Project Summary/Abstract: Immunotherapy for the treatment of various human cancers has drawn great attention, and many immune checkpoint inhibitors are in clinical trials. However, immune checkpoint blockade-based therapy has yet to be fully successful, mainly due to a lack of cancer specificity and immunotherapy-associated side effects. These problems can be overcome by loco-regional delivery of therapeutics and a tumor site?specific immune checkpoint blockade. The primary objective of the present research is to develop a cancer-targeted immune checkpoint blockade therapy for lung cancer by applying a non-toxic nanoparticle-based gene therapy with a novel PD-L1 inhibitory peptide (PD-L1ip) secretory gene. Recently, novel PD-L1ip genes that inhibit the complexation of PD-L1 and PD-1 were generated and effective gene expression was demonstrated in tumor cells as well as significant enhancement of cytotoxic T cell activity in cancer cell growth inhibition in vitro. Independently, in orthotopic mouse models of both murine and human lung cancer, it was demonstrated that intratracheally (IT) administered cell penetrating peptide (CPP) nanoparticle containing genes such as endogenous apoptosis inducer genes (TRAIL and angiotensin II type 2 receptor (AT2R)) or firefly luciferase gene by an aerosolizer caused high expressions of genes in lung tumor cells; relatively lower expression was detected in normal lung epithelial cells. The pulmonary gene therapy with AT2R or TRAIL gene significantly attenuated the growth of lung carcinoma grafts without showing any side effects. These studies suggest that local pulmonary gene delivery effectively distributes CPP nanoparticle containing gene throughout the lung and causes cancer-targeted gene expression. Therefore, it is suggested that the pulmonary delivery of PD-L1ip gene by CPP nanoparticle vector should be an effective local immunotherapy modality for lung cancer. Accordingly, the investigators hypothesize that pulmonary administration of the CPP and PD-L1ip gene complex by an aerosolizer is a feasible, safe and effective treatment for primary and metastatic lung cancer. The specific objectives of the proposed study are; 1) to further study the inhibitory peptide structure and the binding thermodynamics and kinetics with PD-L1 by computational modeling, as well as the function of the peptide in CD8+ T cell-dependent cancer cell lysis in vitro, 2) to evaluate the therapeutic efficacy and adverse event profile of the PD-L1ip gene administered via intratracheal spray in murine models of both murine primary and metastatic lung cancer, 3) to demonstrate the safety and feasibility of intratracheal administration of CPP- PD-L1ip gene complex using normal and lung tumor bearing mice. The proposed study will evaluate the therapeutic and safety profiles of this CPP nanoparticle-based pulmonary immunotherapy, and contains ample training opportunity for graduate/undergraduate students. This pulmonary immunotherapy strategy would be applicable to human patients with primary and/or metastatic lung cancer.