Preparation of Trop2-targeted chimeric antigen receptor T cells and study of their exhaustion status in vitro

To design and construct chimeric antigen receptor (CAR) T cells targeting Trop2, establish an in vitro cell exhaustion model through continuous antigen stimulation, and investigate their anti-tumor activity and exhaustion characteristics.

The second-generation CAR plasmid was constructed based on the single-chain variable fragment (scFv) sequence of Sacituzumab Govitecan targeting Trop2. The viral vector titer was determined by retroviral vector packaging and gradient dilution. Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using Ficoll density gradient centrifugation, and CAR virus vectors were transduced into PBMCs activated with OKT-3/IL-2 to generate Trop2-targeted CAR T cells. CAR expression levels were assessed by flow cytometry using MYC tags. In vitro 3 tumor cell models were established, including human ovarian cancer cells (SKOV3), human breast cancer cells (MDA-MB-453), and human lung cancer cells (A549). The expression of the Trop2 antigen in these models was confirmed using flow cytometry. Additionally, luciferase assay was employed to evaluate the cytotoxic efficiency of Trop2-targeted therapy at various effector-to-target ratios. An in vitro CAR-T exhaustion model was developed, and the long-term killing ability of CAR-T cells was dynamically monitored using the Incucyte live-cell imaging system. The PD-1/TIM-3 phenotype of CAR-T cells was analyzed by flow cytometry, and cytokine secretion levels were quantified using the cytometric bead array (CBA). Transcriptomic sequencing and RT-qPCR were employed to validate the differentially expressed genes associated with exhaustion.

The second-generation CAR T cells targeting Trop2 were successfully constructed. Compared to the P-T group, in vitro experiments demonstrated that these CAR T cells exhibited antigen-specific and dose-dependent cytotoxic effects against tumor cells with high Trop2 expression, such as MDA-MB-453 and SKOV3. A CAR-T cell exhaustion model established through repeated tumor antigen stimulation in vitro revealed that, compared to the initial state, the exhausted Trop2 CAR-T cells exhibited significantly reduced tumor-killing capacity while P-T cells showed almost no killing effect, the expression of inhibitory receptors (PD-1 and TIM-3) was up-regulated on the surface of exhausted CAR-T cells, and the secretion of effector cytokines was diminished. Transcriptomic analysis identified multiple differentially expressed genes in the exhausted CAR-T cells. Pathways related to immune response and T cell receptor signaling were down-regulated, while apoptosis-related pathways were activated. RT-qPCR further confirmed abnormal expression of immunoregulatory genes, including IL3, IL5, and IL13 (P<0. 05).

During continuous in vitro tumor antigen stimulation, the second-generation CAR-T cells targeting the Trop2 antigen demonstrate declined anti-tumor activity, weakened effector function and up-regulated expression of exhaustion-related molecules.