Tae Heung Kang

Epigallocatechin-3-Gallate Enhances CD8+ T Cell–Mediated Antitumor Immunity Induced by DNA Vaccination

Immunotherapy and chemotherapy are generally effective against small tumors in animal models of cancer. However, these treatment regimens are generally ineffective against large, bulky tumors. We have found that a multimodality treatment regimen using DNA vaccination in combination with chemotherapeutic agent epigallocatechin-3-gallate (EGCG), a compound found in green tea, is effective in inhibiting large tumor growth. EGCG was found to induce tumor cellular apoptosis in a dose-dependent manner. The combination of EGCG and DNA vaccination led to an enhanced tumor-specific T-cell immune response and enhanced antitumor effects, resulting in a higher cure rate than either immunotherapy or EGCG alone. In addition, combined DNA vaccination and oral EGCG treatment provided long-term antitumor protection in cured mice. Cured animals rejected a challenge of E7-expressing tumors, such as TC-1 and B16E7, but not a challenge of B16 7 weeks after the combined treatment, showing antigen-specific immune responses. These results suggest that multimodality treatment strategies, such as combining immunotherapy with a tumor-killing cancer drug, may be a more effective anticancer strategy than single-modality treatments.

Oral Administration of High Molecular Mass Poly-γ-Glutamate Induces NK Cell-Mediated Antitumor Immunity

We analyzed the in vivo tumor regression activity of high molecular mass poly-γ-glutamate (γ-PGA) from Bacillus subtilis sups. chungkookjang. C57BL/6 mice were orally administered 10-, 100-, or 2000-kDa γ-PGA or β-glucan (positive control), and antitumor immunity was examined. Our results revealed higher levels of NK cell-mediated cytotoxicity and IFN-γ secretion in mice treated with higher molecular mass γ-PGA (2000 kDa) vs those treated with lower molecular mass γ-PGA (10 or 100 kDa) or β-glucan. We then examined the effect of oral administration of 10- or 2000-kDa γ-PGA on protection against B16 tumor challenge in C57BL/6 mice. Mice receiving high molecular mass γ-PGA (2000 kDa) showed significantly smaller tumor sizes following challenge with the MHC class I-down-regulated tumor cell lines, B16 and TC-1 P3 (A15), but not with TC-1 cells, which have normal MHC class I expression. Lastly, we found that γ-PGA-induced antitumor effect was decreased by in vivo depletion of NK cells using mAb PK136 or anti-asialo GM1 Ab, and that was completely blocked in NK cell-deficient B6 beige mice or IFN-γ knockout mice. Taken together, we demonstrated that oral administration of high molecular mass γ-PGA (2000 kDa) generated significant NK cell-mediated antitumor activity in mice bearing MHC class I-deficient tumors.

Chemotherapy acts as an adjuvant to convert the tumor microenvironment into a highly permissive state for vaccination-induced antitumor immunity

Multiple classes of pharmacologic agents have the potential to induce the expression and release of proinflammatory factors from dying tumor cells. As a result, these cells can in theory elicit an immune response through various defined mechanisms to permanently eradicate disseminated cancer. However, the impact of chemotherapy on the tumor-specific immune response in the context of the tumor microenvironment is largely unknown. Within the tumor microenvironment, the immune response promoted by chemotherapy is antagonized by an immune-suppressive milieu, and the balance of these opposing forces dictates the clinical course of disease. Here, we report that high antigen exposure within the tumor microenvironment following chemotherapy is sufficient to skew this balance in favor of a productive immune response. In elevating antigen exposure, chemotherapy can achieve long-term control of tumor progression without the need of an additional adjuvant. We found that chemotherapy initiated this phenomenon in the tumor microenvironment through an accumulation of dendritic cells, which stimulated CD8(+) T cells and the type I IFN pathway. From this conceptual base, we developed a simple approach to cancer therapy combining chemotherapy and vaccination that may be widely applicable.

