Anna Sokolovska’s research while affiliated with Synlogic, Inc. and other places

Dendritic cells (DCs) have a role in the development and activation of self-reactive pathogenic T cells1,2. Genetic variants that are associated with the function of DCs have been linked to autoimmune disorders3,4, and DCs are therefore attractive therapeutic targets for such diseases. However, developing DC-targeted therapies for autoimmunity requires identification of the mechanisms that regulate DC function. Here, using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies, we identify a regulatory loop of negative feedback that operates in DCs to limit immunopathology. Specifically, we find that lactate, produced by activated DCs and other immune cells, boosts the expression of NDUFA4L2 through a mechanism mediated by hypoxia-inducible factor 1α (HIF-1α). NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs that are involved in the control of pathogenic autoimmune T cells. We also engineer a probiotic that produces lactate and suppresses T cell autoimmunity through the activation of HIF-1α–NDUFA4L2 signalling in DCs. In summary, we identify an immunometabolic pathway that regulates DC function, and develop a synthetic probiotic for its therapeutic activation.

Purpose: SYNB1891 is a live, modified strain of the probiotic E. coli Nissle 1917 (EcN) engineered to produce cyclic dinucleotides under hypoxia leading to stimulator of interferon genes (STING)-activation in phagocytic antigen-presenting cells in tumors and activating complementary innate immune pathways. Patients and methods: This first-in-human study (NCT04167137) enrolled participants with refractory advanced cancers to receive repeat intratumoral (IT) injections of SYNB1891 either alone or in combination atezolizumab, with the primary objective of evaluating the safety and tolerability of both regimens. Results: Twenty-four participants received monotherapy across 6 cohorts, and 8 participants received combination therapy in 2 cohorts. Five cytokine release syndrome events occurred with monotherapy, including one that met the criteria for dose-limiting toxicity at the highest dose; no other SYNB1891-related serious adverse events occurred, and no SYNB1891-related infections were observed. SYNB1891 was not detected in the blood at 6 or 24h after the first IT dose or in tumor tissue 7 days following the first dose. Treatment with SYNB1891 resulted in activation of the STING pathway and target engagement as assessed by upregulation of interferon-stimulated genes, chemokines/cytokines, and T-cell response genes in core biopsies obtained pre-dose and 7 days following the third weekly dose. In addition, a dose‑related increase in serum cytokines was observed, as well as stable disease in 4 participants refractory to prior PD-1/L1 antibodies. Conclusions: Repeat IT injection of SYNB1891 as monotherapy and in combination atezolizumab was safe and well tolerated and evidence of STING pathway target engagement was observed.

Dendritic cells (DCs) control the generation of self-reactive pathogenic T cells. Thus, DCs are considered attractive therapeutic targets for autoimmune diseases. Using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies we identified a negative feedback regulatory pathway that operates in DCs to limit immunopathology. Specifically, we found that lactate, produced by activated DCs and other immune cells, boosts NDUFA4L2 expression through a mechanism mediated by HIF-1a. NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs involved in the control of pathogenic autoimmune T cells. Moreover, we engineered a probiotic that produces lactate and suppresses T-cell autoimmunity in the central nervous system via the activation of HIF-1α/NDUFA4L2 signaling in DCs. In summary, we identified an immunometabolic pathway that regulates DC function, and developed a synthetic probiotic for its therapeutic activation.

The field of cancer immunotherapy began with the injection of pathogenic bacteria intratumorally, but the broad adoption of bacteria in cancer therapeutics was historically limited by difficulty finding a balance between efficacy and safety. Synthetic biology now provides tools to precisely engineer bacteria to address some of the limitations of currently available immunotherapies by genetically modifying bacteria to express one or more immune effectors and deliver them to the tumor microenvironment. Multiple approaches have been developed preclinically, and several have progressed to early phase clinical trials. We describe the available synthetic biology toolkit as well as the current understanding of immune effectors that can be engineered into bacteria. Finally, we describe some of the regulatory considerations for developing engineered microbes for cancer immunotherapy.

