用于CD19靶向T细胞衔接器临床前评估的CD3/CD19双人源化小鼠模型

Background: Immunotherapy has transformed the treatment landscape for cancer and autoimmune diseases, with T-cell engagers (TCEs) emerging as a highly promising therapeutic modality. Among them, CD19-targeting TCEs have achieved notable clinical success in B-cell malignancies such as leukemia and lymphoma. However, despite these advances, the development of more sophisticated preclinical models remains essential to better recapitulate human immune responses and to deepen our understanding of TCE efficacy and safety. To address this need, we established a novel CD3/CD19 double-humanized mouse model specifically designed to provide a more accurate and comprehensive in vivo platform for evaluating CD19-directed TCE therapies. Methods: The CD3 humanized mouse model was generated via ES cell-based gene targeting, in which full-length mouse Cd3e/Cd3d/Cd3g gene clusters were replaced with the corresponding human CD3E/CD3D/CD3G coding sequences. Separately, we developed the CD19 humanized mice using CRISPR/Cas9 engineering and subsequently crossed them with hCD3 strain to obtian the CD3/CD19 dual humanized mouse model. The expression of human CD3 and CD19 protein in hCD3/hCD19 dual knockin mice was confirmed by flow cytometry. We further characterized the model by examining immune cell subsets-including T and B cell populations-as well as basic physiological and biochemical parameters to ensure normal baseline health status. Finally, the hCD3/hCD19 dual knockin mice were treated with Blinatumomab, a first-in-class CD19-targeting BiTE used clinically for B cell malignancies, to evaluate its efficacy in depleting B cells in vivo. Results: As expected, the expression profiles of human CD3 and CD19 in hCD3/hCD19 dual knockin mice were consistent with those observed in human. Additionally, the CD3/CD19 humanized mice displayed a fully functional immune system with well-developed T and B cell populations, and their physiological parameters were comparable to those of wild-type controls. Following Blinatumomab administration, the hCD3/hCD19 mice exhibited robust B cells depletion, confirming the model's suitability for assessing CD19-directed therapeutic activity. Furthermore, in hCD3/hCD19 mice with induced systemic lupus erythematosus (SLE), Blinatumomab treatment reduced anti-dsDNA antibody levels in parallel with B cell depletion, indicating the utility of this dual humanized model for preclinical evaluation of anti-CD19 therapies for autoimmune disease. Conclusion: Our CD3/CD19 double-humanized mouse model represents an advanced and reliable tool for preclinical evaluation of CD19-targeted immunotherapies, including TCEs.