摘要
Cell surface antigen-specific antibodies are of substantial diagnostic and therapeutic importance. The binding properties of such antibodies are usually evaluated by cell-free assays, in particular surface plasmon resonance (SPR) analysis, or flow cytometry. SPR analyses allow the detailed quantitative and dynamic evaluation of the binding properties of antibodies, but need purified, typically recombinantly produced antigens. It can, however, be difficult to produce the required antigen. Furthermore, cellular factors influencing the antigen-antibody interaction are not considered by this method. Flow cytometry-based analyses do not have these limitations, but require elaborated calibration controls for absolute quantification of bound molecules. To overcome the limitations of SRP and flow cytometry in the characterization of cell surface antigen-specific antibodies, we developed Fn14-specific antibody 18D1 as an example of an antibody fusion protein format that includes the luciferase of Gaussia princeps (GpL), which enables very simple and highly sensitive cellular binding studies. We found that GpL-tagging of the C-terminus of the antibody light chain does not affect the interaction of 18D1-IgG1 with its antigen and Fc-gamma receptors (FcγRs). In accordance with this, the GpL(LC-CT)-18D1-IgG1 antibody fusion protein showed basically the same FcγR-dependent agonistic properties as the parental 18D1 antibody. Similar results were obtained with isotype switch variants of 18D1 and antibodies specific for CD95, LTβR and CD40. In sum, we demonstrate that antibody GpL fusion proteins are easily manageable and versatile tools for the characterization of cell surface antigen-antibody interactions that have the potential to considerably extend the instrumentarium for the evaluation of antibodies.
摘要译文
细胞表面抗原特异性抗体具有实质的诊断和治疗重要性。通常通过无细胞测定评估这种抗体的结合特性，特别是表面等离子体共振（SPR）分析或流式细胞术。 SPR分析允许对抗体的结合特性进行详细的定量和动态评估，但需要纯化的，通常是重组产生的抗原。然而，产生所需的抗原可能是困难的。此外，这种方法不考虑影响抗原 - 抗体相互作用的细胞因子。基于流式细胞术的分析没有这些限制，但需要详细的校准控制来对结合分子进行绝对定量。为了克服SRP和流式细胞仪在表征细胞表面抗原特异性抗体方面的局限性，我们开发了Fn14特异性抗体18D1作为抗体融合蛋白形式的实例，其包括高斯葡萄球菌（GpL）的萤光素酶，这使得非常简单和高度敏感的细胞结合研究。我们发现抗体轻链C末端的GpL标记不影响18D1-IgG1与其抗原和Fc-γ受体（FcγR）的相互作用。按照这个，GpL（LC-CT）-18D1-IgG1抗体融合蛋白显示与亲本18D1抗体基本相同的FcγR依赖性激动剂性质。用18D1的同种型转换变体和对CD95，LTβR和CD40具有特异性的抗体获得类似的结果。总共，可用的和多功能的细胞表面抗原 - 抗体相互作用的工具，有可能大大扩展仪器用于评估抗体。
Juliane Kums[1];Johannes Nelke[1];Benedikt Rüth[1];Viktoria Schäfer[1];Daniela Siegmund[1];Harald Wajant[2]. Quantitative analysis of cell surface antigen-antibody interaction using Gaussia princeps luciferase antibody fusion proteins[J]. mAbs, 2017,9(3): 506-520