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1.
Cancer Lett ; 197(1-2): 211-7, 2003 Jul 18.
Article in English | MEDLINE | ID: mdl-12880984

ABSTRACT

The induction of T-cell mediated immunity against neuroblastoma is a challenge due to poor immunogenicity of this malignancy. Here, we demonstrate the induction of protective immunity in a syngeneic murine neuroblastoma model following vaccination with minigenes comprising of three novel natural MHC class I ligands. First, after immunoprecipitation of MHC class I from NXS2 cells, peptides were eluted and examined in tandem-MS analysis which lead to the identification of three novel natural MHC class I peptide ligands, TEALPVKLI from ribonucleotide reductase M2, NEYIMSLI from Ser/Thr protein phosphatase 2A and FEMVSTLI with unknown origin. Second, we constructed two different minigenes, one encoding for the three novel epitopes and the second for three known mTH derived epitopes with high predicted binding affinity to MHC class I by cloning them into the mammalian expression vector pCMV-3FUB. This lead to constructs with an ubiquitin-tag upstream the inserted epitopes in order to facilitate proteasomal degradation. Furthermore the epitopes were separated by a spacer peptide (AAY), which proved to be a preferential proteasome cleavage site. Third, we demonstrate the induction of protective immunity against neuroblastoma using an attenuated strain of Salmonella typhimurium as a carrier harboring pCMV 3FUb vectors encoding for the two minigenes. These findings establish proof of concept that disruption of self tolerance against neuroblastoma associated epitopes may be an effective adjuvant therapeutic strategy.


Subject(s)
Autoantigens/genetics , Liver Neoplasms/prevention & control , Neuroblastoma/prevention & control , Vaccination , Vaccines, DNA , Animals , Binding Sites , CD8-Positive T-Lymphocytes/immunology , Epitopes/immunology , Epitopes/metabolism , Female , Immunity, Cellular , Ligands , Liver Neoplasms/immunology , Liver Neoplasms/secondary , Mice , Neuroblastoma/immunology , Neuroblastoma/secondary , Peptide Fragments/metabolism , Phosphoprotein Phosphatases/chemistry , Plasmids , Protein Phosphatase 2 , Ribonucleoside Diphosphate Reductase/chemistry , Salmonella typhimurium/genetics
2.
Cancer Lett ; 197(1-2): 225-30, 2003 Jul 18.
Article in English | MEDLINE | ID: mdl-12880986

ABSTRACT

Effective chemotherapy in neuroblastoma is limited by poor anti-tumor efficacy, systemic toxicity and the induction of drug resistance. Here, we provide further evidence that a hydrolytic activated prodrug design may overcome these problems. For this purpose, VP-16 was functionally blocked by a carbonate linker to generate two novel chemically stable prodrugs of VP-16, ProVP-16 I and II. We demonstrate profoundly different biological effects in vitro and in vivo of the prodrugs compared to parental VP-16. First, we established an up to >2 log higher in vitro toxicity of the two prodrugs compared to VP-16 on a panel of neuroblastoma cell lines. The highest increase of prodrug mediated cytotoxicity was observed in multi drug resistant cell lines. Second, in vivo studies showed a maximum tolerated dose (MTD) of ProVP-16 II (60 mg/kg), which was at least threefold higher than that of VP-16 (20 mg/kg). Tests of ProVP-16 II in a syngeneic NXS2 neuroblastoma model indicated that mice treated with this prodrug at 1/3 of the MTD was as effective as VP-16 parental compound used at the MTD in suppression of tumor growth. In summary, the etoposide prodrugs proved effective and less toxic and are therefore highly promising new anti-neuroblastoma compounds.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Etoposide/pharmacology , Neuroblastoma/drug therapy , Prodrugs/metabolism , Animals , Biotransformation , Cell Survival/drug effects , DNA Primers/chemistry , Drug Design , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Female , Humans , Hydrolysis , Maximum Tolerated Dose , Mice , Neuroblastoma/metabolism , Neuroblastoma/pathology , Plasmids , Prodrugs/chemical synthesis , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured
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