Background SYNB1891 is a live, modified strain of probiotic E. coli Nissle engineered to produce cyclic dinucleotides under hypoxia leading to stimulator of interferon genes (STING)-activation in phagocytic antigen-presenting cells in tumors and activating complementary innate immune pathways. Methods This first-in-human study ( NCT04167137 ) enrolled patients with refractory advanced solid tumors to receive intratumoral (IT) injections of SYNB1891 monotherapy or in combination atezolizumab. Patients enrolled in the monotherapy arms received doses of 1x10 ⁶ - 3x10 ⁸ live cells on Days 1, 8 and 15 of the first 21-day cycle and then on Day 1 of each subsequent cycle. Patients enrolled in the 2 combination cohorts received doses of 1x10 ⁷ - 3x10 ⁷ live cells in combination with atezolizumab administered on a 21-day cycle. The primary objective of the study was to evaluate safety and tolerability of SYNB1891 alone and in combination with atezolizumab. Other objectives include SYNB1891 kinetics in blood and injected tumor, STING-target engagement as assessed by IT gene expression and serum cytokines, and tumor responses. Results This interim analysis includes 23 patients across 6 monotherapy cohorts dosed at 1x10 ⁶ , 3x10 ⁶ , 1x10 ⁷ , or 3x10 ⁷ , 1x10 ⁸ and 3x10 ⁸ live cells, and 7 patients dosed in 2 combination therapy cohorts (1x10 ⁷ and 3x10 ⁷ live cells). The mean (range) age was 61 (25–82); 19 patients were female. There were 4 cytokine release syndrome events in monotherapy cohorts, including one grade 3 event which met the criterion for dose limiting toxicity at 3x10 ⁸ live cells; there were no other SYNB1891-related serious adverse events. There were no SYNB1891-related infections. SYNB1891 was not detected in the blood at 6 or 24 hours after the first dose or intratumorally 7 days following the first dose. Treatment with SYNB1891 demonstrated activation of the STING pathway and target engagement as assessed by upregulation of interferon-stimulated genes (ISG15, IFIT1, IFIt2), chemokines/cytokines (CXCL9, CXCL10, TNFRS18, TNFSF10) and T-cell response genes (GZMA, CD4, PD-L2) in core biopsies obtained pre-dose and 7 days following the third weekly dose. In addition, there was a dose-response increase in serum cytokines. Durable, stable disease was observed in two patients treated with SYNB1891 monotherapy refractory to prior PD-1/L1 antibodies with vulvar melanoma (1x10 ⁶ live cells; RECIST -28%) and small cell lung cancer (1x10 ⁷ live cells; RECIST -12%). Conclusions Repeat IT injection of SYNB1891 as monotherapy and in combination atezolizumab in this ongoing study is safe and well-tolerated up to at least 1x10 ⁸ live cells, and shows evidence of STING pathway target engagement. Acknowledgements We thank Inessa Vulfova for her clinical support in conduct of this study. Trial Registration clinicaltrials.gov ( NCT04167137 ) Ethics Approval The study protocol, the informed consent form (ICF), and printed subject information materials were reviewed and approved by the institutional review board (IRB) at the investigational site before any study procedures were performed. Written informed consent to participate in the study was obtained from each subject before any study-specific procedures were performed.The Ohio State University Cancer Institutional Review Board; Approval ID: 2020C0194MD Anderson Cancer Center Institutional Review Board; Approval ID: 2019–0576Mary Crawley Medical Research Center Institutional Review Board; Approval ID: 19–31 SYNB1891-CP-001North Texas Institutional Review Board; Approval ID: 2019.040WIRB Approval ID: 20192779University of Pittsburgh Institutional Review Board Approval ID: STUDY20010116

The availability of l-arginine in tumours is a key determinant of an efficient anti-tumour T cell response1–4. Consequently, increases of typically low l-arginine concentrations within the tumour may greatly potentiate the anti-tumour responses of immune checkpoint inhibitors, such as programmed death-ligand 1 (PD-L1)-blocking antibodies⁵. However, currently no means are available to locally increase intratumoural l-arginine levels. Here we used a synthetic biology approach to develop an engineered probiotic Escherichia coli Nissle 1917 strain that colonizes tumours and continuously converts ammonia, a metabolic waste product that accumulates in tumours⁶, to l-arginine. Colonization of tumours with these bacteria increased intratumoural l-arginine concentrations, increased the number of tumour-infiltrating T cells and had marked synergistic effects with PD-L1 blocking antibodies in the clearance of tumours. The anti-tumour effect of these bacteria was mediated by l-arginine and was dependent on T cells. These results show that engineered microbial therapies enable metabolic modulation of the tumour microenvironment leading to enhanced efficacy of immunotherapies.

