PD-1 N58-Glycosylation-Dependent Binding of Monoclonal Antibody Cemiplimab for Immune Checkpoint Therapy

Abstract

Immune checkpoint therapy (ICT) with a monoclonal antibody (MAb) against programmed cell death protein 1 (PD-1) is a powerful clinical treatment for tumors. Cemiplimab is a human IgG4 antibody approved in 2018 and is the first MAb proven to be effective for locally advanced basal cell carcinoma. Here, we report the crystal structure of cemiplimab bound to PD-1 and the effects of PD-1 N-glycosylation on the interactions with cemiplimab. The structure of the cemiplimab/PD-1 complex shows that cemiplimab mainly binds to PD-1 with its heavy chain, whereas the light chain serves as the predominant region to compete with the binding of PD-L1 to PD-1. The interaction network of cemiplimab to PD-1 resembles that of camrelizumab (another PD-1-binding MAb), and the N58 glycan on the BC loop of PD-1 may be involved in the interaction with cemiplimab. The binding affinity of cemiplimab with PD-1 was substantially decreased with N58-glycan-deficient PD-1, whereas the PD-1/PD-L1 blocking efficiency of cemiplimab was attenuated upon binding to the N58-glycosylation-deficient PD-1. These results indicate that both the binding and blocking efficacy of cemiplimab require the N58 glycosylation of PD-1. Taken together, these findings expand our understanding of the significance of PD-1 glycosylation in the interaction with cemiplimab.

Keywords: N58 glycosylation; PD-1; antibody; cemiplimab; immune checkpoint therapy (ICT).

Figure 1

The binding mechanism of cemiplimab to PD-1. (A) Overall structure of cemiplimab bound to PD-1. PD-1 colored in gray is shown as surface representation, while the heavy chain (VH) and light chain (VL) of cemiplimab-scFv are shown as cartoon colored in light blue and light pink. The CDR1, CDR2, and CDR3 loops of the heavy chain are colored in green (HCDR1), blue (HCDR2), and cyan (HCDR3), respectively. The CDR1, CDR2, and CDR3 loops of the light chain are colored in limon (LCDR1), orange (LCDR2), and red (LCDR3), respectively. The BC and FG loops are colored in green. (B) The detailed binding of cemiplimab to the FG and BC loops of PD-1. The residues taking part in forming hydrogen bonds are shown as sticks. The hydrogen bonds between residues are shown as a dashed line in black.

Figure 2

The structure characterization of PD-1 upon binding to MAbs. (A) The key region of PD-1 binding to cemiplimab. The β-strands of PD-1 are represented as the capital characters C, C′, D, F, and G, respectively. The CC′, C′D, and FG loops of PD-1 are highlighted in the blue cartoon, and the key epitopes of PD-1 binding to cemiplimab are shown as orange sticks, respectively. (B) Superposition of PD-1 upon binding to the PD-L1 ligand or different MAbs, including the PD-1 extracted from the complex structures of PD-1/PD-L1 (blue) (PDB code: 4ZQK), PD-1/nivolumab (cyan) (PDB code: 5WT9), PD-1/pembrolizumab (gray) (PDB code: 5JXE), PD-1/toripalimab (green) (PDB code: 6JBT), PD-1/camrelizumab (yellow) (PDB code: 7CU5), PD-1/cemiplimab (magenta) and PD-1/MW11-h317 (light blue) (PDB code: 6JJP). FG loop and BC loop of PD-1 which contributed vital binding to the cemiplimab are shown as a dashed line in black. (C) Flexible conformations of the FG loop of PD-1 upon binding to PD-L1 or different MAbs.

Figure 3

Structural basis of the blockade binding of cemiplimab with PD-L1. (A) Comparison of cemiplimab/PD-1 with PD-L1 extracted from PD-1/PD-L1 complex structure (PDB code: 4ZQK). PD-L1 is shown as smudge cartoon, while PD-1 is shown as surface diagram in white. VH and VL of cemiplimab-scFv are shown as cartoons in light blue and light pink, respectively. (B) The competitive binding surfaces of cemiplimab with PD-L1 on PD-1. The residues bound to cemiplimab alone are colored in deep salmon, while the residues contact with PD-L1 alone are colored in smudge, and the residues contacted by both cemiplimab and PD-L1 are colored in blue. The epitope residues in PD-1 are pointed out in black characters. (C) The binding surface of PD-L1 and structurally known clinically approved MAbs on PD-1 is shown in different colors. The binding surface of PD-L1 and other MAbs, e.g., cemiplimab, camrelizumab, pembrolizumab, nivolumab, toripalimab, and tislelizumab are colored in orange, light pink, limon, purple, yellow, blue, and teal, respectively.

