Skip to Content
Translational Research Program (TRP)
Contact CIP
Show menu
Search this site
Last Updated: 10/10/18

Johns Hopkins University SPORE in Cervical Cancer

Principal Investigator:
T.C. Wu, M.D., Ph.D.

Principal Investigator Contact Information

T.C. Wu, MD, PhD
Professor
Departments of Pathology, Oncology,
Obstetrics and Gynecology and
Molecular Microbiology and Immunology
Johns Hopkins University
School of Medicine
Department of Pathology
1550 Orleans Street, CRB II Room 309
Baltimore, MD 21231
Tel: (410) 614-3899
Fax: (443) 287-4295
Email: wutc@jhmi.edu

Overall Abstract

This competitive renewal application for a Specialized Program of Research Excellence (SPORE) in Cervical Cancer at Johns Hopkins University School of Medicine, University of Alabama at Birmingham, University of Colorado and our new collaborator Mount Sinai Icahn School of Medicine brings together a highly interactive, multidisciplinary, and inter-institutional program of translational research in cervical cancer. The SPORE includes five integrated projects spanning the fields of prevention and treatment cervical cancer and its precursor lesions. Project 1, L1 capsomeres as a next generation preventive HPV vaccine, led by Robert Garcea, M.D. and Warner Huh, M.D. Project 2: Local vaccination and imiquimod for persistent HPV16+ patients with normal cytology, led by Richard Roden, Ph.D., Warner Huh, M.D. and T.-C. Wu, M.D.,Ph.D. Project 3: Treatment of HIV- and HIV+ patients with CIN2/3 using an HPV DNA vaccine administered intramuscularly via electroporation, led by Ronald Alvarez, M.D., Chien-Fu Hung, Ph.D. and Richard Roden, Ph.D. Project 4: Molecular attributes of tissue immune response in HPV disease, led by Cornelia Trimble, M.D. and Michael Donovan, M.D., Ph.D. Project 5: Safety and immunogenicity study of a HPV16-specific therapeutic protein administered intratumorally during chemoradiation, led by Ronald Alvarez, M.D., Richard Roden, Ph.D. and T.-C. Wu, M.D., Ph.D. The five integrated projects are supported by three cores that provide critical supportive infrastructure essential to efficient and effective translational research: the Administrative/Communication Core (Core A), led by T.-C. Wu, M.D., Ph.D. and Edward Partridge, M.D., the Biostatistics/Data Management Core (Core B), led by Sejong Bae Ph.D., Karan Singh, Ph.D., and Chenguang Wang, Ph.D. and the Tissue/Pathology and Immunology Core (Core C), led by Cornelia Trimble, M.D. and Lawrence Lamb, Ph.D. The SPORE also includes a Developmental Research Program (led by T.-C. Wu, M.D., Ph.D. and Drew Pardoll M.D., Ph.D.) to nurture novel research ideas and technologies and a Career Development Program (led by T.-C. Wu, M.D., Ph.D. and Edward Partridge, M.D.) to recruit and facilitate career development of individuals with interest in translational cervical cancer research.

Project 1: L1 Capsomeres as a Next Generation Preventive HPV Vaccine

Project Co-Leaders:
Robert Garcea, M.D. (Basic)
Warner Huh, M.D. (Clinical)

The recent development of prophylactic vaccines protective against “high risk” human papillomaviruses (HPV) is a landmark in medicine. The current vaccines are composed of recombinant virus-like particles (VLPs) of the major capsid protein, L1, including the high-risk HPV types 16 and 18. Phase II/III clinical trials have shown >95% efficacy of these VLP preparations in preventing HPV 16, 18 infection, and thus hopefully preventing the eventual development of about 70% of cervical cancers associated with these types. Despite this success the VLP vaccines have the adverse attributes of high cost ($360 for the initial three immunizations in the USA) and a requirement for refrigeration (i.e., cold chain). We have previously characterized the immunogenic properties of VLP subunits, pentameric L1 capsomeres. Capsomeres can be purified after recombinant expression of L1 in E. coli as untagged native proteins, at levels suggesting a significant reduction in manufacturing expense. The purified protein can be freeze-dried, resuspended, and stored at room temperature without loss of immunogenicity. Capsomeres have been suggested as a “next generation” HPV vaccine that might be ideally suited for production and use in underdeveloped countries of the world where cervical cancer is particularly prevalent. We propose to use an HPV16 L1 capsomere protein that has been GMP produced (BioSidus, S.A.), vialed, and toxicology screened for study in a phase I human trial. In Aims 1 and 2 we propose a dose escalation scheme of 15 subjects at each of three dose levels (10, 50, 250 micrograms), repeated three times, with concomitant analysis of toxicity, development of neutralizing antibodies, and cytotoxic T-cell responses. In the third aim, laboratory and animal experiments will test new vaccine formulation strategies, and evaluate the possibility that capsomere vaccines may be prepared as powders with adjuvants, which are thermostable

SPECIFIC AIMS

Despite the remarkable efficacy of the present VLP HPV vaccines, their cost is prohibitive in low-resource settings. Since Pap smear screening is unaffordable in developing countries, a low cost HPV vaccine is the most economical way to decrease the burden of cervical cancer in this population. Thus, the current imperative for cervical cancer care is the development of a low-cost vaccine that is accessible to underdeveloped areas of the world. The goal of this project is to develop further the concept of a subunit HPV vaccine composed of “capsomeric” subunits of the VLP that can be manufactured after bacterial expression of the HPV L1 protein, at an affordable cost. Successful application of this technology to multiple high-risk HPV types could lead to a universal vaccine reagent capable of preventing HPV infection worldwide.

