Immunotherapy Targeting HPV 16/18 Generates Potent Immune Responses in HPV- Associated Head and Neck Cancer

Author Manuscript Published OnlineFirst on September 21, 2018; DOI: 10.1158/1078-0432.CCR-18-1763 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Immunotherapy targeting HPV 16/18 generates potent immune responses in HPV- Associated Head and Neck Cancer

Charu Aggarwal1, Roger B. Cohen1, Matthew P. Morrow2, Kimberly A. Kraynyak2, Albert J. Sylvester2, Dawson M. Knoblock2, Joshua M. Bauml1, Gregory S. Weinstein3, Alexander Lin4, Jean Boyer2, Lindsay Sakata2, Sophie Tan2, Aubrey Anton2, Kelsie Dickerson2, Drishty Mangrolia2, Russell Vang5, Michael Dallas2, Sandra Oyola2, Susan Duff2, Mark Esser6, Rakesh Kumar6, David Weiner7, Ildiko Csiki2 and Mark L. Bagarazzi2

1 Division of Hematology-Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 2 Inovio Pharmaceuticals, Inc. Plymouth Meeting, PA, USA 3 Department of Otorhinolaryngology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 4 Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 5 Johns Hopkins University, Baltimore, MD, USA 6 MedImmune, Gaithersburg, MD, USA 7 Wistar Institute, Philadelphia, PA, USA

Running Title: DNA immunotherapy in HPV-associated Head and Neck Cancer

Keywords: Immunotherapy, HPV, Head and Neck Cancer, Combination immunotherapy, Oropharyngeal carcinoma

Word Count: 5662

Number of Tables: 1

Number of Figures: 5

Corresponding Author: Charu Aggarwal, MD, MPH, Assistant Professor, Department of Medicine, Hematology-Oncology Division, University of Pennsylvania, 10-137, South Pavilion, 3400 Civic Center Boulevard, Philadelphia, PA 19104. Email: [email protected]. Phone 215-662-6318, Fax: 215-349-5326

Conflict of Interest Disclosures: Charu Aggarwal reported consulting or advisory roles with Genentech, Bristol-Myers Squibb, Lilly, and Celgene; and institutional research funding from Genetech/Roche, Incyte, Macrogenics, and Merck Sharp & Dohme. Roger B. Cohen reported honoraria from Bristol-Myers Squibb; a consulting or advisory role with Heat Biologics, Takeda, Cerulean Pharma, Kolltan Pharmacueticals, Zymeworks, and Pfizer; institutional research funding from Heat Biologics, Macrogenetics, Merck, Takeda, Cleave Biosciences, and Celldex; and travel, accommodations, or expenses from Heat Biologics, Takeda, Kolltan Pharmaceuticals, Cerulean Pharma, Zymeworks, Bristol-Meyers Squibb, and Pfizer. J.M. Bauml reported consulting or advisory roles with Clovis Oncology, Bristol-Myers Squibb, Merck, AstraZeneca, Genentech, Celgene, Boehringer Ingelheim, and Guardant Health; and institutional research funding from Merck, Carevive Systems, Novartis, Incyte, Bayer, and Janssen. GS Weinstein and

Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 21, 2018; DOI: 10.1158/1078-0432.CCR-18-1763 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Alexander Lin have no relevant disclosures. Russell Vang has no relevant disclosures. David B. Weiner received a SRA research grant from Inovio Pharmaceuticals, has received speakers honoraria from Inovio Pharmaceuticals, GeneOne, Astrazeneca, BMGF, he has ownership interest (including patents) in Inovio Pharmaceuticals and is a consultant/advisory board member for Inovio Pharmaceuticals, a consultant for GeneOne and Astrazeneca and Merck. All authors whose listed association is Inovio Pharmaceuticals (MPM, KAK, AJS, DMK, JB, LS, ST, AA, KD, DM, MD, SO, SD, IC, MLB) and Medimmune (ME, RK) are employees of that entity, own stock or stock options relative to that entity and may own one or more patents relating to the drugs being described in this publication.

Funding: Supported by NCI P30 Cancer Center Support Grant # 5-P30-CA-016520-38

Abstract

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.

Experimental Design: We report Phase Ib/II safety, tolerability and immunogenicity results of immunotherapy with MEDI0457 (DNA immunotherapy targeting HPV16/18 E6/E7 with IL-12 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 (AEs). Eighteen of 21 evaluable patients showed elevated antigen specific T cell activity by IFN ELISpot and persistent cellular responses surpassing 100 SFU/106 PBMC were noted out to one year. Induction of HPV-specific CD8+ T cells was observed. MEDI0457 shifted the CD8+/FoxP3+ ratio in 4/5 post-immunotherapy tumor samples and increased the number of perforin+ immune infiltrates in all five 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.

