Improving cytokine-induced killer cell expansion using a gas-permeable culture method for clinical-scale production.

BACKGROUND: Cytokine-induced killer (CIK) cells are a heterogeneous group of immune cells that exert potent MHC-unrestricted cytotoxicity toward various cancer cells in both solid and hematological malignancies. OBJECTIVE: The purposes of this study were to compare the expansion and characteristics of cytokine-induced killer cells between a standard culture method and a gas-permeable culture method and to develop a clinical-scale expansion protocol for cytokine-induced killer cells using a gas-permeable culture method. METHODS: We compared the absolute cell number, fold change, cell subsets, activation markers, cytokine concentrations, and cytotoxicity toward myeloid leukemia cell lines between cytokine-induced killer cells expanded using two different culture methods. Then, we determined the ability to achieve clinical-scale expansion of cytokine-induced killer cells using the gas-permeable culture method. RESULTS: Cytokine-induced killer cells in the gas-permeable culture method group exhibited significantly better expansion but maintained similar cell subsets, activation markers, and cytotoxicity to those in the standard culture method group. In addition, we successfully manufactured cytokine-induced killer cells for clinical use using the gas-permeable culture method. We also showed the clinical efficacy of allogeneic cytokine-induced killer cells produced by the gas-permeable culture method in a patient with acute myeloid leukemia that relapsed after allogeneic hematopoietic stem cell transplantation. This patient maintained ongoing disease remission for 2 years with minimal side effects after cytokine-induced killer cell infusion. CONCLUSIONS: We successfully developed a simple and effective protocol for the ex vivo expansion of cytokine-induced killer cells using the gas-permeable culture method for clinical application.

Novel xeno-free and serum-free culturing condition to improve piggyBac transposon-based CD19 chimeric antigen receptor T-cell production and characteristics.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T cell is a novel therapy for relapse and refractory hematologic malignancy. Characteristics of CAR T cells are associated with clinical efficacy and toxicity. The type of serum supplements used during cultivation affects the immunophenotype and function of viral-based CAR T cells. This study explores the effect of serum supplements on nonviral piggyBac transposon CAR T-cell production. METHODS: PiggyBac CD19 CAR T cells were expanded in cultured conditions containing fetal bovine serum, human AB serum or xeno-free serum replacement. We evaluated the effect of different serum supplements on cell expansion, transduction efficiency, immunophenotypes and antitumor activity. RESULTS: Xeno-free serum replacement exhibited comparable CAR surface expression, cell expansion and short-term antitumor activity compared with conventional serum supplements. However, CAR T cells cultivated with xeno-free serum replacement exhibited an increased naïve/stem cell memory population and better T-cell expansion after long-term co-culture as well as during the tumor rechallenge assay. CONCLUSIONS: Our study supports the usage of xeno-free serum replacement as an alternative source of serum supplements for piggyBac-based CAR T-cell expansion.

An In Vitro Comparison of Costimulatory Domains in Chimeric Antigen Receptor T Cell for Breast Cancer Treatment.

Adoptive cellular therapy with chimeric antigen receptor (CAR) T cells has emerged as a potential novel treatment for various cancers. In this study, we have generated CAR T cells targeting mucin-1 (MUC1), which is an aberrantly glycosylated antigen overexpressed on breast cancer cells. Two different signaling domains, including CD28 and 41BB, were incorporated and directly compared the superiority of different costimulatory signals. Two different CAR MUC1 constructs were transduced into primary T cells and evaluated their characteristics and antitumor activities against MUC1+ cancer cells. CAR MUC1 T cells showed high transduction efficiency and antigen specificity toward MUC1+ cancer cell lines and primary breast cancer cells. When coculturing with target cells, the transduced cells exhibited potent antitumor activity in vitro and secrete proinflammatory cytokines. Upon antigen stimulation, incorporation of the 41BB signaling domain was able to improve T cell proliferation and reduce surface PD1 expression and the upregulation of suppressive cytokines, when compared with CAR MUC1 containing the CD28 domain. Our findings show that CAR T cell targeting MUC1 can be effective against MUC1+ breast cancer cell and support the further development of CAR MUC1 T cells containing 41BB signaling in preclinical and clinical studies of breast cancer treatment.

TIL expansion with high dose IL-2 or low dose IL-2 with anti-CD3/anti-CD28 stimulation provides different quality of TIL-expanded T cell clones.

Tumor infiltrating lymphocytes (TILs) are cells that are present inside the tumor environment, of which include T cells, B cells and natural killer (NK) cells. At present, TILs are used for immunotherapy in various cancers. Knowledge on adoptive transfer of TILs in ovarian cancer is still limited, especially regarding TIL expansion methods. Therefore, the aim of our study was to compare the quality of T cell clones between two expansion methods for ovarian cancer TILs. We show that TILs stimulated with the mitogenic stimulation method (low dose IL-2 with anti-human CD3/CD28) and the standard stimulation method (high dose IL-2 only) both increased total number of T cells. TCR repertoire analyses revealed different TCR repertoire patterns between TIL-expanded T cells that were stimulated with the standard stimulation method (high dose IL-2 only) and the mitogenic stimulation method (low dose IL-2 with anti-human CD3/CD28). Regardless, when TILs were expanded using the standard stimulation method (high dose IL-2 only), the predominant T cell receptor beta variable (TRBV) chains that were used in both TIL-expanded clones of the CD4+ and CD8+ subpopulations were similar. In addition, there were also TIL-expanded CD4+ and CD8+ T cell clones that were dominant in only one or the other subpopulations. These results reveal the bias in TIL quality after being stimulated with different protocols. Further studies are required to understand the selection of TIL expansion, in order for a more efficacy adoptive transfer treatment.