321 PD-L1 blockade synergizes with c-Jun overexpression to boost CAR-T cell activity and mediate dramatic tumor control in an aggressive autochthonous model of lung adenocarcinoma

Background CAR-T cell therapy has been remarkably successful for blood cancers, but efficacy in solid tumors has been limited.1 2 In a phase-1 trial, CAR-T cells targeting the tumor-associated antigen ROR1 induced complete responses in 2 of 3 patients with chronic lymphocytic leukemia, but rapidly lost function in patients with non-small cell lung cancer (NSCLC),3 indicating strategies to preserve CAR-T function are needed to extend efficacy to solid tumors. T cell ‘exhaustion’ is a dysfunctional state resulting from chronic antigen stimulation and an imbalance in AP-1 transcription factors.4–6 Previous studies showed that overexpression of the AP-1 subunit c-Jun restored AP-1 balance and preserved CAR-T function in xenograft models.5 However, xenograft models do not mimic the suppressive tumor microenvironment (TME) of human cancers, and whether c-Jun overexpression is sufficient to preserve CAR-T function in a clinically relevant TME is unknown.7 Methods To develop a clinically relevant model of CAR-T therapy, we adapted the Kras/p53 autochthonous model of NSCLC to express ROR1 (KPROR1). This model mimics the suppressive TME of human NSCLC and recapitulates the dysfunction of ROR1 CAR-Ts observed in patients.3 7 8 We treated tumor-bearing KPROR1 mice with cyclophosphamide to induce lymphodepletion, infused 6x106 ROR1 CAR-T cells overexpressing c-Jun or not (1:1 CD8+:CD4+), and analyzed CAR-T persistence and function over time. Results CAR-T cells overexpressing c-Jun showed improved accumulation, function, and significantly extended survival of KPROR1 mice. However, c-Jun CAR-Ts ultimately underwent exhaustion with delayed kinetics, and all mice eventually succumbed to disease (figure 1). c-Jun CAR-Ts maintained c-Jun overexpression but showed decreased transcription of AP-1 target genes (figure 1 D-E), suggesting c-Jun activity may be impaired in vivo. PD-1 signaling can inhibit c-Jun phosphorylation, which is required for transcriptional activity.9–14 Indeed, treatment with c-Jun CAR-Ts induced greater PD-L1 up-regulation on tumor and myeloid cells, which co-localized with PD-1+ CAR-Ts (figure 2). Administering anti-PD-L1 for 14d following CAR-T treatment had no effect on WT CAR-Ts but dramatically increased tumor-infiltrating c-Jun CAR-Ts by ~100-fold. Additionally, cJun CAR-Ts and anti-PD-L1 induced a synergistic increase in endogenous CD8+PD-1+ T cells, suggestive of epitope spreading (figure 2D-E). Combination treatment with c-Jun CAR-Ts and anti-PD-L1 induced nearly complete eradication of ROR1+ tumor cells, with total tumor burden significantly reduced (figure 3). Conclusions CAR-T cells overexpressing c-Jun show progressive loss of AP-1 activity and function in the TME. Combination of c-Jun CAR-Ts with anti-PD-L1 boosts accumulation of both c-Jun CAR-Ts and endogenous T cells to promote tumor control, suggesting c-Jun overexpression may sensitize CAR-Ts to enhancement with checkpoint blockade. References Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014, (in English), CA: a cancer journal for clinicians, vol. 64, no. 1, pp. 9 - 29, 2014, doi: 10.3322/caac.21208. Srivastava S, Riddell SR. Chimeric Antigen Receptor T Cell Therapy: Challenges to Bench-to-Bedside Efficacy, (in English), Journal of immunology (Baltimore, Md. : 1950), vol. 200, no. 2, pp. 459 - 468, 2018, doi: 10.4049/jimmunol.1701155. Srivastava S, et al. 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