Nanoparticle-formulated mRNA encoding engineered multivalent SIRPα-Fc fusion proteins shows robust anti-cancer activity in preclinical models

The <i>in vivo</i> expression of proteins by mRNA therapeutics is a transformative approach to medicine that involves expressing highly complex and therapeutically relevant molecules utilizing patients' own body. In this study, we engineered complex molecules targeting CD47 with multivalent SIRPα-Fc fusion proteins with a goal to enhance tumor specificity via formulated mRNA administration. Valency allows us to exploit antigen expression level differences between cancer and healthy cells. <i>In vitro</i> analysis showed that NK-mediated cytotoxicity of Tetravalent and Octavalent SIRPα was comparable to a 50,000-fold affinity-improved SIRPα molecule. However, unlike the affinity-improved SIRPα and known anti-CD47 antibodies, the Tetravalent and Octavalent SIRPα showed low to no binding to red blood cells, which also express CD47 albeit at a low level. In addition, we demonstrated <i>in vivo</i> efficacy of mRNAs encoding Tetravalent and Octavalent SIRPα-Fc fusion proteins and observed the complete eradication of established subcutaneous tumors in Raji mice xenograft. Further evaluation of the <i>in-vivo</i>-expressed proteins showed high purity, like that of the recombinant production. Differential scanning fluorimetry analysis revealed excellent thermal stability and resistance to aggregation. These results demonstrate that a significant enhancement in therapeutic window and efficacy could be achieved by engineering complex multivalent molecules.