Immunotherapies represent the next generation of treatments for cancer. The recent clinical successes of immunotherapies have highlighted the potential to achieve stunning efficacy against otherwise intractable cancers. One such clinically validated immunotherapy target is the T-cell surface receptor PD-1 (programmed cell death protein 1), which inhibits T-cell cytotoxicity upon ligation with PD-L1 (programmed death-ligand 1) on a target cell. Antibody therapies targeting PD-1/PD-L1 can be strikingly effective cancer treatments, as demonstrated in the clinic by the recently approved pembrolizumab and nivolumab. However, antibody therapies have significant drawbacks as agents against this pathway. Due to their large size (~150 kDa), antibodies have limited tissue penetrance. Furthermore, due to the presence of the prophagocytic Fc domain, antibodies bound to PD-1 or PD-L1 on the T-cell cause counterproductive lymphocyte depletion. We hypothesized that a small engineered protein capable of blocking PD-1:PD-L1 signaling could avoid these issues and function as a superior immunotherapy. To accomplish this goal, we used yeast display-based directed evolution to affinity-mature a soluble form of the PD-1 ectodomain to increase its affinity for human PD-L1. After several rounds of selection from two generations of mutant libraries, we identified a high-affinity PD-1 variant. By surface plasmon resonance, high-affinity PD-1 binds to human PD-L1 with 110 pM affinity and a half-life of binding of >40 minutes, a dramatic improvement from wild-type PD-1. Direct in vivo binding assays demonstrate that this small protein penetrates solid tissues much more effectively than an anti-PD-L1 monoclonal antibody. Unlike anti-PD-L1 antibodies, high-affinity PD-1 does not lead to the depletion of effector T-cells. Consistent with these advantages, in syngeneic CT26 tumor models, high affinity PD-1 was effective in treating both small (~50 mm3) and large tumors (>150 mm3), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, radiolabeling of high-affinity PD-1 enabled us to utilize our reagent as a non-invasive tracer for PET imaging. Taken together, our data demonstrates the benefits of using small, non-antibody therapeutics as cancer immunotherapy and immune diagnostic agents.
Citation Format: Sydney R. Gordon, Roy Maute, Aaron Mayer, Melissa McCracken, Arutselvan Natarajan, Nan Guo, Richard Kimura, Jonathan M. Tsai, Aashish Manglik, Andrew Kruse, Sanjiv Gambhir, Irving L. Weissman, Aaron M. Ring. Engineered PD-1 variants as immunotherapies for cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B101.
- ©2016 American Association for Cancer Research.