BY-PASSING IMMUNIZATION - HUMAN-ANTIBODIES FROM V-GENE LIBRARIES DISPLAYED ON PHAGE
BY-PASSING IMMUNIZATION - HUMAN-ANTIBODIES FROM V-GENE LIBRARIES DISPLAYED ON PHAGE
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DOI：
10.1016/0022-2836(91)90498-u
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发表时间：
1991-12-05
影响因子：
5.6
通讯作者：
WINTER, G
中科院分区：
文献类型：
--
作者：
MARKS, JD;HOOGENBOOM, HR;WINTER, G
我们模仿免疫选择的特点，在细菌中制造人类抗体。用聚合酶链式反应从未免疫献血员外周血淋巴细胞中扩增出免疫球蛋白重(VH)和轻(Vκ)和Vλ链可变(V)基因的不同文库。利用聚合酶链式反应将重链和轻链V基因随机组合成编码单链抗体片段的基因，并克隆出重组文库(>；107个成员)，用于在噬菌体表面展示。用“抗原”(火鸡蛋白溶菌酶或牛血清白蛋白)或“半抗原”(2-苯并恶唑-5-酮)对噬菌体进行四轮生长和淘洗，筛选出具有“抗原结合”活性的稀有噬菌体，并对其编码重链和轻链基因进行了测序。这些V基因是人类的，其中一些与已知的生殖系V基因几乎相同，而另一些则发生了更严重的突变。制备的可溶性抗体片段与抗原或半抗原特异结合，亲和力良好，KA(TEL)=107M−1，KA(PHOX)=2×106M−1。分离亲和力较高的片段可能需要使用更大的一级文库或从结合剂中构建二级文库。然而，我们的结果表明，单个大型噬菌体展示文库可以用于分离针对任何抗原的人类抗体，绕过杂交瘤技术和免疫。
We have mimicked features of immune selection to make human antibodies in bacteria. Diverse libraries of immunoglobulin heavy (VH) and light (Vκand Vλchain 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 (>107members) cloned for display on the surface of a phage. Rare phage with “antigenbinding” 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) = 107m−1;Ka(phOx) = 2 × 106m−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.