Human papilloma virus associated (HPV+) malignancies are an emerging global epidemic [1
]. HPV associated aerodigestive precancerous lesions and malignancies may occur in the oropharynx, larynx, and upper respiratory tract. While the role of HPV6 in the etiology of a majority of aerodigestive malignancies remains unclear, its role is widely accepted as being causally implicated in recurrent respiratory papillomatosis (RRP) [2
], the most common benign tumor of the laryngeal epithelium. RRP is rare, with an incidence rate estimated at 1.8 per 100,000 adults in the United States [5
]. Although most lesions are benign, some undergo malignant transformation, and patients with RRP have a higher risk of developing laryngeal neoplasias and carcinomas [6
The clinical course of RRP can vary widely amongst affected individuals. Selection of treatment, including active monitoring without treatment, surgery, radiation therapy, or a combination, depends on a number of factors. Usually, repeated surgical removal of papillomas for symptomatic management remains the mainstay of treatment [7
]. In a few cases, malignant transformation may occur, which is usually associated with a dismal prognosis. A few such patients with malignant disease may be candidates for salvage therapies, including potentially definitive surgery [8
]. Selected patients in this setting may benefit from radiation although the morbidity of this approach is substantial [9
Current treatment of HPV6-related RRP and invasive malignant diseases could potentially be improved with the addition of HPV-specific immunotherapy. Available preventive HPV vaccines can generate neutralizing antibodies against the HPV major capsid protein L1, but they have not demonstrated therapeutic effects on HPV infection or existing lesions and are unlikely to engender a cytolytic T-cell response [10
]. HPV-specific immunotherapy, on the other hand, may have therapeutic potential to eliminate preexisting lesions and infections by generating immunity against the HPV virus itself and HPV infected cells. HPV E6 and E7 oncoproteins represent ideal targets for this type of therapeutic intervention because of their constitutive expression in HPV associated tumors and their crucial role in the induction and maintenance of HPV associated diseases [11
]. To that end, our previous clinical studies of HPV-specific immunotherapy engendering T cell responses have shown to translate in clinical benefit for woman exhibiting advanced dysplastic cervical lesions as well as patients with HPV associated squamous cell carcinoma of the head and neck [11
In this study, we evaluated INO-3106, a novel HPV6-specific immunotherapy consisting of synthetic consensus DNA sequences encoding for HPV6 E6 and E7 (Figure 1
), proteins necessary for HPV6 induced transformation of cancers and tumor maintenance. Synthetic DNA plasmids offer several potential advantages as an immunotherapy platform, including the ability to elicit potent immune responses without evidence of genome integration, a favorable safety profile, stability and relative ease in manufacturing [13
]. HPV16/18-specific therapy (VGX-3100, Inovio Pharmaceuticals, Inc.) designed and evaluated based on the same synthetic consensus platform, has demonstrated cellular immune responses that correlated with clinical benefit in the form of dysplastic lesion regression and elimination of HPV16/18 infection in a large, double-blind, randomized placebo-controlled Phase 2b study and now support Phase 3 clinical trials targeting HPV16 and 18 associated diseases [14
Preclinical studies have shown that the immunogenicity of DNA vaccines can be substantially increased by the use of cytokine adjuvants [15
]. Importantly, an engineered plasmid IL-12 genetic adjuvant has been shown to enhance immunogenicity in humans when delivered using the CELLECTRA®
]. We have established in multiple clinical studies targeting both skin delivery as well as local muscle that delivery of optimized DNA via the CELLECTRA®
device is a highly reproducible method of generating immunity in human beings in a rapid fashion for various purposes ranging from induction prophylactic settings as well as therapeutic approaches [20
Here, we present data suggesting that plasmid-encoded HPV6 E6 and E7 antigens are immunogenic in mice when delivered intramuscularly using the CELLECTRA® device and that this immune response is primarily driven by CD8+ T cells. These data supported the initiation of a clinical trial of this plasmid (INO-3106) and we additionally report on the safety and immunogenicity of a pilot study of INO-3106 with or without INO-9012 (IL-12 gene adjuvant) delivered intramuscularly (IM) via EP with the CELLECTRA® device in patients with HPV6 associated RRP. The data from this study suggest that immunotherapy with INO-3106 and IL-12 gene adjuvant could be a non-invasive immune mediated treatment option for patients with RRP and further clinical studies are warranted.
