State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China.
Eur Rev Med Pharmacol Sci. 2020 Dec;24(24):12929-12937. doi: 10.26355/eurrev_202012_24196.
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.
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.
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.
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.
研究 RC48-ADC 的靶向递药特性，RC48-ADC 是一种新型抗体药物偶联物（ADC），由细胞毒剂单甲基澳瑞他汀 E（MMAE）和通过缬氨酸-瓜氨酸接头连接的抗人表皮生长因子受体 2（HER2）抗体组成。
MMAE 从 RC48-ADC 中的解离率被用作其在血清中稳定性的估计。测量了抗体和 RC48-ADC 对多种细胞系的细胞毒性。通过荧光成像确定药物的亚细胞分布。验证了溶酶体靶向的机制。通过荧光标记药物的细胞荧光强度评估 RC48-ADC 的内吞途径。在给荷瘤小鼠尾静脉注射 RC48-ADC 后，分析 MMAE 的细胞内和细胞外分布，以表征 MMAE 的释放。比较了 RC48-ADC 给药后血清和肿瘤中 MMAE 的浓度。
RC48-ADC 在人血清中高度稳定。当 SK-BR-3 细胞系的 HER2 过表达受到 RC48-ADC 抑制时，其增殖能力强于裸抗体。RC48-ADC 和裸抗体均通过小窝蛋白介导和网格蛋白介导的内吞作用内化，并集中在溶酶体中。更高的 HER2 表达与共轭 MMAE 的摄取和细胞内释放增强有关；游离的 MMAE 可以通过旁观者效应杀死肿瘤细胞。尽管血清 RC48-ADC 浓度高于肿瘤，但肿瘤中 MMAE 的暴露量比血清中高约 200 倍，这解释了 RC48-ADC 毒性降低的原因。
体内外实验证实了 RC48-ADC 的靶向输送和释放；它可以将 MMAE 选择性递送至靶向的 HER2 阳性细胞或肿瘤组织，从而降低脱靶毒性并增强人类的抗肿瘤效力。
