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Combinatorial mutagenesis with alternative CDR-L1 and -H2 loop lengths contributes to affinity maturation of antibodies
New Biotechnology ( IF 5.4 ) Pub Date : 2021-01-01 , DOI: 10.1016/j.nbt.2020.09.002
Eeva-Christine Brockmann 1 , Mikko Pyykkö 1 , Heidi Hannula 2 , Kamran Khan 1 , Urpo Lamminmäki 1 , Tuomas Huovinen 1
Affiliation  

Loop length variation in the complementary determining regions (CDRs) 1 and 2 encoded in germline variable antibody genes provides structural diversity in naïve antibody libraries. In synthetic single framework libraries the parental CDR-1 and CDR-2 length is typically unchanged and alternative lengths are provided only at CDR-3 sites. Based on an analysis of the germline repertoire and structure-solved anti-hapten and anti-peptide antibodies, we introduced combinatorial diversity with alternative loop lengths into the CDR-L1, CDR-L3 and CDR-H2 loops of anti-digoxigenin and anti-microcystin-LR single chain Fv fragments (scFvs) sharing human IGKV3-20/IGHV3-23 frameworks. The libraries were phage display selected for folding and affinity, and analysed by single clone screening and deep sequencing. Among microcystin-LR binders the most frequently encountered alternative loop lengths were one amino acid shorter (6 aa) and four amino acids longer (11 aa) CDR-L1 loops leading up to 17- and 28-fold improved affinity, respectively. Among digoxigenin binders, 2 amino acids longer (10 aa) CDR-H2 loops were strongly enriched, but affinity improved anti-digoxigenin scFvs were also encountered with 7 aa CDR-H2 and 11 aa CDR-L1 loops. Despite the fact that CDR-L3 loop length variants were not specifically enriched in selections, one clone with 22-fold improved digoxigenin binding affinity was identified containing a 2 residues longer (10 aa) CDR-L3 loop. Based on our results the IGKV3-20/IGHV3-23 scaffold tolerates loop length variation, particularly in CDR-L1 and CDR-H2 loops, without compromising antibody stability, laying the foundation for developing novel synthetic antibody libraries with loop length combinations not existing in the natural human Ig gene repertoire.

中文翻译:

具有替代 CDR-L1 和 -H2 环长度的组合诱变有助于抗体的亲和力成熟

种系可变抗体基因中编码的互补决定区 (CDR) 1 和 2 中的环长度变化为初始抗体库提供了结构多样性。在合成的单一框架文库中,亲本 CDR-1 和 CDR-2 长度通常不变,并且仅在 CDR-3 位点提供替代长度。基于对种系库和结构解析的抗半抗原和抗肽抗体的分析,我们将具有替代环长度的组合多样性引入到抗地高辛和抗地高辛的 CDR-L1、CDR-L3 和 CDR-H2 环中。共享人类 IGKV3-20/IGHV3-23 框架的微囊藻毒素-LR 单链 Fv 片段 (scFvs)。根据折叠和亲和力选择噬菌体展示文库,并通过单克隆筛选和深度测序进行分析。在微囊藻毒素-LR 结合剂中,最常遇到的替代环长度是短 1 个氨基酸 (6 aa) 和长 4 个氨基酸 (11 aa) 的 CDR-L1 环,分别导致亲和力提高 17 倍和 28 倍。在地高辛结合剂中,长 2 个氨基酸 (10 aa) 的 CDR-H2 环被强烈富集,但亲和力提高的抗地高辛 scFv 也遇到了 7 aa CDR-H2 和 11 aa CDR-L1 环。尽管 CDR-L3 环长度变体在选择中并未特别富集,但鉴定出一个具有 22 倍提高的地高辛结合亲和力的克隆,其中含有 2 个长 (10 aa) 残基的 CDR-L3 环。根据我们的结果,IGKV3-20/IGHV3-23 支架可耐受环长度变化,尤其是在 CDR-L1 和 CDR-H2 环中,而不会影响抗体稳定性,
更新日期:2021-01-01
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