Jang Moon-Sung, Ismail Nurain Syahirah Binti, Yu Yeon Gyu
Department of Biopharmaceutical Chemistry, Kookmin University, Seoul 02707, Republic of Korea.
Antib Ther. 2022 Jul 21;5(3):192-201. doi: 10.1093/abt/tbac016. eCollection 2022 Jul.
CC chemokine receptor 7 (CCR7) is a member of G-protein-coupled receptor family and mediates chemotactic migration of immune cells and different cancer cells induced via chemokine (C-C motif) ligand 19 (CCL19) or chemokine (C-C motif) ligand 21 (CCL21). Hence, the identification of blockade antibodies against CCR7 could lead to the development of therapeutics targeting metastatic cancer.
CCR7 was purified and stabilized in its active conformation, and antibodies specific to purified CCR7 were screened from the synthetic M13 phage library displaying humanized scFvs. The characterization of selected scFvs identified two scFvs that exhibited CCL19-competitive binding to CCR7. IgG's harboring selected scFv sequences were characterized for binding activity in CCR7 cells, inhibitory activity toward CCR7-dependent cAMP attenuation, and the CCL19 or CCL21-dependent migration of CCR7 cells.
Antibodies specifically binding to purified CCR7 and CCR7 cells were isolated and characterized. Two antibodies, IgG(6RG11) and IgG(72C7), showed ligand-dependent competitive binding to CCR7 with K values of 40 nM and 50 nM, respectively. Particularly, IgG(6RG11) showed antagonistic activity against CCR7, whereas both antibodies significantly blocked the ligand-induced migration and invasion activity of CCR7 cancer cells.
Two antibody clones were successfully identified from a synthetic scFv-displaying phage library using purified recombinant CCR7 as an antigen. Antibodies specifically bound to the surface of CCR7 cells and blocked CCR7 cell migration. Particularly, 6RG11 showed antagonist activity against CCR7-dependent cAMP attenuation.
C-C趋化因子受体7（CCR7）是G蛋白偶联受体家族的成员，可介导免疫细胞和不同癌细胞通过趋化因子（C-C基序）配体19（CCL19）或趋化因子（C-C基序）配体21（CCL21）诱导的趋化迁移。因此，鉴定针对CCR7的阻断抗体可能会促进针对转移性癌症的治疗药物的开发。
纯化CCR7并使其稳定在活性构象，从展示人源化单链抗体片段（scFv）的合成M13噬菌体文库中筛选针对纯化CCR7的特异性抗体。对所选scFv的表征鉴定出两个对CCR7表现出CCL19竞争性结合的scFv。对携带所选scFv序列的IgG进行表征，检测其在CCR7细胞中的结合活性、对CCR7依赖性环磷酸腺苷（cAMP）衰减的抑制活性以及CCR7细胞的CCL19或CCL21依赖性迁移。
分离并表征了特异性结合纯化CCR7和CCR7细胞的抗体。两种抗体，即IgG(6RG11)和IgG(72C7)，对CCR7表现出配体依赖性竞争性结合，解离常数（K值）分别为40 nM和50 nM。特别地，IgG(6RG11)对CCR7表现出拮抗活性，而两种抗体均显著阻断CCR7癌细胞的配体诱导的迁移和侵袭活性。
以纯化的重组CCR7为抗原，成功从合成的展示scFv的噬菌体文库中鉴定出两个抗体克隆。抗体特异性结合CCR7细胞表面并阻断CCR7细胞迁移。特别地，6RG11对CCR7依赖性cAMP衰减表现出拮抗活性。
