Production of neutralizing antibody fragment variants in the cytoplasm of E. coli for rapid screening: SARS-CoV-2 a case study

Abstract

Global health challenges such as the coronavirus pandemic warrant the urgent need for a system that allows efficient production of diagnostic and therapeutic interventions. Antibody treatments against SARS-CoV-2 were developed with an unprecedented pace and this enormous progress was achieved mainly through recombinant protein production technologies combined with expeditious screening approaches. A heterologous protein production system that allows efficient soluble production of therapeutic antibody candidates against rapidly evolving variants of deadly pathogens is an important step in preparedness towards future pandemic challenges. Here, we report cost and time-effective soluble production of SARS-CoV-2 receptor binding domain (RBD) variants as well as an array of neutralizing antibody fragments (Fabs) based on Casirivimab and Imdevimab using the CyDisCo system in the cytoplasm of E. coli. We also report variants of the two Fabs with higher binding affinity against SARS-CoV-2 RBD and suggest this cytoplasmic production of disulfide containing antigens and antibodies can be broadly applied towards addressing future global public health threats.

Design of an alternate antibody fragment format that can be produced in the cytoplasm of Escherichia coli

“…The Fab H 3 was found to exhibit a comparable binding affinity (K D = 20 ± 2.6 nM) to its counterpart REGN10987 Fab (K D = 43 ± 4.4 nM) against SARS-CoV-2 RBD. The K D values observed for the REGN10987 Fab are in accordance with previous reports 40 , 41 thereby validating our finding. These results suggest that the replacement of constant domains in a Fab molecule and the C H 3 domain-based heterodimerization does not interfere with the folding and function of the variable domains such that interactions with its antigen are maintained.…”

“…We have previously shown that the wild type REGN10987 Fab produced using the CyDisCo system is functional and binds to the target antigen SARS-CoV-2 RBD 40 . After ensuring that the REGN10987- based Fab H 3 produced in the cytoplasm of E. coli was natively folded, we empirically investigated whether the molecule developed was functionally active and compared its binding affinity with that of the REGN10987 Fab using Biolayer Interferometry (BLI).…”

“…A polycistronic gene for the REGN10987 wild type Fab was synthesized codon optimized (GenScript Biotech Corp.) for E. coli expression. The gene for the heavy chain with a C-terminal hexahistidine tag was placed downstream of the gene for the light chain and cloned using Xba1-EcoR1 based restriction digestion and ligation in a modified pET23-based vector with the T7 promoter replaced by a pTac promoter to generate vector pAAT50 26 , 40 . Genes for the heavy and light chains of REGN10987-based Fab H 3 variants with different linker lengths were synthesized codon optimized (GenScript Biotech Corp.) for E. coli expression.…”

“…For the large-scale cultures in 1 L flasks, the cells were harvested, and the soluble fraction of the lysate was processed for purification as described in 40 . REGN10987 wild type Fab and Fab H 3 (GS-G 4 linker) were purified using a combination of two chromatography steps: Nickel-based Immobilized Metal Affinity Chromatography (IMAC) and Anion Exchange Chromatography (AnEx).…”

“…REGN10987 wild type Fab and Fab H 3 (GS-G 4 linker) were purified using a combination of two chromatography steps: Nickel-based Immobilized Metal Affinity Chromatography (IMAC) and Anion Exchange Chromatography (AnEx). The detailed protocol for both these chromatography steps has been described in 40 . The purified protein obtained after the AnEx step was buffer exchanged into 20 mM phosphate, 150 mM NaCl, pH 6.5 for REGN10987 Fab and pH 6.0 for the Fab H 3.…”