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Recent advances in immunotherapy demonstrate the need to further understand the characteristics of an individual cancer patient’s immune system and how it influences responses to cancer treatment. Here, we developed an immunoprofiling platform to evaluate the features in the blood of cancer patients to test the hypothesis that peripheral immune cell heterogeneity could be used to stratify these patients into different categories or immunotypes to monitor disease progression and treatment response. To that end, we established a unique diagnostic immunoprofiling assay and analytical framework based on the analysis of leukocytes in the peripheral blood using multiparameter flow cytometry. Supervised manual gating of flow cytometry data from a cohort of 50 healthy donors identified 415 cell types and immune activation states that were used to train and later independently validate machine learning models to automatically identify immune cell subsets from raw cytometry data. By applying this tool to peripheral blood samples from a mixed cohort of 299 healthy donors and 323 cancer patients, we developed a machine-learning classification model that can differentiate between these two groups with 93% accuracy. This model was further refined using spectral clustering with bootstrapping, revealing 5 clusters, or immunotypes, characterized by specific physiological immune profiles: (1) Myeloid-derived suppressor/NK cell, (2) Terminally-differentiated CD8+ T cells, (3) Mixed CD4+ T helper cells, (4) CD4+ Th1 & CD8+ T cell memory, and (5) Naive T and B lymphocytes. Interestingly, very few healthy donors could be found in clusters 1 and 2 but were assigned most frequently to cluster 5. Matched RNA-seq was used to further validate these profiles using the cellular deconvolution algorithm, Kassandra, and differential gene expression analysis revealed immunotype-specific signatures that are consistent with immune response potential. Patients in the terminally-differentiated CD8+ T cell cluster had a narrower range of HLA-types than the other clusters, and TCR repertoire analysis indicated significantly increased clonality and reduced clonotype diversity. Within this cluster there was a high degree of overlap between TCR sequences in the peripheral blood and the tumor, indicating a relationship between peripheral blood immunotype and tumor infiltration. Altogether, the establishment of these immunotypes using peripheral blood immunoprofiling represents a promising signature that can be used to identify and stratify cancer patients that will benefit from immune-based therapies. Citation Format: Daniiar Dyikanov, Iris Wang, Tatiana Vasileva, Polina Shpudeiko, Polina Turova, Arseniy A. Sokolov, Olga Golubeva, Evgenii Tikhonov, Anna Kamysheva, Ilya Krauz, Mary Abdou, Madison Chasse, Tori Conroy, Nicholas R. Merriam, Boris Shpak, Anastasia Radko, Anastasiia Kilina, Lira Nigmatullina, Linda Balabanian, Christopher J. Davitt, Alexander A. Ryabykh, Olga Kudryashova, Cagdas Tazearslan, Ravshan Ataullakhanov, Alexander Bagaev, Aleksandr Zaitsev, Nathan Fowler, Michael F. Goldberg. Comprehensive immunoprofiling of peripheral blood reveals five conserved immunotypes with implications for immunotherapy in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6664.

