Identification of Framework Residues in a Secreted Recombinant Antibody Fragment That Control Production Level and Localization inEscherichia coli

Abstract

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 C H 1, C L , 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 V L increased the level almost 15 times, exceeding 500 mg/liter in the growth medium. Here, the substitutions Phe-10 3 Ser, Thr-45 3 Lys, Thr-77 3 Ser, and Leu-78 3 Val were most powerful. In addition, replacing four V H 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 tumorassociated 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.

Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen

“…The fusion proteins were produced at Pharmacia & Upjohn (Stockholm, Sweden) essentially as described (Dohlsten et al, 1994;Abrahmsén et al, 1995;Forsberg et al, 1997). The Fvencoding portions of 5T4 were cloned from the 5T4 hybridoma and fused to sequences coding for the constant regions of the murine IgG1/k antibody C242 lacking the interchain disulphide bond.…”

“…After fermentation, clarified growth medium was applied to a Protein G Sepharose column (Pharmacia Biotech, Uppsala, Sweden) and bound protein eluted with 0.1 M acetic acid, 0.05% Tween 80. Full-length product was separated from a degraded variant lacking the superantigen moiety, 5T4FabV13, on an SP Sepharose HP column (Pharmacia Biotech) using a linear gradient from 60 to 350 mM sodium acetate (Forsberg et al, 1997). SDS-PAGE and chromatographic techniques indicate that the purity of the product was at least 95%.…”

“…Affinities to the 5T4 antigen were measured similarly to Forsberg et al (1997). 5T4FabV13-SEA D227A and 5T4FabV13 respectively were labelled with Na 125 I (NEN, Boston, MA) to obtain a specific activity of 10 to 40 µCi µg -1 protein and an iodine to protein ratio of ≤ 2:1.…”

“…The product was expressed as bicistronic construct with SEA D227A fused to the Cterminus of the Fab heavy chain. The production level was increased 15-fold by replacing 7 amino acid residues in the framework of 5T4Fab, yielding 5T4FabV13 (Forsberg et al, 1997). The yield in the E. coli growth medium of 5T4FabV13-SEA D227A was in the order of 0.5 g l -1 .…”

“…British Journal of Cancer (2000) The product consists of a modified variant of the variable regions from 5T4 antibody (Forsberg et al, 1997) connected to the CL and CH1 domains of the antibody C242 (Dohlsten et al, 1994). The superantigen SEA D227A is fused to the C-terminus of CH1.…”