# Dual-targeting and steric hindrance resolution in HER2 IHC: a novel approach to improve diagnostic sensitivity

## TL;DR

A new method using fusion proteins improves HER2 detection in breast cancer tissues, increasing diagnostic accuracy by targeting different regions and avoiding steric hindrance.

## Contribution

A novel fusion protein (Nby-Aby) that targets HER2 at distinct regions to overcome steric hindrance and improve IHC diagnostic sensitivity.

## Key findings

- The Nby-Aby assay showed higher detection sensitivity for HER2-positive cells compared to conventional methods.
- HER2 scores in tissue microarrays were significantly higher with the Nby-Aby method than with traditional diagnosis.
- Dual-targeting and steric hindrance resolution is a promising strategy for more accurate HER2 IHC assessment.

## Abstract

The HER2 immunohistochemistry (IHC) test is an essential method for detecting breast cancer (BC) and plays a pivotal role in guiding personalized treatment strategies. However, inconsistencies persist among different pathologists using IHC, especially for HER2-low and HER2-negative. This may lead to discrepant clinical decisions, potentially impacting patient outcomes. Since HER2 exists in both dimeric and monomeric forms in cells, certain binding sites of diagnostic antibodies on HER2 dimers may be partially obscured in detection. Therefore, accurately detecting HER2 dimers in IHC is crucial for improving diagnostic precision.

We aligned the structures of HER2 heterodimers and Fabs of pertuzumab and trastuzumab binding to HER2, and found they binding in the same region. To overcome the steric hindrance of HER2 dimers, we employed HER2-binding affibody (Aby) and nanobody (Nby) to construct their fusion protein (Nby-Aby) and human heavy chain ferritin (HFn) based nanoparticles (Nby-HFn, Aby-HFn) for detection. Since the Nby and Aby bind HER2 at two distinct regions that are separate from the HER2 dimerization region, effectively minimizing interference from HER2 dimerization in detection. We assessed the detection performance of Nby-Aby in BC tissues and compared it with conventional HER2 diagnostic antibodies using tissue microarrays (TMAs).

The Nby-Aby assay had higher detection sensitivity for HER2-positive cells in BC tissues compared to the conventional method. Additionally, significantly higher HER2 scores were observed in most BC tissues on tissue microarrays (TMAs) compared to those diagnosed using the traditional method. These findings suggest that dual-targeting and overcoming steric hindrance in HER2 IHC detection is a promising strategy to enhance diagnostic precision.

Dual-targeting different regions and overcoming steric hindrance of HER2 in IHC detection through the Nby-Aby fusion protein enhances diagnostic sensitivity, providing a novel strategy for more accurate HER2 IHC assessment in BC diagnosis.

The online version contains supplementary material available at 10.1186/s12885-025-14553-7.