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. 2017 Feb 6:8:14369.
doi: 10.1038/ncomms14369.

An unexpected N-terminal loop in PD-1 dominates binding by nivolumab

Shuguang Tan  1   2 , Hao Zhang  1   3 , Yan Chai  2 , Hao Song  4 , Zhou Tong  1 , Qihui Wang  1 , Jianxun Qi  2 , Gary Wong  2 , Xiaodong Zhu  5 , William J Liu  6 , Shan Gao  7 , Zhongfu Wang  8 , Yi Shi  2 , Fuquan Yang  9 , George F Gao  2   4   6 , Jinghua Yan  1   2   3
Affiliations

An unexpected N-terminal loop in PD-1 dominates binding by nivolumab

Shuguang Tan et al. Nat Commun. .

Abstract

Cancer immunotherapy by targeting of immune checkpoint molecules has been a research 'hot-spot' in recent years. Nivolumab, a human monoclonal antibody targeting PD-1, has been widely used clinically since 2014. However, the binding mechanism of nivolumab to PD-1 has not yet been shown, despite a recent report describing the complex structure of pembrolizumab/PD-1. It has previously been speculated that PD-1 glycosylation is involved in nivolumab recognition. Here we report the complex structure of nivolumab with PD-1 and evaluate the effects of PD-1 N-glycosylation on the interactions with nivolumab. Structural and functional analyses unexpectedly reveal an N-terminal loop outside the IgV domain of PD-1. This loop is not involved in recognition of PD-L1 but dominates binding to nivolumab, whereas N-glycosylation is not involved in binding at all. Nivolumab binds to a completely different area than pembrolizumab. These results provide the basis for the design of future inhibitory molecules targeting PD-1.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1. Overall structure of PD-1 nivolumab-Fab complex.
(a) Schematic diagram of PD-1 protein. Full-length PD-1 is separated into ectodomain, transmembrane domain and intracellular domain. Ectodomain is composed of the signal peptide, N-loop, IgV domain and stalk region. The N-glycosylation at N58, which could be seen in the complex structure in the ectodomain is indicated with solid green arrowhead, whereas the other three N-glycosylation sites are indicated with hollow green arrowheads. The numbers indicate amino acid positions. (b) Gel filtration profiles of PD-1 (blue), nivolumab-Fab (green) and the PD-1-nivolumab-Fab complex (red) were analysed by size-exclusion chromatography as indicated. (c) The separation profiles of each pooled samples on SDS-PAGE are shown in reducing (+DTT) or non-reducing (-DTT) conditions, 1 for PD-1, 2 for nivolumab-Fab and 3 for PD-1-nivolumab-Fab complex. (d) The complex structure of nivolumab-Fab bound to PD-1. The Fab fragment of nivolumab is shown as cartoon (Heavy chain, cyan; Light chain, pink), and PD-1 is shown as surface representation (light blue). The CDR1, CDR2 and CDR3 loops of the heavy chain (HCDR1, HCDR2, HCDR3) are coloured in marine, yellow and magenta, respectively. The CDR1, CDR2 and CDR3 loops of light chain (LCDR1, LCDR2, LCDR3) are coloured in orange, violet and green, respectively. The N-loop and FG loop of the PD-1 molecule are highlighted in red and blue, respectively.
Figure 2
Figure 2. The atomic interaction details at the binding interface of PD-1 nivolumab-Fab complex.
Detailed interactions of nivolumab binding to the N-loop, BC loop (a) and FG loop (b) of PD-1. Residues involved in the hydrogen bond interaction are shown as sticks and labelled. Hydrogen bonds are shown as dashed black lines.
Figure 3
Figure 3. Structural basis of glycan modification in PD-1.
(a) A cartoon representation of the PD-1 structure. The four N-linked glycosylation sites (N49, N58, N74, and N116) are shown in sticks and coloured orange. The observed glycans are highlighted in sticks and coloured in magenta. (b) The 2 Fo-Fc electron density map of the N58 N-linked glycans contoured at 1.0 sigma is represented in blue. Three glycans consisting of two N-acetylglucosamine (NAGs) and one L-fucose (FUC) can be clearly observed. (c) SDS-PAGE analysis of the molecular reduction of four N-glycosylation single site-mutated PD-1 protein and PD-1 protein expressed in insect cells or refolded PD-1 protein expressed in E. coli cells. Compared with the WT PD-1 protein, each of the four N-glycosylation single site mutations has induced various levels of molecular weight reduction, indicating the N-glycosylation on each of the sites. PD-1 protein expressed in insect cells or E. coli cells showed substantial reduction of molecular weight, indicating the heavy glycosylation of PD-1.
