CAR19-T/NK cell and circular aptamer-drug conjugate (ApDC) combination treatment increases immunotherapy efficacy.

Chimeric antigen receptor (CAR)-based cell therapies have transformed the treatment of haematological malignancies, especially acute lymphoblastic leukaemia (ALL). However, drug resistance limits long-term efficacy. This study aimed to develop a novel combination therapy using aptamer‒drug conjugates and CAR19-T/natural killer (NK) cells to eliminate tumour cells completely and improve the efficacy of CAR-based cell therapies. A novel circular aptamer‒drug conjugate (C-ApDC) targeting protein tyrosine kinase-7 (PTK7) was designed and synthesized, and CD19 CAR-T and CAR-NK cells were constructed. The stability of C-ApDC was analysed by agarose gel electrophoresis, its binding specificity was evaluated by flow cytometry, and its cytotoxicity was measured by a CCK-8 assay. The synergistic effect between C-ApDC and CAR19-T/NK cells was comprehensively assessed through flow cytometry cytotoxicity analysis. To further validate the feasibility of combination therapy, we synthesized a novel C-ApDC-nanobody conjugate and combined it with CAR19-NK/T cells. The stability of the conjugate was analysed by agarose gel electrophoresis, and the cytotoxic effects of the combination regimen on tumour cells were detected by flow cytometry. C-ApDC exhibited greater stability than linear ApDC and specifically bound to and killed PTK7-expressing Nalm6 cells in vitro. C-ApDC significantly enhanced the cytotoxicity of CAR19-T/NK cells to tumour cells. Similarly, the C-ApDC-nanobody conjugate, when used in combination with CAR19-NK/T cells, exhibited high stability. A combination therapy composed of C-ApDC nanobodies and CAR19-T/NK cells was successfully developed. This innovative approach effectively enhances the cytotoxicity of CAR19-T/NK cells against tumour cells, providing a novel therapeutic strategy for tumour treatment and offering a promising solution to overcome CAR-T resistance.