Statement of translational relevance

Human Papilloma Virus (HPV) associated Head and Neck Cancer (HNSCCa) is an emerging global epidemic, where despite the availability of highly curative treatments, some patients will eventually develop recurrent and/or metastatic disease. The availability of checkpoint inhibitors

for metastatic HNSCCa has changed the outcomes for this disease but durable benefit and survival gains occur only in a subset of patients (~15-20%). Tumor HPV status does not seem to be a principal driver of outcomes with PD-1 directed therapies. An approach targeting HPV- specific “non-self” antigens to activate the immune system represents a potential mechanism to improve outcomes. This clinical trial demonstrates that a DNA immunotherapeutic agent targeting HPV 16/18 E6/E7 is safe, with promising antigen-specific immune activation in patients with HNSCCa. Our findings suggest that HPV viral neoantigens can be therapeutically targeted as a complementary immune strategy to PD-1/PD-L1 inhibition in HPV-associated HNSCCa to improve patient outcomes.

Introduction

Squamous cell carcinoma of the head and neck (HNSCCa) is diagnosed in over 500,000 patients worldwide each year, accounting for 5% of all malignancies (1). The immune system plays an important role in head and neck carcinogenesis. Even though HNSCCa in general are among the most highly immune-infiltrated cancer types, certain subsets of HNSCCa are characterized by an immunosuppressive environment, marked by T cell dysfunction, low levels of CD4+ and CD8+ T cells, increased T-regulatory cells (Tregs), cytokine alterations and antigen presentation defects (2-4). Oncogenic human papilloma virus (HPV) infection accounts for a significant number of HNSCCa, most of which are related to the HPV 16 subtype (5, 6). HPV-associated HNSCCa may be particularly dependent on aberrant immune checkpoints that create an immune- privileged site for HPV infection and function as an adaptive resistance mechanism of tumor against host (7, 8).

The availability of checkpoint inhibitors for metastatic HNSCCa has changed outcomes for this disease (9-12) but durable benefit and survival gains occur only in a subset of patients (~15- 20%). Tumor HPV status does not seem to be a principal driver of outcome with this new therapy (13, 14).

Combination strategies may increase the percentage of patients that respond to PD-1 immunotherapy (15, 16). One such potential approach is the addition of HPV-specific immunotherapy, targeting HPV-specific “non-self” antigens to activate the immune system to

recognize the cancer. The HPV E6 and E7 oncoproteins represent ideal targets for an immunotherapeutic agent because of their constitutive expression in HPV-associated tumors and their crucial role in the induction and maintenance of HPV-associated disease (17). HPV-specific immunotherapymay eliminate preexisting lesions and infections by generating cellular immunity against HPV infected cells (18-21). Generation of E6 and E7 specific cellular responses has been demonstrated with the administration of VGX-3100 (Inovio Pharmaceuticals), a novel plasmid based immunotherapy that targets E6/E7 using the SynCon® optimization process to generate synthetic HPV 16/18 E6 and E7 DNA sequences (22, 23). In a pilot study of 18 patients, with previously treated high grade CIN, three doses of VGX-3100 delivered intramuscularly (IM) followed by electroporation with the CELLECTRA® (Inovio Pharmaceuticals) constant current device induced HPV-specific CD8+ T cells that exhibited full cytolytic functionality (22). In a follow-up placebo-controlled, blinded randomized clinical trial of VGX-3100 conducted in 167 patients with HPV16/18-related CIN 2/3, histopathological regression and clearance of HPV16/18 was seen in ~50% of patients that received VGX-3100 alone (n=125) (22, 23). CELLECTRA® delivers three 52 ms controlled electrical pulses in three different orientations directly at the plasmid injection site resulting in significantly enhanced transfection, leading to an increase in overall expression of antigen resulting in more robust immunogenicity than synthetic DNA alone (24-26).

