Listeriolysin O Drives Innate and Adaptive Immune Responses to CD47 Immunotherapy

Abstract

Radioimmunotherapies that combine radiation with antibodies against the "don't eat me" signal CD47 show increasing promise. One opportunity to synergize RT with anti-CD47 is via the Stimulator of Interferon Genes (STING) pathway which facilitates potent immune responses to cytoplasmic DNA. Anti-CD47 should activate STING by increasing macrophage consumption of tumor DNA. However, tumor contents are destroyed in phagolysosomes. Listeria (L.) monocytogenes escape lysosomes by secreting a pore-forming protein Listeriolysin O (LLO). We recently engineered a protein-antibody conjugate linking anti-CD47 to LLO. Here, we demonstrate that LLO-CD47 enhances macrophage STING signaling, tumor cell phagocytosis, and tumor antigen presentation. At doses compatible with minimal toxicity in mice, LLO-CD47 delays the growth of orthotopic breast tumors. By contrast, anti-CD47 fails to activate STING in macrophages or inhibit tumor growth. We further hypothesize that LLO-CD47 requires innate and adaptive immune cells for antitumor immunity. Anti-CD47 was conjugated to LLO using a water-soluble SPDP crosslinker and purified by affinity chromatography. Transmission electron microscopy (TEM) was used to visualize the integrity of macrophage phagolysosomes following treatment. C57B6 mouse bone marrow-derived macrophages (BMDMs) were used to study the impact of LLO-CD47 on M2-to-M1 polarization, tumor cell phagocytosis, STING activation, and antigen presentation. CD8+ T cells or tumor-associated macrophages (TAMs) were depleted from tumor-bearing mice using an anti-CD8 antibody or anti-CSF-1R antibody prior to LLO-CD47 treatment. (1) LLO-CD47 skews BMDMs from M2-to-M1 inflammatory phenotypes and enhances the phagocytosis of E0771 tumor cells. (2) BMDMs visualized by TEM show breaches in phagosome membranes following LLO-CD47, but not anti-CD47, treatment. (3) LLO-CD47 increases levels of phosphorylated STING, IFN, and TNFα relative to cells treated with anti-CD47. (4) LLO-CD47 significantly inhibits the growth of orthotopically implanted E0771 murine breast tumors relative to anti-CD47. (5) The elimination of CD8+ T cells or TAMs abrogates the antitumor effect of LLO-CD47. LLO-CD47 is a de novo protein-antibody conjugate engineered for cGAS-STING pathway activation in innate immune cells. CD8+ T cells and TAMs are required for the antitumor activity LLO-CD47 in orthotopic models of breast cancer. This novel immunotherapy builds on clinical interest in myeloid checkpoint inhibitors and may be studied as a supplemental therapy for patients with metastatic breast cancer.