A Novel Early Memory-Enriched Allogeneic NKG2D CAR-T Cell Therapy Based on CRISPR/Cas9 Technology for Solid Tumors

Background/Objectives: Chimeric Antigen Receptor (CAR)-T cell therapy has demonstrated impressive clinical results against hematological malignancies. However, currently commercialized CAR-T therapies are designed for autologous use, which entails some disadvantages, including high costs, manufacturing delays, complex standardization, and frequent production failures due to patient T cell dysfunction. Moreover, their CARs target one specific antigen, increasing the probability of antigen-negative tumor relapses. To overcome these limitations, we developed a novel NKG2D CAR-T cell therapy for allogeneic use with broad target specificity, as this CAR targets eight different ligands commonly upregulated in both solid and hematological tumors. Additionally, the manufacturing process was optimized to improve the phenotypic characteristics of the final product. Methods: Multiplex CRISPR/Cas9 technology was applied to eliminate the expression of TCR and HLA class I complexes in healthy donor T cells to reduce the risk of graft-versus-host disease and immune rejection, respectively, as well as lentiviral transduction for introducing the second-generation NKG2D-CAR. Moreover, we sought to optimize this manufacturing process by comparing the effect of different culture interleukin supplementations (IL-2, IL-7/IL-15 or IL-7/IL-15/IL-21) on the phenotypic and functional characteristics of the product obtained. Results: Our results showed that the novel CAR-T cells effectively targeted cervicouterine and colorectal cancer cells, and that those manufactured with IL-7/IL-15/IL-21 supplementation showed the most suitable characteristics among the conditions tested, considering genetic modification efficiency, cell proliferation, antitumor activity and proportion of the stem cell memory T cell subset, which is associated with enhanced in vivo CAR-T cell survival, expansion and long-term persistence. Conclusions: In summary, this new prototype of NKG2D CAR-T cell therapy for allogeneic use represents a promising universal treatment for a wide range of tumor types.