Laboratorio de Farmacología Molecular y Modelos Biológicos, Facultad de Ciencias Químicas, Universidad Autónoma de Nuevo León, San Nicolás de los Garza, Mexico.
Laboratorio de Expresión y Purificación de Proteínas, Facultad de Ciencias Químicas, Universidad Autónoma de Nuevo León, San Nicolás de los Garza, Mexico.
Anticancer Res. 2024 May;44(5):1955-1962. doi: 10.21873/anticanres.16998.
BACKGROUND/AIM: The epidermal growth factor receptor (EGFR) is over-expressed in several types of cancer, and monoclonal antibody therapy has been the strategy that has shown the best results. This study focused on the construction of a humanized single chain antibody (huscFv) directed against EGFR (HER1).
The CDR grafting method was used to incorporate murine complementarity determining regions (CDRs) of cetuximab into human sequences. A dot blot assay was used to examine the affinity of the huscFv secreted by HEK293T for EGFR. The inhibitory effect on the viability of A549 cells was evaluated using the WST-1 assay.
The incorporation of murine CDRs of cetuximab into human sequences increased the degree of humanness by 16.4%. The increase in the humanization of scFv did not affect the affinity for EGFR. Metformin had a dose-dependent effect, with an IC of 46 mM, and in combination with huscFv, the cell viability decreased by 45% compared to the 15% demonstrated by huscFv alone.
The CDR grafting technique is efficient for the humanization of scFv, maintaining its affinity for EGFR and demonstrating its inhibitory effect when combined with metformin in A549 cells.
背景/目的：表皮生长因子受体（EGFR）在多种类型的癌症中过度表达，单克隆抗体治疗已成为显示最佳效果的策略。本研究专注于构建针对 EGFR（HER1）的人源化单链抗体（huscFv）。
采用 CDR 移植法将西妥昔单抗的鼠源互补决定区（CDRs）整合到人序列中。点印迹法检测 HEK293T 分泌的 huscFv 对 EGFR 的亲和力。使用 WST-1 测定法评估对 A549 细胞活力的抑制作用。
将西妥昔单抗的鼠源 CDRs 整合到人序列中，使 scFv 的人源化程度提高了 16.4%。scFv 人源化程度的增加并未影响其对 EGFR 的亲和力。二甲双胍具有剂量依赖性作用，IC 为 46mM，与 huscFv 联合使用时，与单独使用 huscFv 相比，细胞活力降低了 45%。
CDR 移植技术可有效实现 scFv 的人源化，保持其对 EGFR 的亲和力，并在与二甲双胍联合使用时在 A549 细胞中显示出抑制作用。
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