Increased Tumor Penetration of Single-Domain Antibody-Drug Conjugates Improves In Vivo Efficacy in Prostate Cancer Models

Cancer Res. 2020 Mar 15;80(6):1268-1278. doi: 10.1158/0008-5472.CAN-19-2295. Epub 2020 Jan 15.


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 in vitro potency resulted in higher in vivo efficacy than other protein-drug conjugates. Biodistribution data, tumor histology images, spheroid experiments, in vivo 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 in vivo efficacy. In conclusion, these results provide evidence that reaching the maximum number of cells with a lethal payload dose correlates more strongly with in vivo efficacy than total tumor uptake or in vitro 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 in vivo than other agents with equal tumor uptake due to improved tissue penetration and cellular distribution.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Antineoplastic Agents, Alkylating / chemistry
  • Antineoplastic Agents, Alkylating / pharmacology*
  • Antineoplastic Agents, Alkylating / therapeutic use
  • Cell Line, Tumor
  • Computer Simulation
  • Female
  • Humans
  • Immunoconjugates / chemistry
  • Immunoconjugates / pharmacokinetics*
  • Immunoconjugates / therapeutic use
  • Male
  • Mice
  • Microscopy, Confocal
  • Models, Biological*
  • Prostatic Neoplasms / diagnostic imaging
  • Prostatic Neoplasms / drug therapy*
  • Prostatic Neoplasms / pathology
  • Single-Domain Antibodies / chemistry
  • Single-Domain Antibodies / pharmacology*
  • Single-Domain Antibodies / therapeutic use
  • Spheroids, Cellular
  • Structure-Activity Relationship
  • Tissue Distribution
  • Xenograft Model Antitumor Assays


  • Antineoplastic Agents, Alkylating
  • Immunoconjugates
  • Single-Domain Antibodies