Methods of producing two chain proteins in bacteria

Abstract

Provided herein are methods of producing a recombinant polypeptide containing two chains, such as an immune mobilizing monoclonal T-cell receptor against cancer (ImmTAC) protein including an alpha chain and a beta chain. In particular, methods are provided for producing heterologous secretory proteins in bacteria through utilization of optimized expression vectors and culture processes.

1 . A method of producing an immune mobilizing monoclonal T-cell receptor against cancer (ImmTAC) comprising a T-cell receptor (TCR) alpha chain and a TCR beta chain in a prokaryotic host cell, the method comprising:
 (a) culturing the host cell to express the TCR alpha chain and the TCR beta chain of the ImmTAC in a culture medium under conditions comprising:   
       a growth phase comprising a growth temperature and a growth agitation rate, and 
       a production phase comprising a production temperature and a production agitation rate, 
       whereby upon expression the TCR alpha chain and the TCR beta chain fold and assemble to form a biologically active ImmTAC in the host cell;
 wherein the host cell comprises a polynucleotide comprising 
 
       (1) a first translational unit encoding the TCR alpha chain of the ImmTAC; 
       (2) a second translational unit encoding the TCR beta chain of the ImmTAC; 
       (3) a third translational unit encoding an FkpA protein; and 
       (4) a fourth translational unit encoding a DsbC protein;
 wherein the growth temperature is from 2 to 10° C. above the production temperature, and the growth agitation rate is from 50 to 250 rpm above the production agitation rate; and 
 (b) recovering the biologically active ImmTAC from the host cell. 
 
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein the polynucleotide further comprises three copies of a promoter, wherein a first copy is in operable combination with the first translational unit, a second copy is in operable combination with the second translational unit, and a third copy is in operable combination with the third translational unit to drive transcription of the first chain, the second chain and the FkpA protein. 
     
     
         4 . The method of  claim 3 , wherein the promoter is an inducible promoter. 
     
     
         5 . The method of  claim 4 , wherein the inducible promoter is an IPTG-inducible promoter that drives transcription of the TCR alpha chain, the TCR beta chain and the chaperone protein in the absence of IPTG induction. 
     
     
         6 . The method of  claim 4 , wherein the inducible promoter is a Pho promoter that drives transcription of the TCR alpha chain, the TCR beta chain and the chaperone protein when phosphate in the culture medium has been depleted. 
     
     
         7 . The method of  claim 1 , wherein the polynucleotide further comprises a selectable marker and the culture medium comprises a selection agent consisting of a single antibiotic to cause the host cell to retain the polynucleotide. 
     
     
         8 . The method of  claim 1 , wherein the first translational unit comprises a first translation initiation region (TIR) in operable combination with a coding region of the TCR alpha chain, and the second translational unit comprises a second translation initiation region (TIR) in operable combination with a coding region of the TCR beta chain, wherein the relative translation strength of the first and second TIR is from about 1.0 to about 3.0. 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein the FkpA protein is  E. coli  FkpA. 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the DsbC protein is  E. coli  DsbC. 
     
     
         15 . The method of  claim 1 , wherein the prokaryotic host cell is a gram-negative bacterium. 
     
     
         16 . The method of  claim 15 , wherein the gram-negative bacterium is  E. coli.    
     
     
         17 . The method of  claim 16 , wherein the  E. coli  is a strain with a degpS210A mutation. 
     
     
         18 . The method of  claim 16 , wherein the  E. coli  is a strain with a genotype of W3110 ΔfhuA ΔphoA ilvG2096 (Val r ) Δprc spr43H1 ΔdegP ΔmanA lacI Q  ΔompT ΔmenE degpS210A. 
     
     
         19 . The method of  claim 1 , wherein the TCR alpha chain comprises a TCR alpha chain variable domain and a TCR alpha chain constant domain, and wherein the TCR beta chain comprises a TCR beta chain variable domain and a TCR beta chain constant domain. 
     
     
         20 . The method of  claim 1 , wherein the two chains of the ImmTAC are linked to each other by at least one disulfide bond. 
     
     
         21 . The method of  claim 1 , wherein the ImmTAC further comprises an antibody fragment that binds a T cell and activates a T cell response. 
     
     
         22 . The method of  claim 21 , wherein the antibody fragment comprises an anti-CD3 single chain antibody fragment. 
     
     
         23 . The method of  claim 1 , wherein the ImmTAC comprises a TCR engineered to possess increased affinity for an antigen, as compared to affinity for the antigen of TCR that has not been engineered. 
     
     
         24 . The method of  claim 1 , wherein the ImmTAC is recovered from the periplasm of the host cell. 
     
     
         25 . The method of  claim 1 , wherein the growth temperature is in the range of about 30° C. to about 34° C. during the growth phase, and the production temperature in the range of about 25° C. to about 29° C. during the production phase. 
     
     
         26 . The method of  claim 1 , wherein the growth agitation rate is sufficient to achieve an oxygen uptake rate in the host cell during the growth phase of from 0.5 to 2.5 mmol/L/min above a peak oxygen uptake rate in the host cell during the production phase. 
     
     
         27 . The method of  claim 1 , wherein the peak oxygen uptake rate of the host cell during the growth phase is in the range of 3.5 to 4.5 mmol/L/min, and the oxygen uptake rate of the host cell during the production phase is in the range of 1.0 to 3.0 mmol/L/min. 
     
     
         28 . The method of  claim 1 , wherein the growth agitation rate is from 10% to 40% higher than the production agitation rate.