Methods and composition for secretion of heterologous polypeptides

Abstract

The present invention relates generally to the fields of molecular biology and protein technology. More specifically, the invention concerns signal sequences for the secretion of heterologous polypeptide from bacteria. The invention also concerns recombinant polypeptides and uses thereof.

1 - 5 . (canceled) 
     
     
         6 . A method of making an antibody, said method comprising culturing a host cell comprising a polynucleotide comprising (1) a first inducible promoter operably linked to a first TIR operably linked to a polynucleotide encoding an antibody heavy chain, wherein the TIR comprises a DsbA variant co-translational prokaryotic secretion signal sequence; and (2) a second inducible promoter operably linked to a second TIR operably linked to a polynucleotide encoding an antibody light chain, wherein the second TIR comprises a co-translational or post-translational prokaryotic secretion signal sequence, inducing expression of the antibody heavy chain and antibody light chain, whereby upon expression of the antibody in the host cell, at least 50% by molar ratio of the heavy and light chains are folded and assembled to form a biologically active antibody, wherein the host cell is an  E. coli  cell. 
     
     
         7 - 8 . (canceled) 
     
     
         9 . The method of  claim 6 , wherein the first translation initiation region comprises sequence of one of SEQ ID NOs: 36-42. 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 6 , wherein the second translation initiation region comprises a STII, DsbA, MalE or PhoA variant signal sequence. 
     
     
         12 . The method of  claim 11 , wherein the second translation initiation region comprises a PhoA or MalE variant signal sequence. 
     
     
         13 . The method of  claim 11 , wherein the second translation initiation region comprises sequence of one of SEQ ID NOs 1-42. 
     
     
         14 . The method of  claim 11 , wherein the second translation initiation region comprises sequence of one of SEQ ID NOs. 1-14, 16-24, 26-39, and 41-42. 
     
     
         15 . The method of  claim 6 , wherein the polynucleotide encoding the antibody further comprises (3) a third translation initiation region operably linked to a polynucleotide encoding a Fc polypeptide, wherein the third translation initiation region comprises a co-translational or post-translational prokaryotic secretion signal sequence. 
     
     
         16 . The method of  claim 15 , wherein the third translation initiation region comprises a PhoA or DsbA variant signal sequence. 
     
     
         17 . The method of  claim 6 , wherein the relative translation strength of the first translational initiation region to the second translational initiation region is about one or two. 
     
     
         18 . The method of  claim 17 , wherein the relative translation strength of the first translational initiation region to the second translational initiation region is about one. 
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 6 , wherein the first and second inducible promoters are each prokaryotic promoters independently selected from the group consisting of phoA, tac, lpp, lac-lpp, lac, ara, and T7 promoter. 
     
     
         21 . The method of  claim 6 , wherein the antibody is a monoclonal antibody. 
     
     
         22 . The method of  claim 21 , wherein the antibody is a chimeric antibody, an affinity matured antibody, a bispecific antibody, humanized antibody, an antibody fragment or a human antibody. 
     
     
         23 . The method of  claim 22 , wherein the antibody fragment is a one-armed antibody. 
     
     
         24 . The method of  claim 22 , wherein the antibody binds c-met. 
     
     
         25 . The method of  claim 24 , wherein the anti-c-met antibody comprises (a) a first polypeptide comprising a heavy chain variable domain having the sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRF NPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSS (SEQ ID NO: 43), CHI sequence, and a first Fc polypeptide; (b) a second polypeptide comprising a light chain variable domain having the sequence: DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR (SEQ ID NO:44), and CLI sequence; and (c) a third polypeptide comprising a second Fc polypeptide, wherein the heavy chain variable domain and the light chain variable domain are present as a complex and form a single antigen binding arm, wherein the first and second Fc polypeptides are present in a complex and form a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm. 
     
     
         26 - 27 . (canceled) 
     
     
         28 . The method of  claim 6 , wherein the  E. coli  is of a strain deficient in endogenous protease activities. 
     
     
         29 . The method of  claim 6 , wherein the genotype of the  E. coli  lacks degP and prc genes and harbors a mutant spr gene. 
     
     
         30 . The method of  claim 6 , wherein the method further comprises recovering the antibody from the host cell culture. 
     
     
         31 . The method of  claim 30 , wherein the antibody is recovered from the host cell culture medium. 
     
     
         32 . The method of  claim 30 , further comprising combining the recovered antibody with a pharmaceutically acceptable carrier, excipient, or carrier to prepare a pharmaceutical formulation comprising the antibody. 
     
     
         33 . The method of  claim 6 , wherein at least 70% by molar ratio of the heavy and light chains are folded and assembled to form a biologically active antibody. 
     
