System for antibody expression and assembly

Abstract

The present invention provides methods and compositions for expression and production of recombinant antibodies in a host cell system, such as prokaryotic and eukaryotic expression systems. Particularly contemplated are recombinant systems for temporally separated expression of light chain and heavy chain of antibodies. The antibody products including antibody fragments can be used in various aspects of biological research, diagnosis and medical treatment.

1 . A process for producing a functional antibody or fragment thereof in a host cell transformed with two separate translational units respectively encoding the light and heavy chains of said antibody or fragment thereof, comprising the steps of: a) culturing the host cell under suitable conditions so that the light chain and heavy chain are expressed in a sequential fashion, thereby temporally separating the production of the light and heavy chains; and b) allowing the assembly of the light and heavy chains to form the functional antibody or fragment thereof.  
     
     
         2 . The process of  claim 1  wherein the host cell is prokaryotic, each translational unit further comprising a nucleotide sequence encoding for a prokaryotic secretion signal or variant thereof operably linked to the N′-terminal of the light or heavy chain.  
     
     
         3 . The process of  claim 2 , wherein the two separate translational units are controlled by different promoters.  
     
     
         4 . The process of  claim 3 , wherein the two translational units are located on a single recombinant vector.  
     
     
         5 . The process of  claim 2 , wherein the secretion signal is selected from the group consisting of STII, OmpA, PhoE, LamB, MBP and PhoA.  
     
     
         6 . The process of  claim 5 , wherein the secretion signal is STII or a variant thereof.  
     
     
         7 . The process of  claim 3 , wherein the promoter for each translational unit is selected from the group consisting of phoA, TacI, TacII, lpp, lac-lpp, lac, ara, trp, trc and T7 promoters.  
     
     
         8 . The process of  claim 7 , wherein one promoter is the phoA promoter and the other promoter is the TacII promoter.  
     
     
         9 . The process of  claim 1 , wherein the antibody fragment is selected from the group consisting of Fab, Fab′, F(ab′) 2 , F(ab′) 2 -leucine zipper, Fv and dsFv.  
     
     
         10 . The process of  claim 1 , wherein the antibody is specific to an antigen selected from the group consisting of VEGF, IgE, CD11, CD18 and tissue factor (TF).  
     
     
         11 . (canceled)  
     
     
         12 . The process of  claim 1 , wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.  
     
     
         13 - 14 . (canceled)  
     
     
         15 . The process of  claim 1 , wherein the host cell is a prokaryotic cell from an  E. coli  strain.  
     
     
         16 . The process of  claim 15 , wherein the  E. coli  strain is genetically engineered to over-express at least one chaperone protein selected from the group consisting of DsbA, DsbC, DsbG and FkpA.  
     
     
         17 . The process of  claim 15 , wherein the  E. coli  strain is deficient for endogenous protease activities.  
     
     
         18 . The process of  claim 15 , wherein the genotype of the  E. coli  strain contains Δprc prc-suppressor.  
     
     
         19 . The process of  claim 1 , wherein the heavy chain of the antibody is full length.  
     
     
         20 . The process of  claim 19 , wherein the light chain is expressed first and the full length heavy chain is subsequently expressed.  
     
     
         21 . A system for sequential expression of a light chain and a heavy chain of an antibody or fragment thereof, comprising a host cell transformed with a recombinant vector, said vector comprising two separate translational units respectively encoding the light chain and heavy chain, each translational unit operably linked to a different promoter, wherein under suitable conditions, the activation of the two translational units are temporally separated, thereby allowing the sequential expression of the light chain and the heavy chain.  
     
     
         22 . The system of  claim 21  wherein the host cell is prokaryotic, wherein each translational unit further comprises a nucleotide sequence encoding for a prokaryotic secretion signal operably linked to the 5′-end of the nucleic acid encoding the light or heavy chain.  
     
     
         23 . The system of  claim 22 , wherein the secretion signal is selected from the group consisting of STII, OmpA, PhoE, LamB, MBP and PhoA.  
     
     
         24 . The system of  claim 23 , wherein the secretion signal is STII or a variant thereof.  
     
     
         25 . The system of  claim 24 , wherein two STII variants are used respectively in the two translational units to provide a translational strength combination of about (7-light, 3-heavy).  
     
     
         26 . The system of  claim 21 , wherein each translational unit is operably linked to a different inducible promoter.  
     
