Dynamic Biosensors GmbH, Munich, Germany.
MAbs. 2023 Jan-Dec;15(1):2149053. doi: 10.1080/19420862.2022.2149053.
The binding properties of bispecific antibodies (bsAb) are crucial for their function, especially when two antigens are targeted on the same cell surface. Dynamic interactions between each of the antibody's arms and its cognate target cause the formation and decay of a biologically functional ternary complex. How association and dissociation processes work cooperatively, and how they influence the avidity of the ternary complex, is still poorly understood. Here, we present a biosensor assay for the simultaneous measurement of the binding kinetics of the therapeutic bsAb emicizumab (Hemlibra®) and its two targets, the blood coagulation factors IX and X (FIX, FX). We describe an automated workflow to characterize binary and ternary-binding modes, utilizing a Y-shaped DNA nanostructure to immobilize the antigens on a sensor and to emulate conditions on a cell or platelet surface by presenting the antigens with optimal accessibility for the bsAb flown over the sensor as analyte. We find that emicizumab binds FX much stronger than FIX (K = 0.05 µM vs. 5 µM, t = 20 s vs. 1 s) with profound consequences on the avidity of the ternary complex, which is dominated by FX's binding properties and a hand-off mechanism from FX to FIX. Moreover, formation and decay of the ternary complex depend on the bsAb concentration during the association phase. Emicizumab's in-vivo mode of action and the catalytic activation of FX can be rationalized from the analyzed binding kinetics. The assay and workflow are well suited for the screening of bispecific binders in drug discovery and provide valuable new kinetic information. bsAb: bispecific antibody; FVIII/FIX/FX: coagulation factors VIII/IX/X; SPR: surface plasmon resonance; k: association rate constant; k: dissociation rate constant; K: equilibrium dissociation constant; t: dissociation half-life.
双特异性抗体 (bsAb) 的结合特性对于其功能至关重要，尤其是当两个抗原在同一细胞表面被靶向时。每个抗体臂与其同源靶标的动态相互作用导致生物功能三元复合物的形成和衰变。结合和解离过程如何协同工作，以及它们如何影响三元复合物的亲和力，仍然知之甚少。在这里，我们提出了一种生物传感器测定法，用于同时测量治疗性 bsAb emicizumab（Hemlibra®）及其两个靶标，即血液凝血因子 IX 和 X（FIX，FX）的结合动力学。我们描述了一种自动化工作流程，用于表征二元和三元结合模式，利用 Y 形 DNA 纳米结构将抗原固定在传感器上，并通过将抗原以最佳可及性呈现给流过传感器作为分析物的 bsAb，模拟细胞或血小板表面的条件。我们发现 emicizumab 与 FX 的结合强度远大于 FIX（K=0.05 µM 对 5 µM，t=20 s 对 1 s），这对三元复合物的亲和力产生了深远的影响，三元复合物的亲和力主要由 FX 的结合特性和从 FX 到 FIX 的递呈机制决定。此外，三元复合物的形成和衰变取决于结合相期间 bsAb 的浓度。从分析的结合动力学可以理解 emicizumab 的体内作用模式和 FX 的催化激活。该测定法和工作流程非常适合药物发现中双特异性结合物的筛选，并提供有价值的新动力学信息。bsAb：双特异性抗体；FVIII/FIX/FX：凝血因子 VIII/IX/X；SPR：表面等离子体共振；k：缔合速率常数；k：解离速率常数；K：平衡解离常数；t：解离半衰期。
