Safety and efficacy of mucosal immunotherapy using human papillomavirus (HPV) type 16 E7-expressing Lactobacillus-based vaccine for the treatment of high-grade squamous intraepithelial lesion (HSIL): the study protocol of a randomized placebo-controlled clinical trial (MILACLE study)

Abstract

We developed an HPV16 E7-expressing Lactobacillus-based therapeutic vaccine, IGMKK16E7, to elicit mucosal E7-specific TH1 cellular immune responses. This study aims to examine the safety and clinical efficacy of IGMKK16E7 on HPV16-positive high-grade squamous intraepithelial lesion (HSIL). This is a multicenter, placebo-controlled, double-blind randomized phase I/II trial to test the safety and efficacy of IGMKK16E7 against HPV16-positive HSIL. The groups will include placebo, low-dose (0.5 g/day), middle-dose (1 g/day), and high-dose (1.5 g/day) IGMKK16E7. The target sample size will be 41 patients per group, and our data on our former agent, GLBL101c, were used to calculate sample size for 70% power and an α level = 0.05. The primary endpoint is IGMKK16E7 safety and pathological regression at week 16, and the secondary endpoints are cytological regression and HPV16 E7 immunological response. This study protocol has been approved by the Japanese Pharmaceuticals and Medical Devices Agency. Patient enrollment will begin in May 2019.

phase I/II clinical trial, design paper, high-grade squamous intraepithelial lesion (HSIL), mucosal immunity, immunotherapy

Background and rationale

High-risk human papillomavirus (HR-HPV) causes cervical cancer and precursor high-grade squamous intraepithelial lesions (HSILs; cervical intraepithelial neoplasia 2–3: CIN2-3). In particular, HPV type 16 (HPV16) is the most common genotype associated with HPV-dependent cancers including cervical, anal, penile, vaginal, vulvar and oropharyngeal cancers, since HPV16 possesses the highest oncogenic features among HR-HPV genotypes (1). However, not all HPV16-infected women experience progression to cancer. Immunological clearance of SILs/CIN is well-reported as the explanation of spontaneous regression (2). Host TH1 cellular immunity against HPV-positive cells involves the clearance of CIN lesions (3). In turn, cellular immunosuppressive status, such as HIV infection and long-term steroid use, are risk factors for progression from CIN to cancer.

Anti-HPV TH1 immune responses are thought to be induced at the cervical mucosa in some HPV-infected women. Numerous studies have reported a natural history of SIL/CIN (4,5). In Japan, Matsumoto et al. (6) reported that the spontaneous regression rate is approximately 70% in CIN1 patients and 50–60% in CIN2 patients within five years after their diagnosis based on a cohort study. Although it is ethically difficult to conduct long-term observational cohort studies of CIN3, several placebo-controlled clinical trials of some therapeutics for HSIL/CIN2-3 have revealed spontaneous regression of CIN3, as shown by placebo group data (7–9).

Considering such rationale, induction of TH1 cellular immunity against HPV by vaccination is a promising strategy for the development of SIL/CIN therapeutics. Many therapeutic vaccine agents are developed and clinical trials have been conducted (3,10,11). A recent phase IIb clinical trial of VGX-3100, a DNA vaccine that can express HPV16/18 E6 and E7, showed clinical efficacy (CIN regression and HPV clearance) for HSIL/CIN2-3 (9), and the development of VGX-3100 may be ongoing. Although many other agents are used in phase I, I/IIa, and IIb clinical trials to treat HSIL/CIN2-3, no efficacious agent has been launched worldwide.

We have developed an immunotherapeutic, HPV16 E7-expressing Lactobacillus casei (L. casei), GLBL101c, and the phase I/IIa clinical trial of GLBL101c has been completed (12). All previous immunotherapy agents for HSIL/CIN2-3 utilize systemic immune responses by intramuscular, subcutaneous or local vaccination of agents. Our strategy provides mucosal immune responses by oral vaccination, because HSIL/CIN2-3 develops in the mucosal epithelium of the cervix, and the mucosal TH1 immune response against the HPV antigen is critical for immunological clearance of the disease within the mucosa (11,12). L. casei is not normal flora in the gut but an immunogenic microbe for humans and has shown an adjuvant effect on TH1 immune responses (13). In our phase I/IIa clinical trial of GLBL-101c, regression from HPV16-related CIN3 to CIN2 was observed in 80% of patients within 9 weeks after the first dose of GLBL101c, and regression from CIN3 to normal was observed in 38% of patients within one year after vaccination (12). The clinical efficacy correlated with local E7-specific TH1 immune response in the cervical lymphocytes. This proof of concept (POC) study demonstrated that oral administration of HPV16 E7-expressing L. casei elicited mucosal TH1 cellular immune responses to HPV E7 at the CIN3 lesion and led to immunological clearance of CIN3.

