Killing tumor-associated bacteria with a liposomal antibiotic generates neoantigens that induce anti-tumor immune responses.

Increasing evidence implicates the tumor microbiota as a factor that can influence cancer progression. In patients with colorectal cancer (CRC), we found that pre-resection antibiotics targeting anaerobic bacteria substantially improved disease-free survival by 25.5%. For mouse studies, we designed an antibiotic silver-tinidazole complex encapsulated in liposomes (LipoAgTNZ) to eliminate tumor-associated bacteria in the primary tumor and liver metastases without causing gut microbiome dysbiosis. Mouse CRC models colonized by tumor-promoting bacteria (Fusobacterium nucleatum spp.) or probiotics (Escherichia coli Nissle spp.) responded to LipoAgTNZ therapy, which enabled more than 70% long-term survival in two F. nucleatum-infected CRC models. The antibiotic treatment generated microbial neoantigens that elicited anti-tumor CD8+ T cells. Heterologous and homologous bacterial epitopes contributed to the immunogenicity, priming T cells to recognize both infected and uninfected tumors. Our strategy targets tumor-associated bacteria to elicit anti-tumoral immunity, paving the way for microbiome-immunotherapy interventions.

Development of an Intranasal Gel for the Delivery of a Broadly Acting Subunit Influenza Vaccine.

Influenza virus is a major cause of death on a global scale. Seasonal vaccines have been developed to combat influenza; however, they are not always highly effective. One strategy to develop a more broadly active influenza vaccine is the use of multiple rounds of layered consensus buildings to generate recombinant antigens, termed computationally optimized broadly reactive antigen (COBRA). Immunization with the COBRA hemagglutinin (HA) can elicit broad protection against multiple strains of a single influenza subtype (e.g., H1N1). We formulated a COBRA H1 HA with a stimulator of interferon genes agonist cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) into a nasal gel for vaccination against influenza. The gel formulation was designed to increase mucoadhesion and nasal retention of the antigen and adjuvant to promote a strong mucosal response. It consisted of a Schiff base-crosslinked hydrogel between branched polyethyleneimine and oxidized dextran. Following a prime-boost-boost schedule, an intranasal gel containing cGAMP and model antigen ovalbumin (OVA) led to the faster generation of serum IgG, IgG1, and IgG2c and significantly greater serum IgG1 levels on day 42 compared to soluble controls. Additionally, OVA-specific IgA was detected in nasal, vaginal, and fecal samples for all groups, except the vehicle control. When the COBRA HA was given intranasally in a prime-boost schedule, the mice receiving the gel containing the COBRA and cGAMP had significantly higher serum IgG and IgG2c at day 41 compared to all groups, and only this group had IgA levels above the background in vaginal, nasal, and fecal samples. Overall, this study indicates the utility of an intranasal gel for the delivery of COBRAs for the generation of serum and mucosal humoral responses.

Polymer and Crosslinker Content Influences Performance of Encapsulated Live Biotherapeutic Products.

INTRODUCTION: Live biotherapeutic products (LBPs), or therapeutic microbes, are an emerging therapeutic modality for prevention and treatment of gastrointestinal diseases. Since LBPs are living, they are uniquely sensitive to external stresses (e.g., oxygen, acid) encountered during manufacturing, storage, and delivery. Here, we systematically evaluate how polymer and crosslinker concentration affects the performance of an encapsulated LBP toward developing a comprehensive framework for the characterization and optimization of LBP delivery systems. METHODS: We encapsulate a model LBP, Lactobacillus casei ATCC 393, in calcium chloride (CaCl2)-crosslinked alginate beads, and evaluate how alginate and CaCl2 concentrations influence LBP formulation performance, including: (i) encapsulation efficiency, (ii) shrinkage upon drying, (iii) survival upon lyophilization, (iv) acid resistance, (v) release, and (vi) metabolite secretion. Approaches from microbiology (e.g., colony forming unit enumeration), materials science (e.g., scanning electron microscopy), and pharmaceutical sciences (e.g., release assays) are employed. RESULTS: LBP-encapsulating alginate beads were systematically evaluated as a function of alginate and CaCl2 concentrations. Specifically: (i) encapsulation efficiency of all formulations was >50%, (ii) all alginate beads shrunk (after lyophilization) and recovered (after rehydration) similarly, (iii) at 10% alginate concentration, lower CaCl2 concentration decreased survival upon lyophilization, (iv) 10% alginate improved acid resistance, (v) sustained release was enabled by increasing alginate and CaCl2 concentrations, and (vi) encapsulation did not impair secretion of l-lactate as compared to free LBP. CONCLUSIONS: This research demonstrates that polymer content and crosslinking extent modulate the performance of polymer-based LBP delivery systems, motivating research into the optimization of material properties for LBP delivery systems.

Enhanced Storage of Anaerobic Bacteria through Polymeric Encapsulation.

