Evaluation of serum-free media formulations in feeder cell expansion of natural killer cells

Abstract

Background & Aim Natural Killer (NK) cells are rapidly evolving as a cellular therapy due to their innate ability to kill a wide range of cancerous cells without prior sensitization and broad safety profile without concerns for causing graft-versus host disease. However, the clinical grade production of NK cell products has historically required the addition of fetal bovine or human serum, which have safety, ethical, and regulatory concerns. Serum-free media have been established for T cell activation and expansion, but little is known about serum-free media in manufacturing NK cells. Here, we sought to assess a broad range of serum-free media in supporting NK cell expansion with K562-based feeder cells for use in adoptive immunotherapy. Methods, Results & Conclusion Cryopreserved expanded human peripheral blood NK cells (ePBNK) were re-stimulated at 1:1 ratio with irradiated K562 expressing mbIL21 and 4-1BBL (CSTX002) and propagated for 7 days in 6 different clinical-grade serum-free media. The three serum-free media that yielded the highest cell numbers (TexMACS, ABS011, and AIM-V/ICSR) were further compared for support of 2-week NK cell expansion starting from freshly-isolated PBNK, and the resulting ePBNK were assessed for phenotype, cytotoxicity, degranulation, and cytokine production. NK cells did not have acceptable expansion kinetics with OpTmizer, SCGM, or StemXVivo. Surprisingly, NK cells had significantly improved expansion with AIM-V/ICSR (5448-fold) compared to RPMI + FBS control (2621-fold). Additionally, the AIM-V/ICSR expanded NK cells showed increased anti-tumor cytotoxicity and TNFa secretion, and had increased expression of several NK cell activating receptors and effector molecules such as DNAM-1, NKG2D, NKp30, FasL, granzyme B, and perforin. These results indicate that the serum-free AIM-V/ICSR expansion medium can support ex vivo expansion of NK cells with high effector function and phenotype compared to the standard RPMI + FBS medium and is an effective alternative for use in adoptive NK cell therapy.