3. Clinical Study
3.1. Clinical Study Population
This was a prospective, open-label, Phase 1 study conducted at the Abramson Cancer Center at the University of Pennsylvania. Male and female patients at least 18 years old were considered for enrollment. To be eligible, patients must have histologically documented HPV6-associated aerodigestive papilloma, premalignant lesion or have aerodigestive invasive malignancy with most recent therapy (e.g., radiation, chemotherapy) completed at least two months prior to first dose of study treatment. Patients must have ECOG 0-1, with adequate liver, renal, hepatic and bone marrow function. Patients were excluded if there was evidence of immunosuppression or anticipated use of immunosuppressive agents, required use of systemic steroids, presence of cardiac pre-excitation syndromes, or were pregnant or breast-feeding. Written informed consent was obtained from each patient prior to performing any assessments. The clinical trial was conducted according to the ethical guidelines of the Declaration of Helsinki and was approved by the University of Pennsylvania IRB.
3.2. Immunotherapy and Electroporation Using CELLECTRA® Device
INO-3106 is a DNA plasmid encoding for the E6 and E7 proteins of HPV type 6, formulated in sterile water for injection. INO-9012 consists of a DNA plasmid encoding for synthetic human IL-12 (p35 and p40 subunits) also formulated in sterile water for injection. Both INO-3106 and INO-9012 were designed using proprietary technology (Inovio Pharmaceuticals, Inc.) as described previously [27
]. The CELLECTRA®
2000 adaptive constant current electroporation device (Inovio Pharmaceuticals, Inc.) delivers three 52 millisecond controlled electric pulses, spaced in 1 s intervals, through a sterile, disposable array to the injection site. When inserted into tissue, the needle array centers around the site of immunotherapy injection and creates transient pores within the cell membrane to enhance cell transfection. INO-3106 with or without INO-9012 was delivered intramuscularly in a 1 mL volume followed immediately by EP with the CELLECTRA®
device. Treatment or dose is defined as injection of DNA plasmids followed by EP.
3.3. Study Design
Following informed consent, each patient was assigned a unique patient identification code. Screening procedures to determine eligibility and collect baseline characteristics were completed within 28 days prior to first dose. Patients received escalating doses of INO-3106, of which the first dose (Day 0) delivered 3 mg of INO-3106, the second dose (Week 3) delivered 6 mg of INO-3106, and the third (Week 6) and fourth (Week 9) doses delivered 6 mg of INO-3106 with 1 mg of INO-9012. Each dose was delivered three weeks apart to allow for observation of development of any grade 2 or higher related systemic adverse events (AEs). In total, participation for all patients included a 9-week treatment period followed by a 6 month long term follow-up period from the last dose.
The primary objective of the study was to evaluate the safety and tolerability of INO-3106 with and without INO-9012. The secondary objective was to determine the humoral and cellular immune responses to INO-3106 with and without INO-9012, and the exploratory objective was to assess preliminary clinical efficacy to the treatment, as well as to associate efficacy with immune cell infiltration in post-dose tissue, if possible.
The study was registered on ClinicalTrials.gov with the identifier NCT02241369. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki and was reviewed and approved by the center’s Institutional Review Board.
3.4. Safety Assessments
Local and systemic adverse events (AEs), vital signs, 12 lead electrocardiograms (ECGs) and the development of laboratory abnormalities were monitored from the date of informed consent through the last follow-up visit. In particular, injection site reactions, including pain, itching, erythema, induration and bruising were assessed on the day of each treatment and for 7 consecutive days post-treatment. Patients were queried at each visit regarding the occurrence of new AEs or disease and use of concomitant medications. All events were graded in accordance with the Common Terminology Criteria for Adverse Events (CTCAE), version 4.03 and coded with MedDRA version 21. Laboratory parameters including hematology, coagulation, serum chemistry (including liver function) and creatine phosphokinase (CPK), were monitored throughout the study and assessed locally at the center.
Further enrollment and treatment was to be halted if one third or more patients experienced a related event requiring expedited reporting: any patient experienced a serious adverse event (SAE), unexpected grade 4 toxicity, potentially life-threatening AE or death assessed as related to study treatment; three or more patients experienced the same related grade 3 or 4 AE; or if any patient reported a grade 3 anaphylaxis.
3.5. HPV6 Specific ELISA
A standardized binding ELISA was performed to measure IgG antibodies from patient’s sera against HPV6 E7 protein. Briefly, plates were coated with 1 ug/mL HPV6 E7 in PBS overnight at 4 °C. The following day, plates were washed and blocked with 3% BSA. After 2h incubation at room temperature, plates were washed again and patient serum samples were added in a 3-fold dilution series from 1:25 to 1:18,225. Each dilution was tested in triplicate. Samples were incubated for 2h at room temperature, followed by a washing step and 1h incubation with detection antibody goat anti-human IgG-HRP at 1:5000. Finally, plates were washed and developed using TMB substrate followed by TMB stop solution. Optical densities (OD) were read on a kinetic microplate reader. Antibody titers were determined and positivity was considered if the average OD of a sample post vaccination was greater than the average OD at baseline plus 2.5 times SD of OD at baseline at the corresponding dilution. Number “1” instead of “0” is used to indicate negative results.
