Marks J D, Hoogenboom H R, Bonnert T P, McCafferty J, Griffiths A D, Winter G
MRC Centre for Protein Engineering, Cambridge, U.K.
J Mol Biol. 1991 Dec 5;222(3):581-97. doi: 10.1016/0022-2836(91)90498-u.
We have mimicked features of immune selection to make human antibodies in bacteria. Diverse libraries of immunoglobulin heavy (VH) and light (V kappa and V lambda) chain variable (V) genes were prepared from peripheral blood lymphocytes (PBLs) of unimmunized donors by polymerase chain reaction (PCR) amplification. Genes encoding single chain Fv fragments were made by randomly combining heavy and light chain V-genes using PCR, and the combinatorial library (greater than 10(7) members) cloned for display on the surface of a phage. Rare phage with "antigen-binding" activities were selected by four rounds of growth and panning with "antigen" (turkey egg-white lysozyme (TEL) or bovine serum albumin) or "hapten" (2-phenyloxazol-5-one (phOx], and the encoding heavy and light chain genes were sequenced. The V-genes were human with some nearly identical to known germ-line V-genes, while others were more heavily mutated. Soluble antibody fragments were prepared and shown to bind specifically to antigen or hapten and with good affinities, Ka (TEL) = 10(7) M-1; Ka (phOx) = 2 x 10(6) M-1. Isolation of higher-affinity fragments may require the use of larger primary libraries or the construction of secondary libraries from the binders. Nevertheless, our results suggest that a single large phage display library can be used to isolate human antibodies against any antigen, by-passing both hybridoma technology and immunization.
我们模拟了免疫选择的特征，以便在细菌中制备人源抗体。通过聚合酶链反应（PCR）扩增，从未免疫供体的外周血淋巴细胞（PBL）中制备了免疫球蛋白重链（VH）和轻链（Vκ和Vλ）可变（V）基因的多样文库。利用PCR随机组合重链和轻链V基因，构建编码单链Fv片段的基因，并将组合文库（超过10^7个成员）克隆用于在噬菌体表面展示。通过四轮生长并用“抗原”（火鸡卵清溶菌酶（TEL）或牛血清白蛋白）或“半抗原”（2-苯基恶唑-5-酮（phOx）进行淘选，筛选出具有“抗原结合”活性的稀有噬菌体，并对编码的重链和轻链基因进行测序。这些V基因是人源的，有些与已知的胚系V基因几乎相同，而另一些则发生了更严重的突变。制备了可溶性抗体片段，结果显示其能特异性结合抗原或半抗原，且亲和力良好，Ka（TEL）=10^7 M^-1；Ka（phOx）=2×10^6 M^-1。分离更高亲和力的片段可能需要使用更大的原始文库或从结合物构建二级文库。然而，我们的结果表明，单个大型噬菌体展示文库可用于分离针对任何抗原的人源抗体，从而绕过杂交瘤技术和免疫过程。
