Improved Potency and Safety of DNA-Encoded Antibody Therapeutics Through Plasmid Backbone and Expression Cassette...

DNA-encoded delivery of antibodies presents a labor- and cost-effective alternative to conventional antibody therapeutics. This study aims to improve the potency and safety of this approach by evaluating various plasmid backbones and expression cassettes. In vitro, antibody levels consistently improved with decreasing sizes of backbone, ranging from conventional to minimal. In vivo, following intramuscular electrotransfer in mice, the correlation was less consistent. While the largest conventional plasmid (10.2 kb) gave the lowest monoclonal antibody (mAb) levels, a regular conventional plasmid (8.6 kb) demonstrated similar levels as a minimal Nanoplasmid (6.8 kb). A reduction in size beyond a standard conventional backbone thus did not improve mAb levels in vivo. Cassette modifications, such as swapping antibody chain order or use of two versus a single encoding plasmid, significantly increased antibody expression in vitro, but failed to translate in vivo. Conversely, a significant improvement in vivo but not in vitro was found with a set of muscle-specific promoters, of which a newly engineered variant gave roughly 1.5- to 2-fold higher plasma antibody concentrations than the ubiquitous CAG promoter. In conclusion, despite the limited translation between in vitro and in vivo, we identified various clinically relevant improvements to our DNA-based antibody platform, both in potency and biosafety.