Sequence plasticity in the antigen-binding site of a therapeutic anti-HER2 antibody
Sequence plasticity in the antigen-binding site of a therapeutic anti-HER2 antibody
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DOI：
10.1016/s0022-2836(02)00677-0
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发表时间：
2002-08-30
影响因子：
5.6
通讯作者：
Lowman, HB
中科院分区：
文献类型：
--
作者：
Gerstner, RB;Carter, P;Lowman, HB
我们检查了高亲和力抗体抗原结合位点的可塑性。在噬菌体展示的 Fab 文库中，人源化抗 Her2 抗体 hu4D5 的选定 CDR 位置和一个 FR 位置被全部 20 个氨基酸取代。使用抗原结合选择来富集高亲和力变体，并且在将所选库收敛到一小组克隆之前获得大量序列。正如预期的那样，与已知的 IgG 序列相比，抗原结合位点的序列变异性总体上减少了；然而，某些位置的可变性比其他位置高得多。将 hu4D5 结合位点的序列变异图与源自先前抗体丙氨酸扫描的图进行比较。可溶性 Fab 片段对抗原的亲和力证实，选择了对抗原具有高亲和力的多个变体，其中包括一种具有单点突变的变体，与亲本 hu4D5 相比，其亲和力提高了约三倍。有趣的是，这种突变是改变侧链化学（Trp 为 Asp）方面最激进的突变之一，并且根据 Wu-Kabat 变异系数计算，发生在最具可塑性的位点。因此，变异性作图产生了关于抗体-抗原相互作用的信息，该信息对于通过丙氨酸扫描诱变获得的信息是有用的和补充的。 (C) 2002 Elsevier Science Ltd. 保留所有权利。
We have examined the plasticity of the antigen-combining site of a high-affinity antibody In phage-displayed Fab libraries, selected CDR positions and one FR position of the humanized anti-Her2 antibody hu4D5 were substituted with all 20 amino acids. Antigen-binding selections were used to enrich for high-affinity variants, and a large number of sequences were obtained prior to convergence of the selected pool to a small set of clones. As expected, sequence variability of the antigen-binding site is overall diminished compared to known IgG sequences; however, certain positions retain much higher variability than others. The sequence variability map of the hu4D5 binding site is compared with a map derived from previous alanine-scanning of the antibody. Affinities of soluble Fab fragments for antigen confirm that multiple variants were selected with high affinity for antigen, including one variant with a single point mutation that was about threefold improved in affinity compared to the parental hu4D5. Interestingly, this mutation is one of the most radical in terms of changing side-chain chemistry (Trp for Asp) and occurs at the most plastic site as calculated by the Wu-Kabat variability coefficient. Thus variability mapping yields information about the antibody-antigen interaction that is useful and complementary to that obtained by alanine scanning mutagenesis. (C) 2002 Elsevier Science Ltd. All rights reserved.