223. Consideration for Quality Control of NKG Cell Line Used for Adoptive Cellular Therapy

Abstract

NK cells play the important role in the adoptive cell therapy. As we know, NK-92 cell, a NK cell line, has been used for the preparation of cell therapy product for clinical trials in some laboratory and had been showed the potent effect in cancer therapy. Except NK-92, another NK cell line named NKG was established by Dr Tian Zhigang in University of Science and Technology of China. NKG cell line was derived from a male patient with non-Hodgkin's lymphoma. It was isolated from patient peripheral blood samples and cultivated through long-lasting period in the medium containing horse and fatal bovine serum and IL-2. Finally it was gradually adopted to grow in serum-free medium suitable for clinical use. The surface market of NKG showed CD56brightCD16- phenotype by FACS and showed the cytotoxicity to several kinds of caner cells by in vitro and in vivo assays. Then it will be potential for applying for clinical trial in the near future. So, it is the first step to qualify the master cell bank of NKG thoroughly before it is into clinical trial.The strategy for qualification of NKG is based on the principle of risk assessment. The parameters which should be considered include the original donor eligibility, culture history and raw materials used during its isolation and cultivation, cell banking, cell characters and its intended use. So the qualification scheme for NKG includes the tests for identity and purity, the adventitious agents, the cell character and its potency.Several methods is used for test of identity and purity of NKG. Except the morphology, isoenzyme analysis is used to confirm its human origin. The STR profile analysis is used to identify it is derived from one human body and has no cross-contamination with other human cell lines. A group of cell surface makers is selected and detected by FACS. These makers will show several feactures of NKG including its NKbright celltype, its high purity with no other immune cell contamination, its low adhesion activity for serum-free adaption and its cytotoxic potency.For adventitious agents test, NKG is tested to confirm there is no presence of bacterial, mycoplasma and viruses contamination. Except for the traditional assays, the viruses is extend detected by induction assay by chemicals. The deep sequencing is also used for virus detection of NKG.Other characters of NKG is also analyzed for qualification, including the growth curve, the cytogenetic analysis, tumorigenicity and oncogenticity using NKG cell protein and DNA.In order to qualify the NKG, three kinds of assays are used for its potency test. The cell surface makers of NKG2D and NKp30 which is related with cytotoxiciey is detected by FACS. Then the cytokine secretion is detected by ELISA for IFN-gand by FACS for Granzyme B and perforin. Finally the cytotoxicity is analyzed by cell-based assay.In conclusion, thorough qualification of NKG cell lines established in China will be important and helpful to support its applying for clinical trial in the future.