P-007: Exploring a safety switch in NKG2D and BCMA CAR NK-92MI immunotherapy

Introduciton Despite impressive preliminary efficacy of CAR-T cells in multiple myeloma (MM), NK cell engineering has emerged as a competitive and safer approach, as they entail an ‘off-the-shelf’ strategy with no graft-versus-host disease. NK-92 is a universal, cheap and fast obtainable cellular therapy previously used in clinical trials. Although modest responses with these cells have been reported in MM, their oncolytic potential can be enhanced by genetic modification. However, there are still reasonable doubts about the efficacy of irradiated NK-92 cells used in clinic. Thus, the aim of our study is to generate a safe and effective CAR NK-92 immunotherapy for MM treatment. Methods NK-92MI cells were transduced with a lentiviral vector expressing NKG2D or BCMA CAR. Single and dual CARs were generated, with same or different combinations of costimulator domains. CAR NK-92MI cells expressing a safety switch were generated by retroviral transduction with an SFGiCasp9.2A.ACD19 construct. All populations were purified by FACS sorting to obtain stable modified effectors. In vitro antitumor activity was analyzed against low and high target ligands expressing MM cell lines: U266, similar expression of both BCMA and NKG2DL; XG-1, BCMAhigh and NKG2DLlow; as well as a BCMA knock-out cell line generated by CRISPR-Cas9 system. For in vivo experiments, NSG mice were intravenously injected with 1×106 U266 ffLuc-GFP cells. 5×106 CAR NK-92MI cells were i.v. administered 48h later, once a week for three weeks. Results CAR NK-92MI cells showed specificity and higher in vitro antitumor activity compared to parental NK-92 cells, as well as lack of hematotoxicity. The combined expression of both NKG2D and BCMA CAR has demonstrated cytotoxic coverage against MM cell lines. In vivo, clinical 10 Gy irradiation dose completely abrogate the efficacy of CAR NK-92 cells in our treatment schedule. Besides, lower irradiation doses are not enough to eliminate NK cells. In order to obtain a safe allogeneic immunotherapy, CAR NK-92MI cells expressing a suicide gene therapy have been generated (99.9% purity) being susceptible to death (near 99% death) upon induction with Rimiducid (AP1903). Currently, iCasp9-CD19 CAR NK-92MI cells are being tested in vivo. Conclusions CAR NK-92MI effectors expressing single and dual CARs have been generated and they all show higher in vitro antitumor efficacy against different MM targets compared to parental NK-92MI cells. In vivo experiments show the inefficacy of irradiated CAR NK-92MI cells as therapeutic strategy in our MM model, leading to the necessity of a combination with a safety switch to ensure an effective and safe off-the-shelf NK immunotherapy for MM treatment.