Live Biotherapeutic Products Research Articles

Spray-Drying Encapsulation of the Live Biotherapeutic Candidate Akkermansia muciniphila DSM 22959 to Survive Aerobic Storage.

Akkermansia muciniphila is regarded as a promising next-generation probiotic or live biotherapeutic candidate. Effective delivery strategies must be developed to ensure high enough viability of the probiotic strain throughout its industrial formulation, distribution chain, shelf-life, and, ultimately, the host’s gastrointestinal tract, where it should exert its beneficial effect(s). Among the possible methodologies, spray-drying is considered industrially attractive regarding its costs, efficiency, and scalability, with the due parameter customization. In this study, spray-drying was explored as a one-step process to encapsulate A. muciniphila DSM 22959, testing the drying settings and three different dairy-based matrices. Microcapsule morphology and size was assessed, and viability throughout storage at 4 or 22 °C and simulated gastrointestinal passage was determined. Akkermansia muciniphila microencapsulation by spray-drying, using 10% skim milk and inlet/outlet temperatures of 150/65 °C, is effective in terms of viability stabilization, both during prolonged aerobic storage and exposure to simulated gastrointestinal passage. Akkermansia muciniphila viability was maintained at around 107 CFU/g up to 28 days at 4 °C under aerobic conditions with viability losses inferior to 1 log reduction. This methodology provides the necessary conditions to efficiently deliver the recommended dose of live A. muciniphila in the human gut as a live biotherapeutic product.

Dosing a synbiotic of human milk oligosaccharides and B. infantis leads to reversible engraftment in healthy adult microbiomes without antibiotics

Predictable and sustainable engraftment of live biotherapeutic products into the human gut microbiome is being explored as a promising way to modulate the human gut microbiome. We utilize a synbiotic approach pairing the infant gut microbe Bifidobacterium longum subspecies infantis (B.infantis) and human milk oligosaccharides (HMO). B. infantis, which is typically absent in adults, engrafts into healthy adult microbiomes in an HMO-dependent manner at a relative abundance of up to 25% of the bacterial population without antibiotic pretreatment or adverse effects. Corresponding changes in metabolites are detected. Germ-free mice transplanted with dysbiotic human microbiomes also successfully engraft with B.infantis in an HMO-dependent manner, and the synbiotic augments butyrate levels both in this invivo model and in invitro coculturesof the synbiotic with specific Firmicutes species. Finally, the synbiotic inhibits the growth of enteropathogensinvitro. Our findings point to a potential safe mechanism for ameliorating dysbioses characteristicof numerous human diseases.

Colonization of the live biotherapeutic product VE303 and modulation of the microbiota and metabolites in healthy volunteers

Manipulation of the gut microbiota via fecal microbiota transplantation (FMT) has shown clinical promise in diseases such as recurrent Clostridioides difficile infection (rCDI). However, the variable nature of this approach makes it challenging to describe the relationship between fecal strain colonization, corresponding microbiota changes, and clinical efficacy. Live biotherapeutic products (LBPs) consisting of defined consortia of clonal bacterial isolates have been proposed as an alternative therapeutic class because of their promising preclinical results and safety profile. We describe VE303, an LBP comprising 8 commensal Clostridia strains under development for rCDI, and its early clinical development in healthy volunteers (HVs). In a phase 1a/b study in HVs, VE303 is determined to be safe and well-tolerated at all doses tested. VE303 strains optimally colonize HVs if dosed over multiple days after vancomycin pretreatment. VE303 promotes the establishment of a microbiota community known to provide colonization resistance.

Finding the right fit: evaluation of short-read and long-read sequencing approaches to maximize the utility of clinical microbiome data.

