CDR WALKING MUTAGENESIS FOR THE AFFINITY MATURATION OF A POTENT HUMAN ANTI-HIV-1 ANTIBODY INTO THE PICOMOLAR RANGE
CDR WALKING MUTAGENESIS FOR THE AFFINITY MATURATION OF A POTENT HUMAN ANTI-HIV-1 ANTIBODY INTO THE PICOMOLAR RANGE
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DOI：
10.1006/jmbi.1995.0626
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发表时间：
1995-12-01
影响因子：
5.6
通讯作者：
BARBAS, CF
中科院分区：
文献类型：
--
作者：
YANG, WP;GREEN, K;BARBAS, CF
我们描述了用于产生非常高亲和力的人抗体的方法学的研究。高亲和力人抗体b4/12针对其对人免疫缺陷病毒1型（HIV-1）的人包膜糖蛋白gp 120的亲和力进行了优化。通过互补决定区（CDR）的饱和诱变构建了5个b4/12文库。抗体Fab片段的文库展示在丝状噬菌体的表面上，并在体外选择与固定的gp 120结合。检查了CDR的顺序和并行优化策略。连续CDR步移策略一致地在所检查的四个不同优化序列中的每一个中产生亲和力改善的b4/12变体。这导致亲和力提高96倍。通过在平行优化策略中组合独立优化的CDR来探索抗体组合位点中的加和效应。构建了含有优化的CDR的六种变体。基于加和效应的亲和力的改善被证明是不可预测的，但确实导致亲和力的适度改善。事实上，六种组合中只有一种表现出加和性。使用该策略制备的最高亲和力Fab的亲和力提高了420倍。与b4/12的6.3nM相比，该Fab的亲和力为15 pM。Fab与gp 120结合的动力学检查显示，亲和力的改善主要由Fab解离速率的减慢所控制。本文提出的方法提供了一种将三级免疫应答的典型抗体的亲和力提高到皮摩尔范围的途径。这样的改进可能对抗体作为治疗剂和抗炎剂的效用具有深远的影响。(C)1995年学术出版社
We describe the investigation of methodologies for the creation of very high affinity human antibodies. The high affinity human antibody b4/12 was optimized for its affinity to the human envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1). Five libraries of b4/12 were constructed by saturation mutagenesis of complementarity-determining regions (CDRs). Libraries of antibody Fab fragments were displayed on the surface of filamentous phage and selected in vitro for binding to immobilized gp120. Sequential and parallel optimization strategies of CDRs were examined. The sequential CDR walking strategy consistently yielded b4/12 variants of improved affinity in each of the four different optimization sequences examined. This resulted in a 96-fold improvement in affinity Additivity effects in the antibody combining site were explored by combining independently optimized CDRs in the parallel optimization strategy Six variants containing optimized CDRs were constructed. Improvement of affinity based on additivity effects proved to be unpredictable but did lead to a modest improvement in affinity. Indeed, only one of the six combinations demonstrated additivity. The highest affinity Fab prepared using this strategy was improved 420-fold in affinity The affinity of this Fab was 15 pM as compared to 6.3 nM for b4/12. Examination of the kinetics of Fab binding to gp120 revealed that improvements in affinity were dominated by a slowing of the off-rate of the Fab. The methodology presented here provides a route for the improvement of the affinities of antibodies typical of tertiary immune responses into the picomolar range. Such improvements may have profound effects on the utility of antibodies as therapeutic and prophylatic agents. (C) 1995 Academic Press Limited