We hypothesized that a cellular immune response similar to that demonstrated in women with HPV-associated CIN would be beneficial by increasing effector T cells and potentially overcoming immune evasion commonly seen in in patients with HPV-associated HNSCCa. We designed a proof of concept study to evaluate the safety and immune effects of MEDI0457 (formerly INO-3112), a combination of synthetic plasmids targeting HPV-16 and HPV-18 E6/E7 antigens (VGX-3100) and a recombinant Interleukin-12 (IL-12) encoding molecular adjuvant (INO-9012), delivered by electroporation with the CELLECTRA® device. One of the major drawbacks of DNA vaccines has been a reduced level of immunogenicity in humans, and addition of a plasmid cytokine adjuvant has been shown to enhance induction of cellular immune responses (27-29). IL-12 is a cytokine that promotes the maturation and function of T cells (30), in pre-clinical models, the inclusion of the IL-12 plasmid as an adjuvant improved the magnitude, quality and breadth of the antigen-specific cellular immune responses (31). Based on these observations, INO-9012, a dual promoter expression plasmid expressing the genes

encoding human IL-12 proteins p35 and p40 was added. . The trial was conducted in two distinct patient populations with locally advanced HPV-associated HNSCCa: patients in Cohort I received immunotherapy before and after definitive surgery while patients in Cohort II received immunotherapy after completion of concurrent chemoradiation. Cohort I was designed as an immunological proof of concept study with unique access to paired tumor samples for analyses of tissue immune responses. Cohort II was designed to determine whether patients could mount an immune response to the antigens encoded by MEDI0457 after cisplatin-based concurrent chemoradiation therapy.

Methods

Patients and Procedures

This was a prospective, single center, open-label, Phase I/IIa study (NCT02163057), Figure 1. This study was conducted in accordance with the ethical guidelines outlined in the Declaration of Helsinki, and was approved by the University of Pennsylvania’s Institutional Review Board. Adult patients with histologically confirmed locally advanced p16+ HNSCCa, with adequate end organ function, ECOG PS of 0-1 were enrolled. Patients must have had a diagnostic surgical core biopsy prior to enrollment. Patients on immunosuppressive medications, including > 10 mg of prednisone/ day, active Hepatitis B, C or HIV and history of active cardiac pre-excitation syndromes were excluded. All patients provided written informed consent. Primary endpoints were safety and tolerability; secondary endpoints were cellular and humoral immune responses. MEDI0457 was administered by IM injection followed immediately by electroporation with the CELLECTRA® device. In Cohort I, patients were seen every 3 weeks before surgery for evaluation and treatment. Patients could receive 1 or 2 immunotherapy doses prior to surgery determined by timing of surgery. Surgery was not delayed to allow for immunotherapy dosing. About 4 weeks post-surgery, if adequate healing was documented by the surgeon, the patient resumed immunotherapy dosing every 3 weeks, for a total of 4 doses. Patients then went on to receive standard adjuvant therapy (if indicated) based on pathologic features. In Cohort II, patients started immunotherapy 2 months after completion of chemoradiation in order to allow recovery from the acute side effects of combined modality therapy. After completion of

immunotherapy, patients in both cohorts were seen every 3 months for total of 6 months for safety follow up and measurement of immune responses. Adverse events were recorded and graded according to NCI Common Terminology Criteria for Adverse Events (CTCAE) v 4.0.

Peripheral correlative analyses

Peripheral blood mononuclear cells (PBMCs) were collected in ACD-A tubes and cells were isolated within 24 hours of draw. Serum was collected in red top tubes. Samples were collected at baseline, at the time of immunotherapy dosing, and at each follow up visit (Figure 1) and cryopreserved for immune analyses in batches. T-cell and antibody responses to HPV16 and HPV18 E6 and E7 were determined by interferon-γ ELISpot, and ELISA as described previously (22). For flow cytometry, PBMCs were recovered after cryopreservation overnight in cell culture medium and spun, washed and re-suspended the following day. After counting, 1 X 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 HPV16 E6 and E7 or HPV18 E6 and E7 that had been pooled at a concentration of 2g/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- APCH7, CD4-PerCPCy5.5, CD14-Pacific Blue, CD-16 Pacific Blue, CD137-APC, PD1-PECy7, Granulysin-FITC (BD Biosciences), CD38-QDot705 and CD69-QDot800 (ThermoFisher) CD8- BV605 and Granzyme A-AF700 (BioLegend), CD-19 Pacific Blue, granzyme B-PETR (Invitrogen) and perforin-PE (Abcam). Staining for extracellular markers (CD4, CD8, CD137, CD69, CD38, PD-1) occurred first, followed by permeablization to stain for the remaining markers. CD3 was stained intracellularly to account for downregulation of the marker following cellular activation. Prepared cells were acquired using an LSR II flow cytometer equipped with BD FACSDiva software (BD Biosciences). Acquired data were analyzed using the FlowJo software version X.0.7 or later (Tree Star).

