NZ733962B2 - Cysteine protease - Google Patents

Abstract

The present invention relates to a novel polypeptide which displays IgG cysteine protease activity, and in vivo and ex vivo uses thereof. Uses of the polypeptide include methods for the prevention or treatment of diseases and conditions mediated by IgG, and methods for the analysis of IgG.

Claims (18)

1. A ptide having IgG cysteine protease activity and comprising a variant of the sequence of SEQ ID NO:4 or 5, which variant: (a) is at least 80% identical to SEQ ID NO: 4 or 5; (b) has a cysteine (C) at the position in said variant sequence which corresponds to position 102 of SEQ ID NO: 3; and (c) has, at the positions in said variant sequence which correspond to positions 92, 272, 294 and 296 of SEQ ID NO: 3, a lysine (K), a ine (H), an aspartic acid (D) and an aspartic acid (D), tively; wherein said polypeptide is more effective at cleaving human IgG than IdeZ and/or is at least as ive at cleaving human IgG as IdeS; and n said variant of the sequence of SEQ ID NO: 4 or 5: (1) has a positively charged amino acid at the position in said variant which corresponds to position 138 of SEQ ID NO: 3; and/or (2) has a positively charged amino acid at the position in said variant which corresponds to on 139 of SEQ ID NO: 3; and/or (3) does not include the contiguous sequence DDYQRNATEA YAKEVPHQIT; and/or (4) has at least one of the following modifications: i. a deletion of the e (L) and threonine (T) residues at the positions in said variant which correspond to positions 64 and 65 of SEQ ID NO: 3; ii. a threonine (T) in place of the arginine (R) at the position in said variant which corresponds to position 70 of SEQ ID NO: 3; iii. a deletion of the tyrosine (Y) at the position in said variant which corresponds to position 71 of SEQ ID NO: 3; iv. a glutamine (Q) in place of the asparagine (N) at the on in said variant which corresponds to position 72 of SEQ ID NO: 3; v. a glycine (G) in place of the asparagine (N) at the position in said variant which corresponds to on 73 of SEQ ID NO: 3; vi. a alanine (A) in place of the glutamic acid (E) at the position in said variant which corresponds to position 67 of SEQ ID NO: 3; vii. a asparagine (N) in place of the glutamine (Q) at the position in said variant which corresponds to position 68 of SEQ ID NO: 3.

2. A polypeptide according to claim 1, wherein the positively charged amino acid at the position in said t which corresponds with position 138 of SEQ ID NO: 3 is arginine (R) or lysine (K), and/or wherein the positively charged amino acid at the position in said variant which corresponds with position 139 of SEQ ID NO: 3 is arginine (R) or lysine (K).

3. A polypeptide according to claim 1 or 2, wherein said variant of the sequence of SEQ ID NO: 4 or 5 is at least 90%, 95% or 99% identical to SEQ ID NO: 4 or 5, tively, and/or wherein said polypeptide is less immunogenic than IdeS.

4. A polypeptide according to claim 3 which is no more immunogenic than IdeZ or , when measured in the same assay.

5. A polypeptide according to any one of the preceding claims, which comprises or consists of the sequence of any one of SEQ ID NOs: 6 to 25.

6. A polypeptide according to any one of the preceding claims, wherein said sequence includes an additional methionine at the N terminus and/or a histidine tag at the C us.

7. A polypeptide according to any one of the ing claims, wherein said polypeptide is at least 2.0 fold more effective than IdeZ at cleaving human IgG, when measured in the same assay.

8. A polypeptide according to any one of the preceding claims which is less immunogenic than IdeS.

9. A polypeptide according to claim 8, wherein the immunogenicity of said polypeptide is no more than 85% of the immunogenicity of IdeS when ed in the same assay.

10. A polynucleotide or expression vector which comprises a nucleic acid sequence ng a polypeptide of any one of the preceding claims.

11. A host cell comprising the polynucleotide or expression vector of claim 10, wherein the host cell excludes a host cell found in vivo in a human.

12. A host cell according to claim 11 which is a bacterial cell.

13. A host cell ing to claim 11 or claim 12 which is a cell of E. coli.

14. A composition comprising a ptide according to any one of claims 1 to 9 and at least one pharmaceutically acceptable carrier or diluent.

15. A use of a polypeptide according to any one of claims 1 to 9 in the manufacture of a medicament for (a) treating a disease or condition mediated in whole or in part by pathogenic IgG antibodies or (b) improving the benefit to a subject of a gene therapy or organ transplant.

16. A use according to claim 15, wherein the disease or condition is ed from Cancer, n’s disease, Anti-GBM glomerulonephritis, Anti-neutrophil cytoplasmic antibody- associated vasculitides, Anti-NMDAR Encephalitis, Anti-phospholipid antibody syndrome (APS), Catastrophic APS, Autoimmune bullous skin diseases, Pemphigus foliaceus, fogo selvagem, pemphigus is, Autoimmune tic anemia, Autoimmune hepatitis, Autoimmune neutropenia, Bullous pemphigoid, Celiac disease, Chronic utricaria, Complete ital heart block, Diabetes type 1A, Epidermolysis bullosa acquisita, Essential mixed cryoglobulinemia, Goodpasture’s me, Goitre, hyperthyroidism, infiltrative exopthalmos and infiltrative dermopathy, Guillain-Barré syndrome, Acute inflammatory demyelinating polyneuropathy, acute motor axonal athy, Hemophilia, Acquired FVIII deficiency, Idiopathic thrombocytopenic purpura, Lambert-Eaton enic syndrome, Mixed Connective Tissue Disease, le a, Myasthenia gravis, Myasthenic crisis, Myocarditis, dilated cardiomyopathy, Neuromyelitis Optica, Primary biliary cirrhosis, Primary Progressive Multiple Sclerosis, Rheumatic heart disease, Rheumatoid Arthritis, Serum-sickness, immune complex hypersensitivity (type III), Systemic Lupus Erythematosis, Lupus nephritis, Stiff-person syndrome, Transplant ion or Thrombotic Thrombocytopenic Purpura..

17. An ex vivo method for the cleavage of IgG, the method comprising contacting a sample containing IgG with a polypeptide according to any one of claims 1 to 9 under conditions which permit IgG cysteine protease activity to occur.

18. A method according to claim 17 which is conducted to generate Fc and Fab fragments and/or wherein the sample is a blood sample taken from a subject ing from a disease or condition as defined in claim 15 or claim 16. HGURE1 Ra?o?fgé??ilzé g; 9.3 (:3 {:33 12:3 HGUREZ 4 SU WWWWWWMWWWWWWu‘mm‘m.wmWWWMWWWWWWWWWWW 6‘91} m 3-50 WW .WWWWWWWWWWM 2.30 WWWWWWWW $3 233,0 WWWWWWWW M m w W a , 3330 mm W_WW_MWMWW pCAR’T1:24 MR?144 pCARYZC?Z {GOAR7333 pCéRTm BX