Contributions
Abstract: EP721
Type: E-Poster Presentation
Session title: Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research
Background
CAR-NK cells represent a safer approach than CAR- T cells. These ground-breaking CAR effectors exhibit some other immunological advantages, 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 Multiple Myeloma (MM), their cytotoxic potential can be enhanced by genetic modification. However, the products obtained so far offer some limitations and could be optimized in MM.
Aims
The aim of our study is to generate a risk-free and effective CAR NK-92MI immunotherapy for MM treatment.
Methods
NK-92MI cells were lentivirally transduced with a single second generation CAR, NKG2D-CAR or BCMA-CAR, both containing identical 4-1BB costimulatory domain, as well as dual BCMA-NKG2D CARs with different combinations of co-stimulators. CAR NK-92MI iCasp9-CD19 populations have been generated by retrovirus transduction with an SFG iCasp9.2A.ΔCD19 construct. All populations were purified by FACS sorting to obtain stable modified cell lines and vector copy number was measured by qPCR to ensure similar CAR expression. In vitro cytotoxicity assays were performed against an ensemble of MM cell lines with high and low expression of target ligands as well as a BCMA knock-out generated by CRISPR-Cas9 system: U266, BCMAhigh and NKG2DLhigh; ARP-1, BCMAdim and NKG2DLdim; XG-1, BCMAhigh and NKG2DLlow; U266bcmaKO, BCMAKO and NKG2DLhigh. In vivo: 5x106 CAR NK-92MI cell infusions were administered i.v. once a week for three weeks.
Results
We have demonstrated specificity, non-toxicity and higher in vitro antitumor activity of CAR NK-92MI cells compared to the parental cell line. Besides, we have noticed that the expression of both CARs in combination has not showed synergy, but a broadening of cytotoxic coverage against MM cell lines in vitro is remarked. In vivo MM orthotopic xenograft mouse model experiments have demonstrated that the irradiation dose used in the clinic completely abrogate the efficacy of this 'off-the-shelf' immunotherapy in our treatment schedule. Decreasing irradiation doses (2.5-3.75 Gy) has no impact on tumor elimination and leads to early NK-92MI-induced death in animals (Figure 1A-B). To settle these drawbacks, CAR NK-92 MI cells expressing suicide gene therapy have been generated (99.8% purity, Figure 1C) and it has been demonstrated that cells can be rapidly eliminated in vitro upon induction with Rimiducid (AP1903). This strategy will assure antitumor efficacy and safety of the immunotherapy, and all generated effectors displayed similar in vitro cytotoxic activity than their CAR NK-92MI counterparts. In vivo experiments are ongoing.
Conclusion
We have generated simple and combined stable CAR NK-92MI cells that improve the cytotoxic activity of the parental cell line. The co-expression of both CARs results in cytotoxic coverage against different MM targets. In vivo, these data show the futility of irradiation as an NK-92 CAR treatment strategy in our MM model and the further need of combinations with iCasp9 safety switch to ensure the safety and efficacy of the therapy.
Keyword(s): Immunotherapy, Multiple myeloma, NK cell, Suicide gene therapy
Abstract: EP721
Type: E-Poster Presentation
Session title: Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research
Background
CAR-NK cells represent a safer approach than CAR- T cells. These ground-breaking CAR effectors exhibit some other immunological advantages, 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 Multiple Myeloma (MM), their cytotoxic potential can be enhanced by genetic modification. However, the products obtained so far offer some limitations and could be optimized in MM.
Aims
The aim of our study is to generate a risk-free and effective CAR NK-92MI immunotherapy for MM treatment.
Methods
NK-92MI cells were lentivirally transduced with a single second generation CAR, NKG2D-CAR or BCMA-CAR, both containing identical 4-1BB costimulatory domain, as well as dual BCMA-NKG2D CARs with different combinations of co-stimulators. CAR NK-92MI iCasp9-CD19 populations have been generated by retrovirus transduction with an SFG iCasp9.2A.ΔCD19 construct. All populations were purified by FACS sorting to obtain stable modified cell lines and vector copy number was measured by qPCR to ensure similar CAR expression. In vitro cytotoxicity assays were performed against an ensemble of MM cell lines with high and low expression of target ligands as well as a BCMA knock-out generated by CRISPR-Cas9 system: U266, BCMAhigh and NKG2DLhigh; ARP-1, BCMAdim and NKG2DLdim; XG-1, BCMAhigh and NKG2DLlow; U266bcmaKO, BCMAKO and NKG2DLhigh. In vivo: 5x106 CAR NK-92MI cell infusions were administered i.v. once a week for three weeks.
Results
We have demonstrated specificity, non-toxicity and higher in vitro antitumor activity of CAR NK-92MI cells compared to the parental cell line. Besides, we have noticed that the expression of both CARs in combination has not showed synergy, but a broadening of cytotoxic coverage against MM cell lines in vitro is remarked. In vivo MM orthotopic xenograft mouse model experiments have demonstrated that the irradiation dose used in the clinic completely abrogate the efficacy of this 'off-the-shelf' immunotherapy in our treatment schedule. Decreasing irradiation doses (2.5-3.75 Gy) has no impact on tumor elimination and leads to early NK-92MI-induced death in animals (Figure 1A-B). To settle these drawbacks, CAR NK-92 MI cells expressing suicide gene therapy have been generated (99.8% purity, Figure 1C) and it has been demonstrated that cells can be rapidly eliminated in vitro upon induction with Rimiducid (AP1903). This strategy will assure antitumor efficacy and safety of the immunotherapy, and all generated effectors displayed similar in vitro cytotoxic activity than their CAR NK-92MI counterparts. In vivo experiments are ongoing.
Conclusion
We have generated simple and combined stable CAR NK-92MI cells that improve the cytotoxic activity of the parental cell line. The co-expression of both CARs results in cytotoxic coverage against different MM targets. In vivo, these data show the futility of irradiation as an NK-92 CAR treatment strategy in our MM model and the further need of combinations with iCasp9 safety switch to ensure the safety and efficacy of the therapy.
Keyword(s): Immunotherapy, Multiple myeloma, NK cell, Suicide gene therapy