Wen Ruixin, Chen Yuewen, Yang Ziwei, Yan Fengying, Li Qing, Wu Weidang
National Key Laboratory of Druggability Evaluation and Systematic Translational Medicine, Tianjin Institute of Pharmaceutical Research (TIPR), Tianjin 300301; School of Pharmacy, Anhui Medical University, Hefei 230032, China.
National Key Laboratory of Druggability Evaluation and Systematic Translational Medicine, Tianjin Institute of Pharmaceutical Research (TIPR), Tianjin 300301; Tianjin Joint Innovation Biotechnology Co., Ltd., Tianjin 300301, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2023;39(11):1024-1031.
Objective To generate the phage display nanobody library immunized by lymphocyte-activation gene 3 (LAG-3) and to validate the functional activity of obtained anti-LAG-3 nanobodies. Methods The peripheral blood cDNA library was isolated from the adult llama which was immunized by human LAG-3 protein. The nanobodies sequences were obtained by nested PCR and cloned into the phagemid vector pComb3XSS, then transformed into Escherichia coli XL1-Blue cells for library generation and quality analysis. Anti-LAG-3 specific nanobodies were screened by phage display and sequenced by next-generation sequencing. Nanobodies were cloned into pET-22b (+) vector and Escherichia coli BL21 (DE3) cells were used for protein expression. The proteins were purified by using the Prism A column, then HPLC-MS, ELISA, Western blot, and surface plasmon resonance technology (SPR) were performed to characterize the nanobodies. Results The library capacity of the nanobody phage immune library with great diversity was 7.20×10 CFU/mL. After four rounds of biopanning, three individual nanobodies with distinct amino acid sequences VHH-L1-3, VHH-L3-2 and VHH-L13-2 were picked. The purity of the purified nanobodies was more than 95%. All of these three nanobodies exhibited high binding affinities with recombinant human LAG-3 specifically, among which the K value of VHH-L13-2 was 3.971×10 mol/L. VHH-L13-2 exhibited the inhibitory effects on the association of LAG-3 and its ligand FGL-1, and the half maximal inhibitory concentration (IC) value was 15.58 nmol/L. Conclusion The anti-LAG-3 phage display nanobody library is generated successfully. The anti-LAG-3 nanobodies possess high specificity and binding affinity and exhibit the inhibitory effects on the association of LAG-3 and its ligand.
目的 构建淋巴细胞激活基因3（LAG-3）免疫的噬菌体展示纳米抗体文库，并验证获得的抗LAG-3纳米抗体的功能活性。方法 从用人LAG-3蛋白免疫的成年羊驼中分离外周血cDNA文库。通过巢式PCR获得纳米抗体序列，并克隆到噬菌粒载体pComb3XSS中，然后转化到大肠杆菌XL1-Blue细胞中进行文库构建和质量分析。通过噬菌体展示筛选抗LAG-3特异性纳米抗体，并通过二代测序进行测序。将纳米抗体克隆到pET-22b(+)载体中，用大肠杆菌BL21(DE3)细胞进行蛋白表达。用Prism A柱纯化蛋白，然后进行HPLC-MS、ELISA、Western blot和表面等离子体共振技术（SPR）对纳米抗体进行表征。结果 构建的具有高度多样性的纳米抗体噬菌体免疫文库库容量为7.20×10 CFU/mL。经过四轮生物淘选，挑选出三个氨基酸序列不同的单个纳米抗体VHH-L1-3、VHH-L3-2和VHH-L13-2。纯化后的纳米抗体纯度均大于95%。这三个纳米抗体均与重组人LAG-3特异性结合亲和力高，其中VHH-L13-2的K值为3.971×10 mol/L。VHH-L13-2对LAG-3与其配体FGL-1的结合具有抑制作用，半数最大抑制浓度（IC）值为15.58 nmol/L。结论 成功构建了抗LAG-3噬菌体展示纳米抗体文库。抗LAG-3纳米抗体具有高特异性和结合亲和力，并对LAG-3与其配体的结合具有抑制作用。
