The Role of Gut Microbiota in Modulating Efficacy and Adverse Effects of CAR T-Cell Therapy in the Pediatric Population

Introduction

The gut microbiome, the trillions of bacteria residing within the human gastrointestinal tract, appear to be critical for modulating cancer immunotherapy efficacy. In fact, adult patients who respond to chimeric antigen receptor T-cell (CAR-T) therapy have distinct gut microbiome signatures compared to non-responders. The role that gut microbiota play in modulating CAR T-cell therapy has not been rigorously studied in the pediatric population. We hypothesize that specific commensal gut bacterial species are required for optimizing CAR-T efficacy and for minimizing adverse effects in the pediatric population.

Methods

Patients aged 6 months to 18 years old with B-lymphoblastic leukemia who met clinical criteria to receive tisagenlecleucel (anti CD19 CAR T-cell therapy) were enrolled in this investigator-initiated non-randomized case control study at Childrens Medical Center in Dallas. Patients with abnormal GI anatomy (that would hinder the ability to obtain a stool sample) were excluded. Fecal samples were collected at apheresis, infusion, and 7, 30, 60, 90, and 180 days post-infusion. Microbiome profiling (16S rRNA sequencing, V4 region) was performed on fecal genomic DNA. Blood was collected at apheresis, infusion, and 30 days post-infusion for analysis of the patients’ systemic immune profile via bulk cytokine profiling. Patients were followed for up to 2 years after infusion to assess response to treatment and monitor adverse effects.

Results

Thirty-five patients were enrolled. Gut microbiome profiling on 96 stool samples collected from 34 patients was completed. A durable response was defined as the lack of relapse or lack of B-cell recovery at 6 months post-CAR-T infusion. Fourteen patients had a durable response and 20 did not have a durable response. Using a linear mixed effect model and linear discriminant analysis Effect Size (LefSE) analyses, we found that Enterococcus spp. was significantly enriched in those patients with a positive, durable response. There was no significant difference in alpha diversity among responders versus non-responders and those who developed cytokine release syndrome (CRS) and/or neurotoxicity versus those who did not have adverse effects. In contrast, the beta diversity among responders was significantly different than those who did not respond, but the amount of variation is largely unexplained (weighted UniFac, R² = 0.05, p-value = 3x10^-4). Interestingly, patients who responded to CAR-T therapy were enriched with Enterococcus species (LDA score = 5.3; adjusted p-value = 0.002) while those who relapsed or had B-cell recovery had enrichment in Ruminococcaceae (LDA = 4.75; adjusted P-value = 0.004) and Lachnospiraceae (LDA = 4.47; adjusted p-value = 0.0008). Enterococcus significantly augmented CAR T-cells’ capacity to produce IFN-gamma, a cytokine critical for anti-tumor immunity, ex vivo -- whereas Lactobacillus acidophilus, a gut microbiota commonly found in yogurt and over the counter probiotics could not. Finally, patients who developed CRS and/or neurotoxicity were enriched in Enterococcus (LDA = 5.29; adjusted p-value = 0.035), Oscillospiraceae (LDA = 3.69; adjusted p-value = 0.0159), and Erysipelotrichaceae (LDA = 2.49; adjusted p-value = 0.044). Cytokine analysis of the blood samples is pending.

Conclusions

In this investigator-initiated non-randomized case control study, pediatric patients with B-lymphoblastic leukemia receiving CAR T-cells (n=34) with a positive, durable response exhibited a significant enrichment of Enterococcus spp. compared to non-responders. Enterococcus has been shown to promote anti-tumor (Th1) immunity in other studies (Viaud et al 2013, Lauté-Caly et al 2018, and Griffin et al 2021), consistent with our ex vivo functional immune cell data. Collectively these data suggest that Enterococcus could be a potential biomarker for CAR-T efficacy and might be leveraged as a CAR-T immune enhancer, but additional studies are merited to confirm these observations and to provide more mechanistic insight.