Increased Tumor Penetration of Single-Domain Antibody-Drug Conjugates Improves <i>In Vivo</i> Efficacy in Prostate Cancer Models.

Abstract

Targeted delivery of chemotherapeutics aims to increase efficacy and lower toxicity by concentrating drugs at the site-of-action, a method embodied by the seven current FDA-approved antibody-drug conjugates (ADC). However, a variety of pharmacokinetic challenges result in relatively narrow therapeutic windows for these agents, hampering the development of new drugs. Here, we use a series of prostate-specific membrane antigen-binding single-domain (Humabody) ADC constructs to demonstrate that tissue penetration of protein-drug conjugates plays a major role in therapeutic efficacy. Counterintuitively, a construct with lower <i>in vitro</i> potency resulted in higher <i>in vivo</i> efficacy than other protein-drug conjugates. Biodistribution data, tumor histology images, spheroid experiments, <i>in vivo</i> single-cell measurements, and computational results demonstrate that a smaller size and slower internalization rate enabled higher tissue penetration and more cell killing. The results also illustrate the benefits of linking an albumin-binding domain to the single-domain ADCs. A construct lacking an albumin-binding domain was rapidly cleared, leading to lower tumor uptake (%ID/g) and decreased <i>in vivo</i> efficacy. In conclusion, these results provide evidence that reaching the maximum number of cells with a lethal payload dose correlates more strongly with <i>in vivo</i> efficacy than total tumor uptake or <i>in vitro</i> potency alone for these protein-drug conjugates. Computational modeling and protein engineering can be used to custom design an optimal framework for controlling internalization, clearance, and tissue penetration to maximize cell killing. SIGNIFICANCE: A mechanistic study of protein-drug conjugates demonstrates that a lower potency compound is more effective <i>in vivo</i> than other agents with equal tumor uptake due to improved tissue penetration and cellular distribution.