Expression system

Abstract

Methods for the production of a bacterial toxin such as diphtheria toxin, and methods for producing conjugates of a bacterial toxins, are provided.

1 . A process for producing a conjugate of a bacterial toxin, comprising:
 (1) growing in a fermentor a culture of gram negative host cells containing a polynucleotide comprising:
 (a) a 3′ toxin sequence encoding a mature bacterial toxin polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO:32, and 
 (b) a 5′ signal sequence encoding a signal peptide, wherein the signal peptide directs transport of said bacterial toxin polypeptide to the bacterial periplasm when expressed in said host cell, and wherein the 5′ signal sequence is not derived from  Corynebacterium diphtheriae  ( C. diphtheriae ); and 
   inducing expression of said polynucleotide such that said bacterial toxin polypeptide is expressed periplasmically; and   (2) purifying said bacterial toxin polypeptide; and   (3) conjugating said purified bacterial toxin to an antigen.   
     
     
         2 . The process of  claim 1  wherein the 3′ toxin sequence encodes a polypeptide selected from the group consisting of:
 (a) a polypeptide having the amino acid sequence of SEQ ID NO: 32, 
 (b) a polypeptide having at least 95% sequence identity to SEQ ID NO: 32; 
 (c) a polypeptide comprising SEQ ID NO:31; and 
 (d) CRM197. 
 
     
     
         3 . The process of  claim 1  wherein the polynucleotide encodes a polypeptide comprising any one of SEQ ID NOs: 33-45. 
     
     
         4 . The process of  claim 1  wherein the 5′ signal sequence is directly 5′ of the 3′ toxin sequence of the polynucleotide. 
     
     
         5 . The process of  claim 1  wherein the 5′ signal sequence encodes a signal peptide having an amino acid sequence selected from:
 (a) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26; 
 (b) variants of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 containing 1, 2 or 3 point mutations, insertions or deletions, which variants direct transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell; and 
 (c) fragments of at least 10 amino acids of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, which fragments direct transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell. 
 
     
     
         6 . The process of  claim 1  wherein the 5′ signal sequence comprises a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25. 
     
     
         7 . The process of  claim 1  where said antigen of step (3) is a capsular bacterial saccharide from a bacterium selected from the group consisting of  Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis , group B  Streptococcus , group A  Streptococcus, Salmonella , Enterococci, and  Staphylococcus aureus.    
     
     
         8 . The process of  claim 7 , where the activated saccharide is conjugated to said bacterial toxin via a linker. 
     
     
         9 . The process of  claim 7 , where conjugation of said purified bacterial toxin to an antigen comprises a process selected from:
 (a) direct reductive amination;   (b) activation of the saccharide by 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP);   (c) activation of the saccharide by cyanogen bromide;   (d) activation of the saccharide by 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) followed by derivitisation of the activated saccharide with adipic acid dihydrazide (ADH); and   (e) activation of the saccharide by cyanogen bromide followed by derivitisation of the activated saccharide with ADH.   
     
     
         10 . A process for producing a conjugate of a bacterial toxin, comprising:
 (1) growing in a fermentor a culture of gram negative host cells containing a polynucleotide comprising:
 (a) a 3′ toxin sequence encoding a mature bacterial toxin polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO:32, and 
 (b) a 5′ signal sequence encoding a signal peptide, wherein the signal peptide directs transport of said bacterial toxin polypeptide to the bacterial periplasm when expressed in said host cell, and wherein the 5′ signal sequence is not derived from  Corynebacterium diphtheriae  ( C. diphtheriae ); and 
   inducing expression of said polynucleotide such that said bacterial toxin polypeptide is expressed periplasmically; and   (2) purifying said bacterial toxin polypeptide; and   (3) conjugating said purified bacterial toxin to an antigen.   
     
     
         11 . The process of  claim 10  wherein the 3′ toxin sequence encodes a polypeptide selected from the group consisting of:
 (a) a polypeptide having the amino acid sequence of SEQ ID NO: 32; 
 (b) a polypeptide having at least 95% sequence identity to SEQ ID NO: 32; 
 (c) a polypeptide comprising SEQ ID NO:31; and 
 (d) CRM197. 
 
     
     
         12 . The process of  claim 10  wherein the polynucleotide encodes a polypeptide comprising any one of SEQ ID NOs: 33-45. 
     
     
         13 . The process of  claim 10  wherein the 5′ signal sequence is directly 5′ of the 3′ toxin sequence of the polynucleotide. 
     
