Cysteine protease

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.

1 . A polypeptide 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 50% 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 optionally   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 histidine (H), an aspartic acid (D) and an aspartic acid (D), respectively; 
 wherein said polypeptide is more effective at cleaving human IgG than IdeZ and/or is at least as effective at cleaving human IgG as IdeS. 
     
     
         2 . A polypeptide according to  claim 1 , wherein 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, optionally wherein said positively charged amino acid is arginine (R) or lysine (K); and/or   (2) has a positively charged amino acid at the position in said variant which corresponds to position 139 of SEQ ID NO: 3, optionally wherein said positively charged amino acid is arginine (R) or lysine (K); 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 leucine (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 position 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 position 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. 
   
     
     
         3 . A polypeptide according to  claim 1 , wherein said variant of the sequence of SEQ ID NO: 4 or 5 is at least 80%, 90%, 95% or 99% identical to SEQ ID NO: 4 or 5, respectively, and/or wherein said polypeptide is less immunogenic than IdeS when measured in the same assay. 
     
     
         4 . A polypeptide according to  claim 1 , which comprises or consists of the sequence of any one of SEQ ID NOs: 6 to 25, optionally wherein said sequence includes an additional methionine at the N terminus and/or a histidine tag at the C terminus. 
     
     
         5 . A polypeptide according to  claim 1 , wherein said polypeptide is at least 2.0 fold more effective than IdeZ at cleaving human IgG, when measured in the same assay. 
     
     
         6 . A polypeptide according to  claim 1  which is less immunogenic than IdeS, wherein the immunogenicity of said polypeptide is no more than 85% of the immunogenicity of IdeS when measured in the same assay. 
     
     
         7 . A polynucleotide or expression vector which comprises a nucleic acid sequence encoding a polypeptide according to  claim 1  or a composition comprising a polypeptide according to  claim 1  and at least one pharmaceutically acceptable carrier or diluent. 
     
     
         8 . A host cell comprising the polynucleotide or expression vector of  claim 7 . 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . A method for the prevention or treatment of a disease or condition in a subject, which method comprises administering to the subject a polypeptide according to  claim 1  to the subject in a prophylactically or therapeutically effective amount or a method for the cleavage of IgG, the method comprising contacting a sample containing IgG with a polypeptide according to  claim 1  under conditions which permit IgG cysteine protease activity to occur. 
     
     
         12 . A method according to  claim 11 , wherein said disease or condition is: 
 a) a disease or condition mediated in whole or in part by pathogenic IgG antibodies; or   b) cancer.   
     
     
         13 . (canceled) 
     
     
         14 . A method for the cleavage of IgG according to  claim 11  which is carried out ex vivo and/or is conducted to generate Fc and Fab fragments and/or wherein the sample is a blood sample taken from a subject suffering from a disease or condition mediated in whole or in part by pathogenic IgG antibodies. 
     
     
         15 . A method to improve the benefit to a subject of a therapy or a therapeutic agent, the method comprising steps (a) and (b), wherein:
 step (a) comprises administering to the subject the polypeptide according to  claim 1 ; and   step (b) comprises subsequently administering to the subject the said therapy or therapeutic agent; 
 optionally wherein steps (a) and (b) are separated by a time interval sufficient for cleavage of substantially all IgG molecules present in the plasma of the subject to take place. 
     
     
         16 . A method according to  claim 12 , wherein the therapy is an organ transplant, or wherein the therapeutic agent is an antibody for the treatment of cancer. 
     
     
         17 . A method for the treatment of cancer or another disease in a subject, the method comprising (a) administering to the subject the polypeptide according to  claim 1 ; and (b) subsequently administering to the subject a therapeutically effective amount of an antibody which is a treatment for said cancer or said other disease; wherein:
 the amount of said polypeptide administered is sufficient to cleave substantially all IgG molecules present in the plasma of the subject; and   steps (a) and (b) are separated by a time interval of at least 2 hours and at most 21 days.   
     
