Jeong-Im Sin

Both antigen optimization and lysosomal targeting are required for enhanced anti‐tumour protective immunity in a human papillomavirus E7‐expressing animal tumour model

DNA immunization is a new approach for cancer immune therapy. In this study, we constructed human papillomavirus (HPV) 16 E7 expression vector cassettes and then compared the abilities of these constructs to induce antitumour protection. Lysosome‐targeted E7 antigens, and to a lesser degree signal sequence‐conjugated and transmembrane region sequence‐conjugated E7 antigens in a DNA form, displayed tumour protection significantly higher than wild‐type E7 antigens. This enhanced tumour protection was mediated by CD8+ cytotoxic T lymphocytes (CTL), as determined by in vivo T‐cell depletion and in vitro interferon‐γ (IFN‐γ) production. Subsequent co‐injection with interleukin‐12‐expressing cDNA showed insignificantly enhanced antitumour protection. However, E7 codon optimization plus lysosomal targeting resulted in a dramatic enhancement in antitumour protection both prophylactically and therapeutically through augmentation of the E7‐specific CTL population, compared to either one of them alone. However, wild‐type or codonoptimized E7 antigens without intracellular targeting displayed no protection against tumour challenge. Thus, these data suggest that antigen codon optimization plus lysosomal targeting strategy could be important in crafting more efficacious E7 DNA vaccines for tumour protection.

CpG-ODN-stimulated dendritic cells act as a potent adjuvant for E7 protein delivery to induce antigen-specific antitumour immunity in a HPV 16 E7-associated animal tumour model.

We previously reported that both E7 and CpG‐oligodeoxynucleotide (ODN) are required for protecting animals from human papillomavirus (HPV) 16 E7‐associated tumour challenge. Here we investigate dendritic cells (DC)‐based approach in this protection. In the study, we isolated bone marrow‐derived DC and stimulated DC with E7 and ODN. In vitro stimulation of DC with E7 plus ODN resulted in more production of interleukin‐12, as compared to that with E7 or ODN alone. Further injection with E7+ODN‐stimulated DC resulted in more significant tumour protection, as compared to stimulation with E7 or ODN alone. We further evaluated the levels of immune responses induced by DC stimulated with E7+ODN. We observed little enhancement of E7‐specific antibody and T helper cell proliferative responses by E7+ODN stimulation, as compared to E7 stimulation. However, there was some enhancement of interferon‐γ (IFN‐γ) production from CD4+ T cells and a more significant production of IFN‐γ from CD8+ T cells by E7+ODN stimulation, as compared to E7 stimulation alone. This was consistent with intracellular IFN‐γ staining levels of CD8+ T cells. Tumour protection further appeared to be mediated by CD8+ T cells, as determined by in vivo T‐cell depletion. Thus, these data suggest that upon ODN stimulation DC might function as a potent adjuvant for E7 protein delivery for induction of protective cellular immunity against HPV E7‐associated tumour challenge.

Adoptive transfer of human papillomavirus E7-specific CTL enhances tumor chemoresponse through the perforin/granzyme-mediated pathway.

Adoptive cytotoxic T lymphocyte (CTL) therapy has an important implication in treating cancer patients. Here, we investigate whether adoptive transfer of human papillomavirus (HPV) E7-specific CTL can enhance tumor chemoresponse using an established cervical cancer animal model. Cisplatin-based chemotherapy plus CTL therapy showed an improved therapeutic effectiveness, along with antitumor protective responses to a parental tumor cell rechallenge. Cisplatin treatment dose-dependently increased the expression of Fas, intercellular adhesion molecule (ICAM)-1, and major histocompatibility complex (MHC) class I antigens (Ags) on tumor cells in vitro. However, CTL-expressing FasL failed to improve antitumor activity in vitro and in animals, resulting from nonfunctional Fas expressed on tumor cells. In contrast, ethylene glycol tetraacetic acid (EGTA) treatment blocked increased sensitivity of cisplatin-treated tumor cells to CTL-mediated killing in vitro, suggesting an important role of the perforin/granzyme-mediated pathway for improved therapeutic effectiveness. This notion was further confirmed by perforin knockout animal studies. Thus, this study shows that (i) modulation of Ag (Fas, ICAM-1) expression by tumor cells has little effect on their increased sensitivity to CTL-mediated killing, (ii) improved therapeutic effectiveness is mediated mainly through the perforin/granzyme-mediated tumor killing pathway, and (iii) a combination of chemotherapy and adoptive E7-specific CTL transfer augments antitumor therapeutic activity in vivo. This finding may have important implications for treating HPV-associated cervical cancer.

