Modified bordetella adenylate cyclase comprising or lacking CD11b/CD18 interaction domain and uses thereof

Abstract

The invention relates to modified Bordetella adenylate cyclase toxins which are deficient for CD11b/CD18 binding and to their use in the preparation of pharmaceutical composition for the treatment of whooping cough and/or for the protection against Bordetella infection. The invention also relates to specific fragments of Bordetella adenylate cyclase comprising the CD11b/CD18 interaction domain and their use, especially for targeting a molecule of interest to CD11b expressing cells.

1 . A protein consisting of a  Bordetella  adenylate cyclase which is modified in the CD11b/CD18 interaction domain by one or more amino acid deletion, substitution or insertion, wherein said protein is deficient for CD11b/CD18 binding and is specifically reactive with antisera recognizing a wild-type  Bordetella  adenylate cyclase.  
     
     
         2 . The protein of  claim 1 , wherein said  Bordetella  adenylate cyclase is modified by the complete deletion of the CD11b/CD18 interaction domain.  
     
     
         3 . The protein of  claim 1  or  2 , wherein the CD11b/CD18 interaction domain is modified by insertion of a peptide therein.  
     
     
         4 . The protein according to any one of  claims 1  to  3 , wherein the  Bordetella  adenylate cyclase is further modified by insertion, deletion or substitution of one or more amino acid in the N-terminal catalytic domain, wherein said modified  Bordetella  adenylate cyclase has a catalytic activity which is decreased as compared to the wild-type  Bordetella  adenylate cyclase activity.  
     
     
         5 . The protein according to  claim 4 , wherein said  Bordetella  adenylate cyclase is modified by deletion of at least amino acid residues 1 to 300 of the N-terminal catalytic domain and preferably the deletion of the amino acid residues 1 to 373.  
     
     
         6 . The protein according to  claim 4 , wherein said  Bordetella  adenylate cyclase is modified in the amino acids which are acylated posttranslationally, these amino acids corresponding to Lys 983 and Lys 860 of the  B. pertussis  adenylate cyclase.  
     
     
         7 . The protein according to any one of  claims 1  to  6 , wherein said  Bordetella  adenylate cyclase is the  Bordetella pertussis  adenylate cyclase.  
     
     
         8 . A pharmaceutical composition comprising the protein according to any one of  claims 1  to  7  in combination with a pharmaceutically acceptable vehicle.  
     
     
         9 . The composition of  claim 8  for use as a vaccine, comprising an immunoprotective and non-toxic quantity of the protein according to any one of  claims 1  to  7 .  
     
     
         10 . The composition of  claim 9 , wherein said composition further comprises one or more suitable priming adjuvants.  
     
     
         11 . The use of the protein according to any one of  claims 1  to  7 , in the preparation of a medicament for the prevention or treatment, in human or in an animal, of disease symptoms associated with whooping cough and/or for protecting a human or an animal against the disease symptoms associated with  Bordetella  infection.  
     
     
         12 . A polypeptide capable of binding to CD11b/CD18, said polypeptide being either 
 a. a fragment of a  Bordetella  adenylate cyclase having from 30 to 500 amino acids, preferably from 50 to 300, and more preferably from 50 to 150 amino acids, said fragment comprising a wild type CD11b/CD18 interaction domain of said  Bordetella  adenylate cyclase, or comprising a fragment of said wild type CD11b/CD18 interaction domain sufficient to retain the capacity to bind to CD11b/CD18,    b. a variant of said fragment having at least 70% identity preferably 80% and more preferably 90% identity with said fragment, wherein said variant retains the capacity to bind to CD11b/CD18.    
     
     
         13 . The polypeptide of  claim 12 , wherein said polypeptide is capable of raising antibodies recognizing specifically  Bordetella  species adenylate cyclase, preferably  Bordetella pertussis  adenylate cyclase.  
     
     
         14 . The polypeptide according to any one of claims  12  or  13 , wherein said polypeptide is a fragment of the  Bordetella pertussis  adenylate cyclase, preferably the fragment extending from amino acid 1166 to amino acid 1281 of the  B. pertussis  adenylate cyclase, and more preferably a fragment comprising the region extending from amino acid 1208 to amino acid 1243 of the  B. pertussis  adenylate cyclase.  
     
     
         15 . The polypeptide according to any one of  claims 12  to  14 , which further comprises an acylation domain of adenylate cyclase and/or the hydrophobic domain.  
     
     
         16 . The polypeptide according to any one of  claims 12  to  15 , wherein said polypeptide is not toxic when administered in vivo to a mammal.  
     
     
         17 . Use of the polypeptide according to any one of  claims 12  to  16 , in the preparation of medicament for the prevention or treatment, in human or in an animal, of disease symptoms associated with whooping cough and/or for protecting a human or an animal against the disease symptoms associated with  Bordetella  infection.  
     
     
         18 . Use of the polypeptide according to any one of  claims 12  to  16 , in the preparation of a vector for targeting a molecule of interest specifically to CD11 b expressing cells.  
     
