single-chain variable fragment (scfv)

Production of functional recombinant antibody fragments in the periplasm of E. coli isa prerequisite step to achieve sufficient reagent for preclinical studies. Thus, the cost-effectiveand lab-scale production of antibody fragments demands the optimization of culture conditions.

The culture conditions such as temperature, optical density (OD600) at induction,induction time, and IPTG concentration were investigated to optimize the functional expressionof a phage-derived scFv molecule using a design of experiment (DoE). Additionally, the effectsof different culture media and osmolyte supplements on the expression yield of scFv wereexamined.

The developed 2FI regression model indicated the significant linear effect of theincubation temperature, the induction time, and the induction OD600 on the expression yieldof functional scFv. Besides, the statistical analysis indicated that two significant interactions ofthe temperature/induction time and the temperature/induction OD600 significantly interplay toincrease the yield. Further optimization showed that the expression level of functional scFvwas the most optimal when the cultivation was undertaken either in the TB medium or in thepresence of media supplements of 0.5 M sorbitol or 100 mM glycine betaine.

In the present study, for the first time, we successfully implemented DoE tocomprehensively optimize the culture conditions for the expression of scFv molecules in aphage antibody display setting, where scFv molecules can be isolated from a tailor-made phageantibody library known as “Human Single Fold scFv Library I.”

Tumor necrosis factor alpha (TNF-α) expression amplifies to excess amounts in several disorders such as rheumatoid arthritis and psoriasis. Although, Anti-TNF biologics have revolutionized the treatment of these autoimmune diseases, formation of anti-drug antibodies (ADA) has dramatically affected their use. The next generation antibodies (e.g. Fab, scFv) have not only reduced resulted immunogenicity, but also proved several benefits including better tumor penetration and more rapid blood clearance. Using affinity selection procedures in this study, a scFv antibody clone was isolated from naïve Tomlinson I phage display library that specifically recognizes and binds to TNF-α. The TNF-α recombinant protein was expressed in genetically engineered Escherichia coli SHuffle® T7 Express, for the first time, which is able to express disulfide-bonded recombinant proteins into their correctly folded states. ELISA-based affinity characterization results indicated that the isolated novel 29.2 kDa scFv binds TNF-α with suitable affinity. In-silico homology modeling study using ‘ModWeb’ as well as molecular docking study using Hex program confirmed the scFv and TNF-α interactions with a scFv-TNF- α binding energy of around -593 kj/mol which is well in agreement with our ELSIA results. The cloned scFv antibody may be potentially useful for research and therapeutic applications in the future.

More than half of the diagnostic and therapeutic recombinant protein production depends on mammalian-based expression system. However, the generation of recombinant antibodies remains a challenge in mammalian cells due to the disulfide bond formation and reducing cytoplasm. Therefore, the production of functional recombinant antibodies in target cell line is necessary to be evaluated before used in therapeutic application such intrabodies against HIV-1.
The work was to test expression of a single-chain variable fragment (scFv) antibody against HIV-1 Capsid p24 protein in a human mammalian-based expression system using HEK293T and Jurkat T cells as a model. Three expression plasmid vectors expressing scFv 183-H12-5C were generated and introduced into HEK293T. Expression of the scFv was analyzed, while ELISA and immunoblotting analysis verified its binding. The evaluation of the recombinant antibody was confirmed by HIV-1 replication and MAGI infectivity assay in Jurkat T cells.
Three plasmid vectors expressing scFv 183-H12-5C was successfully engineered in this study. Recombinant antibodies scFv (~29 kDa) and scFv-Fc (~52 kDa) in the cytoplasm of HEK293T were effectively obtained by transfected the cells with engineered pCDNA3.3-mu-IgGk-scFv 183-H12-5C and pCMX2.5-scFv 183-H12-5C-hIgG1-Fc plasmid vectors respectively. scFv and scFv-Fc are specifically bound recombinant p24, and HIV-1 derived p24 (gag) evaluated by ELISA and Western blot. Jurkat T cells transfected by pCDNA3.3-scFv 183-H12-5C inhibit the replication-competent NL4-3 viral infectivity up to 60%.
Anti-p24 scFv 183-H12-5C antibody generated is suitable to be acted as intrabodies and may serve as a valuable tool for the development of antibody-based biotherapeutics against HIV-1.