Studies on Optimization of Antitumor Effect by Gene-based Immunotherapy

Abstract

The immune system is able to identify and control tumors in a process termed cancer immunosurveillance. Advances in molecular and genetic research have broadened the understandings on the interplay between cancer and the immune system and enable development of cancer immunotherapeutics. Although cancer immunotherapy has greater specificity to tumors than conventional cancer therapeutics, further enhancement of its therapeutic efficacy using versatile approaches including manipulation of immunotherapeutics via genetic engineering, selection of optimal regimen, and invention of delivery system to elicit tumor regression in clinics is required. In the first part of this study, human papillonavirus (HPV) DNA vaccines containing E6 and E7 antigens were generated through codon optimization (Co), fusion of E6 and E7, and addition of a tissue plasminogen activator (tpa) signal sequence, CD40 ligand (CD40L), or fms-like tyrosine kinase-3 ligand (Flt3L). The resulting plasmid DNA constructs were investigated in terms of their antitumor activity as well as their induction of HPV-specific CD8+ T cell responses. When E6Co and E7Co were fused (E67Co), CD8+ T cell responses specific for E6 or E7 antigen decreased, but the preventive antitumor effect improved, demonstrating the importance of broad immunity. Interestingly, Flt3L-fused HPV DNA vaccine exhibited stronger E6- and E7-specific CD8+ T cell responses as well as therapeutic antitumor effect than that of CD40L linked HPV DNA vaccine. Finally, the optimal construct, tFE67Co, was generated by including tpa signal sequence, Flt3L, fusion of E6 and E7, and codon optimization, which induces 23 and 25 times stronger E6- and E7-specific CD8+ T cell responses than those of initial E67 fusion construct. In particular, inclusion of electroporation in intramuscular immunization of tFE67Co further enhances HPV-specific CD8+ T cell responses, leading to complete tumor regression in a therapeutic setting. In the second part of this study, strategies to enhance antitumor efficacy of mesenchymal stem cell (MSC) expressing modified interleukin-12 (MSCs/IL-12M) were investigated because MSC has been considered as an ideal delivery vehicle for cancer gene therapies or therapeutic vaccines due to tumor tropism and prolonged transgene expression. Intratumoral (i.t.) injection of MSCs/IL-12M exhibited stronger tumor-specific T cell responses and antitumor effects as well as more sustained expressions of IL-12 and IFN-g in both sera and tumor sites than did IL-12M-expressing adenovirus (rAd/IL-12M) in mice bearing both solid and metastatic tumors. Subcutaneous (s.c.) injection of MSCs/IL-12M at contra-lateral sites of tumor exhibited similar levels of serum IL-12 and IFN-g as i.t. injection but much weaker antitumor effects in both B16F10 melanoma and TC-1 cervical cancer models than i.t. injection. Although intravenous (i.v.) injection elicited earlier peak serum levels of cytokines, it induced weaker tumor-specific T cell responses and antitumor effects than i.t. injection, indicating that serum cytokine levels are not surrogate indicators of antitumor effects. Taken together, these results indicated that MSC is more efficient than adenovirus as a cytokine gene delivery vehicle and that i.t. injection of MSCs/IL-12M is the best approach to induce strong tumor-specific T cell responses that correlate with anti-metastatic effects as well as inhibition of solid tumor growth, although MSCs themselves have an ability to migrate into the tumor site. In addition, MSCs/IL-12M embedded in Matrigel (MSCs/IL-12M/Matrigel) exhibited significant antitumor effects even in immunodeficient mice such as SCID and BNX mice lacking T, B, and NK cells, but not in IFN-g knock-out mice. These findings provide an optimal approach for designing an efficient clinical protocol of MSC-based cytokine gene therapy to induce strong tumor-specific T cell responses and therapeutic anticancer efficacy.