Figure 2.
Figure 2. Epitope mapping and structural studies of the prioritized anti–PD-1 Ab clones. (A) Ability of anti–PD-1 Abs to block the PD-1–PDL-1 interaction in a Luminex biochemical assay. PD-1–coated beads were incubated in the presence or absence of a competitor anti–PD-1 Ab, and then beads were stained with different concentrations of biotin-labeled PDL-1 protein. Data (n = 2) are mean ± SD. MFI, mean fluorescence intensity. (B) Potency of anti–PD-1 Abs in blocking the PD-1–PDL-1 interaction in a Luminex biochemical assay. PD-1–coated beads were incubated with a fixed concentration of PDL-1 equivalent to the half-maximal inhibitory concentration value for the PD-1–PDL-1 interaction in this assay. The PD-1–PDL-1 complex, bound at 50% in equilibrium, was then treated with increasing concentrations of anti–PD-1 Ab to determine if they were capable of completely disrupting the PD-1–PDL-1 interaction with pembrolizumab used as a positive blocking Ab control. (C) Epitope mapping by site directed mutagenesis of PD-1. Defined epitopes were identified for anti–PD-1 Abs that were either blocking or nonblocking of the PD-1–PDL-1 interaction using HeLa cells transfected with expression vectors encoding PD-1 with substitutions at solvent accessible residues. Amino acid substitutions in PD-1 are indicated in blue lettering above each histogram. Representative data are shown for n = 3 experiments. (D) Ab competitive binding studies for cell-surface PD-1. Jurkat PD-1 cells were incubated with excess of 137F2, 135C12, or 136B4 mouse Abs and then stained with a minimal concentration of the indicated humanized anti–PD-1 Abs (n = 3). (E) hPD-1 and NB01a Fab (humanized version of the mouse 135C12 Ab) complexes were purified by size-exclusion chromatography and crystallized. Crystals diffracted to 2.2 Å resolution, and the structure was solved by molecular replacement. The structure reveals that the binding site of NB01a is adjacent to residues in purple involved in the PD-1 interaction with either PDL-1 or PDL-2. The CC′ loop (residues 70–74) of hPDL-1 is disordered and indicated as a dashed line. Loops connecting β strands BC (57–63), C′D (84–92), and FG (127–133) were also disordered. Strands are named following the canonical designation. Cα superpositioning of the hPD-1 present in the NB01a Fab and hPDL-1 (PDB accession no. 4ZQK) complexes show that NB01a Fab and PDL-1 bind distinct nonoverlapping sites on PD-1. (F) Mapping of variable residues between human PD-1 and monkey, dog, horse, mouse, and rat PD-1 revealed an evolutionarily conserved patch (P1) on the opposite face of PD-1 from the PDL-1 or PDL-2 interaction site (yellow, orange, and light green colored residues). The P1 patch overlaps with the binding epitopes for the 135C12/NB01 (orange residues) and 136B4 (light green residues) antagonistic Abs that are nonblocking of PD-1–PDL-1. Residues N49, N58, N74, and N116 that are predicted N-linked glycosylation sites are show in brown on the PD-1 model to be excluded from the P1 patch, 135C12/NB01, and 136B4 Ab binding epitopes. (G) Cα superpositioning of hPDL-1 coordinates of the NB01a complex with pembrolizumab (PDB accession no. 5GGS) and the nivolumab (PDB accession no. 5GGR) confirms that NB01a Fab binding to PD-1 does not interfere with the binding of either pembrolizumab or nivolumab anti–PD-1 Abs. hPDL-1 is shown as a ribbon diagram in E and F, with the hPDL-1 binding surface (PDB accession no. 4ZQK) colored in purple in G.

Epitope mapping and structural studies of the prioritized anti–PD-1 Ab clones. (A) Ability of anti–PD-1 Abs to block the PD-1–PDL-1 interaction in a Luminex biochemical assay. PD-1–coated beads were incubated in the presence or absence of a competitor anti–PD-1 Ab, and then beads were stained with different concentrations of biotin-labeled PDL-1 protein. Data (n = 2) are mean ± SD. MFI, mean fluorescence intensity. (B) Potency of anti–PD-1 Abs in blocking the PD-1–PDL-1 interaction in a Luminex biochemical assay. PD-1–coated beads were incubated with a fixed concentration of PDL-1 equivalent to the half-maximal inhibitory concentration value for the PD-1–PDL-1 interaction in this assay. The PD-1–PDL-1 complex, bound at 50% in equilibrium, was then treated with increasing concentrations of anti–PD-1 Ab to determine if they were capable of completely disrupting the PD-1–PDL-1 interaction with pembrolizumab used as a positive blocking Ab control. (C) Epitope mapping by site directed mutagenesis of PD-1. Defined epitopes were identified for anti–PD-1 Abs that were either blocking or nonblocking of the PD-1–PDL-1 interaction using HeLa cells transfected with expression vectors encoding PD-1 with substitutions at solvent accessible residues. Amino acid substitutions in PD-1 are indicated in blue lettering above each histogram. Representative data are shown for n = 3 experiments. (D) Ab competitive binding studies for cell-surface PD-1. Jurkat PD-1 cells were incubated with excess of 137F2, 135C12, or 136B4 mouse Abs and then stained with a minimal concentration of the indicated humanized anti–PD-1 Abs (n = 3). (E) hPD-1 and NB01a Fab (humanized version of the mouse 135C12 Ab) complexes were purified by size-exclusion chromatography and crystallized. Crystals diffracted to 2.2 Å resolution, and the structure was solved by molecular replacement. The structure reveals that the binding site of NB01a is adjacent to residues in purple involved in the PD-1 interaction with either PDL-1 or PDL-2. The CC′ loop (residues 70–74) of hPDL-1 is disordered and indicated as a dashed line. Loops connecting β strands BC (57–63), C′D (84–92), and FG (127–133) were also disordered. Strands are named following the canonical designation. Cα superpositioning of the hPD-1 present in the NB01a Fab and hPDL-1 (PDB accession no. 4ZQK) complexes show that NB01a Fab and PDL-1 bind distinct nonoverlapping sites on PD-1. (F) Mapping of variable residues between human PD-1 and monkey, dog, horse, mouse, and rat PD-1 revealed an evolutionarily conserved patch (P1) on the opposite face of PD-1 from the PDL-1 or PDL-2 interaction site (yellow, orange, and light green colored residues). The P1 patch overlaps with the binding epitopes for the 135C12/NB01 (orange residues) and 136B4 (light green residues) antagonistic Abs that are nonblocking of PD-1–PDL-1. Residues N49, N58, N74, and N116 that are predicted N-linked glycosylation sites are show in brown on the PD-1 model to be excluded from the P1 patch, 135C12/NB01, and 136B4 Ab binding epitopes. (G) Cα superpositioning of hPDL-1 coordinates of the NB01a complex with pembrolizumab (PDB accession no. 5GGS) and the nivolumab (PDB accession no. 5GGR) confirms that NB01a Fab binding to PD-1 does not interfere with the binding of either pembrolizumab or nivolumab anti–PD-1 Abs. hPDL-1 is shown as a ribbon diagram in E and F, with the hPDL-1 binding surface (PDB accession no. 4ZQK) colored in purple in G.

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