Background In vivo CAR-T therapy, which relies on the targeted delivery of CAR constructs directly to endogenous T cells, represents a promising next-generation immunotherapy approach. Robust and predictive preclinical animal models are essential for evaluating target specificity, biodistribution, therapeutic efficacy, and safety of in vivo CAR-T. Here, we generated a series of target-humanized mouse models (e.g., CD5, CD8) for preclinical evaluation of in vivo CAR-T.
Methods To systematically validate the utility of these models for in vivo CAR-T evaluation, A rigorous, multi-parameter validation was performed on the CD8-humanized strain, including: 1) Flow cytometric quantification of human CD8 surface protein expression, 2) Comparative immunophenotyping of hematopoietic lineages to assess immune development, and 3) Functional assessment of human CD8 protein. For targeted delivery studies, anti-human CD8 antibody-targeted lipid nanoparticles (tLNPs) encapsulating reporter mRNA were administered systemically.
Results Key findings in CD8 humanized mice: 1) Murine CD8 was replaced by human CD8; the absolute expression level of human CD8 on CD8+ T cells in peripheral blood was comparable to that in human PBMC, with consistent expression profiles. 2) CD8 humanization did not affect murine immune system development or leukocytes cell subpopulation, and human CD8 retained normal biological functions. 3) CD8-tLNPs specifically delivered EGFP to CD8+ T cells in vitro and in vivo. These data confirm the models' applicability for evaluating targeted in vivo CAR-T delivery.
Conclusion We established and validated a panel of humanized models with consistent human target expression and intact immune function, capable of assessing CAR-T delivery. These models provide a robust and versatile platform for preclinical evaluation of in vivo CAR-T delivery, biodistribution, and efficacy. Hybridization further expands their utility for in vivo CAR-T efficacy testing and safety assessment. Overall, our models provide a valuable preclinical platform for the development and evaluation of in vivo CAR-T therapies.
