Hybridoma populations enriched for affinity-matured human IgGs yield high-affinity antibodies specific for botulinum neurotoxins
Hybridoma populations enriched for affinity-matured human IgGs yield high-affinity antibodies specific for botulinum neurotoxins
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DOI：
10.1016/j.jim.2008.01.015
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发表时间：
2008-04-20
影响因子：
2.2
通讯作者：
Dessain, Scott K.
中科院分区：
文献类型：
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作者：
Adekar, Sharad P.;Jones, R. Mark;Dessain, Scott K.
亲和力成熟的人抗体库可以理想地作为针对传染病和生物恐怖剂的抗体治疗剂的来源。用于克隆这些抗体的杂交方法具有许多潜在的优点，包括方便性、高产量抗体表达以及以其天然构型捕获抗体的能力。然而，它们受到杂交瘤不稳定性和抗原特异性、类别转换的人B细胞的有限可及性的阻碍。在这里，我们描述了一种有效的，三步的方法，使用人外周血B细胞，以产生稳定的杂交瘤群体，是高度富集的亲和力成熟的人IgG抗体。（a）选择外周血单核细胞（PBMC）以表达CD 27（一种生发后中心B细胞的标志物），（B）体外培养以促进B细胞增殖和类别转换，和（c）与遗传修饰的骨髓瘤细胞系融合。使用这种策略，我们克隆了5个IgG抗体，结合肉毒杆菌，神经毒素（BoNT），食源性麻痹性疾病，肉毒杆菌中毒，A类选择生物恐怖剂的原因。这些抗体中的两种以低皮摩尔亲和力结合BoNT。一种（30 B）是第一种结合血清型B BoNT的高亲和力人抗体，另一种（6A）能够在暴露前和暴露后模型中体内中和致死剂量的血清型A BoNT。这种优化的杂交瘤方法将广泛地使获得天然人抗体库成为可能。(C)2008 Elsevier B.V.保留所有权利。
The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against infectious diseases and bioterror agents. Hybridoma methods for cloning these antibodies have many potential advantages, including convenience, high-yield antibody expression, and the ability to capture the antibodies in their native configurations. However, they have been hindered by hybridoma instability and limited accessibility of antigen-specific, class-switched human B-cells. Here, we describe an efficient, three-step method that uses human peripheral blood B-cells to produce stable hybridoma populations that are highly-enriched for affinity-matured human IgG antibodies. Peripheral blood mononuclear cells (PBMCs) are (a) selected for expression of CD27, a marker of post-germinal center B-cells, (b) cultured in vitro to promote B-cell proliferation and class-switching, and (c) fused to a genetically modified myeloma cell line. Using this strategy, we cloned 5 IgG antibodies that bind botulinum, neurotoxins (BoNT), the causes of the food-borne paralytic illness, botulism, and Category A Select Bioterror agents. Two of these antibodies bind BoNT with low picomolar affinities. One (30B) is the first high-affinity human antibody to bind serotype B BoNT, and another (6A) is able to neutralize a lethal dose of serotype A BoNT in vivo in pre- and post-exposure models. This optimized hybridoma method will broadly enable access to the native human antibody repertoire. (C) 2008 Elsevier B.V. All rights reserved.