Jayne Knoff

Emerging human papillomavirus vaccines

Introduction: Identification of human papillomavirus (HPV) as the etiologic factor of cervical, anogenital, and a subset of head and neck cancers has stimulated the development of preventive and therapeutic HPV vaccines to control HPV-associated malignancies. Excitement has been generated by the commercialization of two preventive L1-based vaccines, which use HPV virus-like particles (VLPs) to generate capsid-specific neutralizing antibodies. However, factors such as high cost and requirement for cold chain have prevented widespread implementation where they are needed most. Areas covered: Next generation preventive HPV vaccine candidates have focused on cost-effective stable alternatives and generating broader protection via targeting multivalent L1 VLPs, L2 capsid protein, and chimeric L1/L2 VLPs. Therapeutic HPV vaccine candidates have focused on enhancing T cell-mediated killing of HPV-transformed tumor cells, which constitutively express HPV-encoded proteins, E6 and E7. Several therapeutic HPV vaccines are in clinical trials. Expert opinion: Although progress is being made, cost remains an issue inhibiting the use of preventive HPV vaccines in countries that carry the majority of the cervical cancer burden. In addition, progression of therapeutic HPV vaccines through clinical trials may require combination strategies employing different therapeutic modalities. As research in the development of HPV vaccines continues, we may generate effective strategies to control HPV-associated malignancies.

Enhanced cancer radiotherapy through immunosuppressive stromal cell destruction in tumors

Purpose: Radiotherapy kills cancer cells by causing DNA damage, and stimulates a systemic antitumor immune response by releasing tumor antigen and endogenous adjuvant within the tumor microenvironment. However, radiotherapy also induces the recruitment of immunosuppressive myeloid cells, which can interfere with the antitumor immune responses elicited by apoptotic tumor cells. We hypothesized that local delivery of vaccine following radiotherapy will lead to the priming of antigen-specific CTL immune responses and render immunosuppressive myeloid cells susceptible to killing by the activated CTLs. Experimental Design: Using several antigenic systems, we tested whether intratumoral injection of antigenic peptide/protein in irradiated tumors would be able to prime CTLs as well as load myeloid cells with antigen, rendering them susceptible to antigen-specific CTL killing. Results: We show that by combining radiotherapy and targeted antigenic peptide delivery to the tumor, the adjuvant effect generated by radiotherapy itself was sufficient to elicit the priming and expansion of antigen-specific CTLs, through the type I IFN-dependent pathway, leading to synergistic therapeutic antitumor effects compared with either treatment alone. In addition, using two different types of transgenic mice, we demonstrated that CTL-mediated killing of stromal cells in tumors by our approach is important for tumor control. Finally, we confirmed the efficacy of this approach in our preclinical model using two clinically tested therapeutic human papilloma virus (HPV) vaccines. Conclusions: These data serve as an important foundation for the future clinical translation of radiotherapy combined with a clinically tested therapeutic HPV vaccine for the control of HPV-associated cancers. Clin Cancer Res; 20(3); 644–57. ©2013 AACR.

Toll-like Receptor Agonist Imiquimod Facilitates Antigen-Specific CD8+ T-cell Accumulation in the Genital Tract Leading to Tumor Control through IFNγ