We have prepared GMP grade HPV16 L1 capsomeres and completed their toxicological analysis (see Appendix 3 pages 101-680). We plan a dose escalation clinical trial of this monovalent vaccine preparation with concomitant analysis of toxicity and development of neutralizing antibodies. The results of this study will determine whether we can proceed to testing a tetravalent HPV combination vaccine now being prepared under the auspices of the NCI PREVENT program.

In addition to cost, significant barriers to HPV vaccine distribution include a cold chain requirement, general stability over time, and patient compliance with multiple immunizations. Ideally a vaccine should be heat stable, and formulated with an adjuvant that would permit one or at most two administrations for protection. We propose innovative preclinical studies that aim to prepare powder formulations of HPV capsomeres that will have superior physical properties in comparison to current VLP vaccines. Such formulations would solve long-standing barriers in the field of vaccination, such as the preparation of adjuvanted heat stable powders. In summary, this proposal is aimed at developing an L1 capsomere vaccine with equivalent immunogenicity and protective responses as compared to current commercial HPV vaccines, but with superior properties with respect to practical issues that would otherwise prevent global implementation of HPV vaccination.

HYPOTHESIS 1: Vaccination of patients with HPV16 L1 capsomeres in alum is safe.

Specific Aim 1: Evaluate whether vaccination using GMP grade L1 capsomeres in alum is safe in healthy women.

HYPOTHESIS 2: HPV16 L1 capsomeres are immunogenic in patients when formulated in alum.

Specific Aim 2: Determine the immunogenicity and dose response of patients to vaccination with GMP grade L1 capsomeres in alum.

The immune response to L1 capsomeres formulated in alum will be compared to those in placebos and in subjects vaccinated with Gardasil™ for a preliminary analysis of non-inferiority with respect to L1-specific immune responses to HPV type 16. This Phase I trial represents a proof-of-principle study that will facilitate further development of multivalent L1 capsomere vaccines.

HYPOTHESIS 3: Cold chain storage properties are a significant barrier to the distribution and cost of current vaccines. The robust stability of L1 capsomeres should allow development of powdered HPV vaccines that are both thermostable and include adjuvants in their composition.

Specific Aim 3: Develop a freeze-dry protocol for the powder formulation of capsomeres in alum and alum+MPLA. Study the stability, and immunogenicity of these powder formulations in comparison to Gardasil™ and Cervarix™ in murine models.

In preclinical studies we will explore methods to enhance the formulation of capsomeres with respect to stability and adjuvants. We will characterize these formulations by multiple physical measurements and test in murine immunization studies for their ability to elicit neutralizing antibodies.

Project 2: Local TA-CIN/GPI-0100 Vaccination and Imiquimod for Persistent HPV16+/Normal Cytology Patients

Project Co-Leaders:
Richard Roden, Ph.D. (Basic)
T.-C Wu, M.D., Ph.D (Basic)
Warner Huh, M.D. (Clinical)

Pap screening and surgical removal of cervical intraepithelial neoplasia (CIN2/3) has greatly reduced the incidence of cervical cancer, but testing lacks precision and the LEEP/conization procedure to excise these pre-cancerous lesions is associated with cervical incompetence and premature births. Recently co-testing for high risk HPV DNAs and specifically for HPV16 in cytologic samples has been broadly implemented to improve the accuracy of screening, particularly for equivocal cytologic diagnoses, and co-testing in women of 30 or more years. Since half of all persistent HPV16 infections progress to =CIN2, HPV16+ women with normal cytology (negative for intraepithelial lesion and malignancy, NILM) undergo repeat co-testing in one year per ASCCP guidelines. If HPV16+ NILM again, these women undergo colposcopy. This screening identifies large numbers of women 30 years and over with persistent HPV16 infections who are likely to eventually progress and need surgical intervention, with continued potential for transmission or the development of neoplasia at the cervix and other anogenital sites and suffering considerable psychosocial stress. Our overall goal is to develop a regimen to safely and effectively elicit immune clearance of persistent cervical HPV16 infections for secondary prevention of cervical cancer, an important unmet medical need. Intra-muscular administration of an HPV16 E6E7L2 fusion protein (termed TA-CIN) alone had little impact on HPV16+high grade disease. Recent studies suggest that the adjuvant GPI-0100 potently enhances cellular immune responses to TA-CIN vaccination, the importance of their targeting to the disease site, and the impact of imiquimod on the local microenvironment in disease clearance. We hypothesize that local inflammation and antigen-delivery to relevant draining lymph nodes is required for effective genital tract immunity. Thus, we propose to administer TA-CIN formulated with GPI-0100 adjuvant in the cervix of women with persistent HPV16 infections but normal cytology to recruit HPV-specific immune cells to the genital tract, with or without priming the local microenvironment by topical administration of imiquimod on the cervix immediately following vaccination.

