Coversin Variants Lacking C5 Binding

Abstract

The invention is directed to modified Coversin polypeptides which exhibit leukotriene or hydroxyeicosanoid binding activity and reduced or absent C5 binding relative to the unmodified Coversin polypeptide; to nucleic acid molecules encoding said modified Coversin polypeptides; vectors and host cells comprising said nucleic acid molecules; and methods of treating or preventing diseases or conditions mediated by a leukotriene or hydroxyeicosanoid in a subject comprising administering said modified polypeptides or nucleic acids to a subject.

1 - 51 . (canceled) 
     
     
         52 . A method of treating or preventing a disease or condition mediated by a leukotriene or hydroxyeicosanoid in a subject comprising administering to said subject a modified Coversin polypeptide or a fragment thereof in which up to five amino acids are deleted from the N terminus of said modified Coversin polypeptide,
 wherein said modified Coversin polypeptide comprises SEQ ID NO: 3 in which from 1 to 30 amino acid substitutions are made, wherein
 (i) in positions 114 to 124 of SEQ ID NO: 3 the following substitutions are made:
 a. Met116 is replaced with Gln, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr; 
 b. Leu117 is replaced with Ser, Asp, Asn, Glu, Arg, Lys, Gly, Ala, or Pro; 
 c. Gly121 is replaced with Ala, Asp, Asn, Glu, Arg, Lys, Leu, Ile, Phe, Tyr, Met, Pro, or His; 
 d. Leu122 is replaced with Asp, Glu, Asn, Ala, Gln, Arg, Lys, Pro, or His; 
 e. Glu123 is replaced with Ala, Asp, Gln, Asn, Arg, Lys, Gly, Leu, Ser, Ile, Phe, Tyr, Pro, His, or Thr; and wherein 
 
 (ii) Ala44 in SEQ ID NO: 3 is replaced with Asn, Asp, Gln, Glu, Arg, Lys, Leu, Ile, Phe, Tyr, Met, Pro, or His; and wherein 
 (iii) Asp149 in SEQ ID NO: 3 is replaced with Gly, Gln, Asn, Ala, Met, Arg, Lys, Leu, Ser, Ile, Phe, Tyr, Pro, His, or Thr. 
   
     
     
         53 . The method of  claim 52 , wherein said modified Coversin polypeptide exhibits leukotriene or hydroxyeicosanoid binding activity and exhibits reduced or absent C5 binding. 
     
     
         54 . The method of  claim 53 , wherein:
 a. Trp115 is not substituted; or   b. Met114, Trp115, Asp118, Ala119, Gly120 and Val124 are not substituted.   
     
     
         55 . The method of  claim 53 , wherein in positions 114 to 124 of SEQ ID NO: 3:
 a. Met116 is replaced with Gln;   b. Leu117 is replaced with Ser;   c. Gly121 is replaced with Ala;   d. Leu122 is replaced with Asp; and   e. Glu123 is replaced with Ala.   
     
     
         56 . The method of  claim 55 , wherein Ala44 in SEQ ID NO: 3 is replaced with Asn and/or Asp149 in SEQ ID NO: 3 is replaced with Gly. 
     
     
         57 . The method of  claim 56 , wherein:
 a. Trp115 is not substituted; or   b. Met114, Trp115, Asp118, Ala119, Gly120 and Val124 are not substituted.   
     
     
         58 . The method of  claim 53 , wherein the modified Coversin polypeptide or fragment thereof has a loop sequence between amino acid positions 114 to 124 of SEQ ID NO:3 as set out in SEQ ID NO:11. 
     
     
         59 . The method of  claim 58 , wherein the modified Coversin polypeptide or fragment thereof comprises or consists of SEQ ID NO: 6. 
     
     
         60 . The method of  claim 58 , wherein the modified Coversin polypeptide or fragment thereof comprises or consists of a variant of SEQ ID NO: 6 which has 1-20, 2-15, or 3-10 additional substitutions compared to SEQ ID NO:3 beyond those that are set out in SEQ ID NO:6. 
     
     
         61 . The method of  claim 53 , wherein in the six cysteine amino acids at positions 6, 38, 100, 128, 129, 150 of SEQ ID NO: 3 are retained in unmodified form. 
     
     
         62 . The method of  claim 53 , wherein Asn60 and Asn84 are each replaced with Gln. 
     
     
         63 . The method of  claim 53 , wherein:
 a. one or more, or all, of the following amino acids is not substituted: Phe18, Tyr25, Arg36, Leu39, Gly41, Pro43, Leu52, Val54, Met56, Phe58, Thr67, Trp69, Phe71, Gln87, Arg89, His99, His101, Asp103, and Trp115;   b. none of amino acids 5, 6, 11, 13-15, 20-21, 24-27, 29-32, 35-41, 45, 47-48, 50, 52-60, 64, 66, 69-81, 83, 84, 86, 90-94, 97-104, 112-113, 115, 125-129, 132-139, 145, 148, and 150 in SEQ ID NO:3 is substituted;   c. none of amino acids 5, 6, 11, 13-15, 18, 20-21, 24-27, 29-32, 35-41, 43, 45, 47-48, 50, 52-60, 64, 66, 67, 69-81, 83, 84, 86, 87, 89, 90-94, 97-104, 112-113, 115, 125-129, 132-139, 145, 148, and 150 in SEQ ID NO:3 are substituted; and/or   d. none of amino acids 5, 6, 11, 13-15, 18, 20-21, 24-25, 27, 30-32, 35-41, 43, 47-48, 50, 52-60, 64, 66, 67, 69-81, 83, 84, 86, 87, 89, 90-94, 98, 100, 102-104, 112-113, 115, 126, 128-129, 132-139, 145, 148, and 150 in SEQ ID NO:3 is substituted.   
     
