Long-term antigen-specific immune response by an oncolytic adenovirus encoding SP-SA-E7-4-1BBL in HPV-16 cancer model.

BACKGROUND: To describe an oncolytic adenovirus (OAd) encoding SP-SA-E7-4-1BBL that is capable of inducing tumor regression in therapeutic assays. Herein, we tested whether the antitumor effect is given by the induction of a tumor-specific immune response, as well as the minimum dose needed to elicit antitumor protection and monitor the OAd biodistribution over time. METHODS AND RESULTS: C57BL/6 mice (n = 5) per group were immunized twice with OAds encoding SP-SA-E7-4-1BBL, SA-E7-4-1BBL, or SP-SA-4-1BBL and challenged with TC-1 cancer cells. The DNA construct SP-SA-E7-4-1BBL was employed as a control via biolistic or PBS injection. Groups without tumor development at 47 days were rechallenged with TC-1 cells, and follow-up lasted until day 90. The minimum dose of OAd to induce the antitumor effect was established by immunization using serial dilution doses. The cytometry bead assay and the ELISpot assay were used to evaluate cytokine release in response to ex vivo antigenic stimulation. The distribution profile of the OAd vaccine was evaluated in the different organs by histological, immunohistochemical and qPCR analyses. The OAd SP-SA-E7-4-1BBL-immunized mice did not develop tumors even in a rechallenge. A protective antitumor effect was observed from a dose that is one hundredth of most reports of adenoviral vaccines. Immunization with OAd increases Interferon-gamma-producing cells in response to antigen stimulation. OAd was detected in tumors over time, with significant morphological changes, contrary to nontumor tissues. CONCLUSIONS: The OAd SP-SA-E7-4-1BBL vaccine confers a prophylactic, safe, long-lasting, and antigen-dependent antitumor effect mediated by a Th1 antitumor immune response.

Extracellular vesicles produced by immunomodulatory cells harboring OX40 ligand and 4-1BB ligand enhance antitumor immunity.

Genetically modified tumor cells harboring immunomodulators may be used as therapeutic vaccines to stimulate antitumor immunity. The therapeutic benefit of these tumor vaccines is extensively investigated and mechanisms by which they boost antitumor response may be further explored. Tumor cells are large secretors of extracellular vesicles (EVs). These EVs are able to vehiculate RNA and proteins to target cells, and engineered EVs also vehiculate recombinant proteins. In this study, we explore immunomodulatory properties of EVs derived from antitumor vaccines expressing the TNFSF ligands 4-1BBL and OX40L, modulating immune response mediated by immune cells and eliminating tumors. Our results suggest that the EVs secreted by genetically modified tumor cells harboring TNFSF ligands can induce T cell proliferation, inhibit the transcription factor FoxP3, associated with the maintenance of Treg phenotype, and enhance antitumor activity mediated by immune cells. The immunomodulatory extracellular vesicles have potential to be further engineered for developing new approaches for cancer therapy.

Anti-prostate cancer effects of CTL cell induction in vitro by recombinant adenovirus mediated PSMA/4-1BBL dendritic cells: an immunotherapy study.

This study aimed to examine anti-prostate cancer immune response induced by dendritic cells (DCs) transduced with PSMA/4-1BBL recombinant adenoviruses in vitro. Ad-PSMA, Ad-4-1BBL, and Ad-GFP were transfected into DCs derived from peripheral blood of healthy volunteers. Ad-PSMA/4-1BBL-DC, Ad-PSMA-DC, Ad-4-1BBL-DC, Ad-GFP-DC, and normal-DC, PSMA and 4-1BBL protein levels in DCs were detected by western blot. IL-12, IFN-γ and IL-10 were measured by ELISA. Mixed lymphocyte reaction and the cytotoxicity of each group targeted to LNCap, Du145, and 22RV prostate cancer cells were determined by CCK-8 assay. PSMA and 4-1BBL protein could express on DC successfully, the IL-12 supernatant content (134.29 ± 2.22 pg) was higher than others (P < 0.05). The ability to stimulate autologous T lymphocyte proliferation in the co-transfection group was higher than others (P < 0.05). When the DCs were co-cultured with CTLs, the PSMA/4-1BBL-DC-CTL group showed the highest content of IFN-γ (1176.10 ± 14.37pg/5 x 10(6) cells), but the lowest IL-10 content (75.14 ± 2.01 pg/5 x 10(6) cells) (P < 0.05), and the strongest anti-tumor effect when the effector to target ratio was 40:1, along with a higher killing ratio of LNCap cells than others (P < 0.05). Overall, Mature DCs transfected with Ad-PSMA/4- 1BBL not only showed high secretion of IL-12, but also induced CTLs to stimulate and enhance the killing effect of PSMA specific effector cells to PSMA positively expressing prostate cancer cells. Furthermore, the DCs infected with two kinds of tumor-associated antigens would induce more effective tumor-specific CTL induction.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells.

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMax™ Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4%) and CD86 (80.13 ± 2.81%)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD(450) = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.