     
         19 . A vector for targeting a molecule of interest to CD11b/CD18 expressing cells, characterized in that said vector comprises the polypeptide capable of binding to CD11b/CD18 according to any one of  claims 12  to  16 , coupled to a molecule of interest.  
     
     
         20 . The vector according to  claim 19 , wherein said molecule of interest is selected among the group consisting of: peptides, glycopeptides, lipopeptides, polysaccharides, oligosaccharides, nucleic acids, lipids and chemicals.  
     
     
         21 . The vector according to  claim 19  or  20 , wherein said molecule of interest is the active principle of a medicament.  
     
     
         22 . The vector according to any one of  claims 19  to  21 , wherein said molecule of interest is coupled by chemical linkage.  
     
     
         23 . The vector according to any one of  claims 19  to  22 , wherein said molecule of interest comprises an antigen or an epitope.  
     
     
         24 . The vector according to  claim 23 , wherein said molecule of interest is a peptide or a polypeptide comprising an antigen or an epitope.  
     
     
         25 . A nucleic acid which encodes one of the following polypeptides: 
 a. the protein according to any one of  claims 1  to  7 ;    b. the polypeptide according to any one of  claims 12  to  16 ;    c. the vector according to  claim 24 .    
     
     
         26 . A recombinant nucleic acid constituted by the nucleic acid of  claim 25  cloned into an expression vector appropriate for the expression of the encoded polypeptide in a host cell.  
     
     
         27 . The recombinant nucleic acid according to  claim 26 , wherein said expression vector is a plasmid, a cosmid, a phagemid or viral DNA.  
     
     
         28 . A host cell, comprising the nucleic acid according to  claim 25  or the recombinant nucleic acid according to  claim 26  or  27 .  
     
     
         29 . A pharmaceutical composition, comprising the polypeptide according to any one of  claims 12  to  16 , the vector according to any one of  claims 19  to  24 , in combination with a pharmaceutically acceptable vehicle.  
     
     
         30 . The pharmaceutical composition, comprising the vector according to any one of claims  23  or  24 , wherein the molecule of interest is the active principle of a medecine.  
     
     
         31 . The pharmaceutical composition of  claim 30 , wherein said composition is a vaccine.  
     
     
         32 . A polyclonal serum obtainable by the immunization of an animal or a human with the polypeptide according to any one of  claims 12  to  16 , or with a composition according to any one of  claims 29  to  31 .  
     
     
         33 . A monoclonal antibody directed specifically against the polypeptides of  claims 12  to  16 .  
     
     
         34 . The polyclonal serum according to  claim 32 , or the monoclonal antibody according to  claim 33 , wherein said polyclonal serum or said monoclonal antibody is capable of blocking the binding of adenylate cyclase to CD11 b/CD18.  
     
     
         35 . A pharmaceutical composition, comprising the polyclonal serum according to  claim 32  or the monoclonal antibody according to  claim 33 , in combination with a pharmaceutically acceptable vehicle.  
     
     
         36 . The use of a polyclonal serum or a monoclonal antibody according to  claim 35 , in the preparation of medicament for the prevention or treatment, in human or in an animal, of disease symptoms associated with whooping cough and/or for protecting a human or an animal against the disease symptoms associated with  Bordetella  infection.  
     
     
         37 . A method for in vitro targeting a molecule of interest to CD11b expressing cells, said method comprising 
 a. providing CD11b expressing cells extracted from a living organism    b. culturing said CD11b expressing cells with the vector of any of  claims 19  to  24  under appropriate conditions for targeting said vector to said CD11 b expressing cells.    
     
     
         38 . The method of  claim 37 , wherein said molecule of interest comprises an antigen or an epitope.  
     
     
         39 . The method of  claim 37  or  38 , wherein said CD11b expressing cells are myeloid dendritic cells.  
     
     
         40 . CD11 b-expressing cells comprising a molecule of interest obtainable by the method of any of  claims 37  to  39 .  
     
     
         41 . CD11b-expressing cells comprising an antigen or an epitope obtainable by the method of  claim 38 .  
     
     
         42 . A cell therapy product for immunizing a human or an animal against an antigen, characterized in that it comprises an efficient amount of CD11 b expressing cells according to  claim 41 , in combination with a pharmaceutically acceptable vehicle.  
     
     
         43 . A use of CD11b-expressing cells according to  claim 41 , in the preparation of a cell therapy product for immunizing a human or an animal against an antigen.  
     
     
         44 . A method for immunizing a patient against an antigen, said method comprising, 
 a. extracting CD11b expressing cells from said patient,    b. in vitro culturing said CD11 b expressing cells with a vector according to  claim 23  or  24 , under condition appropriate for targeting said vector to said cells,    c. re-administering an efficient amount of said cells comprising said vector into said patient to prime a CD4+ and/or CD8+ response,    thereby immunizing said patient to said antigen.    
     
     
         45 . The method of  claim 44 , wherein said CD11b-expressing cells are myeloid dendritic cells.