Effects Of De Novo Protein Mimic Neo-2/15 On The Function Of NK92 And CAR-NK92 Cell

BackgroundInterleukin-2（IL-2）,an immune-stimulating cytokine,has great potential in tumor immunotherapy.Recent strategies for IL-2-based cancer immunotherapy have generally focused on reducing regulatory T cell development,prolonging half-life,and simplifying preparation through mutations or chemical modifications,but these attempts have not completely eliminated CD25 interactions.Neo-2/15,an IL-2 mimicry protein designed by David Baker of the University of Washington’s Institute for Protein Design,is more stable than natural IL-2 and has a higher affinity for human and mouse IL-2Rβγ_c.NK92 is showing promise in cellular immunotherapy against tumors,having better efficacy and lower cytotoxicity than T cells,while proliferation of NK92 is dependent on IL-2.At present,whether Neo-2/15 protein can normally maintain the proliferation and functional activity of NK92 cells and chimeric antigen receptor modified NK92（CAR-NK92）cells remains to be studied.Objective1.Obtaining IL-2 mimic protein（Neo-2/15）.2.To investigate the effects of Neo-2/15 on maintaining NK92 and CAR-NK92proliferation in vitro.3.To further investigate the effect of Neo-2/15 on the antitumor activity of NK92 and CAR-NK92 in vitro.Methods1.Neo-2/15 protein coding gene was synthesized and inserted into the prokaryotic expression vector p ET-28a,which was introduced into E.coli BL2 receptor cells through thermal excitation,and then induced expression.Finally,Neo-2/15 protein was purified by nickel column.2.The effects of Neo-2/15 and Neo-2/15 after heat treatment on NK92 and CAR-NK92 were determined by CCK-8 method.Flow cytometry was used to analyze the effects of Neo-2/15 on NK92 and CAR-NK92 phenotypes.3.LDH assay was used to detect the killing efficiency of Neo-2/15 treated NK92 and CAR-NK92 after co-incubation with target cells.4.The expression levels of IFN-γ,TNF-α,granulation enzyme B and perforin in Neo-2/15 treated NK92 and CAR-NK92 co-incubated with target cells were determined by ELISA.Results1.In this experiment,E.coli BL2 receptor cells were transformed and induced to express neo-2/15 protein with high purity was obtained by nickel column purification.2.CCK-8 results showed that Neo-2/15 significantly stimulated the proliferation of NK92 and CAR-NK92.3.Flow cytometry showed that Neo-2/15 had no significant effect on the molecular phenotypes of NK92 and CAR-NK92.4.In vitro killing experiments showed that Neo-2/15 had no significant effect on the killing efficiency of NK92 and CAR-NK92.5.ELISA results showed that Neo-2/15 had no significant effect on IFN-γ,TNF-α,granase B and perforin secreted by NK92 and CAR-NK92 during cytotoxicity.ConclusionNeo-2/15 can maintain the normal proliferation and killing effect of NK92 and CAR-NK92,thus providing important evidence for the combination of chimeric antigen receptor-modified NK92 cells and neo-2/15 protein as tumor immunotherapy.