Human Health Therapeutics Research Centre, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada K1A 0R6
Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5.
Biochem J. 2019 Jan 7;476(1):39-50. doi: 10.1042/BCJ20180795.
Up-regulation of epidermal growth factor receptor () is a hallmark of many solid tumors, and inhibition of EGFR signaling by small molecules and antibodies has clear clinical benefit. Here, we report the isolation and functional characterization of novel camelid single-domain antibodies ( or ) directed against human EGFR. The source of these VHs was a llama immunized with cDNA encoding human EGFR ectodomain alone (no protein or cell boost), which is notable in that genetic immunization of large, outbred animals is generally poorly effective. The VHs targeted multiple sites on the receptor's surface with high affinity ( range: 1-40 nM), including one epitope overlapping that of cetuximab, several epitopes conserved in the cynomolgus EGFR orthologue, and at least one epitope conserved in the mouse EGFR orthologue. Interestingly, despite their generation against human EGFR expressed from cDNA by llama cells (presumably in native conformation), the VHs exhibited wide and epitope-dependent variation in their apparent affinities for native EGFR displayed on tumor cell lines. As fusions to human IgG1 Fc, one of the VH-Fcs inhibited EGFR signaling induced by EGF binding with a potency similar to that of cetuximab (IC: ∼30 nM). Thus, DNA immunization elicited high-affinity, functional sdAbs that were vastly superior to those previously isolated by our group through protein immunization.
表皮生长因子受体 () 的上调是许多实体瘤的一个标志，小分子和抗体抑制 EGFR 信号具有明显的临床获益。在这里，我们报告了针对人表皮生长因子受体 () 的新型骆驼科单域抗体 () 的分离和功能表征。这些 VH 的来源是用编码人表皮生长因子胞外结构域的 cDNA 免疫的骆驼（没有蛋白质或细胞增强），这很值得注意，因为对大型、杂交动物进行基因免疫通常效果不佳。VH 以高亲和力针对受体表面的多个位点（范围：1-40 nM），包括与西妥昔单抗重叠的一个表位、在食蟹猴 EGFR 同源物中保守的几个表位，以及在小鼠 EGFR 同源物中至少一个保守的表位。有趣的是，尽管这些 VH 是针对由骆驼细胞表达的 cDNA 中的人 EGFR 产生的（推测为天然构象），但它们对肿瘤细胞系上显示的天然 EGFR 的表观亲和力表现出广泛的、表位依赖性变化。作为与人 IgG1 Fc 的融合物，其中一个 VH-Fc 以与西妥昔单抗相似的效力（IC：约 30 nM）抑制 EGF 结合诱导的 EGFR 信号。因此，DNA 免疫引发了高亲和力、功能的 sdAbs，其性能远远优于我们之前通过蛋白质免疫分离的那些。
