Co-Expression of IL-15 and CCL21 Strengthens CAR-NK Cells to Eliminate Tumors in Concert with T Cells and Equips Them with Higher PI3K/AKT/mTOR Signal Signature

Background: CAR-NK therapy has shown good safety and efficacy in clinical practice. However, the application of CAR-NK therapy still faces huge challenges, such as short-term in vivo persistence and metabolic dysfunction in the microenvironment. Autocrine interleukin (IL)-15 can partially overcome these deficiencies, but further exploration is still needed. CCL21 is an important chemokine for T cells. Herein, we designed a CAR-NK co-expressing IL-15 and CCL21 (15x21 CAR-NK).

Methods: 15 CAR contained CD19 CAR (i.e., CAR) and the IL-15 sequence linked by a 2A peptide, and 15x21 CAR was constructed based on the 15 CAR. The functional validation of 15x21 CAR-NK was performed by in vitro proliferation assays, cytotoxicity assays against CD19⁺ lymphoma cell lines (e.g., Raji and Ramos), cytokine (IFNγ, TNFα, and IL-2) secretion assays, and flow cytometry of effector molecules (e.g., GZMB, CD107a, and Ki67). Transcriptomic data were obtained by RAN sequencing. The chemotaxis and effect of 15x21 CAR-NK on T cells were performed by two transwell chamber models (5um and 0.4um pore size, respectively). The combined impact of 15x21 CAR-NK and T cells in clearing tumors was monitored in real-time by ImageXpress Micro Confocal and NanoLive 3D Explorer. In vivo, experiments were performed using intraperitoneal and subcutaneous luc⁺Raji-bearing NSG mouse models.

Results: CAR-NK, 15 CAR-NK, and 15x21 CAR-NK with >95% CAR expression were obtained by lentiviral transfection of the NK92 cell line and then puromycin screening. In in vitro function experiments, 15x21 CAR-NK showed superior cytotoxicity, higher production of cytokines, and higher expression of effector molecules. Furthermore, 15x21 CAR-NK cells were proven to recruit more T cells and alleviate their exhaustion.

We then explored the cooperation and interactions between 15x21 CAR-NK and T cells. In continuous monitoring, combining 15x21 CAR-NK and T cells could enhance tumor clearing more effectively than monotherapy groups. Further exploration showed that the addition of 15x21 CAR-NK could increase the ratio of CD8⁺T_Naive, CD4⁺ T_CM and decrease the proportion of CD4⁺ T_Eff, as well as downregulate the expression of CD69 and LAG3 and upregulate the expression of GZMB in T cells. In addition, 15x21 CAR-NK showed reduced TIGIT and up-regulated CD158e1 expression in the presence of T cells.

Through GSVA analysis of RNA sequence data of UTD NK, CAR-NK, 15 CAR-NK, and 15x21 CAR-NK and verification by flow cytometry, 15x21 CAR-NK cells were significantly enriched in the PI3K/AKT/mTOR signaling pathway compared to the other three types of NK cells. Further, 15x21 CAR-NK cells were enriched in pathways and functions related to cell survival, such as DNA repair and G2M checkpoints.

We further verified the functions related to the PI3K/AKT/mTOR pathway. 15x21 CAR-NK showed high anti-apoptotic molecules (BCL-2) and low pro-apoptotic molecules (BAD and BID) expression and less enrichment of apoptosis-related pathways (e.g., HSA04210_APOPTOSIS) in transcriptional levels, as well as stronger anti-apoptotic ability under conditions without IL-2 in vitro. Furthermore, 15x21 CAR-NK cells were also characterized by highly expressed mitochondrial-related genes, significantly enriched mitochondrial-associated functions and REACTOME pathways, higher levels of mitochondrial membrane potential, and higher mitochondrial counts.

Conclusions: 15x21 CAR-NK cells have strong cytotoxicity and cytokine secretion functions, as well as recruiting T cells and working with T cells to eliminate CD19⁺ B-cell lymphoma cells. They are also highly enriched in the PI3K/AKT/mTOR signaling pathway and exhibit enhanced anti-apoptosis ability and mitochondrial fitness.