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Updated: Mar 29, 2026
Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression
Published on: February 3, 2023
Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging
Roy L Maute1, Sydney R Gordon1, Aaron T Mayer2
1Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305; Ludwig Center for Cancer Stem Cell Research and Medicine, Stanford University School of Medicine, Stanford, CA 94305; Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305; Department of Pathology, Stanford University Medical Center, Stanford, CA 94305;
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.
Insights
Engineered PD-1 protein acts as a small-molecule drug to block immune checkpoints, improving cancer immunotherapy. This novel approach shows better tumor penetration and efficacy than antibodies, and can also be used for imaging.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Immune checkpoint protein programmed cell death protein-1 (PD-1) signaling promotes tumor growth by suppressing anti-tumor immune responses.
- Monoclonal antibodies targeting the PD-1/programmed cell death ligand-1 (PD-L1) axis are effective cancer treatments but have limitations like poor tumor penetration and T-cell depletion.
Purpose of the Study:
- To engineer a high-affinity, non-antibody PD-1 antagonist for improved cancer immunotherapy.
- To evaluate the therapeutic efficacy and pharmacokinetic properties of the engineered PD-1 antagonist compared to anti-PD-L1 antibodies.
- To assess the potential of the engineered PD-1 as a PET imaging tracer for PD-L1-positive tumors.
Main Methods:
- Directed evolution using yeast-surface display to engineer the PD-1 ectodomain.
- In vitro and in vivo evaluation of high-affinity PD-1 antagonist in CT26 tumor models.
- Radiolabeling of high-affinity PD-1 for PET imaging studies in mice.
Main Results:
- Engineered PD-1 achieved high-affinity (110 pM) competitive antagonism of PD-L1.
- High-affinity PD-1 demonstrated superior tumor penetration and did not deplete peripheral T cells, unlike anti-PD-L1 antibodies.
- High-affinity PD-1 effectively treated both small and large tumors, whereas anti-PD-L1 antibodies were ineffective against large tumors.
- Radiolabeled high-affinity PD-1 successfully distinguished PD-L1-positive from PD-L1-negative tumors in vivo.
Conclusions:
- Small, non-antibody therapeutics targeting the PD-1/PD-L1 axis offer advantages over monoclonal antibodies for cancer immunotherapy.
- Engineered PD-1 exhibits favorable pharmacology, enhanced efficacy against larger tumors, and potential for non-invasive immune diagnostics.