SPECIFIC AIMS:

Several large randomized trials have shown that testing for high risk HPV (hrHPV) DNA in cytologic specimens can improve the identification of women who have or will develop CIN2/3 or cervical cancer (CIN2+), and many countries are examining first line hrHPV DNA testing for their cytologic screening programs. Further, the latest FDA-approved tests detect not only hrHPV as a group, but also specifically HPV16. Despite the licensed preventive HPV vaccines, HPV16 remains as the most prevalent genotype. Half of all persistent HPV16 infections (>12 months) in cytologically normal women progress to CIN2+ (1) . In the United States, the American Cancer Society/American Society of Colposcopy and Cervical Pathology/American Society of Clinical Pathology and the United States Preventive Services Task Force recommends combined pap cytology and hrHPV DNA testing, also known as co-testing, in women 30 and older. It is currently recommended that women who are hrHPV DNA+ and cytologically normal (negative for intraepithelial lesion and malignancy, NILM) undergo repeat co-testing in one year and genotyping for HPV16/18. If positive for 16 or 18, the woman should then be referred for colposcopy. This screening approach can identify women with persistent HPV16 infections who are at substantial risk of developing of high grade CIN which would require surgical intervention, with continued potential for transmission or the development of high grade neoplasia at other anogenital sites, and suffering considerable psychosocial stress. Our overall goal is to develop a treatment regimen that safely and effectively elicits immune clearance of persistent cervical HPV16 infections in women over 30 with normal cytology for secondary prevention of associated cancers. Intra-muscular administration of an HPV16 E6E7L2 fusion protein (termed TA-CIN) has proven immunogenic in several therapeutic vaccine studies but it had little impact on HPV16+ high grade vulvar intraepithelial neoplasia (VIN). However, topical treatment of HPV16+ high grade VIN with the TLR7 agonist imiquimod for 8 weeks followed by three immunizations with TA-CIN over the next 10 weeks elicited complete histologic responses in 32% of patients after imiquimod treatment, but this rose to 58% after vaccination. This trial and our animal studies suggest: 1) the need for an adjuvant to enhance cellular immune responses to protein-based vaccination, 2) the importance of targeting these responses to the disease site and 3) the importance of the intra-lesional immune microenvironment. GPI-0100 is a semi-synthetic Quillaja saponin, a class of adjuvant which elicits potent cellular immune responses to protein vaccines by enhancing immune cell recruitment, maturation and cross-priming. We propose to administer TA-CIN vaccine formulated in GPI-0100 (TA-CIN/GPI-0100) in the cervix to trigger local inflammation and a potent HPV-specific cellular immune response that targets to the genital tract. Finally, we propose to alter the intra-lesional microenvironment and enhance recruitment of HPV-specific immune cells into the cervical mucosa by topical administration of imiquimod on the cervix immediately following vaccination. Immunologic analyses will address whether T cells are programed to traffic to the infected mucosal epithelium based upon the immunization site, or local inflammation, and are both required to clear HPV16 infections.

Hypothesis 1: Intra-cervical administration of an HPV16 E6E7L2 fusion protein (TA-CIN) formulated in the saponin GPI-0100 (TA-CIN/GPI-0100) is well tolerated by women bearing persistent HPV16+, normal cervical cytology and no evidence of CIN2+ on colposcopy.

Aim 1: To perform a dose escalation study of intra-cervical vaccination with TA-CIN/GPI-0100 in women with persistent HPV16+/NILM cytology. In 28 women with NILM cytology and persistent HPV16 infection over 12 months, the safety and tolerability of intra-cervical TA-CIN/GPI-0100 vaccination will be tested in three dose escalating cohorts.

Hypothesis 2: Intra-cervical vaccination with TA-CIN/GPI-0100 can safely be combined with concurrent cervical administration of Imiquimod in women persistent HPV16+, normal cervical cytology and no evidence of CIN2+ on colposcopy.

Aim 2: To vaccinate in the cervix using the selected dose of TA-CIN/GPI-0100 and then apply imiquimod to the cervix of women with persistent HPV16+/NILM cytology. The safety and tolerability will be examined in 14 additional women.

Hypothesis 3: Intra-cervical vaccination with TA-CIN/GPI-0100 potentiates systemic and local immune responses to both L2-specific neutralizing antibody and the E6/E7-specific cellular immunity.

Aim 3: To determine the immunogenicity of intra-cervical TA-CIN/GPI-0100 vaccination in women with HPV16+/NILM cytology with or without topical imiquimod. Humoral and cellular immune responses, both HPV16-specific and non-specific, will be monitored in peripheral blood and at the cervix in each arm.

Hypothesis 4: Intra-cervical vaccination with TA-CIN/GPI-0100 and topical imiquimod impact local immune microenvironment and HPV16 viral load.

Aim 4: To examine virologic, cytologic changes at the cervix upon intra-cervical TA-CIN/GPI-0100 vaccination of women with persistent HPV16+/NILM cytology with and without topical imiquimod. HPV genotype and load, and cytology will be assessed before and after vaccination, and correlated with local inflammation, immune cell subsets and HPV16-specific immunity.