Figure 4
Figure 4. No evidence for N-glycosylation involvement in nivolumab recognition.
(a) A flow cytometric assay of nivolumab binding to PD-1 WT or various N-linked glycosylation sites mutated proteins (N49A, N58A, N74A and N116A) expressed on the cell surface of 293 T cells. Plasmids expressing the full-length PD-1 WT or mutant proteins fused with EGFP at the C terminus were used for transfection. Mock-transfected 293 T cells were used as negative control (NC). (b) SPR assay characterisation of the binding between nivolumab and various PD-1 mutant proteins using a BIAcoreT100 system. The PD-1 N-linked glycosylation sites mutant proteins expressed in 293 T cells were used for the assay. The N-linked glycosylation sites mutations in PD-1 showed no effect to the nivolumab binding. The refolded PD-1 proteins with (L25-R147) or without N-loop (N33-R147) expressed in E. coli and PD-1 protein without N-loop (N33-R147) from insect cells were then analysed for binding affinity with nivolumab. The roles of PD-1 N-loop to the binding of PD-L1 were analysed with response units plotted against protein concentrations. No substantial differences was detected among WT PD-1 expressed in 293 T cells, refolded PD-1 (L25-R147) expressed in E. coli, and N-loop truncated PD-1 (N33-R147) expressed in insect cells or E. coli. The data presented here are a representative of three independent experiments with similar results.
Figure 5
Figure 5. Competitive binding of nivolumab-Fab and PD-L1 with PD-1.
Superposition of the PD-1-nivolumab-Fab complex structure with PD-1-PD-L1 complex structure in side view (a) or top view (b). PD-L1 is shown in yellow and nivolumab-Fab H-chain in cyan, L-chain in pink. (c) Binding surface of PD-1 with PD-L1 or nivolumab. The residues in contact with PD-L1 are coloured in yellow, whereas residues in contact with nivolumab H-chain or L-chain are coloured in cyan or pink, respectively, and the overlapping residues bounded by both PD-L1 and nivolumab are coloured in green.
Figure 6
Figure 6. Distinct blockade binding mode compared with pembrolizumab.
(a) Superposition of PD-1-nivolumab-Fab, PD-1-pembrolizumab-Fab with the PD-1-PD-L1 complex structure. Nivolumab-Fab and pembrolizumab-Fab are coloured in green and magenta, respectively, and PD-L1 is coloured in cyan. PD-1 is shown as surface representation. The N-loop targeted by nivolumab is highlighted in red, and the unavailable pembrolizumab targeted C'D loop is indicated as dashed lines in lemon. Nivolumab and pembrolizumab bind to PD-1 in two different orientations with some clash regions. (b) Binding surface of PD-1 by nivolumab or pembrolizumab. The binding residues in contact with nivolumab are coloured in green, whereas residues in contact with pembrolizumab are coloured in magenta. (c) The Octet competition binding assay of nivolumab or pembrolizumab binding to PD-1. Ni-NTA sensors loaded with PD-1 at stage 1 were first saturated with the indicated MAbs (10 μg ml−1) or PBST buffer at stage 2. The capacity of additional binding was monitored by measuring further shifts after injection of the other antibody (10 μg ml−1) in the presence of the first antibody at stage 3. The result shows that nivolumab binding to PD-1 have partial competition with pembrolizumab.
Figure 7
Figure 7. Targeting the loops of PD-1 for monoclonal antibody-based immune checkpoint blockade.
(a) The loops of PD-1 targeted by nivolumab or pembrolizumab are coloured differently as follows, N-loop, red; BC loop, green; FG loop, blue; C'D loop, magenta. (b) Comparison of PD-1 with other solved PD-1 structures with/without ligands (apo structure, yellow, PDB: 3RRQ; PD-1/PD-L1 structure, green, PDB: 4ZQK; PD-1-pembrolizumab-Fab structure, blue, PDB: 5JXE; PD-1-nivolumab-Fab structure, red).
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