Enhancement of HER2-CAR-CIK Against Ovarian Cancer Cell by the Engineered NK-92MI
WU Guangyuan1#, AN Zhongyao1,2#, LIU Xiaolong1, WANG Hailong1, YU Kuo1, WANG Xin1, LIU Yang1,
QI Pengfei1, FAN Shengjin3,4, WANG Zhenkun1,3*
CAR-CIK therapy is a promising new strategy; however, its efficacy is limited by the functional exhaustion of CIK cells caused by traditional expansion methods. Therefore, increasing the proportion of the critical CD3+CD56+ cell subpopulation and maintaining their function is central for enhancing therapeutic outcomes. This study employed genetic engineering to generate NK-92MI cells co-expressing membrane-bound IL-21 and CD137L (hereinafter referred to as IC-92). A co-culture system combining IC-92 cells with cytokines (IFN-γ, OKT3, IL-1α, and IL-2) was established to expand both CIK and HER2-CAR-CIK cells. Flow cytometry was used to detect the expression of effector molecules, while the Incucyte S3 live-cell analysis system was utilized to evaluate the HER2-specific tumor-killing capacity of CAR-CIK cells. Integrated RNA sequencing and LC-MS-based untargeted metabolomics were applied to preliminarily investigate the mechanisms by which the IC-92 and cytokine co-culture strategy modulates CIK cell function. The results demonstrated that the co-culture significantly enhanced the proliferative activity of CIK cells and increased the proportion of CD3⁺CD56⁺ effector cells, while maintaining the expression of activating receptors such as NKG2D and NKp30 on the cell surface. Compared to the control group, IC-92 combined with cytokines induced a higher proportion of effector memory-like T cell differentiation and markedly strengthened the killing ability of HER2-CAR-CIK cells against the human ovarian cancer cell line SK-OV-3. Sequencing results further confirmed that in the co-cultured and expanded CIK cells, the CD3+CD56+ cell population exhibited significant enrichment of the HIF-1 and AMPK signaling pathways. Additionally, differentially expressed genes associated with glycometabolism and sphingolipid metabolism showed marked enrichment in these pathways. This study success- fully established IC-92 engineered cells that co-express mIL-21 and CD137L. These cells efficiently generated a high proportion of CD3+CD56+ CIK cells, thereby facilitating their differentiation into an effector memory-like phenotype. The research on functions and mechanisms demonstrated that this IC-92-based system enables the expansion of highly potent HER2-CAR-CIK cells with superior tumor-killing activity, an effect attributed to enhanced immunostimulatory signaling and concurrent metabolic reprogramming of glycolytic and sphingolipid pathways.
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