Stable bactericidal/permeability-increasing protein products and pharmaceutical compositions containing the same

Abstract

Disclosed are novel bactericidal/permeability-increasing (BPI) protein products wherein cysteine residue number 132 or 135 is replaced by another amino acid residue, preferably an alanine or serine residue and/or wherein the leucine residue at position 193 is the carboxy terminal residue. Also disclosed are DNA sequences encoding methods for the production of the same in appropriate host cells, and stable homogeneous pharmaceutical compositions containing the analogs suitable for use treatment of gram negative bacterial infection and its sequelae.

1 . A polypeptide analog of bactericidal/permeability-increasing protein or biologically-active fragment thereof wherein a cysteine residue at position 132 or at position 135 is replaced by a different amino acid.  
     
     
         2 . The polypeptide analog of  claim 1  wherein the amino acid replacing said cysteine residue is a non-polar amino acid selected from the group consisting of alanine and serine.  
     
     
         3 . The polypeptide analog of  claim 1  wherein the cysteine residue at position 132 is replaced by alanine.  
     
     
         4 . The polypeptide analog of  claim 1  wherein the cysteine residue at position 135 is replaced by serine.  
     
     
         5 . The polypeptide analog according to claims  1 ,  2 ,  3 , or  4  comprising, except for the cysteine replacement at either position 132 or 135, from 176 to 199 of the amino terminal residues of bactericidal/permeability-increasing protein.  
     
     
         6 . The polypeptide analog according to  claim 5  comprising the initial 193 amino terminal amino acid residues of bactericidal/permeability-increasing protein.  
     
     
         7 . A DNA encoding a polypeptide analog of bactericidal/permeability-increasing protein or a biologically active fragment thereof wherein a cysteine residue at position 132 or at position 135 is replaced by a different amino acid.  
     
     
         8 . The DNA of  claim 7  encoding the thirty-one amino acid leader sequence and the first 193 N-terminal residues of bacterial/permeability-increasing protein and having a stop codon immediately following the codon for the leucine residue at position 193:  
     
     
         9 . The DNA of  claim 7  encoding the thirty-one amino acid leader sequence and the first 199 N-terminal residues of bactericidal/permeability-increasing protein and having a stop codon immediately following the codon for the isoleucine at position 199.  
     
     
         10 . An autonomously replicating DNA vector comprising a DNA according to  claim 7 .  
     
     
         11 . A host cell stably transformed or transfected with DNA according to  claim 7  in a manner allowing expression in said host cell of said polypeptide analog.  
     
     
         12 . A eukaryotic host cell according to  claim 11 .  
     
     
         13 . A host cell according to  claim 12  selected from the group consisting of ATCC CRL 11246 and ATCC HB11247.  
     
     
         14 . A method for producing polypeptide analogs of bactericidal/permeability increasing protein and biologically active fragments thereof comprising growing a host cell according to  claim 11  in a suitable culture medium and isolating said analog from said host cell or said culture medium.  
     
     
         15 . A hybrid fusion protein comprising, at its amino terminal, an analog polypeptide according to  claim 1  and, at its carboxyl terminal, at least one constant domain of an immunoglobulin heavy chain or an allelic variation thereof.  
     
     
         16 . A DNA encoding a hybrid fusion protein according to  claim 15 .  
     
     
         17 . An autonomously replicating DNA vector comprising a DNA according to  claim 16 .  
     
     
         18 . A host cell stably transformed or transfected with a DNA according  claim 16  in a manner allowing expression in said host cell of said fusion polypeptide.  
     
     
         19 . A method for producing hybrid fusion protein according to  claim 15  comprising growing a host cell according to  claim 18  in a suitable culture medium and isolating said analog from said host cell or said culture medium.  
     
     
         20 . A pharmaceutical composition comprising an analog polypeptide according to  claim 1  and a pharmaceutically-acceptable diluent, adjuvant, or carrier.  
     
     
         21 . A pharmaceutical composition comprising a hybrid fusion protein according to  claim 15  and a pharmaceutically acceptable diluent adjuvant, or carrier.  
     
     
         22 . A method of treating bacterial infection or the sequelae thereof comprising administering an effective amount of the pharmaceutical composition of claims  20  or  21 .  
     
     
         23 . A DNA encoding a biologically active fragment of bactericidal/permeability increasing protein having a leucine residue at position 193 as its carboxy terminal residue.  
     
     
         24 . The DNA according to  claim 23  encoding the thirty-one amino acid leader sequence and first 193 N-terminal amino acids of bacterial/permeability-increasing protein and having a stop codon immediately following the codon for the leucine residue at position 193.  
     
     
         25 . An autonomously replicating DNA vector comprising a DNA according to  claim 22 .  
     
     
         26 . A host cell stably transformed or transfected with a DNA according to  claim 23  in manner allowing expression in said host cell.  
     
     
         27 . A method for producing a biologically active bactericid al/permeability increasing protein fragment comprising growing a host cell stably transformed or transfected with DNA according to  claim 23  in a suitable culture medium and isolating said fragment from said host cell or said culture medium.