We have recently developed a next-generation Lactobacillus-based E7-expressing agent, IGMKK16E7, that can elicit 4-fold higher mucosal TH1 cellular immune response to E7 in mice than GLBL101c can (14). IGMKK16E7 is composed of another L. casei strain expressing the full-length HPV16 E7 protein fused to an anchor that transfers the fusion protein onto the cell surface. Our previous data revealed that the number of surface-expressing E7 molecules correlates with number of E7-specific TH1 cells in the intestinal mucosa, and IGMKK16E7 expresses E7 molecules on the cell surface with higher confluence than GLBL101c does.

The aim of this clinical trial is to examine safety and efficacy of IGMKK16E7 for the treatment of HPV16-positive HSIL/CIN2-3, and we will be conducting a phase I/II clinical trial. To do so, we here proposed a study protocol of this clinical trial.

Protocol digest of the MILACLE study

This phase I/II study protocol has been approved by Japanese Pharmaceutics and Medical Devices Agency (PMDA).

Purpose

The purpose of this study is to evaluate the safety of orally administered IGMKK16E7 to patients with HSIL/CIN2-3, to determine the recommended human dose to obtain the optimal efficacy, and to examine the relationship between HPV E7 expression levels and the efficacy of IGMKK16E7.

Study setting

The title of the phase I/II study is mucosal immunotherapy using an HPV-targeting Lactobacillus-based vaccine, IGMKK16E7, for the treatment of HPV16-related, high-grade squamous intraepithelial lesions: the MILACLE study. Condition of this study is high-grade squamous intraepithelial lesions (HSIL/CIN2-3). The study design is a placebo-controlled, parallel, individually randomized, double-blinded study.

Endpoints

Primary outcomes are the safety and efficacy of IGMKK16E7. Adverse events will be evaluated using Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. The efficacy of IGMKK16E7 will be evaluated as follows.

i) Pathological efficacy at 16 weeks after treatment initiation, evaluated by following criteria:

• Complete response (CR): no abnormal findings

• Partial response (PR): regression to CIN1

• Stable disease (SD): no change from CIN2 or CIN3 or regression from HSIL/CIN3 to HSIL/CIN2

• Progression of disease (PD): progression from CIN2 to CIN3 or CIN2/3 to carcinoma

• Clinical benefit is defined as CR + PR

Secondary outcomes are as follows:

• Pathological efficacy at 24 weeks after treatment initiation, evaluated by following criteria:

• Complete response (CR): no abnormal findings

• Partial response (PR): regression to CIN1

• Stable disease (SD): no change from CIN2 or CIN3 or regression from HSIL/CIN3 to HSIL/CIN2

• Progression of disease (PD): progression from CIN2 to CIN3 or carcinoma

• Clinical benefit is defined as CR + PR

• TH1 immune responses against HPV16 E7 at 16 and/or 24 weeks after treatment initiation

• Cytological efficacy at 16 and 24 weeks after treatment initiation, classified by following criteria

• Better: regression from HSIL to low-grade SIL (LSIL), ASC-US or negative for intraepithelial lesion or malignancy (NILM)

• Stable: no change from HSIL

• Worse: progression into squamous cell carcinoma (SCC)

Eligibility criteria

Healthy females aged 20–45 years old who are diagnosed with HSIL/CIN2 or HSIL/CIN3 by histological examination and identified as HPV16 alone or HPV16 with other types infection by genotyping. At eligibility step, each institute use HPV-DNA typing of commercial-base assay. In addition, we confirm pre- and after-HPV types of all enrolled patients by ‘in-house’ HPV typing assay (PGMY method) in the central laboratory (Nihon University). Therefore, we can detect change of HPV types by oral administration of IGMKK16E7 by standardized assay in this study. All the enrolled patients are required to provide written consent.

Exclusion criteria

• Immunocompromised host or a patient who received immunosuppressive therapy

• Patients with SCC on cervical cytology

• Patients willing to be treated by either laser vaporization or conization

• Patients suffering from acute disease

• Patients who have a previous history of hypersensitivity to Lactobacillus-content food/drug or to milk

• Pregnant women or patients who expected to get pregnant

• Breastfeeding mother

• History of medication against CIN within 4 weeks prior to study enrollment

• History of enrollment in the GLBL101c study

• History of pancreatitis

• All patients who are considered nonappropriate for this study by a medical doctor

Pancreatitis is a disease that induce inflammation and produce inflammatory cytokines followed by SIRS. Our immunotherapy might be influenced by these inflammatory immune responses caused by pancreatitis. Therefore, we included pancreatitis as an exclusion criterion.

