T-Regulatory Cells Impair Car T Cell-Mediated Antitumor Activity In A Murine Solid Tumor Model.

Abstract Introduction: Chimeric antigen receptor (CAR) T cell immunotherapy has had only modest success in solid malignancies due in part to tumor antigen heterogeneity and the immunosuppressive tumor microenvironment. We have previously demonstrated that administration of cyclophosphamide (CTX) prior to CAR T infusion promoted the eradication of solid tumors that lacked homogenous expression of the CAR target antigen. Although CTX has multiple effects, we hypothesized that one potential mechanism was depletion of T-regulatory cells (Treg). To test this, we used a genetic model of Treg depletion involving mice with FOXP3-driven diphtheria toxin receptor expression (FOXP3-DTR) and an orally administered chemokine receptor 4 (CCR4) antagonist called CCR4-351. Treg express CCR4 and migrate toward CCL17 and CCL22 which are frequently upregulated by tumors. We have previously shown that the tool compound CCR4-351 decreases Treg accumulation in tumors. Methods: Transgenic FOXP3-DTR C57BL/6 mice were injected s.c. in the flank with a syngeneic murine mesothelioma cell line expressing human mesothelin. When tumors reached ~85 mm3, diphtheria toxin was given i.p. and 24 hours later, 2 doses of 107 transduced human anti-mesothelin CAR T cells (M11) were injected i.v. 2 days apart. In our pharmacologic study, wild type, tumor-bearing C57BL/6 mice were given CCR4-351 daily at 50 mg/kg by oral gavage. Mice were treated with M11 CAR T cells as above, starting one day after CCR4-351 therapy. Tumor growth and mouse survival were determined. Results: Genetic depletion of Treg alone did not significantly slow tumor growth compared to untreated control. However, the combination of M11 CAR T cells plus genetic depletion of Treg significantly slowed tumor growth compared to vehicle control (p<0.05, one-way ANOVA). Compared to untreated controls, treatment with CCR4-351 (p<0.04, log rank) or M11 CAR T cells (p<0.001, log rank) alone significantly improved survival, however there was no significant difference in survival between CAR and CCR4-351 only treated mice. In contrast, the combination of M11 CAR T cells with CCR4-351 resulted in enhanced antitumor activity with 4/9 mice cured and significantly increased survival compared to CAR (p<0.01, log rank) and CCR4-351 (p<0.006, log rank) monotherapies. Conclusions: Treg depletion via systemic ablation or inhibition of trafficking, resulted in augmentation of the antitumor effects of CAR T cells in our pre-clinical immunocompetent solid tumor model. If generalizable, these data support further investigation as to whether the orally bioavailable CCR4 antagonist currently in clinical development, FLX475, may be used to augment human CAR T cell therapy for solid tumors. Citation Format: Michael S. Leibowitz, Nicholas S. Olimpo, Liqing Wang, Aparna Jorapur, Deepa Pookot, Maria Liousia, Evguenia Arguiri, Jing Sun, Astero Klampatsa, Dirk G. Brockstedt, Wayne W. Hancock, Steven M. Albelda. T-regulatory cells impair CAR T cell-mediated antitumor activity in a murine solid tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1585.