Immunogenicity screening assay development for a novel human-mouse chimeric anti-CD147 monoclonal antibody...

The clinical effect of patient immune responses to therapeutic antibodies affect product safety and efficacy, which makes the development of valid, sensitive immune assays a key aspect of antibody drug development. In this paper, we reported the generations of mouse monoclonal and Cynomolgus monkey polyclonal antibodies against the anti-CD147 antibody (Metuzumab) as the internal standards and the positive controls. Seven mouse monoclonal antibodies were shown to recognize both (Fab)2 and full length of Metuzumab, but not the control normal human IgGs, and monoclonal anti-Metuzumab, Clone 2D9 was chosen to be used as the internal standard for anti-Metuzumab study. A Bridging ELISA assay was developed by coating the wells with the antibody drug, and the anti-drug antibody (ADA) in the animal sera were detected by enzyme-labeled antibody. Its limit of detection (LOD) was determined to be 0.39ng/ml of anti-Metuzumab antibody (ADA) with linear range between 0.39-50ng/ml and R(2)=0.994. For normal monkey sera, a minimal dilution was determined to be 1:80. However, very different from peptide or other protein drugs, strong interferences from the residual antibody drugs were observed from most of the testing monkey sera in the preclinical study. It was experimentally determined that the concentration of the residual antibody drug in the assay have to be lower than 1μg/ml, so the assays were carried out at 1:100 dilution of the monkey sera. In the pre-clinical study, 32 monkeys were treated with escalating doses of Metuzumab between 0, 10, 50, 200mg/kg for 13 times over 13weeks of time period. 16 of them were terminated right after the last injection, while the other 16 were rested for additional 4weeks before termination. Afraid to miss any positive response to antibody drug, sera samples were collected at six time points, including 2-, 6- and 10-weeks post 1st dose, prior to last dose, and 2-, 4-weeks into recovery. The highest positive rates were seen with the Medium- and High-dose group 2-weeks post the first injection, 6 out 8 monkeys in the High-dose were positive for free ADA. However, no significant pathologic and clinic adversary effect was observed in those monkeys.