Lo Megan, Kim Hok Seon, Tong Raymond K, Bainbridge Travis W, Vernes Jean-Michel, Zhang Yin, Lin Yuwen Linda, Chung Shan, Dennis Mark S, Zuchero Y Joy Yu, Watts Ryan J, Couch Jessica A, Meng Y Gloria, Atwal Jasvinder K, Brezski Randall J, Spiess Christoph, Ernst James A
From the Departments of Protein Chemistry.
Antibody Engineering.
J Biol Chem. 2017 Mar 3;292(9):3900-3908. doi: 10.1074/jbc.M116.767749. Epub 2017 Jan 11.
The antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-γ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody. We demonstrate that the commonly used N297G (NG) and D265A, N297G (DANG) variants that are efficacious in attenuating effector function in primates retain potent complement activation capacity in mice, leading to safety liabilities in murine studies. In contrast, we found an L234A, L235A, P329G (LALA-PG) variant that eliminates complement binding and fixation as well as Fc-γ-dependent, antibody-dependent, cell-mediated cytotoxity in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an "effectorless" antibody between mice and primates. Further, we show that both human and murine antibodies containing the LALA-PG variant have typical pharmacokinetics in rodents and retain thermostability, enabling efficient knobs-into-holes bispecific antibody production and a robust path to generating highly effector-attenuated bispecific antibodies for preclinical studies.
抗体Fc区域调节抗体的细胞毒性活性和血清半衰期。然而，在治疗环境中，抗体的细胞毒性效应功能通常并不理想，并且可能通过激活针对表达受体抗原的细胞的天然宿主免疫防御而产生安全隐患。据报道，Fc区域的几个氨基酸变化可使抗体的效应功能沉默或降低。这些早期研究主要集中在人抗体与人Fc-γ受体的相互作用上，而Fc区域的这些变化在鼠抗体背景下如何转化仍 largely 未知。我们证明，在灵长类动物中有效减弱效应功能的常用N297G（NG）和D265A、N297G（DANG）变体在小鼠中保留了强大的补体激活能力，在小鼠研究中导致安全隐患。相比之下，我们发现一种L234A、L235A、P329G（LALA-PG）变体，它消除了补体结合和固定以及鼠IgG2a和人IgG1中Fc-γ依赖性、抗体依赖性细胞介导的细胞毒性。这些LALA-PG替换使得在小鼠和灵长类动物之间用“无效应”抗体产生的结果能够更准确地转化。此外，我们表明，含有LALA-PG变体的人源和鼠源抗体在啮齿动物中具有典型的药代动力学，并保留热稳定性，从而能够高效地进行旋钮入孔双特异性抗体制备，并为临床前研究生成高度减弱效应的双特异性抗体提供一条稳健的途径。
