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.

PD-1作为免疫检查点发挥关键作用。信迪利单抗（Sintilimab）是一款新近获批用于癌症免疫治疗的PD-1抗体，其在PD-1上的结合表位尚不明确。为阐明信迪利单抗阻断PD-1活化的分子机制，本研究采用抗体结合表位作图技术（Antibody binding epitope Mapping，AbMap）鉴定信迪利单抗的结合表位，成功获得位于PD-1第127至132位氨基酸的SLAPKA表位。通过构建一系列点突变，结合蛋白质免疫印迹（Western blotting）、生物层干涉术（Biolayer Interferometry，BLI）与流式细胞术，进一步验证了PD-1上的关键残基S127、L128、A129、P130及A132。结构分析显示，该表位部分处于PD-1与PD-L1的结合界面内，且与纳武利尤单抗（Nivolumab）、帕博利珠单抗（Pembrolizumab）的结合表位存在部分重叠。上述结果证实，信迪利单抗可通过结合PD-1上FG环的关键残基，直接竞争PD-1与PD-L1的相互作用，进而减弱PD-1的活化。本研究同时证明了AbMap技术在鉴定治疗性抗体结合表位方面的高效性与准确性。