The human epidermal growth factor receptor (HER/ErbB) family-targeted therapies result in a significant improvement in cancer immunotherapy. Monoclonal antibodies (MAb) against HER2 demonstrated a survival benefit for patients; however, drug resistance unavoidably occurs due to the overexpression of HER3, which leads to treatment failure. Effective inhibition of HER3 besides HER2 is thought to be required to overcome resistance and enhance therapeutic efficacy.

The present study describes the production and characterization of a novel MAb, designated 1G5D2, which acts as a natural bispecific antibody targeting extracellular domains (ECD) of both HER2 and HER3.

In this study, 1G5D2 was produced by hybridoma technology against HER3-ECD, and its structural and functional characteristics were studied by various methodologies, including enzyme linked-immunosorbent assays, flow cytometry, immunoblotting, cell signaling, and cell proliferation assays.

1G5D2 specifically binds to both HER2 (subdomain III + IV) and HER3 (subdomain I + II) expressed on tumor cells, and these receptors compete with each other for binding to this MAb. Competition flow cytometry experiments demonstrated that 1G5D2 does not compete with heregulin and recognizes an epitope out of HER3 ligand-binding site. Evaluation of 1G5D2 inhibitory effects in tumor cell lines co-expressing HER2 and HER3 showed that 1G5D2 synergizes with trastuzumab to inhibit both PI3K/AKT and MAPK/ERK pathways and potently downregulates the proliferation of these tumor cells more efficiently than each MAb alone.

1G5D2 is the first reported hybridoma antibody, which acts as a natural HER2/HER3 bispecific antibody. It might potentially be a suitable therapeutic candidate for HER2/HER3 overexpressing cancer types.