Anti-Her2-CAR CD38 gen locus knock-in enhance the persistence CAR expression and augment the anti-tumor capacity of Natural Killer cells against gastric cancer cells
Chimeric antigen receptor (CAR) natural killer (NK) cells are an emerging form of immunotherapy that combines the innate killing ability of NK cells with the targeted precision of CAR technology. By engineering NK cells to express CARs, these cells can be directed to recognize and attack specific cancer antigens, enhancing their natural cytotoxic response against tumor cells. CAR-NK cells offer several advantages over CAR-T cells, including reduced risk of cytokine release syndrome and graft-versus-host disease, as well as their ability to target a broader range of tumor types. CAR-NK cell therapies are being actively explored for both hematologic malignancies and solid tumors, showing promise in early clinical trials for improving cancer treatment outcomes.
Primary NK cells from peripheral blood were expanded using K562-mIL-21/4-1BBL feeder cells and IL-2. On Day 7, the anti-Her2 second generation CAR cassette was integrated by homologous recombination into the CD38 gene locus of NK cells via Cas9-RNP electroporation combined with AAV6-mediated CAR delivery. CAR expression on NK cells and its phenotyping were analyzed by flow cytometry. Furthermore, CAR-NK cells’ cytotoxicity against Her2+ gastric cancer cell lines, cytokine secretion, and activation markers were also analyzed by flow cytometry. A gastric cancer organoid model was employed to test cytotoxicity, and a xenograft NSG mouse model was used to evaluate the anti-tumor activity of CD38KI-Her2-CAR-NK cells in vivo.
The site-specific integration of the CAR into the CD38 locus via Cas9-RNP and AAV6 may avoided the risks associated with random viral gene insertion. CD38KI-Her2-CAR-NK cells exhibited higher proliferation (221.5 ± 30.35 expansion fold than 156 ± 18.02 for LV-CAR-NK on day 15, n = 6) and comparable CAR+ expression to lentiviral transduction, LV-Her2-CAR (31.88% CD38KI-Her2-CAR-NK v.s. 32.32% LV- Her2-CAR-NK, n = 6). In vitro, CD38KI-Her2-CAR-NK cells showed increased activation molecule expression (NKp44, NKp46, CD107a), enhanced secretion of cytokines (IFN-γ, TNF-α), and superior antigen-specific cytotoxicity against Her2+ gastric cancer cells (MKN-28, AGS) when compared to LV-Her2-CAR NK cells. In the gastric cancer organoid model, CD38KI-Her2-CAR-NK cells demonstrated stronger anti-tumor efficacy compared to lentiviral-transduced CAR-NK cells. In xenograft models, CD38KI-Her2-CAR-NK treatment resulted in improved survival rates and reduced tumor growth compared to the lentiviral-transduced LV-Her2-CAR NK cells group. Flow cytometry revealed that tumor-infiltrating lymphocytes NK cells from the CD38KI-Her2-CAR-NK group expressed more activation markers and secreted higher levels of cytokines.
This study successfully developed CD38KI-Her2-CAR-NK cells using Cas9-RNP electroporation with AAV6, confirming the method is efficiency and safety. The engineered NK cells displayed enhanced proliferation and potent anti-tumor activity against Her2+ gastric cancer cells, presenting a promising strategy for gastric cancer immunotherapy that may extend to other solid tumors.