Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging

Abstract

Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm(3)) and large tumors (150 mm(3)), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.

Keywords: PD-1; PD-L1; PET imaging; cancer immunotherapy; protein engineering.

Fig. 1.

Directed evolution of high-affinity PD-1 with yeast surface display. (A) Model of hPD-1 (green) complexed with hPD-L1 (magenta) constructed by structural alignment of the mPD-1:hPD-L1 complex (PDB ID code 3BIK) with hPD-1 (PDB ID code 3RRQ). Randomized residues of PD-1 are depicted as blue spheres for PD-L1 contact residues and red spheres for core residues. (B) Histogram overlays assessing yeast hPD-L1 staining at each round of selection. For the first-generation selections (Left), all rounds were stained with 100 nM biotinylated hPD-L1. For the second-generation selections (Right), yeast were stained with 1 nM biotinylated hPD-L1. (C) Summary of sequences and hPD-L1 affinities for selected PD-1 variants. The position of each mutated position and the corresponding residue in wild-type PD-1 is indicated at the top of the table. Numbering reflects the amino acid position within the mature PD-1 protein after signal peptide cleavage. Italic font indicates mutations that occurred at nonrandomized sites. Blue column shading indicates PD-L1 contact positions that converged in the HAC consensus sequence, whereas red column shading indicates converging core positions. Gray shading denotes those sequences whose hPD-L1 affinities were determined by surface plasmon resonance (SPR).

Fig. 2.

HAC–PD-1 binds and antagonizes human and mPD-L1 but not PD-L2. (A) Representative surface plasmon resonance (SPR) sensorgrams of wild-type PD-1 (Left) and HAC-V PD-1 (Right) binding to immobilized hPD-L1. (B) Competition binding assays of wild-type hPD-1, HAC-V PD-1, or HACmb on human SK-MEL-28 cells (Left), mouse B16-F10 cells, or yeast displaying hPD-L2. A total of 100 nM hPD-1/SA AlexaFluor 647 tetramer was used as the probe ligand. Error bars represent s.e.m.

Fig. 3.

HAC–PD-1 shows enhanced tumor penetration and does not deplete peripheral T cells. (A) Representative fluorescence microscopy images of sectioned CT26 tumors deficient in PD-L1 (Bottom row) or transgenic for hPD-L1 (Top row) 4 h post-i.p. injection of anti–hPD-L1 AlexaFluor488 (green) and HAC AlexaFluor 594 (red). Nuclei (blue) were labeled with DAPI. (Scale bar, 500 μm.) (B) Representative flow cytometry of dissociated tumors from A showing HAC AlexaFluor 594 staining versus anti–hPD-L1 AlexaFluor 488 staining. Percentages are given in each positive quadrant. (C) Summary of flow cytometry studies from four PD-L1–deficient tumors and four hPD-L1 transgenic tumors. n.s., not significant. ***P < 0.0001, two-way ANOVA. Error bars represent s.e.m. (D) Relative abundance of peripheral CD4+ T cells (Left) or peripheral CD8+ T cells (Right) after 3 d of administration of vehicle (PBS), anti-mouse PD-L1, HACmb, anti-mouse CD47, or combinations of these agents to mice engrafted with CT26 tumors. Significance is indicated relative to the PBS control. ns, not significant. *P < 0.05, ***P < 0.001, one-way ANOVA.

Fig. 4.

Antitumor efficacy of HACmb and anti–PD-L1 antibodies in small and large CT26 syngeneic tumor models. (A, Top) Schematic illustrating the experimental design of the small tumor experiment. Treatment was initiated for all cohorts 7 d after engraftment of tumors. Mice were injected with daily doses of vehicle (PBS), 250 µg anti-mouse PD-L1 (clone 10F.9G2), or 250 µg HACmb for 14 d. (A, Bottom) Relative growth rates of engrafted tumors, calculated as fold-change from displayed individual tumors (Left three panels) or as summary data (Far Right panel) over the course of the treatment period. Error bars represent s.e.m. n.s., not significant. ***P < 0.0001. (B, Top) Schematic illustrating the experimental design of the large tumor experiment. Mice were engrafted with CT26 tumors and monitored daily. When an individual tumor exceeded 150 mm³, the mouse was randomized to a treatment cohort. Tumors were measured daily and received daily treatment with vehicle (PBS), 250 µg anti-mouse PD-L1 (clone 10F.9G2), or 250 µg HACmb for 14 d. Anti-CTLA4 (clone 9D9) was administered as a single dose of 250 µg on day 1 of treatment. (B, Bottom) Summary data for average tumor growth over the 14-d period of treatment. Error bars represent s.e.m. n.s., not significant. ***P < 0.001, two-way ANOVA. Complete statistical analysis at day 14 posttreatment is shown in SI Appendix, Table S3.

Fig. 5.

Micro-PET imaging of hPD-L1 with ⁶⁴Cu–DOTA–HAC. (A) PET-CT images 1 h postinjection of ⁶⁴Cu–DOTA–HAC (230 µCi/25 µg/200 µL) in NSG mice bearing s.c. hPD-L1(+) (red dashed line) or hPD-L1(–) (white dashed line) CT26 tumors or both hPD-L1(+) and hPD-L1(–) CT26 tumors simultaneously (“dual tumor”). Blocking was performed by injection of 500 µg/200 µL of unlabeled HAC–PD-1 2 h before PET tracer. B, bladder; K, kidneys; L, liver; SG, salivary glands; T, tumor. (B) Quantification of tumor uptake in hPD-L1(+) (n = 4), hPD-L1(–) (n = 7), and blocked CT26 tumors (n = 3) 1 h postinjection by ROI analysis, indicated as %ID/g of tissue. There was no PVC correction. Error bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA.