Phase I Trial of a Therapeutic DNA Vaccine for Preventing Hepatocellular Carcinoma from Chronic Hepatitis C Virus (HCV) Infection.

Chronic hepatitis C can lead to cirrhosis and hepatocellular carcinoma. We studied the safety and immunogenicity of a novel therapeutic hepatitis C virus (HCV) genotype 1a/1b consensus DNA vaccine, INO-8000, encoding HCV NS3, NS4A, NS4B, and NS5A proteins alone or co-administered with DNA-encoding IL12 (INO-9012), a human cytokine that stimulates cellular immune function, in individuals with chronic hepatitis C. This was a phase I, multisite dose-escalation trial with an expansion cohort evaluating doses of 0, 0.3, 1.0, and 3.0 mg of INO-9012 (IL12 DNA) as an addition to 6.0 mg of (INO-8000; HCV DNA vaccine). Vaccines were administered by intramuscular injection followed by electroporation at study entry and at weeks 4, 12, and 24. HCV-specific CD4+ and CD8+ T-cell immune responses were measured by IFNγ ELISpot and flow cytometry-based assays. Transient, mild-to-moderate injection site reactions unrelated to IL12 DNA dose were common. Increases in HCV-specific IFNγ production occurred in 15/20 (75%) participants. Increases in the frequency of HCV-specific CD4+ and CD8+ T cells occurred at all dose levels, with the greatest increases seen at 1.0 mg of INO-9012. HCV-specific CD8+ and CD4+ T-cell activities increased in 16/18 (89%) and 14/17 (82%) participants with available data, respectively. The vaccine regimen was safe and induced HCV-specific CD4+ and CD8+ cellular immune responses of modest magnitude in most HCV-infected participants. The addition of 1.0 mg of IL12 DNA provided the best enhancement of immune responses. The vaccine regimen had little effect on controlling HCV viremia. PREVENTION RELEVANCE: The administration of IL12 DNA along with a hepatitis C viral antigen DNA vaccine enhanced the HCV-specific immune responses induced by the vaccine in individuals with chronic hepatitis C, an important cause of hepatocellular carcinoma. IL12 could be an effective adjuvant in vaccines targeting HCV and other oncogenic viruses.

Phase 1 study of safety, tolerability and immunogenicity of the human telomerase (hTERT)-encoded DNA plasmids INO-1400 and INO-1401 with or without IL-12 DNA plasmid INO-9012 in adult patients with solid tumors.

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is frequently classified as a 'universal' tumor associated antigen due to its expression in a vast number of cancers. We evaluated plasmid DNA-encoded hTERT as an immunotherapy across nine cancer types. METHODS: A phase 1 clinical trial was conducted in adult patients with no evidence of disease following definitive surgery and standard therapy, who were at high risk of relapse. Plasmid DNA encoding one of two hTERT variants (INO-1400 or INO-1401) with or without plasmid DNA encoding interleukin 12 (IL-12) (INO-9012) was delivered intramuscularly concurrent with the application of the CELLECTRA constant-current electroporation device 4 times across 12 weeks. Safety assessments and immune monitoring against native (germline, non-mutated, non-plasmid matched) hTERT antigen were performed. The largest cohort of patients enrolled had pancreatic cancer, allowing for additional targeted assessments for this tumor type. RESULTS: Of the 93 enrolled patients who received at least one dose, 88 had at least one adverse event; the majority were grade 1 or 2, related to injection site. At 18 months, 54.8% (51/93) patients were disease-free, with median disease-free survival (DFS) not reached by end of study. For patients with pancreatic cancer, the median DFS was 9 months, with 41.4% of these patients remaining disease-free at 18 months. hTERT immunotherapy induced a de novo cellular immune response or enhanced pre-existing cellular responses to native hTERT in 96% (88/92) of patients with various cancer types. Treatment with INO-1400/INO-1401±INO-9012 drove hTERT-specific IFN-γ production, generated hTERT-specific CD4+ and CD8+ T cells expressing the activation marker CD38, and induced hTERT-specific activated CD8 +CTLs as defined by cells expressing perforin and granzymes. The addition of plasmid IL-12 adjuvant elicited higher magnitudes of cellular responses including IFN-γ production, activated CD4+ and CD8+ T cells, and activated CD8+CTLs. In a subset analysis of pancreatic cancer patients, the presence of immunotherapy-induced activated CD8+ T cells expressing PD-1, granzymes and perforin correlated with survival. CONCLUSIONS: Plasmid DNA-encoded hTERT/IL-12 DNA immunotherapy was well-tolerated, immune responses were noted across all tumor types, and a specific CD8+ phenotype increased by the immunotherapy was significantly correlated with survival in patients with pancreatic cancer.