     
         27 . The system of  claim 26 , wherein the inducible promoter is selected from the group consisting of phoA, TacI, TacII, lpp, lac-lpp, lac, ara, trp, trc and T7 promoters.  
     
     
         28 . The system of  claim 27 , wherein one promoter is the phoA promoter and the other promoter is the TacI promoter.  
     
     
         29 . The system of  claim 21 , wherein the antibody fragment is selected from the group consisting of Fab, Fab′, F(ab′) 2 , F(ab′) 2 -leucine zipper, Fv and dsFv.  
     
     
         30 . The system of  claim 21 , wherein the antibody is specific to an antigen selected from the group consisting of VEGF, IgE, CD11, CD18 and tissue factor (TF).  
     
     
         31 . (canceled)  
     
     
         32 . The system of  claim 21 , wherein the antibody is a chimeric antibody, a humanized antibody, or a human antibody.  
     
     
         33 .- 34 . (canceled)  
     
     
         35 . The system of  claim 21 , wherein the host cell is a prokaryotic cell from an  E. coli  strain.  
     
     
         36 . The system of  claim 35 , wherein the  E. coli  strain is genetically engineered to over-express at least one chaperone protein selected from the group consisting of DsbA, DsbC, DsbG and FkpA.  
     
     
         37 . The system of  claim 35 , wherein the  E. coli  strain is deficient for endogenous protease activities.  
     
     
         38 . The system of  claim 37 , wherein the genotype of the  E. coli  strain contains Δprc prc-suppressor.  
     
     
         39 . The system of  claim 21 , wherein the heavy chain of the antibody is full length.  
     
     
         40 . The system of  claim 39 , wherein the light chain is expressed first and the full length heavy chain is subsequently expressed.  
     
     
         41 . A recombinant vector for production of a functional antibody or fragment thereof in a host cell, said vector comprising a) a first promoter preceding a first translational unit encoding a secretion signal operably linked to a light chain and b) a second promoter preceding a second translational unit encoding a secretion signal operably linked to a heavy chain, said first and second promoters are inducible under different conditions.  
     
     
         42 . The recombinant vector of  claim 41 , wherein the first and second promoters are selected from the group consisting of phoA, TacI, TacII, lpp, lac-lpp, lac, ara, trp, trc and T7 promoters.  
     
     
         43 . The recombinant vector of  claim 42 , wherein the first promoter is the phoA promoter and the second promoter is the TacII promoter.  
     
     
         44 . The recombinant vector of  claim 41 , wherein the secretion signal is selected from the group consisting of STII, OmpA, PhoE, LamB, MBP and PhoA.  
     
     
         45 . The recombinant vector of  claim 44 , wherein the secretion signal is a STII or a variant thereof.  
     
     
         46 . The recombinant vector of  claim 41 , wherein the antibody fragment is selected from the group consisting of Fab, Fab′, F(ab′) 2 , Fv and dsFv.  
     
     
         47 . The recombinant vector of  claim 46 , wherein the antibody fragment is fused to a dimerization domain.  
     
     
         48 . The recombinant vector of  claim 41 , wherein the antibody is specific to an antigen selected from the group consisting of VEGF, IgE, CD11, CD18 and tissue factor (TF).  
     
     
         49 . (canceled)  
     
     
         50 . The recombinant vector of  claim 49 , wherein the antibody is an anti-CD18 F(ab′) 2 -leucine zipper fusion.  
     
     
         51 . The recombinant vector of  claim 50 , said antibody having a light chain of SEQ ID NO:1.  
     
     
         52 . The recombinant vector of  claim 50 , said antibody having a heavy chain of SEQ ID NO:2.  
     
     
         53 . The recombinant vector of  claim 50 , which is a pxCD18-7T3 vector comprising the nucleic acid sequence of SEQ ID NO: 3.  
     
     
         54 . The recombinant vector of  claim 48 , wherein the antibody is an anti-TF antibody.  
     
     
         55 . The recombinant vector of  claim 54 , said antibody having a light chain of SEQ ID NO:4.  
     
     
         56 . The recombinant vector of  claim 54 , said antibody having a heavy chain of SEQ ID NO:5.  
     
     
         57 . The recombinant vector of  claim 54 , which is a pxTF-7T3FL vector comprising the nucleic acid sequence of SEQ ID NO:6.