P-02.02 The influence of fecal supernatants from melanoma patients on CAR T cell cytotoxicity and immune modulation

<h3>Background</h3> Immune checkpoint inhibitors (ICI) have revolutionized melanoma treatment, yet their efficacy is influenced by factors such as the tumor microenvironment and the host microbiome. Emerging evidence suggests that the microbiome can modulate immune response, though the exact mechanisms remain unclear. This study investigates the impact of fecal supernatants from melanoma patients on chimeric antigen receptor (CAR) T cell cytotoxicity, a promising cancer immunotherapy. Supernatants are subdivided regarding response to immunotherapy measured by overall survival (OS) of patients. <h3>Materials and Methods</h3> Fecal supernatants were obtained from six melanoma patients, categorized into two groups: three Elite patients with an outstanding response to immunotherapy reflected by an OS of &gt; 40 months, and three HPP patients who are resistant to treatment and have shown progression of disease. The effects of fecal supernatants on CAR-T cells <i>in vitro</i> were investigated by preincubating human anti-CD19 CAR-T cells with the various supernatants for 72 hours at a 1:100 dilution. Cytotoxicity was assessed by stimulating the T cells with the human B-cell lymphoma cell line NALM6 for 24 hours at an effector-to-target (E:T) ratio of 1:64 using a luciferase-based killing assay. Luminescence data were acquired, and specific lysis was calculated by normalizing sample luminescence to the tumor-only control (1 - (sample luminescence/tumor-only luminescence)). Additional interferon-γ (IFN-γ) and Granzyme B (GRZM B) Enzyme-Linked Immunosorbent Assay (ELISA) assays were performed on the co-culture to assess immune cell functionality. <h3>Results</h3> Compared to MCDA medium used for supernatant culturing and served as reference, fecal supernatants from Elite patients (Elite1-3) did not show significant differences in CAR-T cell cytotoxicity, indicating that these supernatants do not strongly affect CAR-T function. In contrast, fecal supernatants from HPP patients exhibited a significant reduction in CAR-T cytotoxicity. Specifically, supernatants from HPP1 and HPP2 caused a marked decrease in CAR-T activity (p &lt; 0.05), suggesting that fecal supernatants from resistant patients inhibit CAR-T cell function more than those from patients with an outstanding response to immunotherapy. The ELISA results were consistent with the findings of the killing assay, indicating reduced levels of IFN-γ and GRZM B as surrogate markers for functionality in CAR-T cells stimulated with HPP1 and HPP2 supernatants. <h3>Conclusions</h3> The findings indicate that fecal supernatants from ICI-treated melanoma patients modulate CAR-T cell function, with two of the three HPP samples from patients with therapy resistance showing a significant inhibitory effect on CAR-T cytotoxicity. In contrast, Elite samples did not alter CAR-T cell function. These results suggest that microbial metabolites present in the fecal supernatants of resistant melanoma patients may suppress CAR-T cell activity, providing insights into the complex relationship between the gut microbiome and immunotherapies. Further studies are needed to identify the specific metabolites responsible for these effects and explore their potential role in modulating the efficacy of CAR-T cell therapy. Y. Wang: None. R.D. Müller: None. M. Trefny: None. M. Morak: None. L. Arnold: None. S. Kobold: None. L. Heinzerling: None.