Improved IGG-degrading enzymes and methods of use thereof

Abstract

Improved cysteine ​​proteases are provided for specifically cleaving and inactivating immunoglobulin G. The improved cysteine ​​proteases are useful in methods of treating diseases, disorders or conditions characterized by excessive levels of IgG antibodies, including autoimmune disorders, and candidates for organ transplantation sensitized by anti-HLA antibodies, as well as for treating candidates for gene therapy with pre-existing neutralizing antibodies to recombinant vectors and for re-administration of subjects previously treated with gene therapy vectors.

Description

[Technical field]

[0001] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on February 16, 2023, is named PC072839_SequenceListing_ST26.xml, and is 100,559 bytes in size. [Background technology]

[0002] Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) is a natural cysteine ​​protease expressed by the pathogenic bacterium Streptococcus pyogenes that exhibits specificity for its target sequence found in human IgG in addition to several other species. IdeS can cleave IgG below the hinge region, thereby generating F(ab')2 and Fc / 2 fragments. IdeS can cleave IgG in human plasma and can reduce total IgG levels in humans soon after its administration.

[0003] Certain disorders and diseases in humans are mediated by IgG antibodies, particularly autoimmune disorders such as type 1 diabetes and multiple sclerosis, which cause immense suffering. Furthermore, the presence of IgG antibodies can impede the successful administration of established life-saving therapies, such as organ transplants and relatively recently developed procedures such as gene therapy using recombinant viral vectors. Various methods have been attempted to mitigate the deleterious effects of such pathogenic IgG antibodies in patients with autoimmune disorders and candidates for transplantation and gene therapy, but with limited effectiveness.

[0004] Thus, there is a need in the art for compositions and methods capable of degrading, digesting and inactivating pathogenic IgG antibodies in subjects, particularly human subjects with autoimmune disorders or who are candidates for transplantation or gene therapy, and in relation to that need, there is also a need for IdeS protein variants with improved potency and / or stability. Summary of the Invention [Means for solving the problem]

[0005] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein, which equivalents are intended to be encompassed by embodiment (E) below. E1. An isolated cysteine ​​protease that specifically cleaves immunoglobulin G (IgG) antibody molecules. E2. A cysteine ​​protease as described in E1, which has higher potency or thermostability compared to wild-type IdeS. E3.T onset 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0° C. higher compared to the wild-type IdeS protein, as determined using differential scanning calorimetry. E4.T onsetvalues, as determined using differential scanning calorimetry, of at least or about 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45.0, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46.0, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47. 9, 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48.0, 48.1, 48.2, 48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, 49.0, 49.1, 49.2, 49.3, 49.4, 49.5, 49.6, 49.7, 49.8, 49.9 or 50.0°C. E5.T M 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0° C. higher compared to the wild-type IdeS protein, as determined using differential scanning calorimetry. E6.T Mvalues ​​are at least or about 51.0, 51.1, 51.2, 51.3, 51.4, 51.5, 51.6, 51.7, 51.8, 51.9, 52.0, 52.1, 52.2, 52.3, 52.4, 52.5, 52.6, 52.7, 52.8, 52.9, 53.0, 53.1, 53.2, 53.3, 53.4, 53.5, 53.6, 53.7, 53.8, 53.9, 54.0, 54.1, 54.2, 54.3, 54.4, 54.5, 54.6, 54.7, 54.8, 54.9, 55.0, 55.1, 55.2, 55.3, 55.4, 55.5, 55.6, 55.7, 55.8, 55.9, 56.0, 56.1, 56.2, 56.3, 56.4, 56.5, 56.6, 56.7, 56.8, 56.9, 57.0, 57.1, 57.2, 57.3, 57.4, 57.5, 57.6, 57.7, 57.8, 57.9, 58.0, 58.1, 58.2, 58.3, 58.4, 58.5, 58.6, 58.7, 58.8, 58.9, 59.0, 59.1, 59.2, 59.3, 55.9, 54.0, 54.1, 54.2, 54.3, 54.4, 54.5, 54.6, 54.7, 54.8, 54.9, 55.0, 55.1, 55.2, 55.3, 55.4, 55.5, 55.6, 55.7, 55.8, 55.9, 56.0, 56.1, 56.2, 56.3, 56.4, 56.5, 56.6, 56.7, 56.8, 56.9 or 57.0°C. A cysteine ​​protease as described in E2, wherein the cleavage potency of E7.IgG, as determined using ELISA and expressed as an IC50 value, is at least or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5 nM lower compared to wild type IdeS. A cysteine ​​protease as described in E2, having an IgG cleavage efficacy, determined using ELISA and expressed as an IC50 value, of at most 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8 or 3.7 nM. E8. The cysteine ​​protease according to E2, the amino acid sequence of which comprises, consists essentially of, or consists of amino acid numbers 1 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 2 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 3 to 312 of any one of SEQ ID NOs: 3 to 71. E9. The cysteine ​​protease of E9, wherein the amino acid sequence comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 72-82. E10. A pharmaceutical composition comprising the cysteine ​​protease of any one of embodiments E1-E10 and a pharma- ceutically acceptable carrier. E11. A method of treating a subject in need of treatment or prevention of a disease or disorder characterized by excess IgG antibodies, comprising administering to the subject an amount of a cysteine ​​protease described in E10 effective to reduce the concentration of IgG antibodies in a bodily fluid of the subject. E12. The method of E12, wherein said bodily fluid is blood, plasma or serum. E13. The method of E12, wherein the cysteine ​​protease acts by degrading, digesting, or inactivating the IgG antibody. E14. The method of E12, wherein said disease or disorder is an autoimmune disease or disorder, and administering said cysteine ​​protease is effective for treating or preventing said autoimmune disease or disorder. E15. The method of E15, wherein the effective amount of said cysteine ​​protease is at a dose of at least or about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45 or 0.50 mg / kg of subject body weight. E16. The method of E15, wherein said treatment is effective to reduce the concentration of total IgG in a bodily fluid of said subject by at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. E17. The method of E15, wherein said treatment is effective to reduce the concentration of total IgG in a bodily fluid of said subject by up to about 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05 grams / liter. E18. A method of treating a sensitized subject in need of a tissue or organ transplant, comprising administering to said subject an amount of a cysteine ​​protease described in E10 effective to reduce the concentration of anti-HLA antibodies in the subject's body fluids sufficiently to prevent antibody-mediated rejection of said tissue or organ following transplantation. E19. The method of E19, wherein said bodily fluid is blood, plasma or serum. E20. The method of E19, wherein said cysteine ​​protease acts by degrading, digesting or inactivating said anti-HLA antibody. E21. The method according to E19, wherein said organ is a kidney, a liver, a heart, a pancreas, a lung or an intestine. E22. The method of E19, wherein said subject exhibits a calculated panel reactive antibody assay score of at least or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60%. E23. The method of E19, wherein the effective amount of said cysteine ​​protease is at a dose of at least or about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45 or 0.50 mg / kg of subject body weight. E24. The method of E19, wherein said treatment is effective to reduce the concentration of anti-HLA antibodies in the subject's bodily fluids by at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. E25. The method of E19, wherein said treatment is effective to reduce the concentration of total IgG in a bodily fluid of said subject to at most about 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05 grams / liter. E26. The method of E19, wherein said treatment is effective to reduce the calculated panel reactive antibody assay score of said subject's serum by at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. E27. The method of E19, wherein said treatment is effective to reduce the calculated panel reactive antibody assay score of said subject's serum by up to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. E28. The method of E19, wherein the subject subsequently receives a tissue or organ transplant, and the period of time between administration of the cysteine ​​protease and the subsequent transplant is at least or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 hours. E29. A method of treating a subject in need of therapy with a gene therapy vector, comprising administering to the subject an amount of a cysteine ​​protease described in E10 effective to reduce the concentration of IgG antibodies in a body fluid of the subject specific for a component of the gene therapy vector. E30. The method of E30, wherein said bodily fluid is blood, plasma or serum. E31. The method of E30, wherein said cysteine ​​protease acts by degrading, digesting, or inactivating said IgG antibody. E32. The method of E30, wherein said IgG antibody is a neutralizing antibody. E33. The method of E30, wherein said gene therapy vector is a recombinant viral vector. E34. The method of E32, wherein said recombinant viral vector is a recombinant adenoviral vector, a recombinant adeno-associated viral vector, or a recombinant lentiviral vector. E35. The titer of neutralizing antibodies is at least or about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:120, 1:125, 1:150, 1:175, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:65 0, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1700, 1:1800, 1:1900, 1:2000, 1:2100, 1:2200, 1:2300, 1:2400, 1:2500, 1:2600, 1:2700, 1:2800, 1:2900 or 1:3000. E36. The method of E33, wherein the effective amount of said cysteine ​​protease is at a dose of at least or about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45 or 0.50 mg / kg of subject body weight. E37. The method of E33, wherein said treatment is effective to reduce the titer of neutralizing antibodies in a bodily fluid of said subject by at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. E38. The method of E33, wherein said treatment is effective to reduce the reduce titer neutralizing antibodies in a body fluid of the subject to a value of up to 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2 or 1:0.1. E39. The method of E33, wherein said subject is gene therapy treatment naive. E40. The method of E40, wherein said subject subsequently receives treatment with said gene therapy vector. E41. The method of E41, wherein said gene therapy vector is a recombinant viral vector. E42. The method of E42, wherein said recombinant viral vector is a recombinant adenoviral vector, a recombinant adeno-associated viral vector, or a recombinant lentiviral vector. E43. The period between administration of the cysteine ​​protease and subsequent gene therapy is at least or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 10 8, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 52, 54, 55, 56, 58, 60, 62, 64, 65, 66, 68, 70, 72, 74, 75, 76, 78, 80, 84, 85, 90, 95, 96, 100, 108, 110, 120, 125, 130, 132, 135, 140, 144, 145, 150, 155, 156, 160, 165 or 168 hours or 8, 9, 10, 11, 12, 13 or 14 days. E44. The titer of neutralizing antibodies is at least or about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:120, 1:125, 1:150, 1:175, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:6 50, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1700, 1:1800, 1:1900, 1:2000, 1:2100, 1:2200, 1:2300, 1:2400, 1:2500, 1:2600, 1:2700, 1:2800, 1:2900 or 1:3000. E45. The method of E41, wherein the effective amount of said cysteine ​​protease is at a dose of at least or about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45 or 0.50 mg / kg of subject body weight. E46. The method of E41, wherein said treatment is effective to reduce the titer of neutralizing antibodies in the subject's bodily fluid by at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. E47. The method of E41, wherein said treatment is effective to reduce the reduce titer neutralizing antibodies in a body fluid of said subject to a value of up to 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2 or 1:0.1. E48. The method of E33, wherein said subject has been previously treated at least once with the same type of gene therapy vector for which said subject requires treatment. E49. The method of E49, wherein said subject is then treated with said gene therapy vector. E50. The method of E50, wherein said gene therapy vector is a recombinant viral vector. E51. The method of E51, wherein said recombinant viral vector is a recombinant adenoviral vector, a recombinant adeno-associated viral vector, or a recombinant lentiviral vector. E52. The period between administration of the cysteine ​​protease and subsequent gene therapy is at least or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 10 8, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 52, 54, 55, 56, 58, 60, 62, 64, 65, 66, 68, 70, 72, 74, 75, 76, 78, 80, 84, 85, 90, 95, 96, 100, 108, 110, 120, 125, 130, 132, 135, 140, 144, 145, 150, 155, 156, 160, 165 or 168 hours or 8, 9, 10, 11, 12, 13 or 14 days. E53. The titer of neutralizing antibodies is at least or about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:120, 1:125, 1:150, 1:175, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:6 1:50, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1700, 1:1800, 1:1900, 1:2000, 1:2100, 1:2200, 1:2300, 1:2400, 1:2500, 1:2600, 1:2700, 1:2800, 1:2900 or 1:3000. E54. The method of E50, wherein the effective amount of said cysteine ​​protease is at a dose of at least or about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45 or 0.50 mg / kg of subject body weight. E55. The method of E50, wherein said treatment is effective to reduce the titer of neutralizing antibodies in a bodily fluid of said subject by at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. E56. The method of E50, wherein said treatment is effective to reduce the reduce titer neutralizing antibodies in a body fluid of said subject to a value of up to 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2 or 1:0.1. E57. The method of any one of embodiments E12-E57, wherein the subject is a human subject. E58. The method of any one of embodiments E12-E58, wherein said cysteine ​​protease is administered parenterally. E59. The method of E59, wherein said cysteine ​​protease is administered intravenously or intraarterially. E60. The method of any one of embodiments E12, E19, or E30, wherein said step of administering said cysteine ​​protease to said subject is repeated at least once. E61. A kit comprising a container disposed therein, a pharmaceutical composition comprising a cysteine ​​protease, and a label with instructions for carrying out the method of any one of embodiments E12-E61. E62. A polynucleotide encoding a cysteine ​​protease according to any one of embodiments E1-E10. E63. An expression vector comprising the polynucleotide according to E63. E64. A host cell comprising an expression vector according to E64. E65. The host cell of E65, wherein said host cell is a bacterial host cell. E66. A method for producing a cysteine ​​protease, comprising incubating a host cell according to E66 under conditions sufficient to express said cysteine ​​protease and purifying the cysteine ​​protease produced thereby. [Brief description of the drawings]

