Novel antibody molecules and nucleic acids binding to fungal stress protein hsp90

Abstract

An scFv peptide comprising a V H domain and a V L domain linked by an amino acid spacer is disclosed. The V H domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO. 64. The V L domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO. 66. The scFv peptide comprises an additional feature selected from (a) to (e) and combinations thereof. (a) A substitution or deletion of an amino acid in the V H domain at a position corresponding to that selected from the group consisting of: I 29 , H 68 , N 85 , C 97 and combinations thereof. (b) A substitution or deletion of an amino acid in the V L domain at a position corresponding to that selected from the group consisting of: V 2 , V 3 , F 10 , F 14 , A 39 , N 76 and combinations thereof. (c) The amino acid spacer comprises the sequence (GGGGS) n wherein n is between 4 and 6. (d) The V H domain further comprises an N-terminal pelB signal sequence comprising the sequence of SEQ ID NO. 68 or a sequence having at least 80% sequence identity thereto. (e) The V L domain is located at the N-terminal end of the V H domain. (f) The substitution of the cysteine residue in the V H domain corresponding to position 28 with a serine residue.

1 . An scFv peptide comprising a V H  domain and a V L  domain linked by an amino acid spacer, wherein the V H  domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO. 64 and the V L  domain comprises a sequence with at least 80% sequence identity to the sequence of SEQ ID NO. 66 and wherein the scFv peptide comprises an additional feature selected from the group consisting of:
 (a) a substitution or deletion of an amino acid in the V H  domain at a position corresponding to that selected from the group consisting of: I 29 , H 68 , N 85 , C 97  and combinations thereof;   (b) a substitution or deletion of an amino acid in the V L  domain at a position corresponding to that selected from the group consisting of: V 2 , V 3 , F 10 , F 14 , A 39 , N 76  and combinations thereof;   (c) the amino acid spacer comprises the sequence (GGGGS) n  wherein n is between 4 and 6;   (d) the V H  domain further comprises an N-terminal pelB signal sequence comprising the sequence of SEQ ID NO. 68 or a sequence having at least 80% sequence identity thereto;   (e) the V L  domain is located at the N-terminal end of the V H  domain;   (f) the substitution of the cysteine residue in the V H  domain at the position corresponding to position 28 with a serine residue; and   (g) combinations of features (a) to (f).   
     
     
         2 . An scFv peptide according to  claim 1  wherein the scFv peptide comprises the additional feature selected from (a) to (e) or a combination thereof and further comprises the substitution of the cysteine residue in the V H  domain at the position corresponding to position 28 with a tyrosine residue. 
     
     
         3 . An scFv peptide according to  claim 1  or  2  wherein the substitution of the amino acid in the V H  domain is selected from the group consisting of: C 28 S, I 29 S, H 68 R, N 85 S, C 97 Y, C 97 S and combinations thereof. 
     
     
         4 . An scFv peptide according to any one of the preceding claims wherein the substitution of the amino acid in the V L  domain is selected from the group consisting of: V 2 I, V 3 Q, F 10 S, F 14 S, A 39 K, N 76 S and combinations thereof. 
     
     
         5 . An scFv peptide according to any one of the preceding claims further comprising a purification tag, preferably a sequence of 6 histidine residues at the C-terminus. 
     
     
         6 . An scFv peptide according to any one of the preceding claims wherein the amino acid spacer comprises the sequence (GGGGS) n . 
     
     
         7 . An scFv peptide according to any one of the preceding claims comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 12, 16, 20, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 and 62 wherein Xaa denotes an amino acid residue other than cysteine and wherein the N-terminal methionine residue may optionally be cleaved off. 
     
     
         8 . An scFv peptide according to any one of  claims 1  to  6  comprising an amino acid sequence selected from the group consisting of SEQ. ID NO. 8, 10, 14, 18 and 22, wherein Xaa denotes the amino acid residue serine and wherein the N-terminal methionine residue may optionally be cleaved off. 
     
     
         9 . A nucleic acid molecule comprising a sequence encoding an scFv peptide according to any one of the preceding claims. 
     
     
         10 . A nucleic acid molecule according to  claim 9  further comprising the sequence (taa) n  located at the 3′ end of the sequence encoding the scFv peptide wherein n is 1 or 2. 
     
     
         11 . A nucleic acid molecule comprising a sequence encoding a V H  domain comprising a sequence having at least 80% sequence identity to the sequence of SEQ ID NO. 64 and a V L  domain comprising a sequence having at least 80% sequence identity to the sequence of SEQ ID NO. 66 and further comprising the sequence (taa) n  located at the 3′ end of the sequence encoding the V H  or V L  domains wherein n is 1 or 2. 
     
     
         12 . A nucleic acid molecule according to any one of  claims 9  to  11  wherein the molecule comprises a sequence selected from the group consisting of: SEQ ID NO. 3, 5, 11, 15, 19, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59 and 61 wherein nnn denotes a codon coding for an amino acid other than a cysteine residue. 
     
