutations.

245. The polypeptide variant according to claim 244, wherein the 11 or more amino acid mutations are 11 or more amino acid substitutions.

246. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations consist of approximately 5 to approximately 20 amino acid mutations.

247. The polypeptide variant according to claim 246, wherein the aforementioned approximately 5 to approximately 20 amino acid mutations are approximately 5 to approximately 20 amino acid substitutions.

248. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations consist of approximately 5 to approximately 15 amino acid mutations.

249. The polypeptide variant according to claim 248, wherein the aforementioned approximately 5 to approximately 15 amino acid mutations are approximately 5 to approximately 15 amino acid substitutions.

250. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations consist of approximately 8 to approximately 20 amino acid mutations.

251. The polypeptide variant according to claim 250, wherein the aforementioned approximately 8 to approximately 20 amino acid mutations are approximately 8 to approximately 20 amino acid substitutions.

252. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations consist of approximately 10 to approximately 20 amino acid mutations.

253. The polypeptide variant according to claim 252, wherein the aforementioned approximately 10 to approximately 20 amino acid mutations are approximately 10 to approximately 20 amino acid substitutions.

254. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations consist of approximately 16 to approximately 25 amino acid mutations.

255. The polypeptide variant according to claim 254, wherein the aforementioned approximately 16 to approximately 25 amino acid mutations are approximately 16 to approximately 25 amino acid substitutions.

256. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations consist of approximately 16 to approximately 20 amino acid mutations.

257. The polypeptide variant according to claim 256, wherein the aforementioned approximately 16 to approximately 20 amino acid mutations are approximately 16 to approximately 20 amino acid substitutions.

258. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations consist of approximately 16 to approximately 17 amino acid mutations.

259. The polypeptide variant according to claim 258, wherein the aforementioned approximately 16 to approximately 17 amino acid mutations are approximately 16 to approximately 17 amino acid substitutions.

260. The polypeptide variant according to any one of claims 220 to 224, wherein the one or more amino acid mutations consist of approximately 5 to approximately 25 amino acid mutations.

261. The polypeptide variant according to claim 260, wherein the aforementioned approximately 5 to approximately 25 amino acid mutations are approximately 5 to approximately 25 amino acid substitutions.

262. One of I10A and I10Q, one of R11T, Y12K, one of V15H and V15K, one of I35T, one of V46D and V46S, one of N48H and N48G, one of Y52F and Y52S, one of T57K, N102G, L120T, Y127L, K129G, R157G, S159G, L160W, V230G, V230R and V230S, A polypeptide variant according to any one of claims 220 to 261, comprising one amino acid substitution of Sequence ID No. 297, selected from the group consisting of N233S, K250S, A261K, F269N, S274D, A275F, A275S, and A275W, one of D288H and D288K, Q293K, V294A, L300T, and combinations thereof.

263. The polypeptide variant according to any one of claims 220 to 262, wherein the polypeptide variant is selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, T57L, D84E, R88N, E91K, E92K, H93Y, M106L, N117D, H118S, E126D, N162D, E170R, N197Y, F199L, N203T, K213Q, L223I, N233G, S245A, A261P, K286E, and S306I, and does not have one or more amino acid substitutions of SEQ ID NO:

297.

264. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10A, V15H, I35T, and Y52F.

265. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10A, V15H, I35T, and V46S.

266. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10A, V15H, I35T, and V46D.

267. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10A, V15H, I35T, V46D, and T57K.

268. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10A, V15H, I35T, Y52F, and T57K.

269. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10A, V15H, I35T, Y52F, and N102G.

270. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include L120T, V230R, A261K, S274D, and A275F.

271. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include V230S, A261K, S274D, A275F, and D288H.

272. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include V230G, A261K, S274D, A275F, and D288H.

273. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10A, V15H, V230S, A261K, and S274D.

274. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10A, V15H, I35T, V46D, T57K, L120T, V230R, A261K, S274D, A275F, and D288H.

275. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10A, V15H, I35T, V46S, T57K, L120T, V230R, A261K, S274D, A275F, and D288H.

276. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10A, V15H, I35T, N48H, Y52F, T57K, L120T, V230R, A261K, S274D, A275F, and D288H.

277. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10A, V15H, I35T, Y52F, T57K, L120T, V230R, A261K, S274D, A275F, and D288H.

278. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in Sequence ID No. 297 include I10A, V15H, I35T, Y52F, T57K, L120T, V230R, K250S, A261K, S274D, A275F, and D288H.

279. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10A, V15H, I35T, Y52F, T57K, L120T, V230S, A261K, S274D, A275F, and D288H.

280. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10A, V15H, I35T, Y52F, N102G, L120T, V230G, A261K, S274D, A275F, and D288H.

281. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10A, V15H, I35T, Y52F, Y127L, V230G, A261K, S274D, A275F, and D288H.

282. A polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10A, V15H, I35T, N48H, Y52S, T57K, N102G, L120T, S159G, V230R, K250S, A261K, S274D, A275F, D288H, and V294A.

283. The polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in Sequence ID No. 297 include I10A, V15H, I35T, N48H, T57K, N102G, L120T, S159G, L160W, V230R, K250S, A261K, S274D, A275F, D288H, and L300T.

284. A polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10Q, R11T, Y12K, V15K, N48G, Y52S, T57K, L120T, R157G, V230S, K250S, A261K, F269N, A275S, D288K, and Q293K.

285. A polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10Q, R11T, Y12K, V15K, N48G, T57K, L120T, K129G, R157G, V230S, N233S, K250S, A261K, F269N, A275F, D288K, and Q293K.

286. A polypeptide variant according to claim 262 or 263, wherein one or more amino acid substitutions in SEQ ID NO: 297 include I10Q, R11T, Y12K, V15K, N48G, T57K, L120T, K129G, R157G, V230S, N233S, K250S, A261K, F269N, A275W, D288K, and Q293K.

287. A polypeptide variant according to claim 220, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 358 to 430.

288. A polypeptide variant according to claim 220, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 431 to 448.

289. A polypeptide variant according to claim 220, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 449 to 485.

290. A polypeptide variant according to claim 220, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 486 to 489.

291. A polypeptide variant according to claim 220, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 490 to 596.

292. The polypeptide variant according to claim 220, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 358 to SEQ ID NOs. 596, wherein the polypeptide variant comprises amino acid mutations present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO.

297.

293. A polypeptide variant according to claim 220, comprising the amino acid sequence of SEQ ID NO:

303.

294. A polypeptide variant according to claim 220, comprising the amino acid sequence of SEQ ID NO:

304.

295. A polypeptide variant according to claim 220, comprising the amino acid sequence of SEQ ID NO:

305.

296. A polypeptide variant according to claim 220, comprising the amino acid sequence of SEQ ID NO:

306.

297. A polypeptide variant according to claim 220, comprising the amino acid sequence of SEQ ID NO:

307.

298. A polypeptide variant according to claim 220, comprising the amino acid sequence of SEQ ID NO:

308.

299. A polypeptide variant according to claim 220, comprising the amino acid sequence of SEQ ID NO:

309.

300. A polypeptide variant according to claim 220, comprising the amino acid sequence of SEQ ID NO:

310.

301. The polypeptide variant according to claim 220, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 303 to SEQ ID NOs. 310, wherein the polypeptide variant comprises amino acid mutations present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO.

297.

302. A polypeptide variant according to claim 220, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 847 to 1005.

303. A polypeptide variant according to claim 220, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 847 to SEQ ID NOs. 1005, wherein the polypeptide variant comprises an amino acid mutation present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO.

