CD38 deletion of human primary NK cells eliminates daratumumab-induced fratricide and boosts their effector activity

Abstract

Multiple myeloma (MM) is a plasma cell neoplasm that commonly expresses CD38. Daratumumab (DARA), a human monoclonal antibody targeting CD38, has significantly improved the outcome of patients with relapsed or refractory MM, but the response is transient in most cases. Putative mechanisms of suboptimal efficacy of DARA include downregulation of CD38 expression and overexpression of complement inhibitory proteins on MM target cells as well as DARA-induced depletion of CD38high natural killer (NK) cells resulting in crippled antibody-dependent cellular cytotoxicity (ADCC). Here, we tested whether maintaining NK cell function during DARA therapy could maximize DARA-mediated ADCC against MM cells and deepen the response. We used the CRISPR/Cas9 system to delete CD38 (CD38KO) in ex vivo expanded peripheral blood NK cells. These CD38KO NK cells were completely resistant to DARA-induced fratricide, showed superior persistence in immune-deficient mice pretreated with DARA, and enhanced ADCC activity against CD38-expressing MM cell lines and primary MM cells. In addition, transcriptomic and cellular metabolic analysis demonstrated that CD38KO NK cells have unique metabolic reprogramming with higher mitochondrial respiratory capacity. Finally, we evaluated the impact of exposure to all-trans retinoic acid (ATRA) on wild-type NK and CD38KO NK cell function and highlighted potential benefits and drawbacks of combining ATRA with DARA in patients with MM. Taken together, these findings provide proof of concept that adoptive immunotherapy using ex vivo expanded CD38KO NK cells has the potential to boost DARA activity in MM.

Graphical abstract

Figure 1.

Successful generation of CD38^KO NK cells from ex vivo expanded PB-NK cells using Cas9/RNP. (A) Workflow for generating CD38^KO NK cells from ex vivo expanded PB-NK cells using electroporation of Cas9/RNP. (B) CD38 expression in NK cells before and after Cas9/RNP-mediated CD38 deletion (n = 5; mean ± standard deviation [SD]). (C) Representative fluorescence-activated cell sorter (FACS) analyses of the purified CD38^KO NK cells. Each figure indicates the percentage of CD38-expressing NK cells. Isotype controls are depicted with filled histograms. (D) Expansion of CD38^WT and CD38^KO NK cells.

Figure 2.

Resistance of CD38^KO NK cells to DARA-induced fratricide. (A) Representative FACS analyses of the conjugation assay. (B) Summarized data of conjugation assays are shown (n = 3; mean ± SD). (C) Representative FACS analyses of the fratricide assay. (D) Viability of CD38^WT and CD38^KO NK cells treated with DARA compared with that of control (CTR) samples (n = 3; mean ± SD). (E) Representative FACS analyses of PB of NSG mice 7 days after treatment with DARA or saline. (F) Summarized data of NK cell persistence in NSG mice during treatment. The frequency of human NK cells in PB at day 7 and their absolute number in spleen and BM at day 9 are shown (n = 5; mean ± SD).

Figure 3.

Enhanced DARA-mediated ADCC activity of CD38^KO NK cells against MM cell lines and primary MM cells. (A) Representative FACS analyses of CD38 expression of NK cells and myeloma cell lines. Each panel shows the mean fluorescence intensity (MFI). Isotype controls are depicted with filled histograms. (B-C) Representative data of cytotoxicity and DARA-mediated ADCC activity of paired CD38^WT and CD38^KO NK cells against myeloma cell lines and (D) a representative primary MM sample. (E) ADCC activity of paired CD38^WT and CD38^KO NK cells against primary MM samples (effector-to-target [E:T] ratio is 0.1:1) (F) Cytotoxicity of paired CD38^WT and CD38^KO NK cells against primary DARA-resistant MM cells in the presence of DARA.

Figure 4.

Inhibitory effects of ATRA on DARA-mediated NK cell cytotoxicity. (A-B) Cytotoxicity and DARA-mediated ADCC activity of paired CD38^WT and CD38^KO NK cells against myeloma cell lines pretreated with 50 nM ATRA for 48 hours (mean ± SD). (C) Left panel shows representative FACS analyses data for CD38 expression on NK cells (CD3^–CD56⁺) from patients during ATRA treatment or no therapy. Frozen PB mononuclear cells were thawed and analyzed at once. Right panel shows fold increase of MFI (CD38) of NK cells during ATRA therapy compared with no therapy for 3 different patients. (D) Representative FACS analyses data of CD38 expression on CD38^WT and CD38^KO NK cells 48 hours after incubation with 50 nM ATRA or solvent control. Control and ATRA-treated samples are shown with steel blue and red lines, respectively. Unstained controls are depicted with filled histograms. (E) Viability of CD38^WT and CD38^KO NK cells treated with DARA for 48 hours in the presence of 50 nM ATRA or solvent control compared with that of control samples (mean ± SD). (F-G) Cytotoxicity and DARA-mediated ADCC activity of paired CD38^WT and CD38^KO NK cells against myeloma cell lines in a 48-hour cytotoxicity assay in the presence of 50 nM ATRA or solvent control. E:T ratio is 0.25:1 for MM.1S and 0.5:1 for KMS-11 (mean ± SD).

Figure 5.

Favorable metabolic reprogramming of CD38^KO NK cells. (A) Heat map of DEGs of significantly altered pathways (cholesterol biosynthesis and OXPHOS) as determined by IPA, based on normalized RNA-seq data of paired CD38^WT and CD38^KO NK cells (n = 6). (B) Principle components analysis (PCA) of DEGs, showing consistent effect of CD38 deletion for each donor despite wide interdonor variability. (C) Summarized data of metabolic analysis of paired CD38^WT and CD38^KO NK cells (n = 3; mean ± SD). (D) Graphical analysis of basal OCR, ECAR, OCR/ECAR, and spare respiratory capacity (SRC) derived from (C). All experiments were achieved using quintuplicate samples. FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; ROT/AA, rotenone and antimycin A.