Inhibition of the adenosine A2a receptor modulates expression of T cell coinhibitory receptors and improves effector function for enhanced checkpoint blockade and ACT in murine cancer models
- 1k Downloads
Adenosine signaling via the A2a receptor (A2aR) is emerging as an important checkpoint of immune responses. The presence of adenosine in the inflammatory milieu or generated by the CD39/CD73 axis on tissues or T regulatory cells serves to regulate immune responses. By nature of the specialized metabolism of cancer cells, adenosine levels are increased in the tumor microenvironment and contribute to tumor immune evasion. To this end, small molecule inhibitors of the A2aR are being pursued clinically to enhance immunotherapy. Herein, we demonstrate the ability of the novel A2aR antagonist, CPI-444, to dramatically enhance immunologic responses in models of checkpoint therapy and ACT in cancer. Furthermore, we demonstrate that A2aR blockade with CPI-444 decreases expression of multiple checkpoint pathways, including PD-1 and LAG-3, on both CD8+ effector T cells (Teff) and FoxP3+ CD4+ regulatory T cells (Tregs). Interestingly, our studies demonstrate that A2aR blockade likely has its most profound effects during Teff cell activation, significantly decreasing PD-1 and LAG-3 expression at the draining lymph nodes of tumor bearing mice. In contrast to previous reports using A2aR knockout models, pharmacologic blockade with CPI-444 did not impede CD8 T cell persistence or memory recall. Overall these findings not only redefine our understanding of the mechanisms by which adenosine inhibits immunity but also have important implications for the design of novel immunotherapy regimens.
KeywordsImmunotherapy Immune checkpoint A2a PD-1 Lag-3 Treg
Adenosine A2a receptor
American type culture collection
OVA-expressing B16 murine melanoma
Tumor-draining lymph nodes
Inducible nitric oxide synthase
OVA-expressing Listeria monocytogenes
Non-draining lymph nodes
CD8+ effector T cell
OVA class-I tetramer+
We thank members of the Powell lab, especially Chirag Patel, for critical discussion of the manuscript; Corvus pharmaceuticals for their generous gift of CPI-444; and Aduro Biotech for their generous gift of LM-OVA.
RDL designed and conducted the experiments and wrote the manuscript. I-MS, M-HO, I-HS, JW, and JE helped with the experiments. JDP designed the experiments and revised the manuscript.
This work was supported in part by funds from the Bloomberg~Kimmel Institute for Cancer Immunotherapy. In addition, CPI-444 and unrestricted research funds were provided by Corvus.
Compliance with ethical standards
Conflict of interest
Jonathan D. Powell has been a paid consultant for Corvus and has equity in the company. All other authors declare that they have no conflicts of interest.
Ethical approval and ethical standards
All applicable international and national guidelines for the care of animals were followed. All mouse procedures approved by Johns Hopkins University Institutional Animal Care and Use Committee (Protocol #M016M103, approved 4/1/2016).
C57BL/6 obtained from Charles River Laboratories (MC38 experiments) or Jackson Laboratories (ACT; 000664). OT-I and CD90.1, BALB/c mice obtained from The Jackson Laboratory. Male or female mice were used for each experiment; mice were sex and age matched accordingly.
Cell line authentication
MC38 cells were donated by CORVUS pharmaceuticals. The identity and specific pathogen free status of these cells was validated by microsatellite genotype analysis (IDEXX Bioresearch). B16-OVA melanoma cells were a gift from Hyam Levitsky. All other tumor cell lines used were obtained from the ATCC. All cell lines were mycoplasma free via ELISA-based assays performed every 6 months.
- 20.Emens L, Powderly J, Fong L, Brody J, Forde P, Hellmann M, Hughes B, Kummar S, Loi S, Luke J, Mahadevan D, Markman B, McCaffery I, Miller R, Laport G (2017) CPI-444, an oral adenosine A2a receptor (A2aR) antagonist, demonstrates clinical activity in patients with advanced solid tumors. AACR Annual Meeting 2017. Cancer Res 77:Abstract CT119CrossRefGoogle Scholar
- 21.Willingham S, Ho P, Leone R, Piccione E, Choy C, Hotson A, Buggy J, Powell J, Miller R (2016) The adenosine A2A receptor antagonist CPI-444 blocks adenosine-mediated T-cell suppression and exhibits antitumor activity alone and in combination with anti-PD-1 and anti-PD-L1. AACR Annual Meeting 2017. Cancer Res 76:Abstract 2337CrossRefGoogle Scholar
- 33.Koyama S, Akbay EA, Li YY, Herter-Sprie G, Buczkowski KA, Richards WG, Gandhi L, Redig AJ, Rodig SJ, Asahina H, Jones RE, Kulkarni MM, Kuraguchi M, Palakurthi S, Fecci PE, Johnson BE, Janne PA, Engelman JA, Gangadharan SP, Costa DB, Freeman GJ, Bueno R, Hodi FS, Dranoff G, Wong K, Hammerman PS (2016) Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints. Nat Commun 7:10501CrossRefPubMedPubMedCentralGoogle Scholar
- 34.Kamphorst AO, Wieland A, Nasti T, Yang S, Zhang R, Barber DL, Konieczny BT, Daugherty CZ, Koenig L, Yu K, Sica GL, Sharpe AH, Freeman GJ, Blazar BR, Turka LA, Owonikoko TK, Pillai RN, Ramalingam SS, Araki K, Ahmed R (2017) Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent. Science 355:1423–1427CrossRefPubMedPubMedCentralGoogle Scholar
- 44.Ramakers BPC, Riksen NP, Broek PHH, Franke B, Peters WHM, Hoeven JG, Smits P, Pickkers P (2011) Circulating adenosine increases during human experimental endotoxemia but blockade of its receptor does not influence the immune response and subsequent organ injury. Crit Care 15:R3CrossRefPubMedPubMedCentralGoogle Scholar
- 45.Angelin A, Gil-de-Gómez L, Dahiya S, Jiao J, Guo L, Levine MH, Wang Z, Quinn WJ, Kopinski PK, Wang L, Akimova T, Liu Y, Bhatti TR, Han R, Laskin BL, Baur JA, Blair IA, Wallace DC, Hancock WW, Beier UH (2017) Foxp3 reprograms T cell metabolism to function in low-glucose, high-lactate environments. Cell Metab 25:1282–1293.e7CrossRefPubMedPubMedCentralGoogle Scholar
- 47.Hatfield SM, Kjaergaard J, Lukashev D, Schreiber TH, Belikoff B, Abbott R, Sethumadhavan S, Philbrook P, Ko K, Cannici R, Thayer M, Rodig S, Kutok JL, Jackson EK, Karger B, Podack ER, Ohta A, Sitkovsky MV (2015) Immunological mechanisms of the antitumor effects of supplemental oxygenation. Sci Transl Med 7:277ra30CrossRefPubMedPubMedCentralGoogle Scholar
- 49.Beavis PA, Henderson MA, Giuffrida L, Mills JK, Sek K, Cross RS, Davenport AJ, John LB, Mardiana S, Slaney CY, Johnstone RW, Trapani JA, Stagg J, Loi S, Kats L, Gyorki D, Kershaw MH, Darcy PK (2017) Targeting the adenosine 2A receptor enhances chimeric antigen receptor T cell efficacy. J Clin Invest 127:929–941CrossRefPubMedPubMedCentralGoogle Scholar