297.

304. A polypeptide variant according to claim 220, comprising the amino acid sequence of SEQ ID NO:

859.

305. A polypeptide variant according to claim 220, comprising the amino acid sequence of SEQ ID NO:

868.

306. A polypeptide variant according to claim 220, comprising the amino acid sequence of SEQ ID NO:

925.

307. A polypeptide variant according to claim 220, comprising the amino acid sequence of SEQ ID NO:

936.

308. A polypeptide variant according to claim 220, comprising the amino acid sequence of SEQ ID NO:

942.

309. A polypeptide variant according to claim 220, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 859, SEQ ID NO: 868, SEQ ID NO: 925, SEQ ID NO: 936, and SEQ ID NO: 942, wherein the polypeptide variant comprises an amino acid mutation present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO:

297.

310. The polypeptide variant according to any one of claims 220 to 309, wherein the polypeptide variant has at least about 25% of the activity of the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2 in an IgG protease enzyme assay.

311. The polypeptide variant according to any one of claims 220 to 309, wherein the polypeptide variant has at least about 30% of the activity of the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2 in an IgG protease enzyme assay.

312. The polypeptide variant according to any one of claims 220 to 309, wherein the polypeptide variant has at least about 40% activity, at least about 50% activity, at least about 60% activity, at least about 70% activity, at least about 75% activity, at least about 80% activity, at least about 90% activity, at least about 100% activity, at least about 110% activity, and at least about 120% activity of the IdeS enzyme containing the amino acid sequence of SEQ ID NO: 2 in an IgG protease enzyme assay.

313. The polypeptide variant according to any one of claims 220 to 312, wherein the polypeptide variant has at least about 40% activity, at least about 50% activity, at least about 60% activity, at least about 70% activity, at least about 80% activity, at least about 90% activity, at least about 100% activity, at least about 110% activity, or at least about 120% activity of the IdeZ enzyme containing the amino acid sequence of SEQ ID NO: 830 in an IgG protease enzyme assay.

314. The polypeptide variant according to any one of claims 310 to 313, wherein the IgG protease activity in the IgG protease enzyme assay is measured by an enzyme-linked immunosorbent assay including digestion of an immobilized antibody substrate.

315. The polypeptide variant according to any one of claims 220 to 314, wherein the polypeptide variant has one or more T cell epitopes depleted compared to the polypeptide with the amino acid sequence of SEQ ID NO:

297.

316. The polypeptide variant according to any one of claims 220 to 315, wherein the polypeptide variant has substantially the same thermal stability as the IdeS enzyme comprising the amino acid sequence described in SEQ ID NO:

2.

317. The polypeptide variant according to any one of claims 220 to 316, wherein the polypeptide variant has substantially the same thermal stability as the IdeZ enzyme comprising the amino acid sequence described in SEQ ID NO:

830.

318. The polypeptide variant according to any one of claims 220 to 315, wherein the polypeptide variant has higher thermal stability than the IdeS enzyme containing the amino acid sequence described in SEQ ID NO:

2.

319. The polypeptide variant according to any one of claims 220 to 316 and 318, wherein the polypeptide variant has higher thermal stability than the IdeZ enzyme containing the amino acid sequence described in SEQ ID NO:

830.

320. A polypeptide variant according to any one of claims 316 to 319, wherein the thermal stability is measured by differential scanning fluorescence quantitative analysis.

321. A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 to 302.

322. The polypeptide according to claim 321, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37, 39, 41, 50, 51, 54, 56, 60, 61, 65, 67, 137, 140, 144, 150, 159, 285, 286, 287, 288, 289, 292, 293, 294, 295, 296, 297, and 298.

323. The polypeptide according to claim 321, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 140, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 297, and SEQ ID NO:

298.

324. The polypeptide according to claim 323, comprising the amino acid sequence of SEQ ID NO:

54.

325. The polypeptide according to claim 323, comprising the amino acid sequence of SEQ ID NO:

140.

326. The polypeptide according to claim 323, comprising the amino acid sequence of SEQ ID NO:

293.

327. The polypeptide according to claim 323, comprising the amino acid sequence of SEQ ID NO:

294.

328. The polypeptide according to claim 323, comprising the amino acid sequence of SEQ ID NO:

295.

329. The polypeptide according to claim 323, comprising the amino acid sequence of SEQ ID NO:

297.

330. The polypeptide according to claim 323, comprising the amino acid sequence of SEQ ID NO:

298.

331. A polypeptide variant containing an amino acid sequence that is at least approximately 90% to at least approximately 99% identical to one of the amino acid sequences from SEQ ID NOs: 27 to 302.

332. A polypeptide variant according to claim 331, comprising an amino acid sequence that is at least about 90% to at least about 99% identical to one of the amino acid sequences of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 137, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 150, SEQ ID NO: 159, SEQ ID NO: 285, SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, and SEQ ID NO:

298.

333. A polypeptide variant according to claim 331, comprising an amino acid sequence that is at least about 90% to at least about 99% identical to one of the amino acid sequences of SEQ ID NO: 54, SEQ ID NO: 140, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295, SEQ ID NO: 297, and SEQ ID NO:

298.

334. A polypeptide variant of the amino acid sequence of SEQ ID NO: 293, comprising one or more amino acid mutations at one or more amino acid positions: Y12, L31, E34, F36, R37, Y38, N39, I44, A60, D133, N162, F274, A279, H280, V299, wherein the polypeptide variant comprises an amino acid sequence that is at least about 75% identical to SEQ ID NO: 293, and the polypeptide has at least about 25% of the activity of the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2 in an IgG protease enzyme assay.

335. A polypeptide variant according to claim 334, comprising an amino acid sequence that is at least about 80% identical to SEQ ID NO:

293.

336. A polypeptide variant according to claim 334, comprising an amino acid sequence that is at least about 85% identical to SEQ ID NO:

293.

337. A polypeptide variant according to claim 334, comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO:

293.

338. A polypeptide variant according to claim 334, comprising an amino acid sequence that is at least about 95% identical to SEQ ID NO:

293.

339. The polypeptide variant according to any one of claims 334 to 338, wherein the one or more amino acid mutations are one or more amino acid substitutions.

340. A polypeptide variant according to claim 334, comprising the amino acid sequence described in one of sequence numbers 318 to 357.

341. A polypeptide variant according to claim 334, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 318 to SEQ ID NOs. 357, wherein the polypeptide variant comprises an amino acid mutation present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO.

293.

342. A polypeptide variant of the amino acid sequence of SEQ ID NO: 295 [N144], wherein the polypeptide variant has one or more amino acid positions of SEQ ID NO: 295: Y12, S18, I35, I46, A47, Y52, I54, T55, T57, N59, H93, F101, N102, E104, Q105, L120, F125, E126, Y127, T135, Y156, S159, L175, V183, A polypeptide variant comprising one or more amino acid mutations in R200, N201, N205, I207, V230, R231, N233, A241, N244, E246, A261, Y269, V270, S274, A275, I280, S281, A282, I285, D288, and V290, wherein the polypeptide variant comprises an amino acid sequence that is at least about 75% identical to SEQ ID NO:

295.

343. The polypeptide variant according to claim 342, wherein the polypeptide variant does not have an amino acid mutation at one or more of the following amino acid positions of SEQ ID NO: A32, N33, Q36, F41, D84, E91, M106, H118, E170, S195, N197, N203, S245, K286 of SEQ ID NO:

295.