Project 3: Treatment of HIV- and HIV+ patients with HPV16+ CIN2/3 using pNGLV4a-hCRTE6E7L2 DNA vaccine administered intramuscularly via electroporation

Co-Leaders:
Chien-Fu Hung., Ph.D. (Basic)
Richard Roden, Ph.D. (Basic)
Jean Anderson, M.D. (Clinical)

Oncogenic human papillomaviruses (HPVs) are the etiologic agents of 99% of cervical cancer, but HPV16 is the most important type as it causes half of all cervical cancer cases and >90% of HPV-associated cancers at other sites. The inability of licensed vaccines to eliminate existing HPV infections is a major unmet medical need since there is a considerable burden of HPV-associated disease worldwide, and implementation of HPV vaccination in the US has been limited. HPV-associated cancer incidence is also significantly elevated in cervical and at other sites in HIV+ patients. HIV+ patients acquire more frequent multi-type infections, including many genotypes infrequently seen in healthy individuals, and not targeted by the current HPV vaccines. In this SPORE we developed a candidate therapeutic and preventive HPV vaccine, pNGVL4a-hCRTE6E7L2, which comprises a DNA vector encoding calreticulin (CRT) fused genetically with HPV16 E6, E7 (that are expressed in all HPV infected cells) and L2 capsid protein (a broadly protective antigen). Fusion with the heat shock protein CRT has been shown to enhance the potency of DNA vaccines, even in CD4 depleted animals, making the pNGVL4a-hCRTE6E7L2 DNA vaccine particularly promising for use in HIV+ patients, a particularly challenging group to treat. Although DNA vaccines are relatively safe, they generally exhibit suboptimal immunogenicity when administered by conventional intramuscular needle injection, likely reflecting inefficient host cell transduction. We have previously shown that electroporation was the most effective DNA vaccine administration method to generate HPV-specific CD8+ T cell immune responses, compared to conventional intramuscular injection and epidermal delivery via gene gun. Here we propose to use the Ichor TriGrid Electroporation Device, which has been used in multiple clinical trials, for intramuscular administration of pNGVL4a-hCRTE6E7L2 DNA vaccine at escalating doses in HIV- and HIV+ patients with HPV16-associated high-grade cervical intraepithelial neoplasia (CIN 2/3) and to examine the safety, virologic and disease outcomes. The systemic and local immune responses will be correlated with these outcomes as well as lesional expression of PD-L1, a pathway associated with immune escape.

SPECIFIC AIMS

An ideal therapeutic and preventive HPV vaccine would generate cell-mediated immunity to eradicate established infection and/or HPV-associated precancer or cancer lesions as well as protective humoral immunity to prevent new infections. We have previously developed a therapeutic and preventive DNA vaccine, pNGVL4a-hCRTE6E7L2, which encodes calreticulin (CRT) linked HPV16 E6, E7 and L2 proteins. The fusion with the heat shock protein CRT has been shown to enhance the potency of DNA vaccines. We found that vaccination with pNGVL4a-hCRTE6E7L2 DNA vaccine could generate HPV neutralizing antibodies as well as control of HPV16 E6/E7-expressing tumors in a preclinical model. The pNGVL4a-hCRTE6E7L2 DNA vaccine is particularly promising for use in HIV+ patients to treat HPV16-associated lesions as well as to generate a pan-HPV protective effect against new or re-infection for several reasons. First, DNA vaccines are safe, even for immunocompromised individuals, in that they do not contain live pathogen but still elicit cell-mediated and/or humoral immune responses. Second, we have recently shown that CRT profoundly enhances the induction of cellular immunity, and that DNA encoding CRT linked to HPV tumor antigens still elicits an HPV-specific CD8+ T cell response even in CD4-depleted animals. This suggests that pNGVL4a-hCRTE6E7L2 may be active in HIV+ patients with low-level CD4+ T cells, a particularly challenging group to treat. Finally, the broadly protective effects of L2-specific neutralizing antibody have the potential to prevent the unusual HPV types and multi-type HPV infections seen in the HIV+ population. Due to the prevalence and diversity of HPV types and the lack of antiviral agents for HPV, development of broad-spectrum prophylactic vaccines against HPV is an attractive strategy in the prevention of the HPV-associated cancer in HIV+ patients. There are 14 different “oncogenic” types of genital HPV associated with cervical cancer and it is important to protect against all of them as well as the benign types that can have significant morbidity in HIV+ patients.

Although DNA vaccines are relatively safe and well suited for multiple administrations, they generally exhibit suboptimal immunogenicity when administered by conventional intramuscular needle injection, likely reflect inefficient host cell transduction. To overcome this obstacle, we will use in vivo electroporation, which involves DNA plasmid administration to the target tissue followed by brief electrical pulses at the administration site to transiently increase cell membrane permeability. Consequently, this allows the cells to have increased uptake and expression of the DNA plasmid. We have previously shown that electroporation was the most effective DNA vaccine administration method to generate CD8+ T cell immune responses, compared to conventional intramuscular (IM) injection and epidermal delivery via gene gun. We plan to use the Ichor TriGrid Electroporation Device, which has been used in multiple clinical trials (see http://www.ichorms.com/pipeline.shtml). Taken together, intramuscular administration of pNGVL4a-hCRTE6E7L2 DNA vaccine by electroporation will likely not only generate potent cellular immune responses against HPV16-associated high-grade cervical intraepithelial neoplasia (CIN2/3) in HIV- and HIV+ patients but also generate L2-specific neutralizing antibody cross-protective against diverse HPV types, potentially a groundbreaking advance in the field of HPV vaccine development. Therefore, we propose:

Hypothesis 1: Treatment of HIV- and HIV+ patients with HPV16-associated CIN2/3 with pNGVL4a-hCRTE6E7L2 DNA vaccine administered intramuscularly via electroporation is safe and feasible.

