ABSTRACT Chemokines (CK) play critical roles in the recruitment of immune cells into cancer tissues. Progressing tumors are commonly characterized by local production of regulatory chemokines (CKs) and suppressor cells, while production of pro-inflammatory CKs recruits protective CTLs and Th1 cells, dendritic cells (DC) and NK cells into the tumor microenvironment (TME) in association with effective (immuno)therapeutic intervention. We have recently shown that a combination vaccine targeting tumor vascular antigens (TVA) promotes specific CD8+ T cell responses and the coordinate upregulation of stromal CCL5/CXCL9-11 production and therapeutic T cell infiltration, and reduction in CCL22/CXCL12 production and MDSC/Treg content in the TME. Treatment also promotes de novo production of CCR7-ligand CKs CCL19/CCL21 in the TME, and the development of tertiary lymphoid-like structures (TLS). Remarkably, an autologous αDC1/TVA peptide-based vaccine administered with dasatinib to immune checkpoint blockade (ICB)-refractory patients with advanced-stage melanoma (NCT01876212) resulted in objective clinical benefit in 6 of 13 (46%) evaluable patients overall, including 4 of 7 (57%) patients exhibiting primary resistance to anti-PD1 blockade therapy. TCRBseq analyses revealed increased TCR convergence (i.e. immune focus) in the therapy-induced TIL repertoire in advance of objective clinical response. Furthermore, clinical responders exhibited unique TIL clonotypes post-treatment that were not detectable in blood, supporting the TME as a relevant site for treatment-dependent T cell cross-priming and “epitope spreading”. Since new preliminary data in murine B16 melanoma models suggests that therapeutic efficacy of αDC1/TBVA-based vaccines is superior when combined with the Project 1-developed CK modulation (CKM) regimen vs. dasatinib, Project 3, we will test the hypothesis that therapeutic benefits resulting from αDC1/vascular peptide-based immunotherapy in immune checkpoint inhibitor (ICI)-refractory, advanced-stage melanoma patients will be increased when combined with chemokine-modulating regimens (including CKM), Specifically, we will perform a Phase I/II trial of Type-1-polarized dendritic cell (αDC1)/TVA peptide vaccination in combination with tumor-selective chemokine modulation (CKM: Interferon-α2b, Rintatolimod and Celecoxib) in advanced-stage HLA-A2+ melanoma patients with primary PD-1/PD-L1 resistance (Aim 1) and analyze the on-treatment changes in TME and blood of patients to determine clinically-relevant changes in immunological analytes (Aim 2). Animal modeling will then be performed to determine the role of vaccine format in the therapeutic efficacy of combination CKM-based immunotherapy +/- immune regulatory antagonists (Aim 3).