The asparaginyl endopeptidase legumain that is overexpressed in M2-polarized tumor-associated macrophages has been identified as a suitable target for elimination of these cells supporting tumor progression. To enhance the efficacy of DNA immunization against legumain, we performed several modifications in this protein that could improve induction of immune responses. First, we mutated the RGD motif into GGD or RGG sequences. This alteration resulted in diminished maturation of legumain and impaired cellular localization. Then, as tolerance to self-antigens can be broken by the activation of CD4 T-cell help, we tried to enhance the immunogenicity of legumain by the insertion of a foreign helper epitope, namely the p30 epitope from the tetanus toxin. Finally, the 2 modifications were combined. After gene gun DNA immunization of C57BL/6 mice with these constructs, we identified the Lgmn111-119 CD8 T-cell epitope that binds to H-2D molecules. Furthermore, we showed that mutagenesis in the RGD motif significantly enhanced the immune response against legumain. The addition of the p30 helper epitope induced the specific production of IFN-γ by T cells, but did not significantly increase legumain-specific immunity activated after mutagenesis in the RGD motif which might be caused by simultaneous activation of a Th2 response demonstrated by the production of IL-4. However, the beneficial effect of the helper epitope on legumain-specific response was proved after the depletion of regulatory T cells by antibody against CD25 that preferentially stimulated Th1 immunity. The antitumor effect of the modified legumain gene was shown in the immunization against tumors induced by MK16 cells.
- MeSH
- aminokyselinové motivy genetika MeSH
- biolistika MeSH
- buňky NIH 3T3 MeSH
- CD8-pozitivní T-lymfocyty imunologie MeSH
- cysteinové endopeptidasy genetika metabolismus MeSH
- DNA vakcíny * MeSH
- epitopy T-lymfocytární genetika metabolismus MeSH
- experimentální nádory imunologie terapie MeSH
- HEK293 buňky MeSH
- imunoterapie metody MeSH
- interferon gama metabolismus MeSH
- lidé MeSH
- makrofágy imunologie MeSH
- mutace genetika MeSH
- mutageneze cílená MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové biomarkery genetika metabolismus MeSH
- peptidové fragmenty genetika metabolismus MeSH
- protinádorové vakcíny * MeSH
- T-lymfocyty pomocné-indukující imunologie MeSH
- tetanový toxin genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
