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首页> 外文期刊>Journal of Molecular Biology >Sequence Plasticity in the Antigen-binding Site of a Therapeutic Anti-HER2 Antibody.
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Sequence Plasticity in the Antigen-binding Site of a Therapeutic Anti-HER2 Antibody.

机译：治疗性抗HER2抗体的抗原结合位点的序列可塑性。

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摘要

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.
机译：我们已经检查了高亲和力抗体的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位置被所有20个氨基酸取代。使用抗原结合选择来富集高亲和力变异体，并在将选定的集合融合为少量克隆之前获得了大量序列。如所预期的，与已知的IgG序列相比，抗原结合位点的序列变异性总体上降低了;但是，某些位置比其他位置保留更高的可变性。将hu4D5结合位点的序列变异性图谱与先前的抗体的丙氨酸扫描得到的图谱进行比较。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，包括与亲本hu4D5相比亲和力提高约三倍的具有单点突变的一种变体。有趣的是，就改变侧链化学（Asp的Trp）而言，此突变是最根本的突变之一，并且发生在通过Wu-Kabat变异系数计算出的可塑性最高的位置。因此，可变性作图产生了关于抗体-抗原相互作用的信息，该信息与通过丙氨酸扫描诱变获得的信息有用且互补。

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