Background: Systemic immunity can be regulated via transient interactions of non-colonizing bacteria and immune cells in the small intestinal mucosa. The potential to harness these interactions for IO treatment was first described by Sivan in 2015. EDP1503 is a non-colonizing preparation of a single strain of Bifidobacterium animalis lactis that, when administered orally, in preclinical models, invokes an anti-tumor immune response by activation of innate and adaptive immune mechanisms. The pleiotropic effects of EDP1503 are initiated in the small intestine and mediated by interactions with multiple pattern-recognition receptors inducing a proinflammatory signature in human PBMCs, activating CD8 and NK cell IFNγ production and cytolytic activity. Preclinically, EDP1503 inhibits tumor growth as a monotherapy and in combination with anti-PD-1/L1. Single agent PD-1/L1 therapies have showed limited clinical benefit in previously treated triple-negative breast cancer (TNBC) patients. Here, we report the safety, tolerability, and efficacy of EDP1503 in combination with pembrolizumab in patients with TNBC (NCT03775850). Methods: EDP1503-101 is an open label Phase 1/2 study of EDP1503 in combination with pembrolizumab. TNBC subjects who had received ≥ 1 treatment regimen for metastatic disease were enrolled. Prior anti-PD-1/L1 treatment was permitted. Subjects initially receive 14 days of EDP1503 monotherapy orally twice a day (b.i.d) and then EDP1503 b.i.d. in combination with pembrolizumab 200 mg iv every 3 weeks. The initial 3 subjects received 2 capsules of EDP1503 b.i.d. All other subjects received 4 capsules b.i.d. Safety and tolerability were assessed using CTCAE v5.0. Evidence of anti-tumor activity was based on investigator-assessed objective response (OR), by RECIST v1.1 and iRECIST, and disease control rate defined as OR and/or stable disease (SD) after 4 cycles of therapy. Paired biopsies at baseline and day 14 were used to establish PD-L1 status and investigate pharmacodynamic biomarker changes. Results: As of 1 October 2020, 15 TNBC subjects had been treated (median age of 52, median of 2 prior lines of therapy and ECOG of 0-1). Additional subjects are being recruited, up to a maximum of 30. EDP1503 was well-tolerated as monotherapy and in combination with pembrolizumab. 53% of subjects experienced treatment-related adverse events (TRAEs). Most common TRAEs were GI-related: abdominal distension (20%), decreased appetite (20%), diarrhea (13%), flatulence (13%). No Grade 4-5 TRAEs were observed. 1 Grade 3 TRAE (diarrhea) leading to treatment discontinuation was reported. In 12 subjects receiving the 4 capsules b.i.d dose, 2 partial responses (PR) and 1 SD were observed, giving an objective response rate (ORR) of 18% and a disease control rate (DCR) of 27% in evaluable patients (n=11). Both responders had tumor burden reductions of >65% by RECIST. One responder had 4 prior lines of therapy for metastatic disease, including a PD-L1 inhibitor combination, has a PD-L1 combined positive score (CPS) of 30 and has been on study treatment for 192 days with a 66% reduction in target lesions. The only residual lesion is 6 mm which is PET-negative. The other responder had 2 prior lines of therapy for metastatic disease, was checkpoint inhibitor naive, has a PD-L1 CPS of 2 and remained on study treatment for 275 days with a 73% reduction in target lesions before discontinuing due to treatment-related AEs. The patient with SD had also previously relapsed on a PD-L1 inhibitor and was on study treatment for 226 days before progressing. Conclusions: EDP1503 administered with pembrolizumab is safe and well-tolerated with no Grade 4-5 TRAEs or SAEs. Clinical benefit was observed in a subset of TNBC patients treated with the combination of EDP1503 and pembrolizumab. This study is continuing to recruit TNBC patients at the high dose of EDP1503. Citation Format: Loise Francisco-Anderson, Shamira Shariffudin, Humphrey Gardner, Peter Sandy, Michael Goldberg, Shubhra Kashyap, Mary Abdou, Maria Sizova, Valeria Kravitz, Holly Ponichtera, Mark Carlson, Shannon Argueta, Chris Davitt, Pooja Parameswaran, Michael Chisamore, Mark Bodmer, Duncan McHale. A phase I/II clinical trial of EDP1503 with pembrolizumab for triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-27.

Cellular deconvolution algorithms virtually reconstruct tissue composition by analyzing the gene expression of complex tissues. We present the decision tree machine learning algorithm, Kassandra, trained on a broad collection of9,400 tissue and blood sorted cell RNA profiles incorporated into millions of artificial transcriptomes to accurately reconstruct the tumor microenvironment (TME). Bioinformatics correction for technical and biological variability, aberrant cancer cell expression inclusion, and accurate quantification and normalization of transcript expression increased Kassandra stability and robustness. Performance was validated on 4,000 HE slides and 1,000 tissues by comparison with cytometric, immunohistochemical, or single-cell RNA-seq measurements. Kassandra accurately deconvolved TME elements, showing the role of these populations in tumor pathogenesis and other biological processes. Digital TME reconstruction revealed that the presence of PD-1-positive CD8

Introduction The mucosal immune system comprises of a complex network of intestinal epithelial cells and immune cells. We report that an orally delivered microbial strain of Veillonella parvula (EDP1867) that is non-replicating and non-colonizing acts through pattern recognition receptor mediated modulation of small intestinal immune responses to induce potent systemic inflammation resolution. Methods EDP1867 is a strain of Veillonella parvula isolated from mucosal tissue of a human donor and rendered non replicating by gamma irradiation. In vitro experiments were performed in primary human immune cells. In vivo efficacy was done using mouse models of delayed-type hypersensitivity (DTH) and psoriasis. Fluorescently labeled EDP1867 was used in biodistribution experiments. Results EDP1867 induced IL-10 in primary human immune cells. In a DTH model, orally delivered EDP1867 reduced inflammation and diminished production of pro-inflammatory cytokines ex vivo. Fluorescently labeled EDP1867 transited through the gut without systemic exposure in biodistribution studies to demonstrate gut restriction. EDP1867 activity is dependent on TLR2 and migration of immune cells through the gut. In an imiquimod-induced psoriasis model, EDP1867 reduced skin inflammation and psoriasis score and Th17 cytokines in the skin. Conclusions EDP1867 drives broad based resolution of inflammation and can reestablish normal homeostasis. These data demonstrate that EDP1867 can act as a new oral anti-inflammatory drug that is gut restricted and dependent on local immune recognition within the small intestine, to induce systemic anti-inflammatory effects. EDP1867 is currently in clinical development for treatment of inflammatory diseases.