ELISpot assays were performed using separate E6 and E7 peptide pools using a 12 hour stimulation period. Antigenic pools were divided up as follows: HPV16 E6 peptides only, HPV16 E7 peptides only and HPV18 E6 plus E7 peptides pooled into a single stimulation. This latter stimulation was done as a method of preserving available cells for use in other assays.

Results are graphed as individual responses to the combined response of HPV16 E6+HPV16 E7 or the response to the HPV18 E6+E7 peptides. Each patient’s sample received the HPV16 E6 peptide pool as its own triplicate, HPV16 E7 as its own triplicate and HPV18 E6+E7 as a single triplicate. Results are represented as mean SFU/106 PBMC generated from the triplicate media control wells subtracted from mean SFU/106 PBMC generated from HPV antigen triplicate wells. Since there were 2 cohorts in the study (Figure 1), that could vary in their dosing schedule (Cohort I dosing was contingent on the timing of surgery), patients’ results were graphed based on dose and not by study week. CEF (CMV, Epstein Barr, Flu) peptides were included as a control for immune competence during the course of the study; responses to these antigens occur through natural infection/vaccination and are not linked to responses to HPV antigens.

Tissue correlative analyses

As the surgical center was not adjacent to the tissue analytical labs, we were not able to isolate fresh tissue from resection specimens that would survive transport to another location for viable tumor infiltrating lymphocyte (TIL) extraction, isolation and analysis. In place of fresh TIL isolation, tissue was formalin fixed and embedded in paraffin for analysis by immunohistochemistry (IHC). All IHC assays employed a polymer/multimer based secondary detection system. For each IHC staining run, regardless of which platform was used, positive control tissues were included that were treated with the primary antibody and a Buffer Negative control in which the primary antibody was omitted. Samples were initially allocated first for staining CD8 (Dako clone C8/144B, Cat# M7103) and Foxp3 (Ebio clone 236/E7, Cat# 14- 4777). Remaining tissue was then allocated for staining perforin (Abcam clone dG9, Cat# ab194807) and PD-L1 (VENTATA PD-L1 SP263 assay). Whole slide image capture was performed by Histologix (Biocity Nottingham, UK) at x20 magnification with a Hamamatsu Nanozoomer 1.0-HT digital slide scanner. Normal neoplastic regions of interest (ROI) were digitally annotated, where present, onto each section image by the study pathologist. Quantitative image analysis of IHC staining within the annotated ROI was performed by OracleBio (Biocity Scotland, UK) using Definiens Tissue Studio software. An analysis algorithm was developed to detect positive cellular staining across each tissue image. Within the algorithm, image colours were initially separated into respective stain components, e.g. brown and blue. Cells were defined and generated based on the presence of a blue (hematoxylin) stained nucleus. A

threshold level based on identified positive and negative staining in control tissues was then applied to the positive brown color intensity parameter within each cell, above which a cell was defined as positive. The number or area of positive and negative stained cells was then quantified within specific regions of interest across each tissue.

Positive PD-L1 status was assigned to cases with total percentage of tumor cells with membrane staining of any intensity of greater than or equal to 25%. Tissue obtained from the diagnostic biopsy was used to determine HPV 16 or HPV18 positivity via in situ hybridization (ISH) employing proprietary probe sets specific for HPV16 or HPV18 manufactured by DAKO (DAKO Agilent Pathology Solutions, Santa Clara, CA), with a reflex to Roche Linear Array (RLA) if hybridization was not positive for either HPV oncotype. Tissue samples from Cohort I were additionally confirmed for HPV oncotype using RNAscope for HPV16 and HPV18 probes (Advanced Cell Diagnostics, Newark, CA).

Statistical Analysis

The primary analysis was to estimate safety. Toxicities were recorded, graded and summarized. Subjects who received the assigned number of doses were included in the secondary immunogenicity and survival analyses. Changes in immune parameters following immunotherapy including immunoglobulinG (IgG) responses measured by ELISA, number of antigen-specific IFN-γ– secreting cells in response to stimulation with E6 and E7 pools measured by IFN-γ ELISpot assay and induction of antigen specific CD8+ T cell populations expressing granzyme A, granzyme B and perforin were analyzed with Wilcoxon signed-rank tests. In exploratory analyses, disease-free survival (DFS) and overall survival (OS) were measured from the time of administration of the initial immunotherapy dose.