Aim 1: To evaluate the safety and toxicity of pNGVL4a-hCRTE6E7L2 administered via electroporation in HIV- and HIV+ patients with HPV16+ CIN2/3.

Hypothesis 2: Treatment of HPV16-associated CIN2/3 patients with pNGVL4a-hCRTE6E7L2 DNA vaccination via electroporation will generate a significant increase in HPV16 E6 and E7-specific CD8+ T cell immune responses.

Aim 2: To characterize the HPV16 E6- and E7-specific humoral and cell-mediated immune responses in HIV- and HIV+ patients with HPV16+ CIN2/3 vaccinated with pNGVL4a-hCRTE6E7L2 via electroporation.

Hypothesis 3: Treatment of HPV16-associated CIN2/3 in HIV- and HIV+ patients by electroporation of pNGVL4a-hCRTE6E7L2 DNA vaccine will generate L2-specific broadly neutralizing antibodies.

Aim 3: To characterize L2-specific humoral immune responses in HIV- and HIV+ patients with HPV16-associated CIN2/3 upon vaccination with pNGVL4a-hCRTE6E7L2 DNA vaccine via electroporation.

Hypothesis 4: Treatment of HPV16-associated CIN2/3 in HIV- and HIV+ patients by electroporation of pNGVL4a-hCRTE6E7L2 DNA vaccine will reduce HPV viral load and generate histopathological changes in patients’ lesions.

Aim 4: To determine the HPV load and histopathological changes in the lesion and its microenvironment in HIV- and HIV+ patients with HPV16-associated CIN2/3 upon treatment with pNGVL4a-hCRTE6E7L2 DNA vaccine via electroporation.

Project 4: Molecular attributes of tissue immune response in HPV disease

Project Co-Leaders:
Cornelia Trimble, M.D. (Clinical)
Michael Donovan, M.D., Ph.D (Basic)

Clinical success of immunotherapies has been hampered by an incomplete understanding of mechanisms of immune cell homing, function, and retention in the target tissue. Our research team has taken advantage of our unique clinical resources to develop new methods, data, and insights to address these basic questions. Intraepithelial lesions (CIN2/3) caused by human papillomavirus (HPV) present an opportunity to determine how immune responses are generated and maintained in a non-sterile barrier epithelial tissue. CIN2/3 lesions are directly accessible, clinically indolent, and are associated with functionally obligate expression of viral oncoproteins E6 and E7. Although a subset undergo complete regression, peripheral blood T cell responses to viral antigens are weak, and do not correlate with disease outcome. We propose studies to identify mechanisms of immune response sequestered at the site of antigen, in CIN2/3. We have an active immunotherapy program for premalignant HPV disease, testing strategies to enhance T cell responses to viral antigens, and to enable homing and access to the female reproductive tract mucosa. We have developed a constellation of technologies to perform quantitative, tissue-based analyses of human samples obtained before and after study interventions. This proposal is based on maturing data from our two previous funding cycles, including immune therapeutic trials governed by three investigator-sponsored INDs, each of which was generated in collaboration with the NCI RAID program. Our recent preliminary data indicate that systemic therapeutic vaccination can induce a striking effector immune response in the target lesion, despite modest detectable T cell responses to vaccine antigen in the peripheral blood. Our short-term Aims are to identify the molecular signature of these tissue responses; to determine their immune therapeutic relevance; and to develop tissue signatures to predict likelihood of response either to therapeutic vaccination or to direct manipulation of the lesion microenvironment. Our long-term goal is to develop analytic algorithms for tissue-based biomarkers that will provide objective guidelines to inform development of new therapeutic strategies and to guide treatment decisions.

SPECIFIC AIMS:

Previous attempts to translate therapeutic vaccination strategies for intraepithelial HPV disease have yielded limited success, by two standard measures of vaccine efficacy: (1) induction of robust peripheral blood T cell responses to vaccine antigen, and (2) correlation of peripheral blood immune responses with histologic regression of disease. Our recent tissue studies have provided evidence of post-vaccination immunologic changes in target lesions that suggest the induction of clinically relevant tissue-localized immune responses, despite modest detectable responses in circulating T lymphocytes. The identification of post-vaccination changes in target lesions has practical implications for the design and interpretation of immunotherapeutic trials for preinvasive HPV disease. The mechanisms that govern immune cell activation, homeostasis, and retention in the human lower genital tract mucosa are not well understood. However, clinical specimens are small, and are available only in limited quantities, so novel approaches are needed.

