pMDLg/pRRE

Chimeric antigen receptor (CAR)-T cell immunotherapy has changed the concept of treatment of hematological malignancies. Here, researchers found that CB-NK-derived CAR-NK cells were only effective at high doses (high E:T ratio) and that their activity decreased rapidly over time compared with CAR-T cells. In contrast, to exploit the potential of "off-the-shelf" CB-NK, researchers demonstrated that small amounts of CB-NK in CAR-T cell products can promote early activation of CAR-T cells and their migration into MM cells, resulting in enhanced resistance to MM efficacy. Furthermore, there was no increase in cytokines associated with the development of CRS and, importantly, CB-NK enhanced the fitness of CARpos and CARneg T cells, promoting lower levels of fatigue and senescence. This study demonstrates the relevant immunomodulatory effects of CB-NK in collaboration with CAR-T cells to enhance their anti-tumor activity.

For CAR-T cells, pCCL-EF1a-CAR19 or pCCL-EF1a-CARBCMA with packaging plasmids pMDLg-pRRE, pRSV-Rev and envelope plasmid VSV was used. For CAR-NK cells, pCCL-MSCV-CAR19 with pMDLg-pRRE, pRSV-Rev and envelope plasmid BaEV-TR was used to produce viral particles. Production of viral particles for transducing T cells was accomplished in HEK293T cells. Virus production for CAR-NK cells was performed in HEK293T cells transfected with the transfer vector pCCL-MCSV-CAR19 harboring pMDLg-pRRE, pRSV-Rev, and BaEV-TR plasmids. Transfections were performed and viral supernatants were collected at 48 hours, and the virus was concentrated and stored. The obtained virus can be used to transduce CB-NK to obtain CAR-NK cells targeting CD19 (ARI3).

Figure 1. Diagram showing two different protocols used to obtain CAR-NK cells directed against CD19 (ARI3) from CB-NK. (Bachiller, Mireia, et al. 2021)