Identification of framework residues in a secreted recombinant antibody fragment that control production level and localization in Escherichia coli.
The monoclonal antibody 5T4, directed against a human tumor-associated antigen, was expressed as a secreted Fab superantigen fusion protein in Escherichia coli. The product is a putative agent for immunotherapy of non-small cell lung cancer. During fermentation, most of the fusion protein leaked out from the periplasm to the growth medium at a level of approximately 40 mg/liter. This level was notably low compared with similar products containing identical CH1, CL, and superantigen moieties, and the Fv framework was therefore engineered. Using hybrid molecules, the light chain was found to limit high expression levels. Substituting five residues in VL increased the level almost 15 times, exceeding 500 mg/liter in the growth medium. Here, the substitutions Phe-10 --> Ser, Thr-45 --> Lys, Thr-77 --> Ser, and Leu-78 --> Val were most powerful. In addition, replacing four VH residues diminished cell lysis during fermentation. Thereby the product was preferentially located in the periplasm instead of the growth medium, and the total yield was more than 700 mg/liter. All engineered products retained a high affinity for the tumor-associated antigen. It is suggested that at least some of the identified framework residues generally have to be replaced to obtain high level production of recombinant Fab products in E. coli.
Forsberg
G
G
Department of Biology and Biotechnology, Pharmacia and Upjohn, 112 87 Stockholm, Sweden. goran.forsberg@eu.pnu.com
Forsgren
M
M
Jaki
M
M
Norin
M
M
Sterky
C
C
Enhörning
A
A
Larsson
K
K
Ericsson
M
M
Björk
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use*; D000951:Antigens, Neoplasm / Q000276:immunology* / Q000627:therapeutic use; D001667:Binding, Competitive; D014408:Biomarkers, Tumor / Q000276:immunology* / Q000627:therapeutic use; D019496:Cancer Vaccines; D002289:Carcinoma, Non-Small-Cell Lung / Q000628:therapy; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000276:immunology; D005818:Genetic Engineering; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008175:Lung Neoplasms / Q000628:therapy; D008562:Membrane Glycoproteins / Q000276:immunology* / Q000627:therapeutic use; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry* / Q000276:immunology; D013329:Structure-Activity Relationship; D018089:Superantigens / Q000276:immunology
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D019496:Cancer Vaccines; D007140:Immunoglobulin Fab Fragments; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D018089:Superantigens; C063709:trophoblastic glycoprotein 5T4, human