PROJECT SUMMARY The immediate objective of our proposal is to increase immune density within solid malignancies to rectify defects in anti-tumor immunity and maximize response rates to immune checkpoint blockade (ICB) therapy. An important hallmark of cancer is the ability to suppress or exclude innate and adaptive antitumor immune subsets, resulting in a tumor microenvironment less conducive to ICB treatment. Studies using combinatorial strategies have concluded that increased CD8 T cell density during immune checkpoint inhibition significantly enhances anti-tumor efficacy in non-immunogenic “cold” tumors. To increase CD8 T cell infiltration, we have developed a tumor-targeting bacterial platform engineered to secrete chemokines and cytokines, which we refer to as biological beacons or “bio-beacons,” and are meant to recruit and activate select subsets of immune cells. We have completed proof-of-principle experiments to show that bio-beacons, composed of tumor- colonizing E.coli vectors secreting murine C-X-C motif chemokine ligand 9 (CXCL9) and interleukin-15 (IL-15), mediate migration and expansion of murine lymphocytes. While cytotoxic CD8 T cells are thought to mediate the majority of tumor cell killing during immune checkpoint inhibition, it is becoming increasingly clear that overall efficacy of the anti-tumor response depends on a broader spectrum of cell types present within the tumor microenvironment that provide support, such as CD4 T cells, dendritic cells (DCs) and natural killer (NK) cells. Therefore, we propose to generate additional bio-beacons secreting murine CCL3 and CCL4, which are anticipated to recruit a broader spectrum of immune cells including CD4, NK, DCs, and monocytes. We hypothesize that bio-beacons will increase sensitivity of solid tumors to immunotherapy, specifically ICB treatment, by increasing intratumoral density of tumor-reactive immune subsets. We propose to elucidate changes in intratumoral immune composition following bio-beacon treatment and their potential synergy with immune checkpoint blockade therapy as a prelude for clinical development and translation. A series of studies under two Specific Aims will be conducted to: i) develop and evaluate novel bio-beacons intended to recruit supportive immune subsets; ii) determine the changes in intratumoral immune composition mediated by single or combinations of bio-beacons in various solid tumor models; and iii) determine bio-beacon combinations that significantly enhance efficacy of ICB therapy in tumor-bearing mice. These studies will delineate the intratumoral frequency and functional phenotype of immune subsets following bio-beacon administration, which can be used to uncover potential mechanisms contributing to any observed improvements in ICB therapy. If successful, we predict that bio-beacons can be used to increase the frequency of tumor-reactive immune subsets in a variety of solid tumor types and, thus, increase sensitivity to ICB treatment. In futur...