[Pharmacological effects of site specific conjugated anti-human epidermal growth factor receptor 2-antibody drug conjugate using unnatural amino acid technology].

objectiveTo investigate inhibitory activities of a homogenous anti-human epidermal growth factor receptor 2 (HER2)-antibody drug conjugate (ADC) on the proliferation of nine tumor cell lines with different levels of HER2 expressions, and its activities on the tumor growth of five xenograft mouse models.

methodsThe HER2 expression levels of BT-474, Calu-3, MCF-7, MDA-MB-231, MDA-MB-468, SK-BR-3, SK-OV-3, HCC1954, NCI-N87 tumor cell lines were measured using QIFI KIT. For the in vitro anti-proliferation assay, serial diluted anti-HER2-ADC, ado-trastuzumab emtansine, AS269, pAF-AS269 and paclitaxel were added to the seeded cells, and after 72 or 96 hours of incubation, the cell proliferation was analyzed. For the in vivo activity, 5-6 weeks old mice were inoculated with four HER2 positive tumor cell lines HCC1954, BT-474, SK-OV-3, NCI-N87 or one HER2 negative tumor cell line MDA-MB-468. Different amounts of anti-HER2-ADC, ado-trastuzumab emtansine, trastuzumab, paclitaxel and phosphate buffered saline control were injected after the tumor volume reached a certain size, then the tumor growth inhibition was analyzed.

resultsThe expression levels of the six high HER2-expression cell lines SK-OV-3, NCI-N87, SK-BR-3, Calu-3, HCC1954, BT-474 were between 430 000 to 800 000 receptors per cell, which were 50 times higher than those of the other three low HER2 expression tumor cell lines MDA-MB-231, MCF-7, MDA-MB-468. Anti-HER2-ADC had inhibition effects on cell lines with high level of HER2 expression in the in vitro anti-proliferation assay. The half maximal inhibitory concentrations of anti-HER2-ADC on SK-OV-3, NCI-N87, SK-BR-3, Calu-3, HCC1954, BT-474 tumor cell lines were 46 pmol/L, 17 pmol/L, 17 pmol/L, 161 pmol/L, 125 pmol/L, 50 pmol/L, respectively. Anti-HER2-ADC had a dose dependent antitumor activity in vivo in all the HER2 positive xenograft mouse models. In NCI-N87 xenograft tumor model, the same dose of anti-HER2-ADC showed better anti-tumor activity compared with trastuzumab and ado-trastuzumab emtansine, and its relative tumor proliferation rates were about 1/30 to 1/20 of the two. In HCC1954 xenograft tumor model, the complete regression of the tumor was observed. As expected, anti-HER2-ADC had no tumor inhibitory effects on MDA-MB-468 xenograft models with low HER2 expression. The antitumor activities of anti-HER2-ADC in HER2 positive xenograft tumor models were the same as or better than the activities of ado-trastuzumab emtansine.

conclusionThe homogenous site-specific anti-HER2-ADC obtained using unnatural amino acid technology can inhibit the growth of high HER2-expression tumor cells with high potency both in vivo and in vitro.