Methodology Study Of Evaluating Immune Cell Therapeutic Products Cytotoxic Potency In Vitro

Objective To find out a relatively rapid alternative method to the traditional ⁵¹ Cr release assay by optimizing and comparing four cytotoxic assays in vitro.This method is capable of being used to evaluate cytotoxic potency of various immune cells in vitro.Meanwhile,to explore alternative indicator?s?for NK cell cytotoxic potency in vitro.Our aim is to obtain alternative indicator?s?that can be used for the rapid releasing assay of product in relation to cytotoxic potency in vitro as well as improve the safety of released product.Methods NK-92 cell line was used as effect cells and K562 cells line as target cell to optimize the loading condition,killing time and effect to target ratio of four frequently used in vitro cytotoxic potency assays: BATDA assay,CAM assay,Cyto Tox-Glo assay and PKH assay,respectively.Then we analyzed the reproducibility of intra-and inter-tests of each assay and the correlation with ⁵¹ Cr release assay.We also analyzed the applicable extent of these four assays by using different effect or target cells.On one hand,alternative indicator?s?which will substitute cytotoxic potency assay was first choosen by analyzed the secretion activity of IFN-?,perforin and granzyme B using Luminex,and the expression of CD107 a and CD69 surface molecular using flowcytometry.On the other hand,the indicator?s?was choosen by analyzing gene and micro RNA expression difference during NK cell killing progress and further screened out by GO and KEGG enrichment,which has connection with cytotoxic potency.Finally,the chosen alternative indicator?s?was validated by quantitative real time PCR analysis in order to provide a basis for further searching for alternative molecular markers that may be associated with the cytotoxic potency.Results After methodological optimizing,four methods demonstrated effect-to-target-ratio within certain extent in vitro.Comparing to the other methods,CytoTox-Glo assay presented significant hook-effect when effect to target ratio is higher than 40:1.BATDA assay was the short time-consuming method,which took just one hour to observe distinct cytotoxic potency after NK-92 cell interacted with K562 cells,however,CAM and PKH assay took four hours and CytoTox-Glo took six to eight hours.The reproducibility of intra-and interassays is relatively good,and BATDA and CAM assay were performed better than that of the other two assays.The BATDA assay presented the best goodness of fit among all the tested assays when compared to ⁵¹ Cr release assay,R2=0.9281.The BATDA assay was applicable to detect the cytotoxic potency of NK cell to adherent Caov3 cell and other effect cells,such as autologous NK cell and CAR T cell,and represented individual difference.NK-92 cell was cocultured with K562 cell with 10:1 effec to target ratio,sampling with 15 minutes interval until 2 hours.The results showed that the apoptosis of target cells increased in a straight line.Compared with control,IFN-gamma secretion of effector cells in experimental group began to expand from 75 minutes,in the form of index with killing time increasing,and was highly relevant with cytotoxic potency.Granyme B and perforin increased significantly in 75 min,without obvious difference in other time.CD69 continuously high expressed in the surface of NK-92 cell lines,while the expression of CD107 a increased slightly but without significant correlation with cytotoxic potency.There were more than 135 genes and 61 small RNAs showed different expression,based on the results of GO and KEGG enrichment,BIRC3,CSF2 and VCAM1 were chosed those were relevant to cytotoxic potency.Relevant micro RNAs of the differentially expressed genes were hsa-miR-24-2-5p,hsa-miR-624-5p,hsa-miR-532-3p and hsa-miR-545-3.These RNAs and micro RNAs were further confirmed by qPCR,which three differently expressed genes are indeed differently expressed remarkably?p<0.05?,while micro RNAs are not.Conclusion The BATDA assay has the best correlation to ⁵¹ Cr release assay,and is the rapidest and the best reproducibility assay among all tested assays.The BATDA also has advantages over other assays at the extent of effect cells and K562 cells application.Therefore,BATDA assay may be the best candidate to the ⁵¹ Cr release assay to evaluate cytotoxic potency of NK cell and other immune cell in vitro.IFN-? as well as three differentially expressed genes: BIRC3,CSF2 and VCAM1 have the great potential to become alternative indicators or molecular markers for rapid evaluation of cytotoxic potency,but the dose-effect relationship with cytotoxic potency need to be studied further.