     
         14 . The process of  claim 10 , wherein the 5′ signal sequence encodes a signal peptide having an amino acid sequence selected from:
 (a) SEQ ID NO: 24, and 
 (b) variants of SEQ ID NO: 24, varying from the corresponding sequence by 1, 2, or 3 point mutations, amino acid insertions, or amino acid deletions, which variants direct transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell, and 
 (c) fragments of at least 10 amino acids of SEQ ID NO: 24, which fragments direct transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell. 
 
     
     
         15 . The process of  claim 10  wherein the 5′ signal sequence encodes a signal peptide having an amino acid sequence selected from:
 (a) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26; 
 (b) variants of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 containing 1, 2 or 3 point mutations, insertions or deletions, which variants direct transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell; and 
 (c) fragments of at least 10 amino acids of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, which fragments direct transport of said bacterial toxin polypeptide to the periplasm of said bacterial host cell. 
 
     
     
         16 . The process of  claim 10  wherein the 5′ signal sequence comprises a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25. 
     
     
         17 . The process of  claim 10  where said antigen of step (3) is a capsular bacterial saccharide from a bacterium selected from the group consisting of  Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis , group B  Streptococcus , group A  Streptococcus, Salmonella , Enterococci, and  Staphylococcus aureus.    
     
     
         18 . The process of  claim 17 , where the activated saccharide is conjugated to said bacterial toxin via a linker. 
     
     
         19 . The process of  claim 17 , where conjugation of said purified bacterial toxin to an antigen comprises a process selected from:
 (a) direct reductive amination;   (b) activation of the saccharide by 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP);   (c) activation of the saccharide by cyanogen bromide;   (d) activation of the saccharide by 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) followed by derivitisation of the activated saccharide with adipic acid dihydrazide (ADH); and   (e) activation of the saccharide by cyanogen bromide followed by derivitisation of the activated saccharide with ADH.   
     
     
         20 . A process for manufacturing an immunogenic composition comprising the steps of:
 (a) producing a conjugate of a bacterial toxin according to  claim 1 ; and   (b) mixing the said conjugate with a component selected from a pharmaceutically acceptable excipient, a further antigen, and an adjuvant.   
     
     
         21 . A process for manufacturing an immunogenic composition comprising the steps of:
 (a) producing a conjugate of a bacterial toxin according to  claim 17 ; and   (b) mixing the said conjugate with a component selected from a pharmaceutically acceptable excipient, a further antigen, and an adjuvant.   
     
     
         22 . A process for the production of a periplasmically expressed bacterial toxin, comprising:
 (a) in a fed-batch phase, growing a culture of a gram-negative host cell comprising a polynucleotide comprising a 3′ toxin portion encoding a bacterial toxin and a 5′ signal portion encoding a polypeptide that directs transport of said bacterial toxin to the host cell periplasm, wherein the pH is from 6.0-7.0; and   (b) inducing expression of said bacterial toxin at a pH that is, and is maintained at, a pH from 6.5-8.5, wherein said pH is at least 0.5 pH units higher than the pH in step A; and   
       the substrate feed rate is maintained at between 20% and 80% of the substrate feed rate of step A; 
       wherein the bacterial toxin has at least 90% sequence identity to SEQ ID NO:32 and the process is carried out in a fermentor containing from 10-2000 liters of culture. 
     
     
         23 . The process of  claim 22  wherein step (a) is carried out at a temperature of between 20-40° C. and step (b) is carried out at a temperature of between 20-28° C., wherein the temperature of step (a) is higher than step (b). 
     
     
         24 . The process of  claim 22  wherein the pH is maintained using a buffer selected from the group consisting of phosphate buffer, Tris buffer, and histidine buffer. 
     
     
         25 . The process of  claim 22  wherein the pH of step (b) is achieved by the addition of sodium hydroxide or ammonia. 
     
     
         26 . The process of  claim 22  wherein expression of said bacterial toxin in step (b) is induced by the addition of IPTG. 
     
     
         27 . The process of  claim 22  wherein the dissolved oxygen level is between 15-25%. 
     
     
         28 . The process of  claim 22  further comprising: (c) purifying the bacterial toxin. 
     
     
         29 . A process for making a bacterial toxin conjugate comprising the steps of (a) making a bacterial toxin using the process of  claim 22  and (b) conjugating the bacterial toxin produced in step (a) to an antigen. 
     
     
         30 . The process of  claim 29  wherein the antigen is a bacterial saccharide. 
     
     
         31 . The process of  claim 30  wherein the antigen is a capsular saccharide selected from  S. pneumoniae, H. influenzae, N. meningitidis , and  S. aureus.    
     
     
         32 . A process for manufacturing a vaccine comprising the steps of:
 (a) making a bacterial toxin using the process of  claim 22 ;   (b) conjugating the bacterial toxin produced in step a) to an antigen; and   (c) mixing the bacterial toxin conjugate produced in step (b) with a pharmaceutically acceptable excipient.