Autologous Natural Killer Cell-enrichment for Immune Cell Therapy: Preclinical Setting Phase, Shiraz Experience

Somayeh Rezaeifard; Yuji Heike; Jun-Ichi Masuyama; Alireza Rezvani; Reza Vojdani; Nasrollah Erfani

doi:10.18502/ijaai.v20i2.6056

Somayeh Rezaeifard Cancer Immunology and Immunotherapy group, Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
Yuji Heike Laboratory for Joint Research and Development, St. Luke's International Hospital, Tokyo, Japan ³ New City Osaki Clinic, Tokyo, Japan
Jun-Ichi Masuyama New City Osaki Clinic, Tokyo, Japan
Alireza Rezvani Hematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
Reza Vojdani Hematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
Nasrollah Erfani Cancer Immunology and Immunotherapy group, Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

DOI: https://doi.org/10.18502/ijaai.v20i2.6056

Keywords: Breast neoplasms; Cell- and tissue-based therapy; Natural killer cells

Abstract

Natural killer (NK) cell therapy has proven to be a promising approach for the treatment of malignancies. Osaki method for ex-vivo autologous NK cell expansion has been recently introduced in Japan. To start clinical trial phase I at Shiraz University of Medical Sciences in collaboration with the Japanese group, this preclinical setting study aimed to evaluate the proliferative efficacy of the method, the activation status of expanded autologous NK cells, and the likely unwanted contamination of the final cell product. Peripheral blood mononuclear cells (PBMCs) were isolated from 5 healthy individuals' peripheral blood and transferred directly to the specified initial culture bag containing anti-CD52 and anti-CD3 and Interleukin (IL)-2. The cells were cultured for 14-17 days in an incubator, during which the cells received condition media, and underwent several passages into bigger culture bags. All the procedures were carried out in a cleanroom and associated facilities. Before and after activation PBMCs were analyzed for their phenotype and cytotoxic activity; using flow cytometry and cytokine release assay. Our results indicated that NK (CD3-CD16+/-CD56+) cells were expanded 510-fold on average (range 200-1100 fold), and the purity of NK cells per whole lymphocytes exceeded 68%. The expanded cells were highly lytic as indicated by in-vitro cytotoxic assay, with a strong expression of Natural killer group 2 member D (NKG2D) and CD16. The prepared final cell products were negative for HCV, HBV, HIV, mycoplasma, and endotoxin. In the preclinical phase, large numbers of activated and un-contaminated NK cells from healthy individuals' peripheral blood were successfully generated. The method seems to provide ample clean cell product with no contamination and has the potential to be used for NK cell therapy in future clinical trials, suitable to be infused back to the donors in phase I clinical trial.