Abstract
IntroductionIn recent years, immunotherapy including checkpoint blockade acts as potential strategies to cure cancer. A number of recent studies have identified inducible co-stimulator (ICOS) as a crucial player in the immune checkpoints blockade therapy. ICOS, a homodimeric protein which belongs to CD28 superfamily, is expressed on activated T cells and resting memory T cells. Upon inflammation, ICOS and its ligand ICOSL (B7RP-1, B7H-2) have been shown to affect T cell responses via ICOS-mediated PI3-kinase signalling to enhance the T cells development and activity. In addition, previous studies have shown that upon CTLA-4 blockade, the ICOS/ICOSL pathway plays an important role in tumour immunity.In this study, we aim to develop and implement the Fc-fusion recombinant ICOSL proteins to induce T cells activation for therapeutic use. Our data suggested that recombinant ICOSL-Fc can enhance the proliferation and cytotoxicity effect of human Pan T cells by increasing INFg and IL10 secretion. Moreover, ICOSL-Fc can also inhibit tumour growth in xenograft animal models. Based on our study, ICOS/ICOSL pathway could be a promising therapeutic target for the immunotherapy.Material and methodsExpression and Purification of Recombinant ICOSL ProteinsConstruction of Fc-fusion ICOSL proteins:ICOS-mFc,mICOS-mFc, andICOSL-FcExpression and purification of recombinant ICOSL proteinsEmploy Flow Cytometry and ELISA-Based Assay to Confirm ICOSL-Fc Fusion Protein Can Bind to ICOSPerform ICOS binding ELISA assay with fusion ICOSL-Fc protein.Perform Pan T cell flow binding assay with fusion ICOSL-Fc protein.Results and discussionsICOSL-Fc can induce secretion of INFγ and IL-10, and proliferation of human Pan T cells.The tumour specific ICOSL-Fc engagement increases the cytotoxicity of human PBMCConclusionExpressions of PD1, ICOS and CTLA4 can be observed in PBMC under CD3/CD28 stimulation on day 1, day 3, and day 5.Recombinant human ICOSL-Fc protein was able to bind to both human ICOS and mouse ICOS ECD domains.Cell-base immunofluorescence was employed to illustrate recombinant human ICOSL-Fc protein was able to bind activated T cells.3H-Thymidine incorporation study illustrated that recombinant ICOSL-Fc was able to increase T cells proliferation.In vitro efficacy assay suggested that recombinant ICOSL-Fc can remarkable increase IL-10 and INFγ secretion on human pan T cells.The preliminary T cell cytotoxicity assay showed ICOSL-Fc treatment can increase the killing effect.
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