Single domain antibodies or antibody mimetics can function intracellularly and can be engineered to degrade their target protein via fusion to a ubiquitin E3 ligase. These so-called biodegrader intrabodies can modulate intracellular disease-associated proteins, including those that are considered undruggable with small molecules. Multi-targeted biodegrader intrabodies that can simultaneously eliminate several disease-causing proteins have yet to be developed, and the potential to deliver intrabodies into diseased cells using lipid nanoparticle and mRNA technology is an exciting novel therapeutic modality that has yet to be realised.
In this project we developed first-in-class multi-targeted biodegrader intrabodies by harnessing the unique properties of the inhibitor of apoptosis (IAP) E3 ligase RING domain or an IAP binding motif. We fused these motifs with monobodies that inhibit necroptosis, a lytic programmed cell death pathway implicated in diverse inflammatory diseases and cancer metastasis. We showed that the engineered biodegrader intrabodies could simultaneously eliminate the necroptotic effector protein, MLKL, in addition to disease-driving inhibitor of apoptosis (IAP) proteins, via triggering ubiquitin-mediated proteasomal targeting. This multi-targeted protein degradation resulted in the desired functional outcome – biodegrader intrabody expression in cancer cells blocked the inflammatory necroptotic cell death associated with cancer metastasis, while sensitising cancer cells to immunologically silent apoptosis. In vivo, expression of our biodegrader intrabody blocked breast cancer development in an orthotopic mouse model, highlighting the therapeutic potential of delivering biodegrader intrabody mRNA using lipid nanoparticle technology.