Adenosine receptor antagonists A2AR (TT-10) and A2BR (TT-4) demonstrate anti-tumor activity in 4T1-induced syngeneic breast cancer mouse model

Abstract Background: Extracellular adenosine has been shown to negatively regulate anti-tumor immunity. Adenosine is produced at high levels in the TME as a result of hypoxia-driven tumor cell death. As a result, ATP is released from dying cells generating adenosine via enzymatic dephosphorylation, mainly by ectonucleotidases CD39 & CD73. This results in high extracellular adenosine levels, being elevated 100-500x in the TME. The free adenosine proceeds to binding one of its four known receptors A1, A2a, A2b & A3. The binding of adenosine to A2AR & A2BR contributes to immunosuppression via cyclic AMP pathway. It has been observed that bound A2AR suppress T cell function (CD8+T & NK) while A2BR dampens myeloid cell function and promotes chemotaxis. These concerted actions thereby enhance tumor growth and metastasis. Blocking the binding of adenosine to the A2AR & A2BR results in T-cell activation and infiltration of immune cells in the TME. TT-10 & TT-4 are potent and selective antagonists of A2AR & A2BR. Both agents are being developed for the treatment of advanced cancers individually and as dual inhibitors. Methods: Balb/c mice were implanted with 5 x 104 4T1 cells and randomly assigned to 6 groups per study; (1) Vehicle Control-VC, (2) TT-10, 1 mg/kg, PO, BID (3) TT-4, 3 mg/kg, PO, BID (4) Anti-mPD-1, 200 ug, IP (D4, 8, 14) (5) 1 mg/kg TT-10 + 200 ug Anti-mPD-1 (D4, 8, 14) and (6) TT-4, 3 mg/kg, PO, BID + 200 ug Anti-mPD-1 (D4, 8, 14). Mice were dosed for 21 days, and tumor volume was measured 2x a week. After 21 days post initiation of treatment (31 days post 4T1 cell inoculation), mice were sacrificed, TIL populations were isolated from 4T1 induced tumors and interrogated via Flow. Markers for surface staining included CD45, CD3, CD4, CD8, CD11b, CD49, Ly6G. Results: All implanted mice developed measurable tumors. Mean suppression of tumor growth was observed to be greater in single agent TT-10 (p<0.05) & TT-4 (p<0.05), over single agent anti-PD-1 inhibitor when compared to the control on day 31, post 4T1 cell inoculation. Analysis of the TIL at the end of the study further supported the observation of tumor growth suppression, from increased T-cell activity seen for both TT-10 & TT-4 when compared to the VC. Furthermore, striking reductions in MDSC populations were also observed in both TT-10 & TT-4 treated mice, when compared to single agent anti-PD-1 and the VC. Conclusions: TT-10 & TT-4 alone was superior (p<0.05) to the VC as well as single agent anti-PD-1 in slowing tumor growth, which was further supported by the increase in T-cell activity observed in the end of study TIL analysis. Lastly, striking reductions in MDSC populations were also observed in both TT-10 & TT-4 treated mice, when compared to single agent anti-PD-1 and the VC. Citation Format: Desa Rae E. Pastore, Kasim Mookhtiar, Brian Schwartz, Sushant Kumar, Ranganayaki Nagaraj, Ashwinkumar V. Meru. Adenosine receptor antagonists A2AR (TT-10) and A2BR (TT-4) demonstrate anti-tumor activity in 4T1-induced syngeneic breast cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3454.