A tetravalent single chain diabody (CD40/HER2) efficiently inhibits tumor proliferation through recruitment of T cells and anti-HER2 functions.

Our aim was to construct a CD40×HER2 single chain diabody (ScDb) and determine its tumor-specific immune activation and anti-HER2 function. Overlap extension-polymerase chain reaction was applied in the construction of ScDb, and the protein was expressed with the pET28a (+)-Rosetta prokaryotic expression system. Soluble ScDb was purified by a nickel-nitrilotriacetic acid column. Dendritic cells (DC) was stimulated by ScDb and inhibited 4T1 cells proliferation in vitro. In 4T1 tumor mice model, lymphocyte infiltration was prominently detected in ScDb group, Caspase-3 expression was significantly upregulated. ScDb was labeled using quantum dots. Immunofluorescence assay indicated ScDb exhibited high affinity to HER2. T6-17 cells were inhibited by ScDb in vitro. The phosphorylation and expression levels of AKT, ERK were markedly decreased. In T6-17 tumor mice model. Compared to CD40 ScFv, HER2 ScFv and normal saline groups, tumor volume diminished significantly in ScDb group, and tumor cells showed extensive deformation, and pervasive karyopyknosis and karyorrhexis were found. In the present study, we successfully constructed a ScDb fragment and expressed it using a prokaryotic expression system. The in vivo and in vitro experimental results indicated that ScDb could inhibit the proliferation of tumor cells by stimulating the tumor-specific immunoreaction and blocking the HER2-related signaling pathway.