     
         64 . The method of  claim 53 , wherein the modified Coversin polypeptide or fragment thereof binds to LTB 4 . 
     
     
         65 . The method of  claim 53 , wherein the disease comprises: acne, Addison's disease, allergic conjunctivitis, allergic rhinitis, alpha-1 antitrypsin disease (AATD), aneurysms, anterior uveitis, arteritis, arthritis, asthma, atherosclerosis, atopic dermatitis, atopic keratoconjunctivitis, autoimmune blistering diseases, autoimmune uveitis, bacterial keratitis, blepharokeratoconjunctivitis, breast cancer, bronchiolitis obliterans syndrome (BOS), bronchitis, cancer, cancer metastases, chronic B lymphocytic leukaemia, chronic obstructive pulmonary disease (COPD), Churg-Strauss Syndrome, colon cancer, contact dermatitis, contact hypersensitivity, coronary heart disease, Crohn's disease, cystic fibrosis, dementia, diabetic nephropathy, Down's syndrome, exercise and aspirin induced asthma, fibromyalgia, Fuch's corneal dystrophy, fungal keratitis, glomerulonephritis, Goodpasture's Syndrome, gout, graft versus host disease (GVHD), graft versus host syndrome dry eye, hereditary keratoconus, idiopathic pulmonary disease (IPD), inflammatory bowel disease, ischaemia reperfusion injury, juvenile arthritis, keratoconjunctivitis sicca, keratoconus, lattice corneal dystrophy, Leber's congenital amaurosis, lung cancer, malaria, Map-dot-fingerprint corneal dystrophy, mucuous membrane pemphigoid, multiple sclerosis (MS), myocardial infarction, nephritis, neuropathy, obliterative bronchiolitis, obstructive sleep apnea syndrome, ocular herpes simplex or herpes zoster, ocular lupus erythematosus, ocular rosacea, oesophageal cancer, oesophageal adenocarcinoma, Ehlers-Danlos syndrome, osteoarthritis, osteoclastic arthritis, osteogenesis imperfecta, ovarian cancer, pancreatic adenocarcinoma, pancreatic cancer, perennial keratoconjunctivitis, periodontal disease, photokeratitis, post-inflammatory pigmentation, post-menopausal osteoporosis, prostate cancer, psoriasis, psoriatic arthritis, Pterygium, pulmonary arterial hypertension (PAH), radiation induced gastrointestinal inflammation, restenosis after coronary angioplasty, retinitis of prematurity, retinitis pigmentosa, rheumatoid arthritis, sarcoidosis, scleroderma interstitial lung disease, sclerodermia, sepsis, severe persistent asthma, sickle cell disease (SCD), silicosis, Sjogren's dry eye, spondyloarthropathies, stroke, systemic lupus eyrthematosus (SLE), systemic sclerosis, trachoma, trauma, traumatic Keratoconus, tumor metastasis, type 2 diabetes, ulcerative colitis, uveitis, vasculitides, vernal keratoconjunctivitis, or viral keratitis. 
     
     
         66 . The method of  claim 53 , wherein the modified Coversin polypeptide or fragment thereof is genetically or chemically fused to one or more peptides or polypeptides. 
     
     
         67 . The method of  claim 66 , wherein the one or more peptides or polypeptides comprises:
 a. a PAS sequence; or   b. a PAS sequence consisting of 30 copies of SEQ ID NO:13, fused to the N terminus of the modified Coversin polypeptide.   
     
     
         68 . A method of treating or preventing a disease or condition mediated by a leukotriene or hydroxyeicosanoid in a subject comprising administering to said subject a nucleic acid molecule, wherein the nucleic acid molecule encodes a modified Coversin polypeptide or a fragment thereof in which up to five amino acids are deleted from the N terminus of said modified Coversin polypeptide,
 wherein said modified Coversin polypeptide comprises SEQ ID NO: 3 in which from 1 to 30 amino acid substitutions are made, wherein
 (i) in positions 114 to 124 of SEQ ID NO: 3 the following substitutions are made:
 a. Met116 is replaced with Gln, Asp, Asn, Glu, Arg, Lys, Gly, Ala, Pro, His, or Thr; 
 b. Leu117 is replaced with Ser, Asp, Asn, Glu, Arg, Lys, Gly, Ala, or Pro; 
 c. Gly121 is replaced with Ala, Asp, Asn, Glu, Arg, Lys, Leu, Ile, Phe, Tyr, Met, Pro, or His; 
 d. Leu122 is replaced with Asp, Glu, Asn, Ala, Gln, Arg, Lys, Pro, or His; 
 e. Glu123 is replaced with Ala, Asp, Gln, Asn, Arg, Lys, Gly, Leu, Ser, Ile, Phe, Tyr, Pro, His, or Thr; and wherein 
 
 (ii) Ala44 in SEQ ID NO: 3 is replaced with Asn, Asp, Gln, Glu, Arg, Lys, Leu, Ile, Phe, Tyr, Met, Pro, or His; and wherein 
 (iii) Asp149 in SEQ ID NO: 3 is replaced with Gly, Gln, Asn, Ala, Met, Arg, Lys, Leu, Ser, Ile, Phe, Tyr, Pro, His, or Thr. 
   
     
     
         69 . The method of  claim 68 , wherein said modified Coversin polypeptide exhibits leukotriene or hydroxyeicosanoid binding activity and reduced or absent C5 binding. 
     
     
         70 . The method of  claim 69 , wherein the modified Coversin polypeptide or fragment thereof is genetically or chemically fused to one or more peptides or polypeptides.