Abstract

Inhibition of FGFR2 signaling is promising in targeted therapy of FGFR2-related tumors. In this study, anti-FGFR2 nanobodies (Nbs) were isolated through screening of an immune camelid phage display library. Four rounds of biopanning were carried out with commercial human FGFR2 antigen and enrichment was assessed by ELISA and phage titration. The gene of Nb was sub-cloned into the expression vector, and the recombinant vector was transformed into Escherichia coli WK6 cells. The recombinant protein was purified using Ni–NTA affinity chromatography. The anti-FGFR2 Nb (C13) was characterized by SDS-PAGE, western blotting, competitive inhibition ELISA, flow cytometry, MTT, and migration assay. C13 Nb recognized FGFR2 with high specificity and no cross-reactivity was observed with other tested antigens. The affinity of C13 Nb was calculated to be 1.5 × 10⁻⁹ M. Results of cytotoxicity showed that C13 Nb (10 µg/ml) inhibited 85% of the proliferation of T-47D cells (p < 0.001). In addition, C13 inhibited the migration of 68% of T-47D toward the source of the growth factor (p < 0.01). The flow cytometry showed that C13 Nb bound to the surface of FGFR2⁺ cells, T-47D cell line (96%). Results indicate the potential of anti-FGFR2 Nb for targeted therapy of FGFR2-overexpressing tumors after complementary investigations.

摘要

FGFR2 信号传导的抑制在 FGFR2 相关肿瘤的靶向治疗中具有广阔的前景。在本研究中，通过筛选免疫骆驼噬菌体展示文库分离出抗 FGFR2 纳米抗体 (Nbs)。使用商业人 FGFR2 抗原进行四轮生物淘选，并通过 ELISA 和噬菌体滴定评估富集情况。将Nb基因亚克隆至表达载体中，并将​​重组载体转化大肠杆菌WK6 细胞。使用 Ni-NTA 亲和层析纯化重组蛋白。通过 SDS-PAGE、蛋白质印迹、竞争性抑制 ELISA、流式细胞术、MTT 和迁移测定对抗 FGFR2 Nb (C13) 进行表征。C13 Nb 以高特异性识别 FGFR2，与其他测试抗原未观察到交叉反应。C13 Nb的亲和力经计算为1.5 × 10 ^-9 M。细胞毒性结果显示C13 Nb (10 µg/ml)抑制T-47D细胞85%的增殖( p  < 0.001)。此外，C13 抑制 68% 的 T-47D 向生长因子源迁移 ( p  < 0.01)。^{流式细胞术显示C13 Nb与FGFR2 +}表面结合细胞，T-47D 细胞系（96%）。结果表明，经过补充研究后，抗 FGFR2 Nb 有可能用于 FGFR2 过表达肿瘤的靶向治疗。