HSC 人源化小鼠模型：体内 CAR-T 介导的 B 细胞清除及其在靶向药物筛选中的应用

Background : In the research of targeted therapy for B-cell related malignant tumors and autoimmune diseases, traditional animal models have limitations such as significant species differences and inadequate mimicry of human immune function, which hinder the efficiency of drug development. Hematopoietic Stem Cell (HSC)-humanized mice can reconstitute a functional human immune system, providing an ideal in vivo research platform to address these issues. Their application value in the evaluation of in vivo CAR-T (Invivo CAR-T) therapy and screening of innovative drugs urgently needs to be verified. Methods : A standardized protocol was used to construct the HSC-humanized mouse model: Severe immunodeficient NMG mice were preconditioned with sublethal dose irradiation (1.2 Gy) to disrupt the endogenous hematopoietic system and create space for human HSC engraftment. Subsequently, 1.5×10^5 sorted and purified human umbilical cord blood-derived CD34 + HSCs per mouse were transplanted via tail vein injection. After stable model reconstitution (peripheral blood hCD45 + cell ratio ≥25%), the CD19-targeted Invivo CAR-T system was used for intervention. CAR gene/mRNA was delivered via viral vectors (e.g., lentivirus, adeno-associated virus) or targeted lipid nanoparticles (tLNP). Following a single administration, the B-cell depletion efficiency in peripheral blood and spleen, as well as the CAR transfection positive rates in CD4 + T cells and CD8 + T cells, were detected simultaneously. Results : After transplantation, CD34 + HSCs successfully engrafted in the bone marrow of HSC-humanized mice and achieved multi-lineage differentiation, forming a stable population of human immune cells. The immune reconstitution exhibited a time-dependent characteristic: T cells accounted for 40%-50% at 14-20 weeks, B cells were the dominant population in the early stage, accompanied by low-proportion reconstitution of myeloid cells such as NK cells (average proportion ~2%) and monocytes. Human immune checkpoint molecules were expressed on the surface of immune cells. After Invivo CAR-T intervention, autologous B cells in the peripheral blood and spleen of mice were significantly depleted with a near-complete clearance efficiency, and positive CAR expression was detected in both CD4 + T cells and CD8 + T cells. Further analysis showed that B-cell reconstitution was dominated by naive B cells after depletion, suggesting functional resetting of the immune system. By synchronously monitoring CAR transfection efficiency and target cell clearance effect, it provides crucial technical support for the efficacy screening, dosage optimization, and safety evaluation of innovative drugs targeting B cells.