Abstract
BackgroundAdoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells’ ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain.MethodsA second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model.ResultsChimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRvIII expressing glioma cells.ConclusionsOur study demonstrates that the EGFRvIII/CAR-modified T cells can destroy glioma cells efficiently in an EGFRvIII specific manner and release IFN-γ in an antigen dependent manner. The specific recognition and effective killing activity of the EGFRvIII-directed T cells with ICOS signaling domain lays a foundation for us to employ such approach in future cancer treatment.
Highlights
Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy
EGFRvIII/CAR was constructed and T cells were modified successfully by lentiviral EGFRvIII/CAR To generate EGFRvIII-specific T cells, chimeric EGFRvIII/ CAR was constructed As shown in Figure 1A, EGFRvIII/ CAR encodes a fusion protein consist of IgG κ leader peptide, EGFRvIII single chain variable fragment (scFv), the hinge and TM region of human CD8α, intracellular signal domain of inducible costimulator (ICOS) and the CD3ζ chain
No extra linker or space was used between gene fragments since it may increase the immunogenicity of EGFRvIII/CAR leading to immune destruction of the transduced T cells in vivo
Summary
Construction of chimeric antigen receptor and generation of EGFRvIII expressing U87cell line Chimeric EGFRvIII/CAR is composed of EGFRvIII scFv and ICOS-CD3ζ expression cassette. The activated CD3+ T cells were infected with lentiviral EGFRvIII/CAR at MOI of 5 in the presence of IL-2 (30 units/ml). Functional analysis of EGFRvIII/CAR+ T cells Cytotoxicity assay was done as described [15]. In vivo antitumor activity of EGFRvIII/CAR+ T cells Xenograft tumor mouse model was established by subcutaneous (s.c.) flank injections of 5×106 EGFRvIII expressing U87 cells in 6-week-old female BALB/cA-nude mice (Chinese Academy of Science Shanghai Experimental Animal Center). When the tumor burden reached about 500 mm in about 10–14 days after tumor cells inoculation, the mice were assigned to different groups (5 in each group) and injected with 1×107 different T cells/100 μl (EGFRvIII/CAR-transduced T cells, GFP-transduced T cells, and control PBS) either either systemically to tail vein or locally to the tumor mass. P-Values less than 0.05 were considered statistically significant
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