Liu Yinxing, Zheng Mengjie, Lai Zengzu, Xiong Dongsheng, Fan Dongmei, Xu Yuanfu, Peng Hui, Shao Xiaofeng, Xu Yuansheng, Yang Ming, Wang Jinhong, Liu Hanzhi, Xie Yong, Yang Chunzheng, Zhu Zhenping
State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences, 288 Nanjing Road, Tianjin 300020, China.
Cancer Lett. 2004 Mar 18;205(2):143-53. doi: 10.1016/j.canlet.2003.09.038.
Monoclonal antibodies (mAb) directed against CD20, either unmodified or in radiolabeled forms, have been successfully used in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma. Despite all clinical success the exact mechanisms of action of various anti-CD20 antibodies remains mostly unclear. Several mechanisms have been proposed to be responsible for the therapeutic activity of anti-CD20 antibodies, including antibody-dependent cell-mediated cytotoxicity, complement-mediated cytotoxicity, and direct inhibition of tumor growth via induction of apoptosis. We previously produced an anti-CD20 mAb, HI47, and showed that the antibody effectively blocked human B-cell proliferation in vitro and inhibited xenografted B-cell lymphoma in nude mice. In this study, we engineered the chimeric versions of both the Fab and F(ab)'2 fragments of HI47 and produced the fragments in E. coli. Both fragments competed efficiently with HI47 for binding to CD20+ B cells, and inhibited proliferation of B-lymphoma cells in a dose-dependent manner. Mechanistic studies revealed that both antibody fragments induced significant degree of B-cell apoptosis that is independent of any cross-linking agents. Further, both the F(ab)'2 and Fab fragments when administered in vivo significantly inhibited the growth of human B-cell lymphoma xenografts in nude mice. The bivalent F(ab)'2 fragment showed consistently better efficacy compared to its monovalent Fab counterpart in inducing apoptosis and inhibiting B-cell lymphoma growth both in vitro and in vivo. Taken together, these observations suggest that HI47 and its fragments most likely exert their antitumor activity through induction of cell apoptosis, and cross-linking/dimerization of CD20 molecules on B- cell surface is an important, but not essential, process for therapeutic efficacy of HI47 and its fragments.
针对CD20的单克隆抗体（mAb），无论是未修饰的还是放射性标记形式的，都已在临床上成功用作治疗非霍奇金B细胞淋巴瘤的有效治疗剂。尽管取得了所有临床成功，但各种抗CD20抗体的确切作用机制仍大多不清楚。已经提出了几种机制来解释抗CD20抗体的治疗活性，包括抗体依赖性细胞介导的细胞毒性、补体介导的细胞毒性以及通过诱导凋亡直接抑制肿瘤生长。我们之前制备了一种抗CD20单克隆抗体HI47，并表明该抗体在体外有效阻断人B细胞增殖，并抑制裸鼠体内移植的B细胞淋巴瘤。在本研究中，我们构建了HI47的Fab和F(ab)'2片段的嵌合版本，并在大肠杆菌中产生了这些片段。两个片段都能与HI47有效竞争结合CD20+B细胞，并以剂量依赖性方式抑制B淋巴瘤细胞的增殖。机制研究表明，两个抗体片段均诱导了显著程度的B细胞凋亡，且该凋亡与任何交联剂无关。此外，F(ab)'2和Fab片段在体内给药时均显著抑制裸鼠体内人B细胞淋巴瘤异种移植物的生长。在体外和体内诱导凋亡以及抑制B细胞淋巴瘤生长方面，二价F(ab)'2片段始终比其单价Fab对应物表现出更好的疗效。综上所述，这些观察结果表明，HI47及其片段最有可能通过诱导细胞凋亡发挥其抗肿瘤活性，并且B细胞表面CD20分子的交联/二聚化是HI47及其片段治疗效果的一个重要但非必需的过程。