Live microbes such as lactobacilli have long been used as probiotic supplements and, more recently, have been explored as live biotherapeutic products with the potential to treat a range of conditions. Among these microbes is a category of anaerobes that possess therapeutic potential while exhibiting unique oxygen sensitivity and thus requiring careful considerations in the formulation and storage processes. Existing microbial formulation development has focused on facultative anaerobes with natural oxygen tolerance; a few strategies have been reported for anaerobes with demonstrated oxygen intolerance, warranting novel approaches toward addressing the challenges for these oxygen-sensitive anaerobes. Here, we develop a polymeric encapsulation system for the formulation and storage of Bifidobacterium adolescentis (B. adolescentis), a model anaerobe that loses viability in aerobic incubation at 37 °C within 1 day. We discover that this strain remains viable under aerobic conditions for 14 days at 4 °C, enabling formulation development such as solution casting and air drying in an aerobic environment. Next, through a systematic selection of polymer encapsulants and excipients, we show that encapsulation with poly(vinyl alcohol) (PVA) acts as an oxygen barrier and facilitates long-term storage of B. adolescentis, which is partially attributed to reduced generation of reactive oxygen species. Lastly, PVA-based formulations can produce oral capsule-loaded films and edible gummy bears, demonstrating its compatibility with both pharmaceutical and food dosage forms.

Batch Culture Formulation of Live Biotherapeutic Products.

Live biotherapeutic products (LBPs) are an emerging therapeutic modality that are clinically investigated for treating pathogenic infections and inflammatory diseases. A major class of LBPs are feces derived microbial consortiums which require numerous process development steps (e.g. separation, purification, blending) to facilitate LBP formulation into oral dosage forms. A subset of these LBPs circumvent the need for continuous fecal processing by batch culture for individual strains of microbes that are rationally defined and combined in the final LBP formulation. Separately, delivery formulations (e.g. polymer encapsulation) are being developed for LBPs to improve storage and intestinal engraftment; however, formulation requires additional manufacturing processes distinct from fecal processing or batch culture. Here, a streamlined approach termed batch culture formulation (BCF) is developed to combine the individual batch culture and formulation processes into a single-step process. Based on a previously described polymeric film formulation that encapsulates LBPs, BCF is shown to reduce the number of required processes to formulate LBP-films without altering LBP phenotype, function, or storage profiles compared to the standard LBP-film formulation approach. Additionally, it is demonstrated that BCF facilitates scaled-fabrication from the milligram to gram scale with predictable loading, highlighting the potential that BCF has for clinical translation.

Polymeric Films for the Encapsulation, Storage, and Tunable Release of Therapeutic Microbes.

Microbe-based therapeutics (MBTs) are an emerging therapeutic modality for treating gastrointestinal infections and inflammatory bowel diseases. Current formulations for oral delivery of MBTs use capsules to achieve safe gastric transit, but oral formulations that control the spatiotemporal concentration of MBTs are yet to be developed, despite well-established connections between all therapeutics and their location, concentration, and distribution at sites of action. The development of a multi-functional polymer-based encapsulation system to formulate MBTs for enhanced storage and delivery through formulation of a model MBT, Lactobacillus casei ATCC393, is reported here. This approach enables the additive inclusion of excipients and polymers to grant specific functions, toward the development of a modular MBT platform. Through addition of established excipients, the formulation provides long-term storage of the encapsulated MBT. By adding higher molecular weight polymers, the release kinetics of the encapsulated MBTs can be modified. The inclusion of a mucoadhesive polymer significantly increases the adhesion force between the formulation and the intestinal tissue. Together, mucoadhesive and sustained release properties can be used to modulate the spatiotemporal concentration of MBTs. The formulation is compatible with standard oral capsules, thus maintaining existing clinical advantages of oral capsules while providing new functions from film encapsulation.

Trapping of Lipopolysaccharide to Promote Immunotherapy against Colorectal Cancer and Attenuate Liver Metastasis.

The development and progression of colorectal cancer (CRC) is closely related to gut microbiome. Here, the impact of lipopolysaccharide (LPS), one of the most prevalent products in the gut microbiome, on CRC immunotherapy is investigated. It is found that LPS is abundant in orthotopic CRC tissue and is associated with low responses to anti-PD-L1 mAb therapy, and clearance of Gram-negative bacteria from the gut using polymyxin B (PmB) or blockade of Toll-like receptor 4 using TAK-242 will both relieve the immunosuppressive microenvironment and boost T-cell infiltration into the CRC tumor. Further, an engineered LPS-targeting fusion protein is designed and its coding sequence is loaded into a lipid-protamine-DNA (LPD) nanoparticle system for selective expression of LPS trap protein and blocking LPS inside the tumor, and this nanotrapping system significantly relieves the immunosuppressive microenvironment and boosts anti-PD-L1 mAb therapy against CRC tumors. This LPS trap system even attenuates CRC liver metastasis when applied, suggesting the importance of blocking LPS in the gut-liver axis. The strategy applied here may provide a useful new way for treating CRC as well as other epithelial cancers that interact with mucosa microbiome.