Targeted Nanofitin-drug Conjugates Achieve Efficient Tumor Delivery and Therapeutic Effect in an EGFRpos Mouse Xenograft Model.

Adjusting the molecular size, the valency and the pharmacokinetics of drug conjugates are as many leverages to improve their therapeutic window, notably by affecting tumor penetration, renal clearance, and short systemic exposure. In that regard, small tumor-targeting ligands are gaining attention. In this study, we demonstrate the benefits of the small Nanofitin alternative scaffolds (7 kDa) as selective tumor-targeting modules for the generation of drug conjugates, focusing on Nanofitins B10 and D8 directed against the EGFR. Owing to their small size and monovalent format, the two Nanofitins displayed a fast and deep tumor penetration in EGFR-positive A431 xenografts in BALB/c nude mice after intravenous administration, yielding to a targeting of respectively 67.9% ± 14.1 and 98.9% ± 0.7 of the tumor cells as demonstrated by IHC. Conjugation with the monomethyl auristatin E toxin provided homogeneous Nanofitin-drug conjugates, with an overall yield of ≥97%, for in vivo assessment in a curative xenograft model using bioluminescent, EGFR-positive, A431 cells in BALB/c nude mice. Internalization was found critical for efficient release of the toxin. Hence, the intravenous administration of the D8-based construct showed significant antitumor effect in vivo as determined by monitoring tumor volumes and bioluminescence levels over 2 months.

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.