Gao Changshou, Mao Shenlan, Kaufmann Gunnar, Wirsching Peter, Lerner Richard A, Janda Kim D
Department of Chemistry, The Scripps Research Institute and The Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12612-6. doi: 10.1073/pnas.192467999. Epub 2002 Sep 18.
For more than a decade, phage displayed combinatorial antibody libraries have been used to generate and select a wide variety of antibodies. We previously reported that the phage coat proteins pVII and pIX could be used to display the heterodimeric structure of the antibody Fv region. Herein, aspects of this technology were invoked and extended to construct a large, human single-chain Fv (scFv) library of 4.5 x 10(9) members displayed on pIX of filamentous bacteriophage. Furthermore, the diversity, quality, and utility of the library were demonstrated by the selection of scFv clones against six different protein antigens. Notably, more than 90% of the selected clones showed positive binding for their respective antigens after as few as three rounds of panning. Analyzed scFvs were also found to be of high affinity. For example, kinetic analysis (BIAcore) revealed that scFvs against staphylococcal enterotoxin B and cholera toxin B subunit had a nanomolar and subnanomolar dissociation constant, respectively, affording affinities comparable to, or exceeding that, of mAbs obtained from immunization. High specificity was also attained, not only between very distinct proteins, but also in the case of the Ricinus communis ("ricin") agglutinins (RCA(60) and RCA(120)), despite >80% sequence homology between the two. The results suggested that the performance of pIX-display libraries can potentially exceed that of the pIII-display format and make it ideally suited for panning a wide variety of target antigens.
十多年来，噬菌体展示的组合抗体文库一直被用于产生和筛选各种各样的抗体。我们之前报道过，噬菌体外壳蛋白pVII和pIX可用于展示抗体Fv区的异二聚体结构。在此，我们运用并扩展了这项技术的各个方面，构建了一个大型的、展示在丝状噬菌体pIX上的包含4.5×10⁹个成员的人单链Fv（scFv）文库。此外，通过针对六种不同蛋白质抗原筛选scFv克隆，证明了该文库的多样性、质量和实用性。值得注意的是，在仅三轮淘选后，超过90%的所选克隆对其各自的抗原显示出阳性结合。分析发现所选的scFv也具有高亲和力。例如，动力学分析（BIAcore）显示，针对葡萄球菌肠毒素B和霍乱毒素B亚基的scFv分别具有纳摩尔和亚纳摩尔的解离常数，其亲和力与从免疫获得的单克隆抗体相当或超过后者。不仅在非常不同的蛋白质之间，而且在蓖麻（“蓖麻毒素”）凝集素（RCA₆₀和RCA₁₂₀）的情况下，尽管两者之间的序列同源性>80%，也实现了高特异性。结果表明，pIX展示文库的性能可能超过pIII展示形式，使其非常适合筛选各种各样的靶抗原。
