Harnessing a bispecific αGD2 × αCD3 protein engager to target GD2-overexpressing lung tumors

Lung cancer remains one of the most prevalent and lethal malignancies worldwide, with persistently poor 5-year survival rates across all stages. The development of novel therapies is therefore crucial for overcoming treatment resistance, improving survival rates, reducing side effects, and enhancing patient outcomes. Disialoganglioside GD2 has emerged as an attractive tumor-associated antigen for immunotherapy against various cancer types. In our study, we confirmed GD2 expression in lung cancer cells and observed differential expression levels, with A549 cells exhibiting markedly higher GD2 expression compared to NCI-H460 cells. To specifically target GD2-expressing lung cancers, we engineered a bispecific protein engager, termed αGD2 × αCD3 BiPE, designed to redirect T cells toward GD2-overexpressing lung cancer cells. The αGD2 × αCD3 BiPE (55 kDa) was successfully produced in a eukaryotic expression system and purified, as confirmed by Western blot analysis. Binding assays demonstrated that the BiPE specifically recognized GD2 on 143B osteosarcoma cells (which exhibit high GD2 expression) and CD3 on T cells. Functionally, treatment with αGD2 × αCD3 BiPE significantly enhanced T-cell activation and proliferation, as indicated by increased proportion of CD25 + CD3+ and CD69 + CD3+ cells after 5 days compared with untreated controls. In co-culture experiments, the BiPE also enhanced T-cell cytotoxicity, resulting in greater killing of A549 cells compared to NCI-H460 cells, consistent with their differential GD2 expression. These findings demonstrate that αGD2 × αCD3 BiPE effectively enhances T-cell activity and anti-tumor responses in a GD2-dependent manner. This study highlights its potential as a promising therapeutic strategy for GD2-overexpressing cancers, warranting further optimization and evaluation across diverse tumor types.