Activation of Akt as a Mechanism for Tumor Immune Evasion

Immune evasion is an important reason why the immune system cannot control tumor growth. To elucidate the mechanism for tumor immune evasion, we generated an immune-resistant human papillomavirus type 16 (HPV-16) E7-expressing tumor cell line by subjecting a susceptible tumor cell line to multiple rounds of in vivo immune selection with an E7-specific vaccine. Comparison of parental and immune-resistant tumors revealed that Akt is highly activated in the immune-resistant tumors. Retroviral transfer of a constitutively active form of Akt into the parental tumor significantly increased its resistance against E7-specific CD8(+) T-cell mediated apoptosis. The observed resistance against apoptosis was found to be associated with the upregulation of antiapoptotic molecules. We also observed that intratumoral injection of an Akt inhibitor enhanced the therapeutic efficacy of E7-specific vaccine or E7-specific CD8(+) T-cell adoptive transfer against the immune-resistant tumors. Thus, our data indicate that the activation of PI3K/Akt pathway represents a new mechanism of immune escape and has important implications for the development of a novel strategy in cancer immunotherapy against immune-resistant tumor cells.

Nanoparticle-based vaccine delivery for cancer immunotherapy.

Development of nano-sized carriers including nanoparticles, nanoemulsions or liposomes holds great potential for advanced delivery systems for cancer immunotherapy, as such nanostructures can be used to more effectively manipulate or deliver immunologically active components to specific target sites. Successful development of nanotechnology based platform in the field of immunotherapy will allow the application of vaccines, adjuvants and immunomodulatory drugs that improve clinical outcomes for immunological diseases. Here, we review current nanoparticle-based platforms in the efficacious delivery of vaccines in cancer immunotherapy.

Enhancement of dendritic cell-based vaccine potency by anti-apoptotic siRNAs targeting key pro-apoptotic proteins in cytotoxic CD8+ T cell-mediated cell death

Dendritic cells (DCs) have become an important measure for the treatment of malignancies. Current DC preparations, however, generate short-lived DCs because they are subject to cell death from various apoptotic pressures. Antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is one of the main obstacles to limit the DC-mediated immune priming since CTLs can recognize the target antigen expressing DCs as target cells and kill the DCs. CTLs secret perforin and serine protease granzymes during CTL killing. Perforin and serine protease granzymes induce the release of a number of mitochondrial pro-apoptotic factors, which are controlled by members of the BCL-2 family, such as BAK, BAX and BIM. FasL linking to Fas on DCs triggers the activation of caspase-8, which eventually leads to mitochondria-mediated apoptosis via truncation of BID. In this study, we tried to enhance the DC priming capacity by prolonging DC survival using anti-apoptotic siRNA targeting these key pro-apoptotic molecules in CTL killing. Human papillomavirus (HPV)-16 E7 antigen presenting DCs that were transfected with these anti-apoptotic siRNAs showed increased resistance to T cell-mediated death, leading to enhanced E7-specific CD8(+) T cell activation in vitro and in vivo. Among them, siRNA targeting BIM (siBIM) generated strongest E7-specific E7-specific CD8(+) T cell immunity. More importantly, vaccination with E7 presenting DCs transfected with siBIM was capable of generating a marked therapeutic effect in vaccinated mice. Our data indicate that ex vivo manipulation of DCs with siBIM may represent a plausible strategy for enhancing dendritic cell-based vaccine potency.

Cancer vaccination drives Nanog-dependent evolution of tumor cells toward an immune-resistant and stem-like phenotype.

Due to the exquisite specificity and potency of the immune system, vaccination is in theory the most precise and powerful approach for controlling cancer. However, current data from clinical trials indicate that vaccination rarely yields significant benefits for cancer patients in terms of tumor progression and long-term survival. The poor clinical outcomes of vaccination are primarily caused by mechanisms of immune tolerance, especially within the tumor microenvironment. Here, we report that vaccination drives the evolution of tumor cells toward an immune-resistant and stem-like phenotype that promotes tumor growth and nullifies the CTL response. The emergence of this phenotype required the transcription factor Nanog, which is induced as a consequence of immune selection. Nanog expression enhanced the stem-like features of tumor cells and protected them from killing by tumor-reactive CTLs. Delivery of siNanog into tumor-bearing mice rendered the tumor vulnerable to immune surveillance and strongly suppressed its growth. Together, our findings show tumor adaptation to vaccination through gain of an immune-resistant, stem-like phenotype and identify Nanog as a central molecular target in this process. Future vaccination technology should consider Nanog an important target to enhance the immunotherapeutic response.

Ovarian cancer gene therapy using HPV-16 pseudovirion carrying the HSV-tk gene.

