CD38^low Natural Killer Cells Transiently Expressing CD16^F158V m-RNA Potentiates the Therapeutic Activity of Daratumumab Against Multiple Myeloma with Minimal Effector NK Cell Fratricide

Introduction: Multiple Myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (moAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells mediating tumour immunosurveillance in vivo. NK cells also play an important role during moAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their Fcγ RIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring high affinity variant of CD16 harbouring a single point polymorphism (F158V), and this variant has been linked to improved ADCC. However, the contribution of NK cells to the efficacy of Daratumumab remains debatable as clinical data clearly indicate rapid depletion of CD38^high peripheral blood NK cells in patients upon Daratumumab administration. Therefore, we hypothesize that transiently expressing the CD16^F158V receptor using a "safe" mRNA electroporation-based approach, on CD38^low NK cells could significantly enhance therapeutic efficacy of Daratumumab in MM patients. In the present study, we investigate the optimal NK cell platform for generating CD38^low CD16^F158V NK cells which can be administered as an "off-the-shelf"cell therapy product to target both CD38^high and CD38^low expressing MM patients in combination with Daratumumab.

Methods: MM cell lines (n=5) (MM.1S, RPMI-8226, JJN3, H929, and U266) and NK cells (n=3) (primary expanded, NK-92, and KHYG1) were immunophenotyped for CD38 expression. CD16^F158V coding m-RNA transcripts were synthesized using in-vitro transcription (IVT). CD16^F158V expression was determined by flow cytometry over a period of 120 hours (n=5). 24-hours post electroporation, CD16^F158V expressing KHYG1 cells were co-cultured with MM cell lines (n=4; RPMI-8226, JJN3, H929, and U266) either alone or in combination with Daratumumab in a 14-hour assay. Daratumumab induced NK cell fratricide and cytokine production (IFN-γ and TNF-α) were investigated at an E:T ratio of 1:1 in a 14-hour assay (n=3). CD38⁺CD138⁺ primary MM cells from newly diagnosed or relapsed-refractory MM patients were isolated by positive selection (n=5), and co-cultured with mock electroporated or CD16^F158V m-RNA electroporated KHYG1 cells. CD16^F158V KHYG1 were also co-cultured with primary MM cells from Daratumumab relapsed-refractory (RR) patients.

Results: MM cell lines were classified as CD38^hi (RPMI-8226, H929), and CD38^lo (JJN3, U266) based on immunophenotyping (n=4). KHYG1 NK cell line had significantly lower CD38 expression as compared to primary expanded NK cells and NK-92 cell line (Figure 1a). KHYG1 electroporated with CD16^F158V m-RNA expressed CD16 over a period of 120-hours post-transfection (n=5) (Figure 1b). CD16^F158V KHYG1 in-combination with Daratumumab were significantly more cytotoxic towards both CD38^hi and CD38^lo MM cell lines as compared to CD16^F158V KHYG1 alone at multiple E:T ratios (n=4) (Figure 1c, 1d). More importantly, Daratumumab had no significant effect on the viability of CD38^low CD16^F158V KHYG1. Moreover, CD16^F158V KHYG1 in combination with Daratumumab produced significantly higher levels of IFN-γ (p=0.01) upon co-culture with CD38^hi H929 cell line as compared to co-culture with mock KHYG1 and Daratumumab. The combination of CD16^F158V KHYG1 with Daratumumab was also significantly more cytotoxic to primary MM cell ex vivo as compared to mock KHYG1 with Daratumumab at E:T ratio of 0.5:1 (p=0.01), 1:1 (p=0.005), 2.5:1 (p=0.003) and 5:1 (p=0.004) (Figure 1e). Preliminary data (n=2) also suggests that CD16^F158V expressing KHYG1 can eliminate 15-17% of primary MM cells from Daratumumab RR patients ex vivo. Analysis of more Daratumumab RR samples are currently ongoing.

Conclusions: Our study provides the proof-of-concept for combination therapy of Daratumumab with "off-the-shelf" CD38^low NK cells transiently expressing CD16^F158V for treatment of MM. Notably, this approach was effective against MM cell lines even with low CD38 expression (JJN3) and primary MM cells cultured ex vivo. Moreover, the enhanced cytokine production by CD16^F158V KHYG1 cells has the potential to improve immunosurveillance and stimulate adaptive immune responses in vivo.

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