Immune checkpoint Inhibitors (ICIs) are effective immunno-therapeutic agents for cancer. Rapid and sensitive determination of the blocking activity of ICIs is important for ICIs development and immunological research. Among various immune checkpoint (IC) binding assays, cell-based binding assays are widely regarded, and the functional ELISA is a convenient alternative. However, these methodologies are limited by time-consuming preparation of cell lines stably expressing IC molecules, or long turnaround time with high cost. In this study, two magnetic bead based binding assays were developed to evaluate activity of ICIs, which was determined by a soluble ligand/bead immobilized receptor based binding assay (sL/bR binding assay) that assessed efficacy to block binding of one soluble IC ligand on its cognate receptor immobilized beads, or by a soluble receptor/bead immobilized ligand based binding assay (sR/bL binding assay) that assessed efficacy to block binding of soluble IC receptor on its cognate ligand immobilized beads. Half maximal inhibitory concentration (IC50) values of ICIs were calculated to determine ICIs activity. The sL/bR binding assay accurately determined the activity of two TIGIT blocking antibodies, since the relative blocking activity of two TIGIT antibodies determined by the sL/bR binding assay established in this study and that by the cell based binding assay were almost identical. In contrast, the sR/bL binding assay showed significantly improved sensitivity to determine activity of two PD-1 blocking antibodies than the sL/bR binding assay that was tested in this study and previous reports. Moreover, both amount of the used recombinant protein of ICI receptor/ligand and turnaround time of the two binding assays were more than 10 times less than those of the functional ELISA. These data indicate that the two magnetic bead based binding assays are sensitive, rapid and cost-effective methods to determine blocking activity of ICIs.

免疫检查点抑制剂 (ICI) 是有效的癌症免疫治疗剂。快速、灵敏地测定 ICI 的阻断活性对于 ICI 的开发和免疫学研究很重要。在各种免疫检查点 (IC) 结合测定中，基于细胞的结合测定被广泛认为，功能 ELISA 是一种方便的替代方法。然而，这些方法受到制备稳定表达 IC 分子的细胞系耗时长，或周转时间长且成本高的限制。在这项研究中，开发了两种基于磁珠的结合试验来评估 ICI 的活性，这是通过基于可溶性配体/珠子固定化受体的结合试验（sL/bR 结合试验）确定的，该试验评估阻断一种可溶性 IC 配体结合的功效在其同源受体固定珠上，或通过基于可溶性受体/珠子固定配体的结合测定（sR/bL 结合测定）评估阻断可溶性 IC 受体与其同源配体固定珠子结合的功效。计算 ICI 的半数最大抑制浓度 (IC50) 值以确定 ICI 活性。sL/bR 结合测定准确地确定了两种 TIGIT 阻断抗体的活性，因为本研究中建立的 sL/bR 结合测定和基于细胞的结合测定测定的两种 TIGIT 抗体的相对阻断活性几乎相同。相比之下，sR/bL 结合试验在确定两种 PD-1 阻断抗体的活性方面显示出比本研究和先前报告中测试的 sL/bR 结合试验显着提高的灵敏度。而且，两种结合检测的ICI受体/配体重组蛋白用量和周转时间均比功能ELISA少10倍以上。这些数据表明，这两种基于磁珠的结合测定是确定 ICI 阻断活性的灵敏、快速且经济高效的方法。