[0006] [Figure 1A] 1A-1J show an alignment of the amino acid sequences of wild-type IdeS and IdeS mutant proteins engineered based on the published IdeS crystal structure. Among the mutant sequences, the highlighted residues differ from those in the wild-type sequence. [Figure 1B]1A-1J show an alignment of the amino acid sequences of wild-type IdeS and IdeS mutant proteins engineered based on the published IdeS crystal structure. Among the mutant sequences, the highlighted residues differ from those in the wild-type sequence. [Figure 1C] 1A-1J show an alignment of the amino acid sequences of wild-type IdeS and IdeS mutant proteins engineered based on the published IdeS crystal structure. Among the mutant sequences, the highlighted residues differ from those in the wild-type sequence. [Figure 1D] 1A-1J show an alignment of the amino acid sequences of wild-type IdeS and IdeS mutant proteins engineered based on the published IdeS crystal structure. Among the mutant sequences, the highlighted residues differ from those in the wild-type sequence. [Figure 1E] 1A-1J show an alignment of the amino acid sequences of wild-type IdeS and IdeS mutant proteins engineered based on the published IdeS crystal structure. Among the mutant sequences, the highlighted residues differ from those in the wild-type sequence. [Figure 1F] 1A-1J show an alignment of the amino acid sequences of wild-type IdeS and IdeS mutant proteins engineered based on the published IdeS crystal structure. Among the mutant sequences, the highlighted residues differ from those in the wild-type sequence. [Figure 1G] 1A-1J show an alignment of the amino acid sequences of wild-type IdeS and IdeS mutant proteins engineered based on the published IdeS crystal structure. Among the mutant sequences, the highlighted residues differ from those in the wild-type sequence. [Figure 1H] 1A-1J show an alignment of the amino acid sequences of wild-type IdeS and IdeS mutant proteins engineered based on the published IdeS crystal structure. Among the mutant sequences, the highlighted residues differ from those in the wild-type sequence. [Figure 1I]1A-1J show an alignment of the amino acid sequences of wild-type IdeS and IdeS mutant proteins engineered based on the published IdeS crystal structure. Among the mutant sequences, the highlighted residues differ from those in the wild-type sequence. [Figure 1J] 1A-1J show an alignment of the amino acid sequences of wild-type IdeS and IdeS mutant proteins engineered based on the published IdeS crystal structure. Among the mutant sequences, the highlighted residues differ from those in the wild-type sequence. [Diagram 2] FIG. 2 shows a bar graph summarizing stability data for certain IdeS mutants compared to wild-type IdeS (GBT-NCC-0005). [Figure 3A] FIG. 3A shows a Coomassie stained polyacrylamide gel illustrating protein fragments resulting from digestion of IgG protein with wild-type IdeS and certain IdeS mutants. [Figure 3B] FIG. 3B shows a Coomassie stained polyacrylamide gel illustrating the protein fragments resulting from digestion of IgM protein with wild-type IdeS and certain IdeS mutants. [Figure 4] FIG. 4 shows a schematic diagram of the MSD assay format. [Diagram 5] FIG. 5 shows a graph summarizing the amount of intact IgG (% baseline) in rabbits treated with wild-type IdeS (WT) versus IdeS mutant (Var). [Figure 6] FIG. 6 shows a schematic diagram of the MSD LBA assay format. [Figure 7] FIG. 7 shows a graph summarizing the average PK of IdeS mutants (triangles) and wild-type IdeS (squares). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] General Technology The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1998) Academic Press; Culture(RIFreshney,ed.,1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.,1993-1998)J.Wiley and Sons;Methods in Enzymology(Academic Press, Inc.);Handbook of Experimental Immunology(DMWeir and CCBlackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FMAusubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds.,1991);Short Protocols in Molecular Biology(Wiley and Sons,1999);Immunobiology(CA Janeway and P. Travers,1997);Antibodies(P. Finch,1997);Antibodies: a practical approach(D. Catty.,ed.,IRL Press,1988-1989);Monoclonal antibodies: a practical approach(P. Shepherd and C. Dean,eds.,Oxford University Press,2000);Using antibodies: a laboratory manual(E. Harlow and D. Lane(Cold Spring Harbor Laboratory Press,1999);The Antibodies(M. Zanetti and JD Capra,eds.,Harwood Academic Publishers,1995) and other publications.

[0008] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present specification, including definitions, will control. Unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular. Any examples following the terms "eg" or "for example" are not intended to be exclusive or limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice and testing of the present invention, suitable methods and materials are described herein.

[0009] The following terms shall be understood to have the following meanings unless otherwise indicated: the term "isolated molecule", when referring to a molecule (e.g., when the molecule is a protein, polynucleotide, or antibody), refers to a molecule that, by reason of its origin or source of derivation, (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same source, e.g., species, cell in which it is expressed, library, etc., (3) is expressed by cells from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the system in which it is naturally derived is "isolated" from its naturally associated components. A molecule can also be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art. Molecular purity or homogeneity can be assayed by many means well known in the art. For example, the purity of a polypeptide sample can be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, greater resolution may be provided by using HPLC or other means well known in the art for purification.

[0010] As used herein, "mutant," "mutated protein," or "protein variant" refers to a protein that differs from a parent protein by virtue of at least one amino acid modification. A protein variant may refer to the protein itself, a composition comprising the protein, or an amino acid sequence encoding the same. Preferably, a protein variant has at least one amino acid modification compared to the parent protein, e.g., about 1 to about 10 amino acid modifications compared to the parent, preferably about 1 to about 5 amino acid modifications. A protein variant sequence herein preferably retains at least about 80% homology, most preferably at least about 90% homology, more preferably at least about 95% homology, with the parent protein sequence. A mutant protein may refer to the mutant protein itself, a composition comprising the mutant protein, or an amino acid sequence encoding the same. Thus, by "IdeS variant," as used herein, it is meant a protein that differs from wild-type IdeS by at least one amino acid modification. A variant may include a non-natural amino acid. Examples include U.S. Pat. No. 6,586,207; WO 98 / 48032; WO 03 / 073238; U.S. Patent Application Publication No. 2004-0214988 A1; WO 05 / 35727 A2; WO 05 / 74524 A2; JW Hin et al., (2002), Journal of the American Chemical Society 124:9026-9027; JW Hin, & PG Schultz, (2002), ChemBioChem 11:1135-1137; JW Hin, et al., (2002), PICAS United States of America 99:11020-11024; and L. Wang, & PG Schultz, (2002), Chem. 1-10.

[0011] As used herein, "protein" refers to at least two covalently attached amino acids, including proteins, polypeptides, oligopeptides, and peptides. Peptidyl groups can include natural amino acids and peptide bonds or synthetic peptidomimetic structures, i.e., "analogs," such as peptoids (see Simon et al., PNAS USA 89(20):9367 (1992)). Amino acids can be either natural or unnatural, as will be recognized by those skilled in the art. For example, homophenylalanine, citrulline, and norleucine are considered amino acids for the purposes of the present invention, and both D- and L- (R or S) configuration amino acids can be utilized. Variants of the invention can include modifications including the use of unnatural amino acids incorporated using techniques developed by, for example, Schultz and coworkers, including but not limited to, those described in Cropp & Shultz, 2004, Trends Genet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003, Science 301(5635):964-7. Additionally, polypeptides can include synthetic derivatization of one or more side chains or termini, glycosylation, PEGylation, circular permutation, cyclization, linkers to other molecules, fusion to proteins or protein domains, and addition of peptide tags or labels.

[0012] As used herein, "wild-type" or "WT" refers to an amino acid or nucleotide sequence found in nature, including allelic variations. A wild-type protein has an amino acid or nucleotide sequence that has not been intentionally modified.

[0013] An "individual" or "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals (e.g., cows, pigs, horses, chickens, etc.), sport animals, pets, primates, horses, dogs, cats, mice and rats.

[0014] As used herein, an "effective dosage" or "effective amount" of a drug, compound or pharmaceutical composition is an amount sufficient to achieve any one or more beneficial or desired results. In more specific embodiments, an effective amount prevents, alleviates, or ameliorates symptoms of a disease and / or prolongs the survival of the subject being treated. For prophylactic use, beneficial or desired results include eliminating or reducing the risk, reducing the severity, or delaying the onset of a disease, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that appear during the progression of the disease. For therapeutic use, beneficial or desired results include clinical results such as alleviation of one or more symptoms of the disease, reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug, and / or delaying disease progression in a patient. An effective dosage may be administered in one or more administrations. For purposes of this invention, an effective dosage of a drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment, either directly or indirectly. As understood in a clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective dosage" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if a desired result can or will be achieved in combination with one or more other agents.

[0015] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA and antisense DNA, and spliced ​​or unspliced ​​mRNA, rRNA tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-splicing RNA or antisense RNA).

[0016] A "heterologous" nucleic acid sequence refers to a polynucleotide that is inserted into a plasmid or vector for the purpose of vector-mediated transfer / delivery of the polynucleotide to a cell. A heterologous nucleic acid sequence is distinct from, i.e., non-native with respect to, the viral nucleic acid. Once transferred / delivered to a cell, the heterologous nucleic acid sequence contained within the vector may be expressed (e.g., transcribed and translated, as appropriate). Alternatively, a transferred / delivered heterologous polynucleotide in a cell contained within a vector need not be expressed. Although the term "heterologous" is not necessarily used herein in connection with nucleic acid sequences and polynucleotides, reference to a nucleic acid sequence or polynucleotide is intended to include heterologous nucleic acid sequences and polynucleotides, even without or with the qualifier "heterologous."

[0017] "Transgene" is used herein for convenience to refer to a nucleic acid that is intended to be or has been introduced into a cell or organism. Transgene includes any nucleic acid, such as a heterologous polynucleotide sequence or a heterologous nucleic acid that encodes a protein or peptide. The terms transgene and heterologous nucleic acid / polynucleotide sequence are used interchangeably herein.

[0018] A "host cell" includes an individual cell or cell culture that can be or has been a recipient for a vector for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell, due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide of the invention.

[0019] As used herein, "vector" refers to a construct capable of delivering, and preferably expressing, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

[0020] The term "recombinant" refers to compositions that have been manipulated in a manner that does not generally occur in nature, as modifiers of viral vectors, such as recombinant AAV (rAAV) vectors, and as modifiers of sequences, such as recombinant polynucleotides and polypeptides. A particular example of a recombinant AAV vector is when a nucleic acid that is not normally present in the wild-type AAV genome (heterologous polynucleotide) is inserted into the viral genome. An example is when a nucleic acid (e.g., a gene) encoding a therapeutic protein or polynucleotide sequence is cloned into the vector, with or without the 5', 3', and / or intron regions to which genes are normally linked in the AAV genome. Although the term "recombinant" is not necessarily used herein with respect to sequences, such as AAV vectors and polynucleotides, recombinant forms including AAV vectors, polynucleotides, and the like, are expressly included, regardless of any such abbreviations.

[0021] A "rAAV vector" is derived from the wild-type genome of AAV, for example, by using molecular methods to remove all or part of the wild-type AAV genome and replace it with a non-native (heterologous) nucleic acid, such as a nucleic acid encoding a therapeutic protein or polynucleotide sequence. Typically, for a rAAV vector, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained. rAAV is distinguished from the AAV genome because all or part of the AAV genome is replaced with a sequence that is non-native with respect to the AAV genome nucleic acid, such as a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence. Thus, the incorporation of a non-native (heterologous) sequence defines AAV as a "recombinant" AAV vector, which may be referred to as a "rAAV vector."

[0022] Recombinant AAV vector sequences may be packaged and referred to herein as "particles" for subsequent infection (transduction) of cells ex vivo, in vitro or in vivo. When recombinant vector sequences are encapsidated or enclosed in an AAV particle, the particle may also be referred to as "rAAV", "rAAV particle" and / or "rAAV virion". Such rAAV, rAAV particles and rAAV virions contain proteins that encapsidate or encapsulate the vector genome. In some embodiments, such contained proteins are capsid proteins.

[0023] "Vector genome," which may be abbreviated as "vg," refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form the rAAV particle. When a recombinant plasmid is used to construct or produce a recombinant AAV vector, the AAV vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called the "plasmid backbone," which is important for the cloning and amplification of the plasmid, a process required for propagation and recombinant AAV vector production, but is not itself packaged or encapsidated into the rAAV particle. Thus, "vector genome" refers to the nucleic acid that is packaged or encapsidated by the rAAV.

[0024] The term "PEG" refers to polyethylene glycol, a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weight; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. PEGs are commercially available from Sigma Chemical Co. and others, and include, for example, the following functional PEGs: monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethyleneglycol-amine (MePEG-NH2), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES), and monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM). In some embodiments, the PEG can be a polyethylene glycol having an average molecular weight of about 550 to about 10,000 daltons, and is optionally substituted with alkyl, alkoxy, acyl, or aryl. In some embodiments, the PEG may be substituted with methyl at the terminal hydroxyl position. In some embodiments, the average molecular weight of the PEG may be about 750 to about 5,000 daltons, or about 1,000 to about 5,000 daltons, or about 1,500 to about 3,000 daltons, or about 2,000 daltons, or about 750 daltons. The PEG may be optionally substituted with alkyl, alkoxy, acyl, or aryl. In some embodiments, the terminal hydroxyl group may be substituted with a methoxy or methyl group.

[0025] As used herein, the term "serotype" in reference to an AAV vector refers to a capsid that is serologically distinct from other AAV serotypes. Serological distinctiveness is determined by the lack of cross-reactivity between antibodies to one AAV compared to another AAV. Differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in the VP1, VP2 and / or VP3 sequences of the AAV serotypes). Antibodies to one AAV may cross-react with one or more other AAV serotypes due to homology of capsid protein sequences. Under the traditional definition, a serotype means that the virus of interest has been tested for neutralizing activity against sera specific for all existing and characterized serotypes, and no antibodies have been found to neutralize the virus of interest. As more natural virus isolates are discovered and / or capsid mutants are created, there may or may not be serological differences with any of the currently existing serotypes. Thus, if a new virus (e.g., AAV) does not have serological differences, the new virus (e.g., AAV) is a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity has not yet been performed on mutant viruses with capsid sequence modifications to determine whether they are of a different serotype according to the traditional definition of a serotype. Thus, for convenience and to avoid repetition, the term "serotype" refers broadly to both serologically distinct viruses (e.g., AAV) as well as serologically indistinguishable viruses (e.g., AAV) that may fall within a subgroup or variant of a serotype.

[0026] As used herein, the term "effector function" refers to the normal functional characteristics of an antibody. Non-limiting examples of antibody functional characteristics include, for example, binding to an antigen; activating the complement cascade (called complement-dependent cytotoxicity); binding to Fc receptors on effector cells such as macrophages, monocytes, natural killer cells and eosinophils to participate in antibody-dependent cellular cytotoxicity (ADCC); and as a signal for the uptake of bound antigens / pathogens by immune cells such as phagocytes and dendritic cells. Thus, reduction or inhibition of antibody effector function may refer to any one or more of the aforementioned non-limiting functional characteristics. Effector function assays are known in the art and described, for example, in WO2016012285.

[0027] "Fc receptor" refers to any Fc receptor. Specific non-limiting examples of Fc receptors include Fc gamma immunoglobulin receptors (FcyRs) present on cells. In humans, FcyR refers to one, some, or all of a family of Fc receptors including FcyRI (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). FcyR includes naturally occurring polymorphs of FcyRI (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16a), and FcyRIIIB (CD16b).

[0028] Reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter per se. For example, a statement referring to "about X" includes a statement of "X." Numeric ranges are inclusive of the numbers defining the range.

[0029] Whenever an embodiment is described herein using the word "comprising," it is understood that otherwise similar embodiments are also provided that are described with the terms "consisting of" and / or "consisting essentially of." Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising" are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0030] When aspects or embodiments of the invention are described in terms of Markush group terms or other groupings of alternatives, the invention includes each member of the group individually, not just the entire group recited as a whole, and includes all possible subgroups of the main group, as well as the main group absent one or more of the group members. The invention also envisions the explicit exclusion of any one or more of the group members in the claimed invention.

[0031] All patents, patent applications, publications, and other references, GenBank citations, and ATCC citations cited herein are incorporated by reference in their entirety. In the event of a conflict, the present specification, including definitions, will control.

[0032] All features disclosed herein may be combined in any combination. Each feature disclosed in this application may be replaced by an alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are examples of a genus of equivalent or similar features.

