Definitive activation of endogenous antitumor immunity by repetitive cycles of cyclophosphamide with interspersed Toll-like receptor agonists.

Soraya Zorro Manrique,James J Lee,Peter A Cohen,Sandra J Gendler,Noweeda Mirza,Ana L Dominguez,James H Finke,Judy M Bradley,Larry R Pease,Christopher D Spencer

doi:10.18632/oncotarget.10190

Abstract

Many cancers both evoke and subvert endogenous anti-tumor immunity. However, immunosuppression can be therapeutically reversed in subsets of cancer patients by treatments such as checkpoint inhibitors or Toll-like receptor agonists (TLRa). Moreover, chemotherapy can leukodeplete immunosuppressive host elements, including myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs). We hypothesized that chemotherapy-induced leukodepletion could be immunopotentiated by co-administering TLRa to emulate a life-threatening infection. Combining CpG (ODN 1826) or CpG+poly(I:C) with cyclophosphamide (CY) resulted in uniquely well-tolerated therapeutic synergy, permanently eradicating advanced mouse tumors including 4T1 (breast), Panc02 (pancreas) and CT26 (colorectal). Definitive treatment required endogenous CD8+ and CD4+ IFNγ-producing T-cells. Tumor-specific IFNγ-producing T-cells persisted during CY-induced leukopenia, whereas Tregs were progressively eliminated, especially intratumorally. Spleen-associated MDSCs were cyclically depleted by CY+TLRa treatment, with residual monocytic MDSCs requiring only continued exposure to CpG or CpG+IFNγ to effectively attack malignant cells while sparing non-transformed cells. Such tumor destruction occurred despite upregulated tumor expression of Programmed Death Ligand-1, but could be blocked by clodronate-loaded liposomes to deplete phagocytic cells or by nitric oxide synthase inhibitors. CY+TLRa also induced tumoricidal myeloid cells in naive mice, indicating that CY+TLRa's immunomodulatory impacts occurred in the complete absence of tumor-bearing, and that tumor-induced MDSCs were not an essential source of tumoricidal myeloid precursors. Repetitive CY+TLRa can therefore modulate endogenous immunity to eradicate advanced tumors without vaccinations or adoptive T-cell therapy. Human blood monocytes could be rendered similarly tumoricidal during in vitro activation with TLRa+IFNγ, underscoring the potential therapeutic relevance of these mouse tumor studies to cancer patients.

Highlights

The treatment of cancer is currently being revolutionized by interventions which prove that the immune system can control early malignancies, and advanced cancers
The three models we used for initial therapeutic screening contained a strong presence of regulatory T-cells (Tregs) [45,46,47], but ranged widely in myeloid-derived suppressor cells (MDSCs) (CD11b+Gr1+) content, from scant (Panc02) to moderate (CT26) to overwhelming (4T1) [25, 48,49,50]. 4T1 displayed the most aggressive malignant behavior, abruptly metastasizing to multiple organs, and resulting in a tumor burden which has historically proved challenging to cure in wild type (WT) syngeneic mice [50,51,52] (Supplemental Figure S1)
Because activation of macrophage antitumor activity can be associated with inducible nitric oxide synthase [61], we investigated whether Gr1dim myeloid cells from CY+CpG ODN 1826 (CpG)-treated mice employed nitric oxide (NO) to control tumor growth

Summary

Introduction

The treatment of cancer is currently being revolutionized by interventions which prove that the immune system can control early malignancies, and advanced cancers. Successful immunotherapy can range in complexity from simultaneous administration of immune cells, cytokines and chemotherapy [1,2,3,4,5] to single agents, most notably monoclonal antibodies which block signaling of the Programmed death-1 (PD-1) receptor or its ligand, Programmed death-ligand 1 (PD-L1) [6,7,8] Such PD-1 blockade effectively reverses inhibition of the natural effector T-cell response in up to nearly 60% of patients with melanoma as well as subsets of patients with many other cancers, resulting in major and often sustained therapeutic responses [6,7,8]. Sustained effector activation is often or usually subverted by the strongly immunosuppressive environment typically generated by cancer throughout the body, recruiting regulatory T-cells (Tregs) and inducing myeloid-derived suppressor cells (MDSCs) as mediators of tumor escape [18,19,20,21,22,23,24,25,26]

Methods

Results

Conclusion