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.

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