Abstract 5118: STING agonist and TLR7/8 agonist overcome anti-PD-1 resistance mediated by loss of β2M
Authors: Hua Li , Nie Chenpan , Lin Jie , An Annie Xiaoyu , Bourre Ludovic , Wang Jingjing 
Published: April 2023

Introduction: PD-1 blockade has achieved great success in the clinic. However, only a small subset of patients benefits from the treatment and most responders develop acquired resistance thereafter. One of the resistance mechanisms is beta-2-microglobulin (β2M) mutation mediated dysfunction of MHC-I antigen presentation, which contributes to attenuation of cellular cytotoxicity triggered by adaptive CD8+ T cells. Activating innate immunity, such as natural killer (NK) cells, can be used as a strategy to circumvent the attenuation. Immune-stimulating agents like Stimulator of interferon genes (STING) agonists and Toll-like receptor (TLR) 7/8 agonists can elicit NK cell-mediated tumor rejection in preclinical tumor models, independent of CD8+ T cells. Thus, to evaluate novel therapeutic strategies overcoming anti-PD-1 resistance, we established a MC38-OVA-β2M KO tumor model.

Methods: We knocked out β2M genes in a murine MC38-OVA colorectal cancer cell line using CRISPR/Cas9 to generate MC38-OVA-β2M KO cells. β2M knockout was verified by western blot and flow cytometry. For in vitro testing, both MC38-OVA and MC38-OVA-β2M KO cells were treated with IFN-α, IFN-β and IFN-γ for 24h, and then MHC-I and OVA peptide SIINFEKL expression were analyzed by flow cytometry. For in vivo testing, 1 × 106 MC38-OVA cells and MC38-OVA-β2M KO cells were subcutaneously injected into C57BL/6 mice and the models were treated with anti-PD-1. Finally, the MC38-OVA-β2M KO tumor model was treated with anti-PD-1, cyclic nucleotides (CDN), R-848 and anti-PD-1 combined with either CDN or R-848. At the endpoint, the composition of immune cells in spleens were analyzed by flow cytometry.

Results: Following confirmation of β2M knockout, MHC-I expression and MHC-I-OVA peptide presentation were also diminished on MC38-OVA-β2M KO cells, even after treatment of IFN- α, IFN-β, or IFN-γ. In vivo, anti-tumor efficacy of PD-1 blockade was significantly diminished in MC38-OVA-β2M KO model (TGI=31%, p&lt;0.001), compared to MC38-OVA model (TGI=91%). To overcome the resistance, CDN, a STING agonist or R-848, a TLR7/8 agonist was administered alone or combined with anti-PD-1 in the MC38-OVA-β2M KO model. Single treatment of either CDN (TGI=73%) or R-848 (TGI=89%) markedly impeded tumor growth, and combination with anti-PD1 greatly enhanced their anti-tumor effects (TGI of 98% and 99%, respectively). Additionally, flow cytometry analysis showed a decrease of Myeloid-derived suppressor cells (MDSCs) and an increase of NK cells in spleens of the mice treated with CDN and anti-PD-1. In parallel, a decrease of MDSCs was also observed in the mice with combined treatment of R-848 and anti-PD-1.

Conclusion: The successfully established MC38-OVA-β2M KO model is a useful tool for investigating therapeutic strategies to overcome MHC-I down-regulation mediated immunotherapy resistance. Moreover, STING agonists and TLR7/8 agonists may be potential candidates.

Citation Format: Li Hua, Chenpan Nie, Jie Lin, Annie Xiaoyu An, Ludovic Bourre, Jingjing Wang. STING agonist and TLR7/8 agonist overcome anti-PD-1 resistance mediated by loss of β2M. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5118.