Early Solutions, UCB Biopharma UK, Slough, UK.
Methods Mol Biol. 2023;2681:83-97. doi: 10.1007/978-1-0716-3279-6_6.
Phage display is an in vitro technique used in the discovery of monoclonal antibodies that has been used successfully in the discovery of both camelid VHH and shark variable new antigen receptor domains (VNAR). Bovines also contain a unique "ultralong CDRH3" with a conserved structural motif, comprising a knob domain and β-stalk. When removed from the antibody scaffold, either the entire ultralong CDRH3 or the knob domain alone, is typically capable of binding an antigen, to produce antibody fragments that are smaller than both VHH and VNAR. By extracting immune material from bovine animals and specifically amplifying knob domain DNA sequences by PCR, knob domain sequences can be cloned into a phagemid vector producing knob domain phage libraries. Target-specific knob domains can be enriched by panning the libraries against an antigen of interest. Phage display of knob domains exploits the link between phage genotype and phenotype and could prove to be a high throughput method to discover target-specific knob domains, helping to explore the pharmacological properties of this unique antibody fragment.
噬菌体展示是一种体外技术，用于发现单克隆抗体，已成功用于发现骆驼 VH 和鲨鱼可变新抗原受体结构域 (VNAR)。牛也含有独特的“超长 CDRH3”，具有保守的结构模体，由 knob 结构域和 β-茎组成。从抗体支架中去除后，超长 CDRH3 或 knob 结构域本身通常能够结合抗原，产生比 VH 和 VNAR 都小的抗体片段。通过从牛动物中提取免疫物质，并通过 PCR 特异性扩增 knob 结构域 DNA 序列，可以将 knob 结构域序列克隆到噬菌粒载体中，生成 knob 结构域噬菌体文库。通过针对感兴趣的抗原淘选文库，可以富集针对特定靶标的 knob 结构域。利用噬菌体展示 knob 结构域利用了噬菌体基因型和表型之间的联系，可能成为发现针对特定靶标的 knob 结构域的高通量方法，有助于探索这种独特抗体片段的药理学特性。
