Synthetic biology is a powerful tool to create therapeutics which can be rationally designed to enable unique and combinatorial functionalities. Here we utilize non-pathogenic E coli Nissle as a versatile platform for the development of a living biotherapeutic for the treatment of cancer. The engineered bacterial strain, referred to as SYNB1891, targets STING-activation to phagocytic antigen-presenting cells (APCs) in the tumor and activates complementary innate immune pathways. SYNB1891 treatment results in efficacious antitumor immunity with the formation of immunological memory in murine tumor models and robust activation of human APCs. SYNB1891 is designed to meet manufacturability and regulatory requirements with built in biocontainment features which do not compromise its efficacy. This work provides a roadmap for the development of future therapeutics and demonstrates the transformative potential of synthetic biology for the treatment of human disease when drug development criteria are incorporated into the design process for a living medicine.

The EZH2 small-molecule inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase II clinical trials for the treatment of non-Hodgkin lymphoma (NHL). We have previously shown that EZH2 inhibitors display an antiproliferative effect in multiple preclinical models of NHL, and that models bearing gain-of-function mutations in EZH2 were consistently more sensitive to EZH2 inhibition than lymphomas with wild-type (WT) EZH2. Here, we demonstrate that cell lines bearing EZH2 mutations show a cytotoxic response, while cell lines with WT-EZH2 show a cytostatic response and only tumor growth inhibition without regression in a xenograft model. Previous work has demonstrated that cotreatment with tazemetostat and glucocorticoid receptor agonists lead to a synergistic antiproliferative effect in both mutant and wild-type backgrounds, which may provide clues to the mechanism of action of EZH2 inhibition in WT-EZH2 models. Multiple agents that inhibit the B-cell receptor pathway (e.g., ibrutinib) were found to have synergistic benefit when combined with tazemetostat in both mutant and WT-EZH2 backgrounds of diffuse large B-cell lymphomas (DLBCL). The relationship between B-cell activation and EZH2 inhibition is consistent with the proposed role of EZH2 in B-cell maturation. To further support this, we observe that cell lines treated with tazemetostat show an increase in the B-cell maturation regulator, PRDM1/BLIMP1, and gene signatures corresponding to more advanced stages of maturation. These findings suggest that EZH2 inhibition in both mutant and wild-type backgrounds leads to increased Bcell maturation and a greater dependence on B-cell activation signaling.

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The EZH2 inhibitor tazemetostat (EPZ-6438) is emerging as a promising therapeutic agent for the treatment of non-Hodgkin's Lymphoma (NHL). A significant body of work has now demonstrated in vitro and in vivo effects of EZH2 inhibition in preclinical models of lymphoma, in addition to objective clinical responses in early human trials. While EZH2 gain-of-function mutations clearly contribute to lymphomagenesis, patients with lymphomas harboring wild-type EZH2 also show responses to tazemetostat. This suggests a broad role of EZH2 in B-cell oncogenesis. Several recent mouse model studies have demonstrated the importance of wild-type EZH2 catalytic activity in the formation of germinal centers in non-diseased lymph nodes, suggesting a central role for EZH2 in B-lymphocyte maturation. These findings indicate that the importance of EZH2 to B-cell lymphoma likely lies in its ability to regulate B-cell differentiation. To understand the relationship between B-cell maturation and sensitivity to EZH2 inhibition, we evaluated changes in maturation markers and cellular proliferation following treatment of diffuse large B-cell lymphoma (DLBCL) cell lines with tazemetostat in combination with modulators of B-cell activation. Consistent with the importance of EZH2 in the regulation of B-cell differentiation, we observed increased expression of B-cell maturation markers in DLBCL cell lines treated with single agent tazemetostat in vitro. Furthermore, we demonstrate that tazemetostat pre-treatment of subsets of DLBCL cells lines (both EZH2 mutant and wild-type) can sensitize cells to inhibitors of B-cell activation pathways, which include glucocorticoids and BTK, MAPK and PI3K pathway inhibitors. Moreover, the anti-proliferative activity of single agent tazemetostat can be diminished or delayed by co-treatment with biological stimulators of B-cell activation including B-cell receptor ligation, CD40L, LPS and BAFF. Importantly, B-cell receptor ligation and co-stimulation agents have little proliferative effects on DLBCL cell lines on their own, suggesting that the protective function of these agents is directly related to the effects of EZH2 inhibition and not a generic stimulation of proliferation. Our findings suggest that EZH2 inhibition initiates a differentiation program that enables lymphoma cells to proceed through the normal processes of B-cell selection, growth regulation and maturation. Citation Format: Danielle Johnston, Dorothy Brach, Christopher Plescia, Alison Drew, Trupti Lingaraj, Natalie Warholic, Jesse J. Smith, Robert A. Copeland, Heike Keilhack, Elayne Penebre, Sarah K. Knutson, Scott Ribich, Michael J. Thomenius, Alejandra Raimondi. EZH2 plays a critical role in B-cell maturation and in non-Hodgkin's lymphoma: Interplay between EZH2 function and B-cell activation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B85.

TPS2664 Background: High-risk strains of HPV (HPV 16/18) have been associated with the development of multiple cancers, and the associated viral antigens are validated targets from immunotherapy approaches. We engineered red blood cells into allogeneic, off-the-shelf, artificial antigen-presenting cells (aAPCs) that express a human papillomavirus (HPV) 16 E7 peptide bound to human leukocyte antigen (HLA)-A*02:01, the costimulatory molecule 4-1BB ligand (L), and the cytokine interleukin (IL)-12 on the cell surface. This aAPC, RTX-321, activated HPV specific T-cells and promoted effector function in vitro. In animal models using a murine surrogate system, this aAPC approach resulted in robust antigen-specific T-cell expansion, NK cell expansion, tumor control, memory formation and antigen spreading, which led to a broad and robust antitumor immune response . The presence of 4-1BBL and IL-12 induced minimal toxicities in these models due to restriction of the biodistribution of the aAPC to the vasculature and spleen. RTX-321 is a potential in vivo cellular immunotherapy for treating HPV 16-positive cancers including cervical, head and neck and anal cancers. Methods: The RTX-321-01 study is a phase 1 multi-center, dose-escalation study of RTX-321 administered intravenously every 3 weeks in HLA-A*02:01-positive patients with relapsed or refractory HPV 16-positive cancers of the cervix or anal canal, or squamous cell cancers of the head and neck (HNSCC). Patients with cervical cancer or HNSCC will undergo testing for the presence of the HPV 16 virus or provide confirmation from archival tumor tissue prior to enrollment. Patients with anal cancer will not be required to have prospective determination of HPV 16-positive status prior to enrollment given the high incidence in this indication (approximately 80-85 percent of anal cancers). Approximately 18 patients will be enrolled across dose level cohorts to identify the recommended phase 2 dose (RP2D) of RTX-321, followed by RP2D expansion cohorts in specific indications. The starting dose is 1 billion (1x109) cells administered intravenously every 3 weeks (Q3W) and the dose will escalate by half-log increments, following a Bayesian logarithmic regression model (BLRM) with overdose control. Translational studies will investigate the activation and expansion of HPV16 E7 antigen-specific responses as well as broad innate and adaptive responses in multiple peripheral blood samples over the first 3 cycles of therapy as well as in optional paired tumor biopsies. At this time, the study is open and enrolling patients in the first dose escalation cohort (NCT04672980). Clinical trial information: NCT04672980.