The binding epitope of Sintilimab on PD-1 revealed by AbMap

PD-1 plays an important role as immune checkpoint. Sintilimab is a newly approved PD-1 antibody for cancer immunotherapy with unknown binding epitope on PD-1. To elucidate the molecular mechanism for blockade of PD-1 activation by Sintilimab, in this study, we applied Antibody binding epitope Mapping (AbMap) to identify the binding epitope of Sintilimab. An epitope was successfully identified, i.e., SLAPKA, aa127-132. By constructing a serial of point mutations, the dominant residues S127, L128, A129, P130 and A132 of PD-1 were further validated by western blotting, Biolayer Interferometry (BLI), and flow cytometry. Structure analysis showed that the epitope is partially within the binding interface of PD-1 and PD-L1, and this epitope is also partially overlapped with that of Nivolumab and Pembrolizumab. These results demonstrate that Sintilimab can attenuate PD-1 activation by directly competing the interaction between PD-1 and PD-L1 through binding key residues of FG loop on PD-1. This study also proves the high efficiency and accuracy of AbMap for determining the binding epitope of therapeutic antibodies.