gms | German Medical Science

Objective: Glioblastoma multiforme (GBM) represents the most common primary adult brain tumor. Despite surgical resection followed by radiotherapy and chemotherapy, these tumors ultimately recur. One promising strategy for the treatment of GBM is immunotherapy. Yet, several vaccination approaches as well as adoptive transfer of tumor-reactive lymphocytes revealed disappointing responses due to immunosuppressive mechanisms of GBM. Thus far, agonists for toll-like receptors (TLRs) appears promising to reprogram the immunosuppressive environment of GBM. Yet, use of TLR agonists is limited due to unspecific delivery to tumor stroma eventually resulting in pro-tumor effects. We sought to develop a selective delivery system for TLR3 agonists for treatment of GBM. Therefore, we developed immunoconjugate-nanoparticles (IC-NPs) for targeting exclusively GBM cells with surface expression of oncogenic EGFRvIII.

Methods: Mono-biotinylated single variable fragment antibodies (scFv-BAPs) were produced in HEK293T-huBirA cells. The characteristics of scFv-BAPs were assessed by flow cytometry and Western blot analysis. For assembly of tumor-specific and unspecific IC-NPs the respective scFv-BAP was conjugated to (neutr)avidin and mono-biotinylated 50bp dsRNA (TLR3 agonist). Endosomal localization of IC-NPs in treated EGFRvIII-positive cells was examined by confocal microscopy (CLSM). Endosomal activation of TLR3 was investigated using reporter cell lines with ectopic expression of TLR3 and EGFRvIII. The release of type 1 interferons after anti-EGFRvIII IC-NP-treatment in human BS153-resE and in murine SMAvIII tumor cell lines was quantified using ELISA.

Results: Compared to IC-NP conjugated to unspecific control scFv-BAP, the tumor-specific IC-NPs were able to bind to EGFRvIII-expressing target cells, were internalized by receptor-mediated endocytosis and induced apoptosis. The uptake of IC-NPs was confirmed by CSLM and was inhibited using inhibitors of caveolae-mediated endocytosis. Furthermore, upon anti-EGFRvIII-IC-NP treatment TLR3 signaling was detected in HEKBlue-hTLR3-EGFRvIII reporter cell lines. Additionally, anti-EGFRvIII-IC-NPs induced the release of type 1 interferon in murine and human EGFRvIII-expressing glioma cell lines.

Conclusion: Our results demonstrate the feasibility of IC-NPs for targeted delivery of TLR3 agonists to GBM cells. Furthermore the IC-NP-mediated induction of innate immune responses in GBM cells holds promises to improve immunotherapy of GBM.