Antagonist of CD39 and CD73 potentiate Doxycycline repositioning to induce potent antitumor immune response

Abstract

Purpose Studies have reported that cellular metabolism at tumor-immune microenvironment (TiME) serve as a critical checkpoint and perturbs/supports anti-cancer immunity. Extracellular ATP (eATP) may mediate anti-cancer immune response however; its catabolism by ectonucleotidase generates immunosuppressive adenosine. Antagonist of ectonucleotidases: CD39 and CD73 have been explored as potential therapeutic. In the presented work we have tried to repurpose doxycycline for mitigating ATP metabolism with or without antagonist of ectonucleotidase. Methods eATP and adenosine level were quantified. The bone marrow-derived M1 and M2 polarized macrophages were maintained in tumor mimicking condition (TMC). Total or CD4+ Tcells were co-cultured with macrophages to understand the impact of doxycycline and antagonist of ectonucleotidase T cell/subset differentiation. Preclinical efficacy of doxycycline and ectonucleotidase antagonist and their synergy was scored in 4T1 breast carcinoma. Results Doxycycline manipulates macrophage polarization by decreasing the frequency CD206+M2 macrophages that promoted CD4+ directed CD8+ T cell mediated tumor cell lysis. Doxycycline alleviated the expression of CD39 and CD73, rescuing ATP catabolism. Doxycycline delayed tumor growth by enhancing F4/80+ CD86+ M1 macrophages and subsequently anti-tumor Tbet+ CD4+ T-cells, attenuating the frequency of FOXP3+ regulatory T cells which was cooperatively supported by ARL67156 and AMPCP (CD39 and CD73 antagonist). Doxycycline promoted CD8+T cell mediated cytotoxicity which was synergistically enhanced with ARL67156 and AMPCP ensuring a possibility of using doxycycline alone or in combination with antagonist of ectonucleotidase. Conclusion Presented data indicate a prospective usage of doxycycline as novel immune checkpoint blocker (ICB) against ectonucleotidase and may be modified/delivered appropriately as a sole ICB.