Effective immunotherapy options are lacking for patients with advanced NSCLC who progress on a PD-(L)1 inhibitor and for those that EGFR or ALK positive after progression on TKI therapy. One potential approach to improve ICI efficacy is to promote cytolytic T cell infiltration into tumors. This can be accomplished via in situ vaccination with functional antigen presenting cells which can take advantage of the full repertoire of tumor antigens and convert the tumor into a lymph node-like environment promoting both local and systemic T cell activation. The chemokine CCL21 promotes co-localization of naive T cells and antigen-experienced dendritic cells (DCs) to facilitate T cell activation. Our preclinical studies and phase I trial of intratumoral (IT) administration of DC genetically modified to overexpress CCL21 (CCL21-DC) revealed augmentation of tumor antigen presentation in situ, resulting in systemic antitumor immunity. However, increased PD-L1 expression was observed following therapy in some patient tumors, suggesting that tumor-mediated impairment of T cell function may be forestalling a more robust CCL21-DC mediated antitumor response. Therefore, we are conducting a phase I trial, combining IT CCL21-DC with pembrolizumab in patients with advanced NSCLC. Phase I, dose-escalating, trial followed by dose expansion. Maximum of 24 patients (9-12 escalation + 12 expansion) with stage IV NSCLC will be evaluated who have tumors accessible for IT injection and are either (1) EGFR/ALK wild-type after progression on a PD-(L)1 inhibitor or (2) EGFR/ALK mutant after progression on TKI therapy. Three IT injections of autologous CCL21-DC (days 0, 21, 42) will be concurrently administered with pembrolizumab, followed by q3wk pembrolizumab. Primary objective of dose escalation is safety and determination of maximum tolerated dose (MTD) of IT CCL21-DC (5x106, 1x107, or 3x107) when combined with pembrolizumab. Primary objective of dose expansion is objective response rate at MTD. Secondary objectives include adverse event profiling and determination of drug target activity by immune monitoring studies. This trial, NCT03546361, is currently open for enrollment. NCT03546361. The authors.