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.

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