In this Project, we hypothesize that in preinvasive HPV disease, relevant immune responses are sequestered at the site of antigen expression, and that effector cell access to target lesions can be enhanced by direct manipulation of the mucosal microenvironment. Identifying mechanisms of immune regulation in the female lower genital tract mucosa will be predicated on a better understanding of the composition and function of unmanipulated cells within the microenvironment of both normal and pre-malignant tissue. To address this hypothesis we have three Specific Aims:

Aim 1. Determine immunogenicity of peripheral therapeutic vaccination in combination with topical TLR7/8 agonist applied to HPV16+CIN2/3 The goal of this Phase I study is to determine the contributions of therapeutic vaccination and direct manipulation of the lesion with imiquimod, a topical TLR7/8 agonist, in mediating immune clearance of established CIN2/3 lesions. This Aim will test the hypotheses that (1) peripheral therapeutic vaccination can elicit measureable changes in the subset distribution of immune cells sequestered in the target lesion, and (2) imiquimod will induce inflammatory pathways in cervical epithelium,and subsequent cytokine secretion, activation of vascular endothelium, and T cell extravasation to lesional epithelium. In the current funding cycle, we found that either vaccination alone, or imiquimod alone, are well-tolerated by this patient cohort. We expect that the combination will be safe and will enhance immune-mediated clearance of high grade dysplasias. Immune responses to vaccine antigens will be measured in PBMC using conventional methods, including IFN? ELISpot and intracellular cytokine staining and flow cytometry. However, we will also analyze tissue-resident T cells, to determine their phenotype, functional polarization, and expression of surface integrins; and activation markers in lesional endothelium.

Aim 2. Develop digital image analysis algorithms for individual immune expression signatures in CIN2/3 specimens. Tissue specimens banked from patients enrolled in immunotherapy studies will be used to determine immune response signatures. We will perform quantitative phenotypic assessment of in situ immune cell infiltrates in clinically annotated, longitudinally obtained human tissue specimens, using multiplex immunoflourescence, immunohistochemistry, and digital image analysis. These algorithms will be validated prospectively in our ongoing vaccine trials.

Aim 3. Identify the transcriptome of lesional epithelium and subjacent stroma in vaccine “responders”. The mechanisms governing tissue immune cell recruitment, functional polarization, activation, homeostasis, and retention are not well-known. Much of our current understanding is based on studies of circulating cells that have not differentiated to tissue-resident cells. In conjunction with coordinate image analyses on adjacent tissue sections we will isolate epithelial and stromal cell subsets, including T cells, macrophages, and fibroblasts, from primary tissue explants. We will characterize individual cell types in the lesion microenvironment, in tissues obtained on our immunotherapy protocols. This Aim will test the hypothesis that specific cell subsets in the mucosal microenvironment provide signals that direct immune cell activation, proliferation, and polarization.

Project 5: A Phase I safety and immunogenicity study of a HPV16-specific therapeutic protein, TA-CIN, administered intratumorally during chemoradiation, in patients with inoperable HPV16+ cervical cancer

Project Co-Leaders:
Richard Roden, Ph.D (Basic)
T.-C. Wu, M.D., Ph.D. (Basic)
Amanda Fader, M.D. (Clinical)

We have found that chemotherapy and radiation convert the tumor site into an antigen-presenting cell-rich environment, rendering it ideal for the direct injection of a peptide or protein vaccine into the tumor to elicit potent antigen-specific T cell-mediated immune responses. Here, we propose the use of a therapeutic HPV protein vaccine, administered intratumorally, during chemoradiation in patients with inoperable HPV16+ cervical cancer. We have secured the therapeutic HPV protein vaccine TA-CIN from Cancer Research Technology, Ltd. and the adjuvant GPI-0100 from Hawaii Biotech. We have also secured support from the NCI NExT program to formulate and vial clinical grade TA-CIN with GPI-0100. The proposed trial will be performed at the University of Alabama at Birmingham. Specifically we plan to: 1) evaluate the safety and toxicity of TA-CIN with GPI-0100 administered intratumorally in inoperable HPV16+ cervical cancer patients; 2) characterize the HPV-16 E6 and E7 cell-mediated immune responses and L2-specific antibody titers in advanced cervical cancer patients intratumorally vaccinated with TA-CIN/GPI-0100; 3) determine the subset population of immune cells infiltrating the lesion bed, the expression of PD-L1 in the tumor microenvironment and the apoptotic tumor cell death in HPV 16+ advanced cervical cancer patients receiving intratumoral TA-CIN/GPI-0100 vaccination; and 4) characterize the HPV-16 antigen-specific CD8+ T cell-mediated immune responses and therapeutic antitumor effects against E6/E7-expressing tumors in tumor-bearing mice treated with intratumoral vaccination with TA-CIN/GPI-0100, TA-HPV vaccinia or pNGVL4a-hCRTE6E7L2 DNA vaccine in conjunction with cisplatin and/or radiation treatment. The successful implementation of the proposed project will not only improve the treatment of HPV-associated advanced cervical cancer but may also serve as a platform technology for the development of effective therapeutic vaccines for other HPV-associated malignancies including vaginal, vulva, anal, penile and HPV positive oropharyngeal squamous cell carcinoma.

