# Engineering high-efficiency matriptase substrates using E. coli display for applications in prodrug activation

## TL;DR

Researchers engineered highly efficient substrates for the tumor protease matriptase using E. coli display, improving prodrug activation for cancer therapy.

## Contribution

A novel E. coli display platform was developed to screen and optimize high-efficiency matriptase substrates for prodrug activation.

## Key findings

- Engineered substrates showed over 40-fold higher kcat/KM values compared to previously reported sequences.
- Optimized substrates enabled efficient prodrug activation in an antibody-prodrug format in vitro.
- The bacterial display platform is generalizable for discovering substrates of other proteases.

## Abstract

Proteases play a crucial role in biological functions such as tumor progression and tissue homeostasis. Recently, protease-activated prodrugs have gained attention for their potential to enhance selectivity in tumor-targeted therapies. In this study, we report the engineering of substrate sequences for matriptase, a protease overexpressed in tumors and previously explored for prodrug activation in vivo. A peptide library containing millions of potential substrates was displayed on Escherichia coli, and flow cytometric sorting was used to isolate improved substrates based on cleavage efficiency. Hits were ranked by flow cytometry, and the top substrates exhibited kcat/KM values over 40-fold higher than previously reported sequences. These substrates were further evaluated in an antibody-prodrug format, demonstrating exceptional activation. The matriptase substrates hold broad potential for applications such as cleavable linkers in next-generation antibody prodrugs. Furthermore, the developed bacterial display platform shows promise for discovering substrates of other proteases.

•High-throughput E. coli display enables screening of millions of protease substrates•Top hits show >40-fold improved kcat/KM over previously reported matriptase substrates•Optimized substrates enable efficient antibody prodrug activation in vitro

High-throughput E. coli display enables screening of millions of protease substrates

Top hits show >40-fold improved kcat/KM over previously reported matriptase substrates

Optimized substrates enable efficient antibody prodrug activation in vitro

On-target, off-tumor toxicity remains a challenge in targeted cancer therapy. Protease-activated prodrugs offer a strategy to increase selectivity by exploiting proteases enriched in the tumor microenvironment. However, their clinical success depends on the availability of highly efficient and specific protease substrates. In this study, we aimed to develop improved substrate sequences for the tumor-associated protease matriptase and to establish a generalizable method for discovering high-performance substrates. Such advances are valuable for enabling next-generation prodrugs with enhanced selectivity and safety.

Mestre Borras et al. develop an E. coli display platform for high-throughput screening of protease substrates. Using this method, they identify matriptase-cleavable sequences that outperform existing substrates and enable efficient prodrug activation.