[0033] As used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "nucleic acid" includes a plurality of such nucleic acids, reference to a "vector" includes a plurality of such vectors, and reference to a "virus" or "particle" includes a plurality of such viruses / particles.

[0034] The term "about" as used herein refers to a value within 10% of a fundamental parameter. For example, "about 1:10" means 1.1:10.1 or 0.9:9.9, about 5 hours means 4.5 hours or 5.5 hours, etc. The term "about" at the start of a series of values ​​modifies each value by 10%.

[0035] All numerical values ​​or ranges of numbers include integers within such ranges and fractions of values ​​or integers within the ranges, unless the context clearly indicates otherwise. Thus, by way of example, reference to a reduction of 95% or more includes 95%, 96%, 97%, 98%, 99%, 100%, etc., as well as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, etc. Thus, and by way of example, reference to a numerical range such as "1 to 4" includes 2, 3, as well as 1.1, 1.2, 1.3, 1.4, etc. For example, "1 to 4 weeks" includes 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

[0036] Additionally, references to numerical ranges such as "0.01 to 10" include 0.011, 0.012, 0.013, etc., as well as 9.5, 9.6, 9.7, 9.8, 9.9, etc. For example, a dosage of about "0.01 mg / kg to about 10 mg / kg" body weight of a subject includes 0.011 mg / kg, 0.012 mg / kg, 0.013 mg / kg, 0.014 mg / kg, 0.015 mg / kg, etc., as well as 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, etc.

[0037] Reference to an integer with more (greater than) or less than includes every number greater than and less than the reference number, respectively. Thus, for example, reference to more than two includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. For example, administration of a recombinant viral vector, IdeS mutant, "two or more" times includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times. Additionally, reference to a numerical range, such as "1 to 90," includes 1.1, 1.2, 1.3, 1.4, 1.5, etc., as well as 81, 82, 83, 84, 85, etc. For example, "about 1 minute to about 90 days" includes 1.1 minutes, 1.2 minutes, 1.3 minutes, 1.4 minutes, 1.5 minutes, etc., as well as 1 day, 2 days, 3 days, 4 days, 5 days....81 days, 82 days, 83 days, 84 days, 85 days, etc.

[0038] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and not limiting.

[0039] IdeS protein mutants Provided herein are IdeS variants with improved potency and / or stability compared to wild-type IdeS. IdeS was discovered as a protein secreted in its mature form from Streptococcus pyogenes bacteria that specifically cleaves human immunoglobulin G (IgG, including its subclasses IgG1, IgG2, IgG3 and IgG4) at its hinge region, but not other classes of human immunoglobulins. The amino acid sequence of the native IdeS precursor, corresponding to protein RefSeq WP_010922160.1, is provided in Table 1 as SEQ ID NO: 1. The protein comprises a 29 amino acid long secretory signal peptide, the sequence of which is provided as SEQ ID NO: 83, and a 310 amino acid long mature polypeptide, the sequence of which is provided as SEQ ID NO: 84. Further details regarding the structure of IdeS can be found, for example, in von Pawel-Rammingen U, Johansson BP, Bjorck L. IdeS, a novel streptococcal cysteine ​​proteinase with unique specificity for immunoglobulin G. EMBO J. 2002 Apr 2; 21(7):1607-15. doi:10.1093 / emboj / 21.7.1607; Wenig K, et al. Structure of the streptococcal endopeptidase IdeS, a cysteine ​​proteinase with strict specificity for IgG. Proc Natl Acad Sci USA. 2004 Dec 14; 101(50):17371-6. doi:10.1073 / pnas.0407965101.

[0040] Based on a rational protein design approach, the present disclosure provides certain substitution mutants of wild-type IdeS that are surprisingly more thermostable and / or more potent in their ability to cleave IgG molecules when compared to the derived wild-type IdeS protein sequence. The names and amino acid sequences of the novel IdeS mutants are disclosed in Figures 1A-1J along with the reference sequence for the wild-type IdeS protein (RefSeqWP_010922160.1). The wild-type IdeS is exemplified in its precursor form (SEQ ID NO: 1) that includes the natural secretory signal peptide (SEQ ID NO: 83) located at the amino terminus of the mature polypeptide (SEQ ID NO: 84), while the IdeS mutant instead starts with the dipeptide Met-Gly and ends with a His tag that is not present in the wild-type and facilitates small-scale purification (SEQ ID NO: 85). The amino acid differences between the mutants and wild-type IdeS are highlighted. The amino acid sequences of wild-type IdeS and the named variants shown in Figures 1A-1J, along with other amino acid sequences disclosed herein, are further specified by SEQ ID NOs as shown in Table 1 below. Although the variants shown in Figures 1A-1J retain different amino and carboxy termini compared to wild-type IdeS, these differences should be considered exemplary and not limiting. Thus, for example, any variant may not start with Met-Gly, but with the natural IdeS secretion signal peptide or some other sequence of amino acids at its amino terminus. Furthermore, any variant may end with an untagged end, as in the wild-type, or with a different tagged end, or with some other sequence of amino acids at its carboxy terminus, rather than with a His-tag end as exemplified. Thus, for example, in some embodiments, a variant of the present disclosure may include amino acid numbers 1 to 312 of any one of SEQ ID NOs: 2 to 71, or may include amino acid numbers 2 to 312 of any one of SEQ ID NOs: 2 to 71, or may include amino acid numbers 3 to 312 of any one of SEQ ID NOs: 3 to 71, or any other range of amino acids encompassed by SEQ ID NOs: 2 to 71, or a subsequence thereof.

[0041] [Table 1-1]

[0042] [Table 1-2]

[0043] [Table 1-3]

[0044] In some embodiments, the amino acid sequence of an IdeS mutant protein of the present disclosure comprises, consists of, or essentially consists of the amino acid sequence of a mature wild-type IdeS protein (SEQ ID NO: 84) in which one or more amino acids have been replaced with a different amino acid, and / or one or more amino acids have been inserted or deleted, and / or one or more amino acids have been added to its amino terminus, and / or one or more amino acids have been added to its carboxy terminus.

[0045] In some embodiments, the amino acid sequence of the IdeS mutant protein of the present disclosure is: (a) amino acid numbers 1 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 2 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 3 to 312 of any one of SEQ ID NOs: 3 to 71; or (b) a fragment of (a) having Ig endopeptidase activity; or (c) amino acid numbers 1 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 2 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 3 to 312 of any one of SEQ ID NOs: 3 to 71. or (d) a variant of (b) having at least 50% identity to a corresponding portion of amino acid numbers 1 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 2 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 3 to 312 of any one of SEQ ID NOs: 3 to 71, and having Ig endopeptidase activity.

[0046] In some embodiments, the IdeS mutant protein has at least about 60% or more identity (e.g., 60-70%, 70-80%, 80-90% or 90-100% identity) to amino acid numbers 1 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 2 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 3 to 312 of any one of SEQ ID NOs: 3 to 71, or a fragment thereof having Ig endopeptidase activity. In some embodiments, the IdeS mutant protein has at least about 70% or more identity (e.g., 70-80%, 80-90%, or 90-100% identity) to amino acid numbers 1 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 2 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 3 to 312 of any one of SEQ ID NOs: 3 to 71, or a fragment thereof having Ig endopeptidase activity. In some embodiments, the IdeS mutant protein has at least about 80% or more identity (e.g., 80-90% or 90-100% identity) to amino acid numbers 1 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 2 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 3 to 312 of any one of SEQ ID NOs: 3 to 71, or a fragment thereof having Ig endopeptidase activity. In some embodiments, the IdeS mutant protein has at least about 90% or more identity (e.g., 90-100% identity) to amino acid numbers 1-312 of any one of SEQ ID NOs: 2-71, or amino acid numbers 2-312 of any one of SEQ ID NOs: 2-71, or amino acid numbers 3-312 of any one of SEQ ID NOs: 3-71, or a fragment thereof having Ig endopeptidase activity. In some embodiments, the IdeS mutant protein has at least about 95% or more identity (e.g., 95-100% identity) to amino acid numbers 1-312 of any one of SEQ ID NOs: 2-71, or amino acid numbers 2-312 of any one of SEQ ID NOs: 2-71, or amino acid numbers 3-312 of any one of SEQ ID NOs: 3-71, or a fragment thereof having Ig endopeptidase activity.In some embodiments, the IdeS mutant protein has at least about 96% or more identity (e.g., 96-100% identity) to amino acid numbers 1-312 of any one of SEQ ID NOs: 2-71, or amino acid numbers 2-312 of any one of SEQ ID NOs: 2-71, or amino acid numbers 3-312 of any one of SEQ ID NOs: 3-71, or a fragment thereof having Ig endopeptidase activity. In some embodiments, the IdeS mutant protein has at least about 97% or more identity (e.g., 97-100% identity) to amino acid numbers 1-312 of any one of SEQ ID NOs: 2-71, or amino acid numbers 2-312 of any one of SEQ ID NOs: 2-71, or amino acid numbers 3-312 of any one of SEQ ID NOs: 3-71, or a fragment thereof having Ig endopeptidase activity. In some embodiments, the IdeS mutant protein has at least about 98% or more identity (e.g., 98-100% identity) to amino acid numbers 1-312 of any one of SEQ ID NOs: 2-71, or amino acid numbers 2-312 of any one of SEQ ID NOs: 2-71, or amino acid numbers 3-312 of any one of SEQ ID NOs: 3-71, or a fragment thereof having Ig endopeptidase activity. In some embodiments, the IdeS mutant protein has at least about 99% or more identity (e.g., 99-100% identity) to amino acid numbers 1-312 of any one of SEQ ID NOs: 2-71, or amino acid numbers 2-312 of any one of SEQ ID NOs: 2-71, or amino acid numbers 3-312 of any one of SEQ ID NOs: 3-71, or a fragment thereof having Ig endopeptidase activity.

[0047] In some embodiments, the amino acid sequence of an IdeS mutant protein of the present disclosure comprises amino acid numbers 1 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 2 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 3 to 312 of any one of SEQ ID NOs: 3 to 71. In some embodiments, the amino acid sequence of an IdeS mutant protein of the present disclosure consists essentially of amino acid numbers 1 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 2 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 3 to 312 of any one of SEQ ID NOs: 3 to 71. In some embodiments, the amino acid sequence of an IdeS mutant protein of the present disclosure consists essentially of amino acid numbers 1 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 2 to 312 of any one of SEQ ID NOs: 2 to 71, or amino acid numbers 3 to 312 of any one of SEQ ID NOs: 3 to 71.

[0048] In some embodiments, IdeS mutant proteins of the present disclosure comprise, consist of, or consist essentially of an amino acid sequence (SEQ ID NO:84) corresponding to a mature wild-type IdeS protein, lacking a secretory signal peptide sequence located at the amino terminus of the protein or any other type of exogenous peptide sequence located at the amino or carboxy terminus of such protein. Non-limiting examples of such embodiments include the IdeS mutant proteins whose amino acid sequences are provided in SEQ ID NOs:76, 79, and 82. Further examples include IdeS protein variants comprising, consisting of, or consisting essentially of amino acid numbers 3-312 of any one of SEQ ID NOs:3-71.

[0049] In some embodiments, an IdeS mutant protein of the present disclosure comprises a prokaryotic secretory signal peptide sequence located at the amino terminus of the protein, which facilitates export transport of the IdeS protein out of the bacterial cell following its expression. Such a signal peptide sequence can be the native signal peptide sequence of IdeS, such as provided by SEQ ID NO: 83, or a signal peptide sequence from another secreted protein of S. pyogenes. Thus, for example, in some embodiments, IdeS protein variants such as those provided in SEQ ID NOs: 76, 79, and 82 each further comprise the amino acid sequence of SEQ ID NO: 83 at the amino terminus of each such protein. Further examples include IdeS protein variants comprising amino acid numbers 3-312 of any one of SEQ ID NOs: 3-71, each further comprise the amino acid sequence of SEQ ID NO: 83 at the amino terminus of each such protein. In some other embodiments, an IdeS protein variant of the present disclosure can comprise a signal peptide from a secreted protein of a different species of bacteria that is not S. pyogenes, such as E. coli, or another species.Further information on bacterial signal peptides can be found in, for example, Freudl, R. Signal peptides for recombinant protein secretion in bacterial expression systems. Microb Cell Fact 17, 52 (2018). doi:10.1186 / s12934-018-0901-3; and Kaushik S, He H and Dalbey RE (2022) Bacterial Signal Peptides-Navigating the Journey of Proteins. Front. Physiol. 13: 933153. doi:10.3389 / fphys.2022.933153; Green ER, Mecsas J. Bacterial Secretion Systems: An Overview. Microbiol Spectr. 2016 Feb; 4(1):10.1128 / microbiolspec.VMBF-0012-2015.doi:10.1128 / microbiolspec.VMBF-0012-2015; and Kleiner-Grote GRM, Risse JM, Friehs K. Secretion of recombinant proteins from E. coli. Eng Life Sci. 2018 Apr 14; 18(8):532-550.doi:10.1002 / elsc.201700200.

[0050] In some embodiments, such as those in which an IdeS mutant protein is to be expressed in a eukaryotic cell, the IdeS mutant protein of the present disclosure may include a eukaryotic secretory signal peptide sequence that corresponds to the type of cell in which the mutant protein will be expressed. Examples of eukaryotic cells in which the IdeS mutant protein may be expressed include yeast cells, plant cells, insect cells, or mammalian cells, as well as other types.

[0051] In some embodiments, the IdeS mutant proteins of the present disclosure comprise a short peptide sequence located at the amino terminus of the protein beginning with a methionine, which is encoded by a DNA or RNA sequence sufficient to support translation of the IdeS mutant, but does not necessarily target the expressed protein for secretion from the cell in which it is expressed. For example, in some embodiments, the IdeS mutant protein (or wild-type IdeS) may begin with the dipeptide sequence Met-Gly (or MG in single letter code), followed by the mature protein amino acid sequence. In such embodiments in which the IdeS mutant protein is not translated with a secretory signal peptide sequence (e.g., such proteins begin with MG or some other translatable sequence), the protein may be recovered within the cell as it is expressed, released by lysing the cell, and then purifying the protein so released using methods familiar to those of skill in the art. Thus, for example, in some embodiments, the IdeS protein mutants as provided in SEQ ID NOs: 76, 79, and 82 may each further comprise the dipeptide amino acid sequence Met-Gly (MG) at the amino terminus of each such protein. Further examples include IdeS protein variants as provided in SEQ ID NOs: 2-71, each of which begins with Met-Gly(MG), and in some other embodiments may be provided without the last nine amino acids that occur at the respective carboxy termini of SEQ ID NOs: 2-71, each of which corresponds to the His tag of SEQ ID NO: 85. Such embodiments may also be described as IdeS protein variants comprising, consisting of, or consisting essentially of amino acid numbers 1-312 of any one of SEQ ID NOs: 2-71.

