(J-379) Therapeutic macrophage-checkpoint silencing using siRNA-lipid nanoparticles in metastatic ovarian cancer

Friday, October 13, 2023

3:30 PM – 4:30 PM PDT

Location: Exhibit Hall - Row J - Poster # 379

Presenting Author(s)

TR

Theresa M. Raimondo, PhD

Postdoctoral Associate
Massachusetts Institute of Technology, United States

Co-Author(s)

DS

Dennis Shi

Graduate student
Massachusetts Institute of Technology, United States
ST

Sho Toyonaga

Visiting Sicentist
Massachusetts Institute of Technology, United States

Primary Investigator(s)

DA

Daniel Anderson

Professor
Massachusetts Institute of Technology, United States

Introduction:: Inhibiting checkpoints of adaptive immune cell activation (especially T cells) has proven clinical therapeutic efficacy, however only a minority of cancer patients respond. Myeloid cells, especially monocytes and macrophages, are the predominant immune cell in many solid tumors and are potent immunosuppressors. Here, we hypothesized that silencing a macrophage-checkpoint would promote their activation, enhance cancer phagocytosis and antigen presentation, to ultimately promote a therapeutic antitumor T cell response.

Materials and Methods::

Short interfering RNA (siRNA) was designed in silico to silence a macrophage phagocytosis-checkpoint, and siRNAs with various sequences and chemical modification patterns were screened in vitro to assess silencing efficiency, at the level of transcription, using qPCR. Lipid nanoparticles (LNPs) were formulated with a novel, biodegradable, ionizable lipid, as well as standard commercially available lipids, and LNP formulations screened to optimize macrophage transfection in vitro and in vivo. Protein-level silencing of the phagocytosis-checkpoint, as well as cell phenotype, was quantified using flow cytometry.

Results, Conclusions, and Discussions:: Optimized siRNA-LNPs allowed high levels of silencing (~90%) in peritoneal macrophages following low-dose (0.01 mg/kg), intraperitoneal, administration. The novel siRNA-LNPs demonstrated macrophage-tropism in vivo, with uptake mediated by clathrin-mediated endocytosis and macropinocytosis, independent of ApoE and LDLR. siRNA-LNP driven silencing shifted murine and primary human macrophages towards an immunostimulatory phenotype, in vivo and ex vivo, respectively; and, compared to antibody-based checkpoint-blocking, minimized counterproductive mutual phagocytosis (macrophage-macrophage attack often triggered by antibody-opsonization). siRNA-driven macrophage-silencing was synergistic with cancer-opsonizing antibody therapy, the combination yielding ~70% cancer phagocytosis vs ≤10% phagocytosis by cells lacking either opsonizing-treatment or siRNA-silencing, ex vivo. Following macrophage coculture with ovalbumin-expressing melanoma cells, siRNA-LNP lead to a 2-fold increase in MHCI cross-presentation of the ovalbumin antigen. Ultimately, in a murine model of peritoneal metastasized ovarian cancer, intraperitoneal administration of siRNA-LNP + a cancer opsonizing-antibody significantly prolonged survival vs treatment with control siRNA-LNP + antibody. The combination increased both the recruitment and activation of cytotoxic T lymphocytes. Administration of siRNA-LNP with standard chemotherapy (3-rounds), significantly slowed tumor growth following treatment cessation. The slowed growth was concomitant with increases in effector memory CD4 and CD8 T cells in the tumor microenvironment. These data demonstrate that siRNA-LNP elicits potent checkpoint silencing in macrophages, and that such checkpoint silencing ultimately drives the development of antitumor adaptive immunity (T cells). Together this data indicate that RNA-LNPs, and innate immune checkpoint inhibition, are promising platforms for cancer immunotherapy.

Acknowledgements (Optional): :

References (Optional): :