Background Certolizumab pegol (CZP) is a PEGylated, Fc-free anti-TNF that lacks the Fc portion found in monoclonal antibodies. Infliximab (IFX) and adalimumab (ADA) are both antibodies, while etanercept (ETA) is a receptor fusion protein, and all three of these anti-TNFs have an IgG1 Fc. In mothers treated with CZP, it has been reported that lower levels of CZP, compared to ADA or IFX, are transferred to the neonate.1 It has been suggested this transfer differential may be due to the one-way active transport of antibodies across the placenta thought to be mediated by the neonatal Fc receptor (FcRn). However, anti-TNF binding to FcRn, and FcRn-mediated transcytosis across a cell layer, have not been studied.
Objectives To quantify binding of the anti-TNFs CZP, IFX, ADA and ETA to FcRn and to measure FcRn-mediated transcytosis of these agents across a cell layer.
Methods A Biacore™ assay was used to determine the binding of CZP, ADA and IFX to human FcRn. Anti-TNFs were passed over an FcRn-coated chip for 5 minutes at a range of concentrations from 21-670nM to determine the on-binding rate; a buffer at pH 6.0 was used to allow optimum binding. The off-rate was followed for a further 5 minutes by running buffer alone over the chip. MDCK II cells transfected with human FcRn were used to measure FcRn-mediated transcytosis across a cell layer using a pH 5.9 buffer on the apical side and pH 7.2 on the basolateral side. The anti-TNFs and the control antibody (P146), which possessed a Fc modified to prevent binding to FcRn, were biotinylated to allow visualization. The amount of each anti-TNF transcytosed across the cell layer over 4 hours was measured by MSD assay.
Results IFX (132nM) and ADA (225nM) had relatively high binding affinity to FcRn while the binding affinity of ETA to FcRn was approximately 5 to 10-fold lower (1500nM), similar to previously reported results.2 In contrast, CZP did not bind to the FcRn with any measurable affinity. The levels of transcytosis seen with IFX and ADA were 249.6ng/mL and 159.5ng/mL, respectively (mean of 3 experiments), over 4 hours. Transcytosis of ETA (81.3ng/mL) was lower than that of ADA and IFX. In contrast, the level of CZP transcytosis was significantly lower, at 3.2ng/mL, than that observed with the other anti-TNFs tested. The control antibody P146 also showed a low level of transfer at 5.9ng/mL. Since neither the control antibody nor CZP bind to FcRn, the levels detected are probably due to a low level of non-specific leakage across the cell layer.
Conclusions This is the first report to quantify the binding of anti-TNFs to FcRn and their FcRn-mediated transcytosis across a cell layer. CZP does not have an Fc and thus did not bind to FcRn. Moreover, no FcRn-mediated CZP transcytosis was detected. In contrast, ADA and IFX had a relatively high binding affinity to FcRn and were actively transcytosed across the cell layer. ETA showed lower binding affinity to FcRn and subsequent transcytosis, compared to IFX and ADA, but FcRn-mediated transport could still be measured. These results explain the previously observed active transport of anti-TNFs across the placenta seen in patients treated with IFX and ADA, whereas only low levels were observed with CZP.1
Mahadevan U. Clin Gastroenterol Hepatol 2012 [epub ahead of print]; 2. Suzuki T. J Immunol 2010;184(4):1968-1976.
Acknowledgements The authors acknowledge Costello Medical Consulting for writing and editorial assistance which was funded by UCB Pharma.
Disclosure of Interest T. Baker Employee of: UCB Pharma, L. Kevorkian Employee of: UCB Pharma, A. Nesbitt Shareholder of: UCB Pharma, Employee of: UCB Pharma