transfection and transduction of murine t-cells for car-t cell generation

Streamlined Protocol for Mouse CAR-T Cell Generation in Syngeneic Models

Adoptive T cell therapies expressing chimeric antigen receptors (CARs) have revolutionized the field of cancer immunotherapy by harnessing the power of the adaptive immune system to specifically target and eliminate antigen-positive cancer cells1. While the success of CAR-T cell therapies targeting B cell malignancies has been clinically validated, preclinical studies performed in animal models remain vital for the development of new CARs targeting solid tumors. However, limited clinical efficacy has been demonstrated in solid tumor indications thus far, and it is becoming increasingly apparent that individual preclinical models do not accurately predict the pharmacodynamics and clinical efficacy of a living medicine2,3. Therefore, investigators have begun to expand the preclinical study of CAR-T cell products to include parallel assessments in xenograft and syngeneic models of human and murine cancers, respectively.
Unlike xenograft models, where human tumors and T cells are engrafted into immunodeficient mice, syngeneic models enable the examination of CAR-T cell responses in the context of a functional immune system. Specifically, immune-competent mice bearing syngeneic tumors provide a system to study the interaction between adoptively transferred T cells and context-specific milieus - including tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) known to suppress T cell function in the tumor microenvironment (TME)4,5,6. Moreover, syngeneic models offer an additional platform to assess on-target, off-tumor toxicity, and CAR-T cell interaction with host factors that may lead to additional toxicities, including cytokine release syndrome7.
Despite these advantages, the number of syngeneic CAR-T cell studies remains limited. Notably, syngeneic models require autologous engineering of CAR-T cells from the same mouse strain and thus present an additional challenge due to the lack of methodology for efficient murine T cell transduction and ex vivo expansion2,8. This protocol outlines the methods to achieve stable CAR expression through the production of retroviral vectors and optimized T cell transduction. A schematic of the entire process is shown in Figure 1. The use of this approach demonstrates efficient retroviral transduction of murine CAR-T cells and the achievement of high CAR expression without the need for viral concentration through ultracentrifugation. Strategies to change the antigen-specificity of the CAR construct are discussed in addition to the co-expression of additional transgenes.

Author Spotlight: Enhancing CAR-T Cell Function in Syngeneic Tumor Models 

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

Our research explores effective combinations of engineered bacteria and CAR T-cells for solid tumor treatment. It's important for us to study how these two cell therapies function within the context of a complete immune system using syngeneic models of ovarian cancer. Studying the function of CAR T-cells in syngeneic tumor models has been limited by the lack of standardized protocols for generating murine CAR T-cells.Murine T-cells are particularly difficult to transduce in culture ex vivo using traditional methods. We developed this protocol to streamline the production of murine CAR T-cells to enable study in syngeneic models that provide important context to evaluate the activity of new CAR technologies.

Watch this Scientific Journal Video about Transfection and Transduction of Murine T-Cells for CAR-T Cell Generation at JoVE.com

Transfection and Transduction of Murine T-Cells for CAR-T Cell Generation  

transfection-transduction-murine-t-cells-for-car-t-cell

Research

JoVE Journal

Bioengineering

403 visualizações.  Columbia University. Para começar, pipetar 3,75 mililitros de soro reduzido em um tubo marcado A e diluir o meio com 105 microlitros do reagente de transfecção. Vórtice o conteúdo do tubo para misturar bem. Em seguida, adicione 3,75 mililitros do meio de soro reduzido em um tubo marcado B.Dilua os plasmídeos de expressão com 90 microlitros de reagente potencializador.Agora adicione o conteúdo do tubo A ao tubo B e pipetar a solução para misturar bem antes da incubação. Remova cuidadosamente 10 ...

Vídeo: Transfection and Transduction of Murine T-Cells for CAR-T Cell Generation  

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

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