Production of functional antibody Fab fragment by recombinant insect cells

Abstract

The production of an Fab fragment of the catalytic antibody 6D9 in stably transformed lepidopteran insect cells was investigated. On the basis of an expression vector that utilizes the Bombyx mori cytoplasmic actin promoter, from which foreign gene expression is stimulated with the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer, two plasmid vectors were constructed which contain either a neomycin or a blasticidin resistance gene for use as a selectable marker. The genes encoding the heavy chain (Hc; Fd fragment) and light chain (Lc) of the 6D9 Fab fragment were inserted separately into the expression vectors. After cotransfection with the resulting plasmids to introduce the Hc and Lc genes and the two different antibiotic resistance genes, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were cultured in the presence of G418 and blasticidin. Colonies of cells resistant to the antibiotics were obtained around 2 weeks after cotransfection. Western blotting and enzyme-linked immunosorbent assay (ELISA) of the cell culture supernatant suggested that the resistant cells stably secrete an Fab fragment which retains an antigen-binding activity. High yields of over 300 μg/ml of Fab fragment were achieved in simple batch shake-flask culture of transfected insect cells. These results indicate that recombinant insect cells may offer a novel approach for efficient production of antibody molecules.

Lepidopteran cells, an alternative for the production of recombinant antibodies?

“…This combination resulted in a 1,000-fold stimulation of foreign gene expression which, at maximum cell density in a shake-culture, can reach a yield of up to 300 mg/l in High Five cells. 54 Thus methods to obtain relatively high yields of Igs in stable cultures are now available.…”

“…46 Beside these full-length immunoglobulins, many different antibody-derived molecules have been produced e.g., mono-specific single-chain fragments, scFv, [47][48][49][50] bi-specific scFv, 51 Fab. 18,[52][53][54] The expression of scFv is more reliable compared with that of the E. coli system and does not require any re-folding step. Fusion proteins such as chimeric-hormone-antibody molecules that achieved in human cells and may even be immunogenic (see below, "Glycosylation in insect cells").…”

Baculovirus display of functional antibody Fab fragments

“…Culture supernatants containing recombinant baculovirus were analyzed via ELISA, as previously described (Yamaji et al 2008). ELISA plates were coated with a conjugate of hapten (a transition-state analog for the hydrolysis of a chloramphenicol monoester derivative) and bovine serum albumin (BSA) (Takahashi et al 2001) as the antigen, and horseradish peroxidase-conjugated goat anti-mouse IgG (Exalpha Biologicals, Watertown, MA, USA) was used.…”

“…The DNA encoding the BiP signal sequence and the 6D9 Lc gene was amplified from the plasmid pXINSECT-6D9Lc (Yamaji et al 2008) using primers 5 and 9 (Table 1), and was cloned downstream of the polyhedrin promoter in the pFastBac Dual at the EcoRIHindIII site. The resultant plasmid was designated pFastBac -/Lc.…”

“…The DNA encoding the Drosophila immunoglobulin heavy chain binding protein (BiP) signal sequence and the 6D9 Fab Hc gene was PCR amplified from the plasmid pXINSECT-6D9Hc (Yamaji et al 2008) using primers 5 and 6 (Table 1). The plasmid pFastBac Dual (Life Technologies) contained two strong AcNPV promoters, the polyhedrin promoter and the p10 promoter, to allow the simultaneous expression of two proteins (Fig.…”