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Ugel, stefano (2008) Approcci di immunoterapia attiva e passiva basati sull'antigene telomerasi in modelli di carcinogenesi murina. [Tesi di dottorato]

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Abstract (inglese)

Aim of this work was to verify the therapeutic efficacy of active and passive vaccination against m-TERT in mice bearing transplantable tumors and in an autochthonous mouse model of prostate cancer: the TRAMP model. To choose the best vaccination protocol we developed DNA-based antigen-specific expression systems employing eukaryotic plasmid expression vectors and recombinant adenoviruses. We used splenocytes from vaccinated mice to set up mixed leukocyte peptide cultures (MLPC) with the mouse (m)-TERT198-205 peptide. This sequence of mouse TERT was identified as the immunodominant epitope by screening overlapping peptides covering the whole protein. MLPC were tested in IFN?-specific E.L.IS.A. assay against peptide-pulsed cells and unpulsed tumor cell lines of different histotypes, but with the same H-2b haplotype. A persistently high level of recognition was noted against peptidepulsed cell lines, whereas repeated in vitro stimulation with ?-irradiated syngeneic splenocytes pulsed with a low dose of m-TERT198-205 peptide (0.1 ?M) every week led to an increased recognition of unpulsed tumor cell lines. This enhanced recognition was observed only in MLPC from mice vaccinated with DNA plasmid vector and suggested a preferential proliferation of high-avidity CTL clones under these culture conditions. The augment in high-avidity CTLs is supported by TCR analysis by spectratyping, that proved a positive selection of V?-11 chain of TCR during in vitro passages together with TCR clonotype enrichment. We demonstrated an important therapeutic effect by m-TERT198-205 polyclonal CTLs transfer in a metastatic melanoma model, where mice treated with CTLs transfer presented a significant reduction of the lung metastases compared with untreated controls, and in trasplantable tumor models, where TERT198-205 CTLs induced a statistically significant delay in tumor growth and a survival increase in both melanoma- and prostate carcinoma-bearing mice.
To identify and isolate high affinity CTL clones specific for m-TERT198-205 epitope we used the limiting dilution technique starting from the polyclonal CTLs. We obtained a large spectrum of clones, among which clone CTL-7 was the most intriguing. In fact it was able to recognize tumor cell lines of different histotypes (melanoma, prostate carcinoma, colon carcinoma and sarcoma) as well as peptidepulsed cell lines. Transfer of CTL-7 clone exerted a better therapeutic effect on lung metastases generated by melanoma cells.
We also investigated the possibility to elicit a protective antitumor response through an active immunization in TRAMP mice. We performed a cycle of two biweekly i.m. injections of a plasmid encoding m-TERT repeated every 10 weeks for the entire life of the mouse. Mice vaccinated against m-TERT showed a reduction of tumor progression at week 24th and a survival prolongation compared to mock vector-treated groups.
These data represent a good platform to translate more effective vaccines for the therapy of human cancers.

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Tipo di EPrint:Tesi di dottorato
Relatore:Bronte, Vincenzo
Dottorato (corsi e scuole):Ciclo 20 > Corsi per il 20simo ciclo > ONCOLOGIA E ONCOLOGIA CHIRURGICA
Data di deposito della tesi:30 Gennaio 2008
Anno di Pubblicazione:30 Gennaio 2008
Parole chiave (italiano / inglese):Vaccinazione antitumorale, TRAMp, telomrasi, trasferimento adottivo di linfociti citotossici
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/06 Oncologia medica
Struttura di riferimento:Dipartimenti > pre 2012 - Dipartimento di Scienze Oncologiche e Chirurgiche
Codice ID:429
Depositato il:24 Set 2008
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