The most aggressive intracranial primary brain tumor is glioblastoma multiforme (GBM); which is associated with a 5% survival rate five years post-diagnosis. In this work, we uncovered the pathways that lead to the anti-tumoral immune response induced by a combined immunotherapy/conditional cytotoxic approach that utilizes adenoviral vectors (Ad) expressing Fms-like Tyrosine Kinase 3 ligand (Flt3L) and Thymidine Kinase (TK) delivered into the tumor mass in a syngeneic mouse model of GBM. We implanted GL26 cells into the brains of C56/B6 mice and treated tumors 17 days later with AdFlt3L+Ad-TK. This induced infiltration of bone marrow-derived myeloid DC (mDC) into the tumor mass and caused tumor regression and long term survival in ~50% of the mice. This treatment also induced clonal expansion of tumor antigen specific T-lymphocytes, and failed in CD4 and CD8 KO mice. TLR signaling was necessary for the induction of an anti-tumor immune response. In MyD88 and TLR2 KO mice, DCs failed to infiltrate tumors, and the treatment failed. We then identified the endogenous ligand responsible for TLR2 activation as high-mobility-group box 1 (HMGB1), a chromatin protein that acts as a cytokine when released in the extracellular milieu, and as a TLR2 agonist. Cultures of GL26 GBM cells, and other tumor cells (mouse melanoma, small cell lung carcinoma and GL261 glioma cell lines) treated with Ad-TK+GCV, temozolamide or irradiation; all release HMGB1 into the medium. These cell supernatants specifically activated TLR2 signaling; this activation was blocked by glycyrrhizin, which binds and inactivates HMGB1, or antibodies against HMGB1. Increased levels of HMGB1 were detected in the serum of GL26 tumor-bearing mice after the treatment with AdFlt3L+Ad-TK, and the efficacy of the treatment was prevented in vivo by the administration of glycyrrhizin or anti-HMGB1 antibodies. Therapeutic efficacy of Ad-Flt3L and Ad-TK (+GCV) treatment was demonstrated in a second intracranial glioma model and in an intracranial syngeneic melanoma model with concomitant increases in the levels of circulating HMGB1 in response to the therapy. Our work reveals that endogenous HMGB1 released from dying tumor cells in response to the therapy, and subsequent signaling via TLR2, elicit effective anti-tumor immune responses of our novel combined immuno-stimulatory and conditional cytotoxic gene therapy. This approach will be tested in a phase I clinical trial for recurrent glioblastoma to start in 2009. Supported by NIH/NINDS R01 NS44556.01, R21-NSO54143.01; UO1 NS052465.01; RO3 TW006273-01 to M.G.C.; RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309-01, and R21 NS047298-01 to P.R.L; F32 NS058156.01 to M.C.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 364.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

American Association for Cancer Research
2009