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Improving Clinical Manufacturing of IL-15 Activated Cytokine-Induced Killer (CIK) Cells.

ABSTRACT: Cytokine-induced killer (CIK) cells are an immunotherapeutic approach to combat relapse following allogeneic hematopoietic stem cell transplantation (HSCT) in acute leukemia or myelodysplastic syndrome (MDS) patients. Prompt and sequential administration of escalating cell doses improves the efficacy of CIK cell therapy without exacerbating graft vs. host disease (GVHD). This study addresses manufacturing-related issues and aimed to develop a time-, personal- and cost-saving good manufacturing process (GMP)-compliant protocol for the generation of ready-for-use therapeutic CIK cell doses starting from one unstimulated donor-derived peripheral blood (PB) or leukocytapheresis (LP) products. Culture medium with or without the addition of either AB serum, fresh frozen plasma (FFP) or platelet lysate (PL) was used for culture. Fresh and cryopreserved CIK cells were compared regarding expansion rate, viability, phenotype, and ability to inhibit leukemia growth. Cell numbers increased by a median factor of 10-fold in the presence of FFP, PL, or AB serum, whereas cultivation in FFP/PL-free or AB serum-free medium failed to promote adequate CIK cell proliferation (p < 0.01) needed to provide clinical doses of 1 × 10⁶ T cells/kG, 5 × 10⁶ T cells/kG, 1 × 10⁷ T cells/kG, and 1 × 10⁸ T cells/kG recipient body weight. CIK cells consisting of T cells, T- natural killer (T-NK) cells and a minor fraction of NK cells were not significantly modified by different medium supplements. Moreover, neither cytotoxic potential against leukemic THP-1 cells nor cell activation shown by CD25 expression were significantly influenced. Moreover, overnight and long-term cryopreservation had no significant effect on the composition of CIK cells, their phenotype or cytotoxic potential. A viability of almost 93% (range: 89-96) and 89.3% (range: 84-94) was obtained after freeze-thawing procedure and long-term storage, respectively, whereas viability was 96% (range: 90-97) in fresh CIK cells. Altogether, GMP-complaint CIK cell generation from an unstimulated donor-derived PB or LP products was feasible. Introducing FFP, which is easily accessible, into CIK cell cultures was time- and cost-saving without loss of viability and potency in a 10-12 day batch culture. The feasibility of cryopreservation enabled storage and delivery of sequential highly effective ready-for-use CIK cell doses and therefore reduced the number of manufacturing cycles.

SUBMITTER: Bremm M

PROVIDER: S-EPMC6554420 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Improving Clinical Manufacturing of IL-15 Activated Cytokine-Induced Killer (CIK) Cells.

Bremm Melanie M Pfeffermann Lisa-Marie LM Cappel Claudia C Katzki Verena V Erben Stephanie S Betz Sibille S Quaiser Andrea A Merker Michael M Bonig Halvard H Schmidt Michael M Klingebiel Thomas T Bader Peter P Huenecke Sabine S Rettinger Eva E

Frontiers in immunology 20190531

Cytokine-induced killer (CIK) cells are an immunotherapeutic approach to combat relapse following allogeneic hematopoietic stem cell transplantation (HSCT) in acute leukemia or myelodysplastic syndrome (MDS) patients. Prompt and sequential administration of escalating cell doses improves the efficacy of CIK cell therapy without exacerbating graft vs. host disease (GVHD). This study addresses manufacturing-related issues and aimed to develop a time-, personal- and cost-saving good manufacturing p ...[more]

PMID: 31214182

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Project description:Cytokine Induced Killer (CIK) cells are heterogeneous ex vivo expanded T lymphocytes with mixed T-NK phenotype and endowed with a wide MHC-unrestricted antitumor activity against both solid and hematologic malignancies. CIK cells can be easily ex vivo expanded up to clinical relevant rates from circulating peripheral blood mononuclear cells (PBMC), according to a standard protocol involving timed stimulation with IFN- γ (day 0), Ab-antiCD3 OKT3 (day 1) and IL-2 (from day 1 to the end). Nowadays literature provides scanty information on cytokines, chemokines and growth factors secreted by CIK cells. A comprehensive molecular profiling of CIK cells is needed to complete the knowledge of their biologic and functional features. In this perspective we performed a gene expression profile analysis of basal PBMC and ex vivo expanded CIK cells generated from 3 patients with histologic confirmed Gastrointestinal Stromal Tumors (GIST). The principal component analysis (PCA) shows that the CIK cell samples clustered together and were clearly separated from PBMC ones. First, we checked the mRNA expression levels of secreted proteins and we found a significant upregulation of GM-CSF, IL-5, IL-13, and IFN-γ in CIK cells. Moreover, IL-1Ra, IL-1β, IL-6, IL-10, IL-15, IL-8, Eotaxin and MCP-1 are downregulated in CIK cells if compared to PBMC. Noteworthy, among differentially expressed genes (DEGs) we identified other secreted molecules that are upregulated in patient-derived CIK cells, which can contribute to their tumor killing activity: GZMA, GZMB, GZMK, PRF1, IL-32, and LTA. Furthermore, to better characterize the CIK cell phenotype, we studied the surface antigen expression. As expected, we found the downregulation of several myeloid differentiation markers (CD14, CD9, CD93, CSF2RA, CSF2RB, EPB41L3, Receptors for Fc fragments of immunoglobulins, ITGAX, MCEMP1, and TREM1) and B-cell antigens (CD19, CD24, CD79A, CXCR5, and MS4A1). Moreover, microarray data confirmed the upregulation in CIK cells of well-known surface antigens, like CD3D, CD3G, IL2Ra, IL2RG, CD226/DNAM1, ITGAL/LFA-1, KLRK1/NKG2D, NCR3/NKp30, and TRAIL/TNFSF10. Next, in order to identify differentially expressed pathways during CIK cells maturation, we performed a functional analysis by using Ingenuity Pathway Analysis (IPA) software. Among inactivated functions in CIK cells, we found chemotaxis of myeloid cells, phagocytosis, migration of granulocytes and engulfment of leukocytes. Noteworthy, functional gene categories as proliferation of cells, cell death of lymphocytes, cell death of mononuclear leukocytes, apoptosis of B lymphocytes, and quantity of CD4+ T-lymphocytes resulted activated, in agreement with the selection and expansion of CIK cell precursors induced by in vitro treatment of PBMC. Finally, IPA analysis predicted as activated categories cytotoxicity of lymphocytes, cytotoxicity of cells, and cytotoxicity of natural killer cells. In particular, genes involved in cytotoxic mechanism of CIK cells like NCR3/NKp30, KLRK1/NKG2D, TRAIL/TNFSF10, CD226/DNAM1, CD244, CD69, CD96, GZMA, GZMB, PRF1 are differentially expressed. With this work we highlighted previously unknown secretory properties and provided for the first time a comprehensive molecular characterization of CIK cells, opening the possibility to operate on secretory level to improve CIK cell cytotoxic activity against tumors.

Dataset Information

Improving Clinical Manufacturing of IL-15 Activated Cytokine-Induced Killer (CIK) Cells.

Publications

Improving Clinical Manufacturing of IL-15 Activated Cytokine-Induced Killer (CIK) Cells.

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