Abstract 725: Listeria-inspired phagosome escape drives STING responses to CD47 blockade

Abstract Purpose: Through phagocytosis, antigen-presenting cells (APCs) engulf and neutralize malignant cells. ​However, engulfed tumor cells are destroyed in phagolysosomes leaving few peptides to enter cytosol, where cross-presentation of antigens and immune activation occurs. Listeria (L.) monocytogenes escape phagolysosomes by secreting a pore-forming protein Listeriolysin O (LLO). Our breakthrough centers on harnessing LLO to create LLO-CD47, a novel protein-antibody conjugate that fuses the myeloid checkpoint anti-CD47 to LLO. By promoting tumor cell-phagocytosis and the release of tumor peptides and DNA from phagolysosomes, we hypothesize that LLO-CD47 enhances the immune functionalities of APCs. Procedures: 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. C57BL/6 mouse bone marrow-derived macrophages (BMDMs) were used to study M2-to-Ml polarization, tumor cell phagocytosis, STING activation, and antigen presentation. Mice bearing orthotopically implanted spontaneously metastatic murine triple-negative 4T1 tumors and E0771 tumors received intratumoral (IT) or intraperitoneal (IP) injection. 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 treatment. Results: 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 TNFa in BMDMs in vitro and TAMs in vivo, relative to anti-CD47. 4. IT delivery of LLO-CD47 inhibits the growth and metastasis of orthotopically implanted murine breast tumors relative to anti-CD47. Harvested tumors show increased M1 polarized macrophages and CD8+ T cells, which express high levels of PD-1. 5. Co-administration of IP anti-PD-1 and LLO-CD47 delays the progression of breast tumor metastasis and prolongs animal survival relative to animals treated with anti-PD-1 and anti-CD47. 6. The elimination of CD8+ T cells or TAMs abrogates the antitumor effect of LLO-CD47. 7. IP administration of LLO-CD47 showed acceptable toxicity with stable body weight gain and no long-term hematological, renal, or hepatic toxicities. Conclusion: Conjugation of LLO to anti-CD47 enhances macrophage STING activation, antigen presentation, and tumor cell phagocytosis. Our work represents the first antibody-drug conjugate specifically designed to enhance immune activation against cancer.​ Citation Format: Benjamin R. Schrank, Yifan Wang, Abin Antony, Wen Jiang. Listeria-inspired phagosome escape drives STING responses to CD47 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 725.