Comparison of cytotoxic potency between freshly cultured and freshly thawed cytokine-induced killer cells from human umbilical cord blood

Abstract

Immune cell therapy has been incorporated into cancer therapy over the past few years. Chimeric antigen receptor T cells (Car-T cells) transplantation is a novel and promising therapy for cancer treatment and introduces a new age of immune cell therapy. However, the expensive nature of genetic modification procedures limits the accessibility of Car-T cells for cancer treatment. Cytokine-induced killer cells (CIKs) can kill the target cells in an MHC-non-restricted manner; these cells can be developed to "off-the-shelf" immune cell products for cancer treatment. However, the anti-tumor potency of freshly thawed CIKs is not well documented. This study aimed to fill this gap, evaluating the anti-tumor potency of freshly thawed CIKs compared to that of freshly cultured CIKs. CIKs were produced from the human umbilical cord blood in accordance with published protocols. CIKs were cryopreserved in xeno-free cryomedium that contains 5% DMSO, 10% human serum in phosphate buffer saline at - 86 °C. These cells were thawed and immediately utilized in assays (called freshly thawed CIKs) with freshly cultured cells are control. The expression of the surface markers of CIKs, cytokine production, and in vitro anti-tumor cytotoxic cells of freshly thawed CIKs were evaluated and compared to freshly cultured CIKs. Additionally, the freshly thawed CIKs were injected into the breast of tumor-bearing mice to assess the anti-tumor potency in vivo. The results obtained in freshly thawed CIKs and freshly cultured CIKs demonstrated that the expression of CD3, and CD56 were comparable in both cases. The production of TNF-α, IFN-γ, and IL-10 was slightly reduced in freshly thawed cells compared to the freshly cultured cells. The in vitro lysis toward MCF-7 cancer cells was similar between freshly thawed and freshly cultured CIKs. Moreover, the freshly thawed CIKs displayed anti-breast tumor activity in the breast tumor-bearing mice. The volume of tumors significantly reduced in the mice grafted with freshly thawed CIKs while, conversely, the tumor volume in mice of the placebo group gradually increased. This study substantiated that freshly thawed CIKs preserved their anti-tumor potency in both in vitro and in vivo conditions. The results initially revealed the great potential of UCB-CIKs for "off-the-shelf" CIK product manufacturing. However, further studies on the effects of cryomedia, freezing rate, and thawing procedure should be undertaken before freshly thawed off-the-shelf UCB-CIKs are utilized in clinical trials.

Keywords: Cytokine induced killer cells; Immunotherapy; UCB-CIK; Umbilical cord blood; Umbilical cord blood derived cytokine induced killer cells.