Abstract 6679: Deciphering the mechanism of action of a live biotherapeutic product for the treatment of cancer

Abstract Intestinal microbiota is essential in shaping the immune system and its functions. In our effort to harness the potential of beneficial gut microbiota, we identified specific bacterial species that are less abundant in colorectal cancer patients compared to healthy individuals. Oral supplementation of these bacteria causes an effective anti-tumor activity in various cancer models through specific activation of cytotoxic T-cells. Based on these results, we aimed to identify the mechanism of action of this bacterial treatment. In vivo studies were performed in C57BL/6 and BALB/c mice. MC-38, CT-26, B16, LLC1. 1 and 4T1 cells were used for the heterotopic tumor injections. Immunophenotyping was performed using flow cytometry and immunohistochemistry. Metagenomics analysis and whole genome sequence were performed to identify the bacterial strains. Metabolomics analyses were conducted to analyze bacterial supernatant and serum compositions of mice and patients. Since we found that the bacterial treatment led to a systemic anti-tumor effect, we analyzed serum of the mice that received the bacteria and found that a specific metabolite was highly upregulated compared to untreated mice. Analysis of the medium supernatant from cultures of the bacterial strains led to the discovery that metabolite identified in the serum is being produced by our bacteria. Applying this bacterial metabolite alone in the mouse cancer models led to a similar immune activation and anti-tumor effect as the supplementation with our bacterial strains. Furthermore, we identified the receptor that is targeted by this molecule through experiments with known agonists and antagonists for the target. The anti-tumor effect of the bacteria as well as the bacterial metabolite was blocked through the application of a receptor antagonist, proving that activation of this receptor is the mechanism that drives the enhanced anti-tumor immune response. Activation of the receptor, which is mainly expressed on macrophages led to enhanced IL-12 production and recruitment of cytotoxic T-cells into the tumor. Together, we have fully characterized the mechanism of action of our bacterial treatment, including the secreted active metabolite, the target receptor, and the activated immune cells. We are developing a live biotherapeutic product for the treatment of cancer based on oral administration of lyophilized bacteria. The known mechanism of action of our bacteria helps us to optimize the efficacy of our product, define the right dosage and understand possible side effects. Additionally, it enables us to improve patient selection and prediction of treatment response. Since our product is based on commensal bacteria that are commonly present in healthy individuals, we expect a very good safety and tolerability profile. Therefore, our therapy aims to significantly improve the efficacy of cancer treatment as well as the quality of life of cancer patients. Citation Format: Egle Katkeviciute, Francesca Ferraro, Martin Schwill, Philipp Busenhart, Ana Montalban-Arques. Deciphering the mechanism of action of a live biotherapeutic product for the treatment of cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 6679.