Background Tumor DNA is a potent activator of the cGAS-STING pathway.1 CD47 is expressed on various tumor types including breast, prostate, glioblastoma, non-Hodgkin lymphoma, and hepatocellular carcinoma. Interrupting the CD47-SIRP alpha axis by monoclonal antibodies fosters macrophage-mediated phagocytosis of solid and hematopoietic tumor cells.2 The pore-forming protein listeriolysin O (LLO) polymerizes and activates in the acidic vacuoles of antigen presenting cells.3 We hypothesize that LLO conjugation to the anti-CD47 mAb (anti-CD47) enhances the release of tumor DNA within macrophages thereby augmenting cGAS-STING activation, antigen presentation, and tumor phagocytosis.
Methods LLO-CD47 is a protein-antibody conjugate that joins anti-CD47 to LLO using a water-soluble SPDP crosslinker and purified by affinity chromatography. The cytotoxicity of LLO-CD47 relative to anti-CD47 was tested in the mouse breast cancer cell line E0771, the human breast cancer cell line MDA-MB-468, the mouse lung cancer cell line LLC, and the human leukemia monocytic cell line THP-1. C57B6 mouse bone marrow-derived macrophages (BMDM) co-cultured with tumor cell lines were analyzed in the presence of LLO-CD47 and anti-CD47 to determine the efficiency of BMDM tumor cell phagocytosis, STING activation, and antigen presentation. Tumor growth of E0771-bearing wildtype mice was measured following intratumoral injection of anti-CD47 and LLO-CD47.
Results LLO-CD47 enhanced BMDM phagocytosis of LLC and E0771 tumor cells in a dose dependent manner that was significantly higher than cells treated with anti-CD47 alone. LLO-CD47 significantly increased BMDM levels of phosphorylated STING, interferon-stimulated genes, and antigen presentation relative to anti-CD47 treatment. At doses compatible with preserved animal total body weight, red blood cell counts, and lymphocyte counts, intratumoral injection of LLO-CD47 significantly suppressed tumor growth of E0771-bearing wildtype mice relative to anti-CD47. The elimination of CD8+ T cells or tumor associated macrophages in E0771-bearing animals significantly abrogated the antitumor effect of LLO-CD47.
Conclusions Conjugation of the LLO protein to anti-CD47 enhances macrophage cGAS-STING activation, antigen presentation, and tumor cell phagocytosis. This novel LLO-CD47 protein-antibody conjugate builds on clinical interest in targeting macrophages for the treatment of malignancy and may be studied as a supplemental therapy for patients with tumors resistant or refractory to checkpoint therapy.
Ethics Approval Studies involving animals were submitted to, reviewed, and approved by MD Anderson’s Institutional Animal Care and Use Committee in accordance with all applicable animal welfare regulations; protocol number 00002163-RN00.
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Podobnik M, Marchioretto M, Zanetti M, Bavdek A, Kisovec M, Cajnko MM, Lunelli L, Dalla Serra M, Anderluh G. Plasticity of listeriolysin O pores and its regulation by pH and unique histidine [corrected]. Sci Rep. 2015 Apr 8;5:9623. doi: 10.1038/srep09623. Erratum in: Sci Rep. 2015;5:15690.
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