Investigation on the Anti-Cancer Effects of HER2-Targeted CAR-T Cells Engineered Using the PiggyBac Transposon System

Chimeric antigen receptor T (CAR-T) cell therapies have demonstrated significant clinical efficacy in hematological malignancies. However, their application to solid tumors remains substantially limited by multiple challenges, including the risk of off-target effects. Hence, optimizing CAR-T cells for stronger antigen binding is essential.

In this study, we employed a classical anti-human endothelial growth factor receptor 2 (HER2) single-chain variable fragment (scFv) derived from trastuzumab, alongside an anti-HER2-13 scFv identified from a combinatorial cellular CAR library, for the construction of a third-generation CAR-T cell. Meanwhile, the phenotypes and both in vitro and in vivo functions of CAR-T cells transduced with the two scFvs via PiggyBac transposon-mediated gene transfer were compared.

The optimal ratio between the PiggyBac HER2-CAR-puro transposon and the Super PiggyBac transposase plasmid differed during the construction of the two HER2-targeted CAR-T cell types. The expansion abilities, CD3⁺CAR⁺ population, CD4⁺CAR⁺/CD8⁺CAR⁺ proportions, and memory and exhaustion markers between the two CAR-T groups were similar after using the optimized proportion of plasmid. Both CAR-T cell types exhibited significant antitumor activity, with the anti-HER2-13 CAR-T cells demonstrating superior target specificity. Therapeutic effects were observed with both CAR-T cells and trastuzumab in the MDA-MB-231^HER2+ breast tumor xenograft model, with anti-HER2-13 CAR-T cells demonstrating slightly enhanced efficacy and no evident off-target toxicity.

These results highlight the potential of anti-HER2-13 CAR-T cells to serve as a safer and more efficacious alternative in HER2-targeted therapy.