A long-standing challenge in human microbiome research is achieving the taxonomic and functional resolution needed to generate testable hypotheses about the gut microbiota’s impact on health and disease. With a growing number of live microbial interventions in clinical development, this challenge is renewed by a need to understand the pharmacokinetics and pharmacodynamics of therapeutic candidates. While short-read sequencing of the bacterial 16S rRNA gene has been the standard for microbiota profiling, recent improvements in the fidelity of long-read sequencing underscores the need for a re-evaluation of the value of distinct microbiome-sequencing approaches. We leveraged samples from participants enrolled in a phase 1b clinical trial of a novel live biotherapeutic product to perform a comparative analysis of short-read and long-read amplicon and metagenomic sequencing approaches to assess their utility for generating clinical microbiome data. Across all methods, overall community taxonomic profiles were comparable and relationships between samples were conserved. Comparison of ubiquitous short-read 16S rRNA amplicon profiling to long-read profiling of the 16S-ITS-23S rRNA amplicon showed that only the latter provided strain-level community resolution and insight into novel taxa. All methods identified an active ingredient strain in treated study participants, though detection confidence was higher for long-read methods. Read coverage from both metagenomic methods provided evidence of active-ingredient strain replication in some treated participants. Compared to short-read metagenomics, approximately twice the proportion of long reads were assigned functional annotations. Finally, compositionally similar bacterial metagenome-assembled genomes (MAGs) were recovered from short-read and long-read metagenomic methods, although a greater number and more complete MAGs were recovered from long reads. Despite higher costs, both amplicon and metagenomic long-read approaches yielded added microbiome data value in the form of higher confidence taxonomic and functional resolution and improved recovery of microbial genomes compared to traditional short-read methodologies.

A49 PRECLINICAL STUDIES OF TWO NOVEL LIVE BIOTHERAPEUTIC PRODUCTS FOR THE TREATMENT OF ULCERATIVE COLITIS

Abstract Background The therapeutic approaches for ulcerative colitis present a high failure rate. Therefore, more therapeutic options are in the spotlight. Live biotherapeutic products, a more rationalized and tailored approach than probiotics, have a broader effect since they are not limited to a single therapeutic target. Aims To characterize the therapeutic effect of BioColoniz and BioPersist, two live biotherapeutic products derived from Lactobacillus reuteri and E. coli Nissle 1917, respectively, in rodent models of colitis. Methods To test the therapeutic effect of BioColoniz, 5 weeks old Muc2-/- mice, which spontaneously develop colitis, received a single dose of BioColoniz, L. reuteri or Vehicle via oral gavage; mice were monitored weekly until they showed clinical signs of disease and were euthanized. To evaluate the therapeutic effect of BioPersist, 6 weeks old C57BL/6 mice received BioPersist or vehicle via oral gavage, and then were exposed for 7 days to DSS for induction of acute colitis; parallel, mice received 5-ASA or vehicle daily. For both experiments, colons were collected and evaluated for macroscopic and histopathological changes. Results Mice treated with BioColoniz presented lower clinical scores, with a greater weight gain when compared to mice treated with L. retueri or vehicle. The appearance of rectal prolapse, a hallmark of the Muc2-/- model, was only recorded in mice treated with L. reuteri or Vehicle, supporting the protective effect of BioColoniz in the Muc2-/- model. Although the macroscopic analysis showed no changes in colon length among the groups, the histopathological analysis revealed decreased damage in mice treated with BioColoniz, significant when compared to mice who only received the vehicle. In order to evaluate if BioColoniz also protects against the development of comorbid metabolic defects of colitis, mice were also subjected to oral glucose tolerance test, finding that BioColoniz treated mice, especially males, display an improved glucose clearance when compared to L. retueri or vehicle groups. In the DSS model, BioPersist decreased daily clinical scores, significantly delaying the weight loss. The macroscopic analysis showed greater colon length in BioPersist treated mice than in mice treated only with 5-ASA, along with decreased histopathological damage. Conclusions Altogether, BioColoniz and BioPersist reduce the colitic phenotype in the Muc2-/- and DSS colitis models, respectively. Furthermore, BioColoniz protects Muc2-/- mice from the metabolic detriments of colitis. Future studies will focus on describing the mechanisms by which BioColoniz and BioPersist confer their therapeutic effect. Funding Agencies CCCMSFHR

P080 ADS024 attenuates weight loss and decreases disease activity in DSS-induced colitis

