Cheng Ming, Santich Brian H, Xu Hong, Ahmed Mahiuddin, Huse Morgan, Cheung Nai-Kong V
Department of Pediatrics, Memorial Sloan Kettering Cancer Center , New York, NY, USA.
Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
Oncoimmunology. 2016 May 5;5(6):e1168557. doi: 10.1080/2162402X.2016.1168557. eCollection 2016 Jun.
Engineering potent bispecific antibodies from single-chain variable fragments (scFv) remains difficult due to the inherent instability and insufficient binding of scFv's compared to their parental immunoglobulin format. Previously, we described a scFv-based bispecific antibody (scBA) against disialoganglioside (GD2) based on the anti-GD2 murine 5F11-scFv and the anti-CD3 huOKT3-scFv (5F11-scBA). In this study, we substituted the 5F11-scFv with the higher affinity (13-fold) hu3F8-scFv to form hu3F8-scBA. With this modification, hu3F8-scBA redirected T cells to kill GD2(+) cancer cell lines with up to 5,000-fold higher potency (femtomolar EC50) compared with 5F11-scBA (picomolar EC50) in cytotoxicity assays, even against target cells with low GD2 densities. Furthermore, hu3F8-scBA induced stronger T-cell activation than 5F11-scBA, as measured by Ca(2+) flux and cytokine release. Additionally, in vivo, hu3F8-scBA suppressed tumor growth and prolonged mice survival much more effectively than 5F11-scBA, in both neuroblastoma and melanoma xenograft models. We conclude that the functional properties of scBA's can be increased substantially by relatively modest increases in antigen affinity.
相较于其亲本免疫球蛋白形式，单链可变片段（scFv）固有的不稳定性和结合力不足使得工程化高效双特异性抗体仍然具有挑战性。此前，我们报道了一种基于抗GD2鼠源5F11-scFv和抗CD3人源OKT3-scFv的抗双唾液酸神经节苷脂（GD2）双特异性抗体（scBA）（5F11-scBA）。在本研究中，我们用亲和力更高（高13倍）的人源3F8-scFv替换5F11-scFv以形成人源3F8-scBA。通过这种修饰，在细胞毒性试验中，人源3F8-scBA重定向T细胞杀伤GD2(+)癌细胞系的效力比5F11-scBA（皮摩尔EC50）高5000倍（飞摩尔EC50），甚至对低GD2密度的靶细胞也有效。此外，通过Ca(2+)通量和细胞因子释放检测发现，人源3F8-scBA比5F11-scBA诱导更强的T细胞活化。另外，在体内，在神经母细胞瘤和黑色素瘤异种移植模型中，人源3F8-scBA比5F11-scBA更有效地抑制肿瘤生长并延长小鼠存活时间。我们得出结论，通过相对适度地提高抗原亲和力可显著增强scBA的功能特性。
