a Antibody Design Labs , San Diego , CA , USA.
b UDIBI, ENCB, Instituto Politécnico Nacional , México , México.
MAbs. 2019 Apr;11(3):516-531. doi: 10.1080/19420862.2019.1571879. Epub 2019 Feb 26.
We describe here the design, construction and validation of ALTHEA Gold Libraries™. These single-chain variable fragment (scFv), semisynthetic libraries are built on synthetic human well-known IGHV and IGKV germline genes combined with natural human complementarity-determining region (CDR)-H3/J (H3J) fragments. One IGHV gene provided a universal V scaffold and was paired with two IGKV scaffolds to furnish different topographies for binding distinct epitopes. The scaffolds were diversified at positions identified as in contact with antigens in the known antigen-antibody complex structures. The diversification regime consisted of high-usage amino acids found at those positions in human antibody sequences. Functionality, stability and diversity of the libraries were improved throughout a three-step construction process. In a first step, fully synthetic primary libraries were generated by combining the diversified scaffolds with a set of synthetic neutral H3J germline gene fragments. The second step consisted of selecting the primary libraries for enhanced thermostability based on the natural capacity of Protein A to bind the universal V scaffold. In the third and final step, the resultant stable synthetic antibody fragments were combined with natural H3J fragments obtained from peripheral blood mononuclear cells of a large pool of 200 donors. Validation of ALTHEA Gold Libraries™ with seven targets yielded specific antibodies in all the cases. Further characterization of the isolated antibodies indicated K values as human IgG1 molecules in the single-digit and sub-nM range. The thermal stability (Tm) of all the antigen-binding fragments was 75°C-80°C, demonstrating that ALTHEA Gold Libraries™ are a valuable source of specific, high affinity and highly stable antibodies.
我们在这里描述了 ALTHEA Gold Libraries™的设计、构建和验证。这些单链可变片段（scFv）半合成文库是基于合成的人类已知 IGHV 和 IGKV 种系基因与天然人类互补决定区（CDR）-H3/J（H3J）片段构建的。一个 IGHV 基因提供了一个通用的 V 支架，并与两个 IGKV 支架配对，为结合不同的表位提供不同的拓扑结构。支架在与已知抗原-抗体复合物结构中抗原接触的位置进行多样化。多样化方案包括在人类抗体序列中这些位置发现的高使用氨基酸。通过三步构建过程，提高了文库的功能、稳定性和多样性。在第一步中，通过将多样化的支架与一组合成的中性 H3J 种系基因片段组合，生成完全合成的初级文库。第二步是根据 Protein A 结合通用 V 支架的天然能力，选择初级文库以提高热稳定性。在第三步也是最后一步中，将得到的稳定的合成抗体片段与从 200 名供体的大池外周血单核细胞中获得的天然 H3J 片段结合。用七个靶标验证 ALTHEA Gold Libraries™ ，在所有情况下都产生了特异性抗体。对分离出的抗体的进一步表征表明，K 值为人类 IgG1 分子，范围在个位数和亚纳摩尔之间。所有抗原结合片段的热稳定性（Tm）为 75°C-80°C，表明 ALTHEA Gold Libraries™ 是特异性、高亲和力和高度稳定抗体的宝贵来源。