Immune Therapy Targeting E6/E7 Oncogenes of Human Paillomavirus Type 6 (HPV-6) Reduces or Eliminates the Need for Surgical Intervention in the Treatment of HPV-6 Associated Recurrent Respiratory Papillomatosis.

: Background: Recurrent respiratory papillomatosis (RRP) is a rare disorder characterized by the generation of papillomas of the aerodigestive tract, usually associated with human papilloma virus (HPV) subtypes 6, 11. INO-3106 is a DNA plasmid-based immunotherapy targeting E6 and E7 proteins of HPV6, in order to create a robust immune T cell response. METHODS: Testing of INO-3016 in animal models confirmed immunogenicity of the DNA-based therapy. A single-site open-label Phase 1 study was initiated for patients with HPV6-positive RRP. Patients were dosed with INO-3106 with or without INO-9012, a DNA plasmid immunotherapy that encodes IL-12, delivered intramuscularly (IM) in combination with electroporation (EP) with the CELLECTRA® device. Patients received an escalating dose of INO-3106, 3 mg once and then 6 mg for three additional doses, each dose three weeks apart, with the third and fourth doses co-administered with INO-9012. 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 cellular immune responses to INO-3106 with and without INO-9012. Exploratory objectives included preliminary clinical efficacy to the therapy. RESULTS: Three patients were enrolled in this study, of which two had RRP. Study therapy was well-tolerated, with no related serious adverse events and all related adverse events (AEs) were low-grade. Injection site pain was the most common related AE reported. Immunogenicity was evidenced by multiple immune assays showing engagement and expansion of an HPV6-specific cellular response, including cytotoxic T cells. Preliminary efficacy was demonstrated in patients with RRP in the form of reduction in need for surgical intervention for papilloma growth. Prior to intervention, both patients required surgical intervention approximately every 180 days. One patient demonstrated a greater than three-fold increase in surgery avoidance (584 days) and the other patient remains completely surgery-free as of the last contact at 915 days, a greater than 5-fold increase in surgery interval. CONCLUSION: INO-3106 with and without INO-9012 was well tolerated, immunogenic and demonstrated preliminary efficacy in patients with HPV6-associated RRP aerodigestive lesions. Further clinical study is indicated.

Intradermal SynCon® Ebola GP DNA Vaccine Is Temperature Stable and Safely Demonstrates Cellular and Humoral Immunogenicity Advantages in Healthy Volunteers.

BACKGROUND: Nonlive vaccine approaches that are simple to deliver and stable at room temperature or 2-8°C could be advantageous in controlling future Ebola virus (EBOV) outbreaks. Using an immunopotent DNA vaccine that generates protection from lethal EBOV challenge in small animals and nonhuman primates, we performed a clinical study to evaluate both intramuscular (IM) and novel intradermal (ID) DNA delivery. METHODS: Two DNA vaccine candidates (INO-4201 and INO-4202) targeting the EBOV glycoprotein (GP) were evaluated for safety, tolerability, and immunogenicity in a phase 1 clinical trial. The candidates were evaluated alone, together, or in combination with plasmid-encoded human cytokine interleukin-12 followed by in vivo electroporation using either the CELLECTRA® IM or ID delivery devices. RESULTS: The safety profile of all 5 regimens was shown to be benign, with the ID route being better tolerated. Antibodies to EBOV GP were generated by all 5 regimens with the fastest and steepest rise observed in the ID group. Cellular immune responses were generated with every regimen. CONCLUSIONS: ID delivery of INO-4201 was well tolerated and resulted in 100% seroreactivity after 2 doses and elicited interferon-γ T-cell responses in over 70% of subjects, providing a new approach for EBOV prevention in diverse populations. Clinical Trials Registration. NCT02464670.

