# Targeting cancer expressed EGFR with a humanized monoclonal antibody

## TL;DR

This paper describes the development of a humanized antibody, A10, that targets cancer-expressed EGFR and shows strong cell binding and cytotoxic potential.

## Contribution

The novel contribution is the creation and characterization of A10, a humanized antibody with improved binding and therapeutic potential for EGFR-expressing cancers.

## Key findings

- A10 exhibited the strongest cell binding activity among humanized variants of 40H3.
- A10's binding is conformational dependent and unlikely to react with wild-type EGFR.
- An A10-based ADC with MMAE showed potent cytotoxicity against high EGFR-expressing cells.

## Abstract

The humanization of mouse monoclonal antibodies targeting tumor antigens allows for the safe and repeated administration of therapeutic antibodies to humans. Previously, we reported on mouse monoclonal antibody 40H3 and its reactivity with a conformational epitope exposed on EGFR when expressed by cancer cells. To advance 40H3 toward the clinic, we generated various humanized variants, evaluated each for binding to either the peptide epitope or intact cells, and now report on the lead candidate, A10, which exhibited the strongest cell binding activity. A10 binding was characterized in detail on a collection of cancer cell lines each harboring different EGFR mutations or overexpressed EGFR and, where appropriate, compared with 40H3 or Cetuximab. A structural study of the A10 Fab with bound peptide revealed that A10 was unlikely to react with either tethered or untethered forms of wild type EGFR, as the accessibility to the peptide epitope by A10 is EGFR conformational dependent. As a proof of concept to produce therapeutic agents from A10, an antibody drug conjugate (ADC) with monomethyl-auristatin-E (MMAE) was generated and proved potently cytotoxic for cells displaying high levels of EGFR.

The online version contains supplementary material available at 10.1038/s41598-026-46245-y.