MedImmune Research, Granta Park, Cambridge CB21 6GH, UK.
Protein Eng Des Sel. 2011 Apr;24(4):351-60. doi: 10.1093/protein/gzq114. Epub 2010 Dec 14.
Monoclonal antibodies are a commercially successful class of drug molecules and there are now a growing number of antibodies coupled to toxic payloads, which demonstrate clinical efficacy. Determining the precise epitope of therapeutic antibodies is beneficial in understanding the structure-activity relationship of the drug, but in many cases is not done due to the structural complexity of, in particular, conformational protein epitopes. Using the immunotoxin CAT-8015 as a test case, this study demonstrates that a new methodology, hybrid β-lactamase display, can be employed to elucidate a complex epitope on CD22. Following insertion of random CD22 gene fragments into a permissive site within β-lactamase, proteins expressed in Escherichia coli were first screened for correct folding by resistance to ampicillin and then selected by phage display for affinity to CAT-8015. The optimal protein region recognised by CAT-8015 could then be used as a tool for fine epitope mapping, using alanine-scanning analysis, demonstrating that this technology is well suited to the rapid characterisation of antibody epitopes.
单克隆抗体是一类商业上非常成功的药物分子，现在有越来越多的与毒性有效载荷偶联的抗体，这些抗体显示出临床疗效。确定治疗性抗体的精确表位有助于了解药物的结构-活性关系，但在许多情况下，由于特别是构象蛋白表位的结构复杂性，无法做到这一点。本研究以免疫毒素 CAT-8015 作为测试案例，证明了一种新的方法，即杂交β-内酰胺酶展示，可以用于阐明 CD22 上的复杂表位。在将随机的 CD22 基因片段插入β-内酰胺酶的许可位点之后，在大肠杆菌中表达的蛋白质首先通过对氨苄青霉素的抗性筛选来检测正确折叠，然后通过噬菌体展示来选择对 CAT-8015 的亲和力。然后，可以将 CAT-8015 识别的最佳蛋白质区域用作精细表位作图的工具，使用丙氨酸扫描分析，证明该技术非常适合快速表征抗体表位。