Randomization

Dynamic allocation will be carried out by CIN grade (CIN2 or CIN3) and HPV genome (HPV16 alone or HPV16 + other types).

Treatment methods

Patients will be randomly divided into four groups as described, and then low-dose, middle-dose, high-dose of IGMKK16E7 or placebo will be administered daily under double-blinded conditions. All patients will receive four rounds of oral vaccination at weeks 1, 2, 4, and 8. Each dose of IGMKK16E7 or placebo will be administered orally after fasting once each morning for five days during each treatment week.

IGMKK16E7 is an attenuated L. casei bacteria displaying mutated HPV16 E7 molecule on the cell surface with optimal efficiency, which is higher than that of GLBL101c, the formerly tested treatment agent, and we hypothesize that IGMKK16E7 will elicit E7-specific TH1 mucosal immunity (14). In this study, the low-, middle-, and high-doses of IGMKK16E7 will be 0.5, 1.0 and 1.5 g/day of E7-expressing L. casei, respectively.

Follow-up

Patients will be followed-up during weeks 5, 9 and 16. At week 16, pathological and cytological regression will be evaluated as a secondary endpoint. We set an endpoint at week 16 for safety. In general, patients with CIN3 undergo surgical treatment within a few months after their diagnosis. On the other hand, immunotherapy sometimes show the delayed clinical responses after induction of immune responses and thereby the efficacy of the immunotherapy should be evaluated as late as possible. Then, we decided that the pathological evaluation as an endpoint will be done at week 16 for safety and follow-up will be acceptable until week 24 if the lesion will be less than HSIL/CIN2 at week 16. If the patients will be diagnosed as CIN3 or more at week 16, she must receive standard therapy. Patients who exhibit a regression to HSIL/CIN2 or LSIL/CIN1 at week 16 will be followed-up until week 24, since it is well-known that immunotherapy sometimes has a ‘delayed’ clinical response. Indeed, our previous study on GLBL101c also showed a CR in some patients after week 16 (12). Therefore, we will follow-up patients with less than HSIL/CIN2 until week 24.

Immunological evaluation

For the evaluation of immunological response, peripheral blood mononuclear cells (PBMCs) will be collected in BD vacutainer tubes (BD bioscience, San Jose, CA). The immunological response will be evaluated as described previously (12). In brief, using an HPV16 E7 overlapping peptides set, interferon-γ (IFNγ) will be examined.

Target sample size and statistical methods

Our previous study showed that 1.0 g/day of GLBL101c was an optimal dose for the treatment of CIN3 with 38% CR within one year (12). On the other hand, approximately 10% of CIN3 has shown spontaneous regression within 6 to 9 months in the placebo group (7–9). Therefore, we hypothesize that the expected response rates of the low-, middle-, high-doses of IGMKK16E7 and will be approximately 20%, 30%, 40%, and 10%, respectively, within the follow-up period. The linear trends among the study arms will be assessed by the Cochran Armitage trend test, and the target sample size was calculated as 31 patients per group to provide 80% power at an α level of 0.05. Adding 10 patients for each as a margin, 41 patients for each group will be enrolled. Besides determining the linearity, the dose-response relationship will also be assessed to select the most efficient dose.

Interim analysis and monitoring

We will not conduct an interim analysis for the clinical efficacy of IGMKK16E7. However, the safety of IGMKK16E7 will be independently evaluated by the Safety Monitoring Committee at the time point when 80 patients have been enrolled or when any severe adverse event associated with this agent occurs.

The endpoint data will be independently reviewed by the academic research organization, CReSCent, at the University of Tokyo and by a Clinical Research Support Center.

Acknowledgments

We thank to American Journal Expert for grammatical edition.

Funding

The MILACLE study is funded by the Practice Research for Innovative Cancer Control from the Japan Agency for Medical Research and Development (AMED: No. 18ck12R2-202-004) and by GLOVACC Inc.

Conflict of interest statement

GLOVACC Inc. gifted IGMKK16E7 and placebo and partially supported this clinical trial.

Clinical trials registry

Trial registration number: UMIN000034253, jRCT2031190034

Participating institutes

This study will take place across multiple institutions, including Nihon University Itabashi Hospital, Keio University Hospital, Kyushu University Hospital, and Tsukuba University Hospital. Central pathological review will occur at Osaka University Graduate School of Medicine. The central laboratory for HPV typing and E7 expression level will be at Nihon University School of Medicine.