Engineering of a Bispecific Nanofitin with Immune Checkpoint Inhibitory Activity Conditioned by the Cross-Arm Binding to EGFR and PDL1

Re-education of the tumor microenvironment with immune checkpoint inhibitors (ICI) has provided the most significant advancement in cancer management, with impressive efficacy and durable response reported. However, low response rates and a high frequency of immune-related adverse events (irAEs) remain associated with ICI therapies. The latter can be linked to their high affinity and avidity for their target that fosters on-target/off-tumor binding and subsequent breaking of immune self-tolerance in normal tissues. Many multispecific protein formats have been proposed to increase the tumor cell’s selectivity of ICI therapies. In this study, we explored the engineering of a bispecific Nanofitin by the fusion of an anti-epidermal growth factor receptor (EGFR) and anti-programmed cell death ligand 1 (PDL1) Nanofitin modules. While lowering the affinity of the Nanofitin modules for their respective target, the fusion enables the simultaneous engagement of EGFR and PDL1, which translates into a selective binding to tumor cells co-expressing EGFR and PDL1 only. We demonstrated that affinity-attenuated bispecific Nanofitin could elicit PDL1 blockade exclusively in an EGFR-directed manner. Overall, the data collected highlight the potential of this approach to enhance the selectivity and safety of PDL1 checkpoint inhibition.