Streamlining bispecific antibody production in E. coli with scFv-Constant domain fusions.

The single-chain Fragment variable (scFv) antibody format is a common format used for phage display. The solubility of identified scFv clones in phage format in instances does not translate the same solubility as independent recombinant proteins. To overcome this bottleneck, we propose the fusion of scFv clones to the antibody constant domains for improved solubility. It has been reported that immunoglobulins devoid of the heavy constant domain 1 (CH1) will have reduced solubility allowing accumulation in Russell Bodies (RB). Here we propose the introduction of CH1 and kappa constant domain (Cκ) with and without skp chaperone to improve soluble expression. Utilizing heavy and light constant domains facilitates the spontaneous assembly of a Fab-like structure. This configuration successfully presents two distinct scFVs on each chain, thereby achieving bispecificity. This system is specifically designed to optimize the expression of the scFv-constant domain fusion necessary for bispecific formation. Crucially, its modular design allows for simple and rapid scFv swapping, enabling the seamless assessment of different antibody pairings for effective bispecific antibody generation.