NK cell expansion with EX21-exosomes

      NK cells as part of innate immune system provide immunity against tumors. However, the paucity of NK cells in blood can be as low as 5% of total lymphocytes. Thus, for the intent of adoptive immunotherapy, a novel method to enhance anti-tumor activity and expand numbers of NK cells is desirable. Our group developed a NK cell expansion method that utilizes plasma membrane (PM) nanoparticles derived from engineered K562 cells (CSTX-002) expressing 41BBL and membrane-bound IL-21. Eliminatiing the need for feeder cells improves safety and may allow greater wide adoption of the method. Exosomes, which are cell-derived nanovesicles naturally secreted by cancer cells, may be another novel way to expand NK cells. EX21-exosomes were prepared from CSTX-002 cells and tested for their ability to expand NK cells. The exosomes were characterized by Nanosight for their size and concentration, and presence of exosomal markers (CD63 and HSP70) was confirmed by Western blot. CD3-depleted PBMCs were cultured with EX21 (20 ng of protein/mL of culture) for 14 days. NK cell counts were monitored, and media changed every 2–3 days. NK cells cultured with EX21 expanded 530 fold (344–710) over 14 days and 735 fold (667–802) in presence of PM21-particles. Furthermore, EX21 exosomes were preferentially taken up by the NK cells rather than T cells during expansion as determined by using Alexa647-labelled exosomes. EX21-NK and PM21-NK cells had comparable in vitro cytotoxicity against K562 cells. Comparative in vivoefficacy of EX21-NK cells and PM21-NK cells was assessed in NSG mice implanted ip with 1x106 SKOV-3Luc ovarian tumor cells, seeded for 4 days. SKOV-bearing mice were treated with vehicle or two doses (10x106, 5 days apart)of EX21-NK cells cultured for 14 days with EX21, and co-administered with or without in vivo EX21-exosomes (10 µg, 3x/week). The control group received PM21-NK cells (same dosage) with in vivo administration of PM21-particles (600 µg, 3x/week). Treatment of SKOV engrafted NSG mice with either EX21-NK cells or with PM21-NK cells allowed significantly (<0.0001) increased survival compared to untreated animals (41–44 vs 29 days post treatment). Administration of ip EX21-exosomes to SKOV-3 bearing mice had no effect on survival in neither untreated control or EX21-NK cell treated groups. In conclusion, EX21-exosmes efficiently expand NK cells in vitro, and EX21-NK cells are equally effective as PM21-NK cell at anti-tumor activity, both in vitro and in vivo.
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