Abstract Background Ulcerative colitis (UC) is a chronic, idiopathic inflammatory disease characterised by a dysregulated innate and adaptive immune response to gut microbiota that contributes to the perpetuation of colonic inflammation. ADS024 is an orally administered live biotherapeutic product (LBP), comprised of a single strain of Bacillius velezensis, being developed for potential application in the treatment of patients with UC. Methods Colitis was induced in 39 mice by exposure to 3% dextran sodium sulfate (DSS)-treated drinking water from Day 0 to Day 5; 6 control animals did not receive DSS. Animals were then dosed with vehicle (PBS; n=15) or ADS024 (n=12) twice daily for 14 days via oral gavage or with comparator anti-p40 mAb (n=12) once daily (IP) on Days 6, 9, 12, 15. All animals were monitored daily for body weight loss, diarrhea, blood in stool, and activity level. Each of these parameters was individually scored according to the disease activity index (DAI) scoring scheme. Animals underwent video endoscopy on Day 19 to assess colitis severity and stool consistency was scored during endoscopy. Following endoscopy on Day 19, all surviving animals were sacrificed, and blood was collected and processed for serum and analysed for metabolomic profile. Results Animals in which colitis was induced by exposure to 3% DSS demonstrated significant weight loss, most profoundly from Days 8 to 11, an increase in the composite DAI and increased mean colon weight:length ratios as compared to control animals. Animals treated with ADS024 had improved body weight, starting at 72 hours post-treatment and decreased DAI scoring over the course of the study compared to the vehicle control group (p<0.01 and p<0.05 respectively), comparable to treatment with anti-p40 mAb which has a mechanism of action similar to ustekinumab (Stelara®) approved for the treatment of UC. A trend towards a decrease in the mean colon weight:length ratio was observed in animals treated with ADS024, but there was no difference in mean endoscopy scoring compared to the vehicle control group. DSS treatment altered the serum biochemical profile at day 19 and ADS024 induced changes suggesting a reversion towards the control state. Conclusion Oral treatment with ADS024 improved body weight loss, reduced DAI, and resulted in a trend towards reduction in mean colon weight:length ratios in the DSS-induced mouse colitis model. In addition, ADS024 treatment led to a rebalancing of the systemic metabolomic shift caused by DSS exposure.

Probiotic supplementation demonstrates therapeutic potential in treating gut dysbiosis and improving neurocognitive function in age-related dementia.

Probiotics, as live microorganisms that improve intestinal microbial balance, have been implicated in the modulation of neurodegenerative diseases via the microbiome-gut-brain axis by improving gut dysbiosis. This review examines the association between probiotics and neurocognitive function in age-related dementia. We searched MEDLINE, Embase, Scopus, Web of Science and Cochrane library for in vivo studies using equivalent combinations of "probiotics" and "dementia" as per PRISMA. From the 52 in vivo studies identified, 5 human and 22 animal studies with comparable quantitative outcomes on neurocognitive/behavioural function were meta-analysed by forest plots, subgroup analysis and meta-regression. The analysis of biomarkers, risk of bias and publication bias were also performed. In elderly humans, probiotics correlates with a non-significant difference of neurocognitive function in Mini-Mental State Examination, but with significant improvement only in those diagnosed with Alzheimer's disease. In animals, probiotics significantly improved neurocognitive function as measured by Morris Water Maze, Y-Maze, and Passive Avoidance. Further analysis by subgrouping and meta-regression found that the probiotics-neurodegeneration association is age dependent in humans but is neither dose dependent nor duration dependent in animals or humans. Analysis of biomarkers suggested that the neurocognitive effect of probiotics is associated with an altered gut microbiome profile, downregulated proteinopathic, inflammatory and autophagic pathways, and upregulated anti-oxidative, neurotrophic, and cholinergic pathways. Overall, we report promising results in animal studies but limited evidence of probiotics leading to neurocognitive improvement in humans. More research into probiotics should be conducted, especially on live biotherapeutic products for targeted treatment of gut dysbiosis and age-related dementia.

Development and Validation of a Novel Microbiome-Based Biomarker of Post-antibiotic Dysbiosis and Subsequent Restoration.

