PDF(Pubmed)
Abstract：
Rationale: T-cell-redirecting bispecific antibodies (bsAbs) and trispecific antibodies (tsAbs) designed to recognize different epitopes or antigens have emerged as promising cancer therapies. Current approaches are all designed to include another antibody specific to the site of the primary antibody, and the molecular structures are generally established. However, the dimensions of target molecule and epitope location play a key role in the efficiency of the immunological synapse (IS) formation and subsequent T-cell-redirecting activities, therefore the connection flexibility of these antibodies determines the geometries of different formats of these molecules and will have a major impact on the efficacy. Methods: We describe a novel recombination strategy using various linker designs to site-specifically fuse anti-Her2 (2Rs15) or anti-VEGFR2 (3VGR19) nanobodies to different positions of the anti-CD3 antibody fragment (Fab, SP34). Based on the comparison among the various antigen-specific bsAbs, we could determine the desired fusion site of each nanobody to SP34, and further ensure the optimal structure of tsAbs with synergistic dual-antigen enhanced T-cell-redirecting activities. Results: This approach allows precise control of the formation of IS between Her2- and/or VEGFR2-expressing cancer cells and T cells, to obtain the optimal structure of the Her2/VEGFR2/CD3 tsAb without the need to map antibody-binding epitopes. Optimization of Her2/VEGFR2/CD3 tsAb results in enhanced T-cell-redirecting in vitro and in vivo antitumor efficacy compared with the corresponding bsAbs alone or in combination, and the potency to overcome tumor relapse due to antigen escape or resistance to Herceptin and Cyramza therapy. Conclusion: The novel design strategy for developing tsAbs using a site-specific recombination approach represents a promising platform for immuno-oncology and in applications other than cancer therapy.
摘要：
原理:设计用于识别不同表位或抗原的T细胞重定向双特异性抗体(bsAbs)和三特异性抗体(tsAbs)已经成为有希望的癌症疗法。目前的方法都被设计为包括对第一抗体位点具有特异性的另一种抗体，和分子结构一般建立。然而,靶分子和表位位置的大小在免疫突触（IS）形成和随后的T细胞重定向活动的效率中起关键作用，因此,这些抗体的连接灵活性决定了这些分子不同形式的几何形状,并将对疗效产生重大影响.方法：我们描述了一种新的重组策略，使用各种接头设计将抗Her2（2Rs15）或抗VEGFR2（3VGR19）纳米抗体位点特异性地融合到抗CD3抗体片段的不同位置（Fab，SP34)。基于各种抗原特异性bsAb之间的比较，我们可以确定每个纳米抗体与SP34的融合位点，并进一步确保具有协同双抗原增强的T细胞重定向活性的tsAbs的最佳结构。结果：这种方法可以精确控制表达Her2-和/或VEGFR2的癌细胞和T细胞之间的IS形成，获得Her2/VEGFR2/CD3tsAb的最佳结构，而无需绘制抗体结合表位。与单独或联合使用相应的bsAb相比，Her2/VEGFR2/CD3tsAb的优化导致T细胞重定向在体外和体内抗肿瘤功效增强，以及克服由于抗原逃逸或对赫赛汀和Cyramza疗法的抗性而引起的肿瘤复发的效力。结论：使用位点特异性重组方法开发tsAb的新设计策略代表了免疫肿瘤学和癌症治疗以外的应用的有希望的平台。
