[Preparation of Trop2-Targeted CAR-T Cells Based on Nanobodies and Their Antitumor Effects Against Non-Small Cell Lung Cancer].

Objective: To investigate whether chimeric antigen receptor (CAR) T cells constructed with nanobodies based on trophoblast cell-surface antigen 2 (Trop2) can be used to treat Trop2-positive non-small cell lung cancer.

Methods: A Trop2-specific phage display nanobody library was constructed to screen for Trop2-specific nanobodies. The antigen-binding capacities of three Trop2 nanobodies (8^#, 14^#, and 48^#) were assessed using indirect enzyme-linked immunosorbent assay (ELISA), and their binding affinities were analyzed through surface plasmon resonance (SPR) analysis. CAR-T cells were constructed with Trop2-specific nanobodies and were then co-cultured with the Trop2-positive NCI-H292 cell line expressing luciferase and the Trop2-negative A549 cell line. Luciferase values at effector-to-target ratios of 4∶1, 2∶1, 1∶1, and 1∶2 were measured using a multifunctional microplate reader to assess the killing efficiency. The levels of interleukin (IL)-2, interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α) cytokines in the supernatant at an effector-to-target ratio of 4∶1 were measured using the ELISA method. We also established in this study an NCI-H292 xenograft model in NCG immunodeficient mice, which were divided into three groups, a phosphate-buffered saline (PBS) control group, a Mock-T group, and a Trop2 CAR-T group (n = 5). A total of 1×10⁷ Trop2 CAR-T cells were administered via tail vein injection. Throughout the experimental period, the growth and survival status of the mice were observed daily, and tumor sizes were measured once every three days to analyze the survival time.

Results: A Trop2-specific nanobody was successfully screened from the nanobody library, and indirect ELISA initially indicated that nanobody 48^# had the strongest affinity. Subsequently, surface plasmon resonance analysis revealed that nanobody 48^# exhibited an affinity in the range of 2.49×10^－8 M, indicating that it was a high-affinity antibody. Based on this nanobody, Trop2 CAR-T cells were successfully constructed. Furthermore, in vitro experiments demonstrated that Trop2 CAR-T cells killed Trop2-positive NCI-H292 non-small cell lung cancer cells in a dose-dependent manner. ELISA showed a significant increase in the secretion of cytokines (IL-2, IFN-γ, and TNF-α) in the co-culture system, further validating their antitumor activity. In the NCI-H292 xenograft mouse model, the Trop2 CAR-T group exhibited reduced tumor size (P < 0.001) and prolonged survival time of tumor-bearing mice (P < 0.05) compared to the PBS and Mock-T groups.

Conclusion: These findings demonstrate that CAR T cells constructed with Trop2 nanobodies can effectively treat Trop2-positive non-small cell lung cancer.