Immunotherapy Targeting HPV16/18 Generates Potent Immune Responses in HPV-Associated Head and Neck Cancer.

PURPOSE: Clinical responses with programmed death (PD-1) receptor-directed antibodies occur in about 20% of patients with advanced head and neck squamous cell cancer (HNSCCa). Viral neoantigens, such as the E6/E7 proteins of HPV16/18, are attractive targets for therapeutic immunization and offer an immune activation strategy that may be complementary to PD-1 inhibition. PATIENTS AND METHODS: We report phase Ib/II safety, tolerability, and immunogenicity results of immunotherapy with MEDI0457 (DNA immunotherapy targeting HPV16/18 E6/E7 with IL12 encoding plasmids) delivered by electroporation with CELLECTRA constant current device. Twenty-two patients with locally advanced, p16+ HNSCCa received MEDI0457. RESULTS: MEDI0457 was associated with mild injection site reactions, but no treatment-related grade 3-5 adverse events (AE) were noted. Eighteen of 21 evaluable patients showed elevated antigen-specific T-cell activity by IFNγ ELISpot, and persistent cellular responses surpassing 100 spot-forming units (SFUs)/106 peripheral blood mononuclear cells (PBMCs) were noted out to 1 year. Induction of HPV-specific CD8+ T cells was observed. MEDI0457 shifted the CD8+/FoxP3+ ratio in 4 of 5 post immunotherapy tumor samples and increased the number of perforin+ immune infiltrates in all 5 patients. One patient developed metastatic disease and was treated with anti-PD-1 therapy with a rapid and durable complete response. Flow-cytometric analyses revealed induction of HPV16-specific PD-1+ CD8+ T cells that were not found prior to MEDI0547 (0% vs. 1.8%). CONCLUSIONS: These data demonstrate that MEDI0457 can generate durable HPV16/18 antigen-specific peripheral and tumor immune responses. This approach may be used as a complementary strategy to PD-1/PD-L1 inhibition in HPV-associated HNSCCa to improve therapeutic outcomes.

Clinical and Immunologic Biomarkers for Histologic Regression of High-Grade Cervical Dysplasia and Clearance of HPV16 and HPV18 after Immunotherapy.

Purpose: As previously reported, treatment of high-grade cervical dysplasia with VGX-3100 resulted in complete histopathologic regression (CR) concomitant with elimination of HPV16/18 infection in 40.0% of VGX-3100-treated patients compared with only 14.3% in placebo recipients in a randomized phase IIb study. Here, we identify clinical and immunologic characteristics that either predicted or correlated with therapeutic benefit from VGX-3100 to identify parameters that might guide clinical decision-making for this disease.Experimental Design: We analyzed samples taken from cervical swabs, whole blood, and tissue biopsies/resections to determine correlates and predictors of treatment success.Results: At study entry, the presence of preexisting immunosuppressive factors such as FoxP3 and PD-L1 in cervical lesions showed no association with treatment outcome. The combination of HPV typing and cervical cytology following dosing was predictive for both histologic regression and elimination of detectable virus at the efficacy assessment 22 weeks later (negative predictive value 94%). Patients treated with VGX-3100 who had lesion regression had a statistically significant >2-fold increase in CD137+perforin+CD8+ T cells specific for the HPV genotype causing disease. Increases in cervical mucosal CD137+ and CD103+ infiltrates were observed only in treated patients. Perforin+ cell infiltrates were significantly increased >2-fold in cervical tissue only in treated patients who had histologic CR.Conclusions: Quantitative measures associated with an effector immune response to VGX-3100 antigens were associated with lesion regression. Consequently, these analyses indicate that certain immunologic responses associate with successful resolution of HPV-induced premalignancy, with particular emphasis on the upregulation of perforin in the immunotherapy-induced immune response. Clin Cancer Res; 24(2); 276-94. ©2017 AACR.

Augmentation of cellular and humoral immune responses to HPV16 and HPV18 E6 and E7 antigens by VGX-3100.

