Abstract 5237: Engineered probiotics direct the antigen specificity of CAR-T cells in situ

Abstract Antigen-targeting therapies such as chimeric antigen receptor (CAR)-T cells have achieved unparalleled success in the treatment of hematological malignancies. However, broad success has remained bottlenecked by the lack of targets that are specifically, and uniformly, expressed on heterogenous solid tumors. In contrast, certain strains of bacteria are gaining recognition as a new class of antigen-agnostic cell therapies due to their selective growth within the immuno-suppressive niche of the solid tumor microenvironment (TME). Bridging these approaches, we developed a platform of probiotic-guided CAR-T cells (ProCARs) - in which tumor-colonizing probiotics release synthetic targets that efficiently tag tumor tissue for CAR-mediated lysis. Here, we engineered a well-characterized strain of probiotic bacteria, E. coli Nissle 1917, to sustain the intratumoral production of genetically encoded CAR targets and T cell attracting chemokines in multiple xenograft and syngeneic models of human and murine tumors. We design orthogonal CAR targets (Tags) as modified dimers of GFP that broadly anchor to collagens, fibronectins, and heparin sulfates found in high abundance within the solid TME to achieve antigen-agnostic tumor targeting. We demonstrate that Tags robustly coat the surface of cancer cell lines through interaction with cell surface matrix proteins, thus leading to the activation of GFP CAR-T cells and Tag-dependent killing across a panel of genetically distinct target cells. We additionally show that injected probiotics selectively grow within the tumor core and maintain the intratumoral production of Tags and human chemokines - leading to therapeutic efficacy across multiple subcutaneous and orthotopic tumor models. Moreover, we demonstrate a systemic antitumor benefit in immune-competent hosts, whereby unilateral treatment of primary tumors leads to a significant reduction in the growth rate of distal, untreated tumors. Finally, we show that intratumoral bacteria provide natural TLR agonists that trigger substantial activation of human and murine ProCAR-T cells. Our findings highlight the potential of the ProCAR platform to address the critical roadblock of identifying suitable CAR targets by providing an antigen in situ that is orthogonal to both healthy tissue and tumor genetics. Altogether, the use of a bacterial delivery platform in the ProCAR system offers a partner organism that naturally enhances CAR-T cell effector function and broadens the scope of CAR-T cell therapy to include previously difficult to target tumors. Citation Format: Rosa L. Vincent, Fangda Li, Ana Vardoshvili, Candice Gurbatri, Nicholas Arpaia, Tal Danino. Engineered probiotics direct the antigen specificity of CAR-T cells in situ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5237.