# A Cathepsin B‐Triggered CO‐Releasing Molecule with a Non‐Toxic Metal Core for Targeted Tumor Delivery

## TL;DR

A new CO-releasing molecule activated by a tumor enzyme is attached to an antibody, enabling targeted delivery of carbon monoxide to cancer cells.

## Contribution

A non-toxic, iron-based CO-releasing molecule triggered by cathepsin B is site-specifically conjugated to an antibody for targeted tumor delivery.

## Key findings

- The ET-CORM–antibody conjugate delivers CO selectively to HER2-overexpressing and CatB-expressing cells.
- The conjugate has a drug-to-antibody ratio of 6.8, corresponding to about 20 CO molecules per conjugate.
- The ET-CORM is built on a biocompatible iron core and activated by tumor-associated cathepsin B.

## Abstract

Carbon monoxide (CO) has shown therapeutic potential across various diseases, including cancer. To enable controlled delivery, many CO‐releasing molecules (CORMs) have been developed. However, their clinical translation has been limited due to concerns about stability, potential toxicity, and insufficient targeting ability. In this study, we report the synthesis and characterization of an enzyme‐triggered CO‐releasing molecule (ET‐CORM) that can be site‐specifically conjugated to antibodies. This novel ET‐CORM is built on a biocompatible iron core, and releases CO upon cleavage by the cancer‐associated protease cathepsin B (CatB). The incorporation of a bioorthogonal handle into ET‐CORM enabled its efficient and site‐specific conjugation to the clinically used antibody trastuzumab via the interchain disulfide bonds. The resulting ET‐CORM–antibody conjugate (ET‐CORM‐Ab) exhibited an average drug‐to‐antibody ratio (DAR) of 6.8, corresponding to approximately 20 CO molecules per conjugate. This construct allowed for selective intracellular CO delivery to HER2‐overexpressing and CatB‐expressing cells in vitro. This study represents a metal‐based CORM–antibody conjugate activated by a tumor‐associated enzymatic trigger, opening new avenues for investigating CO‐mediated effects and advancing CO‐based cancer therapies to the clinics.

An iron‐based, enzyme‐triggered CO‐releasing molecule (ET‐CORM) is site‐specifically conjugated to the antibody trastuzumab. This novel ET‐CORM‐antibody conjugate releases carbon monoxide upon cleavage by the tumor‐associated protease cathepsin B. The construct enables selective intracellular CO delivery to HER2‐overexpressing and CatB‐expressing cells, presenting a new strategy for targeted, CO‐based cancer therapies.