We have previously demonstrated the immunogenicity of VGX-3100, a multicomponent DNA immunotherapy for the treatment of Human Papillomavirus (HPV)16/18-positive CIN2/3 in a phase 1 clinical trial. Here, we report on the ability to boost immune responses with an additional dose of VGX-3100. Patients completing our initial phase 1 trial were offered enrollment into a follow on trial consisting of a single boost dose of VGX-3100. Data show both cellular and humoral immune responses could be augmented above pre-boost levels, including the induction of interferon (IFN)γ production, tumor necrosis factor (TNF)α production, CD8+ T cell activation and the synthesis of lytic proteins. Moreover, observation of antigen-specific regulation of immune-related gene transcripts suggests the induction of a proinflammatory response following the boost. Analysis of T cell receptor (TCR) sequencing suggests the localization of putative HPV-specific T cell clones to the cervical mucosa, which underscores the putative mechanism of action of lesion regression and HPV16/18 elimination noted in our double-blind placebo-controlled phase 2B trial. Taken together, these data indicate that VGX-3100 drives the induction of robust cellular and humoral immune responses that can be augmented by a fourth "booster" dose. These data could be important in the scope of increasing the clinical efficacy rate of VGX-3100.

Safety, efficacy, and immunogenicity of VGX-3100, a therapeutic synthetic DNA vaccine targeting human papillomavirus 16 and 18 E6 and E7 proteins for cervical intraepithelial neoplasia 2/3: a randomised, double-blind, placebo-controlled phase 2b trial.

BACKGROUND: Despite preventive vaccines for oncogenic human papillomaviruses (HPVs), cervical intraepithelial neoplasia (CIN) is common, and current treatments are ablative and can lead to long-term reproductive morbidity. We assessed whether VGX-3100, synthetic plasmids targeting HPV-16 and HPV-18 E6 and E7 proteins, delivered by electroporation, would cause histopathological regression in women with CIN2/3. METHODS: Efficacy, safety, and immunogenicity of VGX-3100 were assessed in CIN2/3 associated with HPV-16 and HPV-18, in a randomised, double-blind, placebo-controlled phase 2b study. Patients from 36 academic and private gynaecology practices in seven countries were randomised (3:1) to receive 6 mg VGX-3100 or placebo (1 mL), given intramuscularly at 0, 4, and 12 weeks. Randomisation was stratified by age (<25 vs ≥25 years) and CIN2 versus CIN3 by computer-generated allocation sequence (block size 4). Funder and site personnel, participants, and pathologists were masked to treatment. The primary efficacy endpoint was regression to CIN1 or normal pathology 36 weeks after the first dose. Per-protocol and modified intention-to-treat analyses were based on patients receiving three doses without protocol violations, and on patients receiving at least one dose, respectively. The safety population included all patients who received at least one dose. The trial is registered at ClinicalTrials.gov (number NCT01304524) and EudraCT (number 2012-001334-33). FINDINGS: Between Oct 19, 2011, and July 30, 2013, 167 patients received either VGX-3100 (n=125) or placebo (n=42). In the per-protocol analysis 53 (49·5%) of 107 VGX-3100 recipients and 11 (30·6%) of 36 placebo recipients had histopathological regression (percentage point difference 19·0 [95% CI 1·4-36·6]; p=0·034). In the modified intention-to-treat analysis 55 (48·2%) of 114 VGX-3100 recipients and 12 (30·0%) of 40 placebo recipients had histopathological regression (percentage point difference 18·2 [95% CI 1·3-34·4]; p=0·034). Injection-site reactions occurred in most patients, but only erythema was significantly more common in the VGX-3100 group (98/125, 78·4%) than in the placebo group (24/42, 57·1%; percentage point difference 21·3 [95% CI 5·3-37·8]; p=0·007). INTERPRETATION: VGX-3100 is the first therapeutic vaccine to show efficacy against CIN2/3 associated with HPV-16 and HPV-18. VGX-3100 could present a non-surgical therapeutic option for CIN2/3, changing the treatment outlook for this common disease. FUNDING: Inovio Pharmaceuticals.

Genome-wide profiling of oral squamous cell carcinoma by array-based comparative genomic hybridization.