3.6. HPV6 Specific Flow Cytometry
PBMCs were recovered after cryopreservation overnight in cell culture medium and spun, washed and re-suspended the following day. After counting, 1 × 106 PBMCs were plated into a 96-well plate in R10 medium from patients with sufficient sample. For antigen specific responses, cells were stimulated 5 days with a combination of peptides corresponding to HPV6 E6 and E7 that had been pooled at a concentration of 2 µg/mL, while an irrelevant peptide was used as a negative control (OVA) and concanavalin A was used as a positive control (Sigma-Aldrich). No co-stimulatory antibodies or cytokines were added to cell cultures at any point. At the end of the 5 day incubation period, cells were stained for CD3-BUV737, CD4-APC-Cy7, CD14-BUV395, CD-16-BUV395, CD137-APC, Granulysin-AF488 CD-19-BUV395, CD38-BV786, CD8-BV650, granzyme B-AF700 (BD Biosciences), Granzyme A-PECy7 (ThermoFisher), PD-1-PEDazzle, perforin-BV421, Ki67-BV605 and CD69-BV711 (BioLegend). Staining for extracellular markers (CD4, CD8, CD137, CD69, CD38, PD-1) occurred first, followed by permeabilization to stain for the remaining markers. CD3 was stained intracellularly to account for downregulation of the marker following cellular activation. Acquired data were analyzed using the FlowJo software version X.0.7 or later (Tree Star).
3.7. HPV6 Specific PBMC Stimulation for Gene Expression Analysis
For short term stimulation, Cryopreserved PBMCs were thawed, rested overnight, and stimulated for 22 h at 37 °C, 5% CO2 and 95% humidity with either DMSO (negative control) or HPV6 E6 and E7 overlapping peptide pools (OLPs). Following stimulation, culture supernatants were collected and stored at −20 °C. Cells were then lysed using Buffer RLT (Qiagen) and stored at −80 °C.
For long term stimulation, Cryopreserved PBMCs were thawed, rested overnight, and stimulated at 37 °C, 5% CO2 and 95% humidity for 11 days with HPV6 E6 and E7 OLPs. On days 1, 4, 6 and 8, fresh media containing IL-2 and IL-7 was added at 10 U/mL and 10 ng/mL, respectively. On day 11, PBMCs were washed and rested overnight at 37 °C, 5% CO2 and 95% humidity. Following overnight rest, PBMCs were re-stimulated with HPV6 E6 and E7 OLPs for 22 h with either DMSO (negative control) or HPV6 E6 and E7 OLPs. At the end of the 22 h stimulation, cell supernatants were collected and stored at −20 °C. Cells were then lysed and stored at −80 °C.
3.8. Multiplexed Gene Expression Analysis
Cell lysates were thawed in batches of 12 as per manufacturer instructions and hybridized to capture probes and fluorophore-barcoded reporter probes using the nCounter (NanoString) GX Human Immunology V2 panel, which consists of 594 genes plus 15 internal reference controls. Samples were then placed in the automated nCounter Prep Station (Nanostring) for hybridization of capture probes to a translucent cartridge, after which gene expression was measured by the nCounter Digital Analyzer (Nanostring) via direct counts of reporter probes in each sample lane.
3.9. Statistical Methods
Regarding the CD8+ mediated cellular immune response induced by INO-3106 in C57BL/6 mice, the frequencies of HPV6 E6 and E7-specific IFN-γ spot forming units (SFU) per million determined by ELISpot assay total splenocytes were compared to CD8 depleted splenocytes using the Wilcoxon signed-rank test.