Antitumor therapeutic effects of Salmonella typhimurium containing Flt3 Ligand expression plasmids in melanoma-bearing mouse

An attenuated strain of Salmonella typhimurium has been tested in animals and clinically as an anticancer agent due to its in vivo tumor-targeting and tumoricidal properties. We exploited a genetically-engineered S.typhimurium harboring Flt3 Ligand (Flt3L) expression vectors as a tumoricidal agent to enhance its therapeutic efficacy. Flt3L showed tumoricidal effects when expressed in tumor cells in vitro. When melanoma-bearing mice were treated locally with Salmonella, S. typhimurim with Flt3L expression vectors inhibited tumor growth more than Salmonella controls (50% vs. 0% in tumor regression rates). Moreover, it prolonged survivals of animals without induction of memory antitumor protective responses to a parental tumor re-challenge (50% vs. 0% in survival rates). These results suggest that a genetically engineered S. typhimurium with Flt3L expression vectors has the potential to be applicable as a safer and more effective tumor-targeting and tumoricidal agent.

Survival Benefits of Neoadjuvant Chemotherapy Followed by Radical Surgery versus Radiotherapy in Locally Advanced Chemoresistant Cervical Cancer

The aim of this study was to analyze long-term survivals in patients with stage IB to IIA cervical cancer treated by neoadjuvant chemotherapy setting. Between February 1989 and January 1998, 94 women with previously untreated stage IB to IIA carcinoma of the uterine cervix who received cisplatin based neoadjuvant chemotherapy were enrolled in this study. All of patients with chemoresponse (complete response, n=15; partial response, n=47) and 16 patients with chemoresistance received radical surgery (RS group). The other 16 patients with chemoresistance received radiotherapy for definite treatment (RT group). In the RS group, the 10 yr survival estimation in patients with bulky tumors (diameter ≥4 cm, n=26) was similar to that with non-bulky tumors (83.3% vs. 89.3%, p=NS). In selected patients with chemoresistance, those treated by radiotherapy (n=16) showed significantly poorer survivals than those treated by radical surgery (n=16) [10 yr survival rates of RT (25%) vs. RS (76.4%), p=0.0111]. Our results support that a possible therapeutic benefit of neoadjuvant chemotherapy plus radical surgery is only in patients with bulky stage IB to IIA cervical cancer. In cases of chemoresistance, radical surgery might be a better definite treatment option.

Human papillomavirus vaccines for the treatment of cervical cancer.

Human papillomavirus (HPV) infection is a major cause of cervical cancer, the second most common cancer in women worldwide. Currently, a HPV L1-based virus-like particle has been approved as a prophylactic vaccine against HPV infection, which will probably lead to a reduction in cervical cancer incidence within a few decades. Therapeutic vaccines, however, are expected to have an impact on cervical cancer or its precursor lesions, by taking advantage of the fact that the regulatory proteins (E6 and E7) of HPV are expressed constantly in HPV-associated cervical cancer cells. Vaccine types targeting these regulatory proteins include the recombinant protein and DNA vaccines, peptide vaccines, dendritic-cell vaccines, and viral and bacterial vector deliveries of vaccines, and these may provide an opportunity to control cervical cancer. Further approaches incorporating these vaccine types with either conventional therapy modalities or the modulation of CD4+ regulatory T cells appear to be more promising in achieving increased therapeutic efficacy. In this review, we summarize current and future therapeutic vaccine strategies against HPV-associated malignancies at the animal and clinical levels.

Suppression of antitumour protective cytotoxic T lymphocyte responses to a human papillomavirus 16 E7 DNA vaccine by coinjection of interleukin-12 complementary DNA: involvement of nitric oxide in immune suppression

Interleukin‐12 (IL‐12) has been shown to enhance cellular immunity in vitro and in vivo. The beneficial roles of IL‐12 as a DNA vaccine adjuvant have been commonly observed. Here the impact of IL‐12 complementary DNA (cDNA) as an adjuvant for a human papillomavirus (HPV) type 16 E7 DNA vaccine is investigated in a mouse tumour model. Coinjection of E7 DNA vaccine with IL‐12 cDNA completely suppressed antigen‐specific cytotoxic T‐lymphocyte (CTL) responses, leading to a complete loss of antitumour protection from a tumour cell challenge. In addition, antigen‐specific antibody and T helper cell proliferative responses were also suppressed by IL‐12 cDNA coinjection. This inhibition was observed over different IL‐12 cDNA doses. Furthermore, separate leg injections of IL‐12 and E7 cDNAs suppressed antigen‐specific CTL and tumour protective responses, but not antibody and T helper cell proliferative responses, suggesting different pathways for suppression of these two separate responses. Further knockout animal studies demonstrated that interferon‐γ and nitric oxide are not directly associated with suppression of antigen‐specific antibody responses by IL‐12 cDNA coinjection. However, nitric oxide was found to be involved in suppression of antigen‐specific CTL and tumour protective responses by IL‐12 cDNA coinjection. These data suggest that coinjection of IL‐12 cDNA results in suppression of E7‐specific CTL responses through nitric oxide, leading to a loss of antitumour resistance in this DNA vaccine model. This study further shows that the adjuvant effect of IL‐12 is dependent on the antigen types tested.

Promises and challenges of human papillomavirus vaccines for cervical cancer

Cervical cancer is the leading cause of death in women living in developing countries, mostly in Asia, South America, Africa and Europe. Owing to improved personal hygiene and regular Pap smear scr...