Activated and Expanded Natural Killer Cells Expressing an NKG2D-CAR Efficiently Target Multiple Myeloma Cells

Background:

Multiple myeloma (MM) remains an incurable disease and new therapeutic venues are required. Immune-based therapies represent a new weapon in the fight against cancer. Natural killer (NK) cells have an important role as natural control of tumor cells. Based on that, we carried out a proof-of-concept phase I clinical trial to make multiple infusions of autologous activated and expanded NK cells (NKAE) together with anti-myeloma drugs in MM (NCT02481934). In spite of the good clinical results, better responses are needed to erradicate MM. Chimeric antigen receptors (CARs) are emerging as a very good treatment strategy against MM. CAR studies have focused on their expression in T cells. However, CAR-NK cells could have lower toxicity and provides a method to redirect these cells more specifically to target refractory cancer. As our group has previously demonstrated an essential role of NKG2D receptor in NK cell cytotoxic activity, the objective of this work was to combine NK cell activation with the expression of an NKG2D-CAR in primary NK cells from MM patients.

Methods:

NKAE cells were generated by co-culture of peripheral blood mononuclear cell (PBMCs) with the genetically modified and previously irradiated K562 CL9 mIL21 cell line (kindly given by the Nationwide Children's Hospital) at a 1:1.5 ratio, and interleukin-2 at 100 IU/ml. NKAE cells were transduced with NKG2D CARs containing 4-1BB and CD3z signaling domains (kindly provided by the Perelman School of Medicine). The viral supernatant was produced by transient transfection of HEK293T cells with the vector genome plasmid and lentiviral packaging helper plasmids. NKAE expansion and NKG2D-CAR expression were evaluated by flow cytometry based on the percentage of the NK cell population and on the expression of the activating receptor NKG2D. The gating strategy was based on dead/live cells and doublet discrimination. NKAE-CAR in vitro cytotoxicity against MM cell lines was tested by performing 2-hour europium-TDA release assays.

Results:

NKAE cells were expanded during 10 days, after that transduction was performed using MOI=5. Better transduction efficiency was found on purified NKAE cells (100% purity) than on NKAEs which were not purified (purity higher than 80%). One day after transduction purified NKAE cells show an increase of NKG2D positive cells (62,6%±12,2%) when compare with untransduced NKAE cells (n=4). NKAE-CAR cells had similar viability as the untransduced NKAE cells. NKG2D-CAR expression was monitored 72 hours after transduction, 24% of NKG2D positive cells was due to episomal expression (38,5%±1,54%) but remain stable up to 7 days (36,8±11,06%). NKAE-CAR in vitro cytotoxic activity was tested at different NKAE-CAR:MM cells ratio (1:8 up to 32:1) using MM L-363 cell line as target (n=4). NKG2D CAR-expressing NKAE cells showed significantly higher cytotoxic activity against myeloma cells than untransduced NKAE cells even at low ratio. NKAE-CAR cells showed maximum cytotoxic capacity at 8:1 ratio (112%±19,2% lysed MM cells), however untransduced NKAE cells showed maximum activity at 32:1 ratio (67,5%±15,8%). At same ratio (8:1), NKAE-CAR cells were able to destroy 112% (±19,2%) MM cells, 53,75% more than untransduced NKAE cells. At very low ratio (1:8) NKG2D-CAR expressing NKAE cells could destroy 42,4% (±2,4%) MM cells.

Conclusions:

Primary NK cells from MM patients can be activated and transduced in vitro with an NKG2D CAR showing higher cytotoxic activity that untransduced NKAE cells. These results warrant further development of NKAEs-CAR as a new treatment modality for refractory MM.