The PD-L1/PD-1 immune checkpoint pathway, consisting of the inhibitory receptor PD-1 on T cells, and its ligand PD-L1 on tumor cells and antigen presenting cells (APCs), is a major mechanism for tumors to escape immune attack. Blocking antibodies of PD-1 and PD-L1 have demonstrated unprecedented clinical activities against an array of human cancers, yet durable benefit is limited to a small subset of patients. Expanding the clinical benefit to a larger population of patients has met critical challenges due to our incomplete understanding of the PD- L1/PD-1 pathway and how their blockade antibodies work. Our long-term goal is to fill these mechanistic gaps through the use of novel, unique and robust approaches. We recently uncovered two novel aspects of PD-L1/PD- 1 signaling. Intracellularly, we showed that the T cell costimulatory receptor CD28 is a primary target of PD-1 associated phosphatases. Extracellularly, we discovered that PD-L1 can be neutralized in cis by PD-1 expressed on the same cells, i.e., APCs. In this proposal, we will integrate and extend these two prior findings to elucidate a poorly defined extracellular crosstalk between PD-L1/PD-1 and CD28 pathways. Our preliminary experiments revealed that the CD28 ligand B7.1 binds and neutralizes PD-L1 in cis, but not in trans. Guided by this new finding, we propose to pursue three specific aims to determine the biochemical and functional consequences of the B7.1/PD-L1 cis-interaction and how this interaction might underlie unreported mechanisms of actions for PD- L1 and PD-1 blockade antibodies.
In Aim -1, we will determine whether B7.1 neutralizes the ability of PD-L1 to bind and activate PD-1 through cis-interaction.
In Aim -2, we will ask the reciprocal question of whether cis-PD- L1 inhibits the ability of B7.1 to bind and activate CD28.
In Aim -3, we will use well-defined co-culture assays and mouse tumor models to determine the effects of PD-1 and PD-L1 blockade antibodies in the context of B7.1/PD- L1 cis-interaction. The key for achieving these goals is to decouple and quantitate cis and trans interaction at the cell-cell interface. To this end, we plan to integrate classical approaches and our recently developed membrane reconstitution, live cell imaging (TIRF) and cell-specific antibody blockade assays. Completion of the project will reveal in-depth insights into the regulatory mechanism of the PD-L1/PD-1 immune checkpoint axis, its crosstalk with the B7/CD28 pathway, and the mechanism of actions of therapeutic antibodies. These mechanistic insights will likely yield novel targets and biomarkers of PD-1 targeted cancer immunotherapy.

Public Health Relevance

Development of PD-1 and PD-L1 inhibitors is a major breakthrough in cancer immunotherapy, yet clinical success is restricted to a small subset of cancer patients. Our proposed studies aim to uncover a novel regulatory mechanism of the PD-L1/PD-1 pathway and to elucidate the mechanisms of action of their therapeutic inhibitors, using novel, unique and precise assays. Execution of the proposed research will lay the foundation for designed more effective and durable PD-1 targeted cancer immunotherapy.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Method to Extend Research in Time (MERIT) Award (R37)
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Molecular and Integrative Signal Transduction Study Section (MIST)
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Mccarthy, Susan A
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University of California, San Diego
Schools of Arts and Sciences
La Jolla
United States
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