OBJECTIVES: Array-based comparative genomic hybridization (aCGH) was used to develop a genome-wide molecular profile of oral squamous cell carcinoma (OSCC). Copy number alterations (CNAs) were identified by chromosomal region, mapped to specific genes, and compared with several previously documented CNAs associated with head and neck squamous cell carcinoma (HNSCC). The status of 512 commonly altered cancer genes was assessed and evaluated as potential correlates of tumor behavior. METHODS: Tumor tissue DNA was isolated for aCGH from 21 prospectively collected fresh-frozen OSCC specimens. aCGH was performed at 0.9-Mb resolution to identify distinct regions of genomic alteration and their associated genes. Cancer genes commonly altered were then correlated with clinicopathologic tumor data. RESULTS: Genomic regions most frequently amplified (>35%) were located on 3q, 5p, 8q, 9q, and 20q, although regions most frequently deleted (>40%) involved chromosomes 3p, 8p, 13q, and 18q. Minimal regions of CNA identified, by aCGH narrowed larger, previously documented CNAs associated with HNSCC to significantly smaller regions, yielding shorter lists of candidate genes. Cancer-related genes altered in greater than 25% OSCC samples were identified (22 amplified, 17 deleted). Several genes associated with the Fanconi anemia DNA-damage response pathway were frequently altered, including BRCA1, BRCA2, FANCD2, and FANCG. Other cancer-related genes linked to hereditary cancer syndromes include VHL, MLH1, XPC, and RB1. CONCLUSIONS: Genome-wide aCGH can be used to detect and map CNAs in OSCC tissue specimens with high resolution. These data implicate several candidate genes and gene pathways in the tumorigenesis of sporadic OSCC.

Microarray analysis of mouse ear tissue exposed to bis-(2-chloroethyl) sulfide: gene expression profiles correlate with treatment efficacy and an established clinical endpoint.

Bis-(2-chloroethyl) sulfide (sulfur mustard; SM) is a potent alkylating agent. Three treatment compounds have been shown to limit SM damage in the mouse ear vesicant model: dimercaprol, octyl homovanillamide, and indomethacin. Microarrays were used to determine gene expression profiles of biopsies taken from mouse ears after exposure to SM in the presence or absence of treatment compounds. Mouse ears were topically exposed to SM alone or were pretreated for 15 min with a treatment compound and then exposed to SM. Ear tissue was harvested 24 h after exposure for ear weight determination, the endpoint used to evaluate treatment compound efficacy. RNA extracted from the tissues was used to generate microarray probes for gene expression profiling of therapeutic responses. Principal component analysis of the gene expression data revealed partitioning of the samples based on treatment compound and SM exposure. Patterns of gene responses to the treatment compounds were indicative of exposure condition and were phenotypically anchored to ear weight. Pretreatment with indomethacin, the least effective treatment compound, produced ear weights close to those treated with SM alone. Ear weights from animals pretreated with dimercaprol or octyl homovanillamide were more closely associated with exposure to vehicle alone. Correlation coefficients between gene expression level and ear weight revealed genes involved in mediating responses to both SM exposure and treatment compounds. These data provide a basis for elucidating the mechanisms of response to SM and drug treatment and also provide a basis for developing strategies to accelerate development of effective SM medical countermeasures.

Genomic analysis of rodent pulmonary tissue following bis-(2-chloroethyl) sulfide exposure.

Bis-(2-chloroethyl) sulfide (sulfur mustard, SM) is a carcinogenic alkylating agent that has been utilized as a chemical warfare agent. To understand the mechanism of SM-induced lung injury, we analyzed global changes in gene expression in a rat lung SM exposure model. Rats were injected in the femoral vein with liquid SM, which circulates directly to the pulmonary vein and then to the lung. Rats were exposed to 1, 3, or 6 mg/kg of SM, and lungs were harvested at 0.5, 1, 3, 6, and 24 h postinjection. Three biological replicates were used for each time point and dose tested. RNA was extracted from the lungs and used as the starting material for the probing of replicate oligonucleotide microarrays. The gene expression data were analyzed using principal component analysis and two-way analysis of variance to identify the genes most significantly changed across time and dose. These genes were ranked by p value and categorized based on molecular function and biological process. Computer-based data mining algorithms revealed several biological processes affected by SM exposure, including protein catabolism, apoptosis, and glycolysis. Several genes that are significantly upregulated in a dose-dependent fashion have been reported as p53 responsive genes, suggesting that cell cycle regulation and p53 activation are involved in the response to SM exposure in the lung. Thus, SM exposure induces transcriptional changes that reveal the cellular response to this potent alkylating agent.