NKG2D CAR-Expressing Lymphocytes Target Acute Myeloid Leukemia Cells
Authors: Leivas Alejandra , Córdoba Laura , Valeri Antonio , Rio Paula , Primo Daniel , Ballesteros Joan , Fernández Lucía , Perez-Martinez Antonio , Lee Dean Anthony , Powell Daniel J. , Martinez-Lopez Joaquin 
Published: November 2019

Background:
Acute myeloid leukemia (AML) is a hematological malignancy with a very low overall survival. Among the new treatment modalities, chimeric antigen receptor (CAR) therapy is showing promising results in other hematological malignancies. Since AML exhibits high heterogeneity and does not have specific differential antigens of the hematopoietic stem cell, using NKG2D-CAR cells could be an appropriate therapeutic strategy against AML. NKG2D receptor has a wide range of specific tumor cell ligands (MICA, MICB, ULBP-1, ULBP-2 and ULBP-3) which are expressed in more than 80% of all tumors. For this reason, the objective of this work was to evaluate the anti-tumor activity of activated and expanded natural killer cells (NKAE) and T cells expressing an NKG2D CAR.
Methods:
T cells and NK cells were isolated from the healthy donor´s peripheral blood mononuclear cells ring (n = 5) by immunomagnetic depletion. NKAE cells were obtained by co-culture with subletally irradiated CSTX002 cells. The purified NKAEs and T cells were transduced with an NKG2D CAR with 4-1BB and CD3z signaling domains. The efficiency of transduction was evaluated by flow cytometry detecting NKG2D expression. Also, the immunoprofiling of surface molecules and the cytotoxicity against primary blasts of AML, as well as the expression of NKG2D ligands in tumor cells (AML cell lines and primary blasts) were analyzed by flow cytometry. The cytotoxicity of untransduced NKAE, CAR-NKAE cells, untransduced T cells and CAR-T cells was evaluated by 4 hour europium release assay. Toxicity on healthy tissue (healthy lung cells and PBMCs from third party) was analyzed in the same way. The safety of NKG2D-CAR transduced cells was evaluated using CGH arrays to detect chromosomal abnormalities.
Results:
Both the AML cell lines and primary blasts from AML patients showed expression of the MICA/B and ULBPs-1 to 3 ligands. NKG2D ligands expression was highly variable from a cell line to another. However, all the cell lines and samples showed high expression of at least 1 of the 5 analyzed ligands. Four hour europium release assays revealed that untransduced T cells had higher cytotoxicity than untransduced NKAE cells at the same ratio (32: 1) against both OCI-AML-3 cytarabine-resistant cell line (52.7% ± 14.2% vs. 32.5% ± 7.2%) as to the cytarabine-sensitive cell line (56.42% ± 5.6% vs. 50.14 % ± 5.9%). T cells showed a better transduction efficiency than NKAE cells at a multiplicity of infection (MOI) of 5. It was observed that both the CAR-NKAE cells and the CAR-T cells had higher cytotoxicity against these two lines (OCI-AML-3R and OCI-AML-3S) after being transduced with our NKG2D-CAR. However, the antitumor activity of CAR-T cells always remained superior to that of the CAR-NKAE for both OCI-AML-3R (54.26% ± 3.8% vs. 35.3% ± 6.7%) and for OCI-AML-3S (63.36% ± 3.5% vs. 54.3% ± 3.7%) cell lines, with a greater difference compared to drug resistant cells. The antitumor activity of CAR-T cells and CAR-NKAE cells was always superior on the sensitive cell line. The antitumor activity of NKG2D CAR-T cells was evaluated against primary blasts from AML patients (n = 4), observing a nearly complete destruction of the blasts after 24 hours, at a very low target: effector ratio of 4:1.
CAR-T cells populations were analyzed by flow cytometry and we found that CAR-T cells were highly positive to CD45RO and reduced expression of CD45RA, on the other hand, they showed high IL-2 production exhibiting a central memory phenotype.
Slight toxicity was observed against lung cells in both cell types. However, CAR-T cells exhibited some toxicity on third party PBMCs being null in the case of CAR-NKAE. CGH arrays studies showed no variation in the copy number, resulting from the introduction of the CAR, of genes which their variation has been associated with chromosomal instability. No significant changes associated with transduction process were observed in the surface phenotype of CAR-NKAE cells or CAR-T cells.
Conclusions:
We have demonstrated that AML cells could be target with an NKG2D-CAR. Primary NKAE cells and T cells can be transduced with an NKG2D-CAR at a very low MOI to enhance their antileukemic activity. CD3+ CAR-T cells are more effective than CAR-NKAE cells. Moreover, CAR-T cells were able to completely destroy AML blasts. Although further studies are needed, these results show the potential of NKG2D-CAR T and NK cell therapy in AML.

Disclosures
Rio: Rocket Pharmaceuticals: Equity Ownership, Patents & Royalties, Research Funding. Lee:Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.