Center for Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands.
Department of Dermatology/Allergology, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands.
Front Immunol. 2021 May 4;12:660037. doi: 10.3389/fimmu.2021.660037. eCollection 2021.
Human monoclonal antibodies (mAbs) are valuable tools to link genetic information with functional features and to provide a platform for conformational epitope mapping. Additionally, combined data on genetic and functional features provide a valuable mosaic for systems immunology approaches. Strategies to generate human mAbs from peripheral blood have been described and used in several studies including single cell sequencing of antigen-binding B cells and the establishment of antigen-specific monoclonal Epstein-Barr Virus (EBV) immortalized lymphoblastoid cell lines (LCLs). However, direct comparisons of these two strategies are scarce. Hence, we sought to set up these two strategies in our laboratory using peanut 2S albumins (allergens) and the autoantigen anti-Rho guanosine diphosphate dissociation inhibitor 2 (RhoGDI2, alternatively 'ARHGDIB') as antigen targets to directly compare these strategies regarding costs, time expenditure, recovery, throughput and complexity. Regarding single cell sequencing, up to 50% of corresponding V(D)J gene transcripts were successfully amplified of which 54% were successfully cloned into expression vectors used for heterologous expression. Seventy-five percent of heterologously expressed mAbs showed specific binding to peanut 2S albumins resulting in an overall recovery of 20.3%, which may be increased to around 29% by ordering gene sequences commercially for antibody cloning. In comparison, the establishment of monoclonal EBV-LCLs showed a lower overall recovery of around 17.6%. Heterologous expression of a mAb carrying the same variable region as its native counterpart showed comparable concentration-dependent binding abilities. By directly comparing those two strategies, single cell sequencing allows a broad examination of antigen-binding mAbs in a moderate-throughput manner, while the establishment of monoclonal EBV-LCLs is a powerful tool to select a small number of highly reactive mAbs restricted to certain B cell subpopulations. Overall, both strategies, initially set-up for peanut 2S albumins, are suitable to obtain human mAbs and they are easily transferrable to other target antigens as shown for ARHGDIB.
人源单克隆抗体 (mAbs) 是将遗传信息与功能特征联系起来的有价值的工具，并为构象表位作图提供了一个平台。此外，遗传和功能特征的综合数据为系统免疫学方法提供了有价值的镶嵌图。已经描述了从外周血中产生人源 mAbs 的策略，并在包括抗原结合 B 细胞的单细胞测序和建立抗原特异性单克隆 Epstein-Barr 病毒 (EBV) 永生化淋巴母细胞系 (LCL) 在内的几项研究中使用。然而，这两种策略的直接比较很少。因此，我们试图在我们的实验室中使用花生 2S 白蛋白（过敏原）和自身抗原抗 Rho 鸟苷二磷酸解离抑制剂 2（RhoGDI2，也称为 'ARHGDIB'）作为抗原靶标建立这两种策略，直接比较这些策略在成本、时间支出、回收率、通量和复杂性方面的差异。关于单细胞测序，成功扩增了相应 V(D)J 基因转录本的高达 50%，其中 54%成功克隆到用于异源表达的表达载体中。75%的异源表达 mAbs 特异性结合花生 2S 白蛋白，总回收率为 20.3%，通过商业订购基因序列进行抗体克隆，回收率可提高到 29%左右。相比之下，建立单克隆 EBV-LCL 的总回收率约为 17.6%。与天然对应物携带相同可变区的 mAb 的异源表达显示出可比的浓度依赖性结合能力。通过直接比较这两种策略，单细胞测序可以以中等通量的方式广泛检查抗原结合 mAbs，而建立单克隆 EBV-LCL 是选择少数高度反应性 mAb 的有力工具，这些 mAb 仅限于某些 B 细胞亚群。总体而言，最初为花生 2S 白蛋白建立的这两种策略都适合获得人源 mAbs，并且如 ARHGDIB 所示，它们很容易转移到其他靶抗原。
