Abstract P4-06-19: The effect of relieving adenosine-mediated immunosuppression on trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (T-ADCC) against HER2+ breast cancer cell lines
Authors: Gaynor N , Noone J , Monedero J , Murphy EE , O'Gorman DJ , Crown J , Collins DM 
Published: May 2019

Introduction: Trastuzumab (T) is a monoclonal antibody therapy used in the treatment of HER2+ breast cancer. T inhibits HER2 intracellular signalling and is capable of engaging the immune system through ADCC. Adenosine is an important negative regulator of the immune response through its interaction with the A2A receptor (A2AR, ADORA2A). Relieving adenosine-mediated immunosuppression by inhibiting A2AR may improve NK cell-mediated T-ADCC against HER2+ breast cancer cells. In addition, we have previously shown that SKBR3 cells resistant to the EGFR/HER2 tyrosine kinase inhibitor (TKI) lapatinib are less sensitive to T-ADCC and showed increased A2AR protein levels. This study examines the effects of inhibiting A2AR signalling on NK cell-mediated T-ADCC against treatment naïve HER2+ breast cancer cell lines HCC1954 and SKBR3 and lapatinib and afatinib (irreversible pan-HER-family TKI)-resistant sublines of HCC1954 and SKBR3.

Methods: HER2+ breast cancer cell lines SKBR3 and HCC1954 were exposed to afatinib (150nM) or lapatinib (1μM) for 6 months to generate TKI-resistant SKBR3-A and HCC1954-L cell lines. Acid-phosphatase-based proliferation assays were used to confirm resistance to TKI treatment. Western blotting was used to examine A2AR and HER2 protein levels in cell lines. NK cells were isolated from healthy volunteer whole blood by MACSxpress isolation kits. Immune cell-mediated cytotoxicity was determined at a 1:1 (NK cell: TC) ratio over 12 hours using a flow cytometry-based method. Direct cytotoxicity and T-ADCC were determined +/- A2AR agonist CGS21680 (1 μM) and/or A2AR antagonist preladenant (100 nM) for all cell lines. Experiments were carried out three times with three separate volunteer samples with representative results presented.

Results: HCC1954-L cells were 5.3-fold resistance to lapatinib (IC50 1.65 μM +/- 0.22 μM) vs. HCC1954 (IC50 0.31 μM +/- 0.15 μM). SKBR3-A cells were 33-fold resistant to afatinib (IC50 0.28 μM +/- 0.006 nM) vs. the parental SKBR3 cell line (IC50 0.009 μM +/- 0.006 μM). SKBR3 and HCC1954 expressed detectable protein levels of A2AR. A2AR and HER2 levels were not significantly changed between parental and resistant cell lines. Levels of direct cytotoxicity and T-ADCC elicited by NK cells were higher against SKBR3-A (p=0.002) and HCC1954-L cells (p=0.0004) than parental cell lines. The A2AR agonist CGS21680 alone had inconsistent effects on direct cytotoxicity and T-ADCC in all cell lines tested. The addition of A2AR antagonist preladenant to CGS21680, but not preladenant alone, increased T-ADCC against the parental HCC1954 cells by 12.7 +/- 3.4% and parental SKBR3 cells by 9.5 +/- 3.6%. T-ADCC levels in the targeted therapy-resistant HCC1954-L and SKBR3-A cell lines were not impacted by the CGS21680/preladenant combination.

Conclusions: A HER2-targeted therapy resistance phenotype is associated with increased T-ADCC in the models tested. Inhibition of activated A2AR can increase T-ADCC elicited by NK cells against treatment naïve HER2+ breast cancer cell lines but not TKI-resistant sublines. Further work is warranted to examine the impact of targeting A2AR in HER2+ breast cancer.

Citation Format: Gaynor N, Noone J, Monedero J, Murphy EE, O'Gorman DJ, Crown J, Collins DM. The effect of relieving adenosine-mediated immunosuppression on trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (T-ADCC) against HER2+ breast cancer cell lines [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-19.