Purpose: Imiquimod is a Toll-like receptor 7 agonist used topically to treat external genital warts and basal cell carcinoma. We examined the combination of topical imiquimod with intramuscular administration of CRT/E7, a therapeutic human papillomavirus (HPV) vaccine comprised of a naked DNA vector expressing calreticulin fused to HPV16 E7. Experimental Design: Using an orthotopic HPV16 E6/E7+ syngeneic tumor, TC-1, as a model of high-grade cervical/vaginal/vulvar intraepithelial neoplasia, we assessed if combining CRT/E7 vaccination with cervicovaginal deposition of imiquimod could result in synergistic activities promoting immune-mediated tumor clearance. Results: Imiquimod induced cervicovaginal accumulation of activated E7-specific CD8+ T cells elicited by CRT/E7 vaccination. Recruitment was not dependent upon the specificity of the activated CD8+ T cells, but was significantly reduced in mice lacking the IFNγ receptor. Intravaginal imiquimod deposition induced upregulation of CXCL9 and CXCL10 mRNA expression in the genital tract, which are produced in response to IFNγ receptor signaling and attract cells expressing their ligand, CXCR3. The T cells attracted by imiquimod to the cervicovaginal tract expressed CXCR3 as well as CD49a, an integrin involved in homing and retention of CD8+ T cells at mucosal sites. Our results indicate that intramuscular CRT/E7 vaccination in conjunction with intravaginal imiquimod deposition recruits antigen-specific CXCR3+ CD8+ T cells to the genital tract. Conclusions: Several therapeutic HPV vaccination clinical trials using a spectrum of DNA vaccines, including vaccination in concert with cervical imiquimod, are ongoing. Our study identifies a mechanism by which these strategies could provide therapeutic benefit. Our findings support accumulating evidence that manipulation of the tumor microenvironment can enhance the therapeutic efficacy of strategies that induce tumor-specific T cells. Clin Cancer Res; 20(21); 5456–67. ©2014 AACR.

Control of HPV-associated tumors by innovative therapeutic HPV DNA vaccine in the absence of CD4+ T cells

Human papillomavirus (HPV) infections are particularly problematic for HIV + and solid organ transplant patients with compromised CD4+ T cell-dependent immunity as they produce more severe and progressive disease compared to healthy individuals. There are no specific treatments for chronic HPV infection, resulting in an urgent unmet need for a modality that is safe and effective for both immunocompromised and otherwise normal patients with recalcitrant disease. DNA vaccination is attractive because it avoids the risks of administration of live vectors to immunocompromised patients, and can induce potent HPV-specific cytotoxic T cell responses. We have developed a DNA vaccine (pNGVL4a-hCRTE6E7L2) encoding calreticulin (CRT) fused to E6, E7 and L2 proteins of HPV-16, the genotype associated with approximately 90% vaginal, vulvar, anal, penile and oropharyngeal HPV-associated cancers and the majority of cervical cancers. Administration of the DNA vaccine by intramuscular (IM) injection followed by electroporation induced significantly greater HPV-specific immune responses compared to IM injection alone or mixed with alum. Furthermore, pNGVL4a-hCRTE6E7L2 DNA vaccination via electroporation of mice carrying an intravaginal HPV-16 E6/E7-expressing syngeneic tumor demonstrated more potent therapeutic effects than IM vaccination alone. Of note, administration of the DNA vaccine by IM injection followed by electroporation elicited potent E6 and E7-specific CD8+ T cell responses and antitumor effects despite CD4+ T cell-depletion, although no antibody response was detected. While CD4+ T cell-depletion did reduce the E6 and E7-specific CD8+ T cell response, it remained sufficient to prevent subcutaneous tumor growth and to eliminate circulating tumor cells in a model of metastatic HPV-16+ cancer. Thus, the antibody response was CD4-dependent, whereas CD4+ T cell help enhanced the E6/E7-specific CD8+ T cell immunity, but was not required. Taken together, our data suggest that pNGVL4a-hCRTE6E7L2 DNA vaccination via electroporation warrants testing in otherwise healthy patients and those with compromised CD4+ T cell immunity to treat HPV-16-associated anogenital disease and cancer.

Intratumoral Injection of Therapeutic HPV Vaccinia Vaccine Following Cisplatin Enhances HPV-specific Antitumor Effects

Despite the conventional treatments of radiation therapy and chemotherapy, the 5-year survival rates for patients with advanced-stage cervical cancers remain low. Cancer immunotherapy has emerged as an alternative, innovative therapy that may improve survival. Here, we utilize a preclinical HPV-16 E6/E7-expressing tumor model, TC-1, and employ the chemotherapeutic agent cisplatin to generate an accumulation of CD11c+ dendritic cells in tumor loci making it an ideal location for the administration of therapeutic vaccines. Following cisplatin treatment, we tested different routes of administration of a therapeutic HPV vaccinia vaccine encoding HPV-16 E7 antigen (CRT/E7-VV). We found that TC-1 tumor-bearing C57BL/6 mice treated with cisplatin and intratumoral injection of CRT/E7-VV significantly increased E7-specific CD8+ T cells in the blood and generated potent local and systemic antitumor immune responses compared to mice receiving cisplatin and CRT/E7-VV intraperitoneally or mice treated with cisplatin alone. We further extended our study using a clinical grade recombinant vaccinia vaccine encoding HPV-16/18 E6/E7 antigens (TA-HPV). We found that intratumoral injection with TA-HPV following cisplatin treatment also led to increased E7-specific CD8+ T cells in the blood as well as significantly decreased tumor size compared to intratumoral injection with wild type vaccinia virus. Our study has strong implications for future clinical translation using intratumoral injection of TA-HPV in conjunction with the current treatment strategies for patients with advanced cervical cancer.

Treatment of Tumors with Vitamin E Suppresses Myeloid Derived Suppressor Cells and Enhances CD8+ T Cell-Mediated Antitumor Effects

Vitamin E has been shown to have strong anticarcinogenic properties, including antioxidant characteristics, making it an ideal candidate for use in combination with immunotherapies that modify the tumor microenvironment. The tumor microenvironment contains immunosuppressive components, which can be diminished, and immunogenic components, which can be augmented by immunotherapies in order to generate a productive immune response. In the current study, we employ the α-tocopherol succinate isomer of vitamin E to reduce immunosuppression by myeloid derived suppressor cells (MDSCs) as well as adoptive transfer of antigen-specific CD8+ T cells to generate potent antitumor effects against the HPV16 E7-expressing TC-1 tumor model. We show that vitamin E alone induces necrosis of TC-1 cells and elicits antitumor effects in TC-1 tumor-bearing mice. We further demonstrate that vitamin E reverses the suppression of T cell activation by MDSCs and that this effect is mediated in part by a nitric oxide-dependent mechanism. Additionally, treatment with vitamin E reduces the percentage of MDSCs in tumor loci, and induces a higher percentage of T cells, following T cell adoptive transfer. Finally, we demonstrate that treatment with vitamin E followed by E7-specific T cell adoptive transfer experience elicits potent antitumor effects in tumor-bearing mice. Our data provide additional evidence that vitamin E has anticancer properties and that it has promise for use as an adjuvant in combination with a variety of cancer therapies.

Histone deacetylase inhibitor AR-42 enhances E7-specific CD8+ T cell-mediated antitumor immunity induced by therapeutic HPV DNA vaccination

We have previously created a potent DNA vaccine encoding calreticulin linked to the human papillomavirus (HPV) oncogenic protein E7 (CRT/E7). While treatment with the CRT/E7 DNA vaccine generates significant tumor-specific immune responses in vaccinated mice, the potency with the DNA vaccine could potentially be improved by co-administration of a histone deacetylase inhibitor (HDACi) as HDACi has been shown to increase the expression of MHC class I and II molecules. Thus, we aimed to determine whether co-administration of a novel HDACi, AR-42, with therapeutic HPV DNA vaccines could improve the activation of HPV antigen-specific CD8+ T cells, resulting in potent therapeutic antitumor effects. To do so, HPV-16 E7-expressing murine TC-1 tumor-bearing mice were treated orally with AR-42 and/or CRT/E7 DNA vaccine via gene gun. Mice were monitored for E7-specific CD8+ T cell immune responses and antitumor effects. TC-1 tumor-bearing mice treated with AR-42 and CRT/E7 DNA vaccine experienced longer survival, decreased tumor growth, and enhanced E7-specific immune response compared to mice treated with AR-42 or CRT/E7 DNA vaccine alone. Additionally, treatment of TC-1 cells with AR-42 increased the surface expression of MHC class I molecules and increased the susceptibility of tumor cells to the cytotoxicity of E7-specific T cells. This study indicates the ability of AR-42 to significantly enhance the potency of the CRT/E7 DNA vaccine by improving tumor-specific immune responses and antitumor effects. Both AR-42 and CRT/E7 DNA vaccines have been used in independent clinical trials; the current study serves as foundation for future clinical trials combining both treatments in cervical cancer therapy.Key messageAR-42, a novel HDAC inhibitor, enhances potency of therapeutic HPV DNA vaccinesAR-42 treatment leads to strong E7-specific CD8+ T cell immune responsesAR-42 improves tumor-specific immunity and antitumor effects elicited by HPV DNA vaccineAR-42 is more potent than clinically available HDACi in combination with HPV DNA vaccine

