Preparation and characterization of a high-affinity monoclonal antibody against nerve growth factor

• Published in: Protein expression and purification 2022-01, Vol.189, p.105966-105966, Article 105966
• Main Authors: Liu, Shuang, Shen, Yunlong, Chen, Pengyu, Guo, Cuiyu, Zhang, Guangbing, Jiang, Xiaohua, He, Jianxiong, Yang, Jinliang
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - isolation & purification; Antibodies, Monoclonal - pharmacology; Antibodies, Monoclonal, Humanized - pharmacology; Antibody Affinity; Antibody Specificity; Arthritis - drug therapy; Arthritis - genetics; Arthritis - immunology; Arthritis - pathology; Disease Models, Animal; Female; Gene Expression; HEK293 Cells; Humans; Hybridoma; Hybridomas - chemistry; Hybridomas - immunology; Immunization; Immunoglobulin Fc Fragments - genetics; Immunoglobulin Fc Fragments - immunology; Lymphocytes - chemistry; Lymphocytes - immunology; Mammalian expression; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Monoclonal antibody; Nerve growth factor; Nerve Growth Factor - antagonists & inhibitors; Nerve Growth Factor - genetics; Nerve Growth Factor - immunology; Pain - genetics; Pain - immunology; Pain - pathology; Pain - prevention & control; Receptor, trkA - antagonists & inhibitors; Receptor, trkA - genetics; Receptor, trkA - immunology; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2021.105966

Nerve growth factor (NGF) is produced and released in injured tissues or chronic pain tissues caused by other diseases. Studies have shown that monoclonal antibodies targeting NGF have a good efficacy in the treatment of osteoarthritis (OA), low back pain and chronic pain, which may be a promising therapy. In this study, DNA sequences of NGF-his and NGF-hFc were synthesized using eukaryotic expression system and subcloned into pTT5 expression vector. After that, NGF proteins were expressed by transient expression in HEK293E cells. We immunized mice with NGF-hFc protein and fused mouse spleen cells to prepare hybridomas. NGF-His protein was used to screen out the hybridoma supernatant that could directly bind to NGF. Antibodies were purified from hybridioma supernatant. Futhermore, via surface plasmon resonance (SPR) screening, six anti-NGF mAbs were screened to block the binding of NGF and TrkA receptor in the treatment of chronic pain. Among them, 58F10G10H showed high affinity (KD = 1.03 × 10−9 M) and even better than that of positive control antibody Tanezumab (KD = 1.53 × 10−9 M). Moreover, the specific reactivity of 58F10G10H was demonstrated by TF-1 cell proliferation activity experiments, competitive binding Enzyme-linked immunosorbent assay (ELISA) and the arthritis animal models in mice, respectively. In conclusion, in this study, a method for the preparation of high-yield NGF-HFC and NGF-His proteins was designed, and a high-affinity monoclonal antibody against NGF with potential for basic research and clinical application was prepared. •Expression of NGF-his and NGF-hFc protein with high yield.•Screen candidate hybridomas for high-affinity monoclonal antibodies.•Anti-NGF antibody for the treatment of chronic pain.