Generation and characterization of mAb 61H9 against junctional adhesion molecule-a with potent antitumor activity
Kang Liu,
Hang Yang,
Rong Xiong,
Yunlong Shen,
Guiqin Song,
Jinliang Yang,
Zhenling Wang
Affiliations
Kang Liu
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Sichuan University, Chengdu, Sichuan, China
Hang Yang
Institute of Tissue Engineering and Stem Cells, Nanchong Central Hospital, The Second Clinical Medical College, North Sichuan Medical College, Nanchong, Sichuan, China
Rong Xiong
Institute of Tissue Engineering and Stem Cells, Nanchong Central Hospital, The Second Clinical Medical College, North Sichuan Medical College, Nanchong, Sichuan, China
Yunlong Shen
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Sichuan University, Chengdu, Sichuan, China
Guiqin Song
School of Basic Medicine and Forensic Medicine, North Sichuan Medical College, Nanchong, Sichuan, China
Jinliang Yang
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Sichuan University, Chengdu, Sichuan, China
Zhenling Wang
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Sichuan University, Chengdu, Sichuan, China
Junctional adhesion molecule-A (JAM-A) is an adhesion molecule that exists on the surface of certain types of cells, including white blood cells, endothelial cells, and dendritic cells. In this study, the cDNA sequences of JAM-A-Fc were chemically synthesized with optimization for mammalian expression. Afterward, we analyzed JAM-A protein expression through transient transfection in HEK293 cell lines. Mice were immunized with JAM-A-Fc protein, and hybridoma was prepared by fusing myeloma cells and mouse spleen cells. Antibodies were purified from the hybridoma supernatant and four monoclonal strains were obtained and numbered 61H9, 70E5, 71A8, and 74H3 via enzyme-linked immunosorbent assay screening. Immunofluorescence staining assay showed 61H9 was the most suitable cell line for mAb production due to its fluorescence signal being the strongest. Flow cytometric analysis proved that 61H9 possessed high affinity. Moreover, antagonism of JAM-A mAb could attenuate the proliferative, migrative, and invasive abilities of ESCC cells and significantly inhibit tumor growth in mice. By examining hematoxylin-eosin staining mice tumor tissues, we found inflammatory cells infiltrated lightly in the anti-JAM-A group. The expression of BCL-2 and IκBα in the anti-JAM-A group were decreased in mice tumor tissues compared to the control group. Ultimately, a method for preparing high-yield JAM-A-Fc protein was created and a high affinity mAb against JAM-A with an antitumor effect was prepared.