Ovarian cancer is the leading cause of death from all gynecological cancers and conventional therapies such as surgery, chemotherapy, and radiotherapy usually fail to control advanced stages of the disease. Thus, there is an urgent need for alternative and innovative therapeutic options. We reason that cancer gene therapy using a vector capable of specifically delivering an enzyme-encoding gene to ovarian cancer cells will allow the cancer cell to metabolize a harmless prodrug into a potent cytotoxin, which will lead to therapeutic effects. In the current study, we explore the use of a human papillomavirus (HPV) pseudovirion to deliver a herpes simplex virus thymidine kinase (HSV-tk) gene to ovarian tumor cells. We found that the HPV-16 pseudovirion was able to preferentially infect murine and human ovarian tumor cells when administered intraperitoneally. Furthermore, intraperitoneal injection of HPV-16 pseudovirions carrying the HSV-tk gene followed by treatment with ganciclovir led to significant therapeutic anti-tumor effects in murine ovarian cancer-bearing mice. Our data suggest that HPV pseudovirion may serve as a potential delivery vehicle for ovarian cancer gene therapy.

Enhancing dendritic cell vaccine potency by combining a BAK/BAX siRNA-mediated antiapoptotic strategy to prolong dendritic cell life with an intracellular strategy to target antigen to lysosomal compartments

Dendritic cell (DC)‐based vaccines have become important in immunotherapeutics as a measure for generating antitumor immune responses. We have previously demonstrated that linkage of the antigen gene to a lysosomal targeting signal, a sorting signal of the lysosome‐associated membrane protein type 1 (LAMP‐1), enhances the potency of DC‐based vaccines. DCs have a limited life span, hindering their long‐term ability to prime antigen‐specific T cells. In this study, we attempted to further improve the potency of a DC vaccine that targets human papilloma virus 16 (HPV16) E7 to a lysosomal compartment (DC‐Sig/E7/LAMP‐1) by combining a strategy to prolong DC life. We show that small interfering RNA‐targeting Bak and Bax proteins can be used to allow transfected DCs to resist being killed by T cells. This is done by downregulating these proapoptotic proteins, which have been known as so‐called gate keepers in mitochondria‐mediated apoptosis. DCs expressing intact E7 or Sig/E7/LAMP‐1 became resistant to attack by CD8+ T cells after transfection with BAK/BAX siRNA, leading to enhanced E7‐specific T cell activation in vitro and in vivo. More importantly, vaccination with E7‐presenting DCs transfected with BAK/BAX siRNA generated a strong therapeutic effect against an E7‐expressing tumor in vaccinated mice, compared with DCs transfected with control siRNA. Our data indicate that a combination of strategies to enhance intracellular Ag processing and to prolong DC life may offer a promising strategy for improving DC vaccine potency.

Innovative DNA vaccine for human papillomavirus (HPV)-associated head and neck cancer.

Human papillomavirus (HPV), particularly type 16, has been associated with a subset of head and neck cancers. The viral-encoded oncogenic proteins E6 and E7 represent ideal targets for immunotherapy against HPV-associated head and neck cancers. DNA vaccines have emerged as attractive approaches for immunotherapy due to its simplicity, safety and ease of preparation. Intradermal administration of DNA vaccine by means of gene gun represents an efficient method to deliver DNA directly into dendritic cells for priming antigen-specific T cells. We have previously shown that a DNA vaccine encoding an invariant chain (Ii), in which the class II-associated Ii peptide (CLIP) region has been replaced by a Pan-DR-epitope (PADRE) sequence to form Ii-PADRE, is capable of generating PADRE-specific CD4+ T cells in vaccinated mice. In the current study, we hypothesize that a DNA vaccine encoding Ii-PADRE linked to E6 (Ii-PADRE-E6) will further enhance E6-specific CD8+ T cell immune responses through PADRE-specific CD4+ T-helper cells. We found that mice vaccinated with Ii-PADRE-E6 DNA generated comparable levels of PADRE-specific CD4+ T-cell immune responses, as well as significantly stronger E6-specific CD8+ T-cell immune responses and antitumor effects against the lethal challenge of E6-expressing tumor compared with mice vaccinated with Ii-E6 DNA. Taken together, our data indicate that vaccination with Ii-E6 DNA with PADRE replacing the CLIP region is capable of enhancing the E6-specific CD8+ T-cell immune response generated by the Ii-E6 DNA. Thus, Ii-PADRE-E6 represents a novel DNA vaccine for the treatment of HPV-associated head and neck cancer and other HPV-associated malignancies.