Thymidine hat

Erythromycin, clarithromycin, roxithromycin, diltiazem, ritonavir, ketoconazole, fluconazole, cimetidine, ticlopidine, omeprazole, fluvoxamine, paroxetine, fluoxetine, quinidine, aflatoxin B1, and sterigmatocystin were obtained from Wako, Japan. Gestodene and mifepristone were obtained from Tokyo Chemical Industry, Japan. G418 was obtained from Funakoshi, Japan. Ethinylestradiol, rifampicin, tienilic acid, terbinafine, hypoxanthine, aminopterin, and thymidine (HAT) and 4,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma Aldrich, USA. All other chemicals were of the highest grade commercially available.

TF‐1 cells were purchased from American Tissue Culture Collection (ATCC, Rockville, MD, USA), and were cultured with RPMI1640 (Gibco, Waltham, MA, USA) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C and 5% CO₂. The Cell Counting Kit‐8 (CCK8) was purchased from Dojindo Laboratories (Tokyo, Japan). Human antibody germline sequences and primers were synthesized by Sangon Biotech (Shanghai, China). Tetramethylbenzidine (TMB) substrate was purchased from Thermo Fisher Scientific, PEG400 and thymidine (HAT) were from Sigma‐Aldrich. The human NGF sequences was fused into 6xHis‐tag and cloned into the pCAT1.0 vector (constructed in Darsbio Ltd.), followed by transient transfection of HEK293 cells for protein production. Amino acid sequences of tanezumab light and heavy chains were obtained from the international Immunogenetics information system (IMGT/mAb‐DB), and huAb45 were generated in‐house as described below.

NGF antibodies were generated by the hybridoma technique. BALB/c mice (Beijing Vital River Laboratory Animal Technology Co. Ltd., Beijing, China) were immunized by NGF‐6xHis and keyhole limpet hemocyanin. To form hybridomas, immunized mouse spleen cells were fused with myeloma cell SP2/0 at a 5:1 ratio using PEG400 (Sigma‐Aldrich, Darmstadt, Germany) and grown in 100 mm hypoxanthine, 0.4 mm aminopterin, and 1.6 mm thymidine (HAT) (Sigma‐Aldrich,) selection medium. Enzyme‐linked immunoassay (ELISA) was used to test positive hybridoma cells 12 days after infusion, and then the cells were continually subcloned at least three times with limiting dilution to ensure clonality. Hybridoma cells capable of producing high‐sensitivity monoclonal antibodies against NGF in cell culture supernatants were recognized by ELISA. HuAb45 used in this study was screened out from a pool of neutralizing antibodies as the most potent blocker of NGF binding to TrkA. The variable regions of heavy (VH) and light (VL) chains were cloned into the human IgG2 backbone by “framework shuffling” technology, and then transiently expressed in HEK293F cells.