Background Metastatic colorectal cancer (mCRC) expresses high levels of the adenosine-generating enzyme CD73 and represents a significant unmet medical need, with an ~14% five-year survival rate. Adenosine-mediated signaling via A2a and A2b receptors impairs activation, proliferation, and cytotoxic activity of effector T cells, resulting in reduced anti-tumor immunity. Etrumadenant (etruma; AB928) is an orally bioavailable, selective A2a and A2b receptor antagonist capable of reversing adenosine-mediated immunosuppression and enhancing the clinical activity of chemo/immunotherapy. Etruma, in combination with zimberelimab (Z) (anti-PD-1 antibody), FOLFOX, and bevacizumab (bev) (EZFB regimen) demonstrated an impressive overall survival (OS) benefit compared to the regorafenib (rego) control arm in third-line, chemo-resistant mCRC in the primary analysis of the ARC-9 study (NCT04660812).

Methods We investigated the mechanism of action (MoA) of etruma in mCRC patients from ARC-9 by assessing the transcriptomic profiles of 10 tumor biopsies taken on treatment compared to matched baseline tumor samples. We also examined the protein levels of the adenosine-generating enzyme CD73 in 92 baseline tumor tissues from ARC-9 by immunohistochemistry (IHC).

Results Treatment with EZFB led to a significant reduction in the expression levels of the adenosine-regulated NR4A gene family (NR4A1, NR4A2, and NR4A3). Decrease in tumor NR4A gene expression after treatment with EZFB was accompanied by a significant increase in T-cell activation gene expression signature, consistent with etruma-mediated reversal of adenosine immunosuppression in the tumor. Of the 92 tumor tissues assessed by IHC, 68 cases (~74%) expressed CD73. Patients with CD73-positive tumors (≥1% tumor cell (TC) staining) experienced significantly longer progression free survival (PFS) and overall survival (OS) on EZFB compared to CD73 IHC-positive cases from the rego arm (PFS HR = 0.34; CI 0.19, 0.61; OS HR = 0.20; CI 0.07, 0.75), suggesting a link between clinical benefit on EZFB and the adenosine-producing enzyme CD73.

Conclusions We demonstrate that treatment of mCRC patients with EZFB is associated with a reduction in tumor expression levels of the adenosine-regulated NR4A gene family and an increase in tumor inflammation, presumably reflecting a reversal of tumor immunosuppression. Consistent with an adenosine-targeting MoA, EZFB provided significantly improved PFS and OS compared to rego for patients with CD73 IHC-positive tumors.

Trial Registration ARC-9 study (NCT04660812).

Ethics Approval All patients were provided the most recent copy of the Informed Consent form describing the study and associated interventions, which the patient or their legally authorized representative were required to sign and date prior to participating in the study.