344. A polypeptide variant according to claim 342 or 343, comprising an amino acid sequence that is at least about 80% identical to SEQ ID NO:

295.

345. A polypeptide variant according to claim 342 or 343, comprising an amino acid sequence that is at least about 85% identical to SEQ ID NO:

295.

346. A polypeptide variant according to claim 342 or 343, comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO:

295.

347. A polypeptide variant according to claim 342 or 343, comprising an amino acid sequence that is at least about 95% identical to SEQ ID NO:

295.

348. The polypeptide variant according to any one of claims 342 to 347, wherein the one or more amino acid mutations are one or more amino acid substitutions.

349. The polypeptide variant according to any one of claims 342 to 347, wherein the one or more amino acid mutations consist of approximately 10 to approximately 30 amino acid mutations.

350. The polypeptide variant according to claim 349, wherein the approximately 10 to approximately 30 amino acid mutations are approximately 10 to approximately 30 amino acid substitutions.

351. The polypeptide variant according to any one of claims 342 to 347, wherein the one or more amino acid mutations consist of approximately 10 to approximately 25 amino acid mutations.

352. The polypeptide variant according to claim 351, wherein the aforementioned approximately 10 to approximately 25 amino acid mutations are approximately 10 to approximately 25 amino acid substitutions.

353. The polypeptide variant according to any one of claims 342 to 347, wherein the one or more amino acid mutations consist of approximately 12 to approximately 24 amino acid mutations.

354. The polypeptide variant according to claim 353, wherein the aforementioned approximately 12 to approximately 24 amino acid mutations are approximately 12 to approximately 24 amino acid substitutions.

355. The polypeptide variant according to any one of claims 342 to 347, wherein the one or more amino acid mutations consist of approximately 12 to approximately 20 amino acid mutations.

356. The polypeptide variant according to claim 355, wherein the aforementioned approximately 12 to approximately 20 amino acid mutations are approximately 12 to approximately 20 amino acid substitutions.

357. The polypeptide variant according to any one of claims 342 to 347, wherein the one or more amino acid mutations consist of approximately 12 to approximately 16 amino acid mutations.

358. The polypeptide variant according to claim 357, wherein the aforementioned approximately 12 to approximately 16 amino acid mutations are approximately 12 to approximately 16 amino acid substitutions.

359. The polypeptide variant according to any one of claims 342 to 347, wherein the one or more amino acid mutations are 12, 13, or 14 amino acid mutations.

360. The polypeptide variant according to claim 359, wherein the 12, 13, or 14 amino acid mutations are 12, 13, or 14 amino acid substitutions.

361. The polypeptide variant according to any one of claims 342 to 346, wherein the one or more amino acid mutations consist of approximately 21 to approximately 24 amino acid mutations.

362. The polypeptide variant according to claim 361, wherein the approximately 21 to approximately 24 amino acid mutations are approximately 21 to approximately 24 amino acid substitutions.

363. The polypeptide variant according to any one of claims 342 to 346, wherein the one or more amino acid mutations consist of 21, 22, 23, or 24 amino acid mutations.

364. The polypeptide variant according to claim 363, wherein the 21, 22, 23, or 24 amino acid mutations are 21, 22, 23, or 24 amino acid substitutions.

365. One of Y12N, S18K, I35E, I35K, and I35S; one of I46D, A47E, I54T, T55K, T55H, and T55R; one of T57L, N59D, H93Y, N102G, E104R, one of L120T, and L120S; one of F125W, T135H, S159K, L175A, L175N, and L175S; one of V183W, R200D, R200G, and R200T; one of N201E, N205D, I207T, V230H, and V230Q; R231W, N233G, A241G, E246G, and E246K A polypeptide variant according to any one of claims 342 to 364, comprising one amino acid substitution of SEQ ID NO: 295, selected from the group consisting of one of A261K, A261I, A261F, A261G, A261Q, and A261S, one of Y269D, Y269S, and Y269W, one of V270T, V272T, and V272Y, one of S274D, and S274G, one of A275S, and A275W, one of I280M, S281G, A282G, one of D288K, and D288T, V290F, and combinations thereof.

366. The polypeptide variant according to any one of claims 342 to 365, wherein the polypeptide variant is selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, F199L, N203T, K213Q, L223I, S245A, N246E, K250E, K286E, and S306I, and does not have one or more amino acid substitutions of SEQ ID NO:

295.

367. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I46D, A47E, T55H, H93Y, E104R, L120T, F125W, T135H, S159K, and A275S.

368. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I46D, A47E, T55H, H93Y, E104R, L120T, F125W, T135H, S159K, A261I, and A275S.

369. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261F, and A275S.

370. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261G, and A275S.

371. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261Q, and A275S.

372. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, A261S, and A275S.

373. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I46D, A47E, T55R, H93Y, E104R, L120S, F125W, T135H, S159K, A261K, and A275S.

374. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I46D, A47E, T55R, H93Y, E104R, L120T, F125W, T135H, S159K, A261K, and A275S.

375. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, and A275S.

376. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N201E, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

377. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

378. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.

379. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261Q, V270T, A275W, I280M, and V290F.

380. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, A261S, Y269W, A275W, A282G, and V290F.

381. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, A261S, Y269W, A275W, A282G, and D288T.

382. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35E, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N201E, N233G, E246K, A261Q, Y269W, A275W, A282G, and D288T.

383. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35E, I46D, A47E, T55R, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, A261K, Y269W, A275W, A282G, and D288T.

384. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35E, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N201E, R231W, E246G, Y269W, A275W, A282G, and D288T.

385. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35E, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, Y269W, A275W, A282G, and D288T.

386. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

387. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261S, V270T, A275W, I280M, and V290F.

388. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.

389. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, T55K, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246K, A261S, Y269W, S274G, A275S, A282G, and D288K.

390. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.

391. A polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, R200G, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

392. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N201E, N233G, E246G, V270T, A275W, I280M, and V290F.

393. A polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N201E, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

394. A polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.

395. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, V270T, A275W, I280M, and V290F.

396. A polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246G, A261K, V270T, A275W, I280M, and V290F.

397. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, V270T, A275W, I280M, and V290F.

398. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in Sequence ID No. 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

399. A polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in Sequence ID No. 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, R200T, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

400. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175S, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.

401. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, V230H, E246K, Y269W, A275W, A282G, and D288T.

402. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175A, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F.

403. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, V183W, N205D, N233G, E246K, Y269W, S274D, A275S, A282G, and D288T.

404. A polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, V183W, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F.

405. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, V230H, E246K, A261K, Y269W, S274G, A275S, A282G, and D288T.

406. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, V230Q, E246G, A261K, Y269W, S274G, A275S, A282G, and D288T.

407. A polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246K, A261K, Y269W, S274G, A275S, A282G, and V290F.

408. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, Y269W, S274G, A275S, A282G, and V290F.

409. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, Y269W, A275W, A282G, and D288T.

410. A polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246G, Y269W, S274G, A275S, A282G, and V290F.

411. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, Y269W, S274G, A275S, A282G, and D288K.

412. A polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261K, Y269W, S274D, A275S, A282G, and V290F.

413. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261K, Y269W, S274D, A275S, A282G, and D288K.

414. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions of SEQ ID NO: 295 include Y12N, S18K, I35K, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175S, N205D, N233G, E246G, Y269W, A275W, A282G, and D288T.

415. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35S, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

416. The polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35S, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.