Background The small intestinal axis (SINTAX) is a network of anatomic and functional connections between the small intestine and the rest of the body. It acts as an immunosurveillance system, integrating signals from the environment that affect physiological processes throughout the body. The impact of events in the gut in the control of tumor immunity is beginning to be appreciated. We have previously shown that an orally delivered single strain of commensal bacteria induces anti-tumor immunity preclinically via pattern recognition receptor-mediated activation of innate and adaptive immunity. Some bacteria produce extracellular vesicles (EVs) that share molecular content with the parent bacterium in a particle that is roughly 1/1000th the volume in a non-replicating form. We report here an orally-delivered and gut-restricted bacterial EV which potently attenuates tumor growth to a greater extent than whole bacteria or checkpoint inhibition. Methods EDP1908 is a preparation of extracellular vesicles produced by a gram-stain negative strain of bacterium of the Oscillospiraceae family isolated from a human donor. EDP1908 was selected for its immunostimulatory profile in a screen of EVs from a range of distinct microbial strains. Its mechanism of action was determined by ex vivo analysis of the tumor microenvironment (TME) and by in vitro functional studies with murine and human cells. Results Oral treatment of tumor-bearing mice with EDP1908 shows superior control of tumor growth compared to checkpoint inhibition (anti-PD-1) or an intact microbe. EDP1908 significantly increased the percentage of IFNγ and TNF producing CD8+ CTLs, NK cells, NKT cells and CD4+ cells in the tumor microenvironment (TME). EDP1908 also increased tumor-infiltrating dendritic cells (DC1 and DC2). Analysis of cytokines in the TME showed significant increases in IP-10 and IFNg production in mice treated with EDP1908, creating an environment conducive to the recruitment and activation of anti-tumor lymphocytes. Conclusions This is the first report of striking anti-tumor effects of an orally delivered microbial extracellular vesicle. These data point to oral EVs as a new class of immunotherapeutic drugs. They are particularly effective at harnessing the biology of the small intestinal axis, acting locally on host cells in the gut to control distal immune responses within the TME. EDP1908 is in preclinical development for the treatment of cancer. Ethics Approval Preclinical murine studies were conducted under the approval of the Avastus Preclinical Services’ Ethics Board. Human in vitro samples were attained by approval of the IntegReview Ethics Board; informed consent was obtained from all subjects.

The small intestinal axis (SINTAX) is a network of anatomic and functional connections between the small intestine and the rest of the body. It acts as an immunosurveillance system, integrating signals from the environment that affect physiological processes throughout the body. The impact of events in the gut in the control of tumor immunity is beginning to be appreciated. We have previously shown that an orally delivered single strain of commensal bacteria induces anti-tumor immunity via activation of innate and adaptive immunity. Some bacteria produce extracellular vesicles (EVs) that share molecular content with the parent bacterium in a particle that is roughly 1/1000th the volume in a non-replicating form. We report here an orally-delivered and gut-restricted bacterial EV which potently attenuates tumor growth to a greater extent than whole bacteria or checkpoint inhibition. EDP1908 is a preparation of EVs produced by a bacterium of the Oscillospiraceae family isolated from a human donor. Treatment of tumor-bearing mice with EDP1908 shows superior control of tumor growth compared to anti-PD-1 or an intact microbe. EDP1908 significantly increases the percentage of IFNγ producing CD8+, NK, NKT and CD4+ cells in the tumor microenvironment (TME). EDP1908 also increases tumor-infiltrating dendritic cells (DC1 and DC2) and production of IP-10 and IFNg in the TME, creating an environment conducive to the recruitment and activation of anti-tumor lymphocytes. These data point to oral EVs as a new class of immunotherapeutic drugs that harness the biology of the small intestinal axis, acting locally on host cells in the gut to control distal immune responses within the TME. EDP1908 is in preclinical development for the treatment of cancer.