A serum-free protocol for the ex vivo expansion of Cytokine-Induced Killer cells using gas-permeable static culture flasks

Abstract

Cytokine-Induced (CIK) cells represent an attractive approach for cell-based immunotherapy, as they show several advantages compared with other strategies. Here we describe an original serum-free protocol for CIK cell expansion that employs G-Rex devices and compare the resulting growth, viability, phenotypic profile and cytotoxic activity with conventional culture in tissue flasks. CIK cells were obtained from buffy coats, seeded in parallel in G-Rex and tissue flasks, and stimulated with clinical-grade IFN-g, anti-CD3 antibody and IL-2. G-Rex led to large numbers of CIK cells, with a minimal need for technical interventions, thus reducing the time and costs of culture manipulation. CIK cells generated in G-Rex showed a less differentiated phenotype, with a significantly higher expression of naive-associated markers such as CD62L, CD45RA and CCR7, which correlates with a remarkable expansion potential in culture and could lead to longer persistence and a more sustained anti-tumor response in vivo. The described procedure can be easily translated to large-scale production under Good Manufacturing Practice. Overall, this protocol has strong advantages over existing procedures, as it allows easier, time-saving and cost-effective production of CIK effector cells, fostering their clinical application.

Innovative therapeutic strategy for B-cell malignancies that combines obinutuzumab and cytokine-induced killer cells

“…In particular, in the autologous setting, the challenge is to generate sufficient effectors starting from blood samples containing mostly tumor cells and very low CD3 + counts. To this aim, we have upgraded a culture protocol 16 that was developed for an immediate translation into GMP as it employs pharmaceutical grade reagents, the elimination of animal derivatives (such as FBS) and serum-free media, according to the guidelines for ATMP production, 45 and already allows the generation of clinically-relevant numbers of CIK cells. In the BL-CIK protocol, the early addition of Blina in the culture that simultaneously targets CD3 on effectors and CD19 on target cells, led to the concomitant expansion of CIK cells and the elimination of the malignant B cell fraction, without any magnetic selection or cell sorting at the beginning of the expansion.…”

“…PBMCs were obtained from peripheral blood samples of adult patients (age, 66.6±4.4 years) and were expanded in G-Rex gas-permeable flasks (Wilson Wolf Saint Paul, Massachusetts, USA). 16 At day 0, PBMCs were seeded in G-Rex6M at a density of 5×10 5 cells/cm 2 (5×10 6 cells/ well) in 40 mL of X-VIVO 10 medium (Lonza, Switzerland) supplemented with 1% Penicillin/Streptomycin (Lonza, Switzerland) and rhIFN-γ (R&D, MN, USA) at 1000 U/mL. After 24 hours, anti-CD3 pure humanfunctional grade antibody (OKT-3, Miltenyi Biotec, CA, USA) at 50 ng/mL and rhIL-2 (Proleukin, Novartis, Switzerland) at 500 IU/mL were added.…”

Cord-Blood-Derived Professional Antigen-Presenting Cells: Functions and Applications in Current and Prospective Cell Therapies

“…In a different approach, UCB-derived DC are being employed as effector cells for the development of cytokine-induced killer cells (CIK) [ 168 ]. CIKs describe a heterogeneous population of ex vivo expanded T cells that acquire CD56 expression during their expansion and boast the capabilities of both T and NK cells [ 191 ]. In contrast to other adoptive cell therapies, CIKs can be relatively easily obtained through cytokine stimulation of PBMCs or UCB cells and do not require antigen-specific stimuli to recognize and act on tumor cells [ 191 ].…”

“…CIKs describe a heterogeneous population of ex vivo expanded T cells that acquire CD56 expression during their expansion and boast the capabilities of both T and NK cells [ 191 ]. In contrast to other adoptive cell therapies, CIKs can be relatively easily obtained through cytokine stimulation of PBMCs or UCB cells and do not require antigen-specific stimuli to recognize and act on tumor cells [ 191 ]. Co-culture of UCB-derived DC and CIKs was shown to produce an almost 27-fold increase in CIK proliferation in comparison with CIKs by themselves and be superior to a co-culture with adult peripheral blood-derived DC [ 168 ].…”

Breast cancer treatment by transplantations of dendritic cells and cytokine-induced killer cells: An update on clinical trials

“…The clinical study further demonstrated the antitumor potential of UCB-CIK with minimal toxicities 113,114 . Further, large-scale production of GMP-grade CIK is under vigorous study; Castiglia S et al and Palmerini P et al suggested the significant impact of culture systems on CIK cell quality 115,116 . Serum-free conditions were studied to abrogate the in-consistent quality of human serum and human pool plasma.…”