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M. Gurney et al.
cells. To assess whether CD38 CAR targeting has an addi- tional cytotoxic effect, we tested CD38 CAR-KHYG-1 cells and mock-transduced KHYG-1 cells against CD38- positive AML cell lines (Figure 1B). CD38 CAR-KHYG-1 cells demonstrated greater cytotoxic effects against CD38-positive cell lines relative to mock-transduced KHYG-1 cells at all E:T ratios tested, with relatively greater increases seen against the strongly CD38-positive THP-1 cells (specific cytotoxicity 58% vs. 28% for THP- 1, 10:1 E:T ratio; P<0.0001).
ATRA has been shown to upregulate CD38 expression across all AML subtypes, mediated by a retinoic acid response element in the first intron of the CD38 gene.20 Pretreatment with ATRA at 10 nM for 48 h led to marked induction of CD38 expression on KG1a cells, which do not express detectable levels of CD38 in resting condi- tions (Figure 1C). CD38 CAR-KHYG-1 cells were cyto- toxic to ATRA-pretreated KG1a cells, while mock-trans- duced KHYG-1 cells showed little cytotoxicity despite ATRA pretreatment (Figure 1D).
To better mimic the CD38 expression profile encoun- tered in AML, we tested the efficacy of CD38 CAR- KHYG-1 cells against primary bone marrow mononuclear cells from AML patients (Figure 2A, B). CD38 CAR- KHYG-1 cells displayed greater specific cytotoxicity against AML blasts relative to mock-transduced KHYG-1 cells across a range of blast cell CD38 expression, with the degree of specific cytotoxicity correlating with blast cell CD38 expression (Figure 2C).
CRISPR/Cas9 gene editing of CD38 in primary NK cells reduces NK cell fratricide upon CD38 chimeric antigen receptor expression
While alloreactive NK cell approaches have shown some success in treating AML, we hypothesized that increased expression of CD38 during ex vivo NK cell expansion could be sufficient to trigger effector cell fratri- cide after expression of a CD38 CAR, despite affinity optimization. Indeed, we observed a consistent, mean 4- fold increase in CD38 expression during feeder-free expansion of NK cells in interlueukin-2-containing media. Increases in CD38 from baseline (mean fluorescence intensity [MFI] 11,903) were detectable by day 5 (MFI 40,948) and persisted to at least day 13 (MFI 38,600) (Figure 3A). Extrapolating from our previous work on THP-1 cells (MFI 31,866), we concluded that this degree of CD38 expression would lead to a fratricidal effect upon CD38 CAR expression thus limiting the cytotoxic capacity of ex vivo-expanded CD38 CAR-NK cells.
We, therefore, set out to use CRISPR/Cas9 gene edit- ing technology to disrupt the CD38 gene in primary NK cells. We used a multi-sgRNA format, introducing sgRNA-Cas9 complexes using a high-efficiency, electro- poration-based approach on a platform scalable to Good Manufacturing Practice (GMP) grade development. CD38 KD and mock-electroporated cells were further expanded for use in functional assays. A consistent KD effect was achieved across all NK cell donors (mean 84%; range, 75-92%) (Figure 3B). CD38 KD was detectable 48 h after CRISPR/Cas9 gene editing, peaked by day 3-7 after electroporation and was stable across the duration of expansion suggesting minimal differences in the growth potential of CD38 KD and mock-electroporated NK cells in this expansion system (Online Supplementary Figure S1).
To confirm that CD38 KD eNK cells showed greater resistance to fratricide than wild-type eNK cells, we intro- duced mRNA coding for an affinity-optimized CD38 CAR. CAR expression was confirmed by complementary staining techniques – an anti-human IgG with light chain specificity, and biotinylated protein L, with control (back- ground) and CAR staining depicted in Figure 3C. CD38 KD eNK cells displayed significantly less cell death than wild-type eNK cells, measured 18 h after CD38 CAR mRNA electroporation in the absence of target cells (18% vs. 37%, P=0.002) (Figure 3D), confirming a greater resist- ance to fratricide. Furthermore, the biphasic CD38 expression pattern (representing the small residual CD38- positive NK cell population after CRISPR/Cas9 gene edit- ing) was lost in the CD38 KD population after CD38 CAR mRNA transfection, but not after non-specific (CD16) mRNA electroporation (Figure 3E). This empha- sized the tendency of the CD38 CAR-NK cells to target CD38-positive eNK cells despite affinity-optimization of the CD38 CAR binding domain.
CD38 knockdown - CD38 chimeric antigen receptor-NK cells efficiently target primary acute myeloid leukemia blasts
To confirm that CD38 CAR expression in CD38 KD eNK cells enhances the activity of alloreactive NK cells against AML, CD38 KD eNK cells were electroporated with CD38 CAR mRNA or mock-electroporated prior to co-culture with bone marrow mononuclear cells from AML patients with a variety of molecular AML subtypes (Online Supplementary Table S1). CD38 KD - CD38 CAR- NK cells showed enhanced cytotoxicity relative to mock- electroporated CD38 KD cells, with the effect being most prominent at the highest E:T ratios tested (Figure 4A, B). Enhanced cytotoxicity was observed for all AML patients and cytotoxicity at the 5:1 E:T ratio correlated with blast cell CD38 expression (R2=0.81) (Figure 4C).
We investigated the potential of ATRA pretreatment as a means of modulating CD38 expression and potentiating the effects of CD38 CAR targeting using CD38 KD - CD38 CAR-NK cells. ATRA pretreatment induced a mean 5-fold upregulation of surface CD38 expression in blast cells (Figure 4D). The increased CD38 expression was associated with greater sensitivity to CD38 KD - CD38 CAR-NK cells compared to dimethylsulfoxide-treated bone marrow mononuclear cell samples tested at the 2:1 and 5:1 E:T ratios (Figure 4E).
Discussion
We set out to augment the potential of NK cell adoptive transfer strategies in AML through expression of an affin- ity-optimized CD38 CAR. We demonstrated two poten- tial approaches to CD38 CAR-NK cell therapy in this set- ting. We confirmed that CD38 KD eNK cells show reduced fratricide after CD38 CAR expression, allowing effective targeting of primary AML blasts. As an alterna- tive approach we modified the NK cell line KHYG-1 to express a CD38 CAR, successfully targeting AML cell lines and primary samples. Both approaches could be enhanced by induction of CD38 expression using ATRA. We chose a NK cell line with naturally low CD38 expres- sion to ensure viability after introducing a CD38 CAR. KHYG-1 cells have previously been shown to maintain
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haematologica | 2022; 107(2)