Low-dose metronomic cyclophosphamide complements the actions of an intratumoral C-class CpG TLR9 agonist to potentiate innate immunity and drive potent T cell-mediated anti-tumor responses

Abstract

The synthetic oligonucleotide SD-101 is a potent and specific agonist for tolllike receptor 9. Intratumoral injection of SD-101 induces significant anti-tumor immunity in preclinical and clinical studies, especially when combined with PD-1 blockade. To build upon this strategy, we studied the enhancement of SD-101 activities by combination with low-dose cyclophosphamide, a well-characterized agent with potentially complementary activities. In multiple mouse tumor models, we demonstrate substantial anti-tumor activity of the combination, compared to each single agent. Combination therapy generated CD8+ T cell dependent immunity leading to rejection of both non-injected and injected tumors and long-term survival, even in very large tumors. Mechanistic studies encompassing global gene expression changes and characterization of immune cell infiltrates show the rapid, sequential induction of innate and adaptive responses and identify discrete contributions of SD-101 and cyclophosphamide. Importantly, these changes were prominent in tumors not injected directly with SD-101. Combination treatment resulted in creation of a permissive environment for a systemic anti-tumor immune response, including a reduction of intratumoral regulatory T cells (Tregs) and an increase in "M1" versus "M2" tumorassociated macrophage (TAM) phenotypes. Additionally, we observed increased immunogenic cell death as well as antigen processing in response to combination treatment.

CpG Adjuvant in Allergen-Specific Immunotherapy: Finding the Sweet Spot for the Induction of Immune Tolerance

“…These observations sparked the research on bacterial compounds such as CpG-ODN for cancer treatment ( 209 ). It has been shown that CpG-ODN potentiates both innate and adaptive anti-tumor immunity, mainly by enhancing NK cell and CD8 T cell cytotoxic activity ( 81 , 210 ). Among other successful applications in the cancer field ( Table 3 ), CpG-ODN was used as prophylactic treatment for reducing brain metastasis through microglia activation ( 211 ), or in combination with M362, a TLR6 ligand, with which it potentiates CD8 T cell response against breast cancer cells ( 212 ).…”

Immune System Alterations in Multiple Myeloma: Molecular Mechanisms and Therapeutic Strategies to Reverse Immunosuppression

“…The alkylating agent cyclophosphamide has been used in the treatment of MM for over 60 years and, at low doses, it also presents significant immunomodulatory activity [ 193 ]. In this sense, treatment of different tumor models with mafosfamide (a chemical compound related to cyclophosphamide) or cyclophosphamide induces CALR translocation [ 181 , 182 ] and the release of HMGB1 [ 181 ], both of them being surrogate ICD markers. Accordingly, cyclophosphamide-treated mice showed an increase in tumor infiltrating DCs with an activated phenotype [ 181 ].…”

“…Moreover, type I IFN, a cytokine known to stimulate DC activation and T-cell priming, has a synergistic antitumor effect with cyclophosphamide [ 181 ]. In addition to ICD induction, cyclophosphamide also depleted Tregs [ 182 , 183 ], promoted Th1 polarization [ 183 ], increased activated NK cells [ 182 ] and modulated the myeloid population [ 182 ] in different tumor models.…”

Modulating the Tumour Microenvironment by Intratumoural Injection of Pattern Recognition Receptor Agonists

“…Low-dose metronomic cyclophosphamide complements the actions of an intratumoural CpG-ODN to potentiate innate immunity and drive anti-tumour responses [ 31 ]. In this setting, the chemotherapy shaped the immune response, with a reduction in Tregs, and an increase in M1-type macrophages, a phenotype typically associated with antitumour effects through direct tumouricidal activity and release of factors such as IL-12, CXCL9, and CXCL10 that enhance T cell and NK cell function and migration to the tumour.…”

“…While PRR agonists have been investigated as systemic therapies, advancement to the clinic has been hampered by systemic toxicities [ 25 , 26 , 27 ]. However, studies suggest that an intratumoural route of administration can lead to high local concentrations of PRR agonists in the TME while limiting systemic exposure that can potentially lead to toxicity [ 6 , 28 ], even when used in a repetitive dosing regimen [ 29 , 30 , 31 ].…”