# Tumor-specific drug activation by pericellular proteases

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2023 · $177,630

## Abstract

ABSTRACT
Dysregulated pericellular proteolysis is a major driver of the malignant transformation of normal epithelial cells
to cancer cells. The increased proteolytic activity in the tumor microenvironment has been exploited for drug
development purposes. For example, antibody-drug conjugates (ADCs) consist of a highly potent anti-cancer
payload conjugated to a tumor-targeting antibody. This coupling renders the cargo inactive until it is released by
proteolytic cleavage in the pericellular space or upon degradation of the antibody within the target cell. ADCs
are still handicapped by dose-limited toxicities, most likely due to off-target cell binding or premature drug release
by proteases in circulation. The off-target binding can be overcome by engineering protease-activated pro-
antibodies (pADCs) that can bind only after being activated at the site of disease by a protease while the
premature drug release can be improved by utilizing peptide linker sequences that are stable in the presence of
blood proteases but are efficiently cleaved by proteases at the site of disease. Our hypothesis is that pADCs that
require two proteolytic activation steps, may improve our ability to selectively target anti-cancer drugs to the
tumor microenvironment. The pADC will be converted into an ADC by a pericellular protease and subsequently
bind to the tumor cell. The payload will be released by either an extracellular protease or an intracellular
(endosomal or lysosomal) protease. The objectives of this proposal are to design ADCs and pADCs that have
superior plasma stability and improved release kinetics by cancer-associated proteases. Our long-term
objectives are to develop pADCs with enhanced anti-cancer efficacy compared to current therapeutics.
In Aim 1, we will generate a substrate specificity profile for proteases secreted by patient-derived organoids of
breast cancer. In addition, we will isolate lysosomes and endosomes and characterize the proteolytic activity in
these organelles. Finally, we will evaluate protease activity in serum and plasma to understand the circulating
proteolytic activity. In Aim 2, we will develop ADCs and pADCs that are selectively activated by cancer proteases
and stable in plasma and serum.

## Key facts

- **NIH application ID:** 10554408
- **Project number:** 5R21CA256460-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** Anthony John O'Donoghue
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $177,630
- **Award type:** 5
- **Project period:** 2022-02-01 → 2025-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10554408

## Citation

> US National Institutes of Health, RePORTER application 10554408, Tumor-specific drug activation by pericellular proteases (5R21CA256460-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10554408. Licensed CC0.

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