RESUMO
A bispecific ligand-directed toxin (BLT) consisting of human interleukin-13, epithelial growth factor, and the first 389 amino acids of diphtheria toxin was assembled in order to target human glioblastoma. In vitro, DTEGF13 selectively killed the human glioblastoma cell line U87-luc as well as other human glioblastomas. DTEGF13 fulfilled the requirement of a successful BLT by having greater activity than either of its monospecific counterparts or their mixture proving it necessary to have both ligands on the same single chain molecule. Aggressive brain tumors established intracranially (IC) in nude rats with U87 glioma genetically marked with a firefly luciferase reporter gene were treated with two injections of DTEGF13 using convection enhanced delivery resulting in tumor eradication in 50% of the rats which survived with tumor free status at least 110 days post tumor inoculation. An irrelevant BLT control did not protect establishing specificity. The bispecific DTEGF13 MTD dose was measured at 2 microg/injection or 0.5 microg/kg and toxicity studies indicated safety in this dose. Combination of monospecific DTEGF and DTIL13 did not inhibit tumor growth. ELISA assay indicated that anti-DT antibodies were not generated in normal immunocompetent rats given identical intracranial DTEGF13 therapy. Thus, DTEGF13 is safe and efficacious as an alternative drug for glioblastoma therapy and warrants further study.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Glioblastoma/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/toxicidade , Animais , Especificidade de Anticorpos , Neoplasias Encefálicas/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Genes Reporter , Glioblastoma/patologia , Humanos , Imunoglobulina G/sangue , Ligantes , Fígado/efeitos dos fármacos , Luciferases/genética , Transplante de Neoplasias , Ratos , Ratos Nus , Proteínas Recombinantes de Fusão/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A novel bivalent single chain fusion protein, Bic3, was assembled consisting of the catalytic and translocation domains of diphtheria toxin (DT(390)) fused to two repeating sFv molecules recognizing human CD3 epsilon of the human T-cell receptor. Historically, problems with these constructs include low yield, toxicity, and reduced efficacy. Instead of using conventional Gly(4)Ser linkers to connect heavy/light chains, aggregation reducing linkers (ARL) were used which when combined with a new SLS-based refolding method reduced aggregation and enhanced the yield of final product. Toxicity was reduced at least 25-fold by repeating the two sFv molecules and adding a portion of the hinge-CH2-CH3 human constant regions. The resulting Bic3 was just as cytotoxic to HPB-MLT.UM T leukemia cells in vitro (IC(50)=4 pmol) as a monovalent construct made with the same DT and sFv. In vivo, Bic3 was effective in a new and aggressive therapy model in which it significantly prolonged survival of scid mice with established human T-cell leukemia (p<0.0001 compared to controls). Importantly, no toxicity measured by weight loss, enzyme function, or histology was observed at the highest dose of Bic3 tested (2000 ug/kg). Bic3 warrants investigation as a new drug for treating T-cell malignancy and other T-cell related disorders.
Assuntos
Imunotoxinas/efeitos adversos , Imunotoxinas/química , Leucemia de Células T/tratamento farmacológico , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/síntese química , Animais , Anticorpos Monoclonais , Complexo CD3/imunologia , Modelos Animais de Doenças , Humanos , Imunoglobulina G , Rim/patologia , Camundongos , Camundongos SCID , Receptores de Antígenos de Linfócitos T , Proteínas Recombinantes de Fusão/química , Linfócitos T/efeitos dos fármacosRESUMO
A gene splicing technique was used to create a hybrid fusion protein DTAT encoding the 390 amino acid portion of diphtheria toxin (DT(390)), a linker, and the downstream 135-amino terminal fragment portion of human urokinase plasminogen activator. DTAT was assembled to target human glioblastoma cell lines in a murine intracranial model. Previously published in vitro studies demonstrated that DTAT was highly selective and toxic to human glioblastoma cell lines in a flank tumor model. The purpose of this study was to determine the toxicity, specificity and possible therapeutic efficacy of DTAT in an intracranial model. Convection enhanced delivery of DTAT resulted in about a 16-fold increase in maximum tolerated dose. Intracranial administration of DTAT on an every-other-day basis in nude mice with established U87 MG brain tumors resulted in significant reductions in tumor volume and significantly prolonged survival (p < 0.0001). Magnetic resonance imaging proved to be a powerful tool in mice and rats for demonstrating tumor growth in a xenograft intracranial model, assessing the efficacy of DTAT in tumor volume reduction and detecting DTAT-associated intracranial toxicity and vascular damage. These results suggest that the DTAT recombinant fusion protein is highly effective in an intracranial model and DTAT might be an effective treatment for glioblastoma.