Here, we report that a plasmid encoding HPV6 E6 and E7 antigens (INO-3106) delivered by the CELLECTRA® device was immunogenic in preclinical models, allowing for the support of a clinical trial for HPV6 associated recurrent respiratory papillomatosis. The resulting Phase 1 clinical trial of INO-3016 with and without IL-12 DNA adjuvant administered intramuscularly and delivered via electroporation by the CELLECTRA® device treated two patients with HPV6 associated recurrent respiratory papillomatosis. Administration of the immunotherapy was well-tolerated. There were no treatment-related SAEs and the most frequent treatment-emergent AEs were injection site reactions. All patients showed induction of cellular responses to the HPV6 E6 and E7 antigens as demonstrated by at least one immunological assessment. Notably, both evaluable RRP patients derived clinical benefit from treatment with INO-3106, mainly in the form of delayed treatment intervention (e.g., surgery) relative to their pre-study surgery frequencies. Moreover, the fact that the patient who exhibited more robust cellular activity after INO-3106 treatment remains surgery free while the patient with less robust cellular activity delayed but did not completely avoid surgery suggests a possible causal relationship between the induction of an HPV6-specific cellular response and the type/duration of clinical benefit. With the understanding that the evaluation of two patients does not sufficiently power a study to reject a null hypothesis, these results are nonetheless encouraging and present the idea that additional dosing to continue to boost the cellular response may be preferable in this treatment setting. Such a hypothesis requires further testing in order to validate, however.
Treatment with INO-3106 resulted in the induction of HPV6-specific cellular responses across a variety of immunoassays. The confirmation of production of IFN-γ using ELISpot as well as the confirmation of expression of activation markers concomitant with synthesis of granzyme and perforin on CD8+ T cells via flow cytometry suggests that INO-3106 drove the induction of a proinflammatory immune response that included T cells with hallmarks of highly activated cytotoxic lymphocytes. These results are further underscored by the observation of dynamic regulation of pro-inflammatory as well as regulatory gene transcripts in PBMCs after completion of treatment. Specifically, we observed increased expression of Granzyme B and TNFRSF9/CD137 transcripts, confirming the activity of cytotoxic lymphocytes on the transcriptomic level. The necessity for a T cell response of this nature in combating HPV-driven disease has been exemplified in two of our previous clinical trials for DNA-based immunotherapy, both of which were delivered using the CELLECTRA®
device. In the context of HPV-associated cervical dysplasia in a large, double-blind, placebo-controlled Phase 2b trial, clinical response to treatment with VGX-3100 (DNA immunotherapy for targeting E6/E7 of HPV16/18) in the form of regression of lesions concomitant with elimination of HPV infection was statistically associated with the presence of a robust cellular response that included IFN-γ and CD8+ T cells exhibiting phenotypic markers of cytoxicity [25
]. Additionally, in another trial investigating treatment of HPV-associated squamous cell cancer of the oropharynx, a patient with metastatic cancer who achieved a complete response to treatment with nivolumab after treatment with MEDI0457 (VGX-3100 + INO-9012) was noted as having a therapy-driven robust expansion of PD1+
cytotoxic T cells [27
]. Thus, the current study provides further evidence that HPV-specific immunotherapies delivered by the CELLECTRA®
device induce the generation of potent T cell responses that have the potential to clinically impact HPV-associated tumorigenesis.
The data collected from this study are the first to suggest that an HPV specific immunotherapy may be able to impact the clinical status of patients with HPV6 associated recurrent respiratory papillomatosis and act as an additional or alternative adjuvant therapy. These findings are complementary to data presented earlier this year, where administration of pembrolizumab was associated with a reduced need for routine surgical interventions [34
]. Together, these findings offer early data to support the use of immunotherapeutic approaches in the management of these patients. The current standard of care for treatment for this disease is repeated surgical intervention, which presents a number of complications and is unlikely to completely eradicate lesion recurrence as latent virus may reside in adjacent tissue [34
]. Other non-surgical adjuvant interventions are indicated in patients with rapid regrowth of lesions or aggressive disease, but such therapies also carry inherent risks and require further evaluation to determine optimal treatment regimens [7
]. Current treatment limitations highlight the need to identify non-invasive and immune mediated approaches to treating patients with HPV positive areodigestive disease. Indeed, preventive HPV vaccines have been reported to reduce papilloma growth and extend time between interventions, however, determination of therapeutic efficacy requires continued evaluation [33
]. Similarly, PD-1/PD-L1 inhibition represents a rational approach to treating RRP, but expression and impact on clinical outcome is less characterized [34
Our study has a few important limitations that should be noted. First, the small size of the study precludes our ability to formally test a number of hypotheses. The small size of this study is likely due to the limited enrollment criteria, i.e. this was limited to patients over the age of 18 years and therefore could not capture a population of patients with Juvenile RRP, a condition that is perhaps slightly more common than in the adult population. Additionally, this was a single center study, with a small sample size, and therefore was limited in its catchment: the study was designed as a small Phase 1P study, wherein individual patients, specifically those with more severe RRP disease with inevitable continued resections and potential malignant transformation, are treated with investigational products (i.e., INO-3106 and INO-9012) otherwise not obtainable.