(J-369) Optimization and scale-up production of oral inulin gel for modulation of the gut microbiome for cancer immunotherapy

Introduction:: The immune-checkpoint inhibitors (ICIs) have witnessed great success in cancer treatment; however, ICIs could associate with serious immune-related adverse events and only benefits a subset of patients due to the heterogeneities in tumors, patients, and implications of gut microbiota. This underscores the need for strategies that can engineer systemic antitumor immunity through gut microbiome manipulation. We have recently shown that inulin formulated into oral hydrogel modulates the gut commensal microbes and exerts a synergistic effect with immune checkpoint inhibitors in various murine tumor models ^[1]. Toward the goal of initiating a human clinical study, we have optimized the scale-up manufacturing of inulin gel.

Materials and Methods::

Inulin gel scale-up production was conducted by first blending inulin powder with preservatives and acidifiers in water. The inulin suspension was then filled into mylar bags and the packages were subjected to heat-cooling cycles at various temperatures and durations for gelling optimization. The resulting inulin gel was characterized by its rheological, and physicochemical properties and shelf-life. The synergistic effect with immune checkpoint inhibitors of the inulin gel was verified in the CT26 tumor model by oral administration of the gel during anti-PD-1 therapy. Microbiome DNA isolation and 16S rRNA gene sequencing were performed to analyze the gut microbiome changes resulting from the ICB treatment and inulin gel supplementation.

Results, Conclusions, and Discussions::

Inulin gel was manufactured at the one-kilogram scale and dispensed as 10 grams per serving in food-grade packages suitable for human consumption. Viscoelastic parameters measured by the rheometer indicated that the gel strength and viscosity were improved by 10-fold for the new inulin gel formulation, compared to the original formulation. Inulin remained stable under such conditions, with the same degree of polymerization as the non-heating reference, as evidenced by gel permeation chromatography (GPC). However, the storage modulus (G’) and loss modulus (G’’) decreased markedly when heating was extended to 90 minutes, suggesting potential polysaccharides degradation. The microbial safety tests found no growth of anaerobes, molds, yeasts, and spores in the inulin gel upon 210 days of storage, and the packages demonstrated resistance to the known food pathogen challenges, showing great shelf stability. Oral gavage of inulin gel in mice significantly improved the antitumor efficacy of anti-PD-1 and slowed the growth of CT26 tumors with a 2-fold increase in the complete regression rate compared with the a-PD-1 monotherapy.  The 16S rRNA sequencing revealed that the inulin gel not only restored the decreased richness and diversity in the gut resulting from a-PD-1 treatment but also reshaped the microbiota composition and increased the abundance of the butyrogenic species such as Ruminococcus.

 

Overall, we have successfully produced one kilogram of inulin gel sufficient for 100 human servings. The resulting inulin gel shows increased gel strength, which contributes to the colon retentive properties and benefits the prebiotic fermentation in the colon. Furthermore, the shelf-life is extended to over 30 weeks, rendering inulin gel safe for human consumption. The scaled-up inulin gel can modulate the gut microbiome, ameliorates dysbiosis associated with immune checkpoint inhibitors, and enriches the prebiotics in the gut. Short-chain fatty acids generated from inulin fermentation enhanced the antitumor CD8+ T cell responses and increased the efficacy of anti-PD-1 in the CT26 mouse model. In conclusion, we have optimized and scaled up the inulin gel formulation for future human studies of gut microbiome modulation for engineering antitumor immunity and improving the clinical outcomes of ICIs.

Acknowledgements (Optional): :

References (Optional): : [1] Han, Kai, et al. "Generation of systemic antitumour immunity via the in situ modulation of the gut microbiome by an orally administered inulin gel." Nature biomedical engineering 5.11 (2021): 1377-1388.