A single-chain variable fragment (scFv) targeting vascular endothelial growth factor receptor 2 was previously generated from a phage display library in our laboratory. However, it has shortened half-life and lacks Fc fragment for effector cell recognition. To address these challenges, a ligand of NK-cell receptor NKG2D was fused to the scFv and created a fusion protein scFv-major histocompatibility complex class I-related chain A (MICA), which is expected to recognize tumor cells through the scFv moiety and stimulate NK cells through the MICA. The fusion protein demonstrated specific binding to both vascular endothelial growth factor receptor 2 and NKG2D in protein-based and cell-based assays. In addition, it demonstrated antiangiogenic activities including restraining the proliferation, migration, transwell invasion, and tube formation of human umbilical vein endothelial cells. Furthermore, the fusion protein exhibited significant cytotoxicity on K562, MDA-MB-435, and B16F10 cells and triggered NK92 cell-mediated cytotoxicity on MDA-MB-435 cells by stimulating the release of significant cytokines. The fusion protein targeting strategy, therefore, provides a means to engage lymphocyte effector cells against tumor specific antigen overexpressing tumor cells.
*State Key Laboratory of Natural Medicines, School of Life Science & Technology, China Pharmaceutical University, Nanjing
†ImmuneOnco BiopharmCo., Ltd, Shanghai, P.R. China
D.O.A., M.T., and Y.W. contributed equally.
Reprints: Juan Zhang, State Key Laboratory of Natural Medicines, China Pharmaceutical University, 154#, Tongjiaxiang 24, Nanjing 210009, P.R. China (e-mail: firstname.lastname@example.org).
Received July 1, 2016
Accepted January 4, 2017