High-affinity CD16 integration into a CRISPR/Cas9-edited CD38 locus augments CD38-directed antitumor activity of primary human natural killer cells

Abstract

BackgroundAdoptive transfer of natural killer (NK) cells with augmented antibody-dependent cellular cytotoxicity (ADCC) capabilities and resistance to CD38 targeting has the potential to enhance the clinical anti-myeloma activity of daratumumab (DARA). Therefore, we sought to develop an efficient CRISPR/Cas9-based gene editing platform to disrupt CD38 expression (CD38 knockout (KO)) in ex vivo expanded NK cells and simultaneously arm CD38KO NK cells with a high-affinity CD16 (CD16-158V) receptor.MethodsCD38KO human NK cells were generated using Cas9 ribonucleoprotein complexes. The platform was expanded by incorporating messenger RNA (mRNA) transfection of CD38KO NK cells and targeted gene insertion at the CD38 locus to mediate gene knockin (KI). The capacity of these gene-edited NK cells to persist and mediate ADCC in the presence of DARA was tested in vitro and in a MM.1S xenograft mouse model.ResultsHighly efficient CD38 gene disruption was achieved in ex vivo expanded NK cells without affecting their proliferative or functional capacity. CD38 KO conferred resistance to DARA-induced NK cell fratricide, enabling persistence and augmented ADCC against myeloma cell lines in the presence of DARA in vitro and in a MM.1S xenograft mouse model. CD38KO NK cells could be further modified by transfection with mRNA encoding a CD16-158V receptor, resulting in augmented DARA-mediated ADCC. Finally, we observed that a homology-directed repair template targeted to the CD38 locus facilitated an efficient 2-in-1 CD38 KO coupled with KI of a truncated CD34 reporter and CD16-158V receptor, with CD38KO/CD16KI NK cells demonstrating a further enhancement of DARA-mediated ADCC both in vitro and in vivo.ConclusionsAdoptive immunotherapy using ex vivo expanded CD38KO/CD16KI NK cells has the potential to boost the clinical efficacy of DARA. By incorporating complementary genetic engineering strategies into a CD38 KO manufacturing platform, we generated NK cells with substantially augmented CD38-directed antitumor activity, establishing a strong rationale for exploring this immunotherapy strategy in the clinic.

Overcoming tumor resistance mechanisms in CAR-NK cell therapy

“…In MM, where CD38 is highly expressed in malignant plasma cells, daratumumab treatment together with a concomitant CD38 expression in NK cells leads to a marked decrease in NK cell numbers due to fratricide. Consequently, CD38 KO NK cells blocked daratumumab-induced fratricide, showing an improved metabolic profile and consequently enhanced cytotoxic activity against CD38 expressing MM cell lines and primary cells ( 377 , 378 ). In this setting, it is worth mentioning that a clinical trial in MM is already ongoing to test FT576, iPSC-derived BCMA CAR NK cells in which CD38 has been ablated to avoid mAb-mediated fratricide, in combination with other drugs (NCT05182073).…”

Current advances of CRISPR-Cas technology in cell therapy

“…CD38 disruption by CRISPR could increase antibody-dependent cellular cytotoxicity and enhance persistence. Several studies of iPSC-NK cell therapies with CRISPR genome editing to enhance innate immunity have been initiated for treating leukemia ( Clara et al., 2022 ).…”

Natural Killer Cells: A Promising Kit in the Adoptive Cell Therapy Toolbox

“…CD38 KO cells successfully resisted DARA-induced fratricide with potent ADCC activity and cytotoxicity, indicating a novel strategy to reinforce the therapeutic effect of DARA [ 184 ]. A recent study simultaneously endowed CD38 KO NK cells with a high-affinity CD16a-158V receptor which further elevated DARA-induced ADCC against myeloma cells [ 185 ]. In addition, triple-gene-edited CD38 KO iPSC-derived NK cells, co-expressing non-cleavable CD16a and IL-15/IL-15R fusion protein, have achieved desirable results in the tests of their in vivo persistence and tumor-directed cytotoxicity [ 200 ].…”