Combinatorial Immunotherapy of Mcam Targeting Chimeric Antigen Receptor Modified Expanded Natural Killer Cells and IL-15 Agonist Against Neuroblastoma

Abstract

Objectives

Patients with recurrent metastatic neuroblastoma (NB) have a dismal survival. Natural Killer (NK) cells have long been recognized as important in treating pediatric solid tumors, including NB. To enhance the cytotoxicity of NK cells and facilitate specific targeting of tumor cells, we and others have engineered NK cells to express chimeric antigen receptors (CARs) against molecular targets. The melanoma cell adhesion molecule (MCAM) is expressed in NB and constitutes a novel target for immunotherapy. NKTR-255 is polymer-conjugated recombinant human IL-15 receptor agonist that stimulates proliferation and survival of NK cells. Here we develop an ex-vivo expanded CAR NK cell targeting MCAM and combine it with NKTR-255 to facilitate increased anti-tumor efficacy against NB.

Methods

PBMCs were expanded into NK cells using K562-mbIL21-41BBL feeder cells. The anti-MCAM CAR NK cells were generated by electroporation of CAR mRNA into expanded NK cells. NK cell cytotoxicity was evaluated by luciferase based cytotoxicity assay. IFN-γ and perforin secretion were analyzed by ELISA. CRISPR-Cas9 approach was utilized to knockout MCAM in NB cells. A NB xenograft mouse model in NSG mice was utilized for evaluating in vivo tumor growth and animal survival.

Results

We found that electroporation resulted in CAR expression in >60% of NK cells with a duration for about 6 days. Compared to mock NK cells, expression of MCAM CAR significantly enhanced NK cytotoxic activity against MCAM^high SKNFI and CHLA255 cells but not MCAM^low Be2C cells at the effector to target (E:T) ratios of 0.2:1 and 0.5:1 (p<0.05). CAR NK cells had significantly higher interferon γ and perforin secretion than mock NK when incubated with SKNFI cells (p<0.05). The enhanced cytotoxic activity of CAR NK cells was due to specific targeting of MCAM because we did not observe the enhanced cytotoxicity in MCAM^KO NB cells. Furthermore, CAR NK significantly decreased tumor growth and prolonged animal survival in vivo (p<0.05). NKTR-255 markedly increased the expression levels of NK activating receptors NKp30, NKG2D, and NKp44 and significantly improved NK cell survival and maintained NK cell expansion in the absence of IL-2 in vitro (p<0.01). NKTR-255 significantly enhanced the cytotoxic activity of CAR NK cells targeting SKNFI cells at an E:T ratio of 0.5:1 (p<0.01) (Fig 1A) with significantly enhanced secretion of interferon γ and perforin (p<0.01) (Fig 1B, C). Combination with NKTR-255 further enhanced the anti-tumor effects of CAR NK cells (p < 0.001 compared to vehicle control, p < 0.05 compared to CAR NK), and further prolonged animal survival (100% vs 30% survival at day 126 compared to CAR NK, p < 0.05) (Fig 1D, E).

Conclusions

Our studies demonstrate that ex vivo expanded and modified anti-MCAM CAR NK cells alone and/or in combination with NKTR-255 are promising novel alternative therapeutic approaches to targeting MCAM^high malignant NB.