Adams Jarrett J, Nelson Bryce, Sidhu Sachdev S
Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.
Methods Mol Biol. 2014;1060:149-70. doi: 10.1007/978-1-62703-586-6_9.
In order to comprehensively manipulate the human proteome we require a vast repertoire of pharmacological reagents. To address these needs we have developed repertoires of synthetic antibodies by phage display, where diversified oligonucleotides are used to modify the complementarity-determining regions (CDRs) of a human antigen-binding fragment (Fab) scaffold. As diversity is produced outside the confines of the mammalian immune system, synthetic antibody libraries allow us to bypass several limitations of hybridoma technology while improving the experimental parameters under which pharmacological reagents are produced. Here we describe the methodologies used to produce synthetic antibody libraries from a single human framework with diversity restricted to four CDRs. These synthetic repertoires can be extremely functional as they produce highly selective, high affinity Fabs to the majority of soluble human antigens. Finally we describe selection methodologies that allow us to overcome immuno-dominance in our selections to target a variety of epitopes per antigen. Together these methodologies allow us to produce human monoclonal antibodies to manipulate the human proteome.
为了全面操控人类蛋白质组，我们需要大量的药理学试剂。为满足这些需求，我们通过噬菌体展示技术开发了合成抗体库，其中使用多样化的寡核苷酸来修饰人源抗原结合片段（Fab）支架的互补决定区（CDR）。由于多样性是在哺乳动物免疫系统之外产生的，合成抗体库使我们能够绕过杂交瘤技术的几个局限性，同时改善生产药理学试剂的实验参数。在这里，我们描述了从单一人类框架产生合成抗体库的方法，其多样性仅限于四个CDR。这些合成文库具有极高的功能性，因为它们能产生针对大多数可溶性人类抗原的高选择性、高亲和力的Fab。最后，我们描述了选择方法，这些方法使我们能够在选择过程中克服免疫优势，从而针对每个抗原靶向多种表位。这些方法共同使我们能够生产人类单克隆抗体来操控人类蛋白质组。