[0052] In some embodiments, the amino terminal methionine (M) may be removed during expression or subsequent purification, such as through the action of endogenous aminopeptidases or other mechanisms, such that the predominant species of IdeS mutant protein lacks methionine (Met, M) at its amino terminus. Instead, the following amino acid located immediately following the methionine prior to its removal is present at the amino terminus. Thus, for example, in some embodiments, IdeS protein variants as provided in SEQ ID NOs: 76, 79, 82, and 84 may each further comprise a single amino acid Gly (G) at the amino terminus of each such protein. Further examples include IdeS protein variants comprising amino acid numbers 3-312 of any one of SEQ ID NOs: 2-71, each further comprising a single amino acid Gly (G) at the amino terminus of each such protein. Such embodiments may also be described as IdeS protein variants comprising, consisting of, or consisting essentially of amino acid numbers 2-312 of any one of SEQ ID NOs: 2-71. Still further examples include IdeS protein variants such as those provided in SEQ ID NOs: 73, 75, 78 and 81, each of which begins with Gly (G).

[0053] In some embodiments, such as affinity capture using antibody metal ion resins, the IdeS protein variants of the present disclosure contain a short sequence of amino acids located at the amino- or carboxy-terminus of the protein that confers some desired function. Commonly used peptide and protein tags include those known as CBP, FLAG, GST, HA, HBH, MBP, Myc, Poly-His, S-tag, SUMO, TAP, TRX and V5, among others. Such tags can also be provided with protease cleavage sites, if desired, to allow post-translational removal of the tag, non-limiting examples include those known as TEV, thrombin and PreScission sites, among others. Tags and other functional peptides and protein moieties that can be fused to the IdeS protein variants of the present disclosure are described in Kimple ME, Brill AL, Pasker RL. Overview of affinity tags for protein purification. Curr Protoc Protein Sci. 2013 Sep 24; 73:9.9.1-9.9.23. doi:10.1002 / 0471140864.ps0909s73.

[0054] Modification of polypeptides is routine in the art and need not be detailed herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, deletions or additions of one or more amino acids or the use of chemical analogs that do not significantly adversely alter the functional activity or that mature (increase) the affinity of the polypeptide for its ligand.

[0055] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an IdeS protein with an N-terminal methionyl residue or an IdeS protein fused to an epitope tag. Other insertional variants of the IdeS protein include fusions to the N- or C-terminus of the IdeS protein of enzymes or polypeptides that increase the half-life of the IdeS protein in the blood circulation.

[0056] Substitutional variants have at least one amino acid residue in the IdeS protein removed and a different residue inserted in its place. Conservative substitutions are shown in Table 2 under the heading of "conservative substitutions." If such substitutions result in altered biological activity, more substantial changes, such as those designated "representative substitutions" in Table 2 or further described below for amino acid classes, can be introduced and the products screened.

[0057] [Table 2]

[0058] Significant modifications of the biological properties of the IdeS protein can be accomplished by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone, e.g., ?-sheet or helix structure, in the region of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the maintenance of the bulk of the side chains. Natural residues are divided into groups based on shared side chain properties: (1) Nonpolar: Norleucine, Met, Ala, Val, Leu, Ile; (2) uncharged polar: Cys, Ser, Thr, Asn, Gln; (3) Acidic (negatively charged): Asp, Glu; (4) Basic (positively charged): Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe, His.

[0059] Non-conservative substitutions are made by exchanging a member of one of these classes for another class.

[0060] The IdeS variant of interest can be sequenced and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the IdeS variant of interest can be maintained in a vector in a host cell, which can then be expanded and frozen for future use.

[0061] In some embodiments, the IdeS mutants of the present disclosure are more thermostable when compared to wild-type IdeS. In some embodiments, improved thermostability is desirable because it may improve manufacturability, formulation, long-term storage, patient delivery and efficacy. Many methods for quantifying protein stability are known in the art and can be used to measure the thermostability of IdeS mutant proteins in comparison to wild-type IdeS, including cyclic dichroism (CD), dynamic and static light scattering (DLS and SLS), size exclusion chromatography with multi-angle light scattering (SEC-MALS), Fourier transform infrared spectroscopy (FTIR), analytical ultrafiltration (AUC), size exclusion chromatography (SEC), differential scanning fluorescence (DSF), autofluorescence (IF) and differential scanning calorimetry (DSC).

[0062] In some embodiments, the thermal stability of IdeS variants is quantified using DSC, which generates two values ​​that can be conveniently used to express thermal stability: Tonset is the temperature at which the pure protein begins to unfold, and TM is the thermal transition temperature at which 50% of the protein is in its native conformation and 50% is denatured.

[0063] In some embodiments, the Tonset value of an IdeS variant of the present disclosure is at least or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0°C higher than the wild-type IdeS protein, or any value in a range between or including any of the specifically recited values ​​above. In some embodiments, the Tonset value of an IdeS variant of the disclosure is at least or about 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45.0, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46.0, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48.0, 48.1, 48.2, 48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, 49.0, 49.1, 49.2, 49.3, 49.4, 49.5, 49.6, 49.7, 49.8, 49.9, 50.0, 50.1, 50.2, 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, 51.0, 51.1, 51.2, 51.3, 51.4, 51.5, 51.6, 51.7, 51.8, 51.9, 52.0, 5 7.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48.0, 48.1, 48.2, 48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, 49.0, 49.1, 49.2, 49.3, 49.4, 49.5, 49.6, 49.7, 49.8, 49.9 or 50.0°C, or greater, or any value in a range between or including any of the aforesaid specifically recited values.

[0064] In some embodiments, the TM value of an IdeS variant of the present disclosure is at least or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0° C. higher than the wild-type IdeS protein, or any value in a range between or including any of the specifically recited values ​​above. In some embodiments, the TM value of an IdeS variant of the disclosure is at least or about 51.0, 51.1, 51.2, 51.3, 51.4, 51.5, 51.6, 51.7, 51.8, 51.9, 52.0, 52.1, 52.2, 52.3, 52.4, 52.5, 52.6, 52.7, 52.8, 52.9, 53.0, 53.1, 53.2, 53.3, 53.4, 53.5, 53.6, 53.7, 53.8, 53.9, 54.0, 54.1, 54.2, 54.3, 54.4, 54.5, 54.6, 54.7, 54.8, 54.9, 55.0, 55.1, 55.2, 55.3, 55.4, 55.5, 55.6, 55.7, 55.8, 55.9, 56.0, 56.1, 56.2, 56.3, 56.4, 56.5, 56.6, 56.7, 56.8, 56.9, 57.0, 57.1, 57.2, 57.3, 57.4, 57.5, 57.6, 57.7, 57.8, 57.9, 58.0, 58.1, 58.2, 58.3, 58.4, 58.5, 58.6, 58.7, 58.8, 58.9, 59.0, 59 2, 54.3, 54.4, 54.5, 54.6, 54.7, 54.8, 54.9, 55.0, 55.1, 55.2, 55.3, 55.4, 55.5, 55.6, 55.7, 55.8, 55.9, 56.0, 56.1, 56.2, 56.3, 56.4, 56.5, 56.6, 56.7, 56.8, 56.9 or 57.0°C or greater, or any value in a range between or including any of the aforesaid specifically recited values.

[0065] In some embodiments, the IdeS variants of the present disclosure are more potent when compared to wild-type IdeS. In some embodiments, improved potency is desired because it allows the same amount of target IgG to be cleaved with a lower mass, concentration, or dose of the IdeS variant when compared to wild-type IdeS. Several methods for quantifying IdeS enzymatic potency have been described and can be used to measure the potency of IdeS mutant proteins in comparison to wild-type IdeS, such as ELISA-based methods, visualization of the products of IdeS digestion of IgG, or mass spectrometry methods, such as those described in Hess, JL, et al., Immunoglobulin cleavage by the streptococcal cysteine ​​protease IdeS can be detected using protein G capture and mass spectrometry, J.Microbiol.Meths.,70(2):284-291(2007)(doi.org / 10.1016 / j.mimet.2007.04.017).

[0066] In some embodiments, the potency of the IdeS variants of the present disclosure may be measured using an ELISA-based sandwich assay. In one version of this assay, human IgG is immobilized to an ELISA plate via a capture antibody (such as mouse anti-human) specific for the human IgG F(ab) region. IdeS protein (mutant or wild type) is then added to the wells of the plate at various concentrations and incubated for a predetermined time, which acts to cleave the human IgG below the hinge in successive steps to generate a single cleaved IgG (scIgG), then a F(ab')2 fragment and two Fc monomers. Vindebro, R, et al., Rapid IgG heavy chain cleavage by the streptococcal IgG endopeptidase IdeS is mediated by IdeS monomers and is not due to enzyme dimerization, FEBS Letters 587(12):1818-1822(2013)(doi.org / 10.1016 / j.febslet.2013.04.039). A detection antibody specific for the dimeric human IgG Fc domain can then be used to measure the amount of intact human IgG versus partially or completely cleaved IgG. Results can be expressed as IC50 values, with lower numbers indicating stronger potency.

[0067] In some embodiments, the human IgG cleavage potency of an IdeS variant of the present disclosure, when determined using ELISA and expressed as an IC50 value, is at least or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5 nM lower or lower than the IC50 value of wild-type IdeS in the same type of assay, or the IC50 value is in a range between or including any of the specifically recited values ​​above. In some embodiments, the human IgG cleavage potency of an IdeS variant of the disclosure, as determined using ELISA and expressed as an IC50 value, is at most 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8 or 3.7 nM or lower, or the IC50 value is a range between or including any of the specifically recited values ​​above.

[0068] Methods for Producing IdeS Proteins The mutant IdeS proteins of the present disclosure can be produced using any technique known in the art for producing recombinant proteins. In some embodiments, the coding sequence for the IdeS protein mutant can be cloned into an expression vector, which is introduced into a suitable host cell, where the sequence is then transcribed and translated into protein. The coding sequence can be optimized for the type of host cell in which they are to be expressed. The IdeS mutant proteins expressed by the host cell can then be purified using techniques known in the art for purifying recombinant proteins.

[0069] In some embodiments, the expression vector is a bacterial expression vector that contains a promoter (such as lac, PL or T7 promoter) and a terminator (such as T7 terminator) for initiating and terminating transcription of the protein coding sequence, respectively, as well as other functional elements, such as a multiple cloning site into which the coding sequence can be conveniently inserted, a ribosome binding site for improving translation efficiency, a bacterial origin of replication, a sequence encoding an affinity tag and a protease cleavage site to allow removal of the tag, and an antibiotic resistance gene (such as for ampicillin or kanamycin). The expression vector can be replicated and purified in a bacterial host (such as DH10B and DH5-alpha strains), and then used to transform other bacterial strains suitable for recombinant protein expression (such as E. coli BL21, BL21(DE3) and K-12 strains). After transformation with the expression vector, the bacteria are grown in a medium, usually with the addition of an antibiotic to prevent the growth of non-transformed cells. Depending on the nature of the promoter, protein expression can then be induced, such as by adding nutrients or drugs to the medium (such as lactose or IPTG if the lac promoter is used) or by changing environmental variables such as temperature or pH. The bacteria are then maintained in the medium under conditions that promote protein expression. After a sufficient time, the protein expressed from the vector is recovered and purified. Proteins with secretion signals can be recovered directly from the medium. Other proteins that remain within the bacterial cells can be recovered after concentrating the cells and lysing them using physical methods, such as enzymes to digest the cell wall or sonication. In any case, the expressed protein in the medium or the protein released from the cells can be purified using methods familiar to those skilled in the art, such as filtration to remove cell debris, salt precipitation, chromatographic methods such as affinity, ion exchange or hydrophobic interaction chromatography and buffer exchange, among other possible methods.Bacterial expression of recombinant proteins has also been described, for example, in Langlais, C., Korn, B. (2005). Recombinant Protein Expression in Bacteria. In: Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine. Springer, Berlin, Heidelberg. https: / / doi.org / 10.1007 / 3-540-29623-9_4800; Overton TW. Recombinant protein production in bacterial hosts. Drug Discov Today. 2014 May; 19(5):590-601. doi:10.1016 / j.drudis.2013.11.008; Rosano GL and Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges.Front.Microbiol.5:172.doi:10.3389 / fmicb.2014.00172;Rosano,GL,Morales,ESand Ceccarelli,EA(2019),New tools for recombinant protein production in Escherichia coli:A 5-year update.Protein Science, 28:1412-1422. Described at https: / / doi.org / 10.1002 / pro.3668.

[0070] In other embodiments, the IdeS protein variants of the present disclosure may be expressed in non-bacterial host cells, such as yeast cells, plant cells, insect cells or mammalian cells, using appropriate expression vectors and methods familiar to those of skill in the art for protein expression in such host cells.

[0071] In addition to the IdeS mutant proteins, the present disclosure provides nucleic acids (e.g., DNA or RNA) comprising nucleotide sequences encoding the IdeS mutant proteins (which may be codon optimized according to the type of host cell in which expression is to be achieved), expression vectors comprising such nucleotide sequences, host cells comprising such expression vectors, methods for producing the IdeS mutant proteins of the present disclosure, as well as methods for purifying such proteins after they have been expressed.

[0072] Treatment and prevention methods In some embodiments, the disclosure provides methods of administering to a subject in need of prevention or treatment of a disease or disorder characterized by an excessive concentration of certain IgG antibodies in such subject's blood, plasma, or serum, an IdeS mutein of the disclosure in an amount sufficient to prevent or treat the disease or disorder. In some embodiments, the disease or disorder is treated or prevented by reducing the concentration of IgG antibodies in the subject's blood, plasma, or serum. In some embodiments, the subject is a human subject, and the IgG antibodies can be of the IgG1, IgG2, IgG3, or IgG4 subclass.

[0073] As used herein, with respect to a disease or disorder manifested in a subject, "treat" or "treatment" means reducing or arresting its severity or rate of progression, at least partially alleviating at least one symptom or sign associated with such disease or disorder, or altering the value of a diagnostic assessment or biomarker to a value indicative of a less severe disease state. A therapeutically effective amount of an IdeS variant of the present disclosure is an amount sufficient to treat the disease or disorder in a subject.

[0074] As used herein with respect to a disease or disorder that is not yet manifest in a subject, but which such subject is thought to be predisposed to, "prevent" or "prevention" means to avert or delay (even temporarily) the onset of the disease process or the onset of symptoms or signs associated with such disease or disorder. A prophylactically effective amount of an IdeS variant of the present disclosure is an amount sufficient to prevent a disease or disorder in a subject.

