Coupling brain perfusion screens and next generation sequencing to identify blood-brain barrier binding antibodies
Coupling brain perfusion screens and next generation sequencing to identify blood-brain barrier binding antibodies
复制标题
DOI：
10.1002/aic.16360
复制
发表时间：
2018-12-01
期刊：
影响因子：
3.7
通讯作者：
Shusta，Eric V.
中科院分区：
文献类型：
--
作者：
Stutz，Charles C.;Georgieva，Julia V.;Shusta，Eric V.
体内靶向血脑屏障（BBB）的抗体对于治疗神经系统疾病特别有意义。在这里，我们通过脑灌注筛选了噬菌体展示单链抗体（scFv）文库，试图分离出靶向大鼠血脑屏障的scFv。经过四轮筛选后，所得的抗体池仍然高度复杂，并且离散克隆取样没有鉴定出任何能够与大鼠 BBB 结合的 scFv。因此，对所得池中的重链 CDR3 进行下一代测序 (NGS)，所得数据用于鉴定 12 个 scFv 克隆，这些克隆具有高丰度和/或从第 3 轮到第 4 轮富集，表明潜在的命中。其中，两个 scFv（表示为 scFv 4 和 scFv 40）被鉴定为结合大鼠 BBB。这些 scFv 都不是通过离散采样鉴定的，这促使 NGS 成为从复杂的体内筛选中鉴定先导抗体的工具。 © 2018 美国化学工程师学会AIChE J, 64: 4229–4236, 2018
Antibodies that target the blood–brain barrier (BBB) in vivo are of particular interest for the treatment of neurological diseases. Here, we screened a phage display single‐chain antibody (scFv) library by brain perfusion in an attempt to isolate scFv that target the rat BBB. After four rounds of screening, the resulting antibody pool remained highly complex and discrete clonal sampling did not identify any scFvs capable of binding to the rat BBB. Thus, the heavy chain CDR3 in the resulting pools was subjected to next generation sequencing (NGS), and the resulting data was used to identify 12 scFv clones that were of high abundance and/or enriched from Round 3 to 4, signifying potential hits. Of these, two scFv, denoted scFv 4 and scFv 40, were identified that bound the rat BBB. Neither of these scFvs was identified by discrete sampling, motivating NGS as a tool to identify lead antibodies from complex in vivo screens. © 2018 American Institute of Chemical EngineersAIChE J, 64: 4229–4236, 2018