Background: The human gut microbiota are important to health and wellness, and disrupted microbiota homeostasis, or “dysbiosis,” can cause or contribute to many gastrointestinal disease states. Dysbiosis can be caused by many factors, most notably antibiotic treatment. To correct dysbiosis and restore healthier microbiota, several investigational microbiota-based live biotherapeutic products (LBPs) are in formal clinical development. To better guide and refine LBP development and to better understand and manage the risks of antibiotic administration, biomarkers that distinguish post-antibiotic dysbiosis from healthy microbiota are needed. Here we report the development of a prototype Microbiome Health Index for post-Antibiotic dysbiosis (MHI-A).Methods: MHI-A was developed and validated using longitudinal gut microbiome data from participants in clinical trials of RBX2660 and RBX7455 – investigational LBPs in development for reducing recurrent Clostridioides difficile infections (rCDI). The MHI-A algorithm relates the relative abundances of microbiome taxonomic classes that changed the most after RBX2660 or RBX7455 treatment, that strongly correlated with clinical response, and that reflect biological mechanisms believed important to rCDI. The diagnostic utility of MHI-A was reinforced using publicly available microbiome data from healthy or antibiotic-treated populations.Results: MHI-A has high accuracy to distinguish post-antibiotic dysbiosis from healthy microbiota. MHI-A values were consistent across multiple healthy populations and were significantly shifted by antibiotic treatments known to alter microbiota compositions, shifted less by microbiota-sparing antibiotics. Clinical response to RBX2660 and RBX7455 correlated with a shift of MHI-A from dysbiotic to healthy values.Conclusion: MHI-A is a promising biomarker of post-antibiotic dysbiosis and subsequent restoration. MHI-A may be useful for rank-ordering the microbiota-disrupting effects of antibiotics and as a pharmacodynamic measure of microbiota restoration.

FunHoMic: A Marie Skłodowska-Curie Innovative Training Network for the study of the Fungus-Host-Microbiota interplay

Introduction The FunHoMic project is a Marie Skłodowska-Curie Innovative Training Network comprising 13 PhD students, 8 academic partners and 3 industry partnersaimingto understand the interplay between fungi, hostsand microbiota to improve prevention and treatment of fungal infections. Importance About 2 billion people suffer fungal infections, which have a mortality rate close to that of malaria or breast cancer. Candida albicans has a high clinical and economic burden, making it of particular interest to the FunHoMic project. 70% of women experience at least one episode of vulvovaginal candidiasis (“thrush”) during their lifetime; 8% suffer recurring infections. C. albicans may live as a commensal but can cause symptoms when the fungus-host-microbiota equilibrium is disrupted. Infections by C. albicans have a significant clinical impact, with fatalities in severe cases. Many factors are associated with C. albicans infections; intensive care, neutropenic and diabetic patients are most at risk of systemic infection. Rising antifungal drug resistance has led to certain C. albicans infections having no treatment option. Aim The FunHoMic consortium combines projectson fungal pathogenesis, immunology, microbial ecology and’omics technologies to understand and exploit interactions between fungus, host and microbiota. Identification of novel bio markers on the fungal side such as genetic polymorphisms or on the host side such asmicrobiota profiles, metabolites and/or immune markers can lead to patient classification based on relative risk of infection. This could be the beginning of personalised management for fungal infections using preventive or therapeutic interventions like new antifungals, immune modulators or Live Biotherapeutic Products (LBPs). This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the MarieSklodowska-Curie grant agreement No 812969.

A Randomized Open Label Pilot Study of Clostridium Butyricum Miyairi 588 (CBM588) in Recipients of Allogeneic Hematopoietic Cell Transplantation

A Randomized Open Label Pilot Study of Clostridium Butyricum Miyairi 588 (CBM588) in Recipients of Allogeneic Hematopoietic Cell Transplantation

Live Biotherapeutic Products and Probiotics for the Skin

The skin microbiome is essential for the proper barrier functions of the skin, preventing the invasion of pathogens and regulating immune responses. When disrupted, recurrent conditions including atopic dermatitis, acne, or chronic wounds can result. Live biotherapeutic products (LBPs), an emerging class of therapeutics based on live bacteria, are promising for treating these challenging conditions. Several topical LBPs have entered clinical trials for the skin and are quickly reporting positive data from early trials. Herein, a selection of these clinical trials are analyzed to highlight the potential of topical LBPs and provide insights into their identification and development. In addition, the outstanding challenges that need to be addressed for the full translation of these LBPs into clinical therapeutics are commented. In doing so, an overview of the existing formulation landscape for microbial‐based topical products, including direct‐to‐consumer probiotics, is provided. Once the outstanding formulation and delivery challenges are addressed for LBPs and the first topical LBPs reach the market, they will be a promising modality that provides novel therapeutic mechanisms for addressing previously difficult‐to‐treat conditions.