[0075] In some embodiments, the disease or disorder is an autoimmune disorder in which the subject produces IgG antibodies (autoantibodies) that bind to unknown self-antigens expressed by cells or tissues of the body. Exemplary autoimmune disorders that may be treated using the IdeS muteins of the present disclosure include Addison's disease, dermatomyositis, Hashimoto's thyroiditis, anti-glomerular basement membrane disease, antineutrophil cytoplasmic antibody vasculitis, vasculitis, Wegener's granulomatosis, Churg-Strauss syndrome, microscopic polyangiitis, anti-N-methyl-D-aspartate receptor encephalitis, antiphospholipid syndrome, autoimmune bullous skin diseases, pemphigus, pemphigus foliaceus, pemphigus brazilianus, pemphigus vulgaris, autoimmune hemolytic anemia, pernicious anemia, autoimmune hepatitis, autoimmune neutropenia, Bullous pemphigoid, celiac disease, chronic utricaria, complete congenital heart block, type 1 diabetes mellitus, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, Goodpasture's syndrome, anti-glomerular basement membrane disease, Graves' disease, Graves' disease, Guillain-Barre syndrome, acute inflammatory demyelinating polyneuropathy, acute motor axonal neuropathy, acquired factor FVIII deficiency hemophilia, idiopathic thrombocytopenic purpura, Lambert-Eaton myasthenic syndrome, mixed connective tissue disease, myasthenia gravis, dilated cardiomyopathy These include, but are not limited to, myocarditis, neuromyelitis optica, primary biliary cirrhosis, multiple sclerosis, systemic sclerosis, CREST syndrome, rheumatic heart disease, rheumatoid arthritis, reactive arthritis, serum sickness, immune complex type III hypersensitivity, Sjogren's syndrome, systemic lupus erythematosus, lupus nephritis, stiff-body syndrome, vitiligo, scleroderma, transplant rejection, and thrombotic thrombocytopenic purpura, among others possible others.

[0076] In some embodiments, antigens against which IgG autoantibodies are produced include Ro-RNP complex, La antigen, small nuclear ribonucleoproteins (snRNPs), double-stranded DNA, histones, topoisomerase I, centromere, myeloperoxidase, proteinase 3, cardiolipin, citrullinated proteins, carbamylated proteins, rheumatoid factor, phospholipids, alpha 3 chain of basement membrane collagen (type IV collagen), Rh blood group antigens, I antigen, platelet integrin GpIIB:IIIa, epidermal cadherin, ribosomes, pancreatic beta cell antigen, myelin basic protein, carboxypeptidase H, chromogranin A, glutamatergic receptor β ... Examples of proteins that may be involved in the induction of inflammatory bowel disease include, but are not limited to, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), proinsulin, phospholipid-beta-2 glycoprotein I complex, poly(ADP-ribose) polymerase, Sm antigen of the U-1 small ribonucleoprotein complex, alpha enolase, aquaporin-4, beta arrestin, myelin oligodendrocyte glycoprotein, proteolipid protein, S100 beta, collagen II, heat shock proteins, human cartilage glycoprotein 39, and others that may be involved.

[0077] In some embodiments, the disease or disorder is monoclonal gammopathy of undetermined significance (MGUS), in which the paraprotein produced by an abnormal clonal population of plasma cells is IgG.

[0078] In some embodiments, subjects with an autoimmune disease and / or subjects who produce autoantibodies or have MGUS are administered a dose of an IdeS variant of the disclosure that is at least or about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45 or 0.50 mg / kg of the subject's body weight, or a dose or range between and including any of the foregoing specifically recited values. In some embodiments, treatment of a subject having an autoimmune disease or MGUS with an IdeS protein variant of the disclosure at the recited dosages is effective to treat or prevent autoimmune disease or MGUS. In some embodiments, treatment of a subject having an autoimmune disease or MGUS with an IdeS protein variant of the disclosure at the recited dosages is effective to reduce the concentration of total IgG in the subject's blood, plasma or serum by at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or a percentage reduction or range between and including any of the foregoing specifically recited values, or up to about 7.0, 6.5, 7.0, 8.5, 9.6, 9.7, 9.8, 9.9, 100%, ... , 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05 grams per liter (g / L), or less, or to a concentration or range between and including any of the specifically recited values ​​above. In some embodiments, treatment of a subject with an autoimmune disease with an IdeS protein variant of the present disclosure at the recited dosages is effective to inhibit or reduce effector function, even if unknown, of IgG autoantibodies that bind to autoantigens. In some embodiments, the effector function is mediated by an Fc receptor.

[0079] In some embodiments, a subject with an autoimmune disease and / or who produces autoantibodies or has MGUS may be administered a second or subsequent dose of an IdeS variant of the present disclosure, where the further dose may be the same or a different dose than the first dose of the IdeS variant. In some embodiments, the second or subsequent dose may be administered at any appropriate interval to maintain an acceptably low level of autoantibodies. The interval may be regular, such as weekly or biweekly or monthly or bimonthly or every 3, 4, 5 or 6 months or some other regular interval. Alternatively, in some embodiments, the subject may be monitored by periodically taking samples, such as blood, plasma or serum, to determine the concentration or titer of the autoantibodies over time, and administering further doses of the IdeS protein variant as desired or necessary to maintain the concentration or titer of the autoantibodies within a predetermined range. In some embodiments, the subject is a human subject, and the IgG antibodies may be of the IgG1, IgG2, IgG3 or IgG4 subclass.

[0080] In some embodiments, the present disclosure provides a method of administering to a human subject sensitized to human leukocyte antigens (HLA) in need of a tissue or organ transplant an amount of an IdeS mutein of the present disclosure sufficient to reduce or deplete the concentration of anti-HLA IgG antibodies in the blood of such subject, thereby preventing antibody-mediated rejection after transplantation. In some embodiments, the transplanted organ is the kidney, liver, heart, pancreas, lung or intestine or other organ. In some embodiments, the anti-HLA IgG antibodies may be of the IgG1, IgG2, IgG3 or IgG4 subclass.

[0081] As known in the art, HLA sensitization can occur when a human is exposed to the cells of another individual, for example as a result of blood transfusion, pregnancy, or previous organ transplantation. Upon such exposure, the immune system of the sensitized individual recognizes the foreign cells as foreign and produces anti-HLA antibodies. If the titers of such circulating antibodies are high enough, they can cause the sensitized host to reject transplanted tissues or organs through mechanisms such as antibody-mediated rejection (AMR), such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0082] Whether a subject is sensitized can be determined by any suitable method. For example, a panel reactive antibody (PRA) test can be used to determine whether a subject is sensitized. A calculated PRA score that is at or above a certain threshold means that the subject is "high immune risk" or "sensitized" and at risk of AMR if the transplant proceeds. Alternatively, a cross-match test can be performed in which a sample of the transplant donor's blood is mixed with the subject's blood sample. A positive cross-match means that the subject has antibodies that react with the donor sample, indicating that the subject is sensitized and should not proceed with the transplant. In some embodiments, a sensitized subject at risk for AMR exhibits a calculated panel reactive antibody test score of at least or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% or more.

[0083] In some embodiments, a sensitized subject is administered a dose of an IdeS variant of the present disclosure at least or about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45 or 0.50 mg / kg of subject body weight, or a dose or range between and including any of the foregoing specifically recited values, and thereafter said subject receives a tissue or organ transplant. In some embodiments, treatment of sensitized subjects with an IdeS protein variant of the present disclosure at the recited dosages is effective to reduce or eliminate the risk of AMR upon tissue or organ transplantation.

[0084] In some embodiments, the period of time between administration of the IdeS variant and transplantation is at least or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 hours or more, or a period or range between and including any of the specifically recited values ​​above.

[0085] In some embodiments, the sensitized subject may be administered a second or subsequent dose of an IdeS variant of the present disclosure prior to transplantation, where the additional dose may be the same or a different dose than the first dose of the IdeS variant. In some embodiments, the second dose is administered 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 hours or more after the first dose, or after a period or range of time between and including any of the specifically recited values ​​above. In some embodiments, multiple administrations of an IdeS protein variant may be desirable if the subject has developed nAbs to IdeS due to previous infection with S. pyogenes, previous administration of IdeS, the presence of particularly high titers of anti-HLA antibodies, as well as other reasons. In some embodiments, prior to a second or subsequent administration of an IdeS protein variant of the present disclosure, the subject may be monitored by taking at least one sample, e.g., blood, plasma or serum, and testing such sample for the concentration or titer of nAbs or other types of IgG to determine whether further administration of an IdeS protein variant is desirable or necessary.

[0086] In some embodiments, the dose of the IdeS variant and / or the time period between administration of the IdeS variant and transplantation is sufficient time for the subject to convert from a positive crossmatch to a negative crossmatch using an assay to detect the presence of donor-specific anti-HLA antibodies (DSA) in a blood, plasma or serum sample from the subject. DSA in such samples can be quantified by a variety of methods known in the art, such as complement dependent cytotoxicity (CDC), flow cytometric crossmatch (FCXM) and / or multiplexed single antigen bead (SAB) assays. Such methods are further described, for example, in Mulley, WR, and J Kanellis, Understanding crossmatch testing in organ transplantation: A case-based guide for the general nephrologist, Nephrology 16:125-133(2011)(doi:10.1111 / j.1440-1797.2010.01414.x); Lorant T, et al., Safety, immunogenicity, pharmacokinetics, and efficacy of degradation of anti-HLA antibodies by IdeS(imlifidase) in chronic kidney disease patients, Am J Transplant.18:2752-2762(2018)(DOI:10.1111 / ajt.14733).

[0087] In some embodiments, the dose of the IdeS variant and / or the period of time between administration of the IdeS variant and transplantation is sufficient to reduce the concentration of total IgG in the subject's blood, plasma or serum by at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or a percentage or range between and including any of the values ​​specifically recited above. In some embodiments, the dose of the IdeS variant and / or the period of time between administration of the IdeS variant and transplantation is sufficient to reduce the concentration of total IgG in the subject's blood, plasma or serum to a maximum of about 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05 grams per liter (g / L) or less, or to a concentration or range between and including any of the values ​​specifically recited above.

[0088] In some embodiments, the dose of the IdeS variant and / or the time period between administration of the IdeS variant and transplantation is sufficient to reduce the concentration of DSA in the subject's blood, plasma or serum by at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or a percentage reduction or range between and including any of the foregoing specifically recited values. The concentration of DSA in a subject's blood, plasma or serum sample may be determined using CDC, FCXM, SAB or any other suitable assay known in the art.

[0089] In some embodiments, the dose of the IdeS variant and / or the period of time between administration of the IdeS variant and transplantation is sufficient to reduce the subject's calculated serum panel reactive antibody (PRA) assay score by at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more, or a percentage reduction or range between and including any of the values ​​specifically recited above. In some embodiments, the dose of the IdeS variant and / or the time period between administration of the IdeS variant and transplantation is sufficient time to reduce the calculated panel reactive antibody (PRA) assay score of the subject's serum by up to about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, or less, or to a concentration or range between and including any of the specifically recited values ​​above. The calculated panel reactive antibody (PRA) assay score of the subject's serum may be determined using a complement dependent cytotoxicity (CDC) assay or any other suitable assay known in the art.

[0090] In some embodiments, a second or subsequent dose of an IdeS protein variant of the present disclosure may be administered to a sensitized subject who has received a transplant after transplantation, and the additional dose may be the same or different from the first dose of an IdeS variant administered before transplantation. In some embodiments, the second dose is administered at least 12, 24, 36, or 48 hours, or 5, 6, 7, 8, 9, 10, 11, 12, or 13 days, or 2, 3, or 4 weeks or more after transplantation, or after a period or range between and including any of the specifically recited values ​​above. In some embodiments, prior to the second or subsequent administration of an IdeS protein variant of the present disclosure, the subject may be monitored by taking at least one sample, such as blood, plasma, or serum, and testing such sample for the concentration or titer of nAb or other type of IgG to determine whether further administration of an IdeS protein variant is desired or required.

[0091] In some embodiments, the IdeS mutants of the present disclosure may be used to reduce the concentration of neutralizing IgG antibodies in a subject prior to gene therapy. In certain methods of gene therapy, natural viruses are modified by modifying their genomes to both prevent replication in the subject's target cells and introduce sufficient heterologous genetic information to express RNA or proteins with the desired function. Such modified viruses are called vectors in recognition of their ability to carry new genetic information to the patient's target cells for the purpose of effecting therapy. Examples of types of viruses that have been used to modify gene therapy vectors include adenoviruses, adeno-associated viruses, and lentiviruses (such as HIV-1 or HIV-2).

[0092] As known in the art, recombinant AAV vectors are derived from natural adeno-associated viruses, but are extensively modified both to prevent their replication and to include heterologous non-viral gene sequences intended to provide a therapeutic effect. AAV vectors generally comprise an AAV capsid and a modified AAV genome. The capsid is a proteinaceous shell that contains three viral proteins, AAV VP1, VP2 and VP3, involved in specific binding to target cells, and encloses and protects the genome. AAV vectors may use any natural serotype or variant, as well as non-natural capsids that have been derivatized, modified, altered or evolved to improve their function to some extent compared to the natural parent capsid. The vector genome is then derived from the AAV viral genome, but is extensively modified. More specifically, the only viral sequences that are retained are the inverted terminal repeats (ITRs), one of which is located at each of the 5' and 3' ends of the single-stranded DNA molecule that represents the genome. However, the AAV viral rep and cap genes, which are responsible for encoding the four Rep proteins (in AAV2) and three viral capsid proteins, respectively, involved in such important functions as replication and packaging, are completely removed. In the vector, these genes are replaced by a transgene expression cassette. The latter sequence is intended to express in the transduced target cells a transgene product that is intended to have some therapeutic effect. In some vectors, the transgene encodes a protein that is missing or defective in the patient due to an underlying genetic mutation, and the treatment is intended to provide a functional copy of that gene. For example, the transgene may encode clotting factor VIII, which is missing or defective in hemophilia A, or clotting factor IX, which is missing or defective in hemophilia B. Many other types of protein encoding transgenes useful in AAV vectors are possible. In other vectors, the transgene may encode an RNA or a protein that is intended to change the function of the transduced target cell.Examples include regulatory RNAs, such as those involved in the mechanism of RNA interference, or Cas nucleases expressed for the purpose of effecting gene editing. To support the expression of the transgene, the expression cassette often also contains transcriptional control sequences active in the target cell, such as a promoter to initiate transcription of the transgene and a polyadenylation signal sequence to terminate transcription. As known in the art, the expression cassette may contain other functional subsequences, such as enhancers, introns, stuffers, miRNA sequences, which are intended to improve desired properties, such as expression levels, tissue specificity of expression, message stability, packaging fidelity, etc. AAV vectors may contain other properties, such as the use of self-complementary genomes, which are intended to improve or affect function in some desired way. The type of AAV vector used in connection with the methods of treatment or prevention described herein should not be considered limiting.