417. A polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35S, I46D, A47E, T55K, H93Y, N102G, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, A261Q, V270T, A275W, I280M, and V290F.

418. A polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35S, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, V230Q, A241G, Y269W, A275W, A282G, and V290F.

419. A polypeptide variant according to claim 365 or 366, wherein one or more amino acid substitutions in SEQ ID NO: 295 include Y12N, S18K, I35S, I46D, A47E, I54T, T57L, N59D, H93Y, E104R, L120T, F125W, T135H, S159K, L175N, N205D, R231W, E246K, Y269W, A275W, A282G, and V290F.

420. A polypeptide variant according to claim 342, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 597 to 657.

421. A polypeptide variant according to claim 342, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 597 to SEQ ID NOs. 657, wherein the polypeptide variant comprises an amino acid mutation present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO.

295.

422. A polypeptide variant according to claim 342, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1006 to 1490 and SEQ ID NOs: 1952 to 2590.

423. The polypeptide variant according to claim 342, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs: 1006 to 1490 and SEQ ID NOs: 1952 to 2590, wherein the polypeptide variant comprises an amino acid mutation present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO:

295.

424. Sequence ID 1118, Sequence ID 1120, Sequence ID 1122, Sequence ID 1123, Sequence ID 1125, Sequence ID 1126, Sequence ID 1127, Sequence ID 1129, Sequence ID 1282, Sequence ID 1962, Sequence ID 1963, Sequence ID 1964, Sequence ID 1965, Sequence ID 1970, Sequence ID 1971, Sequence ID 1983, Sequence ID 2004, Sequence ID 2024, Sequence ID 2028, Sequence ID 2371, Sequence ID 2372, Sequence ID 2373, Sequence ID 2390, Sequence ID 2421, Sequence ID 2427, Sequence ID 2428, Sequence ID 2429, Sequence ID 2430, Sequence ID 2431, Sequence ID 2432, Sequence ID 2433, Sequence ID 2434, Sequence ID 2437, Sequence ID 2439, Sequence ID 2446, Sequence ID 2450, Sequence ID A polypeptide variant according to claim 342, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of 2463, SEQ ID NO: 2464, SEQ ID NO: 2476, SEQ ID NO: 2478, SEQ ID NO: 2485, SEQ ID NO: 2486, SEQ ID NO: 2487, SEQ ID NO: 2489, SEQ ID NO: 2491, SEQ ID NO: 2493, SEQ ID NO: 2494, SEQ ID NO: 2511, SEQ ID NO: 2524, SEQ ID NO: 2526, SEQ ID NO: 2528, SEQ ID NO: 2574, and SEQ ID NO: 2575, wherein the polypeptide variant comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, with respect to the amino acid sequence of SEQ ID NO: 295, wherein the polypeptide variant comprises an amino acid mutation present in the reference amino acid sequence.

425. Sequence ID 1118, Sequence ID 1120, Sequence ID 1122, Sequence ID 1123, Sequence ID 1125, Sequence ID 1126, Sequence ID 1127, Sequence ID 1129, Sequence ID 1282, Sequence ID 1970, Sequence ID 1971, Sequence ID 1983, Sequence ID 2004, Sequence ID 2024, Sequence ID 2028, Sequence ID 2390, Sequence ID 2421, Sequence ID 2446, Sequence ID 2450, Sequence ID 2463, Sequence ID 2464, Sequence ID 2476, Sequence ID 2478, Sequence ID 2485, Sequence ID 2486, Sequence ID 2487, Sequence ID A polypeptide variant according to claim 342, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of 2489, SEQ ID NO: 2491, SEQ ID NO: 2493, SEQ ID NO: 2494, SEQ ID NO: 2511, SEQ ID NO: 2574, and SEQ ID NO: 2575, wherein the polypeptide variant comprises an amino acid mutation present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO:

295.

426. A polypeptide variant of the amino acid sequence of SEQ ID NO: 295 [N144], wherein the polypeptide variant comprises a first set of amino acid substitutions of SEQ ID NO: 295 and a second set of amino acid substitutions of SEQ ID NO:

295. The first set of amino acid substitutions in Sequence ID No. 295 consists of Y12N, S18K, I46D, A47E, H93Y, E104R, F125W, T135H, and S159K, and the second set of amino acid substitutions in Sequence ID No. 295 consists of one of I35K, I35E, and I35S, one of I54T, T55K, T55R, and T55H, one of T57L, N59D, N102G, L120T, and L120S, one of L175N, L175A, and L175S, and V183W, R200G, and R200T. It is a set of approximately 3 to 15 amino acid substitutions selected from the group consisting of one of N201E, N205D, V230H and V230Q, one of R231W, N233G, A241G, E246K and E246G, one of A261K, A261Q, A261S, A261I, A261F and A261G, one of Y269W, V270T, S274G and S274D, one of A275W and A275S, one of I280M, A282G, D288T and D288K, and V290F. The polypeptide variant is a polypeptide variant that includes an amino acid sequence that is at least about 90% identical to SEQ ID NO:

295.

427. The polypeptide variant according to claim 426, wherein the polypeptide variant does not have an amino acid mutation at one or more of the following amino acid positions of SEQ ID NO: A32, N33, Q36, F41, D84, E91, M106, H118, E170, S195, N197, N203, S245, K286 of SEQ ID NO:

295.

428. The polypeptide variant according to claim 426 or 427, wherein the polypeptide variant is selected from the group consisting of A32T, N33D, T35I, Q36Y, F41L, V46K, D84E, R88N, E91K, E92K, M106L, N117D, H118S, E126D, E170R, S195N, N197Y, F199L, N203T, K213Q, L223I, S245A, N246E, K250E, K286E, and S306I, and does not have one or more amino acid substitutions of SEQ ID NO:

295.

429. A polypeptide variant according to any one of claims 426 to 428, comprising an amino acid sequence that is at least about 91% identical to SEQ ID NO:

295.

430. A polypeptide variant according to any one of claims 426 to 428, comprising an amino acid sequence that is at least about 92% identical to SEQ ID NO:

295.

431. A polypeptide variant according to any one of claims 426 to 428, comprising an amino acid sequence that is at least about 93% identical to SEQ ID NO:

295.

432. A polypeptide variant according to any one of claims 426 to 428, comprising an amino acid sequence that is at least about 94% identical to SEQ ID NO:

295.

433. A polypeptide variant according to any one of claims 426 to 428, comprising an amino acid sequence that is at least about 95% identical to SEQ ID NO:

295.

434. A polypeptide variant according to any one of claims 426 to 428, comprising an amino acid sequence that is at least about 96% identical to SEQ ID NO:

295.

435. A polypeptide variant according to any one of claims 426 to 428, comprising an amino acid sequence that is approximately 90% to approximately 96% identical to SEQ ID NO:

295.

436. A polypeptide variant according to any one of claims 426 to 428, comprising an amino acid sequence that is approximately 92% to approximately 96% identical to SEQ ID NO:

295.

437. The polypeptide variant according to any one of claims 426 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of about 3 to about 15 amino acid substitutions selected from the group consisting of one of I35K and I35E, one of I54T, T55K, T57L, N59D, N102G, L120T, L175N, N205D, R231W, N233G, one of E246K and E246G, one of A261K and A261Q, one of Y269W, V270T, A275W and A275S, I280M, A282G, D288T, and V290F.

