Department of Neurological Surgery and The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL.
Department of Biomedical Engineering and Health Systems, Kungliga Tekniska Högscholan (Royal Institute of Technology), Sweden.
Transl Res. 2023 Jan;251:27-40. doi: 10.1016/j.trsl.2022.06.016. Epub 2022 Jul 3.
Inflammasomes are multiprotein complexes of the innate immune response that recognize a diverse range of intracellular sensors of infection or cell damage and recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into an inflammasome signaling complex. The recruitment, polymerization and cross-linking of ASC is upstream of caspase-1 activation and interleukin-1β release. Here we provide evidence that IC 100, a humanized IgG4κ monoclonal antibody against ASC, is internalized into the cell and localizes with endosomes, while another part is recycled and redistributed out of the cell. IC 100 binds intracellular ASC and blocks interleukin-1β release in a human whole blood cell inflammasome assay. In vitro studies demonstrate that IC 100 interferes with ASC polymerization and assembly of ASC specks. In vivo bioluminescence imaging showed that IC 100 has broad tissue distribution, crosses the blood brain barrier, and readily penetrates the brain and spinal cord parenchyma. Confocal microscopy of fluorescent-labeled IC 100 revealed that IC 100 is rapidly taken up by macrophages via a mechanism utilizing the Fc region of IC 100. Coimmunoprecipitation experiments and confocal immunohistochemistry showed that IC 100 binds to ASC and to the atypical antibody receptor Tripartite motif-containing protein-21 (TRIM21). In A549 WT and TRIM21 KO cells treated with either IC 100 or IgG4κ isotype control, the levels of intracellular IC 100 were higher than in the IgG4κ-treated controls at 2 hours, 1 day and 3 days after administration, indicating that IC 100 escapes degradation by the proteasome. Lastly, electron microscopy studies demonstrate that IC 100 binds to ASC filaments and alters the architecture of ASC filaments. Thus, IC 100 readily penetrates a variety of cell types, and it binds to intracellular ASC, but it is not degraded by the TRIM21 antibody-dependent intracellular neutralization pathway.
炎症小体是先天免疫反应的多蛋白复合物，可识别多种感染或细胞损伤的细胞内传感器，并将衔接蛋白含有半胱氨酸蛋白酶募集结构域（ASC）的凋亡相关斑点样蛋白募集到炎症小体信号复合物中。ASC 的募集、聚合和交联是 caspase-1 激活和白细胞介素-1β释放的上游事件。在这里，我们提供的证据表明，针对 ASC 的人源化 IgG4κ 单克隆抗体 IC 100 被内化到细胞内，并与内体定位，而另一部分则被回收并重新分布出细胞。IC 100 在人全血细胞炎症小体测定中结合细胞内 ASC 并阻断白细胞介素-1β的释放。体外研究表明，IC 100 干扰 ASC 聚合和 ASC 斑点的组装。体内生物发光成像显示，IC 100 具有广泛的组织分布，可穿过血脑屏障，并容易穿透大脑和脊髓实质。荧光标记的 IC 100 的共聚焦显微镜显示，IC 100 被巨噬细胞通过利用 IC 100 的 Fc 区域的机制快速摄取。共免疫沉淀实验和共聚焦免疫组织化学显示，IC 100 与 ASC 和非典型抗体受体三部分基序含有蛋白-21（TRIM21）结合。在用 IC 100 或 IgG4κ 同种型对照处理的 A549 WT 和 TRIM21 KO 细胞中，与 IgG4κ 处理对照相比，给药后 2 小时、1 天和 3 天，细胞内 IC 100 的水平更高，表明 IC 100 逃避蛋白酶体的降解。最后，电子显微镜研究表明，IC 100 与 ASC 丝结合并改变 ASC 丝的结构。因此，IC 100 容易穿透多种细胞类型，并且与细胞内 ASC 结合，但它不受 TRIM21 抗体依赖性细胞内中和途径的降解。
