B. R. Schrank1, Y. Wang2, and W. Jiang Jr3; 1Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 2Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, 3Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX
Purpose/Objective(s): Antitumor immune responses in antigen-presenting cells (APCs) are stimulated by the phagocytosis of tumor cells and the detection of tumor DNA by cytosolic nucleic acid sensors. However, these processes are impeded by phagocytosis checkpoints and inefficient cytosolic release of nucleic acids from phagolysosomes. Tumor DNA functions as a potent immunostimulant in APCs. However, no current therapeutic agents target phagolysosomal release of tumor DNA or other tumor cell components. Here, we developed a novel antibody-drug conjugate (ADC), CD47-LLO, by combining an antibody targeting the "don’t eat me" signal (CD47) with a bacterial toxin (listeriolysin O, LLO) from Listeria monocytogenes. CD47-LLO promotes cancer cell phagocytosis while creating openings in phagolysosomal membranes that allow for cytosolic escape of tumor-derived contents without damaging the APC. CD47-LLO treatment in vivo inhibits breast tumor growth and metastasis and improves animal survival as monotherapy or in combination with T cell checkpoint blockade. We hypothesize that CD47-LLOs antitumor activity requires activated intratumoral macrophages, dendritic cells, and lymphocytes that bridge innate and adaptive responses. Materials/
Methods: Anti-CD47 was conjugated to LLO using a water-soluble SPDP crosslinker and purified by affinity chromatography. To define the identities and activities of innate and adaptive immune cells that accumulate in tumors upon CD47-LLO treatment, single-cell RNA sequencing (scRNA-seq) was performed on CD45+ cells isolated from EO771 murine models of triple-negative breast cancer. The spatial organization of tumor-immune cell neighborhoods was examined using in situ confocal microscopy. Results: CD47-LLO induces a 30-fold increase in CD45+ cells in EO771 tumors relative to tumors treated with anti-CD47. In tumor-associated macrophages, CD47-LLO drives preferential expression of pathways supporting cGAS-STING signaling, phagocytosis, lymphocyte activation, and antigen presentation. CD47-LLO reorganizes tumor-immune neighborhoods as compared with anti-CD47 through the clustering of CD8 and CD4 T cells, which colocalize with dendritic cells. Depletion of tumor-associated macrophages and CD8 T cells significantly diminishes the antitumor effect of CD47-LLO. Conclusion: Despite dozens of CD47 checkpoint inhibitors in clinical trials, their variable tumor-type impact has spurred a search for complementary biologics to augment tumor inflammation. CD47-LLO devises a new therapeutic strategy that harnesses tumor-derived peptides and nucleic acids for antitumor activity. CD47-LLO is a first-in-class immunostimulatory ADC designed to bridge innate and adaptive immunity through the recruitment and activation of intratumoral macrophages.
