Enhancement of Interleukin-12 Gene-Based Tumor Immunotherapy by the Reduced Secretion of p40 Subunit and the Combination with Farnesyltransferase Inhibitor

Abstract

Interleukin-12 (IL-12) gene was shown to produce both IL-12 and p40 subunit. The excess production of the p40 subunit as a natural antagonist of IL-12 is a major obstacle of IL-12 gene-based cancer therapy. We previously reported that IL-12N220L gene, which selectively reduces the secretion of the p40 subunit, induces long-lasting stronger type 1 helper T cells (T(H)1) and cytotoxic T lymphocyte (CTL) immunity in hepatitis C virus (HCV) E2 DNA vaccination model and higher protection from challenge with tumor cells expressing E2 than IL-12 in a prophylactic setting. Here, we demonstrated that intratumoral injection of IL-12N220L-expressing adenovirus showed better tumor growth inhibition and higher survival rate than that of IL-12 or granulocyte macrophage-colony stimulating factor (GM-CSF)-expressing adenovirus in a therapeutic setting. In particular, the mice cured by IL-12N220L treatment were protected against intravenous rechallenge of the same tumor cells better than those by IL-12 treatment. In addition, the enhanced antitumor activity of IL-12N220L was confirmed in B16F10 lung metastasis model, which correlated with the frequency of tumor-specific interferon (IFN)-gamma-secreting cells. When tested in CT26/NP tumor that expresses influenza nucleoprotein (NP) as a tumor antigen, IL-12N220L induced stronger NP-specific T(H)1 and CTL responses than IL-12, particularly at a later time point, indicating the generating long-term tumor-specific memory T-cell responses. Moreover, the potent antitumor effects of IL-12N220L were further augmented by combination with chemotherapy using farnesyltransferase inhibitor (FTI), LB42908. Taken together, our results suggest that IL-12N220L is superior to IL-12 in cancer immunotherapy, which can be further enhanced by combination with chemotherapy.

Marked enhancement of antigen‐specific T‐cell responses by IL‐7‐fused nonlytic, but not lytic, Fc as a genetic adjuvant

“…The AdEasy Vector System (Obiogene) was used to generate rAd [34]. Briefly, rAd expressing OVA was generated as described previously [32].…”

Enhanced delivery efficiency of recombinant adenovirus into tumor and mesenchymal stem cells by a novel PTD

“…IL-12N220L was previously shown to have a stronger antitumor effect than wild-type IL-12. [33][34][35] When the serum IL-12 level was determined by enzyme-linked immunosorbent assay at 24 h after tumor injection, the expression level of this transgene was increased 33-fold by HP4 compared with Tat 48À60 (Figure 5a). Consistent with the IL-12 levels, co-treatment of rAd/IL-12N220L with HP4 significantly increased the survival rate (80%) compared to Tat 48À60 and IL-12N220L alone (40 and 20% respectively; Figure 5b).…”

Adenovirus-mediated gene transfer of interleukin-23 shows prophylactic but not therapeutic antitumor effects

“…rAd/IL-12N220L treatment elicited a potent effect on tumor regression in all of the tumor models (Figure 2), which is in agreement with our previous report. 15 However, the rAd/IL-23N220L treatment showed only a marginal antitumor effect, which was statistically insignificant as compared to the rAd/Mock treatment (PX0.05). It is worth noting that similar results were observed using rAd encoding native IL-23 (data not shown), indicating that this mild antitumor effect was indeed attributable to IL-23 itself rather than to the mutated form of IL-23.…”

“…15 Briefly, the cDNA of murine IL-12N220L, 14 IL-23, IL-23N220L 13 and chicken ovalbumin (OVA) 16 were subcloned into the adenoviral shuttle vector, pShuttleCMV. After recombination with the adenoviral backbone vector, pAdEasy in Escherichia coli BJ5183, the recombinant adenoviruses were generated and expanded in 293 cells.…”

“…15 Briefly, after harvesting splenocytes from the mice on indicated days, the cells were co-incubated with OVA 257-264 peptide (SIINFEKL) or mitomycin C-treated tumor cells on an IFN-g capture antibody (BD Pharmingen, San Diego, CA)-coated 96-well ELISPOT plate (Millipore, Bedford, MA) for 24 h. After subsequent incubation with biotinylated rat anti-mouse IFN-g antibody (BD Pharmingen), the number of spots was counted using the AID ELISPOT Reader System (Autoimmun Diagnostika, Strassberg, Germany). The average number of IFN-g secreting cells was shown per 1 Â 10 6 splenocytes.…”