438. The polypeptide variant according to any one of claims 426 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of about 3 to about 15 amino acid substitutions selected from the group consisting of one of I35K, I54T, T55K, T57L, N59D, N102G, L120T, L175N, N205D, N233G, E246K, Y269W, V270T, A275W, and A275S, I280M, A282G, and V290F.

439. The polypeptide variant according to any one of claims 426 to 438, wherein the second set of amino acid substitutions in Sequence ID No. 295 is a set of approximately 3 to approximately 5 amino acid substitutions.

440. The polypeptide variant according to any one of claims 426 to 431 and 435 to 438, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of about 12 to about 15 amino acid substitutions.

441. The polypeptide variant according to any one of claims 426 to 433 and 435 to 438, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of about 5 to about 15 amino acid substitutions.

442. The polypeptide variant according to any one of claims 426 to 433 and 435 to 438, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of about 5 to about 12 amino acid substitutions.

443. The polypeptide variant according to any one of claims 426 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of about 3 to about 13 amino acid substitutions selected from the group consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F.

444. The polypeptide variant according to claim 443, wherein the second set of amino acid substitutions in Sequence ID No. 295 is a set of approximately 3 to approximately 5 amino acid substitutions.

445. The polypeptide variant according to claim 443, wherein the second set of amino acid substitutions in Sequence ID No. 295 is a set of approximately 12 to approximately 13 amino acid substitutions.

446. The polypeptide variant according to claim 443, wherein the second set of amino acid substitutions in Sequence ID No. 295 is a set of approximately 5 to approximately 13 amino acid substitutions.

447. The polypeptide variant according to any one of claims 426 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of three amino acid substitutions consisting of T55H, L120T, and A275S.

448. The polypeptide variant according to any one of claims 426-433 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of four amino acid substitutions consisting of T55H, L120T, A261I, and A275S.

449. The polypeptide variant according to any one of claims 426-433 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of four amino acid substitutions consisting of T55K, L120T, A261F, and A275S.

450. The polypeptide variant according to any one of claims 426-433 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of four amino acid substitutions consisting of T55K, L120T, A261G, and A275S.

451. The polypeptide variant according to any one of claims 426-433 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of four amino acid substitutions consisting of T55K, L120T, A261Q, and A275S.

452. The polypeptide variant according to any one of claims 426-433 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of four amino acid substitutions consisting of T55K, L120T, A261S, and A275S.

453. The polypeptide variant according to any one of claims 426-433 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of four amino acid substitutions consisting of T55R, L120S, A261K, and A275S.

454. The polypeptide variant according to any one of claims 426-433 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of four amino acid substitutions consisting of T55R, L120T, A261K, and A275S.

455. The polypeptide variant according to any one of claims 426-433 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of five amino acid substitutions consisting of I54T, T57L, N59D, L120T, and A275S.

456. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35E, T55K, N102G, L120T, L175N, N201E, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

457. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35E, T55K, N102G, L120T, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

458. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35E, T55K, N102G, L120T, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.

459. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35E, T55K, N102G, L120T, L175N, N205D, R231W, E246K, A261Q, V270T, A275W, I280M, and V290F.

460. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 12 amino acid substitutions consisting of I35E, T55K, L120T, L175A, N205D, N233G, E246K, A261S, Y269W, A275W, A282G, and V290F.

461. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 12 amino acid substitutions consisting of I35E, T55K, L120T, L175A, N205D, N233G, E246K, A261S, Y269W, A275W, A282G, and D288T.

462. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 12 amino acid substitutions consisting of I35E, T55K, L120T, L175N, N201E, N233G, E246K, A261Q, Y269W, A275W, A282G, and D288T.

463. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 12 amino acid substitutions consisting of I35E, T55R, L120T, L175A, N205D, N233G, E246K, A261K, Y269W, A275W, A282G, and D288T.

464. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35E, I54T, T57L, N59D, L120T, L175A, N201E, R231W, E246G, Y269W, A275W, A282G, and D288T.

465. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35E, I54T, T57L, N59D, L120T, L175A, N205D, N233G, E246K, Y269W, A275W, A282G, and D288T.

466. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, T55K, N102G, L120T, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

467. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, T55K, N102G, L120T, L175N, N205D, N233G, E246G, A261S, V270T, A275W, I280M, and V290F.

468. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, T55K, N102G, L120T, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.

469. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, T55K, L120T, L175N, N205D, N233G, E246K, A261S, Y269W, S274G, A275S, A282G, and D288K.

470. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175A, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.

471. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, R200G, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

472. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N201E, N233G, E246G, V270T, A275W, I280M, and V290F.

473. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N201E, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

474. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.

475. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N205D, N233G, A241G, V270T, A275W, I280M, and V290F.

476. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N205D, R231W, E246G, A261K, V270T, A275W, I280M, and V290F.

477. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N205D, R231W, E246K, V270T, A275W, I280M, and V290F.

478. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, N205D, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

479. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175N, R200T, R231W, E246K, A261K, V270T, A275W, I280M, and V290F.

480. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, N102G, L120T, L175S, N205D, N233G, E246G, V270T, A275W, I280M, and V290F.

481. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175A, N205D, V230H, E246K, Y269W, A275W, A282G, and D288T.

482. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175A, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F.

483. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, V183W, N205D, N233G, E246K, Y269W, S274D, A275S, A282G, and D288T.

484. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, V183W, N205D, N233G, E246K, Y269W, A275W, A282G, and V290F.

485. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, V230H, E246K, A261K, Y269W, S274G, A275S, A282G, and D288T.

486. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, V230Q, E246G, A261K, Y269W, S274G, A275S, A282G, and D288T.

487. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, N233G, E246K, A261K, Y269W, S274G, A275S, A282G, and V290F.

488. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, N233G, A241G, Y269W, S274G, A275S, A282G, and V290F.

489. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, N233G, A241G, Y269W, A275W, A282G, and D288T.

490. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, R231W, E246G, Y269W, S274G, A275S, A282G, and V290F.

491. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 14 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, R231W, E246K, Y269W, S274G, A275S, A282G, and D288K.

492. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, R231W, E246K, A261K, Y269W, S274D, A275S, A282G, and V290F.

493. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 15 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175N, N205D, R231W, E246K, A261K, Y269W, S274D, A275S, A282G, and D288K.

494. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35K, I54T, T57L, N59D, L120T, L175S, N205D, N233G, E246G, Y269W, A275W, A282G, and D288T.

495. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35S, T55K, N102G, L120T, L175N, N205D, N233G, E246G, A261Q, V270T, A275W, I280M, and V290F.

496. The polypeptide variant according to any one of claims 426 to 431 and 435 to 436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35S, T55K, N102G, L120T, L175N, N205D, N233G, A241G, A261Q, V270T, A275W, I280M, and V290F.

497. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35S, T55K, N102G, L120T, L175N, N205D, R231W, E246K, A261Q, V270T, A275W, I280M, and V290F.

498. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35S, I54T, T57L, N59D, L120T, L175N, N205D, V230Q, A241G, Y269W, A275W, A282G, and V290F.

499. The polypeptide variant according to any one of claims 426-431 and 435-436, wherein the second set of amino acid substitutions of Sequence ID No. 295 is a set of 13 amino acid substitutions consisting of I35S, I54T, T57L, N59D, L120T, L175N, N205D, R231W, E246K, Y269W, A275W, A282G, and V290F.

500. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1118.

501. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1120.

502. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1122.

503. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1123.

504. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1125.

505. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1126.

506. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1127.

507. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1129.

508. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1282.

509. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1962.

510. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1963.

511. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1964.

512. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1965.

513. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1970.

514. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1971.

515. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 1983.

516. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2004.

517. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2024.

518. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2028.

519. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2371.

520. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2372.

521. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2373.

522. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2390.

523. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2421.

524. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2427.

525. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2428.

526. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2429.

527. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2430.

528. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2431.

529. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2432.

530. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2433.

531. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2434.

532. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2437.

533. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2439.

534. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2446.

535. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2450.

536. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2463.

537. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2464.

538. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2476.

539. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2478.

540. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2485.

541. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2486.

542. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2487.

543. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2489.

544. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2491.

545. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2493.

546. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2494.

547. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2511.

548. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2524.

549. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2526.

550. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2528.

551. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2574.

552. A polypeptide variant according to claim 426, comprising the amino acid sequence of SEQ ID NO: 2575.

553. The polypeptide variant according to any one of claims 342 to 552, wherein the polypeptide variant has at least about 25% of the activity of the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2 in an IgG protease enzyme assay.

554. The polypeptide variant according to any one of claims 342 to 552, wherein the polypeptide variant has at least about 30% of the activity of the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2 in an IgG protease enzyme assay.

555. The polypeptide variant according to any one of claims 342 to 552, wherein the polypeptide variant has at least about 40% activity, at least about 50% activity, at least about 60% activity, at least about 70% activity, at least about 75% activity, at least about 80% activity, at least about 90% activity, at least about 100% activity, at least about 110% activity, and at least about 120% activity of the IdeS enzyme containing the amino acid sequence of SEQ ID NO: 2 in an IgG protease enzyme assay.

556. The polypeptide variant according to any one of claims 342 to 555, wherein the polypeptide variant has at least about 25% activity, at least about 40% activity, at least about 50% activity, at least about 60% activity, at least about 70% activity, at least about 80% activity, at least about 90% activity, at least about 100% activity, at least about 110% activity, and at least about 120% activity of the IdeZ enzyme containing the amino acid sequence of SEQ ID NO: 830 in an IgG protease enzyme assay.

557. The polypeptide variant according to any one of claims 553 to 556, wherein the IgG protease activity in the IgG protease enzyme assay is measured by an enzyme-linked immunosorbent assay including digestion of an immobilized antibody substrate.

558. The polypeptide variant according to any one of claims 342 to 557, wherein the polypeptide variant has one or more T cell epitopes depleted compared to the polypeptide with the amino acid sequence of SEQ ID NO:

295.

559. The polypeptide variant according to any one of claims 342 to 558, wherein the polypeptide variant has substantially the same thermal stability as the IdeS enzyme comprising the amino acid sequence described in SEQ ID NO:

2.

560. The polypeptide variant according to any one of claims 342 to 559, wherein the polypeptide variant has substantially the same thermal stability as the IdeZ enzyme comprising the amino acid sequence described in SEQ ID NO:

830.

561. The polypeptide variant according to any one of claims 342 to 558, wherein the polypeptide variant has higher thermal stability than the IdeS enzyme containing the amino acid sequence described in SEQ ID NO:

2.

562. The polypeptide variant according to any one of claims 342 to 559 and 561, wherein the polypeptide variant has higher thermal stability than the IdeZ enzyme containing the amino acid sequence described in SEQ ID NO:

830.

563. A polypeptide variant according to any one of claims 559 to 562, wherein the thermal stability is measured by differential scanning fluorescence quantitative analysis.

564. A polypeptide variant of the amino acid sequence of SEQ ID NO: 298 [N30], wherein the polypeptide variant includes one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: I10, R11, Y12, V15, Y18, V46, Y52, F101, N102, Q105, L120, F125, E126, Y127, Y156, R157, S159, V230, R231, N233, A261, V270, A275, and F269, wherein the polypeptide variant includes an amino acid sequence that is at least about 75% identical to SEQ ID NO: 298, and wherein the polypeptide variant has at least about 25% of the activity of the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2 in an IgG protease enzyme assay.

565. A polypeptide variant according to claim 564, comprising an amino acid sequence that is at least about 80% identical to SEQ ID NO:

298.

566. A polypeptide variant according to claim 564, comprising an amino acid sequence that is at least about 85% identical to SEQ ID NO:

298.

567. A polypeptide variant according to claim 564, comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO:

298.

568. A polypeptide variant according to claim 564, comprising an amino acid sequence that is at least about 95% identical to SEQ ID NO:

298.

569. The polypeptide variant according to any one of claims 564 to 568, wherein the one or more amino acid mutations are one or more amino acid substitutions.

570. A polypeptide variant according to claim 564, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 658 to 703.

571. A polypeptide variant according to claim 564, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 658 to SEQ ID NOs. 703, wherein the polypeptide variant comprises an amino acid mutation present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO.

298.

572. A polypeptide variant of the amino acid sequence of SEQ ID NO: 294 [N31], wherein the polypeptide variant includes one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: I10, R11, Y12, E14, V15, Y18, V46, N48, Y52, D130, A132, Y157, N159, Y161, K162, V232, R233, N235, D260, A263, V272, S276, A277, the polypeptide variant includes an amino acid sequence that is at least about 75% identical to SEQ ID NO: 294, and the polypeptide variant has at least about 25% of the activity of the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2 in an IgG protease enzyme assay.

573. A polypeptide variant according to claim 572, comprising an amino acid sequence that is at least about 80% identical to SEQ ID NO:

294.

574. A polypeptide variant according to claim 572, comprising an amino acid sequence that is at least about 85% identical to SEQ ID NO:

294.

575. A polypeptide variant according to claim 572, comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO:

294.

576. A polypeptide variant according to claim 572, comprising an amino acid sequence that is at least about 95% identical to SEQ ID NO:

294.

577. The polypeptide variant according to any one of claims 572 to 576, wherein the one or more amino acid mutations are one or more amino acid substitutions.

578. A polypeptide variant according to claim 572, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 704 to 750.

579. A polypeptide variant according to claim 572, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 704 to SEQ ID NOs. 750, wherein the polypeptide variant comprises an amino acid mutation present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO.

294.

580. A polypeptide variant of the amino acid sequence of SEQ ID NO: 54, wherein the polypeptide variant includes one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: I10, I11, Y12, E14, Y18, H19, V143, Y157, Y161, and K162 of SEQ ID NO: 54, wherein the polypeptide variant includes an amino acid sequence that is at least about 75% identical to SEQ ID NO: 54, and wherein the polypeptide variant has at least about 25% of the activity of the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2 in an IgG protease enzyme assay.

581. A polypeptide variant according to claim 580, comprising an amino acid sequence that is at least about 80% identical to SEQ ID NO:

54.

582. A polypeptide variant according to claim 580, comprising an amino acid sequence that is at least about 85% identical to SEQ ID NO:

54.

583. A polypeptide variant according to claim 580, comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO:

54.

584. A polypeptide variant according to claim 580, comprising an amino acid sequence that is at least about 95% identical to SEQ ID NO:

54.

585. The polypeptide variant according to any one of claims 580 to 584, wherein the one or more amino acid mutations are one or more amino acid substitutions.

586. A polypeptide variant according to claim 580, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 751 to 784.

587. A polypeptide variant according to claim 580, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 751 to SEQ ID NOs. 784, wherein the polypeptide variant comprises an amino acid mutation present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO.

54.