     
         18 . A method according to  claim 10 , wherein the cancer is Acute lymphoblastic leukemia, Acute myeloid leukemia, Adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, Anal cancer, Appendix cancer, Astrocytoma, childhood cerebellar or cerebral, Basal cell carcinoma, Bile duct cancer, extrahepatic, Bladder cancer, Bone cancer, Osteosarcoma/Malignant fibrous histiocytoma, Brainstem glioma, Brain cancer, Brain tumor, cerebellar astrocytoma, Brain tumor, cerebral astrocytoma/malignant glioma, Brain tumor, ependymoma, Brain tumor, medulloblastoma, Brain tumor, supratentorial primitive neuroectodermal tumors, Brain tumor, visual pathway and hypothalamic glioma, Breast cancer, Bronchial adenomas/carcinoids, Burkitt lymphoma, Carcinoid tumor, Carcinoid tumor, gastrointestinal, Carcinoma of unknown primary, Central nervous system lymphoma, Cerebellar astrocytoma, Cerebral astrocytoma/Malignant glioma, Cervical cancer, Chronic lymphocytic leukemia, Chronic myelogenous leukemia Chronic myeloproliferative disorders, Colon Cancer, Cutaneous T-cell lymphoma, Desmoplastic small round cell tumor, Endometrial cancer, Ependymoma, Esophageal cancer, Ewing’s sarcoma in the Ewing family of tumors, Extracranial germ cell tumor, Childhood, Extragonadal Germ cell tumor, Extrahepatic bile duct cancer, Eye Cancer, Intraocular melanoma, Eye Cancer, Retinoblastoma, Gallbladder cancer, Gastric (Stomach) cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal stromal tumor (GIST), Germ cell tumor: extracranial, extragonadal, or ovarian, Gestational trophoblastic tumor, Glioma of the brain stem, Glioma, Childhood Cerebral Astrocytoma, Glioma, Childhood Visual Pathway and Hypothalamic, Gastric carcinoid, Hairy cell leukemia, Head and neck cancer, Heart cancer, Hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, Hypothalamic and visual pathway glioma, Intraocular Melanoma, Islet Cell Carcinoma (Endocrine Pancreas), Kaposi sarcoma, Kidney cancer (renal cell cancer), Laryngeal Cancer, Leukemias, Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia), Leukemia, acute myeloid (also called acute myelogenous leukemia), Leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia), Leukemia, chronic myelogenous (also called chronic myeloid leukemia), Leukemia, hairy cell, Lip and Oral Cavity Cancer, Liposarcoma, Liver Cancer (Primary), Lung Cancer, Non-Small Cell Lung Cancer, Small Cell, Lymphomas, Lymphoma, AIDS-related, Lymphoma, Burkitt, Lymphoma, cutaneous T-Cell, Lymphoma, Hodgkin, Lymphomas, Non-Hodgkin (an old classification of all lymphomas except Hodgkin’s), Lymphoma, Primary Central Nervous System, Macroglobulinemia, Waldenström, Malignant Fibrous Histiocytoma of Bone/Osteosarcoma, Medulloblastoma, Melanoma, Melanoma, Intraocular (Eye), Merkel Cell Carcinoma, Mesothelioma, Adult Malignant, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Mouth Cancer, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Diseases, Myelogenous Leukemia, Chronic, Myeloid Leukemia, Adult Acute, Myeloid Leukemia, Childhood Acute, Myeloma, Multiple (Cancer of the Bone-Marrow), Myeloproliferative Disorders, Nasal cavity and paranasal sinus cancer, Nasopharyngeal carcinoma, Neuroblastoma, Non-Hodgkin lymphoma, Non-small cell lung cancer, Oral Cancer, Oropharyngeal cancer, Osteosarcoma/malignant fibrous histiocytoma of bone, Ovarian cancer, Ovarian epithelial cancer (Surface epithelial-stromal tumor), Ovarian germ cell tumor, Ovarian low malignant potential tumor, Pancreatic cancer, Pancreatic cancer, islet cell, Paranasal sinus and nasal cavity cancer, Parathyroid cancer, Penile cancer, Pharyngeal cancer, Pheochromocytoma, Pineal astrocytoma, Pineal germinoma, Pineoblastoma and supratentorial primitive neuroectodermal tumors, Pituitary adenoma, Plasma cell neoplasia/Multiple myeloma, Pleuropulmonary blastoma, Primary central nervous system lymphoma, Prostate cancer, Rectal cancer, Renal cell carcinoma (kidney cancer), Renal pelvis and ureter, transitional cell cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma, Ewing family of tumors, Kaposi Sarcoma, Sarcoma, soft tissue, Sarcoma, uterine, Sézary syndrome, Skin cancer (nonmelanoma), Skin cancer (melanoma), Skin carcinoma, Merkel cell, Small cell lung cancer, Small intestine cancer, Soft tissue sarcoma, Squamous cell carcinoma, Squamous neck cancer with occult primary, metastatic, Stomach cancer, Supratentorial primitive neuroectodermal tumor, T-Cell lymphoma, cutaneous - see Mycosis Fungoides and Sézary syndrome, Testicular cancer, Throat cancer, Thymoma, Thymoma and Thymic carcinoma, Thyroid cancer, Thyroid cancer, Transitional cell cancer of the renal pelvis and ureter, Trophoblastic tumor, Ureter and renal pelvis, transitional cell cancer Urethral cancer, Uterine cancer, endometrial, Uterine sarcoma, Vaginal cancer, Visual pathway and hypothalamic glioma, Vulvar cancer, Waldenström macroglobulinemia and Wilms tumor (kidney cancer);
 optionally wherein the cancer is prostate cancer, breast cancer, bladder cancer, colon cancer, rectal cancer, pancreatic cancer, ovarian cancer, lung cancer, cervical cancer, endometrial cancer, kidney (renal cell) cancer, oesophageal cancer, thyroid cancer, skin cancer, lymphoma, melanoma or leukemia. 
 
     
     
         19 . The polypeptide of  claim 3 , wherein said polypeptide is no more immunogenic than IdeZ or IdeS/Z when measured in the same assay. 
     
     
         20 . The host cell of  claim 8 , wherein the cell is a bacterial cell. 
     
     
         21 . The host cell of  claim 20 , wherein the bacterial cell is E. coli. 
     
     
         22 . The method of  claim 12 , wherein said disease or condition is listed in Table D. 
     
     
         23 . The method of  claim 14 , wherein said disease or condition is listed in Table D. 
     
     
         24 . The method of  claim 15 , wherein the polypeptide is administered to the subject in an amount sufficient to cleave substantially all IgG molecules present in the plasma of the subject.