Conjugating MMAE to a novel anti-HER2 antibody for selective targeted delivery.

OBJECTIVE: To investigate the target delivery properties of RC48-ADC, a novel antibody drug conjugate (ADC) comprising cytotoxic monomethyl auristatin E (MMAE) and an anti-human epidermal growth factor receptor 2 (HER2) antibody tethered via valine-citrulline linker, in vitro and in vivo. MATERIALS AND METHODS: Dissociation rate of MMAE from RC48-ADC was used as an estimate of its stability in serum. Cytotoxicity of the antibody and RC48-ADC towards multiple cell lines was measured. Subcellular distribution of the drug was determined by fluorescence imaging. The mechanism of lysosome targeting was verified. Endocytic pathways of RC48-ADC were assessed by the cellular fluorescence intensity of fluorescently-labelled drugs. Intracellular and extracellular distribution of MMAE was analysed after RC48-ADC or MMAE administration to characterize MMAE release. The serum and tumour concentration of MMAE was compared after tail-vein injection of RC48-ADC into tumour-bearing mice. RESULTS: RC48-ADC was highly stable in human serum. HER2-overexpressed cell line SK-BR-3 proliferation was stronger when suppressed by RC48-ADC than by the naked antibody. Both RC48-ADC and naked antibody were internalized via caveolae-mediated and clathrin-mediated endocytosis and concentrated in lysosomes. Higher HER2 expression was associated with enhanced uptake and intracellular release of conjugated MMAE; free MMAE could kill tumour cells via the bystander effect. Although serum RC48-ADC concentration was higher than that in tumours, exposure of MMAE in tumours was ~200 times higher than in serum, which rationalized the reduced toxicity of RC48-ADC. CONCLUSIONS: In vitro and in vivo experiments confirmed the targeted transport and release of RC48-ADC; it could selectively deliver MMAE to the targeted HER2-positive cell or tumour tissue, which could reduce off-target toxicity and enhance anti-tumour potency in humans.