构建三特异性IgG样抗体需要至少结合三种不同的结合模体，从而形成复杂的结构并具有挑战性的生产这些分子。在这里，我们首次报道了基于以前报道的二合一抗体与酵母表面展示鸡来源免疫库鉴定的新型抗CD16a常见轻链模块结合而成的三特异性自然杀伤细胞激活剂的构建。由此产生的抗体同时针对表皮生长因子受体（EGFR）、程序性死亡配体1（PD-L1）和CD16a，具有两个Fab片段，从而在EGFR/PD-L1双阳性肿瘤细胞上具有特异的细胞结合特性和强效的ADCC作用。这项研究为基于鸟免疫获得所需的靶标组合、价数、分子对称性和结构的多特异性治疗抗体的进一步发展铺平了道路。版权所有©2023 Harwardt，Carrara，Bogen，Schoenfeld，Grzeschik，Hock和Kolmar。

To construct a trispecific IgG-like antibody at least three different binding moieties need to be combined, which results in a complex architecture and challenging production of these molecules. Here we report for the first time the construction of trispecific natural killer cell engagers based on a previously reported two-in-one antibody combined with a novel anti-CD16a common light chain module identified by yeast surface display (YSD) screening of chicken-derived immune libraries. The resulting antibodies simultaneously target epidermal growth factor receptor (EGFR), programmed death-ligand 1 (PD-L1) and CD16a with two Fab fragments, resulting in specific cellular binding properties on EGFR/PD-L1 double positive tumor cells and a potent ADCC effect. This study paves the way for further development of multispecific therapeutic antibodies derived from avian immunization with desired target combinations, valencies, molecular symmetries and architectures.Copyright © 2023 Harwardt, Carrara, Bogen, Schoenfeld, Grzeschik, Hock and Kolmar.