The steadfast advance of the synthetic biology field has enabled scientists to use genetically engineered cells, instead of small molecules or biologics, as the basis for the development of novel therapeutics. Cells endowed with synthetic gene circuits can control the localization, timing and dosage of therapeutic activities in response to specific disease biomarkers and thus represent a powerful new weapon in the fight against disease. Here, we conceptualize how synthetic biology approaches can be applied to programme living cells with therapeutic functions and discuss the advantages that they offer over conventional therapies in terms of flexibility, specificity and predictability, as well as challenges for their development. We present notable advances in the creation of engineered cells that harbour synthetic gene circuits capable of biological sensing and computation of signals derived from intracellular or extracellular biomarkers. We categorize and describe these developments based on the cell scaffold (human or microbial) and the site at which the engineered cell exerts its therapeutic function within its human host. The design of cell-based therapeutics with synthetic biology is a rapidly growing strategy in medicine that holds great promise for the development of effective treatments for a wide variety of human diseases.

Introduction: SYNB1891 is a live, modified strain of the probiotic E. coli Nissle engineered to produce cyclic dinucleotides under hypoxia leading to stimulator of interferon genes (STING)-activation in phagocytic antigen-presenting cells in tumors and activating complementary innate immune pathways. Methods: This first-in-human study (NCT04167137) is enrolling patients with refractory advanced solid tumors or lymphoma to receive an intratumoral (IT) injection of SYNB1891 on Days 1, 8 and 15 of the first 21-day cycle and then on Day 1 of each subsequent cycle. Dose escalation is planned across 7 cohorts (1x106 - 1x109 live cells) with Arm 1 consisting of escalating doses of SYNB1891 as monotherapy, and Arm 2 in combination with atezolizumab. The primary objective is to determine the single-agent maximum tolerated dose as monotherapy and the recommended Phase 2 dose in combination with atezolizumab. Other objectives include SYNB1891 kinetics in blood and the injected tumor, STING-target engagement as assessed by IT gene expression and serum cytokines, and tumor responses. Results: This interim analysis includes the first 11 patients across 4 cohorts dosed at 1x106, 3x106, 1x107, or 3x107 live cells, with a total of 59 doses administered. The mean (range) age was 56 (25-70); 9 patients were female. There were no dose-limiting toxicities, SYNB1891-related infections or discontinuations due to adverse events. There was one SYNB1891-related serious adverse event in a patient who experienced a grade 2 cytokine release syndrome that resolved within one day, and one patient experienced a grade 2 injection site reaction of erythema which resolved. SYNB1891 was not detected in the blood at 6 or 24 hours after the first dose or intratumorally 7 days following the first dose. Treatment with SYNB1891 demonstrated activation of the STING pathway and target engagement as assessed by upregulation of interferon-stimulated genes (ISG15, IFIT1, IFIt2), chemokines/cytokines (CXCL9, CXCL10, TNFRS18, TNFSF10) and T-cell response genes (GZMA, CD4, PD-L2) in core biopsies obtained pre-dose and 7 days following the third weekly dose. In addition, there was a dose-response increase in serum cytokines at dose levels of 1x107 and 3x107 live cells (IL-6, TNFα, IFNγ, IL-1Ra) at 6 hours post-dose. Durable, stable disease was observed in two patients refractory to prior PD-1/L1 antibodies with vulvar melanoma (1x106 live cells) and small cell lung cancer (1x107 live cells). Conclusions: Repeat IT injection of SYNB1891 as monotherapy is safe and well-tolerated up to at least 3x107 cells and shows evidence of STING pathway target engagement. These data support the continued dose escalation of SYNB1891 as monotherapy, and initiation of Arm 2 in combination with atezolizumab. Citation Format: Filip Janku, Jason J. Luke, Aoife Brennan, Richard Riese, Mary Varterasian, Michael B. Armstrong, Karen L. Kuhn, Anna Sokolovska, James F. Strauss. Intratumoral injection of SYNB1891, a synthetic biotic designed to activate the innate immune system, demonstrates target engagement in humans including intratumoral STING activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT110.

The availability of L-arginine in tumors is a key determinant of an efficient anti-tumor T cell response. Consequently, elevation of typically low L-arginine levels within the tumor may greatly potentiate the anti-tumor responses of immune checkpoint inhibitors, such as PD-L1 blocking antibodies. However, currently no means are available to locally increase intra-tumoral L-arginine levels. Here, we used a synthetic biology approach to develop an engineered probiotic Escherichia coli Nissle 1917 strain that colonizes tumors and continuously converts ammonia, a metabolic waste product that accumulates in tumors4, into L-arginine. Colonization of tumors with these bacteria elevated intra-tumoral L-arginine concentrations, increased the amount of tumor-infiltrating T cells, and had striking synergistic effects with PD-L1 blocking antibodies in the clearance of tumors. The anti-tumor effect of the living therapeutic was mediated by L-arginine and was dependent on T cells. These results show that engineered microbial therapies enable metabolic modulation of the tumor microenvironment leading to enhanced efficacy of immunotherapies.