[0093] Further information on AAV vectors can be found, for example, in Pupo, A, et al., AAV vectors: The Rubik's cube of human gene therapy, Mol Ther 30(12):3515-41(2022)(doi.org / 10.1016 / j.ymthe.2022.09.015); Li, C and RJ Samulski, Engineering adeno-associated virus vectors for gene therapy,Nat Revs Genetics 21:255-72(2020)(doi.org / 10.1038 / s41576-019-0205-4);Wang,D,et al.,Adeno-associated virus vector as a platform for gene therapy delivery.Nat Rev Drug Discov 18,358-378(2019)(doi.org / 10.1038 / s41573-019-0012-9);Bulcha,JT,et al.,Viral vector platforms within the gene therapy landscape.Sig Transduct Target Ther 6,53(2021)(doi.org / 10.1038 / s41392-021-00487-6). Further information on lentiviral vectors can be found, for example, in Wolff, JH and Mikkelsen, JG, Delivering genes with human immunodeficiency virus-derived vehicles: still state-of-the-art after 25 years. J Biomed Sci 29, 79(2022)(doi.org / 10.1186 / s12929-022-00865-4); Poletti, V and Mavilio, F. Designing Lentiviral Vectors for Gene Therapy of Genetic Diseases. Viruses. 2021 Aug 2; 13(8):1526(doi:10.3390 / v13081526).

[0094] One challenge to the use of modified viruses as gene therapy vectors is that candidates for gene therapy may have been previously infected with the same or similar type of virus from which the vector is produced, generating an antibody response against one or more components of the virus carried in the vector. Depending on the titer and specificity of the antibodies, their presence in gene therapy candidates may interfere with the ability of vector particles to bind to target cells, preventing target cell transduction and expression of the therapeutic transgene carried by the vector. At best, this may reduce the effective dose of the vector, requiring higher doses to compensate, or at worst, rendering the intended gene therapy ineffective and requiring the candidate to be removed from treatment. For example, many adeno-associated virus (AAV) serotypes and variants naturally infect humans, causing seroconversion in some humans and generating neutralizing antibodies (nAbs) against capsid proteins used in certain AAV-based vectors.This phenomenon can be seen, for example, in Rasko, J, et al., Global Seroprevalence of Neutralizing Antibodies Against Adeno-Associated Virus (AAV) Serotypes of Relevance to Gene Therapy, Blood (2022) 140 (Supplement 1):10668-10670(doi.org / 10.1182 / blood-2022-158305);Kruzik, A, et al., Prevalence of Anti-Adeno-Associated Virus Immune Responses in International Cohorts of Healthy Donors,Mol Ther Meths Clin Devel,Vol.14,P126-133(2019)(doi.org / 10.1016 / j.omtm.2019.05.014);Weber,T,Anti-AAV Antibodies in AAV Gene Therapy:Current Challenges and Possible Solutions, Front Immunol, 12: 658399 (2021) (doi:10.3389 / fimmu.2021.658399).

[0095] Although various strategies have been proposed to mitigate the challenge of nAbs to AAV vectors, such as treating gene therapy candidates with drugs to suppress B cell function, one approach that shows particular promise involves administering an IgG-degrading enzyme, such as IdeS or IdeZ, to patients prior to administration of the gene therapy vector. Such approaches are described, for example, in Leborgne C, et al., IgG-cleaving endopeptidase enables in vivo gene therapy in the presence of anti-AAV neutralizing antibodies. Nat Med. (2020) 26: 1096-101. doi: 10.1038 / s41591-020-0911-7; Elmore ZC, et al., Rescuing AAV gene transfer from neutralizing antibodies with an IgG-degrading enzyme. JCI Insight. (2020) 5: e139881. doi: 10.1172 / jci.insight.139881. The IdeS mutants of the present disclosure, because of their superior properties compared to the wild-type IdeS used in earlier experiments, can be advantageously used to reduce the concentration of neutralizing IgG antibodies in the blood of candidates for gene therapy sufficiently so that those who might otherwise be ineligible can be successfully treated with the vector.

[0096] Thus, in some embodiments, the present disclosure provides a method of preparing or pretreating a subject for gene therapy by administering to such a subject an amount of an IdeS protein variant of the present disclosure sufficient to at least temporarily reduce or eliminate the concentration of nAbs to a level that does not interfere with, reduce or prevent vector transduction in such a subject, where such subject has a pre-existing titer of neutralizing IgG antibodies against a component of the gene therapy vector that is high enough to interfere with, reduce or prevent the ability of the vector to transduce its intended target cells when administered to such a subject. In some embodiments, such treatment is effective to allow successful administration of a gene therapy vector to a subject that would otherwise be considered ineligible for gene therapy due to the presence of excessively high titers of nAbs against a component of the gene therapy vector. In some embodiments, such treatment is effective to reduce the concentration of nAbs in the blood of the subject to allow the gene therapy vector to successfully transduce its intended target cells after its subsequent administration. In some embodiments, the subject is gene therapy treatment naive, meaning that the subject has never previously undergone a particular type of gene therapy or been administered a particular type of gene therapy vector.

[0097] The titer of neutralizing antibodies already present in a serum sample from a subject can be determined using various assays known in the art. In general, such assays are designed to detect which dilutions of serum or other samples suspected to contain nAbs are effective to inhibit 50% of some physiologically relevant signal generated by the assay without the addition of sample, defined as 100%. For example, assays are designed to detect and quantify nAbs against a particular capsid of a recombinant AAV vector, the signal being the amount of reporter protein (such as luciferase) generated by cells transduced in vitro. Serial dilutions of serum samples are mixed with a predetermined amount of reporter vector using the capsid in question. After incubation, the mixture is added to cells in culture known to support vector binding and expression of the reporter transgene. As expression proceeds, the amount of signal from the reporter (e.g., light output from luciferase) indicates transduction efficacy, which is inversely proportional to the amount of nAb in the mixture. From a graph relating sample dilution factor to signal intensity, the dilution factor that results in 50% signal inhibition compared to no serum addition is defined as the nAb titer. A similar assay is designed to quantify the number of vector particles (as genome copies) that bind to target cells in culture after exposure to diluted serum samples. The dilution factor that prevents 50% of vector particles from binding to target cells is then defined as the titer.The assay is further described, for example, in Meliani, A, et al., Determination of anti-adeno-associated viral vector neutralizing antibody titer with an in vitro reporter system. Hum. Gene Ther. Methods 26, 45-53 (2015) (doi.org / 10.1089 / hgtb.2015.037); Guo, P, et al., Rapid AAV-Neutralizing Antibody Determination with a Cell-Binding Assay, Mol Ther Meths Clin Devel. 13: 40-46 (2019) (doi.org / 10.1016 / j.omtm.2018.11.007).

[0098] In some embodiments, a subject to be pre-treated with an IdeS protein variant of the present disclosure prior to administration of a gene therapy vector has a neutralizing antibody titer against the vector of at least or about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:120, 1:125, 1:150, 1:175, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:60 0, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1700, 1:1800, 1:1900, 1:2000, 1:2100, 1:2200, 1:2300, 1:2400, 1:2500, 1:2600, 1:2700, 1:2800, 1:2900 or 1:3000 or higher titers, or titers or ranges between and including any of the aforesaid specifically recited values. In the preceding section, this ratio represents the serum sample dilution factor calculated to inhibit 50% of gene therapy vector activity in an appropriate assay, such as a transduction efficiency assay using a reporter vector or a vector and target cell binding assay or other appropriate assay. Further, in the preceding section, a higher dilution factor, as indicated by a higher value for the second number in the ratio, represents a higher concentration of nAb in the sample and thus a higher titer of such nAb.

[0099] In some embodiments, the nAb is specific for a component of a recombinant viral vector, such as an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector, or another vector derived from a virus, hi some embodiments, the nAb is specific for a protein comprising the capsid of an AAV vector, such as the AAV VP1, VP2, or VP3 capsid protein. In some embodiments, the capsid recognized by the nAb can be any naturally occurring serotype or variant of AAV, non-limiting examples of which include AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVRh74 or AAVRh10 or others, or from any non-naturally occurring modified, altered or evolved AAV or AAV derivative, non-limiting examples of which include the indicated AAV-2i8, Spark100, AAV-Voy801, AAV-DJ, AAV PHP.B or many others.

[0100] In some embodiments, subjects with pre-existing titers of neutralizing IgG antibodies against components of a gene therapy vector that are high enough to interfere with, reduce or block the ability of the vector to transduce its intended target cells are primed or pre-treated for gene therapy with a vector by administering to such subject a dose of an IdeS variant of the present disclosure effective to reduce the concentration of nAbs to a level that no longer interferes with, reduces or blocks vector transduction in the subject. In some embodiments, an effective dose of an IdeS protein variant of the present disclosure is at least or about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45 or 0.50 mg / kg of subject body weight, or a dose or range between and including any of the foregoing specifically recited values. In some embodiments, treatment of a subject previously administered a gene therapy vector with an IdeS protein variant of the present disclosure at the listed dosages is effective to inhibit or reduce effector function of neutralizing IgG antibodies produced by the subject against components of the gene therapy vector.

[0101] In some embodiments, pre-treating a subject with pre-existing nAb titers to a gene therapy vector is effective to reduce the titer of nAbs to the vector in the subject's blood, plasma or serum by at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or a percentage reduction or range between and including any of the foregoing specifically recited values. In some embodiments, pre-treating a subject with a pre-existing nAb titer to a gene therapy vector is effective to reduce the titer of nAbs to the vector in the subject's blood, plasma or serum to up to 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, 1:0.1, or less, or to a titer or range of values ​​between and including any of the specifically recited values ​​above.

[0102] In some embodiments, a method of priming or pretreating a subject with a pre-existing nAb titer to a gene therapy vector comprises administering to such subject a single dose of an IdeS protein variant of the present disclosure. In other embodiments, the method comprises administering multiple doses of an IdeS variant protein, such as 2, 3, 4 or more doses of such an IdeS variant protein. Each dose of the multiple doses may be the same dose or may be a different dose. The time between each of the several doses, more than two doses, may be the same time or may be a different time. Exemplary non-limiting time periods between administration of any two doses of the IdeS protein variant include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 26, 28, 30, 32, 34, 35, 36, 38, 40, 42, 44, 45, 46, 48 hours or more, or time periods or ranges between and including any of the specifically recited values ​​above. In some embodiments, administering multiple doses of the IdeS protein variant may be desirable if the subject has developed nAbs to IdeS due to previous infection with S. pyogenes, previous IdeS administration, the presence of particularly high titers of antibodies to the gene therapy vector, as well as other reasons. In some embodiments, prior to a second or subsequent administration of an IdeS protein variant of the present disclosure, the subject is monitored by obtaining at least one sample, such as blood, plasma or serum, and testing such sample for the concentration or titer of nAbs or other types of IgG to determine whether further administration of an IdeS protein variant is desirable or required.

[0103] In some embodiments, the methods of the present disclosure further comprise a second step of pretreating a subject with a sufficient dose of an IdeS protein variant of the present disclosure to reduce nAb titers against a gene therapy vector, followed by administering such a gene therapy vector to such subject. In some embodiments, the period between administration of the IdeS variant (or the last dose thereof, if multiple) and administration of the gene therapy vector is at least or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 10 41, 42, 43, 44, 45, 46, 47, 48, 50, 52, 54, 55, 56, 58, 60, 62, 64, 65, 66, 68, 70, 72, 74, 75, 76, 78, 80, 84, 85, 90, 95, 96, 100, 108, 110, 120, 125, 130, 132, 135, 140, 144, 145, 150, 155, 156, 160, 165, 168 hours or 8, 9, 10, 11, 12, 13, 14 days or more, or a period or range between and including any of the specifically recited values ​​aforesaid.

[0104] In some embodiments, in the second step of administering the gene therapy vector to the subject, the gene therapy vector can be any suitable gene therapy vector, given the nature and severity of the subject's underlying health condition, such as a diagnosis with a disease or disorder suitable for treatment or prevention with the gene therapy vector. The choice of which gene therapy vector to administer is within the knowledge of one of skill in the art, as are other aspects of successfully administering such treatment to achieve a desired therapeutic or preventative outcome, such as application of any exclusion criteria for subject selection and treatment, the dose and administration form of the gene therapy vector, the number and frequency of dose administration, the route of administration, management of adverse events or side effects (such as immune responses to the vector) and follow-up, such as monitoring the subject for changes in health indicative of efficacy or side effects, evaluation of clinical endpoints or taking tissue or fluid samples to detect and measure changes in biomarkers, or other steps. In some embodiments, the gene therapy vector is a recombinant AAV vector comprising any suitable capsid and transgene expression cassette, given the nature of the subject's underlying health condition, disease or disorder, and other considerations familiar to one of skill in the art. In some embodiments, administration of an IdeS protein variant of the present disclosure prior to gene therapy is effective to reduce the dose of gene therapy vector that would otherwise need to be administered to a subject to achieve a given level of transduction efficiency, level of therapeutic or prophylactic efficacy, or to express a given level of a transgene product.

[0105] In some embodiments, the AAV vector expresses a functional coagulation factor VIII protein for the treatment of hemophilia A, or a functional coagulation factor IX protein for the treatment of hemophilia B, or a truncated but functional (at least partially) dystrophin protein for the treatment of Becker or Duchenne muscular dystrophy. The use of other AAV vectors expressing other types of transgenes for the treatment or prevention of other types of diseases or disorders is also possible. In some other embodiments, the gene therapy vector is a different recombinant viral vector, such as an adenovirus (e.g., AdV5), lentivirus (e.g., HIV-1, HIV-2), retrovirus, herpes simplex virus (e.g., HSV1, HSV2), alphavirus, favivirus, and others are possible.

[0106] In some embodiments, the methods of the present disclosure further include a third step of pretreating a subject with an IdeS protein variant of the present disclosure to reduce nAb titers to a gene therapy vector, and then administering one or more doses of an IdeS protein variant of the present disclosure to the subject after administration of the gene therapy vector to maintain low levels of nAbs in the subject. In some embodiments, the dose of IdeS protein variant to be administered following administration of a gene therapy vector is at least or about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45 or 0.50 mg / kg of subject body weight, or a dose or range between and including any of the specifically recited values ​​aforesaid, which is effective to maintain low or undetectable levels of nAb in the subject. In some embodiments, the IdeS protein variant is administered to the subject at least or about 3, 6, 9, 12, 15, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72 hours or 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or 2.5, 3, 3.5, 4 weeks or more after administration of the gene therapy vector, or after a period or range of time between and including any of the specifically recited values ​​above, which is effective to maintain low or undetectable levels of nAb in the subject.

[0107] In some embodiments, a method of post-treating a subject after gene therapy to maintain low or undetectable levels of nAbs comprises administering a single dose of an IdeS protein variant of the present disclosure to such a subject. In other embodiments, the method comprises administering multiple doses of an IdeS variant protein, such as 2, 3, 4 or more doses of such an IdeS variant protein. Each dose of the multiple doses may be the same dose or a different dose. The time between each of the more than two doses may be the same time or a different time. Exemplary non-limiting time periods between administration of any two doses of an IdeS protein variant include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 26, 28, 30, 32, 34, 35, 36, 38, 40, 42, 44, 45, 46, 48 hours or more, or time periods or ranges between and including any of the specifically recited values ​​above. In some embodiments, prior to the second or subsequent administration of an IdeS protein variant of the present disclosure, the subject may be monitored by obtaining at least one sample, such as blood, plasma or serum, and testing such sample for nAb or other type of IgG concentration or titer, to determine whether further administration of an IdeS protein variant is desired or required.

