Abstract 1825: A bacterial consortium engineered from feces of patients responding to anti-PD1 treatment mediates tumor response through IFNγ in non-small cell lung cancer
Authors: Newsome Rachel C. , Oh Joonseok , Jian Wenya , Gharaibeh Raad Z. , Crawford Jason , Jobin Christian 
Published: April 2023

Abstract
Introduction: We previously showed that non-small cell lung cancer (NSCLC) patients that respond (R) to immune checkpoint blockade (ICB) have a different microbial community structure than non-responders (NR) pre-treatment (Genome Med 2022). Importantly, we showed that pooled R microbiota transplantation into gnotobiotic xenograft mice decreased tumor growth compared to NR colonized mice following anti-PD1 therapy, a phenomenon associated with enrichment of the Bacteroides genus. However, the bacterial strains driving this synergistic effect with anti-PD1 therapy and mechanism of action are unknown.
Method: Feces collected from R mice were used as source material for high-throughput microbial isolation performed with the GALT Prospector technology. Bacterial identification was performed with MALDI-TOF Biotyper and Sanger sequencing. The cell free supernatants and under 3 kDa small molecules of 183 Bacteroides isolates were screened for their ability to stimulate IFNγ production by primary CD8+ T cells by flow cytometry. A consortium composed of 6 IFNγ-stimulating isolates or NR feces was transplanted into a gnotobiotic mouse model of lung cancer treated with anti-PD1, with or without anti-IFNγ monoclonal antibody depletion. Tumors were harvested at endpoint for flow cytometric analysis, and blood serum for IFNγ ELISA. Bioactivity-guided fractionation coupled with LC-MS analysis was used to identify small molecules from the cell free supernatant of a single stimulating Bacteroides isolate. Potential bioactive small molecule characterization was performed using LC-MS/MS supported by computational pipelines for structural prediction followed by NMR for structural confirmation.
Results: We cultured and identified 679 isolates from 30 unique species. The cell free supernatant from 6 out of 183 Bacteroides isolates stimulated IFNγ production from primary CD8+ T cells. Small molecules from the six stimulatory isolates’ supernatants significantly induced IFNγ production compared to 6 taxonomy-matched non-stimulatory isolates (P=0.039). A defined consortium composed of the six stimulatory isolates (R-consortium) was able to colonize germ free mice, and decreased tumor growth compared to NR feces-colonized mice (P=0.041). IFNγ depletion of R-consortium mice significantly increased tumor growth (P=0.012) compared to non-depleted mice. Intratumor IFNγ+ CD8+ T cell frequency and circulating serum IFNγ was elevated only in R-consortium mice. Bioassay guided fractionation revealed 5 small molecule metabolites produced by Bacteroides and their structure and bioactivity are currently being investigated.
Conclusion: A microbial consortium engineered from R patients’ feces synergizes with anti-PD1 therapy to reduce lung cancer growth through an IFNγ-dependent mechanism which may be mediated by small molecule metabolites.
Citation Format: Rachel C. Newsome, Joonseok Oh, Wenya Jian, Raad Z. Gharaibeh, Jason Crawford, Christian Jobin. A bacterial consortium engineered from feces of patients responding to anti-PD1 treatment mediates tumor response through IFNγ in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1825.