48P - The A2AR antagonist inupadenant promotes humoral responses in preclinical models

Background

A₂A receptor (A_2AR) is a key mediator of adenosine immunosuppression. Inupadenant is a highly selective A_2AR antagonist, which maintains full potency at the high levels of adenosine found in tumors. We recently reported that B cells are a novel cellular target of inupadenant (Martinoli et al., AACR 2023). Here, we further investigate how inupadenant modulates humoral responses in vitro, ex vivo and in vivo.

Methods

Inupadenant was assessed through: in vitro generation of antibody secreting cells (ASCs) from memory B cells isolated from human PBMCs; ex vivo tumor culture of tumor slices derived from human lung cancer; and single cell RNA sequencing (scRNAseq) on isolated healthy tonsil B cells. The A_2AR agonist CGS-21680 (in vitro and scRNAseq) or ATP (ex vivo) were added to trigger A_2AR signaling, and B cells were stimulated with CpG (scRNAseq) or CD40L (ex vivo). Cell cultures were assessed by flow cytometry and Legendplex. The CT26-OVA model was used to study tumor-targeted immunoglobulin (Ig) production and immune infiltration by ELISA and RNAseq.

Results

Inupadenant rescued the A_2AR-mediated inhibition of B cell maturation into ASCs and Ig production in both in vitro and ex vivo systems. In CT26-OVA mice, inupadenant increased OVA-specific IgGs in blood and tumor, as well as B cells and ASCs in spleen and tumor. Besides ASCs, germinal center (GC) B cells were the main B cell subset affected by A_2AR signaling in human tonsil, as supported by (i) the highest number of differentially expressed genes observed in these subsets after CGS-21680 stimulation, and (ii) by their highest expression of the A_2AR gene, ADORA2A. Inupadenant completely reverted the effect of CGS-21680 in ASCs and GC B cells, including the expression of AICDA, a critical gene for somatic hypermutation and class-switch recombination in GCs.

Conclusions

These data support the notion that inupadenant restores or even potentiates B cell maturation toward ASCs and GC reactions in both secondary lymphoid organs as well as tumor in the presence of A_2AR signaling. This process is essential to produce high affinity antibodies and potentially for sustained anti-tumor immunity. Additional work is ongoing to confirm this novel mechanism in inupadenant-treated patients.

Legal entity responsible for the study

iTeos Therapeutics.

Funding

iTeos Therapeutics.

Disclosure

P. Tieppo, M. Mercier, S. Jung, D. Carbonez, L. Chaible, R. Marillier, F. Strozzi, H. Shehade, Y. McGrath, M. Rossetti: Financial Interests, Personal, Full or part-time Employment: iTeos Therapeutics; Financial Interests, Personal, Stocks/Shares: iTeos Therapeutics. M. Dieu-Nosjean: Financial Interests, Personal, Speaker, Consultant, Advisor: iTeos Therapeutics.