[0108] In other embodiments, the disclosed method allows the subject to be re-administered or re-medicated with a gene therapy vector. Sometimes, in some subjects, the first treatment with gene therapy may result in suboptimal therapeutic effect or suboptimal duration of therapeutic effect. There may be various reasons. For example, the transduction efficiency of target cells may not be as high as desired due to the presence of nAbs against vector components; the cellular immune system gradually eliminates transduced cells; gene therapy is administered to pediatric subjects, and as the subjects mature their organs and improve in body size, the relative amount of therapeutic transgene product from the vector gradually decreases, or for some other reason. In such a situation, it may be desirable to re-administer the same or similar type of vector to the subject at least a second time. However, one challenge to successful re-administration arises from the fact that the first administration of gene therapy vector may stimulate an immune response against one or more of its components, including the generation of nAbs that may prevent the second administration of gene therapy vector from being successful. Because of their proven effectiveness in cleaving and inactivating IgG antibodies, the present disclosure also provides, in some embodiments, methods for administering IdeS protein variants to a subject previously treated with a gene therapy vector that caused the subject to produce neutralizing IgG antibodies in order to reduce or eliminate the titer of such antibodies, thereby allowing a second or subsequent administration of the same type of vector, or a different vector that still shares the antigenic properties of the first vector and is adapted to be recognized by at least some of the same nAbs.

[0109] In some embodiments, the neutralizing antibody titer against a gene therapy vector in a subject to be treated with an IdeS protein variant of the present disclosure before the vector is re-administered is at or about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:120, 1:125, 1:150, 1:175, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1: 600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1700, 1:1800, 1:1900, 1:2000, 1:2100, 1:2200, 1:2300, 1:2400, 1:2500, 1:2600, 1:2700, 1:2800, 1:2900, 1:3000 or more, or a titer or range between and including any of the aforesaid specifically recited values.

[0110] In some embodiments, the gene therapy vector to be readministered is a recombinant viral vector, such as an adenoviral vector, an adeno-associated viral (AAV) vector, or a lentiviral vector, or another vector derived from a virus, hi some embodiments, the gene therapy vector to be readministered is an AAV vector that uses the same type of capsid as the initially administered vector, or a different capsid that is recognized by the same nAb raised against the same type of capsid as the initially administered vector.

[0111] In some embodiments, prior to readministering a gene therapy vector in which the subject has produced sufficient titers of a previous gene therapy nAb to impede, reduce or block the ability of the readministered vector to transduce its intended target cells, the subject is administered a dose of an IdeS protein variant of the present disclosure effective to reduce the concentration of the nAb to a level that no longer interferes with, reduces or blocks vector transduction in the subject. In some embodiments, an effective amount of an IdeS variant of the disclosure is at least or about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / kg of subject body weight, or a dose or range between and including any of the foregoing specifically recited values.

[0112] In some embodiments, treating a subject with an IdeS protein variant of the present disclosure is effective to reduce the titer of nAbs against the vector to be re-administered by at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or a percentage reduction or range between and including any of the foregoing specifically recited values. In some embodiments, treating a subject with an IdeS protein variant of the present disclosure is effective to reduce the titer of nAbs to the vector to be re-administered to up to 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, 1:0.1, or less, or to a titer or range of values ​​between and including any of the values ​​specifically recited above.

[0113] In some embodiments, treating a subject with an IdeS mutant protein prior to re-administration of a gene therapy vector comprises administering a single dose of an IdeS mutant protein to such subject, while in other embodiments, the treatment comprises administering multiple doses of an IdeS mutant protein, such as two, three, four or more doses of such an IdeS mutant protein. Each dose of the multiple doses may be the same dose or a different dose. The time between each of the more than two doses may be the same or a different time. Exemplary non-limiting time periods between administration of any two doses of an IdeS protein variant include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 26, 28, 30, 32, 34, 35, 36, 38, 40, 42, 44, 45, 46, 48 hours or more, or time periods or ranges between and including any of the specifically recited values ​​above. In some embodiments, prior to the second or subsequent administration of an IdeS protein variant of the present disclosure, the subject may be monitored by obtaining at least one sample, e.g., a blood, plasma or serum sample, and testing such sample for nAb or other type of IgG concentration or titer, to determine whether further administration of an IdeS protein variant is desired or required.

[0114] In some embodiments, the methods of the present disclosure further comprise a second step of, after administration to a subject of an IdeS protein variant of the present disclosure in an amount sufficient to reduce the titer of nAbs to a gene therapy vector initially administered to such subject, re-administering to such subject the same type of gene therapy vector or administering to such subject a different vector (such as an AAV vector using a different capsid) that is still recognized by nAbs produced by the subject to the initially administered gene therapy vector. In some embodiments, the period between administration of the IdeS variant (or the last administration, if multiple administrations) and re-administration of the gene therapy vector is at least or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 10 41, 42, 43, 44, 45, 46, 47, 48, 50, 52, 54, 55, 56, 58, 60, 62, 64, 65, 66, 68, 70, 72, 74, 75, 76, 78, 80, 84, 85, 90, 95, 96, 100, 108, 110, 120, 125, 130, 132, 135, 140, 144, 145, 150, 155, 156, 160, 165, 168 hours or 8, 9, 10, 11, 12, 13, 14 days or more, or a period or range between and including any of the specifically recited values ​​aforesaid.

[0115] In some embodiments, the period of time between the first administration of the gene therapy vector and the re-administration of the gene therapy vector is at least or about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years, or 10, 20, 30, 40, 50 or more decades, or a period or range between and including any of the specifically recited values ​​above. In some embodiments, the gene therapy vector is re-administered to the subject at least a second time, prior to which the subject is treated with an IdeS protein variant of the present disclosure in an amount effective to reduce the concentration of nAbs to the vector to a level that does not interfere with, reduce or prevent vector transduction in the subject.

[0116] After undergoing gene therapy, certain subjects may develop an immune response against the product of the gene therapy vector, such as the product of the therapeutic transgene contained by the vector. As a result, subjects who have undergone gene therapy may experience suboptimal or limited duration of treatment effects. To address these issues, a method is further provided in which the IdeS protein variant of the present disclosure is administered to a subject who has been treated with a gene therapy vector to treat or prevent an antibody response against the product expressed by the gene therapy vector.

[0117] In some embodiments, subjects previously administered a gene therapy vector are administered a dose of an IdeS variant of the present disclosure at least or about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / kg of subject body weight, or a dose or range between and including any of the specifically recited values ​​above. In some embodiments, treatment with an IdeS variant protein at the recited dosages is effective to treat or prevent an antibody response to a product expressed by the gene therapy vector. In some embodiments, treatment of a subject previously administered a gene therapy vector with an IdeS protein variant of the present disclosure at the dosages listed is effective to inhibit or reduce effector function of neutralizing IgG antibodies produced by the subject against the product expressed by the gene therapy vector.

[0118] In some embodiments, treatment of a subject previously administered a gene therapy vector with an IdeS protein variant of the present disclosure at the recited dosages increases the titer of nAbs to the product expressed by the gene therapy vector in the subject's blood, plasma or serum by at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or a percentage between and including any of the foregoing specifically recited values. a percent reduction or range, reduce or is effective to reduce the titer of a nAb to a product expressed by a gene therapy vector up to 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, 1:0.1, or less, or to a titer or range value between and including any of the foregoing specifically recited values.

[0119] In some embodiments, a subject previously administered a gene therapy vector may be administered a second or subsequent dose of an IdeS variant of the present disclosure, where the additional dose may be the same as the first dose of the IdeS variant or may be a different dose. In some embodiments, the second or subsequent dose may be administered at any suitable interval to maintain an acceptably low level of nAbs to the product expressed by the gene therapy vector in the subject's blood, plasma, or serum. The interval may be regular, such as weekly or biweekly, or monthly or bimonthly, or every 3, 4, 5, or 6 months, or any other regular interval. Alternatively, in some embodiments, the subject may be monitored by periodically taking samples, such as blood, plasma, or serum, to determine the concentration or titer of antibodies to the product expressed by the gene therapy vector over time, and administering additional doses of the IdeS protein variant as desired or necessary to maintain the concentration or titer of nAbs within a predetermined range. In some embodiments, the subject is a human subject, and the IgG antibodies may be of the IgG1, IgG2, IgG3, or IgG4 subclass.

[0120] In some embodiments, administration of the IdeS protein variant of the present disclosure for treatment or prophylactic purposes as described herein may be performed in conjunction with at least a second type of immunosuppressive therapy. In some embodiments, the second type of immunosuppressive therapy may suppress the innate immune system, B cell activity or function, T cell activity or function, or some other attribute or aspect of the immune system, such as complement activity, antigen presentation, or some other aspect of immune function. The second type of immunosuppressive therapy may be non-specific, such as treatment with a steroid, or may be specific, such as treatment with a monoclonal antibody, one example being rituximab, that targets a specific cell surface protein involved in immune response mechanisms. In some embodiments, the IdeS protein variant of the present disclosure is administered to the subject before, substantially simultaneously with, or after administration of the second type of immunosuppressive therapy to the subject. Often, but not necessarily, the second type of immunosuppressive therapy is administered first before administration of the IdeS protein variant, and such first immunosuppressive therapy relies on administering an immunosuppressant, such as a monoclonal IgG antibody that is itself cleaved in the presence of the IdeS protein variant. Representative, non-limiting examples of immunosuppressants that may be administered in conjunction with an IdeS protein variant of the present disclosure include corticosteroids such as prednisone, calcineurin inhibitors such as tacrolimus or cyclosporine, IMDH inhibitors such as azathioprine, leflunomide or mycophenolate, JAK inhibitors such as tofacitinib, mTOR inhibitors such as sirolimus, everolimus or rapamycin, or others such as abatacept, adalimumab, anakinra, basiliximab, certolizumab, daclizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab or vedolizumab, among others possible.

[0121] subject The subject to which the IdeS protein variants of the present disclosure may be administered as described herein may be any suitable subject. In some embodiments, such subjects include, but are not limited to, humans and non-human primates, such as gibbons, gorillas, chimpanzees, orangutans, or macaques, and other mammals or animals, such as domestic animals (e.g., dogs and cats), livestock animals (e.g., poultry, such as chickens and ducks, or horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). Human subjects may be of any age, including fetuses, newborns, infants, children, juveniles, and adults.

[0122] Pharmaceutical Compositions The present disclosure further provides pharmaceutical compositions comprising an IdeS protein variant of the present disclosure and at least one pharma- ceutically acceptable excipient, vehicle, carrier or diluent. The formulation of such compositions is within the knowledge of one of ordinary skill in the art, taking into consideration factors such as storage conditions, stability, dosage and form, convenience, route of administration and other factors known to those of ordinary skill in the art. Representative non-limiting excipients include pH buffers such as TRIS, phosphate or citrate buffers; antioxidants or reducing agents such as ascorbic acid, methionine or histidine; preservatives such as benzalkonium chloride; stabilizers such as PEG, albumin, gelatin or polyvinylpyrrolidone; monosaccharides, disaccharides or other carbohydrates such as glucose, sucrose, trehalose, mannose or dextran; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; inorganic or organic salts; wetting agents such as glycerol; and surfactants such as Tween®, Triton® or Pluronic®. Representative non-limiting diluents include water, isotonic saline, PBS or Ringer's solution. In some embodiments, the pharmaceutical composition may be provided as an aqueous suspension or solution, as a liposomal formulation, or as a biodegradable polymer system, in lyophilized form for subsequent reconstitution with a diluent. The pharmaceutical composition may be formulated for immediate or sustained release. The pharmaceutical compositions of the present disclosure may be placed into any suitable single-use or multi-use container or packaged for long-term storage, distribution to an end user, or convenient administration to a subject, non-limiting examples of which include vials and plastic IV bags.

[0123] Method of administration The present disclosure further provides a method of administering the pharmaceutical composition of the present disclosure to a subject by any suitable route of administration, including systemic, topical and local delivery routes. Non-limiting examples include administering the pharmaceutical composition continuously or in one or more boluses by intravenous injection or infusion. Other examples include delivery of the pharmaceutical composition comprising the IdeS protein variant to a subject by routes such as parenteral, intradermal, subcutaneous, transdermal, intramuscular, intraarterial, intraperitoneal, intraarticular, intraosseous, intrathecal, intraorbital, intramucosal, intraparenchymal, intrapleural, intrahepatic, via the portal vein, or any other suitable administration route.

[0124] kit The present disclosure further provides a kit comprising packaging material and one or more components therein. The kit may comprise a label or package insert comprising a description of the components or instructions for in vitro, in vivo or ex vivo use of the components therein. The kit may comprise a collection of such components, for example, a vial containing a pharmaceutical formulation comprising an IdeS protein variant of the present disclosure, a separate vial containing a diluent, and a tube and needle for injection. In some embodiments, a kit refers to a physical structure that houses one or more components of the kit. The packaging material may keep the components sterile and may be made from materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.). The label or insert may variously include information indicating the identity of one or more components therein; amount to be administered; clinical pharmacology of the active ingredients, including mechanism of action, pharmacokinetics and pharmacodynamics; indications; possible therapeutic or prophylactic benefits of the active ingredients, compositions or components of the kit; possible adverse side effects, complications or reactions; manufacturer; place and date of manufacture; lot number; expiration date; and warnings to the subject or clinician regarding contraindications or drug interactions. The label or insert may include instructions to the clinician or subject for using one or more of the kit components in a method, use, treatment protocol or treatment regime. The instructions may include dosage, frequency or duration of administration and instructions for carrying out any of the methods, uses, treatment protocols or prophylactic or treatment regimes described herein. The label or insert may include "printed matter", such as paper or cardboard, or may be separate or may be affixed to the component, kit or packaging material (e.g., box) or attached to the ampule, tube or vial containing the kit component.

[0125] The following examples are given for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. EXAMPLES

[0126] Example 1: IdeS mutant optimization This example illustrates the generation and testing of IdeS mutants with improved properties compared to wild-type IdeS.

[0127] Over multiple rounds of optimization, single and multiple substitution mutants were designed based on the published crystal structure of the wild-type IdeS protein (Wenig et.al. PNAS December 14,2004 101(50)17371-76). After expressing and purifying the mutants, the proteins were tested to identify those with improved thermostability and / or potency compared to the wild-type. The results of these screens are shown in Table 3, where the position numbers of the substituted amino acids refer to the wild-type IdeS precursor protein (SEQ ID NO: 1) containing the native secretion signal. The corresponding SEQ ID NOs are listed in Table 1. At least the optimized mutant proteins, designated GBT-IdeS-0037, GBT-IdeS-0045 and GBT-IdeS-0085, showed both higher thermostability and potency compared to the wild-type IdeS. Mutant and control wild-type IdeS (designated GBT-NCC-0005) were expressed with an amino-terminal Met-Gly (MG) rather than the native secretion signal and a carboxy-terminal His tag (SEQ ID NO: 85) to facilitate purification. ND = not determined.