Precise quantification of bacterial strains after fecal microbiota transplantation delineates long-term engraftment and explains outcomes

Fecal microbiota transplantation (FMT) has been successfully applied to treat recurrent Clostridium difficile infection in humans, but a precise method to measure which bacterial strains stably engraft in recipients and evaluate their association with clinical outcomes is lacking. We assembled a collection of >1,000 different bacterial strains that were cultured from the fecal samples of 22 FMT donors and recipients. Using our strain collection combined with metagenomic sequencing data from the same samples, we developed a statistical approach named Strainer for the detection and tracking of bacterial strains from metagenomic sequencing data. We applied Strainer to evaluate a cohort of 13 FMT longitudinal clinical interventions and detected stable engraftment of 71% of donor microbiota strains in recipients up to 5 years post-FMT. We found that 80% of recipient gut bacterial strains pre-FMT were eliminated by FMT and that post-FMT the strains present persisted up to 5 years later, together with environmentally acquired strains. Quantification of donor bacterial strain engraftment in recipients independently explained (precision 100%, recall 95%) the clinical outcomes (relapse or success) after initial and repeat FMT. We report a compendium of bacterial species and strains that consistently engraft in recipients over time that could be used in defined live biotherapeutic products as an alternative to FMT. Our analytical framework and Strainer can be applied to systematically evaluate either FMT or defined live bacterial therapeutic studies by quantification of strain engraftment in recipients.

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.

1024P Baseline biomarkers associated with clinical benefit in patients with solid tumors refractory to immune checkpoint inhibitors (ICIs) treated with live biotherapeutic MRx0518 in combination with pembrolizumab

MRx0518 is a novel, gut microbiome derived, single strain oral Live Biotherapeutic Product (LBP) with potent anti-tumour efficacy in multiple cancer models. MRx0518 has been shown to induce activation of CD8+ T-cells and suppress differentiation of Treg cells in vitro, and to increase the CD8/Treg cell ratio in murine models of cancer. In an ongoing phase I/II study (NCT03637803), preliminary antitumor activity has been observed in patient’s refractory to ICIs when MRx0518 is administered in combination with pembrolizumab.

The nasal mutualist Dolosigranulum pigrum AMBR11 supports homeostasis via multiple mechanisms

SummaryComparing the nasal microbiome of healthy individuals and chronic rhinosinusitis (CRS) patients revealed Dolosigranulum pigrum as a species clearly associated with nasal health, although isolates obtained from healthy individuals are scarce. In this study, we explored the properties of this understudied lactic acid bacterium by integrating comparative genomics, habitat mining, cultivation, and functional characterization of interaction capacities. Mining 10.000 samples from the Earth Microbiome Project of 17 habitat types revealed that Dolosigranulum is mainly associated with the human nasal cavity. D. pigrum AMBR11 isolated from the nose of a healthy individual exerted antimicrobial activity against Staphylococcus aureus, decreased proinflammatory cytokine production in airway epithelial cells, and Galleria mellonella larvae mortality induced by this important nasal pathobiont. Furthermore, the strain protected the nasal barrier function in a mouse model using interleukin-4 as disruptive cytokine. Hence, D. pigrum AMBR11 is a mutualist with high potential as topical live biotherapeutic product.

Entering First-in-Human Clinical Study With a Single-Strain Live Biotherapeutic Product: Input and Feedback Gained From the EMA and the FDA.

During the last decade, a plethora of novel therapies containing live microorganisms as active substance(s) has emerged with the aim to treat, prevent, or cure diseases in human beings. Both the Food and Drug Administration (FDA) and the European Directorate for the Quality of Medicines and Health Care (EDQM) codified these biotherapies as Live Biotherapeutic Products (LBPs). While these innovative products offer healthcare opportunities, they also represent a challenge for developers who need to set the most suitable designs for non-clinical and clinical studies in order to demonstrate a positive benefit/risk ratio through relevant quality, safety, and efficacy data that are expected by the drug competent authorities. This article describes how YSOPIA Bioscience, supported by the Pharmabiotic Research Institute (PRI), addressed the regulatory challenges during the early development phase of their single-strain LBP, Xla1, in order to obtain the necessary authorizations to bring this drug to the clinical stage.

