In vitro functional evaluation of soluble single domain antibodies targeting the HER2 dimerization domain

Treatment of HER2-overexpressing breast cancer remains a significant challenge. Previously, we selected single-domain antibodies (dAbs) against the HER2 dimerization domain using a phage display antibody library. In this study, we aimed to produce those dAbs in a phage-free manner and evaluate their antitumor effects in vitro.

The DNA fragment of antibodies was transferred to the pET32a expression vector to use a prokaryotic system and purified via affinity chromatography based on the histidine tag. The MTT assay was used to demonstrate the effect of the produced single-domain antibodies on the proliferation and survival of SKBR3 cancer cells. The expression of apoptosis-related genes was examined by qPCR, and the level of apoptosis induced by the single-domain antibodies was assessed using the Annexin V and PI methods.

Our single-domain antibodies markedly diminished the proliferation and viability of the SKBR3 cell line. They also increased the expression of apoptosis-associated genes, such as BAX and Caspase-3, while only within a 48-hour period decreasing the expression of the anti-apoptotic gene BCL2 significantly. Moreover, the expression of STAT3 decreased.

Our results demonstrated that the single-domain antibodies produced exhibited an acceptable level of functionality in vitro. Given the small size of the single-domain antibodies compared to the traditional whole antibodies, these data suggest that the soluble recombinant sdAbs retain in vitro activity in SKBR3 cells and support further evaluation of their HER2-binding specificity and therapeutic potential.