MUTANT CyaA POLYPEPTIDES AND POLYPEPTIDE DERIVATIVES SUITABLE FOR THE DELIVERY OF IMMUNOGENIC MOLECULES INTO A CELL

Abstract

The invention relates to mutant CyaA/E570Q+K860 polypeptides suitable for use as proteinaceous vectors for delivering one or more molecules of interest into a cell, in particular into a cell expressing the COM receptor. The invention further relates to polypeptide derivatives suitable for eliciting an immune response in a host. The invention is more particularly directed to polypeptides derived from an adenylate cyclase protein (CyaA) either under the form of a toxin or of a toxoid, which are mutant polypeptides. Said mutant polypeptides are capable of retaining the binding activity of native CyaA to a target cell and preferably of also retaining the translocating activity of native CyaA through its N-terminal domain into target cells and furthermore have a pore-forming activity which is reduced or suppressed as compared to that of the native CyaA toxin.

1 . A Polypeptide which is a mutant of an adenylate cyclase protein (mutant polypeptide) and whose amino add sequence comprises or consists of one of the following sequences:
 a) the amino acid sequence of the adenylate cyclase (CyaA) of  Bordetella pertussis, Bordetella parapertussis  or  Bordetella hinzii  wherein the following mutations have been performed:
 (i) the substitution of the glutamic acid residue at position 570 by a glutamine residue (E570Q) or by a conservative amino acid residue, and 
 (ii) the substitution of the lysine residue at position 860 by an arginine residue (K860R) or by a conservative amino acid residue, or; 
   b) an amino acid sequence of a fragment of the adenylate cyclase of  Bordetella pertussis, Bordetella parapertussis  or  Bordetella hinzii,  which fragment has the capacity of the CyaA protein of  Bordetella pertussis  to bind to a target cell and the capacity to translocate its N-terminal adenylate cyclase enzyme domain or part thereof into said cell, wherein said fragment further contains the following mutated amino acid residues located at positions 570 and 860 in said adenylate cyclase: E570Q and K860R, or   c) an amino acid sequence which differs from the amino add sequence as defined in a) or b) by one or more amino acid residue substitutions and/or insertions and which has the capacity of the CyaA protein of  Bordetella Pertussis  to bind to a target cell and the capacity to translocate its N-terminal adenylate cyclase enzyme domain or part thereof into said cell, wherein said amino acid sequence further contains the following mutated amino acid residues located at positions 570 and 860 in said adenylate cyclase: E570Q and K860R, or   d) the amino acid sequence of the adenylate cyclase (CyaA) of  Bordetella bronchiseptica  wherein the following mutations have been performed:
 (i) the substitution of the glutamic acid residue at position 569 by a glutamine residue (E569Q) or by a conservative amino acid residue, and 
 (ii) the substitution of the lysine residue at position 859 by an arglnine residue (K859R) or by a conservative amino acid residue, or 
   e) an amino acid sequence of a fragment of the adenylate cyclase of  Bordetella bronchiseptica,  which fragment has the capacity of the CyaA protein of  Bordetella pertussis  to bind to a target cell and the capacity to translocate its N-terminal adenylate cyclase enzyme domain or part thereof into said cell, wherein said fragment further contains the following mutated amino acid residues located at positions 569 and 859 in said adenylate cyclase: E569Q and K859R, or   f) an amino acid sequence which differs from the amino acid sequence as defined in d) or e) by one or more amino acid residue substitutions and/or insertions and which has the capacity of the CyaA protein of  Bordetella Pertussis  to bind to a target cell and the capacity to translocate its N-terminal adenylate cyclase enzyme domain or part thereof into said cell, wherein said amino acid sequence further contains the following mutated amino acid residues located at positions 569 and 859 in said adenylate cyclase: E569Q and K859R.   
     
     
         2 . Polypeptide according to  claim 1 , wherein the amino acid of said adenylate cyclase of  Bordetella Pertussis  adenylate cyclase is disclosed as SEQ ID N° 1. 
     
     
         3 . Polypeptide according to  claim 2 , wherein the amino acid sequence of said polypeptide further differs from that of SEQ ID N° 1 by 1 to 200 amino acid residue substitutions, deletions, and/or insertions. 
     
