Enhancement of DNA Vaccine Potency Against Legumain

Abstract

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.

Combined Cancer Immunotherapy Against Aurora Kinase A

“…33,48 This observation is just the opposite of the results obtained for DNA vaccination against Aurka, indicating that the nature of the tumor (self-) antigen could play a role in the antitumor effect after depletion of CD25 + cells. 33,48 This observation is just the opposite of the results obtained for DNA vaccination against Aurka, indicating that the nature of the tumor (self-) antigen could play a role in the antitumor effect after depletion of CD25 + cells.…”

“…45,46 Using the newly identified H-2K b Aurka 220-228 epitope, we showed that both the PADRE epitope and the LAMP-1 sequences augmented Aurka-specific response after DNA vaccination by means of a gene gun. 47 In our previous studies with the Sox2 48 and legumain self-antigens, 33 we have shown that a single administration of antibody against CD25 before the first DNA vaccination enhanced the activation of splenocytes against both a self-antigen and a helper epitope. These data correspond with our results obtained for the HPV16 E7 antigen.…”

“…The PADRE peptide (AKFVAAWTLKAAA; >81% pure), 32 the Lgmn 108-116 peptide derived from mouse legumain (EDVTPENFL; >94% pure), 33 the OVA 257-264 peptide derived from chicken ovalbumin (SINFEKL, >97% pure), 34 the E7 49-57 peptide derived from HPV16 E7 oncoprotein (RAHYNIVTF, > 96% pure), 35 …”

Reprogramming of Tumor-Associated Macrophages with Anticancer Therapies: Radiotherapy versus Chemo- and Immunotherapies

“…This discovery led Smahel and his team to modify the gene sequence of legumain to enhance the efficacy of immunization against legumain. These modifications induced reduced legumain maturation and impaired cellular localization, and resulted in T helper (CD4 + ) and cytotoxic (CD8 + ) lymphocyte-dependent elimination of TAMs (43). …”

Tumor-associated macrophages, potential targets for cancer treatment

“…A single-chain peptide bound to the CD206 receptor was attached to nanobodies that can selectively target CD206 + TAMs [ 11 ]. Legumain, a stress protein and a member of the asparagine endopeptidase family, can serve as an efficient therapeutic target when overexpressed in TAMs [ 12 ]. Targeting surface markers such as scavenger receptor A and CD52 by using immunotoxin-conjugated monoclonal antibodies (mAbs) has been investigated in ovarian cancer [ 13 ].…”