Gut Microbiota Tuning Promotes Tumor-Associated Antigen Cross Presentation and Enhances CAR T Antitumor Effects

Background: Chimeric Antigen Receptor (CAR) T cell immunotherapy has revolutionized the treatment of B-cell malignancies. However, a significant subset of these patients either fails to respond or eventually relapses. Moreover, in solid cancers, CAR T immunotherapy has had little to no success in the clinic so far. In recent years, several studies have shown the influence of commensal gut microbes on T cell function, in particular in the setting of checkpoint immunotherapy. Our group has recently demonstrated that modulation of the gut microbiota using antibiotics such as oral vancomycin (vanco) can enhance the efficacy of tumor-specific T cells in animal models. In this study, we sought to study the effect of vanco-induced dysbiosis on CART immunotherapy using murine models and clinical correlates.

Methods and Results: We used the CD19+ A20 lymphoma and the B16 melanoma (transduced with CD19) murine models. Lymphoma- and melanoma-bearing mice were randomized to received oral vanco or vehicle alone (CTR), or in combination with either control untransduced murine T cells (UTD) or murine CART19 (4-1BB). Oral vanco or vehicle treatments started on the day of A20 cells injection and throughout the duration of the experiment (40-45 days). A20-bearing mice treated with CART19+vanco showed strikingly improved tumor control compared to either vanco alone or UTD+vanco (day 40 tumor volume in mm ³ (mean ± s.e.m): CTR=1,678.8±279.4, UTD=1,803.2±180, UTD+vanco=1,477±174, CART19=1,219±208, CART19+vanco=439.5±122.5 , CART versus CART+vanco Two Way Anova P <0.0001). Of note, CART19+vanco also displayed a longer overall survival as compared to controls (UTD= 0/7 alive at day 45 (0%), UTD+vanco= 2/7 (28.6%), CART19= 4/8 (50%), CART+vanco= 8/8 (100%)). To evaluate whether gut microbiota modulation improves CART therapy against solid tumors, we engrafted mice with CD19+ B16 melanoma cells and treated them with murine CART19 or control T-cells with or without vanco. Mice receiving CART+vanco displayed increased tumor control as compared to CART alone (day 21 tumor volume in mm ³ mean ± s.e.m. CTR=1,820.7±131.3, UTD=1,315.9±360.8, UTD+vanco=1,223.6±297.3, CART=1,315±360.8, CART+vanco=443.8±131.9, Two Way Anova CART versus CART+vanco P <0.0001). To investigate the mechanisms responsible for the improved anti-tumor activity, we analyzed gene-expression (nanoString) of 770 immune-oncology targets in tumor samples collected at day 5 after CART. The Ingenuity analysis showed up-regulation of the cross-presentation pathway in tumors of vanco+CART mice but not in CART alone. The functional validation of this mechanism was performed exploiting the physiological expression in A20 cells of the endogenous ecotropic murine leukemia provirus antigen gp70, also expressed in the colorectal cancer cell line CT26, which, however, lacks CD19 expression. Hence, purified T cells from CART19-treated A20-bearing mice were transferred into mice engrafted with CT26 tumors. These adoptively transferred T cells from CART+vanco group - but not the T cells obtained from mice treated with CART alone - displayed significant anti-tumor activity, (day 19 tumor volume in mm ³ (mean ± s.e.m): CTR= 1,360.6±123.3, CT26+CART= 932.9±234.9, CART+vanco= 402.5±139.4, Two Way Anova CART versus CART+vanco P<0.0044). To validate these data, we generated patient-derived gut microbiota avatars, performing a "human to mouse FMT" and observed increased antigen presentation in avatars treated with CART19+vanco. Lastly, in a cohort of 30 B-cell acute lymphoblastic leukemia patients treated with CART19 (CTL019, NCT02030847), 4 patients exposed to oral vanco in the first 3 weeks after CART infusion showed higher CART19 peak expansion and higher peak cytokine levels (IL6, IL10, IL1Ra) in 3/4 patients exposed to oral vanco compared with matched unexposed patients.

Conclusions: These results suggest that the modulation of the gut microbiota using vancomycin affects the outcome of CART therapy in preclinical models with better anti-tumor effect via cross-priming and enhanced CART expansion in tumor samples. In a retrospective cohort of patients with B-ALL receiving vancomycin after CART19 therapy, we observed higher CART expansion and serum inflammatory cytokines. Based on these observations, a clinical trial of oral vanco in patients receiving CD19-directed CAR T cells for B-cell lymphomas is planned.