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首页> 外文期刊>Journal of Molecular Biology >Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides [Review]
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Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides [Review]

机译：基于模块化共有框架和随机分配有三核苷酸的CDR的全合成人组合抗体文库（HuCAL）[综述]

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摘要

By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V-H and seven V-L (four V kappa and three V lambda) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs. Molecular modeling verified that all canonical classes were present. We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A first set of antibody phage display libraries totalling 2 x 10(9) members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question. Diversity was created by replacing the V-H and V-L CDR3 regions of the master genes by CDR3 Library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences. The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 %. Selection experiments against many antigens yielded a diverse set of binders with high affinities. Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes. (C) 2000 Academic Press. [References: 118]
机译：通过在结构，氨基酸序列多样性和种系使用方面分析人类抗体库，我们发现七个V-H和七个V-L（四个V kappa和三个V lambda）种系覆盖了所用人类抗体多样性的95％以上。得出每个家族的共有序列，并针对在大肠杆菌中的表达进行了优化。为了使所有六个互补决定区（CDR）易于多样化，通过引入CDR两侧的独特限制性核酸内切酶位点，将合成基因设计为模块化且相互兼容的。分子模型验证了所有经典分类均存在。我们可以证明，所有主控基因均在大肠杆菌的周质中表达为可溶性蛋白。在将所有49种组合的基因克隆到噬菌粒载体中后，创建了第一批共有2 x 10（9）个成员的抗体噬菌体展示文库，而噬菌粒载体本身没有相关的限制性酶切位点。通过用混合三核苷酸产生并偏向天然人抗体CDR3序列的CDR3文库盒替换主基因的V-H和V-L CDR3区来创建多样性。未选择文库的257个成员的测序表明，正确的序列（因此可能具有功能）的频率为61％。针对许多抗原的选择实验产生了一组具有高亲和力的结合物。由于所有主基因的模块化设计，现在可以使用预先构建的CDR盒文库在不了解特定序列的情况下快速优化单个结合物或什至结合物库。少量的49个主基因将使未来的改进能够迅速纳入，而框架的分离可能有助于分析自然界为何进化出了这些不同的抗体种系基因亚家族。 （C）2000学术出版社。 [参考：118]

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