Abstract
BackgroundRecently, allogeneic natural killer (NK) cells have gained considerable attention as promising immunotherapeutic tools due to their unique biological functions and characteristics. Although many NK expansion strategies have been reported previously, a deeper understanding of cryopreserved allogeneic NK cells is needed for specific therapeutic approaches.MethodsWe isolated CD3−CD56+ primary natural killer (pNK) cells from healthy donors and expanded them ex vivo using a GMP-compliant method without any feeder to generate large volumes of therapeutic pNK cells and cryopreserved stocks. After validation for high purity and activating phenotypes, we performed RNA sequencing of the expanded and cryopreserved pNK cells. The pNK cells were used against various cancer cell lines in 7-AAD/CFSE cytotoxicity assay. For in vivo efficacy study, NSG mice bearing subcutaneous cisplatin-resistant A2780cis xenografts were treated with our pNK cells or cisplatin. Antitumor efficacy was assessed by measuring tumor volume and weight.ResultsCompared to the pNK cells before expansion, pNK cells after expansion showed 2855 upregulated genes, including genes related to NK cell activation, cytotoxicity, chemokines, anti-apoptosis, and proliferation. Additionally, the pNK cells showed potent cytolytic activity against various cancer cell lines. Interestingly, our activated pNK cells showed a marked increase in NKp44 (1064-fold), CD40L (12,018-fold), and CCR5 (49-fold), and did not express the programmed cell death protein 1(PD-1). We also demonstrated the in vitro and in vivo efficacies of pNK cells against cisplatin-resistant A2780cis ovarian cancer cells having a high programmed death-ligand 1(PD-L1) and low HLA-C expression.ConclusionsTaken together, our study provides the first comprehensive genome wide analysis of ex vivo-expanded cryopreserved pNK cells. It also indicates the potential use of expanded and cryopreserved pNK cells as a highly promising immunotherapy for anti-cancer drug resistant patients.
Highlights
Allogeneic natural killer (NK) cells have gained considerable attention as promising immunotherapeutic tools due to their unique biological functions and characteristics
The increase of some activating receptors, chemokines, and anti-apoptotic genes, such as NKp44 (1064-fold), TNFSF10 (24.5-fold), CCR2 (130.1-fold), CCR3 (1427.5fold), CCR5 (49.9-fold), CCR6 (124.1-fold), and BCL2 (10.5-fold), was more significant in selectively expanded primary natural killer (pNK) cells than in non-selectively expanded pNK cells (6.3, 9.7, 6.6, NA, 16.7, NA, and 1.7-fold, respectively) (Fig. 3 and Additional file: Fig. S3). These results indicated that the NK cell culture method from Peripheral blood mononuclear cells (PBMCs) with or without CD3−CD56+ selection increased the expression of various activating NK receptors and cytotoxicity-related genes and when pNK cells were cultured with CD3−CD56+ selection, CD40 ligand (CD40L), NKp44, and migration-related genes were more significantly expressed
As CD158b (KIR2DL2/3) was increased in expanded pNK cells (Fig. 2c), we investigated the effect of human leukocyte antigen (HLA)-C1 or C2 expression on the cytotoxicity of pNK cells
Summary
Allogeneic natural killer (NK) cells have gained considerable attention as promising immunotherapeutic tools due to their unique biological functions and characteristics. Methods: We isolated CD3−CD56+ primary natural killer (pNK) cells from healthy donors and expanded them ex vivo using a GMP-compliant method without any feeder to generate large volumes of therapeutic pNK cells and cryopreserved stocks. The NK cell activating signal is mediated by several NK receptors, including natural killer group 2D (NKG2D), natural cytotoxicity receptors (NCRs; NKp46, NKp44, and NKp30), Ig-like receptors (2B4), and costimulatory receptors (DNAM-1). NK cell inhibition is regulated by two major histocompatibility complex class I (MHC-1; known as human leukocyte antigen (HLA))specific inhibitory receptors, killer cell immunoglobulin-like receptors (KIRs) and the CD94-NKG2A heterodimer. Virus-infected or tumor cells lose surface MHC class I expression, leading to a low inhibitory NK cell signaling and allowing NK cells to become activated to kill target cells [2, 9]
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