Ding Li, Tian Caiping, Feng Song, Fida Guissi, Zhang Congying, Ma Yuxiang, Ai Guanhua, Achilefu Samuel, Gu Yueqing
1. Department of Biomedical Engineering, China Pharmaceutical University, School of Life Science and Technology, Nanjing, China.
2. Department of Radiology, Washington University, School of Medicine, St. Louis, USA.
Theranostics. 2015 Jan 21;5(4):378-98. doi: 10.7150/thno.10084. eCollection 2015.
Targeting tumors using miniature antibodies is a novel and attractive therapeutic approach, as these biomolecules exhibit low immunogenicity, rapid clearance, and high targeting specificity. However, most of the small-sized antibodies in existence do not exhibit marked anti-tumor effects, which limit their use in targeted cancer immunotherapy. To overcome this difficulty in targeting multiple biomarkers by combination therapies, we designed a new bifunctional antibody, named MaAbNA (multivalent antibody comprised of nanobody and affibody moieties), capable of targeting EGFR1 and HER2, which are widely overexpressed in a variety of tumor types. The small-sized (29 kDa) MaAbNA, which was expressed in E.coli, consists of one anti-EGFR1 nanobody and two anti-HER2 affibodies, and possesses high affinity (KD) for EGFR1 (4.1 nM) and HER2 (4.7 nM). In order to enhance its anti-tumor activity, MaAbNA was conjugated with adriamycin (ADM) using a PEG2000 linker, forming a new complex anticancer drug, MaAbNA-PEG2000-ADM. MaAbNA exhibited high inhibitory effects on tumor cells over-expressing both EGFR1 and HER2, but displayed minimal cytotoxicity in cells expressing low levels of EGFR1 and HER2. Moreover, MaAbNA-PEG2000-ADM displayed increased tumoricidal effects than ADM or MaAbNA alone, as well exhibited greater antitumor efficacy than EGFR1 (Cetuximab) and HER2 (Herceptin) antibody drugs. The ability of MaAbNA to regulate expression of downstream oncogenes c-jun, c-fos, c-myc, as well as AEG-1 for therapeutic potential was evaluated by qPCR and western-blot analyses. The antitumor efficacy of MaAbNA and its derivative MaAbNA-PEG2000-ADM were validated in vivo, highlighting the potential for use of MaAbNA as a highly tumor-specific dual molecular imaging probe and targeted cancer therapeutic.
使用微型抗体靶向肿瘤是一种新颖且有吸引力的治疗方法，因为这些生物分子具有低免疫原性、快速清除和高靶向特异性。然而，现有的大多数小型抗体并未表现出显著的抗肿瘤作用，这限制了它们在靶向癌症免疫治疗中的应用。为了通过联合疗法克服靶向多种生物标志物的这一困难，我们设计了一种新的双功能抗体，名为MaAbNA（由纳米抗体和亲和体部分组成的多价抗体），它能够靶向在多种肿瘤类型中广泛过度表达的EGFR1和HER2。在大肠杆菌中表达的小型（29 kDa）MaAbNA由一个抗EGFR1纳米抗体和两个抗HER2亲和体组成，对EGFR1（4.1 nM）和HER2（4.7 nM）具有高亲和力（KD）。为了增强其抗肿瘤活性，使用PEG2000接头将MaAbNA与阿霉素（ADM）偶联，形成一种新的复合抗癌药物MaAbNA-PEG2000-ADM。MaAbNA对同时过度表达EGFR1和HER2的肿瘤细胞表现出高抑制作用，但对表达低水平EGFR1和HER2的细胞显示出最小的细胞毒性。此外，MaAbNA-PEG2000-ADM比单独的ADM或MaAbNA表现出更高的杀瘤效果，并且比EGFR1（西妥昔单抗）和HER2（赫赛汀）抗体药物表现出更强的抗肿瘤功效。通过qPCR和蛋白质免疫印迹分析评估了MaAbNA调节下游癌基因c-jun、c-fos、c-myc以及AEG-1表达的治疗潜力。MaAbNA及其衍生物MaAbNA-PEG2000-ADM的抗肿瘤功效在体内得到验证，突出了MaAbNA作为高度肿瘤特异性双分子成像探针和靶向癌症治疗药物的应用潜力。