     
         4 . Polypeptide according to  claim 1  or  2 , which is capable of binding to cells and of translocating its N-terminal adenylate cyclase enzyme domain into said cells wherein said cells express the CD11b/CD18 receptor and wherein binding to said cells occurs through binding to said CD11b/CD18 receptor. 
     
     
         5 . Polypeptide according to  claim 1 ,  2 ,  3  or  4 , which is a mutant of an adenylate cyclase toxoid whose adenylate cyclase activity in cells is partly or totally suppressed as compared to that of the  Bordetella pertussis  CyaA toxin. 
     
     
         6 . Polypeptide according to  claim 5 , wherein said partial or total suppression of adenylate cyclase activity is achieved by insertion of a dipeptide between the amino acid residues at positions 188 and 189 of the adenylate cyclase of  Bordetella Pertussis,  especially of SEQ ID N° 1. 
     
     
         7 . Polypeptide derivative comprising or consisting of the polypeptide according to any of  claims 1  to  6  and which is further combined with one or more molecules of interest. 
     
     
         8 . Polypeptide derivative according to  claim 7 , wherein each of said one or more molecules of interest consists of an amino acid sequence suitable for eliciting an immune response. 
     
     
         9 . Polypeptide derivative according to  claim 8 , wherein the amino acid sequence of each of said molecule(s) suitable for eliciting an immune response consists of 5 to 800 especially 300 to 600, or 400 to 500 amino acid residues. 
     
     
         10 . Polypeptide derivative according to  claim 8  or  9 , wherein the amino acid sequence of each of said molecule(s) suitable for eliciting an immune response comprises or consists of an amino acid sequence of a poliovirus antigen, an HIV virus antigen, an influenza virus antigen, a choriomeningitis virus sequence, a tumor antigen, or comprises or consists of a part of an amino acid sequence of any these antigens which comprises at least one epitope. 
     
     
         11 . Polypeptide derivative according to  claims 8  to  10 , which is a recombinant polypeptide wherein the amino acid sequence of each of said molecule(s) suitable for eliciting an immune response is inserted into a permissive site of the adenylate cyclase amino acid sequence of the mutant polypeptide, thereby preserving the capacity of said mutant polypeptide to translocate its N-terminal adenylate cyclase enzyme domain into target cells. 
     
     
         12 . Polypeptide derivative according to  claim 8  or  9 , wherein each of said amino acid sequences suitable for eliciting an immune response is grafted, especially chemically grafted, onto an amino acid residue of said mutant polypeptide. 
     
     
         13 . Polypeptide according to  claims 1  to  6  or polypeptide derivative according to any of  claims 7  to  12  for use in therapy. 
     
     
         14 . Polypeptide according to  claims 1  to  6  or polypeptide derivative according to any of  claims 7  to  12  for use in therapy in order to elicit a T-cell immune response and/or in order to elicit a B-cell immune response in a host in need thereof. 
     
     
         15 . Polypeptide derivative according to anyone of  claims 7  to  14  for the prevention or the treatment of a disease selected from neoplasia, cancers and infectious diseases selected from viral-, retroviral-, bacterial-, parasite- or fungal-induced diseases. 
     
     
         16 . Polypeptide or polypeptide derivative according to  claim 13 ,  14  or  15 , wherein said polypeptide is to be administered in combination with an adjuvant and/or in combination with another therapeutically active molecule. 
     
     
         17 . Polypeptide or polypeptide derivative according to  claim 13  or  14 , wherein said polypeptide is not to be administered in combination with an adjuvant. 
     
     
         18 . Pharmaceutical composition comprising a polypeptide according to any of  claims 1  to  6  or a polypeptide derivative according to any one of  claims 7  to  17 , a pharmaceutically acceptable carrier, and optionally an adjuvant and/or a therapeutically active molecule. 
     
     
         19 . Use of a polypeptide derivative according to any one of  claims 7  to  12 , for the preparation of a therapeutic composition for the treatment of a disease selected from neoplasia, cancers and infectious diseases selected from viral- or retroviral-induced diseases. 
     
     
         20 . Method for the preparation of a proteinaceous vector suitable for the delivery of a molecule into a cell comprising binding said molecule to a polypeptide according to any of  claims 1  to  6 .