Antisense oligonucleotide targeting CD39 improves anti-tumor T cell immunity

Abhishek S Kashyap,Tamara Thelemann,Frank Jaschinski,Alfred Zippelius,Monika Schell,Lisa Hinterwimmer,Julia Festag,Sven Michel,Sandra M Kallert,Richard Klar,Melanie Buchi

doi:10.1186/s40425-019-0545-9

Abstract

BackgroundCancer cells are known to develop mechanisms to circumvent effective anti-tumor immunity. The two ectonucleotidases CD39 and CD73 are promising drug targets, as they act in concert to convert extracellular immune-stimulating ATP to adenosine. CD39 is expressed by different immune cell populations as well as cancer cells of different tumor types and supports the tumor in escaping immune recognition and destruction. Thus, increasing extracellular ATP and simultaneously reducing adenosine concentrations in the tumor can lead to effective anti-tumor immunity.MethodsWe designed locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) with specificity for human or mouse CD39 that do not need a transfection reagent or delivery system for efficient target knockdown. Knockdown efficacy of ASOs on mRNA and protein level was investigated in cancer cell lines and in primary human T cells. The effect of CD39 knockdown on ATP-degrading activity was evaluated by measuring levels of ATP in tumor cell supernatants and analysis of T cell proliferation in the presence of extracellular ATP. The in vivo effects of CD39-specific ASOs on target expression, anti-tumor immune responses and on tumor growth were analyzed in syngeneic mouse tumor models using multi-color flow cytometry.ResultsCD39-specific ASOs suppressed expression of CD39 mRNA and protein in different murine and human cancer cell lines and in primary human T cells. Degradation of extracellular ATP was strongly reduced by CD39-specific ASOs. Strikingly, CD39 knockdown by ASOs was associated with improved CD8+ T cell proliferation. Treatment of tumor-bearing mice with CD39-specific ASOs led to dose-dependent reduction of CD39-protein expression in regulatory T cells (Tregs) and tumor-associated macrophages. Moreover, frequency of intratumoral Tregs was substantially reduced in CD39 ASO-treated mice. As a consequence, the ratio of CD8+ T cells to Tregs in tumors was improved, while PD-1 expression was induced in CD39 ASO-treated intratumoral CD8+ T cells. Consequently, CD39 ASO treatment demonstrated potent reduction in tumor growth in combination with anti-PD-1 treatment.ConclusionTargeting of CD39 by ASOs represents a promising state-of-the art therapeutic approach to improve immune responses against tumors.

Highlights

Cancer cells are known to develop mechanisms to circumvent effective anti-tumor immunity
CD39 protein expression is reduced in human CD4+ and CD8+ T cells after hCD39-specific Antisense oligonucleotide (ASO) treatment ASOs with specificity for hCD39 were initially screened for target mRNA suppression in HDLM-2 cells without the use of a transfection reagent, a human Hodgkin lymphoma cell line with high endogenous expression of CD39
We demonstrate that CD39-specific ASOs reinstate ATP supply through potent and sustained suppression of CD39 expression on T cells, which improves T cell proliferation

Summary

Introduction

Cancer cells are known to develop mechanisms to circumvent effective anti-tumor immunity. Dying cancer cells release ATP which may enhance anti-tumor immune responses e.g. through recruitment and activation of dendritic cells (DCs), macrophages and their precursors [7, 8]. Binding of ATP to P2X7 receptors on DCs results in DC activation and the release of pro-inflammatory cytokines such as IL-1β or IL-18 [9] These cytokines in turn activate NK cells, T cells, and macrophages and enhance their proliferation and cytolytic functions [5]. Taken together, targeting CD39 will decrease the degradation of ATP and result in increased levels of immune stimulatory extracellular ATP This will concurrently lead to the suppression of adenosine through the prevention of AMP generation, the substrate of CD73

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Results

Conclusion