Milking the Cow: Cattle-Derived Chimeric Ultralong CDR-H3 Antibodies and Their Engineered CDR-H3-Only Knobbody Counterparts Targeting Epidermal Growth Factor Receptor Elicit Potent NK Cell-Mediated Cytotoxicity.
Milking the Cow: Cattle-Derived Chimeric Ultralong CDR-H3 Antibodies and Their Engineered CDR-H3-Only Knobbody Counterparts Targeting Epidermal Growth Factor Receptor Elicit Potent NK Cell-Mediated Cytotoxicity.
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DOI：
10.3389/fimmu.2021.742418
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发表时间：
2021
影响因子：
7.3
通讯作者：
Zielonka S
中科院分区：
文献类型：
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作者：
Pekar L;Klewinghaus D;Arras P;Carrara SC;Harwardt J;Krah S;Yanakieva D;Toleikis L;Smider VV;Kolmar H;Zielonka S
在这项工作中，我们已经产生了表皮生长因子受体（EGFR）特异性牛源性超长CDR-H3抗体结合牛免疫酵母表面展示。免疫后，特异性扩增超长CDR-H3区并通过同源重组移植到IGHV 1 -7支架上以促进Fab展示。通过荧光激活细胞分选（FACS）容易地获得抗原特异性克隆并重新格式化为嵌合抗体。分箱实验揭示了EGFR的结构域I、II和IV的表位靶向，没有产生的结合剂与西妥昔单抗、马妥珠单抗或EGF竞争结合EGFR。牛源性嵌合抗体能够有效诱导针对EGFR过表达肿瘤细胞的抗体依赖性细胞介导的细胞毒性（ADCC），其效力（EC 50杀伤）在皮摩尔范围内。此外，大多数抗体能够显著抑制EGFR介导的下游信号传导。此外，我们证明，一小部分的CDR-H3旋钮产生的抗体能够独立地作为互补位促进EGFR结合时，移植到Fc部分的人IgG 1。除了轻微至中度降低的能力外，这些工程化的节体在很大程度上保留了其亲本抗体的主要特性，例如细胞结合和ADCC的触发。因此，Knobbodies可能会出现作为有前途的工具，生物技术应用后，进一步优化。
In this work, we have generated epidermal growth factor receptor (EGFR)-specific cattle-derived ultralong CDR-H3 antibodies by combining cattle immunization with yeast surface display. After immunization, ultralong CDR-H3 regions were specifically amplified and grafted onto an IGHV1-7 scaffold by homologous recombination to facilitate Fab display. Antigen-specific clones were readily obtained by fluorescence-activated cell sorting (FACS) and reformatted as chimeric antibodies. Binning experiments revealed epitope targeting of domains I, II, and IV of EGFR with none of the generated binders competing with Cetuximab, Matuzumab, or EGF for binding to EGFR. Cattle-derived chimeric antibodies were potent in inducing antibody-dependent cell-mediated cytotoxicity (ADCC) against EGFR-overexpressing tumor cells with potencies (EC50 killing) in the picomolar range. Moreover, most of the antibodies were able to significantly inhibit EGFR-mediated downstream signaling. Furthermore, we demonstrate that a minor fraction of CDR-H3 knobs derived from generated antibodies was capable of independently functioning as a paratope facilitating EGFR binding when grafted onto the Fc part of human IgG1. Besides slightly to moderately diminished capacities, these engineered Knobbodies largely retained main properties of their parental antibodies such as cellular binding and triggering of ADCC. Hence, Knobbodies might emerge as promising tools for biotechnological applications upon further optimization.