Multi-faceted influence of immunomodulatory metabolites on CAR-T cell therapy

Abstract Background:Chimeric antigen receptor (CAR) T cell therapy has revolutionized treatment for hematologic malignancies achieving high complete remission rates up to 80% in refractory patients. However, long-term durable responses are observed in only 50% of patients with a substantial proportion experiencing severe toxicities Munshi, N. C. et al., 2021; Locke, F. L. et al., 2022. Emerging evidence shows gut microbiome and antibiotic exposure is associated with clinical outcomes profoundly modulating systemic immunity which in turn impacts CAR-T cell expansion, persistence, and toxicity Stein-Thoeringer, C. K. et al., 2023. Elucidating the impact of microbiome on CAR-T cell therapy is therefore critical to optimize treatment outcomes. Recent research suggests that immunomodulatory metabolites (IMMs) derived from gut microbiota play a pivotal role in directly shaping immunotherapy outcomes Luu, M. et al., 2021; Thiele Orberg, E. et al., 2024; Perl, M. et al.,2025. To assess the impact of IMMs on CAR-T cell therapy outcomes, we performed an in-depth analysis of the gut microbiome using shotgun metagenomics and metabolite profiling in CAR-T cell recipients and validated our results through preclinical experiments. Methods:Pre-treatment fecal content (n= 56 samples) was profiled by shotgun metagenomic sequencing and fecal as well as serum (n= 111/98 samples for fecal/serum respectively) metabolite concentrations were quantified using targeted mass spectrometry (n= 44/25 metabolites after QC for fecal/serum respectively) in 129 patients receiving CAR-T cell therapy for hematologic and solid malignancies across three German centers (Heidelberg, Munich, Regensburg). The impact of IMMs on CAR-T cell cytotoxicity and phenotype was evaluated using in vitro cell lysis assays and flow cytometry. Syngeneic B-cell lymphoma mouse models were employed to confirm the roles of these metabolites in vivo. Furthermore, single-cell RNA sequencing (scRNA-Seq) of artificially stimulated human CAR-T cells provided insights into treatment-specific, phenotypic and functional changes. SCENITH assay was further employed to functionally assess the altering energy metabolism profile of IMMs. Results:Unsupervised hierarchical clustering of both fecal and serum IMMs revealed that a loss of these metabolites was associated with poorer clinical outcomes and reduced complete response rates compared to patients with preserved metabolite levels. While individual bacteria were not predictive, depletion of obligate anaerobes and short-chain fatty acids (SCFAs) such as valeric acid (VA) was associated with increased risk of disease progression. Notably, we identified differentially regulated metabolites including the indole derivatives namely indole-3-carboxyaldehyde (ICA) as well as the branched-chain fatty acid (BCFA) like isovaleric acid (IVA) to be significantly higher in patients with disease progression. ICA impaired, whereas VA enhanced CAR-T cell functions both in vitro and in a syngeneic B-cell lymphoma model following oral administration. Remarkably, IVA showed opposing effects- enhancing CAR-T cell fitness and expansion but also stimulating tumor cell growth, ultimately reducing survival in vivo. scRNA-Seq combined with CRISPR/Cas9-mediated knockout of the aryl hydrocarbon receptor (AHR) identified an AHR-dependent inhibitory pathway through which ICA impairs CAR-T cell efficacy. In contrast, VA enhanced the metabolic fitness and cytolytic function of CAR-T cells confirmed via SCENITH assay. These findings enabled to form a four-parameter clinical risk score that accurately predicts progression free survival (HR=2.94 for patients with 4 points vs. ≤ 3) and overall survival (HR=2.0 for patients with 4 points vs. ≤ 3) of CAR-T cell therapy patients. Discussion:Our study uncovers a critical and multi-faceted role of gut-derived IMMs in modulating CAR-T cell function and clinical outcomes revealing that specific metabolites can either enhance or inhibit CAR-T cell efficacy, but simultaneously affect cancer progression, through defined molecular pathways. These insights highlight the potential of metabolite profiling as a superior biomarker for patient stratification and risk assessment compared to fecal taxonomic composition alone. Furthermore, our findings pave the way for the development of metabolite-guided microbiome-based therapeutics that could significantly improve outcomes in CAR-T cell therapy and potentially other cancer immunotherapies.