Department of Medicinal Chemistry, University of Washington, Seattle, Washington.
Biologics Research, Janssen Research & Development, Spring House, Pennsylvania.
Proteins. 2020 May;88(5):689-697. doi: 10.1002/prot.25853. Epub 2019 Nov 20.
Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen-binding fragments (Fabs) of an mAb allow for high-avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half-life. To augment the effector functions or half-life of an IgG1 mAb, we constructed a novel "2Fc" mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral-like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6- to 130-fold, resulting in apparent affinity increases of 7- to 42-fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild-type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.
单克隆抗体（mAbs）已成为一类重要的治疗药物，特别是在癌症免疫治疗领域。虽然 mAb 的两个抗原结合片段（Fabs）允许与分子靶标高亲和力结合，但可结晶片段（Fc）则与免疫效应元件结合。IgG 类 mAb 用于治疗自身免疫性疾病，不仅可以通过几种机制（包括通过 Fab 臂直接与抗原结合）引发抗肿瘤免疫功能，还可以通过 Fab 与肿瘤结合并与补体成分 C1q 和 Fcγ 受体结合的 Fc 结合来引发。此外，IgG 与新生儿 Fc 受体（FcRn）的结合允许内体循环和延长血清半衰期。为了增强 IgG1 mAb 的效应功能或半衰期，我们构建了一种新型的“2Fc”mAb，除了正常的两个 Fab 结构域外，它还含有两个 Fc 结构域。该 2Fc mAb 的结构和功能表征表明，它存在于类似四面体的几何形状中，并通过 Fab 结构域保留结合能力。此外，Fc 区的重复显著增强了对 Fc 受体 FcγRI、FcγRIIIa 和 FcRn 的亲和力，表现为复合物解离率降低，在受体密度较高时更为明显。在中等受体密度下，Fc 受体的解离速率降低了 6 至 130 倍，导致表观亲和力增加了 7 至 42 倍。计量分析证实，每个 2Fc mAb 可以同时结合两个 FcγRI 分子或四个 FcRn 分子，这是野生型 mAb 结合量的两倍。总之，IgG Fc 区的重复允许增加对 Fc 受体的亲和力，这可能转化为临床相关的效应功能或药代动力学增强。
