Chimeric antigen receptor (CAR)-modified natural killer (NK) cells show antileukemic activity against acute myeloid leukemia (AML) in vivo. However, NK cell-mediated tumor killing is often impaired by the interaction between human leukocyte antigen (HLA)-E and the inhibitory receptor, NKG2A. Here, we describe a strategy that overcomes CAR-NK cell inhibition mediated by the HLA-E-NKG2A immune checkpoint. We generate CD33-specific, AML-targeted CAR-NK cells (CAR33) combined with CRISPR/Cas9-based gene disruption of the NKG2A-encoding KLRC1 gene. Using single-cell multi-omics analyses, we identified transcriptional features of activation and maturation in CAR33-KLRC1^ko-NK cells, which are preserved following exposure to AML cells. Moreover, CAR33-KLRC1^ko-NK cells demonstrate potent antileukemic killing activity against AML cell lines and primary blasts in vitro and in vivo. We thus conclude that NKG2A-deficient CAR-NK cells have the potential to bypass immune suppression in AML.

While chimeric antigen receptor (CAR) T cell therapy has shown promising outcomes among patients with hematologic malignancies, it has also been associated with undesirable side-effects such as cytokine release syndrome (CRS). CRS is triggered by CAR T-cell-based activation of monocytes, which are stimulated via the CD40L–CD40R axis or via uptake of GM-CSF to secrete proinflammatory cytokines. Mouse models have been used to model CRS, but working with them is labor-intensive and they are not amenable to screening approaches. To overcome this challenge, we established two simple cell-based CRS in vitro models that entail the co-culturing of leukemic B cells with CD19-targeting CAR T cells and primary monocytes from the same donor. Upon antigen encounter, CAR T cells upregulated CD40L and released GM-CSF which in turn stimulated the monocytes to secrete IL-6. To endorse these models, we demonstrated that neutralizing antibodies or genetic disruption of the CD40L and/or CSF2 loci in CAR T cells using CRISPR-Cas technology significantly reduced IL-6 secretion by bystander monocytes without affecting the cytolytic activity of the engineered lymphocytes in vitro. Overall, our cell-based models were able to recapitulate CRS in vitro, allowing us to validate mitigation strategies based on antibodies or genome editing.

The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5′-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated, for the first time, the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.

Chimeric Antigen Receptor (CAR) T cell therapy has achieved high response rates in patients with relapsed or refractory hematologic malignancies. However, comparable efficacy in solid tumors remains limited, partly due to poor CAR T cell persistence and immune-mediated rejection. A major contributor, which has hampered the clinical efficacy of CAR T cells in clinical practice, is the immunogenicity of the murine-derived single-chain variable fragments (scFvs) commonly used in CAR constructs. Cell and humoral immune responses to the murine parts of CARs have been implicated in CAR T cell rejection. Here, we describe the generation and in vitro characterization of humanized CAR T cells targeting prostate-specific membrane antigen (PSMA) on prostate cancer cells, based on two distinct murine scFvs (A5 and D7). Humanization improved the germinality index and successfully preserved CAR surface expression. Functional assays demonstrated that humanized PSMA-CAR T cells retained antigen-specific binding, activation and cytotoxicity, differentiation, exhaustion and cytokine secretion profiles comparable to their murine counterparts. These results support the feasibility of humanization as a strategy to reduce immunogenicity without compromising CAR T cell capabilities, providing a foundation for further in vivo validation in solid tumor settings.