Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines
Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines
复制标题
DOI：
10.1038/nbt752
复制
发表时间：
2002-11-01
影响因子：
46.9
通讯作者：
Neuberger, MS
中科院分区：
文献类型：
--
作者：
Cumbers, SJ;Williams, GT;Neuberger, MS
我们表明，迭代抗原介导的选择B细胞系，组成型超突变的免疫球蛋白V基因在培养过程中，可以利用在体外产生抗体。从拉莫斯（一种表达特异性未知IgM的超突变人类B细胞系），我们衍生出表现出逐步改善的与链霉亲和素结合的后代。结合最初由互补决定区（CDR）中的突变赋予，但成熟是由于战略框架突变。一个更强大的系统是由一个超突变的鸡B淋巴瘤线，由于其快速增殖，高速率的突变积累，和遗传易处理。从单个细胞开始，我们选择了衍生物的平行谱系，使突变抗体对独立的测试抗原的亲和力增加。选择以极低的亲和力阈值开始，但抗体可以以纳摩尔亲和力递送。该策略可以证明对于体外产生抗原特异性单克隆抗体有用，并且可以扩展到其他蛋白质-配体相互作用的成熟。
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.