     
         34 - 38 . (canceled) 
     
     
         39 . The method of  claim 6 , wherein the host cell further comprises a polynucleotide encoding at least one prokaryotic polypeptide selected from the group consisting of DsbA, DsbC, DsbG, and FkpA. 
     
     
         40 . The method of  claim 39 , wherein the polynucleotide encodes both DsbA and DsbC. 
     
     
         41 . A method of making an antibody, said method comprising culturing a host cell comprising a polynucleotide comprising (1) a first inducible promoter operably linked to a first TIR operably linked to a polynucleotide encoding an antibody heavy chain, wherein the TIR comprises a DsbA variant co-translational prokaryotic secretion signal sequence; and (2) a second inducible promoter operably linked to a second TIR operably linked to a polynucleotide encoding an antibody light chain, wherein the second TIR comprises a co-translational or post-translational prokaryotic secretion signal sequence, inducing expression of the antibody heavy chain and antibody light chain, and lysing the host cell to form a whole cell lysate, whereby upon expression of the antibody in the host cell, at least 50% by molar ratio of the heavy and light chains in the whole cell lysate are folded and assembled to form a biologically active antibody. 
     
     
         42 . The method of  claim 41 , wherein the first translation initiation region comprises sequence of one of SEQ ID NOs: 36-42. 
     
     
         43 . The method of  claim 41 , wherein the second translation initiation region comprises a STII, DsbA, MalE or PhoA variant signal sequence. 
     
     
         44 . The method of  claim 43 , wherein the second translation initiation region comprises a PhoA or MalE variant signal sequence. 
     
     
         45 . The method of  claim 43 , wherein the second translation initiation region comprises sequence of one of SEQ ID NOs 1-42. 
     
     
         46 . The method of  claim 43 , wherein the second translation initiation region comprises sequence of one of SEQ ID NOs. 1-14, 16-24, 26-39, and 41-42. 
     
     
         47 . The method of  claim 41 , wherein the polynucleotide encoding the antibody further comprises (3) a third translation initiation region operably linked to a polynucleotide encoding a Fc polypeptide, wherein the third translation initiation region comprises a co-translational or post-translational prokaryotic secretion signal sequence. 
     
     
         48 . The method of  claim 47 , wherein the third translation initiation region comprises a PhoA or DsbA variant signal sequence. 
     
     
         49 . The method of  claim 41 , wherein the relative translation strength of the first translational initiation region to the second translational initiation region is about one or two. 
     
     
         50 . The method of  claim 49 , wherein the relative translation strength of the first translational initiation region to the second translational initiation region is about one. 
     
     
         51 . The method of  claim 41 , wherein the first and second inducible promoters are each prokaryotic promoters independently selected from the group consisting of phoA, tac, lpp, lac-lpp, lac, ara, and T7 promoter. 
     
     
         52 . The method of  claim 41 , wherein the antibody is a monoclonal antibody. 
     
     
         53 . The method of  claim 52 , wherein the antibody is a chimeric antibody, an affinity matured antibody, a bispecific antibody, humanized antibody, an antibody fragment or a human antibody. 
     
     
         54 . The method of  claim 53 , wherein the antibody fragment is a one-armed antibody. 
     
     
         55 . The method of  claim 53 , wherein the antibody binds c-met. 
     
     
         56 . The method of  claim 55 , wherein the anti-c-met antibody comprises (a) a first polypeptide comprising a heavy chain variable domain having the sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRF NPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSS (SEQ ID NO: 43), CHI sequence, and a first Fc polypeptide; (b) a second polypeptide comprising a light chain variable domain having the sequence: DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR (SEQ ID NO:44), and CLI sequence; and (c) a third polypeptide comprising a second Fc polypeptide, wherein the heavy chain variable domain and the light chain variable domain are present as a complex and form a single antigen binding arm, wherein the first and second Fc polypeptides are present in a complex and form a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm. 
     
     
         57 . The method of  claim 41 , wherein the host cell is a prokaryotic cell. 
     
     
         58 . The method of  claim 57 , wherein the prokaryotic cell is deficient in endogenous protease activities. 
     
     
         59 . The method of  claim 41 , wherein the method further comprises recovering the antibody from the whole cell lysate. 
     
     
         60 . The method of  claim 59 , wherein the method further comprises combining the recovered antibody with a pharmaceutically acceptable carrier, excipient, or carrier to prepare a pharmaceutical formulation comprising the antibody. 
     
     
         61 . The method of  claim 41 , wherein at least 70% by molar ratio of the heavy and light chains are folded and assembled to form a biologically active antibody. 
     
     
         62 . The method of  claim 41 , wherein the host cell further comprises a polynucleotide encoding at least one prokaryotic polypeptide selected from the group consisting of DsbA, DsbC, DsbG, and FkpA. 
     
     
         63 . The method of  claim 62 , wherein the polynucleotide encodes both DsbA and DsbC.