Engineered NIR light-responsive bacteria as anti-tumor agent for targeted and precise cancer therapy

Engineered anaerobic bacteria known as live biotherapeutic products (LBPs) have shown great advances in cancer therapy. One advantage of anaerobic bacteria as drug carrier is that it spontaneously target to tumor and persistently release anti-tumor factors. To realize effective anti-cancer therapeutics, one essential premise is to improve the controllability of treatment. Here, we designed near-infrared (NIR)-light responsive bacteria as anti-tumor agent, which is based on a blue-light responsive module and upconversion nanoparticles. The upconversion nanoparticles converted external NIR light to local blue light to noninvasively activate blue-light responsive module (EL222) in engineered LBPs. The activated LBPs then produce tumor necrosis factor α (TNFα) for precise tumor ablation. In vitro and in vivo results have proven that this engineered NIR-light-responsive bacteria could efficiently inhibit tumor growth. We anticipate that this controllable and safe bacteria-based therapy can facilitate the application of LBPs to accurately and effectively regulate diseases.

Rationally designed bacterial consortia to treat chronic immune-mediated colitis and restore intestinal homeostasis

Environmental factors, mucosal permeability and defective immunoregulation drive overactive immunity to a subset of resident intestinal bacteria that mediate multiple inflammatory conditions. GUT-103 and GUT-108, live biotherapeutic products rationally designed to complement missing or underrepresented functions in the dysbiotic microbiome of IBD patients, address upstream targets, rather than targeting a single cytokine to block downstream inflammation responses. GUT-103, composed of 17 strains that synergistically provide protective and sustained engraftment in the IBD inflammatory environment, prevented and treated chronic immune-mediated colitis. Therapeutic application of GUT-108 reversed established colitis in a humanized chronic T cell-mediated mouse model. It decreased pathobionts while expanding resident protective bacteria; produced metabolites promoting mucosal healing and immunoregulatory responses; decreased inflammatory cytokines and Th-1 and Th-17 cells; and induced interleukin-10-producing colonic regulatory cells, and IL-10-independent homeostatic pathways. We propose GUT-108 for treating and preventing relapse for IBD and other inflammatory conditions characterized by unbalanced microbiota and mucosal permeability.

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

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. 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. 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. 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.

Preclinical monotherapeutic and anti-PD-1 synergistic anti-tumor efficacy of MNC-168, a novel human-derived live biotherapeutic product.

e14581 Background: Human gut microbiome has been extensively demonstrated as a critical causative factor in determining the efficacy of cancer immunotherapy. Cumulative clinical evidences indicate the prevalent presence of Enterococcus species in the responder of anti-PD-1 treated patients. We here demonstrated the preclinical anti-tumor efficacy of a novel human derived Enterococcus strain MNC-168. Methods: Using multiple murine syngeneic cold tumor models and in vivo pharmacokinetics mouse model, we characterized the anti-tumor activity and pharmacokinetics of MNC-168 via oral-delivered route in monotherapy and immune checkpoint inhibitor (ICI) anti-mPD-1 combination. Systematic multi-omics analyses and in vitro mechanistic study were conducted to reveal the biomarkers and mode of action of MNC-168. Results: We found MNC-168 significantly and mono-therapeutically counteract tumor development in diverse tumor models, including colorectal cancer (with tumor inhibition ratio of 65.5%), hepatocellular carcinoma (27.4%), fibrosarcoma (33.5%) and renal carcinoma (56.4%). Notably, MNC-168 in combination with anti-mPD-1 synergistically exerted the tumor inhibition ratio up to 68.3-89.2% and significantly reverse anti-mPD-1 non-responsiveness. Integrated multi-omics analyses and in vitro validation study revealed the mechanism by which MNC-168 reinvigorates the immune system through targeting multiple routes, including activating systemic and tumor-resident innate and adaptive immunity, removal of tumor physical barrier, reinforced recruitment of tumor-infiltrating lymphocytes, and maintenance of immuno-stimulant activity via production of immune-simulating metabolites. Activation and boosting of interferon-γ production of CD8 T cells and CD4 T cells are the vital mediators for the anti-tumor ability of MNC-168. In vivo pharmacokinetics study shows that the anti-tumor efficacy of MNC-168 is positively correlated with gut-restricted abundance of MNC-168. Conclusions: MNC-168 is highly potently used as a first-in-class live biotherapeutic product for cancer monotherapy and promoting efficacy of ICI-mediated cancer immunotherapy.