SPECIFIC AIMS:

Our overall hypothesis is that intratumoral TA-CIN/GPI-0100 vaccination combined with chemoradiotherapy will induce HPV antigen-specific immune responses that correlate with histopathological changes in the tumor microenvironment. The current standard of care for advanced cervical cancer includes the chemotherapeutic drug, cisplatin, in conjunction with local radiation therapy. However, as the five-year survival in most patients affected by advanced cervical cancer is approximately 30%, an innovative treatment strategy, such as immunotherapy, that can improve patient outcomes is urgently needed. We have recently discovered that chemoradiation converts the tumor microenvironment into a site permissive for the activation of an adaptive immune response, creating an opportunity for the direct injection of a peptide or protein vaccine into the tumor to elicit potent antigen-specific cell-mediated immune responses. Because cervical cancer is accessible for intratumoral injection and concurrent chemoradiation serves as the standard care for advanced cervical cancer, our finding has direct translational potential. Our results to date indicate that intratumoral injection of the protein-based HPV vaccine TA-CIN, with the adjuvant GPI-0100, generates antitumor effects in mouse models sufficiently promising to initiate a clinical trial in advanced cervical cancer patients in conjunction with concurrent chemoradiation. We have secured support from the NCI NExT program to formulate and vial clinical grade TA-CIN with GPI-0100, a semi-synthetic saponin adjuvant (see Letter from Dr. Yovandich). Here we propose to exploit the temporally permissive tumor microenvironment induced by chemoradiation and the availability of clinical grade TA-CIN/GPI-0100 to potentially enhance E6/E7 antigen-specific immune responses and antitumor effects in inoperable HPV16 associated cervical cancer patients. Our specific aims are as follows:

Specific Aim 1: To evaluate the safety and toxicity of TA-CIN with GPI-0100 administered intratumorally in HPV16+ advanced cervical cancer patients.

A Phase I clinical trial will be conducted in patients with inoperable HPV16+ cervical tumors. Patients will receive chemoradiotherapy and a total of three intratumoral injections of TA-CIN/GPI-0100. The vaccine will be administered in a dose/volume-escalating manner. Cohorts of 7 patients will receive vaccinations beginning at a dose of 0.25 mL per vaccination. Doses will increase to 0.5 mL and 1 mL per vaccination if no toxicities are observed. If no toxicities are observed at the maximum dose, we will expand the cohort to a total of 14 patients.

Specific Aim 2: To characterize the HPV-16 E6 and E7 cell-mediated immune responses and L2-specific antibody titers in advanced cervical cancer patients intratumorally vaccinated with TA-CIN/ GPI-0100.

The E6 and E7-specific CD8+ T cell immune responses will be characterized using quantitative T cell immunological assays including IFN-g ELISPOT assay and intracellular cytokine staining followed by flow cytometry analysis utilizing HPV-16 E7 and E6 overlapping peptides. In addition, in patients with the HLA-A*0201 haplotype, we will also perform E7 and E6-peptide loaded HLA-A2 tetramer staining analysis. Furthermore, the L2-specific humoral immune responses will be characterized by ELISA and in vitro neutralization assays using different types of HPV pseudovirions.

Specific Aim 3: To determine the subset population of immune cells infiltrating the tumor bed, the expression of PD-L1 in the tumor microenvironment and the apoptotic tumor cell death in HPV 16+ advanced cervical cancer patients receiving intratumoral TA-CIN/ GPI-0100 vaccination.

Immunocytochemical staining will be performed to determine the number and distribution of the subset populations of immune cells in the tumor bed. We are particularly interested in examining the presence of CD11b+ MDSCs. We will also characterize the apoptotic tumor cell death in the cervical lesions. The data generated in Aim 2 will be correlated with the apoptotic tumor cell death observed in HPV 16+ advanced cervical cancer patients receiving intratumoral TA-CIN/GPI-0100 vaccination.

Specific Aim 4: To characterize the HPV-16 antigen-specific CD8+ T cell-mediated immune responses and therapeutic antitumor effects against E6/E7-expressing tumors in tumor-bearing mice treated with intratumoral vaccination with TA-CIN/GPI-0100, TA-HPV vaccinia or pNGVL4a-hCRTE6E7L2 DNA vaccine in conjunction with cisplatin and/or radiation treatment.

Mice will be will be inoculated subcutaneously with TC-1 tumor cells and treated with cisplatin and/or radiation in conjunction with TA-CIN/GPI-0100, TA-HPV or pNGVL4a-hCRTE6E7L2 DNA vaccine. The proposed experiments will not only serve to provide support for the use of the alternative therapeutic HPV vaccines in conjunction with conventional chemoradiation in the case that the TA-CIN/GPI-0100 trial encounters any issues (toxicity, etc.), but will also create opportunities for combination regimens with TA-CIN/GPI-0100 vaccine for intratumoral vaccination (such as a prime-boost regimen) to further improve therapeutic effects of conventional chemoradiation therapy.

Core A: Administration and Communication

Core Co-Directors:
T.-C. Wu, M.D., Ph.D.
Warner Huh, M.D.

