efficient-generation-of-murine-chimeric-antigen-receptor-car-t-cells

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological ...

Columbia University

Columbia College

Living bacteria therapies have been proposed as an alternative approach to treating a broad array of cancers. In this study, we developed a genetically encoded microbial encapsulation system with tunable and dynamic expression of surface capsular polysaccharides that enhances systemic delivery. Based on a small RNA screen of capsular biosynthesis pathways, we constructed inducible synthetic gene circuits that regulate bacterial encapsulation in Escherichia coli Nissle 1917. These bacteria are capable of temporarily evading immune attack, whereas subsequent loss of encapsulation results in effective clearance in vivo. This dynamic delivery strategy enabled a ten-fold increase in maximum tolerated dose of bacteria and improved anti-tumor efficacy in murine models of cancer. Furthermore, in situ encapsulation increased the fraction of microbial translocation among mouse tumors, leading to efficacy in distal tumors. The programmable encapsulation system promises to enhance the therapeutic utility of living engineered bacteria for cancer.

A programmable encapsulation system improves delivery of therapeutic bacteria in mice.

Tumors use multiple mechanisms to actively exclude immune cells involved in antitumor immunity. Strategies to overcome these exclusion signals remain limited due to an inability to target therapeutics specifically to the tumor. Synthetic biology enables engineering of cells and microbes for tumor-localized delivery of therapeutic candidates previously unavailable using conventional systemic administration techniques. Here, we engineer bacteria to intratumorally release chemokines to attract adaptive immune cells into the tumor environment. Bacteria expressing an activating mutant of the human chemokine CXCL16 (hCXCL16) offer therapeutic benefit in multiple mouse tumor models, an effect mediated via recruitment of CD8 T cells. Furthermore, we target the presentation of tumor-derived antigens by dendritic cells, using a second engineered bacterial strain expressing CCL20. This led to type 1 conventional dendritic cell recruitment and synergized with hCXCL16-induced T cell recruitment to provide additional therapeutic benefit. In summary, we engineer bacteria to recruit and activate innate and adaptive antitumor immune responses, offering a new cancer immunotherapy strategy.

Chemokines expressed by engineered bacteria recruit and orchestrate antitumor immunity.

A major challenge facing tumor-antigen targeting therapies such as chimeric antigen receptor (CAR)-T cells is the identification of suitable targets that are specifically and uniformly expressed on heterogeneous solid tumors. By contrast, certain species of bacteria selectively colonize immune-privileged tumor cores and can be engineered as antigen-independent platforms for therapeutic delivery. To bridge these approaches, we developed a platform of probiotic-guided CAR-T cells (ProCARs), in which tumor-colonizing probiotics release synthetic targets that label tumor tissue for CAR-mediated lysis in situ. This system demonstrated CAR-T cell activation and antigen-agnostic cell lysis that was safe and effective in multiple xenograft and syngeneic models of human and mouse cancers. We further engineered multifunctional probiotics that co-release chemokines to enhance CAR-T cell recruitment and therapeutic response.

Probiotic-guided CAR-T cells for solid tumor targeting.

Traditional 2D/3D co-culture models typically do not reflect the cellular heterogeneity of pancreatic ductal adenocarcinoma (PDAC) tumors, while in vivo models can be slow and ill-suited to mechanistic investigations. Here, we present a protocol for culturing murine PDAC explants and a corresponding human PDAC model using tissue slice explants. We describe steps for sponge production, preparation of media and materials, tissue collection, and sectioning. We then detail procedures for explant plating, daily culture, and collection of samples.

Generation and ex vivo culture of murine and human pancreatic ductal adenocarcinoma tissue slice explants.

The sparse vascularity of Pancreatic Ductal Adenocarcinoma (PDAC) presents a mystery: what prevents this aggressive malignancy from undergoing neoangiogenesis to counteract hypoxia and better support growth? An incidental finding from prior work on paracrine communication between malignant PDAC cells and fibroblasts revealed that inhibition of the Hedgehog (HH) pathway partially relieved angiosuppression, increasing tumor vascularity through unknown mechanisms. Initial efforts to study this phenotype were hindered by difficulties replicating the complex interactions of multiple cell types in vitro. Here we identify a cascade of paracrine signals between multiple cell types that act sequentially to suppress angiogenesis in PDAC. Malignant epithelial cells promote HH signaling in fibroblasts, leading to inhibition of non-canonical WNT signaling in fibroblasts and epithelial cells, thereby limiting VEGFR2-dependent activation of endothelial hypersprouting. This cascade was elucidated using human and murine PDAC explant models, which effectively retain the complex cellular interactions of native tumor tissues.

Article	Year
A programmable encapsulation system improves delivery of therapeutic bacteria in mice. Nature biotechnology\| PubMed ID: 35301496	2022
Chemokines expressed by engineered bacteria recruit and orchestrate antitumor immunity. Science advances\| PubMed ID: 36888717	2023
Probiotic-guided CAR-T cells for solid tumor targeting. Science (New York, N.Y.)\| PubMed ID: 37824640	2023
Generation and ex vivo culture of murine and human pancreatic ductal adenocarcinoma tissue slice explants. STAR protocols\| PubMed ID: 37950862	2023
Tumor Explants Elucidate a Cascade of Paracrine SHH, WNT, and VEGF Signals Driving Pancreatic Cancer Angiosuppression. Cancer discovery\| PubMed ID: 37966260	2023

Fangda Li

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