Optimization of anti-CD20 humanized antibody hu8E4 by site-directed mutation based on epitope analysis
Optimization of anti-CD20 humanized antibody hu8E4 by site-directed mutation based on epitope analysis
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基于表位分析的定点突变优化抗CD20人源化抗体hu8E4
DOI：
10.1016/j.bbrc.2015.02.158
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发表时间：
2015-04-17
影响因子：
3.1
通讯作者：
Guo, Yajun
中科院分区：
文献类型：
--
作者：
Chen, Yalin;Chen, Lin;Guo, Yajun
尽管抗CD 20嵌合抗体（mAb）利妥昔单抗在治疗B细胞淋巴瘤中有效，但其疗效仍然可变且通常适中。Hu 8 E4是一种抗CD 20人源化抗体，与利妥昔单抗相比，其显示出显著更高的抗肿瘤活性。以前的研究表明，利妥昔单抗和几乎所有已知的抗CD 20鼠单抗识别CD 20的大细胞外环内的A170/P172基序。在这项研究中，我们证明了hu 8 E4也识别A170/P172基序，这表明利妥昔单抗和hu 8 E4识别的表位非常相似。在此基础上，将重链可变区的三个可以提高利妥昔单抗亲和力的单突变（D57 E、Y102 K和Y102 T）转移到hu 8 E4中。结果显示，D57 E和Y102 T而不是Y102 K成功地增强了hu 8 E4与CD 20的结合。在这些hu 8 E4突变体中，hu 8 E4（D57 E）表现出最高的亲和力。进一步研究了hu 8 E4（D57 E）的体外和体内抗肿瘤活性。我们的数据表明，hu 8 E4（D57 E）在介导CDC和诱导B淋巴瘤细胞凋亡方面与hu 8 E4一样有效，但在ADCC方面比hu 8 E4更有效。重要的是，hu 8 E4（D57 E）在延长携带播散性B淋巴瘤细胞的SCID小鼠的存活方面显示出比Hu 8 E4显著更有效，表明其可能是用于B细胞淋巴瘤的有希望的治疗剂。此外，本研究还表明，可以提高利妥昔单抗的亲和力的突变可以转移到其他抗CD 20鼠单抗，以增强其与CD 20的结合。(C)2015 Elsevier Inc. All rights reserved.
Despite the effectiveness of the anti-CD20 chimeric antibody (mAb), rituximab, in treating B-cell lymphomas, its efficacy remains variable and often modest. Hu8E4 is an anti-CD20 humanized antibody which exhibits markedly higher antitumor activity compared with rituximab. Previous studies have indicated that rituximab and almost all known anti-CD20 murine mAbs recognize the A170/P172 motif within the large extracellular loop of CD20. In this study, we demonstrated that hu8E4 also recognized the A170/P172 motif, suggesting that the epitopes recognized by rituximab and hu8E4 are very similar. Based on this, three single mutations (D57E, Y102K and Y102T) at the heavy chain variable region that can improve the affinity of rituximab were transferred to hu8E4. The results showed that D57E and Y102T but not Y102K successfully enhanced the binding of hu8E4 to CD20. Out of these hu8E4 mutants, hu8E4(D57E) exhibited the highest affinity. The in vitro and in vivo antitumor activity of hu8E4(D57E) was further investigated. Our data indicated that hu8E4(D57E) was as effective as hu8E4 in mediating CDC and inducing apoptosis in B-lymphoma cells, but it was more potent in ADCC than hu8E4. Importantly, hu8E4(D57E) was shown to be significantly more effective than Hu8E4 in prolonging the survival of SCID mice bearing disseminated B-lymphoma cells, suggesting that it might be a promising therapeutic agent for B-cell lymphomas. Moreover, this study also suggests that the mutations that can improve the affinity of rituximab may be transferred to other anti-CD20 murine mAbs to enhance their binding to CD20. (C) 2015 Elsevier Inc. All rights reserved.