Systemic Ad-Flt3L delivery coupled with tumor killing elicited by intratumoral delivery of Ad-HSV1-TK eradicates intracranial GBM in a syngeneic rat model

MUHAMMAD; PUNTEL; CANDOLFI; LIU; KROEGER; NICHOLS; LOWENSTEIN; CASTRO

Boston, Massachusetts, USA

Congreso; American Society of Gene Therapy; 2008

Systemic Ad-Flt3L delivery coupled with tumor killing elicited by intratumoral delivery of Ad-HSV1-TK eradicates intracranial GBM in a syngeneic rat modelAKM Ghulam Muhammad1, Mariana Puntel1, Marianela Candolfi1, Chunyan Liu1, Kurt Kroeger1, W. Stephen Nichols2, Pedro R. Lowenstein1,3, Maria G. Castro1,31Gene Therapeutics Research Institute and 2Dept. of Pathology, Cedars Sinai Medical Center, 8700 Beverly Boulevard, Davis 5090; Los Angeles, CA 90048; 3Depts. of Molecular and Medical Pharmacology, and Medicine; The Brain research Institute; and Jonsson Comprehensive Cancer Center, UCLA, CA, USAAdenoviral vector (Ad) expressing fms-like tyrosine kinase 3 ligand (Flt3L) was delivered systemically with the aim to render less invasive the recently developed gene therapy strategy which combines immune stimulation utilizing Flt3L and conditional cytotoxicity elicited by herpes simplex type-1 thymidine kinase (HSV1-TK) for the treatment of GBM (Ali et al, Cancer Res 2005, 65:7194-7204; Candolfi et al, Mol Ther 2006, 14:371-81; King et al, Neuro Oncology 2007, Dec 13). Six days following stereotactic implantation of 4,500 CNS-1 cells in Lewis rats striatum (+1mm anterior from bregma, +3mm lateral and -5mm from dura), intravenous pre-dosing with an empty Ad (Ad-0; 4x1010vp) was performed in order to saturate the liver reticulo-endothelium system/Kuffer cells; within 4~6 hours, Ad-Flt3L (5x109pfu) was delivered via the tail vein followed by intratumoral delivery of Ad-TK (1x108pfu). The controls received either Ad-Flt3L or Ad-0 in the tail vein followed by intratumoral saline or Ad-TK. After 24 hours, ganciclovir (GCV, 25 mg/kg) was injected i.p. daily for 10 days. The GBM bearing control rats succumbed by ~day 22, whereas, ~80% of the Ad-Flt3L (systemic)+Ad-TK (intratumoral) treated rats survived over 60 days (P<0.01; log-rank test). The long-term survivors were rechallenged with 4,500 CNS-1 cells in the contralateral striatum and 70% of them survived long-term (over 120 days) without additional treatment. H&E stained liver specimens did not reveal any signs of hepatotoxicity. Nissl staining on the brains of Ad-Flt3L+Ad-TK treated long-term survivors displayed no residual tumor. Immunohistochemistry with antibodies against myelin basic protein or tyrosine hydroxylase showed absence of demyelination or striatal damage, respectively; CD68+ macrophages, few CD8+ T cells, and MHC II immunopositive cells were also detected. Positive delayed type hypersensitivity reaction in the long-term survivors indicated the presence of a systemic anti-GBM cellular immune response. These findings raise the possibility of treatment of human GBM by systemic Ad-Flt3L alone in the current clinical setting in which the availability of GBM antigens from dead/dying GBM tumor cells is elicited as a consequence to surgical manipulation, radiotherapy and or chemotherapy (e.g., temozolomide treatment) in human patients. This treatment strategy will render this combined gene therapy strategy even safer and less invasive. In conclusion, gene therapy strategy of immune stimulation using systemic Ad-Flt3L coupled with conditional cytotoxic treatment (Ad-TK/GCV) elicits tumor regression and immunological memory inhibiting tumor recurrence highlighting its prospect as a novel adjuvant treatment strategy for human GBM.Support: NIH/NINDS Grants 1R01 NS44556.01; 1R21-NSO54143.01; 1UO1 NS052465.01 to M.G.C.; NIH/NINDS Grants 1RO1 NS 054193.01; RO1 NS 42893.01 and 1R21 NS047298-01 to P.R.L.