Figure 4

Interaction between MAbs and N58 glycosylation on BC loop. (A) The comparison of the overall binding of cemiplimab, nivolumab, and pembrolizumab to PD-1. Superimposition of cemiplimab/PD-1 complex with that of pembrolizumab/PD-1 (PDB: 5JXE) and nivolumab/PD-1 (PDB: 5WT9). The cemiplimab, pembrolizumab, and nivolumab are shown as ribbon and colored in cyan, magenta, and orange, respectively. PD-1 extracted from camrelizumab/PD-1 (PDB code: 7CU5) complex is shown as surface representation colored in white. (B) Superimposition of cemiplimab/PD-1 complex with that of camrelizumab (PDB: 7CU5), MW11-h317 (PDB: 6JJP) and mAb059c (PDB: 6K0Y). The VH domains of the MAbs are shown as ribbons while the VL domains are not shown. PD-1, cemiplimab, camrelizumab, MW11-h317, and mAb059c are colored in gray, cyan, magenta, orange and blue, respectively. The CC′, C′D, and FG loops of PD-1, which participate in binding to Mabs are highlighted in blue. (C) Structure-based sequence alignment of cemiplimab and other anti-PD-1 MAbs. Coils above the sequences indicate α-helices, and the lines with arrowhead represent the β sheets. Residues highlighted in yellow are highly conserved. The sequence alignment was generated with ClustalX and ESPript. (D, E) The interaction of N-glycosylation N58 with MW11-h317 (D) or camrelizumab (E). The amino acid residues involved in hydrogen bond interaction and N58 glycans are shown as sticks, with amino acids in MW11-h317 colored in orange, camrelizumab colored in magenta, and the glycans in PD-1 colored in green. Hydrogen bonds are labeled by yellow dashed lines. (F) Comparison of cemiplimab/PD-1 complex with that of camrelizumab (PDB: 7CU5), and the amino acids in cemiplimab are colored in cyan and residues in camrelizumab are colored in magenta.

Figure 5

N-glycosylation of N58 remotes the binding to cemiplimab. (A) SPR assay characterization of the binding profiles of cemiplimab with PD-1-WT (left), PD-1-N58A (middle) proteins expressed in 293F cells, and PD-1-E. coli (right) expressed in E. coli cells. (B) SPR assay characterization of the binding of camrelizumab with PD-1-WT (left), PD-1-N58A (middle) expressed in 293F cells, and PD-1-E. coli (right) expressed in E. coli cells. The mean value of the KD was recorded after repeating each experiment three times.

Figure 6

Reduced blocking efficiency of cemiplimab to N58 glycosylation-deficient PD-1. (A) Untransfected 293T cells and transfected 293T cells incubated with isotype antibody as negative control. (B) The blocking of the binding of His-tagged PD-1-WT (blank) or PD-1-N58A (blue) proteins to PD-L1 expressing 293T cells is analyzed with varying concentrations (0, 2, 4, 10, 20, 40, and 80 μg/ml) of full-length cemiplimab (left) or camrelizumab (right). The PD-L1 expressing 293T cells staining with His-tagged PD-1-WT or PD-1-N58A are prepared as a positive control. (C, D) The frequencies of the His-tagged PD-1-WT or PD-1-N58A protein staining positive subpopulations in the absence (0 μg/ml) or presence (20 μg/ml) of cemiplimab (C) or camrelizumab (D). At the same concentration (20 μg/ml) of cemiplimab or camrelizumab, the frequency of His-tagged PD-1-WT or PD-1-N58A staining positive cells was calculated based on PD-L1-GFP-positive cells. The experiment was repeated twice in the results averaged.