Cumbers Sarah J, Williams Gareth T, Davies Sarah L, Grenfell Richard L, Takeda Shunichi, Batista Facundo D, Sale Julian E, Neuberger Michael S
Medical Research Laboratory of Biology, Hills Road, Cambridge CB2 2QH, UK.
Nat Biotechnol. 2002 Nov;20(11):1129-34. doi: 10.1038/nbt752. Epub 2002 Oct 15.
We show that iterative antigen-mediated selection of B-cell lines that constitutively hypermutate their immunoglobulin V genes during culture can be exploited to generate antibodies in vitro. From Ramos, a hypermutating human B-cell line expressing IgM of unknown specificity, we derived descendants that exhibit stepwise improved binding to streptavidin. Binding is initially conferred by mutations in complementarity-determining regions (CDRs), but maturation is due to strategic framework mutations. A more powerful system is provided by a hypermutating chicken B-lymphoma line, owing to its rapid proliferation, high rate of mutation accumulation, and genetic tractability. Starting from a single cell, we selected parallel lineages of derivatives, making mutated antibodies of increasing affinity to independent test antigens. Selection is initiated at an exceedingly low affinity threshold, but antibodies can be delivered with nanomolar affinities. The strategy could prove useful for in vitro generation of antigen-specific monoclonal antibodies and may be extendable to the maturation of other protein-ligand interactions.
我们表明，在培养过程中组成性地对其免疫球蛋白V基因进行高频率突变的B细胞系的迭代抗原介导选择，可用于在体外产生抗体。从Ramos（一种表达特异性未知的IgM的高频率突变人B细胞系）中，我们获得了后代，它们对链霉亲和素的结合能力逐步提高。结合最初是由互补决定区（CDR）中的突变赋予的，但成熟是由于策略性的框架突变。一种高频率突变的鸡B淋巴瘤细胞系提供了一个更强大的系统，这得益于其快速增殖、高突变积累率和遗传易处理性。从单个细胞开始，我们选择了平行的衍生物谱系，产生了对独立测试抗原有越来越高亲和力的突变抗体。选择在极低的亲和力阈值下启动，但抗体可以以纳摩尔亲和力产生。该策略可能被证明对体外产生抗原特异性单克隆抗体有用，并且可能扩展到其他蛋白质 - 配体相互作用的成熟。
