Antibody gene transfer aims to administer to patients the monoclonal antibody (mAb)- encoding nucleotides rather than the mAb protein, enabling prolonged in vivo production at low cost. By leveraging the advantages of gene therapy, it can potentially innovate the entire mAb field. Using plasmid DNA (pDNA), our group previously achieved pre-clinical proof of concept for intramuscular antibody gene transfer in mice. However, the currently attained mAb expression levels (μg/ml range) are likely too low to gain widespread application. Further innovation is subsequently required.
This study outlines our preliminary efforts in the engineering of a more potent and safe DNA platform for intramuscular antibody gene transfer.
4D5, the murine equivalent of trastuzumab (Herceptin®), was used as a model for mAb expression. As DNA backbone, we used plasmids that contain either no (minicircle), minimal (Nanoplasmid®), or varying sizes of bacterial sequences (conventional plasmids). 4D5 light chain (LC) and heavy chain (HC) were either jointly cloned in one plasmid (using a dual cassette system) or individually into two separate plasmids (with a single cassette each). Both ubiquitous (CAG and CMV) and muscle-specific promoters were evaluated. Constructs were transfected in vitro (C2C12 murine myoblast cells) and in BALB/c mice, using lipofection and intramuscular electroporation, respectively. 4D5 concentrations were quantified with ELISA.
In vivo evaluation was limited to the dual CAG cassette constructs. Minicircles thereby provided no advantage over a conventional plasmid. Considering its more laborious production, the use of minicircles was abandoned. Nanoplasmids® demonstrated a size-dependent improvement in expression; the larger the bacterial backbone of the conventional plasmids, the more pronounced the benefit of the Nanoplasmid® was. In vitro data demonstrated that the two individual CAG HC and LC plasmids outperformed a single dual CAG cassette plasmid. This increase was significantly higher for Nanoplasmids® than for conventional plasmids. CMV and muscle-specific promoters gave a similar 4D5 expression in vitro, but were both outperformed by CAG. A selection of promoters will be further evaluated in vivo.
Our preliminary results indicate that both backbone and cassette engineering can affect antibody expression. Moving forward, this provides multiple leads towards building a more robust mAb expression platform."