NON-LEAKY, INDUCIBLE ADENOVIRAL-MEDIATED DELIVERY OF A MUTATED FORM OF IL-13 (IL13. E13.K) FUSED TO PSEUDOMONAS EXOTOXIN INDUCES REGRESSION OF INTRACRANIAL HUMAN GLIOBLASTOMA XENOGRAFTS

CANDOLFI; XIONG; MUHAMMAD; PUNTEL; LIU; KROEGER; MONDKAR; RODRIGUEZ; DEBINSKI; LOWENSTEIN; CASTRO

Dallas, Texas, USA

Congreso; Society for Neuro-Oncology; 2007

ET-43. NON-LEAKY, INDUCIBLE ADENOVIRAL-MEDIATEDDELIVERY OF A MUTATED FORM OF IL-13 (IL13.E13.K) FUSED TO PSEUDOMONAS EXOTOXIN INDUCESREGRESSION OF INTRACRANIAL HUMAN GLIOBLASTOMAXENOGRAFTSMarianela Candolfi,1 Weidong Xiong,1 Akm Muhammad,1 MarianaPuntel,2 Chunyan Liu,1 Kurt Kroeger,2 Sonali Mondkar,1 RonaldRodriguez,3 Waldemar Debinski,4 Pedro Lowenstein,2 and MariaCastro2; 1Cedars Sinai Medical Center-UCLA, Los Angeles, CA, USA;2Gene Therapeutics Research Institute, Cedars Sinai Medical Center,Los Angeles, CA, USA; 3Department of Urology, Medical Oncology,Radiation Oncology and Molecular Radiation Sciences, Viral Oncology,Cellu, Johns Hopkins University School of Medicine, MD, USA; 4BrainTumor Center of Excellence, Wake Forest University, NC, USAGlioblastomas (GBMs) overexpress IL13alpha2R, which is absent inthe normal brain. Attempts to target this receptor and treat recurrent GBMin human patients have used native human IL-13 fused to Pseudomonasexotoxin A (PE), hIL-13–PE38QQR, or Cintredekin Besudotox, administeredlocally as protein formulation (Kunwar et al., J Clin Oncol. 2007(25): 837–844). We wished to test the hypothesis that adenovirus-mediatedexpression of the mutated form of human IL13 (IL13.E13K) fused to PE,which has been previously shown to target specifically human GBM (NatBiotechnol. 1998 16(5):449–453), would result in increased survival andtumor regression in human xenograft GBM models. We developed an adenovirusvector (Ad) expressing a chimeric toxin (muIL-13–PE) composedof PE fused to a mutated form of hIL13 (IL13.E13.K, muIL-13). muIL13binds to IL13alpha2R with high affinity and it has negligible binding for thephysiological IL13/IL4R, which is present in several areas of the brain andcan be targeted by the wild type hIL13–PE. We constructed the therapeuticAd-muIL4–TRE-muIL13–PE that expresses the cytotoxin muIL-13–PE;specific for GBM cells, and, as an extra safety feature, a mutated IL-4 (IL4.Y124D, muIL-4) that blocks the binding of IL13 to the physiological IL13/IL4 receptor (Int J Oncol. 1999 15(3): 481–486). We also constructed acontrol vector that expresses muIL-4 and muIL-13 (i.e., Ad-muIL4–TREmuIL13).Transgene expression is driven by the bidirectional TRE promoter,which is activated by the transactivator (TetON, expressed withinAd-TetON), in the presence of the inducer (i.e., DOX). We also encoded thetetracycline controlled transcriptional silencer, tTSKid, which inhibits basallevels of gene expression from the tetracycline inducible promoter. Inducibleexpression of muIL-4, as well as muIL-13–PE toxin was detected byimmunocytochemistry in COS-7 and human GBM cells. Cell viability wasreduced by 70% when the human GBM cells were incubated in the presenceof Ad-muIL4–TRE-muIL13–PE in the “ON” state (Dox1), but remainedunaffected in the “OFF” state, indicating that the expression of the chimerictoxin can be tightly regulated. Ad-muIL4–TRE-muIL13–PE did notaffect the viability of COS-7 cells, which do not express the IL13alpha2receptor. These results suggest that the cytotoxic effect of the chimeric isspecific to glioma cells expressing IL13alpha2R and does not target thephysiological IL13/IL4R expressed in control COS-7 cells. We also administeredAd-muIL4–TRE-muIL13–PE1Ad-TetON in the striatum of nudemice, which were fed chow containing Dox to activate transgene expression.muIL4, muIL13 and muIL13–PE toxin were detected in the mousebrain 7 days after the intracranial injection, with no toxicity, as determinedby Nissl staining and immunocytochemistry of CD3 (T cells) and F4/80(macrophages). Also, we administered Ads intratumorally into intracranialhuman U251 GBM xenografts in nude mice. Ad-muIL4–TRE-muIL13–PEsignificantly increased the survival of tumor-bearing mice when comparedto saline-treated or mice treated with the control virus or with mice treatedwith the hIL13–PE used in the human GBM trial. All Ad-muIL4–TREmuIL13–PE–treated mice survived for over 100 days after tumor implantation.Our results suggest that regulated adenoviral delivery of muIL13–PEtoxin to glioblastomas will lead to specific Dox-dependent cytotoxicityin IL-13alpha2R expressing-GBM cells and not to the surrounding nonneoplasticbrain. Supported by National Institutes of Health/NationalInstitute of Neurological Disorders & Stroke (NIH/NINDS) Grant 1R01NS44556.01, Minority Supplement NS445561; 1R21–NSO54143.01; 1UO1NS052465.01 to M.G.C.; NIH/NINDS Grants 1 RO1 NS 054193.01; RO1NS 42893.01; U54 NS045309–01, and 1R21 NS047298–01 to P.R.L. M.C.is supported by NIH/NINDS 1F32 NS058156.01.