The Administration/Communication Core (Core A) is responsible for facilitating the coordination and oversight of all Program activities and for disseminating information within the SPORE and for external interactions. The Core is designed for low-cost, yet efficient administration and communication in order to focus funds on research activities. This core includes a basic science director (Dr. T.-C. Wu) and a clinical research director (Dr. Edward Partridge). Dr. Wu is responsible for coordinating basic scientific efforts and the coordination of individual projects. Dr. Partridge oversees patient identification, enrollment, and patient monitoring in the context of the Core. The administrative component of the Core follows an organizational diagram for management activities. Monitoring of research will occur via 1) Research Project Teams, 2) Committee of Research Project Leaders, 3) Core Investigators Committee, 4) Developmental Research Projects Program, 5) Career Development Committee, and 6) the central SPORE Steering Committee. Furthermore, the Internal and External Advisory Boards provide formal evaluations and reports to the Steering Committee. This resource funds a Clinical Research Coordinator who interacts with the other cores as well as with personnel from each individual project to ensure that all patient information, specimens, and results are properly collected and recorded in the computerized database. Core A also funds a patient advocate, who provides invaluable feedback on various SPORE activities from a unique perspective. Appropriate monitoring of patient safety, adverse events, and data management and confidentiality will be provided by the data safety monitoring boards. The communication component of the Core is directed at intra-SPORE, inter-SPORE, and National Cancer Institute research activities. The Core coordinates essential Program interactions including preparing applications and progress reports for the SPORE, all planning and evaluation activities, arranging and publicizing SPORE activities, coordinating advisory committee meetings, producing annual reports and performing analysis of budgetary matters.

Core B: Biostatistics and Data Management Core

Core Co-Directors:
Chenguang Wang, Ph.D.
Sejong Bae, Ph.D.

The primary objective of the Biostatistics/Data Management Core is to contribute to the science and operation of the Cervical SPORE by participating fully in its activities, in addition to providing assistance and direction in experimental design, data systems, quality control and statistical data analysis through consultation and collaboration; to build an infrastructure with the ability to share and manage data at Johns Hopkins University, University of Alabama at Birmingham, University of Colorado at Boulder and now Mount Sinai School of Medicine. The core will provide centralized statistical services as well as collaborative research and data management support for the research projects of the SPORE. The Core will serve as the focal point from which the SPORE investigators and career development candidates can draw statistical expertise for their research projects. The specific aims of the Core include biostatistical consultation and support to all projects in the program, by assisting in the study design, data collection, quantitative modeling, publication, as well as interpretation, visualization and analysis of data arising in the course of program activities. The Core will also provide assistance with the identification and solution of complex database tasks arising in the course of project activities -this includes integration of clinical and basic research databases and interfaces for data entry, data retrieval, patient or sample tracking, and procedures to ensure data quality, integrity, and confidentiality at JHU and UAB. This web-based database will provide a centralized means to produce interim reports of projected progress, patient accrual, processing of specimens, completeness of data gathering, and monitoring of patient drop out or loss to follow-up. The Biostatistics/Data Management Core is led by Chenguang Wang, Ph.D., from Johns Hopkins University and Karan Singh, Ph.D., and Sejong Bae Ph.D. from University of Alabama at Birmingham. The Core is comprised of biostatisticians and supporting personnel from both JHU and UAB. All projects and other cores of the SPORE will be supported by the Biostatistics/Data Management Core.

Core C: Tissue/Pathology and Immunology Core

Core Co-Directors:
Cornelia Trimble, M.D. (Clinical, Basic)
Lawrence Lamb, Ph.D. (Basic)

Project Summary:

The purpose of this shared resource is to collect and distribute clinically annotated human biospecimens related to cervical cancer and HPV disease, and to provide pathologic and immunologic expertise and support for tissue-based analyses for investigators in the Johns Hopkins/University of Alabama/University of Colorado/Mount Sinai Cervical Cancer SPORE. The tissue core has been in existence since 1998 and has expanded with the support of the Cervical Cancer SPORE. To date, we have banked frozen tissue samples from 810 benign gynecologic tissues, 2,220 tumor resections, and 178 resections of preinvasive cervical HPV lesions. Longitudinally obtained exfoliated cell and peripheral blood specimens from 245 subjects enrolled in our CIN2/3 protocols have also been obtained, as well as one-time subject-matched peripheral blood samples for benign tissues (206), and from 632 tumor samples. Formalin fixed blocks are available for virtually all of these specimens. In addition, this shared resource has also banked specimens from patients enrolled in therapeutic vaccination protocols for HPV-associated squamous cancers of the head and neck. All biospecimens are harvested and banked in accordance with the National Cancer Institute’s Best Practice Guidelines for Biorepositories. The core has been expanded to include expert pathologic and immunologic consultation to investigators, including guidance for quantitative digital image analyses-guided molecular studies of tissues. Specimens will be collected under the supervision of pathologists with expertise in gynecologic pathology, in close collaboration with clinical colleagues in these areas. Clinical information for subjects enrolled in our clinical protocols is entered into a password-protected web-based tracking system. This internal web-based system follows the recommendations of the National Research Council, and includes user authentication, encryption, audit trails, and disaster recovery. A review mechanism is in place for prioritization of distribution of requested resources to investigators within and external to the Johns Hopkins Cervical Cancer SPORE. Biosamples have been shared with collaborators at academic institutions across the country.