588. A polypeptide variant of the amino acid sequence of SEQ ID NO: 140, wherein the polypeptide variant includes one or more amino acid mutations at one or more amino acid positions of SEQ ID NO: M10, R11, Y12, V15, Y42, V46, A47, N48, Q49, N102, V104, I113, Y118, L120, D121, K123, Y127, Y156, R157, Y201, Q202, V230, F269, S274, A275, D288, and G296, wherein the polypeptide variant includes an amino acid sequence that is at least about 75% identical to SEQ ID NO: 140, and wherein the polypeptide variant has at least about 25% of the activity of the IdeS enzyme containing the amino acid sequence described in SEQ ID NO: 2 in an IgG protease enzyme assay.

589. A polypeptide variant according to claim 588, comprising an amino acid sequence that is at least about 80% identical to SEQ ID NO:

140.

590. A polypeptide variant according to claim 588, comprising an amino acid sequence that is at least about 85% identical to SEQ ID NO:

140.

591. A polypeptide variant according to claim 588, comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO:

140.

592. A polypeptide variant according to claim 588, comprising an amino acid sequence that is at least about 95% identical to SEQ ID NO:

140.

593. The polypeptide variant according to any one of claims 588 to 592, wherein the one or more amino acid mutations are one or more amino acid substitutions.

594. A polypeptide variant according to claim 588, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 785 to 829.

595. A polypeptide variant according to claim 588, comprising an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 785 to SEQ ID NOs. 829, wherein the polypeptide variant comprises an amino acid mutation present in the reference amino acid sequence with respect to the amino acid sequence of SEQ ID NO.

140.

596. The polypeptide or polypeptide variant according to any one of claims 1 to 595, wherein the polypeptide or polypeptide variant is isolated.

597. A polypeptide or polypeptide variant according to any one of claims 1 to 596, further comprising a purified tag at the C-terminus.

598. A polypeptide or polypeptide variant according to any one of claims 1 to 597, further comprising a purified tag at the N-terminus.

599. The polypeptide or polypeptide variant according to claim 597 or 598, wherein the purified tag is a polyhistidine tag.

600. The polypeptide or polypeptide variant according to any one of claims 1 to 599, wherein the polypeptide or polypeptide variant is not pegylated.

601. The polypeptide or polypeptide variant according to any one of claims 1 to 599, wherein the polypeptide or polypeptide variant is pegylated.

602. A fusion protein comprising a first domain and a second domain, wherein the first domain comprises a polypeptide or polypeptide variant as described in any one of claims 1 to 601, and the second domain comprises a first random coil polypeptide domain comprising at least about 100 amino acids.

603. The fusion protein according to claim 602, wherein the first domain is the C-terminus of the second domain.

604. The fusion protein according to claim 602, wherein the first domain is the N-terminus of the second domain.

605. The fusion protein according to any one of claims 602 to 604, wherein an amino acid linker is present between the first domain and the second domain.

606. The fusion protein according to claim 605, wherein the amino acid linker is approximately 2 amino acids long to approximately 5 amino acids long.

607. The fusion protein according to claim 606, wherein the amino acid linker is 2 amino acid long.

608. The fusion protein according to claim 607, wherein the amino acid linker is Gly-Ser.

609. The fusion protein according to any one of claims 602 to 608, further comprising a third domain, the third domain comprising a second random coil polypeptide domain comprising at least about 100 amino acids.

610. The fusion protein according to claim 609, wherein the first domain is the N-terminus of the second domain and the C-terminus of the third domain.

611. The fusion protein according to claim 610, wherein an amino acid linker is present between the first domain and the second domain.

612. The fusion protein according to claim 610 or 611, wherein an amino acid linker is present between the first domain and the third domain.

613. The fusion protein according to claim 611 or 612, wherein the amino acid linker is approximately 2 amino acids long to approximately 5 amino acids long.

614. The fusion protein according to claim 613, wherein the amino acid linker is 2 amino acid long.

615. The fusion protein according to claim 614, wherein the amino acid linker is Gly-Ser.

616. The fusion protein according to any one of claims 602 to 615, wherein the second domain comprises a Pro-Ala-Ser (PAS) polypeptide.

617. The fusion protein according to any one of claims 609 to 616, wherein the third domain comprises a Pro-Ala-Ser (PAS) polypeptide.

618. The fusion protein according to claim 616 or 617, wherein the PAS polypeptide has the amino acid sequence described in SEQ ID NO:

831.

619. The fusion protein according to claim 616 or 617, wherein the PAS polypeptide has the amino acid sequence described in SEQ ID NO:

832.

620. The fusion protein according to claim 616 or 617, wherein the PAS polypeptide has the amino acid sequence described in SEQ ID NO:

833.

621. The fusion protein according to claim 616 or 617, wherein the PAS polypeptide comprises the amino acid sequence described in SEQ ID NO: 835, SEQ ID NO: 837, SEQ ID NO: 839, SEQ ID NO: 841, SEQ ID NO: 843, or SEQ ID NO:

845.

622. The fusion protein according to any one of claims 602 to 621, wherein the second domain comprises an elongated recombinant (XTEN) polypeptide.

623. The fusion protein according to any one of claims 609 to 622, wherein the third domain comprises an elongated recombinant (XTEN) polypeptide.

624. The fusion protein according to claim 622 or 623, wherein the XTEN polypeptide has the amino acid sequence described in SEQ ID NO:

834.

625. The fusion protein according to any one of claims 602 to 624, wherein the second domain comprises a Pro-Ala (PA) polypeptide.

626. The fusion protein according to any one of claims 609 to 625, wherein the third domain comprises a Pro-Ala (PA) polypeptide.

627. The fusion protein according to any one of claims 602 to 626, wherein the first random coil polypeptide domain comprises about 100 amino acids to about 800 amino acids.

628. The fusion protein according to any one of claims 609 to 627, wherein the second random coil polypeptide domain comprises about 100 amino acids to about 800 amino acids.

629. The fusion protein according to any one of claims 602 to 626, wherein the first random coil polypeptide domain comprises about 100 amino acids to about 700 amino acids.

630. The fusion protein according to any one of claims 609 to 629, wherein the second random coil polypeptide domain comprises about 100 amino acids to about 700 amino acids.

631. The fusion protein according to any one of claims 602 to 626, wherein the first random coil polypeptide domain comprises about 100 amino acids to about 600 amino acids.

632. The fusion protein according to any one of claims 609 to 631, wherein the second random coil polypeptide domain comprises about 100 amino acids to about 600 amino acids.

633. The fusion protein according to any one of claims 602 to 626, wherein the first random coil polypeptide domain comprises about 100 amino acids to about 500 amino acids.

634. The fusion protein according to any one of claims 609 to 633, wherein the second random coil polypeptide domain comprises about 100 amino acids to about 500 amino acids.

635. The fusion protein according to any one of claims 602 to 626, wherein the first random coil polypeptide domain comprises about 100 to about 400 amino acids.

636. The fusion protein according to any one of claims 609 to 635, wherein the second random coil polypeptide domain comprises about 100 amino acids to about 400 amino acids.

637. The fusion protein according to any one of claims 602 to 626, wherein the first random coil polypeptide domain comprises about 100 to about 300 amino acids.

638. The fusion protein according to any one of claims 609 to 637, wherein the second random coil polypeptide domain comprises about 100 to about 300 amino acids.

639. A fusion protein comprising a first domain and a second domain, wherein the first domain comprises a polypeptide or polypeptide variant as described in any one of claims 1 to 601, and the second domain comprises a human immunoglobulin class G (IgG) immunoglobulin fragment crystallizable (Fc) domain.

