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.protein engineering | cancer immunotherapy | PET imaging | PD-1 | PD-L1

Practical Immuno-PET Radiotracer Design Considerations for Human Immune Checkpoint Imaging

“…2639) was obtained from the American Type Culture Collection. CT26 genetic variants expressing human PD-L1 were generated using the methods previously reported (27). All cells were grown in RPMI supplemented with 10% fetal bovine serum, glutamine (2 mmol/L), penicillin (100 units/mL), streptomycin (100 mg), and fungizone (0.25 mg/mL) and maintained in a humidified, 5% CO 2 incubator at 37°C.…”

“…Using the ectodomain of wild-type PD-1 protein, we previously affinity-matured a 14-kDa PD-L1 binder with a 100 pM dissociation constant, which exhibited high immunoreactivity in vitro and in pilot in vivo PET imaging studies. This protein, termed high-affinity consensus (HAC) PD1, is to our knowledge the first engineered binder to be used for human PD-L1 immune checkpoint imaging (27). As a therapeutic, HAC-PD1 significantly enhanced survival in mice bearing large PD-L1-positive tumors compared with treatment with anti-PD-L1 monoclonal antibodies.…”

The Positive Relationship Between γH2AX and PD-L1 Expression in Lung Squamous Cell Carcinoma

“…Several anti-PD-L1 antibodies have been used to evaluate PD-L1 expression in clinical trials, but the cut-off values were not consistent, partly because of the low sensitivity of the antibodies used for immunohistochemistry (38,39). Furthermore, PD-L1 protein expression was not ubiquitous in the tumor tissue samples; some exhibited peripheral staining, another exhibited patchy staining (data not shown), as already reported both in clinical samples and in animal studies (38,40). Because of the heterogeneity of PD-L1 staining intensity/distribution in tumors, we adopted the Hscore instead of using the tumor proportion score (TPS), which is used as a biomarker for the anti-PD-1 therapeutic response (6, 9).…”

Advances in Molecular Imaging Strategies in Immune Checkpoint Therapy

“…In addition, many preclinical studies have been done to support their targeted synergistic effects on multiple types of immune checkpoints, such as PD-L1, TIM-3, LAG-3, etc. These results shed light on the potential of small-protein mAbs in research on immune checkpoint blockades, as well as their applicability in the modulation of the immune response (Maute et al, 2015). Therefore, these molecular imaging methods are able to establish a basis for the non-invasive quantification of temporal or spatial features of immune checkpoints.…”