Cervical Cancer: Development of Targeted Therapies Beyond Molecular Pathogenesis

It is well-known that the human papillomavirus (HPV) is the causative agent of cervical cancer. The integration of HPV genes into the host genome causes the upregulation of E6 and E7 oncogenes. E6 and E7 proteins inactivate and degrade tumor suppressors p53 and retinoblastoma, respectively, leading to malignant progression. HPV E6 and E7 antigens are ideal targets for the development of therapies for cervical cancer and precursor lesions because they are constitutively expressed in infected cells and malignant tumors, but not in normal cells, and they are essential for cell immortalization and transformation. Immunotherapies are being developed to target E6/E7 by eliciting antigen-specific immune responses. siRNA technologies target E6/E7 by modulating the expression of the oncoproteins. Proteasome inhibitors and histone deacetylase inhibitors are being developed to indirectly target E6/E7 by interfering with their oncogenic activities. The ultimate goal for HPV-targeted therapies is the progression through clinical trials to commercialization.

Combination of proteasome and HDAC inhibitor enhances HPV16 E7-specific CD8+ T cell immune response and antitumor effects in a preclinical cervical cancer model.

BackgroundBortezomib, a proteasome inhibitor and suberoylanilide hydroxamic acid (SAHA, also known as Vorinostat), a histone deacetylase inhibitor, have been recognized as potent chemotherapeutic drugs. Bortezomib and SAHA are FDA-approved for the treatment of cutaneous T cell lymphoma and multiple myeloma/mantle cell lymphoma, respectively. Furthermore, the combination of the bortezomib and SAHA has been tested in a variety of preclinical models and in clinical trials and may be ideal for the treatment of cancer. However, it remains unclear how this treatment strategy affects the host immune response against tumors.ResultsHere, we used a well-defined E6/E7-expressing tumor model to examine how the immune system can be motivated to act against tumor cells expressing tumor antigens. We demonstrate that the combination of bortezomib and SAHA elicits potent antitumor effects in TC-1 tumor-bearing mice. Additionally, we are the first to show that treatment with bortezomib and SAHA leads to tumor-specific immunity by rendering tumor cells more susceptible to killing by antigen-specific CD8+ T cells than treatment with either drug alone.ConclusionsThe current study serves an important foundation for the future clinical application of both drugs for the treatment of cervical cancer.

Cancer immunotherapy employing an innovative strategy to enhance CD4+ T cell help in the tumor microenvironment.

Chemotherapy and/or radiation therapy are widely used as cancer treatments, but the antitumor effects they produce can be enhanced when combined with immunotherapies. Chemotherapy kills tumor cells, but it also releases tumor antigen and allows the cross-presentation of the tumor antigen to trigger antigen-specific cell-mediated immune responses. Promoting CD4+ T helper cell immune responses can be used to enhance the cross-presentation of the tumor antigen following chemotherapy. The pan HLA-DR binding epitope (PADRE peptide) is capable of generating antigen-specific CD4+ T cells that bind various MHC class II molecules with high affinity and has been widely used in conjunction with vaccines to improve their potency by enhancing CD4+ T cell responses. Here, we investigated whether intratumoral injection of PADRE and the adjuvant CpG into HPV16 E7-expressing TC-1 tumors following cisplatin chemotherapy could lead to potent antitumor effects and antigen-specific cell-mediated immune responses. We observed that treatment with all three agents produced the most potent antitumor effects compared to pairwise combinations. Moreover, treatment with cisplatin, CpG and PADRE was able to control tumors at a distant site, indicating that our approach is able to induce cross-presentation of the tumor antigen. Treatment with cisplatin, CpG and PADRE also enhanced the generation of PADRE-specific CD4+ T cells and E7-specific CD8+ T cells and decreased the number of MDSCs in tumor loci. The treatment regimen presented here represents a universal approach to cancer control.