Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Alarich-Weiss-Straße 4, Darmstadt, D-64287, Germany.
Ferring Darmstadt Laboratory, Biologics Technology and Development, Alarich-Weiss-Straße 4, Darmstadt, D-64287, Germany.
Biotechnol J. 2021 Mar;16(3):e2000240. doi: 10.1002/biot.202000240. Epub 2020 Oct 8.
The phylogenetic distance between chickens and humans accounts for a strong immune response and a broader epitope coverage compared to rodent immunization approaches. Here the authors report the isolation of common light chain (cLC)-based chicken monoclonal antibodies from an anti-epidermal growth factor receptor (EGFR) immune library utilizing yeast surface display in combination with yeast biopanning and fluorescence-activated cell sorting (FACS). For the selection of high-affinity antibodies, a yeast cell library presenting cLC-comprising fragment antigen binding (Fab) fragments is panned against hEGFR-overexpressing A431 cells. The resulting cell-cell-complexes are sorted by FACS resulting in gradual enrichment of EGFR-binding Fabs in three sorting rounds. The isolated antibodies share the same light chain and show high specificity for EGFR, resulting in selective binding to A431 cells with notable EC50 values. All identified antibodies show very good aggregation propensity profiles and thermostabilities. Additionally, epitope binning demonstrates that these cLC antibodies cover a broad epitope space. Isolation of antibodies from immunized chickens by yeast cell biopanning makes an addition to the repertoire of methods for antibody library screening, paving the way for the generation of cLC-based bispecific antibodies against native mammalian receptors.
与啮齿动物免疫方法相比，鸡与人类在系统发育上的距离较远，这导致针对鸡的免疫反应更强，表位覆盖范围更广。作者在这里报告了一种从抗表皮生长因子受体（EGFR）免疫文库中分离基于常见轻链（cLC）的鸡单克隆抗体的方法，该方法利用酵母表面展示技术结合酵母生物淘选和荧光激活细胞分选（FACS）。为了选择高亲和力的抗体，作者构建了一个展示包含 cLC 的片段抗原结合（Fab）片段的酵母细胞文库，并用其筛选过表达 hEGFR 的 A431 细胞。通过 FACS 对形成的细胞-细胞复合物进行分选，在三轮分选过程中逐渐富集 EGFR 结合的 Fab。分离得到的抗体共享相同的轻链，对 EGFR 具有高度特异性，导致对 A431 细胞的选择性结合，具有显著的 EC50 值。所有鉴定出的抗体均表现出很好的聚集倾向谱和热稳定性。此外，表位聚类表明这些 cLC 抗体覆盖了广泛的表位空间。通过酵母细胞生物淘选从免疫鸡中分离抗体，为抗体文库筛选方法的组合增添了一种方法，为针对天然哺乳动物受体的 cLC 双特异性抗体的生成铺平了道路。