640. The fusion protein according to claim 639, wherein the first domain is the C-terminus of the second domain.

641. The fusion protein according to claim 639, wherein the first domain is the N-terminus of the second domain.

642. The fusion protein according to any one of claims 639 to 641, wherein the Fc domain comprises an Fc polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2591 to 2606.

643. The fusion protein according to any one of claims 639 to 641, wherein the Fc domain comprises an Fc polypeptide having an amino acid sequence that is at least about 50%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a reference amino acid sequence selected from the group consisting of SEQ ID NOs. 2591 to SEQ ID NOs. 2606.

644. The fusion protein according to any one of claims 639 to 641, wherein human IgG is human IgG1.

645. The fusion protein according to any one of claims 639 to 641, wherein the human IgG is human IgG2.

646. The fusion protein according to any one of claims 639 to 641, wherein the human IgG is human IgG3.

647. The fusion protein according to any one of claims 639 to 641, wherein the human IgG is human IgG4.

648. The fusion protein according to any one of claims 639 to 647, wherein the polypeptide or polypeptide variant is a monomer.

649. The fusion protein according to any one of claims 639 to 647, wherein the polypeptide or polypeptide variant is a monomer and exists as a homodimer.

650. The fusion protein according to any one of claims 639 to 647, wherein the polypeptide or polypeptide variant is a monomer and exists as a heterodimer.

651. A fusion protein comprising a first domain and a second domain, wherein the first domain comprises a polypeptide or polypeptide variant as described in any one of claims 1 to 601, and the second domain comprises an albumin protein.

652. The fusion protein according to claim 651, wherein the albumin protein is human albumin.

653. The fusion protein according to claim 651 or 652, wherein the first domain is the C-terminus of the second domain.

654. The fusion protein according to claim 651 or 652, wherein the first domain is the N-terminus of the second domain.

655. The fusion protein according to any one of claims 651 to 654, wherein an amino acid linker is present between the first domain and the second domain.

656. The fusion protein according to claim 655, wherein the amino acid linker is approximately 2 amino acids long to approximately 5 amino acids long.

657. The fusion protein according to claim 656, wherein the amino acid linker is 2 amino acid long.

658. The fusion protein according to claim 657, wherein the amino acid linker is Gly-Ser.

659. A composition comprising a polypeptide or polypeptide variant according to any one of claims 1 to 601, and a pharmaceutically acceptable excipient.

660. A composition comprising a fusion protein according to any one of claims 602 to 658, and a pharmaceutically acceptable excipient.

661. An isolated nucleic acid encoding a polypeptide or polypeptide variant according to any one of claims 1 to 601, or a fusion protein according to any one of claims 602 to 658.

662. A nucleic acid vector comprising the nucleic acid described in claim 661.

663. A host cell comprising the nucleic acid vector according to claim 662.

664. A method for treating a disease that is entirely or partially mediated by a pathogenic immunoglobulin G (IgG) antibody in a patient requiring treatment, comprising administering to the patient the composition according to claim 659 or 660.

665. The diseases that are entirely or partially mediated by pathogenic immunoglobulin G (IgG) antibodies include Addison's disease, anti-glomerular basement membrane (anti-GBM) glomerulonephritis, Goodpasture syndrome, anti-neutrophil cytoplasmic antibody-associated vasculitis (ANCA-associated vasculitis), Churg-Strauss syndrome, microscopic polyangiitis, anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis, antiphospholipid syndrome (APS) (e.g., catastrophic APS), and self- Autoimmune bullous skin diseases, pemphigus (e.g., pemphigus foliaceus, endemic pemphigus, pemphigus vulgaris), autoimmune hemolytic anemia (AIHA), autoimmune hepatitis (AIH), autoimmune neutropenia (AIN), bullous pemphigoid (BP), celiac disease, chronic urticaria, complete congenital heart block (CCHB), type 1A diabetes, epidermolysis bullosa, essential mixed cryoglobulinemia, Graves' disease, Guillain-Barré syndrome (GBS), chronic inflammation Symptomatic demyelinating polyneuropathy (CIDP), acute inflammatory demyelinating polyneuropathy (AIDP), acute motor axonal neuropathy (AMAN), acquired factor VIII deficiency, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), Lambert-Eaton myasthenic syndrome (LEMS), mixed connective tissue disease (MCTD), multiple myeloma, myasthenia gravis, myasthenic crisis, myocarditis, dilated cardiomyopathy (DCM), stasis The method according to claim 664, wherein the condition is hemorrhagic cardiomyopathy, neuromyelitis optica (NMD), primary biliary cirrhosis (PBC), primary progressive multiple sclerosis (PPMS), systemic lupus erythematosus (SLE) (e.g., lupus nephritis), stiff person syndrome (SPS), Sjögren's syndrome (SS), systemic sclerosis (scleroderma), rheumatoid arthritis (RA), rheumatic heart disease (RHD), serum sickness syndrome, or type III immune complex hypersensitivity.

666. The method according to claim 665, wherein the disease, which is entirely or partially mediated by a pathogenic immunoglobulin G (IgG) antibody, is chronic inflammatory demyelinating polyneuropathy (CIDP).

667. The method according to claim 665, wherein the disease, which is entirely or partially mediated by a pathogenic immunoglobulin G (IgG) antibody, is acute inflammatory demyelinating polyneuropathy (AIDP).

668. A method for preventing or treating antibody-mediated rejection (AMR) of an organ allograft in an organ transplant patient requiring treatment, comprising administering the composition according to claim 659 or 660 to the organ transplant patient.

669. The method according to claim 668, wherein the administration is performed before transplantation.

670. The method according to claim 669, wherein the administration is performed approximately three weeks to approximately one hour before transplantation.

671. The method according to claim 668, wherein the administration is performed during organ transplantation.

672. The method according to claim 668, wherein the administration is performed after organ transplantation.

673. The method according to any one of claims 668 to 672, wherein the organ is a solid organ.

674. The method according to claim 673, wherein the solid organ is a kidney, small intestine, pancreas, heart, lung, or liver.

675. The method according to claim 674, wherein the solid organ is a kidney.

676. The method according to any one of claims 664 to 675, wherein administration includes parenteral administration.

677. The method according to claim 676, wherein the parenteral administration is intravenous administration.

678. The method according to claim 677, wherein the intravenous administration is intravenous infusion.

679. The method according to any one of claims 664 to 675, wherein the administration includes intradermal, subcutaneous, transdermal, intramuscular, intra-arterial, intraperitoneal, intra-articular, or intraosseous administration.

680. A method for recombinantly producing a polypeptide or polypeptide variant according to any one of claims 1 to 601, (i) Culturing a host cell comprising a nucleic acid vector comprising a nucleic acid sequence encoding a polypeptide or polypeptide variant according to any one of claims 1 to 601, wherein the nucleic acid sequence is operably linked to a heterologous promoter under conditions that enable expression of the nucleic acid sequence encoding the polypeptide or polypeptide variant and recombinant production of the polypeptide or polypeptide variant by the host cell, (ii) A method comprising isolating the polypeptide or polypeptide variant produced by recombinant.

681. A method for producing a fusion protein according to any one of claims 602 to 658 by recombination, (i) culturing a host cell comprising a nucleic acid vector comprising a nucleic acid sequence encoding a fusion protein according to any one of claims 602 to 658, wherein the nucleic acid sequence is operably linked to a heterologous promoter under conditions that enable expression of the nucleic acid sequence encoding the fusion protein and recombinant production of the fusion protein by the host cell, (ii) A method comprising isolating the fusion protein produced by recombination.

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