[0128] [Table 3-1]

[0129] [Table 3-2]

[0130] [Table 3-3]

[0131] Example 2: Stability and potency of specific IdeS mutants This example illustrates the improved characteristics of certain IdeS mutants compared to wild-type IdeS.

[0132] Mutant IdeS proteins were generated and evaluated for stability and potency. Enzyme activity was quantified to determine potency by activity ELISA assay. Stability was quantified by differential scanning calorimetry (DSC) after thermal forced degradation in PBS pH 7.2 at 37°C (20 mM sodium phosphate, 400 mM NaCl; neat, 10 microliter injection, samples analyzed in parallel using a YMC-Pack Diol-120, 300 x 8 mm). The results are summarized in Figure 2. The data show that IdeS mutants GBT-IdeS-0037, GBT-IdeS-0045 (same substitutions as PF-07899856), GBT-IdeS-0068 and GBT-IdeS-0085 are more stable than wild-type IdeS (GBT-NCC-0005) at physiological temperatures.

[0133] Further studies were performed to compare the potency and stability of IdeS mutants GBT-IdeS-0045 and GBT-IdeS-0085 compared to GBT-NCC-0005 (similar to wild-type IdeS except that it contains a Met-Gly dipeptide at its N-terminus and a His tag at its C-terminus). Enzyme activity was assessed to determine potency using an activity ELISA assay. Stability was assessed by DSC after thermally forced degradation at 37° C. in PBS pH 7.2 formulation over 7 days. Results are summarized in Tables 4 and 5 below. HMMS = high molecular weight species.

[0134] [Table 4]

[0135] [Table 5]

[0136] Compared to GBT-NCC-0005 (similar to wild-type IdeS), both IdeS protein variants GBT-IdeS-0045 and GBT-IdeS-0085 were more potent against IgG cleavage and showed higher thermal stability as well as reduced aggregation tendency (or improved colloidal stability) at physiological temperatures in PBS.

[0137] Example 3: Ig cleavage specificity of certain IdeS mutants This example illustrates that certain IdeS mutants, like wild-type IdeS, specifically cleave IgG.

[0138] Preparations of purified IgG and IgM proteins were incubated with IdeS mutants GBT-IdeS-0045 and GBT-IdeS-0085, which are identical to wild-type IdeS in the region corresponding to the mature protein (both expressed with an N-terminal Met-Gly dipeptide, GBT-NCC-0005 additionally containing a C-terminal His tag), and with IdeS proteins GBT-NCC-0005 and PF-07826653. After incubation, the reactions were quenched with SDS buffer and loaded onto a denaturing polyacrylamide gel to separate the protein fragments resulting from the antibody digestion, followed by Coomassie dye staining using standard methods. The results of digestion of IgG proteins are shown in Figure 3A. The control lane shows only protein bands corresponding to intact IgG and IdeS. Digestion with wild-type IdeS and each of the IdeS mutants converted the single high molecular weight band corresponding to intact IgG into two smaller bands characteristic of the F(ab')2 and Fc fragments. These results indicate that, like wild-type IdeS, the two IdeS mutants tested also efficiently cleaved IgG. Similar experiments were performed to test whether wild-type IdeS and the IdeS mutants had any activity against IgM antibodies, and the results are shown in FIG. 3B. As expected, wild-type IdeS was unable to digest IgM protein, and each of the two IdeS mutants tested produced similar results. These results indicate that the IdeS mutants GBT-IdeS-0045 and GBT-IdeS-0085 have similar specificity as wild-type IdeS for binding and cleaving IgG, but not other immunoglobulins (specifically IgM).

[0139] Example 4: Pharmacodynamic studies of wild type and IdeS mutants This example illustrates the effectiveness of IdeS mutants compared to wild-type IdeS in degrading IgG in an animal model.

[0140] In this study, the in vivo potency of an IdeS mutant, PF-07899856, was compared to PF-07826653, which is identical to wild-type IdeS in the portion corresponding to the mature protein. The IdeS mutant PF-07899856 has the same substitutions as the mutant GBT-IdeS-0045 compared to wild-type IdeS, but lacks the C-terminal His tag present in the latter. Both PF-07899856 and PF-07826653 were expressed with an amino-terminal Met-Gly dipeptide in place of the native secretory signal peptide, but after expression and purification, the N-terminal Met was missing from the major species of both proteins, presumably removed by an endogenous aminopeptidase. The amino acid sequence of PF-07899856 as originally expressed with Met-Gly is provided by SEQ ID NO: 74, and the amino acid sequence of PF-07826653 is provided by SEQ ID NO: 72. After removal of the N-terminal Met, the amino acid sequence of PF-07899856 is provided by SEQ ID NO: 75, and the amino acid sequence of PF-07826653 is provided by SEQ ID NO: 73. PF-07826653 is referred to as "wild type" for convenience here and in Example 5, even though it differs from mature wild-type IdeS by one or two residues at the amino terminus. Male New Zealand White rabbits were administered a single dose of either PF-07899856 (1 mg / kg IV, n=3) or wild-type IdeS (1 mg / kg IV, n=3) and IgG levels were assessed at various time points post-dose.

[0141] Intact IgG and scIgG were evaluated using the Meso Scale Discovery (MSD) assay platform (Figure 4). In this assay, F(ab')2 goat anti-rabbit IgG specific for F(ab')2 is used as the capture reagent and biotinylated F(ab')2 goat anti-rabbit IgG (Fc fragment specific) is used as the detection agent. Streptavidin ruthenium is used as the detection reagent. This assay detects intact IgG and scIgG, but not F(ab')2 or Fc fragments. For this assay, the standard curve range is 300-0.00508 ng / mL in buffer using purified rabbit IgG control. Upper limit of quantification (ULOQ)=100 ng / mL and lower limit of quantification (LLOQ)=0.015 ng / mL in buffer. In the qualification of the assay, intrinsic QCs (EQCs) were determined and are summarized in Table 6.

[0142] [Table 6]

[0143] Intact IgG was reduced with both wild type IdeS and PF-07899856 treatment, with the average maximum reduction observed 6-24 hours post-dose (Figure 5). Animals treated with IdeS mutant PF-07899856 had less intact IgG when compared to animals treated with wild type IdeS (Figure 5). IgG recovery was observed at 48 and 168 hours post-dose. The percent efficiency of cleavage by wild type IdeS (rabbits 1-3) and IdeS mutant PF-07899856 (rabbits 4-6) is summarized in Table 7.

[0144] [Table 7]

[0145] These results demonstrate that the IdeS mutant PF-07899856 is more effective than wild-type IdeS in reducing IgG in vivo.

[0146] Example 5: Pharmacokinetics (PK) of IdeS mutants This example illustrates the PK of IdeS mutants against IgG in an animal model study.

[0147] The PK of IdeS variant PF-07899856 and wild-type IdeS was evaluated using the Meso Scale Discovery LBA assay (Figure 6). In this assay, goat anti-IdeS polyclonal antibody is used as the capture reagent and ruthenium-labeled goat anti-IdeS polyclonal antibody is used as the detection reagent. For this assay, the ROQ at 100% was 10.0 ng / ml to 1,280 ng / ml. Tables 8 and 9 provide PK data in male New Zealand White rabbits after a single 1 mg / kg IV dose of wild-type IdeS and IdeS variant PF07899856, respectively. The time course of IdeS protein plasma concentrations after single dose administration is provided graphically in Figure 7.

[0148] [Table 8]

[0149] [Table 9]

[0150] These results demonstrate that the PK parameters for the IdeS mutant PF-07899856 and wild-type IdeS were similar.

[0151] Although the disclosed teachings have been described with respect to various applications, methods, kits and compositions, it will be appreciated that various changes and modifications can be made without departing from the scope of the teachings herein and the claims below. The foregoing examples are provided to better illustrate the disclosed teachings, but are not intended to limit the scope of the teachings provided herein. While the teachings of the present invention have been described with respect to these representative embodiments, those skilled in the art will readily appreciate that many variations and modifications of these representative embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the present teachings.

[0152] All references cited herein (including patents, patent applications, articles, textbooks, etc.), and the references cited therein, to the extent they have not already been cited, are incorporated herein by reference in their entirety. In the event that one or more of the incorporated literature and similar material, including but not limited to defined terms, terminology usage, described techniques, etc., differs from or conflicts with this application, this application controls.

[0153] The foregoing description and examples detail certain specific embodiments of the invention and set forth the best mode contemplated by the inventors. However, no matter how detailed the foregoing may appear in text, it will be understood that the invention can be practiced in many ways and that the invention should be construed in accordance with the appended claims and any equivalents thereof.

Claims

1. A cysteine ​​protease that specifically cleaves immunoglobulin G (IgG) antibody molecules, wherein the cysteine ​​protease has higher efficacy or thermal stability compared to wild-type IdeS.

2. T ｏｎｓｅｔ If the value is determined using differential scanning calorimetry, Compared to wild-type IdeS protein, it is at least or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0°C higher, or At least or about 44.0, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45.0, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46.0, 46.1, 46.2, 46.3, 46.4, 46.5, 46.6, 46.7, 46.8, 46.9, 47.0, 47.1, 4 The cysteine ​​protease according to claim 1, wherein the temperature is 7.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48.0, 48.1, 48.2, 48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, 49.0, 49.1, 49.2, 49.3, 49.4, 49.5, 49.6, 49.7, 49.8, 49.9, or 50.0°C.

3. T Ｍ If the value is determined using differential scanning calorimetry, Compared to wild-type IdeS protein, it is at least or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0°C higher, or At least or about 51.0, 51.1, 51.2, 51.3, 51.4, 51.5, 51.6, 51.7, 51.8, 51.9, 52.0, 52.1, 52.2, 52.3, 52.4, 52.5, 52.6, 52.7, 52.8, 52.9, 53.0, 53.1, 53.2, 53.3, 53.4, 53.5, 53.6, 53.7, 53.8, 53.9, 54.0, 54.1, 5 The cysteine ​​protease according to claim 1, wherein the temperature is 4.2, 54.3, 54.4, 54.5, 54.6, 54.7, 54.8, 54.9, 55.0, 55.1, 55.2, 55.3, 55.4, 55.5, 55.6, 55.7, 55.8, 55.9, 56.0, 56.1, 56.2, 56.3, 56.4, 56.5, 56.6, 56.7, 56.8, 56.9, or 57.0°C.

4. If the IgG cleavage efficacy is determined using ELISA and expressed as the IC50 value, Compared to wild-type IdeS, it is at least or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 nM lower, or The cysteine ​​protease according to claim 1, wherein the maximum is 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, or 3.7 nM.

5. The cysteine ​​protease according to claim 1, wherein the amino acid sequence includes, basically consists of, or consists of, any one of the amino acid numbers 1 to 312 of SEQ ID NOs. 2 to 71, or any one of the amino acid numbers 2 to 312 of SEQ ID NOs. 2 to 71, or any one of the amino acid numbers 3 to 312 of SEQ ID NOs. 3 to 71.

6. The cysteine ​​protease according to claim 5, wherein the amino acid sequence contains, basically consists of, or consists of any one of the amino acid sequences of sequence numbers 72 to 82.

7. A pharmaceutical composition comprising a cysteine ​​protease according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

8. A composition for treating a subject requiring treatment or prevention of a disease or disorder characterized by excessive IgG antibodies, comprising the cysteine ​​protease described in claim 6, wherein the composition is administered to the subject in an amount of cysteine ​​protease effective in reducing the concentration of IgG antibodies in the subject's body fluids.

9. The composition according to claim 8, wherein the disease or disorder is an autoimmune disease or disorder, and administration of the composition is effective in treating or preventing the autoimmune disease or disorder.

10. The composition according to claim 9, wherein the effective amount of the cysteine ​​protease is at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / kg of target body weight.

11. The above treatment Effective in reducing the total IgG concentration in the body fluids of the subject by at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or The composition according to claim 9, which is effective in reducing the total IgG concentration in the body fluid of the subject to a maximum of approximately 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, and 0.05 grams / liter.

12. A composition for treating a sensitized subject requiring tissue or organ transplantation, comprising the cysteine ​​protease described in claim 6, wherein the composition is administered to the subject in an amount of cysteine ​​protease that is effective in sufficiently reducing the concentration of anti-HLA antibodies in the subject's body fluids to prevent antibody-mediated rejection of the transplanted tissue or organ.

13. The composition according to claim 12, wherein the organ is a kidney, liver, heart, pancreas, lung, or intestine.

14. The composition according to claim 12, wherein the subject exhibits at least or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the calculated panel reaction antibody assay scores.

15. The above treatment Effective in reducing the concentration of anti-HLA antibodies in the body fluids of the subject by at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or Effective in reducing the calculated panel reaction antibody assay score of the aforementioned serum by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or The composition according to claim 12, which is effective in reducing the calculated panel reaction antibody assay score of the target serum to a maximum of approximately 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

16. The composition according to claim 12, wherein the subject subsequently undergoes a tissue or organ transplant, and the period between the administration of the composition and the subsequent transplant is at least or about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 hours.

17. A composition for treating a subject requiring treatment with a gene therapy vector, comprising the cysteine ​​protease described in claim 6, wherein the composition is administered to the subject in an amount of cysteine ​​protease effective in reducing the concentration of IgG antibodies in the subject's body fluids that are specific to the components of the gene therapy vector.

18. The composition according to claim 17, wherein the IgG antibody is a neutralizing antibody.

19. The composition according to claim 17, wherein the gene therapy vector is a recombinant viral vector, a recombinant adenovirus vector, a recombinant adeno-associated virus vector, or a recombinant lentivirus vector.

20. The titer of the neutralizing antibody is at least or about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:120, 1:125, 1:150, 1:175, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1 :650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1700, 1:1800, 1:1900, 1:2000, 1:2100, 1:2200, 1:2300, 1:2400, 1:2500, 1:2600, 1:2700, 1:2800, 1:2900 or 1:3000, and The treatment is effective in reducing the titer of neutralizing antibodies in the body fluids of the subject by at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or The composition according to claim 18, wherein the treatment is effective in reducing the titer of the neutralizing antibody in the body fluid of the subject to a maximum value of 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.

1.

21. The composition according to claim 18, wherein the effective amount of the cysteine ​​protease is at least 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45, or 0.50 mg / kg of target body weight.

22. The composition according to claim 18, wherein the subject is gene therapy treatment naive or has been previously treated at least once with the same type of gene therapy vector in need of treatment.

23. The composition according to any one of claims 8, 12, or 17, wherein the administration of the composition to the subject is repeated at least once.

24. A polynucleotide encoding a cysteine ​​protease according to any one of claims 1 to 6.

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