     
         13 . A nucleic acid molecule according to any one of  claims 9  to  11  wherein the molecule comprises a sequence selected from the group consisting of: SEQ. ID NO. 7, 9, 13, 17 and 21, wherein nnn denotes a codon coding for a serine residue. 
     
     
         14 . A nucleic acid molecule according to any one of  claims 9  to  13  wherein the molecule is a DNA or RNA molecule. 
     
     
         15 . A pharmaceutical composition comprising an scFv peptide according to any one of  claims 1  to  8  in combination with a pharmaceutically acceptable excipient, diluent or carrier. 
     
     
         16 . A combined preparation comprising an scFv peptide according to any one of  claims 1  to  8  and an antifungal agent or an anticancer agent. 
     
     
         17 . A composition comprising an scFv peptide according to any one of  claims 1  to  8  and an antifungal agent or an anticancer agent. 
     
     
         18 . A method of treating a patient with a fungal infection comprising administering to the patient an effective amount of an scFv peptide according to any one of  claims 1  to  8 . 
     
     
         19 . A method according to  claim 18  further comprising the step of administering to the patient an effective amount of an antifungal agent. 
     
     
         20 . An scFv peptide according to any one of  claims 1  to  8  for use in the treatment of a fungal infection. 
     
     
         21 . The method according to  claim 18  or  19  or the scFv peptide according to  claim 20  wherein the fungal infection is due to a  Candida, Cryptococcus, Histoplasma, Aspergillus, Torulopsis, Mucormycosis, Blastomycosis, Coccidioidomycosis or Paracoccidioidomycosis organism.    
     
     
         22 . The combined preparation according to  claim 16 , the composition according to  claim 17 , the method according to  claim 18 ,  19  or  21  or the scFv peptide according to  claim 20  or  21  wherein the antifungal agent is an azole antifungal, preferably selected from the group consisting of: itraconazole, voriconazole isavuconazole, fluconazole, miconazole, ketoconazole; and posaconazole. 
     
     
         23 . The combined preparation according to  claim 16 , the composition according to  claim 17 , the method according to  claim 18 ,  19  or  21  or the scFv peptide according to  claim 20  or  21  wherein the antifungal agent is selected from the group consisting of: a polyene antifungal agent and an echinocandin antifungal agent. 
     
     
         24 . The combined preparation, composition, method or scFv peptide according to  claim 23  wherein the polyene antifungal agent comprises amphotericin B or a derivative thereof. 
     
     
         25 . A method of treating a patient with a cancer disease comprising administering to the patient in need an effective amount of a scFv peptide according to any one of  claims 1  to  8 . 
     
     
         26 . A method according to  claim 25  further comprising the step of administering to the patient an effective amount of at least one other anticancer agent. 
     
     
         27 . An scFv peptide according to any one of  claims 1  to  8  for use in the treatment of a cancer disease. 
     
     
         28 . The combined preparation according to  claim 16 , the composition according to  claim 17 , the method according to  claim 25  or  26  or the scFv peptide according to  claim 27  wherein the anticancer agent is selected from the group consisting of: Doxorubicin, Daunorubicin, Epirubicin, Herceptin, Docetaxel, Cisplatin, Imatinib, Paclitaxel, Docetaxel, Hydroxyurea, 5-fluorouracil, Oxaliplatin, Irinotecan, Cytarabine and Raltitrexed. 
     
     
         29 . A method of treating a patient with of a condition involving raised levels of TNFα and/or IL-6 comprising administering to the patient an effective amount of a scFv peptide according to any one of  claims 1  to  8 . 
     
     
         30 . An scFv peptide according to any one of  claims 1  to  8  for use in the treatment of a condition involving raised levels of TNFα and/or IL-6. 
     
     
         31 . A method according to  claim 29  or an scFv peptide according to  claim 30  wherein the condition involving raised levels of TNFα and/or IL-6 is selected from the group consisting of: sepsis, SIRS and an autoimmune disease. 
     
     
         32 . A method or scFv peptide according to  claim 31  wherein the autoimmune disease is selected from the group consisting of: Crohn's disease, rheumatoid arthritis, ulcerative colitis and systemic lupus erythematosus. 
     
     
         33 . A vector molecule comprising a nucleotide sequence according to any one of  claims 9  to  14 . 
     
     
         34 . A host cell comprising a vector molecule according to  claim 33 . 
     
     
         35 . A method for producing a scFv peptide according to any one of  claims 1  to  8  which comprises culturing a host cell having incorporated therein an expression vector containing, under control of suitable transcriptional control elements, a nucleic acid molecule according to any one of  claims 9  to  14  under conditions sufficient for expression of said peptides in the host cell thereby causing the production of said peptide and recovering the peptide produced by said cell.