Identification of a monoclonal antibody that targets PD-1 in a manner requiring PD-1 Asn58 glycosylation

Abstract

Programmed cell death 1 (PD-1) is inhibitory receptor and immune checkpoint protein. Blocking the interaction of PD-1 and its ligands PD-L1/ L2 is able to active T-cell-mediated antitumor response. Monoclonal antibody-based drugs targeting PD-1 pathway have exhibited great promise in cancer therapy. Here we show that MW11-h317, an anti-PD-1 monoclonal antibody, displays high affinity for PD-1 and blocks PD-1 interactions with PD-L1/L2. MW11-h317 can effectively induce T-cell-mediated immune response and inhibit tumor growth in mouse model. Crystal structure of PD-1/MW11-h317 Fab complex reveals that both the loops and glycosylation of PD-1 are involved in recognition and binding, in which Asn58 glycosylation plays a critical role. The unique glycan epitope in PD-1 to MW11-h317 is different from the first two approved clinical PD-1 antibodies, nivolumab and pembrolizumab. These results suggest MW11-h317 as a therapeutic monoclonal antibody of PD-1 glycosylation-targeting which may become efficient alternative for cancer therapy.

Keywords: Cancer; Cancer immunotherapy; Structural biology.

Fig. 1

The complex structure of MW11-h317 Fab bound to PD-1. The Fab fragment of MW11-h317 and PD-1 are shown as cartoon (MW11-h317 Fab heavy chain in green, light chain in cyan, PD-1 in gray). The glycans in PD-1 are shown as sticks in yellow

Fig. 2

Loops of PD-1 in MW11-h317 Fab recognition. a The C′D loop, BC loop, and FG loop of PD-1 are represented in skyblue, pink, and lime, respectively. The CDR1, CDR2, and CDR3 loops of MW11-h317 light chain (LCDR1, LCDR2, LCDR3) are shown in yellow, blue, and magenta, respectively. The CDR1, CDR2, and CDR3 loops of MW11-h317 heavy chain (HCDR1, HCDR2, HCDR3) are represented in red, deep teal, and orange, respectively. Detailed interactions of MW11-h317 Fab binding to the PD-1 BC loop b, C′D loop c, and FG loop d are presented. Residues involved in the hydrogen bond interaction are shown as sticks and labeled. Hydrogen bonds are shown as dashed red lines

Fig. 3

Glycosylation of PD-1. a N-glycosylation of N49, N58, and N116 in PD-1. b Diagrammatic representation of N-link glycans of N58, where blue squares represent N-acetylglucosamines, red triangle is fucose, and green circles are mannose. c The Fo−Fc simulated annealing omit map of N-linked glycans of N58 contoured at 3.0σ. The stick model of glycans was superposed in the image. d Interactions between N-linked glycans of N58 and MW11-h317. The participating amino acid residues and glycans are shown with a stick model, with amino acids in MW11-h317 colored in green and glycans in PD-1 colored in yellow. Hydrogen bonds are indicated with magenta dashed lines

Fig. 4

N-glycosylation of N58 participates in MW11-h317 recognition. A flow cytometric analysis of MW11-h317 binding to WT PD-1 or various glycosylation sites mutated proteins (N49A, N58A, N74A, and N116A) expressed on the cell surface of HEK 293 cells. Plasmids expressing WT PD-1 or mutant proteins fused with EGFP were used for transfection. Mock-transfected HEK 293 cells were used as negative control (NC)

Fig. 5

Competitive binding of MW11-h317 Fab with PD-1 ligand PD-L1. a Superposition of the PD-1/MW11-h317 Fab complex structure with PD-1/PD-L1 complex structure (PDB ID: 4ZQK). MW11-h317 H chain is shown in green, L chain in cyan and PD-L1 in pink. PD-1 is shown as surface representation in gray. b Binding surface of PD-1 with MW11-h317 or PD-L1. The residues in contact with MW11-h317 are shown in green, whereas residues in contact with PD-L1 are shown in pink, and the overlapping residues bounded by both MW11-h317 and PD-L1 are shown in orange

Fig. 6

Distinct blocking mechanism of MW11-h317 compared with nivolumab and pembrolizumab. a, b Competitive binding of MW11-h317 and nivolumab (PDB ID: 5WT9) with PD-1. MW11-h317 and nivolumab are shown in green and blue, respectively, and the overlapping residues bounded by both MW11-h317 and nivolumab are shown in cyan. c, d Competitive binding of MW11-h317 and pembrolizumab (PDB ID: 5GGS) with PD-1. MW11-h317 and pembrolizumab are shown in green and red, respectively, and the overlapping residues bounded by both MW11-h317 and pembrolizumab are shown in yellow. e Antibodies competitive ELISA indicated that MW11-h317 was partly competitive with nivolumab or pembrolizumab. n = 3 independent experiments; data are mean ± SEM

Fig. 7

Comparable activities of MW11-h317 to nivolumab. a Affinities of MW11-h317 and nivolumab to PD-1. b MW11-h317 and nivolumab blocked the binding between PD-1 and PD-L1 (or PD-L2). c IL-2 or IFN-γ production in mixed lymphocytes stimulated by MW11-h317 and nivolumab. d Volumes and weights of subcutaneous xenograft tumors in PD-1 transgenic mice. Administration dose 10, 2, and 0.5 mg kg⁻¹ indicated as 10, 2, and 0.5 following antibodies’ name (n = 8 mice/tumor experiments)