From the Departments of Therapeutic Discovery and.
J Biol Chem. 2014 Feb 7;289(6):3571-90. doi: 10.1074/jbc.M113.513366. Epub 2013 Dec 5.
Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fcγ receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. The co-crystal structure of FcγRIII in complex with Fc revealed that Fc binds to FcγRIII asymmetrically with two Fc chains contacting separate regions of the FcγRIII by utilizing different residues. To fully explore this asymmetrical nature of the Fc-FcγR interaction, we screened more than 9,000 individual clones in Fc heterodimer format in which different mutations were introduced at the same position of two Fc chains using a high throughput competition AlphaLISA® assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcγRIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. Compared with previously identified Fc variants in conventional IgG format, Fc heterodimers with asymmetrical mutations can achieve similar or superior potency in ADCC-mediated tumor cell killing and demonstrate improved stability in the CH2 domain. Fc heterodimers also allow more selectivity toward activating FcγRIIA than inhibitory FcγRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcγRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.
抗体依赖的细胞毒性 (ADCC) 是通过抗体的 Fc 片段与免疫细胞上的 Fcγ 受体 (FcγRs) 结合，在结合肿瘤或病毒抗原后介导的。FcγRIII 与 Fc 复合物的共晶结构表明，Fc 通过利用不同的残基，不对称地与 FcγRIII 结合，两条 Fc 链与 FcγRIII 的不同区域结合。为了充分探索 Fc-FcγR 相互作用的这种不对称性质，我们使用高通量竞争 AlphaLISA®测定法，在 Fc 异二聚体形式中筛选了超过 9000 个单独的克隆，其中两条 Fc 链的同一位置引入了不同的突变。为此，我们鉴定了一组新型 Fc 变体，它们与 FcγRIIIA（Phe-158 和 Val-158 同种型）的结合得到显著改善，体外 ADCC 活性增加，并在人肿瘤异种移植小鼠模型中强烈抑制肿瘤生长。与传统 IgG 形式中已鉴定的 Fc 变体相比，具有不对称突变的 Fc 异二聚体在 ADCC 介导的肿瘤细胞杀伤中可以达到相似或更高的效力，并在 CH2 结构域中表现出更好的稳定性。Fc 异二聚体也允许对激活型 FcγRIIA 比对抑制型 FcγRIIB 具有更高的选择性。Fc 变体的去岩藻糖基化进一步增加了 Fc 与 FcγRIIIA 的亲和力，导致 ADCC 活性大大提高。这些 Fc 变体的发现将为开发具有增强 ADCC 效应功能的下一代治疗性抗体提供新的机会，用于治疗癌症和传染病。
