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

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 improvemen in vivo but no 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.