Phase I/II Trial of Electroporation-Delivered pBI-4 Vaccine (TriGrid™ PVX4) for Treatment of Women With HPV16-Positive CIN1 or Persistent Infection

STUDY OVERVIEW AND TRIAL DESIGN This study is a randomized, double-blind, placebo-controlled, multicenter Phase II clinical trial in Taiwan to evaluate the safety, efficacy, tolerability, and immunogenicity of the pBI-4 therapeutic DNA vaccine administered via the clinical-stage Intramuscular TriGrid™ Delivery System version 2.0 (TDS-IM v2.0) [1-4]. The target study population consists of adult female subjects aged 19 years and older who have biopsy-confirmed Grade 1 cervical intraepithelial neoplasia (CIN1) with concurrent Human Papillomavirus type 16 (HPV16) infection, or who exhibit persistent (defined as 6 months or longer) HPV16 infection with normal cytology (CIN<1) [5-8].

A maximum target of 94 eligible subjects will be enrolled and randomized in a 1:1 ratio into two parallel groups: 47 subjects in the active pBI-4 vaccine group and 47 subjects in the placebo group [1, 4, 9].

THERAPEUTIC VACCINE AND ELECTROPORATION DELIVERY MECHANISM The investigational drug, pBI-4, is a clinical-grade, 6,999 base-pair circular plasmid DNA vaccine [10-12]. It is engineered to express human calreticulin (CRT) linked to codon-optimized HPV16 E6, E7, and L2 residues (amino acids 11-200) [11]. To ensure clinical safety, the gene sequences encoding the HPV16 E6 and E7 proteins incorporate specific mutations that disable their native oncogenic functions [10, 11, 13].

The vaccine (at a dose of 1.0 mg in 1.0 mL volume) or placebo (1.0 mL of phosphate-buffered saline) is administered intramuscularly into the vastus lateralis (thigh) muscle [1, 14, 15]. To overcome the historical limitation of low cellular DNA uptake in humans, the trial utilizes the automated TDS-IM v2.0 delivery system [14, 16-18]. During the automated administration sequence (which takes approximately 10 seconds), the device deploys an injection needle and four surrounding electrodes into the muscle [19]. Following fluid delivery, short, mild electrical pulses are applied to the local tissue [19]. This electroporation process transiently opens pores in cell membranes, enhancing intracellular plasmid DNA uptake and antigen expression by 2 to 3 orders of magnitude compared to standard needle injections [16, 19, 20].

TWO-STAGE ADAPTIVE REGIMEN MODIFICATION The trial employs a two-stage group-sequential design featuring a pre-specified, non-binding adaptive regimen modification [3, 21]. A single interim analysis will be conducted by an independent Data Safety Monitoring Board (DSMB) once approximately 50% of the randomized participants (approximately 42 patients) complete their Month 6 primary assessment [9, 22].

At the interim analysis, the DSMB will calculate the conditional power (CP) based on the primary efficacy endpoint (HPV16 clearance rate) under the design alternative (assuming a 60% clearance rate in the vaccine group versus a 30% clearance rate in the placebo group) [9, 21, 23].

• If the conditional power is calculated to be less than 20% (indicating suboptimal efficacy of the single-dose regimen), the trial will trigger a non-binding futility rule and adaptively switch to a two-dose (prime-boost) regimen for all remaining participants enrolled in Stage 2 [21]. Under this adapted schedule, Stage 2 participants will receive their first dose at Month 0 and a booster dose at Month 2, while maintaining double-blind placebo control and 1:1 randomization [21, 24].
• If the conditional power is 20% or higher, the trial will continue with the original single-dose regimen for the rest of the study [21].

CLINICAL EVALUATIONS AND FOLLOW-UP TIMELINE

All participating subjects will undergo structured clinical assessments across multiple visits:

