Affinity improvement of single antibody VH domains: Residues in all three hypervariable regions affect antigen binding
Affinity improvement of single antibody VH domains: Residues in all three hypervariable regions affect antigen binding
复制标题
DOI：
10.1016/s1380-2933(96)00045-0
复制
发表时间：
1996-09-01
期刊：
影响因子：
--
通讯作者：
Riechmann, L
中科院分区：
文献类型：
--
作者：
Davies, J;Riechmann, L
背景：通过抗体工程，免疫球蛋白可以针对其特定的应用而定制。在这方面，需要较小的识别单元来将抗原靶向于阻塞的位置，如实体肿瘤。目的：为了设计高效、最小尺寸的识别单元，重链可变区(VH)已被修饰为抗原特异性的单域抗体片段。为了开发一种合理的方法来提高亲和力，本文通过分析半抗原和蛋白质配体的VH结构域中CDR1和2残基的随机化的影响来研究抗原结合。研究设计：随机展示噬菌体和亲和力，以改善和分析抗原结合。以可溶形式测定新选择的VH结构域的亲和力，以评估修饰残基在结合中的作用。结果：在5个随机试验中，有4个新的VH与原VH相比具有更好的抗原亲和力。解离常数从160 nM下降到25 nM或47 nM(抗Ox VH的CDR1或CDR2随机化)，从300 nM下降到31 nM(抗NLP VH的CDR2随机化)，以及从3.1mM下降到1.6mM(抗溶菌酶VH的CDR2随机化)。结论：通过对CDR1和CDR2进行位点特异性二次随机化、噬菌体展示和抗原选择，可以提高VH结构域的亲和力。由于CDR3序列的差异形成了本研究中使用的主要VH结构域之间的唯一差异，CDR1和CDR2突变对亲和力的影响与这三个CDR都参与了单个VH结构域的抗原结合是一致的。
Background: Through antibody engineering, immunoglobulins can be tailored for their particular application. In this respect, small recognition units are desired for the targeting of antigens in obstructed locations like solid tumors. Objectives: To design efficient, minimum size recognition units, heavy chain variable regions (VH) had previously been modified for the use as antigen specific, single domain antibody fragments. To develop a rational approach to improve affinity, antigen binding is investigated here by analysing the effect of randomisations of CDR1 and 2 residues in VH domains specific for hapten and protein ligands. Study design: Randomised repertoires were displayed on phage and affinity selected to improve and analyse antigen binding. Affinities of newly selected VH domains were determined in their soluble format to assess the role of modified residues in binding. Results: In four of five randomisation experiments, a new VH with an improved antigen affinity compared to the primary VH was selected. Dissociation constants decreased from 160 nM to 25 nM or 47 nM (CDR1 or CDR2 randomisation of an anti-Ox VH), from 300 nM to 31 nM (CDR2 randomisation of an anti-NLP VH) and from 3.1 mu M to 1.6 mu M (CDR2 randomisation of an anti-lysozyme VH). Conclusions: Thus the affinity of VH domains can be improved after site specific, secondary randomisations in CDR1 and CDR2, phage display and antigen selection. As differences in the CDR3 sequences had formed the only difference between the primary VH domains used in this study, the effect of CDR1 and CDR2 mutations on affinity is consistent with a participation of all three CDRs in antigen binding by single VH domains.