Department of Discovery Biology, Bristol Myers Squibb, Princeton, NJ 08543;
J Immunol. 2014 May 1;192(9):4083-92. doi: 10.4049/jimmunol.1303239. Epub 2014 Mar 26.
CD40-CD40L interactions play a critical role in regulating immune responses. Blockade of CD40L by Abs, such as the anti-CD40L Ab 5c8, demonstrated positive clinical effects in patients with autoimmune diseases; however, incidents of thromboembolism (TE) precluded further development of these molecules. In this study, we examined the role of the Fc domain interaction with FcγRs in modulating platelet activation and potential for TE. Our results show that the interaction of the 5c8 wild-type IgG1 Fc domain with FcγRs is responsible for platelet activation, as measured by induction of PAC-1 and CD62P. A version of 5c8 with a mutated IgG1 tail was identified that showed minimal FcγR binding and platelet activation while maintaining full binding to CD40L. To address whether Fc effector function is required for immunosuppression, a potent Ab fragment, termed a "domain Ab" (dAb), against murine CD40L was identified and fused to a murine IgG1 Fc domain containing a D265A mutation that lacks Fc effector function. In vitro, this dAb-Fc demonstrated comparable potency to the benchmark mAb MR-1 in inhibiting B cell and dendritic cell activation. Furthermore, the anti-CD40L dAb-Fc exhibited a notable efficacy comparable to MR-1 in various preclinical models, such as keyhole limpet hemocyanin-induced Ab responses, alloantigen-induced T cell proliferation, "heart-to-ear" transplantation, and NZB × NZW F1 spontaneous lupus. Thus, our data show that immunosuppression and TE can be uncoupled and that a CD40L dAb with an inert Fc tail is expected to be efficacious for treating autoimmune diseases, with reduced risk for TE.
CD40-CD40L 相互作用在调节免疫反应中起着关键作用。Abs（如抗 CD40L Ab 5c8）阻断 CD40L，在自身免疫性疾病患者中显示出积极的临床效果；然而，血栓栓塞（TE）事件排除了这些分子的进一步发展。在这项研究中，我们研究了 Fc 结构域与 FcγRs 的相互作用在调节血小板活化和 TE 潜力中的作用。我们的结果表明，5c8 野生型 IgG1 Fc 结构域与 FcγRs 的相互作用是导致血小板活化的原因，如通过诱导 PAC-1 和 CD62P 来测量。鉴定出 5c8 的一种突变 IgG1 尾部版本，该版本显示出最小的 FcγR 结合和血小板活化，同时保持对 CD40L 的完全结合。为了解决 Fc 效应功能是否需要免疫抑制，鉴定出一种针对小鼠 CD40L 的有效 Ab 片段，称为“结构域 Ab”（dAb），并将其融合到含有缺乏 Fc 效应功能的 D265A 突变的小鼠 IgG1 Fc 结构域中。在体外，这种 dAb-Fc 显示出与基准 mAb MR-1 相当的抑制 B 细胞和树突状细胞活化的效力。此外，抗 CD40L dAb-Fc 在各种临床前模型中表现出与 MR-1 相当的显著疗效，例如钥孔血蓝蛋白诱导的 Ab 反应、同种抗原诱导的 T 细胞增殖、“心耳”移植和 NZB×NZW F1 自发性狼疮。因此，我们的数据表明，免疫抑制和 TE 可以分离，并且具有惰性 Fc 尾部的 CD40L dAb 有望治疗自身免疫性疾病，血栓栓塞风险降低。