• Baseline/Screening (Month -1 to 0): Obtaining written informed consent, checking full eligibility, medical and gynecological history, physical examination with ECOG performance status, vital signs, clinical laboratory tests (including complete blood count with differential, serum chemistry, HIV, Hepatitis B and C screenings), urine pregnancy test, Pap smear, Roche Cobas HPV genotyping, and colposcopy with endocervical curettage (ECC) and mandatory tissue biopsy [6, 25-27].
• Vaccination Visit (Month 0 Day 1): Pre-dose physical exam, vital signs, urine pregnancy test, and a research blood draw (serum and peripheral blood mononuclear cells) for baseline HPV16-specific humoral and cell-mediated immune assessments [14, 28-30]. Following the pBI-4 or placebo injection via TDS-IM v2.0, subjects are observed on-site for up to 60 minutes, with vital signs recorded at 30 and 60 minutes post-dose to monitor for potential immediate hypersensitivity or vasovagal reactions [14, 30-32].
• Post-Vaccination Phone Call (Week 1 / Day 8): A telephone interview to record any local reactogenicity, systemic adverse events, and concomitant medications, alongside the administration of the Procedure Tolerability Questionnaire to measure pain (using a 10-point Visual Analog Scale) and device acceptability [29, 31-33].
• Month 6 Follow-Up Visit: Physical exam, vital signs, safety laboratory tests, research blood draw for immunologic response, Pap smear, Roche Cobas HPV genotyping, and colposcopy with ECC (with biopsy performed only if clinically indicated) [29, 34-36].
• Month 12 Follow-Up Visit (Final Evaluation): A replication of the Month 6 visit assessments, representing the completion of the 12-month post-vaccination follow-up period.

If the adaptive two-dose regimen is triggered, Stage 2 participants will undergo an additional vaccination visit at Month 2 Day 1 (with pre-dose safety labs and a pregnancy test) and a subsequent post-vaccination telephone call at Week 9 to document tolerability and safety parameters [24, 40].

Inclusion criteria

• Patients with CIN1 on biopsy and whose cytologic sample is HPV16+ by Roche Cobas test in the past 30 days, OR patients with CIN<1 on biopsy whose cytologic sample is HPV16+ by Roche Cobas test in the past 30 days and a history of a second HPV16+ test at least 6 months prior

• (Co-infections with HPV types other than HPV16 are permissible for study entry)
• Age ≥ 19 years
• Baseline Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 at the time of study treatment administration
• Adequate organ function at the time of enrollment as defined by:

White blood cell count > 3,000/mcL Absolute lymphocyte number > 500/mcL Absolute neutrophil count > 1,000/mcL Platelets > 90,000/mcL Hemoglobin > 9 g/dL Total bilirubin < 3 x institutional limit of normal AST(SGOT)/ALT(SGPT) < 3 x institutional limit of normal Creatinine < 2.5 x institutional limit of normal Women of child-bearing potential must agree to use effective contraception (hormonal and/or barrier) prior to study entry and for the duration of the study

• Ability to understand and willingness to sign a written informed consent document
• Subject is able to adhere to the study visit schedule and other protocol requirements

Exclusion criteria

• Patients with AIS (adenocarcinoma in situ) or cancer determined by cervical cytology at study entry

• Histologic evidence of CIN2 or above
• Patients with a diagnosis of immunosuppression or prolonged, active use of immunosuppressive medications such as steroids
• Prior treatment with any HPV therapeutic agent (e.g., Imiquimod, Veregen, Podophylotoxin)
• Patients who are receiving any other investigational agents or blood products within 30 days prior to the first dose
• Patients with an uncontrolled intercurrent illness including, but not limited to, ongoing or active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance
• Patients with a history of autoimmune disease such as multiple sclerosis (exclusive of a history of thyroiditis, psoriasis, Sjogren's, or inflammatory bowel disease)
• Patients with a history of allergic reactions attributed to compounds used in agent preparation
• Patients who are pregnant or breast feeding
• Patients with active or chronic infection of HIV, HCV, or HBV
• Patients who have had a prior LEEP or cervical conization procedure
• History of prior malignancy (permitted only if the patient has been disease-free for ≥ 5 years, or has completely resected basal cell or squamous cell carcinoma of the skin)
• Less than 1 acceptable potential injection site for intramuscular injection and electroporation (considering the left/right medial deltoid, and anterolateral quadriceps muscles)
• A site is unacceptable if there is inadequate muscle mass to support at least a 19 mm (0.75 inch) injection depth or a skinfold thickness of ≥ 50 mm
• (Note: For participants weighing ≤ 65 kg, acceptable sites are confined to the outer aspect of the upper thigh, and the deltoids are not eligible)
• Contraindication to intramuscular injections and blood draws
• A metal implant or implantable device within the area of the electroporation injection site at > 2 of the eligible injection sites .A nonremovable electronic stimulation device (such as cardiac demand pacemakers, automatic implantable cardiac defibrillators, nerve stimulators, or deep brain stimulators).