A novel vesicular stomatitis virus armed with IL-2 mimic for oncolytic therapy

Oncolytic virus(OV)is increasingly being recognized as a novel vector in cancer immunotherapy.Increasing evidence suggests that OV has the ability to change the immune status of tumor microenvironment,so called transformation of‘cold’tumors into‘hot’tumors.The improved anti-tumor immunity can be induced by OV and further enhanced through the combination of various immunomodulators.The Neo-2/15 is a newly de novo synthesized cytokine that functions as both IL-2 and IL-15.However,it specifically lacks the binding site of IL-2 receptorαsubunit(CD25),therefore unable to induce the Treg proliferation.In present study,a recombinant vesicular stomatitis virus expressing the Neo-2/15(VSVM51R-Neo-2/15)was generated.Intratumoral delivery of VSVM51R-Neo-2/15 efficiently inhibited tumor growth in mice without causing the IL-2-related toxicity previously observed in clinic.Moreover,treatment with VSVM51R-Neo-2/15 increased the number of activated CD8t T cells but not Treg cells in tumors.More tumor-bearing mice were survival with VSVM51R-Neo-2/15 treatment,and the surviving mice displayed enhanced protection against tumor cell rechallenge due to the induced anti-tumor immunity.In addition,combination therapy of OV and anti-PD-L1 immune checkpoint inhibitors further enhanced the anti-tumor immune response.These findings suggest that our novel VSVM51R-Neo-2/15 can effectively inhibit the tumor growth and enhance the sensitivity to immune checkpoint inhibitors,providing promising attempts for further clinical trials.

Development of a novel platform for recombinant protein production in Corynebacterium glutamicum on ethanol

Corynebacterium glutamicum represents an emerging recombinant protein expression factory due to its ideal features for protein secretion,but its applicability is harmed by the lack of an autoinduction system with tight regulation and high yield.Here,we propose a new recombinant protein manufacturing platform that leverages ethanol as both a delayed carbon source and an inducer.First,we reanalysed the native inducible promoter PICL from the acetate uptake operon and found that its limited capacity is the result of the inadequate translation initial architecture.The two strategies of bicistronic design and ribozyme-based insulator can ensure the high activity of this promoter.Next,through transcriptional engineering that alters transcription factor binding sites(TFBSs)and the first transcribed sequence,the truncated promoter PA256 with a dramatically higher transcription level was generated.When producing the superfolder green fluorescent protein(sfGFP)under 1%ethanol conditions,PA256 exhibited substantially lower protein accumulation in prophase but an approximately 2.5-fold greater final yield than the strong promoter PH36.This superior expression mode was further validated using two secreted proteins,camelid antibody fragment(VHH)and endoxylanase(XynA).Furthermore,utilizing CRISPRi technology,ethanol utilization blocking strains were created,and PA256 was shown to be impaired in the phosphotransacetylase(PTA)knockdown strains,indicating that ethanol metabolism into the tricarboxylic acid cycle is required for PA256 upregulation.Finally,this platform was applied to produce the“de novo design”protein NEO-2/15,and by introducing the N-propeptide of CspB,NEO-2/15 was effectively secreted with the accumulation 281 mg/L obtained after 24 h of shake-flask fermentation.To the best of our knowledge,this is the first report of NEO-2/15 secretory overexpression.