Lymphocytic leukemias and non-Hodgkin lymphomas (NHL) associated with B-cell neoplasms shown clinical and biological heterogeneity. Despite therapeutic advances, many cases relapse, highlighting the need for new approaches. T cells modified with Chimeric Antigen Receptors (CAR) targeting CD19, a B-cell surface antigen, have shown remarkable clinical efficacy in immunotherapy against hematologic malignancies. However, CD19-CAR-T cell therapy presents limitations, such as adverse effects including cytokine release syndrome. In this context, Natural Killer (NK) cells emerge as a promising alternative for CAR-based therapy due to reduced toxicity and the feasibility of allogeneic applications. Considering NK cell adoptive transfer, the NK-92 cell line has been evaluated in clinical settings for CAR-based therapy. NK cells express Natural Cytotoxicity Receptors (NCRs), including NKp30, which recognize tumor-associated ligands. The NKp30-dependent cytotoxicity requires association with adaptor proteins bearing ITAM motifs, such as CD3ζ. However, NK-92 cells lack endogenous CD3ζ, impairing signal transduction through NKp30. Thus, engineering NK-92 cells to express CD3ζ and CD19-CAR containing NKp30 as a co-stimulatory domain may enhance their cytotoxic function against malignant B cells.

Modification of NK-92 cells with CD3ζ followed by CD19-CAR containing NKp30 as a co-stimulatory domain, aiming to enhance cytotoxic activity against malignant B cells.

(i) Construction of lentiviral vectors encoding CD19-NKp30-CAR and CD3ζ, separately; (ii) production of lentiviral particles carrying CD3ζ through transfection of HEK-293T cells; (iii) transduction of NK-92 cells via spinoculation.

CD19-NKp30-CAR construct was assembled by cloning the domains followed by and ligation using Gibson assembly. The construct was validated by restriction enzyme digestion and PCR amplification of the CAR transgene. This construct will be used to transduce NK-92 cells that were previously modified with CD3ζ via lentiviral transduction. Lentiviral vectors encoding CD3ζ were produced by transfection of HEK-293T cells, associated with helper plasmids, resulting in a titer of 1.335 × 10⁸ TU/mL. Transduction of NK-92 cells reached 17.4% of CD3ζ-positive cells detected by flow cytometry on day three post-transduction. These modified cells were enriched via cell sorting.

NK-92 cells can be engineered to the stable expression of CD3ζ, enabling further modification with CD19-NKp30-CAR, thereby enhancing cytotoxic activity against B cells malignancies.