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M. Gurney et al.
ment of multiple myeloma.28 Indeed, overcoming the frat- ricidal effect of daratumumab through combination with ex vivo eNK cells is actively under investigation in multi- ple myeloma. In keeping with prior reports, we observed CD38 upregulation during NK cell expansion, which was sufficient to lead to a fratricidal effect despite the use of an optimized-affinity CD38 CAR design.18 While it has been considered difficult to apply genetic engineering approaches to primary NK cells, we achieved a consis- tent, and high-efficiency disruption of the CD38 gene using a multi-sgRNA approach coupled with a flow trans- fection system. Our findings are comparable to recent descriptions of CRISPR/Cas9 editing in primary NK cells but using a different sgRNA design and expansion approach.27,29,30 The resulting CD38 KD eNK cells contin- ued to expand and displayed reduced fratricide after CD38 CAR expression. With the availability of CRISPR/Cas9 and the relative ease of application to pri- mary NK cells using clinically adaptable platforms now demonstrated by multiple groups, there are vast possibil- ities for this technology across NK cell therapeutics.
One potential limitation to CD38 targeting in AML is the limited capacity to target LSC populations, question- ing the ‘curative’ potential of the therapies. LSC in AML are well-established, and while our understanding has evolved to include the existence of some CD38-positive LSC populations, it is likely that many LSC do reside within the traditional CD34-positive, CD38-negative compartment.31 Considering this feature of AML LSC, a CAR-NK cell targeting CD38 could be expected to have greater LSC targeting potential than a CAR-T cell, because of the presence of the innate activating pathways of NK cells above and beyond the CD38-specific CAR. Indeed the potential for long-term disease control, and thus LSC targeting capabilities can be inferred from data establishing the importance of NK cell KIR-ligand mis- match in the efficacy of allogeneic stem cell transplanta- tion.16 Furthermore, a tandem CAR approach including a LSC-specific antigen and/or a variant of TRAIL (tumor necrosis factor related apoptosis inducing ligand) could be incorporated to augment LSC targeting.32 Tailored approaches using CAR modified NK cells targeting com- binations based on the specific identified LSC immunophenotype in each case may ultimately be required given the absence of an identified universal LSC marker. This approach is becoming feasible with current and emerging technologies.
Antibody- and protein-based approaches have been considered previously in attempts to overcome fratricide in a CD38-directed CAR-T cell platform.33 CRISPR/Cas9- generated, CD38 KD eNK cells have recently and success- fully been applied to reducing the NK cell fratricidal effects of daratumumab with a focus on multiple myeloma.27 Interestingly, while a magnetic separation step was uti- lized to enhance the purity of the KD population in this innovative study, our data suggest that expression of a CD38 CAR combined with a highly efficient CRISPR/ Cas9 KD will likely lead to a self-selecting KD population without additional processing. While not the focus of our experiments, Kararoudi et al.27 also explored the cellular bioenergetic benefit of deletion of CD38 in eNK cells. CD38 converts nicotinamide adenine dinucleotide (NAD+) to cyclic adenosine diphosphate-ribose through an enzy- matic function. Additional NAD+ availability due to loss of CD38 supplies an important co-factor favoring oxidative
phosphorylation within NK cells. FT538, a NK cell prod- uct derived from induced pluripotent stem cells being developed by FATE Therapeutics, incorporates a CD38 deletion to overcome fratricide when combined with daratumumab. The group also demonstrated greater resistance to oxidative stress conferred by deletion of CD38, a characteristic likely to be favorable within the tumor microenvironment.34 These enhancements to NK cell biology suggest a broad range of applications for CD38 KD eNK cells beyond CD38 targeting and fratricide concerns. Simple and consistent approaches to their gen- eration will likely be of clinical utility.
In conclusion, we present two viable approaches to CD38 CAR-NK cell therapies applied to AML. Both our CD38 CAR-KHYG-1 cells and CD38 KD eNK cell plat- forms overcome effector cell fratricide relating to NK cell CD38 expression. Furthermore, we report an efficient approach to CRISPR/Cas9 genome editing adapted to pri- mary eNK cells and suitable for GMP expansion.
Disclosures
MG has received educational funding from Janssen Pharmaceuticals and Takeda. AS and SS have received research funding from ONK Therapeutics Limited. LKM is an employee of ONK Therapeutics Limited. SK and RS are employees of Maxcyte Inc. SZ has received research funding from Takeda, Celgene, and Janssen and is a member of the board of directors or an advisory committee for Takeda, Celgene, and Janssen. NWCJvdD has received research funding from Janssen Pharmaceuticals, Amgen, Celgene, Novartis, and BMS and has participated in advisory boards for Janssen Pharmaceuticals, Amgen, Celgene, BMS, Takeda, Roche, Novartis, Bayer, and Servier. TM has received research funding from Gilead, Celgene, Novartis, ONK Therapeutics Limited, Genmab, Janssen and has been a member of an advisory board for Janssen. ES has collaborated in research projects with Janssen, Roche, Celgene, and Takeda. MOD has received research funding from ONK Therapeutics Limited, BMS, Celgene, and Glycomimetics; is a member of the board of direc- tors or an advisory committee for Janssen, Abbvie, and ONK Therapeutics Limited; and owns equity in ONK Therapeutics Limited. EN has no potential conflicts of interest to disclose.
Contributions
SS and MOD conceived the research. EN, MG, AS and LKM performed functional assays. SK and RS contributed to electroporation optimization. ES contributed to acquisition of patients’ samples and the primary AML assay design. TM, SZ and NVD developed CD38 CAR-KHYG1 cells and associated functional assays. MG, SS and AS wrote the manuscript and prepared the figures. All authors contributed to editing and reviewing the final manuscript prior to submission.
Acknowledgments
Reagents for electroporation were contributed by Maxcyte inc.
Funding
This research was supported by Irish Clinical Academic Training (ICAT) Programme fellowship funding to MG. ICAT is supported by the Wellcome Trust and the Health Research Board (grant num- ber 203930/B/16/Z), the Health Service Executive National Doctors Training and Planning and the Health and Social Care, Research and Development Division, Northern Ireland. Work per- formed in collaboration with Blood Cancer Network Ireland was funded by Science Foundation Ireland and the Irish Cancer Society (Blood Cancer Network Ireland, 14/ICS/B3042).
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haematologica | 2022; 107(2)