CD56-targeted in vivo genetic engineering of natural killer cells mediates immunotherapy for acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy that starts from bone marrow and spreads to other organs. At the time of diagnosis, both innate and defective natural killer (NK) cells are present in AML patients. The dysfunction of the NK cells is due to the absence of NK cell receptors such as NKG2D on tumor cells that help with tumor immune escape, and also the polycomb protein, EzH2, which plays an important role in the commitment and differentiation of NK cells. The inhibition of EzH2 activates NK cells towards enhanced lytic activity. However, the adoptive transfer of NK cells for cancer treatment is still under scrutiny due to limitations like production cost, vein-to-vein time, and complicated experimental procedures. In order to circumvent these issues, here, in vivo CD56⁺ NK cell genetic engineering is hypothesized through the CD56-directed delivery of the pSMP-EzH2 shRNA plasmid encapsulated in chitosan nanoparticles (pEzH2@CSNPs@CD56). The pSMP-EzH2 shRNA plasmid was encapsulated in chitosan nanoparticles followed by CD56 antibody conjugation through EDC–NHS chemistry. CD56 antibody-conjugated nanoparticles selectively target CD56⁺ NK cells and downregulate EzH2 expression in CD56⁺ NK cells of human PBMCs. The in vitro CD56⁺ CD3⁻ NK cells were enriched and stably suppressed EzH2 expression to prepare adoptive CD56⁺ CD3⁻ NK (EzH2⁻) cells for anti-AML immunotherapy. The in vitro NK (EzH2⁻) cells and pEzH2@CSNPs@CD56 reduced splenomegaly while immunophenotyping revealed in vivo downregulation of the c-Kit⁺ leukemia stem cell population along with upregulation of the differentiation markers CD11b and Gr-1 in the peripheral blood and bone marrow of AML1-ETO9a-induced xenograft nude mice. CD56⁺CD3⁻ and CD56⁺CD38⁺ cell populations were significantly increased in the peripheral blood and bone marrow, which indicated NK cell-mediated AML cell killing took place suggesting that use of pEzH2@CSNPs@CD56 is a safe and viable strategy for NK cell-mediated anti-AML immunotherapy.