Banting and Best Department of Medical Research and Department of Molecular Genetics, The Donnelly Centre, University of Toronto, 160 College Street, Toronto, Ontario, Canada M5S 3E1.
J Mol Biol. 2013 Feb 22;425(4):803-11. doi: 10.1016/j.jmb.2012.11.037. Epub 2012 Dec 3.
A synthetic phage-displayed antibody repertoire was constructed with equivalent chemical diversity in the third complementarity-determining regions of the heavy (CDR-H3) and light (CDR-L3) chains, which contrasts with natural antibodies in which CDR-H3 is much more diverse than CDR-L3 due to the genetic mechanisms that generate antibody encoding genes. Surprisingly, the synthetic repertoire yielded numerous functional antibodies that contained mutated CDR-L3 sequences but a fixed CDR-H3 sequence. Alanine-scanning analysis of antibodies that recognized 10 different antigens but contained a common CDR-H3 loop showed that, in most cases, the fixed CDR-H3 sequence was able to contribute favorably to antigen recognition, but in some cases, the loop was functionally inert. Structural analysis of one such antibody in complex with antigen showed that the inert CDR-H3 loop was nonetheless highly buried at the antibody-antigen interface. Taken together, these results show that CDR-H3 diversity is not necessarily required for the generation of antibodies that recognize diverse protein antigens with high affinity and specificity, and if given the chance, CDR-L3 readily assumes the dominant role for antigen recognition. These results contrast with the commonly accepted view of antigen recognition derived from the analysis of natural antibodies, in which CDR-H3 is presumed to be dominant and CDR-L3 is presumed to play an auxiliary role. Furthermore, the results show that natural antibody function is genetically constrained, and it should be possible to develop more functional synthetic antibody libraries by expanding the diversity of CDR-L3 beyond what is observed in nature.
构建了一个合成噬菌体展示抗体库，其重链（CDR-H3）和轻链（CDR-L3）的第三互补决定区（CDR）具有相同的化学多样性，这与天然抗体形成鲜明对比，由于产生抗体编码基因的遗传机制，CDR-H3 的多样性远大于 CDR-L3。令人惊讶的是，该合成库产生了许多功能抗体，其包含突变的 CDR-L3 序列但具有固定的 CDR-H3 序列。对识别 10 种不同抗原但具有共同 CDR-H3 环的抗体进行丙氨酸扫描分析表明，在大多数情况下，固定的 CDR-H3 序列能够有利于抗原识别，但在某些情况下，该环是无功能的。对与抗原结合的一种此类抗体的结构分析表明，无功能的 CDR-H3 环尽管如此，仍高度埋藏在抗体-抗原界面中。总之，这些结果表明，生成能够高亲和力和特异性识别各种蛋白质抗原的抗体不一定需要 CDR-H3 多样性，如果有机会，CDR-L3 将很容易承担抗原识别的主导作用。这些结果与从天然抗体分析中得出的关于抗原识别的普遍接受观点形成对比，在该观点中，CDR-H3 被认为是主导的，而 CDR-L3 被认为起着辅助作用。此外，这些结果表明天然抗体功能受到遗传限制，通过将 CDR-L3 的多样性扩展到超出自然界中观察到的范围，应该有可能开发出更具功能的合成抗体库。
