Glycosylation-independent binding of monoclonal antibody toripalimab to FG loop of PD-1 for tumor immune checkpoint therapy

Abstract

Monoclonal antibody (mAb)-based blockade of programmed cell death 1 (PD-1) or its ligand to enable antitumor T-cell immunity has been successful in treating multiple tumors. However, the structural basis of the binding mechanisms of the mAbs and PD-1 and the effects of glycosylation of PD-1 on mAb interaction are not well understood. Here, we report the complex structure of PD-1 with toripalimab, a mAb that is approved by China National Medical Products Administration as a second-line treatment for melanoma and is under multiple Phase 1-Phase 3 clinical trials in both China and the US. Our analysis reveals that toripalimab mainly binds to the FG loop of PD-1 with an unconventionally long complementarity-determining region 3 loop of the heavy chain, which is distinct from the known binding epitopes of anti-PD-1 mAbs with structural evidences. The glycan modifications of PD-1 could be observed in three potential N-linked glycosylation sites, while no substantial influences were detected to the binding of toripalimab. These findings benefit our understanding of the binding mechanisms of toripalimab to PD-1 and shed light for future development of biologics targeting PD-1. Atomic coordinates have been deposited in the Protein Data Bank under accession code 6JBT.

Keywords: PD-1; Toripalimab; complex structure; glycosylation.

Figure 1.

Antitumor efficacy of toripalimab in a MC38 bearing mouse model. (a,b) Blocking of the binding of PD-1 to PD-L1 or PD-L2 using protein-based ELISA assay (a) or cell-based flow cytometry assay (b). (c) Flow chart of the animal study. MC38 was inoculated into human PD-1 knock-in mice of the C57BL/6 background. Toripalimab was administered via intraperitoneal (i.p.) injection every 3 or 4 d from day 7 (D7) after MC38 tumor inoculation. The size of the tumor was monitored every 3 or 4 d after injection of toripalimab or control mAb. Saline and control mAb were enrolled as negative control. (d) Mice bearing subcutaneous MC38 palpable tumors for 7 d were treated i.p. with four doses of toripalimab, 0.3, 1, 3, and 10 mg/kg, or saline or control IgG. The data with each dot show the average tumor volume of the group while the SE was presented as longitudinal bars. (e–j) Individual follow-up of tumor sizes is presented for each experimental group with each line showing the changes of the tumor size of each mouse.

Figure 2.

The complex structure of toripalimab and PD-1. (a) Gel filtration profiles of PD-1 (red), toripalimab-Fab (black), and the PD-1/toripalimab-Fab complex (green) were analyzed by size-exclusion chromatography as indicated. The SDS-PAGE analyses are shown in reducing (+DTT) or nonreducing (-DTT) conditions, one for PD-1, two for toripalimab-Fab, and three for toripalimab-Fab/PD-1 complex. (b) The complex structure of toripalimab and PD-1. The V fragment of toripalimab is shown as cartoon (heavy chain (VH), wheat; light chain (VL), lemon), and PD-1 is shown as surface representation (light blue). The CDR1, CDR2, and CDR3 loops of the heavy chain (HCDR1, HCDR2, and HCDR3) are colored in light pink, marine, and hot pink, respectively. The CDR1, CDR2, and CDR3 loops of light chain (LCDR1, LCDR2, and LCDR3) are colored in yellow, orange, and magenta, respectively. The FG loop of the PD-1 molecule is highlighted in cyan. The right panel showed the detailed binding of toripalimab to the FG loop of PD-1. Residues involved in the hydrogen bond interaction are shown as sticks and labeled. Hydrogen bonds are shown as dashed black lines.

Figure 3.

Competitive binding of toripalimab and PD-L1 with PD-1. (a) Superposition of the toripalimab/PD-1 complex structure with PD-1/PD-L1 complex structure (PDB code: 4ZQK). PD-L1 is shown in light pink while VH of toripalimab is colored in wheat and VL in lemon. (b) Binding surface of PD-1 with PD-L1 or toripalimab. The residues in contact with PD-L1 are colored in light pink, whereas residues in contact with toripalimab are colored in light teal, and the overlapping residues bound by both PD-L1 and toripalimab are colored in marine.

Figure 4.

Glycan modifications of PD-1 and glycosylation independent binding of toripalimab. (a) Complex structure of toripalimab-Fab and PD-1 with glycan depicted as sticks in orange. Four potential N-linked glycosylation sites, N49, N58, N74, and N116, were shown as sticks in light teal. (b). SPR assay characterization of the binding between toripalimab and PD-1 proteins using a BIAcoreT100 system. The refolded PD-1 protein (L25-R147), which is expressed in E. coli and refolded in vitro, and PD-1 protein obtained from 293T cells were analyzed for binding affinity with toripalimab, with toripalimab immobilized on the chip. The binding characteristics (K_a, K_d, and K_D) were labeled accordingly. The data presented here are a representative of three independent experiments with similar results.

Figure 5.

Comparative binding of PD-1 targeting mAbs. (a) Superimposition of the PD-1 from complex structures of PD-1/PD-L1 (cyan) (PDB code: 4ZQK), PD-1/pembrolizumab (light pink) (PDB code: 5JXE), PD-1/nivolumab (lemon) (PDB code: 5WT9), and PD-1/toripalimab (light blue) (PDB code: 6JBT). The loops contributed major binding to the mAbs were highlighted in dashed circles. (b) Comparison of the FG loop of the PD-1s from the complex structures. The FG loop of PD-1 shifted 8.19 Å upon the binding to nivolumab or toripalimab. (c) Superimposition of the complex structures of PD-1/pembrolizumab, PD-1/nivolumab and PD-1/toripalimab with the mAbs shown as ribbon in red, blue, and lemon, respectively. The PD-1 from PD-1/toripalimab complex is shown as surface in light blue with the FG loop highlighted in cyan.