# Design and Characterization of Prodrugged Anti‐CTLA‐4 Antibodies

## TL;DR

Researchers designed a modified anti-CTLA-4 antibody that reduces off-target effects and improves safety by attaching a PEG molecule, which is cleaved in the tumor environment.

## Contribution

A novel prodrug strategy for anti-CTLA-4 antibodies using site-specific PEGylation to enhance safety and pharmacokinetics.

## Key findings

- PEGylation at specific antibody sites reduces binding to CTLA-4 and Fc gamma receptors.
- The prodrugged antibody shows high stability and extended half-life in a mouse model.
- Cleavage in the tumor microenvironment restores antibody function.

## Abstract

Therapeutic antibodies are widely used to treat diseases like cancer and inflammatory conditions by binding with high specificity to their molecular targets. Masking is a strategy to mitigate undesirable activity in nontarget tissues, improving safety and pharmacokinetic (PK) profiles by reducing target‐mediated drug disposition. In this article, masking of an anti‐CTLA‐4 antibody by conjugating a large PEG molecule to specific sites on the antibody is explored. While anti‐CTLA‐4 immunotherapy benefits solid tumor treatment, its adverse events limit its utility. Multiple conjugation sites within the complementarity‐determining regions and adjacent framework regions are evaluated to attenuate CTLA‐4 binding. The optimal site for maximizing masking efficiency is identified, allowing for efficient bioconjugation and functional restoration upon exposure to a cleaving enzyme in the tumor microenvironment. The prodrugged antibody exhibits reduced binding to CTLA‐4 and Fc gamma receptors, high stability, and an extended half‐life in a mouse model. This technology has the potential to improve the PK profile and safety attributes of therapeutic antibodies.

Masking an anti‐CTLA‐4 antibody by conjugating a large PEG molecule to specific sites is explored as a way of mitigating undesirable activity in nontarget tissues. This prodrugged antibody exhibits reduced binding to CTLA‐4 and Fc gamma receptors, demonstrated high stability, and had an extended half‐life in a mouse model, potentially improving the pharmacokinetic profile and safety attributes of therapeutic antibodies.© 2025 WILEY‐VCH GmbH