Results

Baseline Characteristics and Safety

Between May 2014 and August 2016, 27 patients were screened, and 22 were enrolled and treated: 6 in Cohort I and 16 in Cohort II. Each patient received 4 total doses of MEDI0457. Demographics are summarized in Table 1. In keeping with the global epidemiology of HPV-

associated HNSCCa, patients on this study were predominantly male with median age 57.5 years, and about half were never smokers. HPV genotyping was available for all patients. The majority of the patients had HPV 16 (n=19, 86%); 3 patients had other genotypes (HPV 26, 33 and 35). MEDI0457 was well-tolerated. All adverse events regardless of attribution are detailed in Supplementary Table 1. There were no Grade 3 or higher related AEs. The most common related AEs were injection site pain (all grade 1, 21 events), which were evenly distributed amongst cohorts I and II. All patients are alive with median follow up of 15.9 months; 3 patients developed progressive disease, and 12 month DFS rate was 89.4%.

MEDI0457 induces strong, long-lived antibody responses

Generation of antibodies to both the E6 and E7 antigens of HPV16 and HPV18 was observed (Fig 2A) following MEDI0457 treatment. Peak seroreactivity was highest against the HPV18 E7 antigen (88.2% of subjects) followed by the HPV16 E7 antigen (64.7%). Across both cohorts, 100% of patients showed seroreactivity to at least one antigen. HPV16 E6 and HPV18 E6 antigens had lower seroreactivity compared to E7 antigens (Supplementary Table 2), which is consistent with previous studies of VGX-3100. Antibody titers could be detected against at least one of the four HPV antigens 3 months after the last dose of immunotherapy, indicating that administration of MEDI0457 could induce antibodies that persisted for at least 6 months after the start of immunotherapy. The longest persistence of seroreactivity against all four antigens in a single patient was noted 9 months following the final dose. Persistent reactivity against any antigen was noted in a single patient 23 months following the final dose of MEDI0457, when the patient was discharged from the study.

MEDI0457 induces robust HPV-specific Interferon Gamma production from T cells

Assessment of cellular immune responses induced by MEDI0457 was done by performing an overnight IFN ELISpot without the addition of supportive cytokines on directly isolated peripheral blood mononuclear cells (PBMC) obtained prior to and following MEDI0457 dosing. Cellular reactivity against HPV16 and HPV18 antigens across the study population prior to dosing was generally low (<25 SFU/106 PBMC against all four HPV antigens) with the

exception of two patients in Cohort I (patient 9 and patient 24) who had minimal baseline reactivity against HPV16 (45.8 and 43.3 SFU/106 PBMC, Fig 2B). After immunotherapy, both cohorts displayed peak ELISpot responses that exceeded pre-dose levels against both HPV16 and HPV18 antigens (Fig 2B). Specifically, patients in Cohort I developed median peak increases above baseline for HPV16 and HPV18 antigens of 63 and 75 SFU/106 PBMC, while those in Cohort II exhibited median increases of 75 and 55 SFU/106 PBMC, respectively (Fig 2C). As HPV16-associated HNSCCa constituted the majority of the study population and no patients with HPV18 genotype associated malignancy were enrolled, we focused on ELISpot analysis of responses against individual HPV16 antigens. The HPV16 E6 and E7 antigens were similarly immunogenic, as shown by a comparison of peak median ELISpot responses. Cohort I showed median increases above baseline for HPV16 E6 antigen of 30 SFU/106 PBMC and 10 SFU/106 PBMC for HPV16 E7 (Fig 2D). Similarly, Cohort II showed median increases above baseline of 32 SFU/106 PBMC for HPV16 E6 and 20 SFU/106 PBMC for HPV16 E7 (Fig 2D). Importantly, 8 of 21 patients showed a peak response of ≥100 SFU/106 PBMC against HPV16 E6 or E7 suggesting robust HPV-specific cellular immune response. Responses appeared to be diminished when assayed three months after the completion of dosing in both cohorts, but remained above baseline for most patients (Fig 2E) and persistent long-term reactivity against any antigen was noted in one patient 23 months following the final treatment, when the patient completed the study.

MEDI0457 induces antigen specific cytotoxic T cells (CTLs)

We and others have shown previously that the production of Interferon gamma is suggestive of a Th1 immune response but does not correlate 1:1 with lytic activity(23, 24, 32-34). It is generally accepted that a cytolytic response by CD8+ T cells is likely to be of importance for control and elimination of neoplastic cells. We therefore performed flow cytometry on PBMCs from patients with sufficient sample isolated prior to the first dose and following the last dose of MEDI0457 to assay the ability of HPV16 and HPV18 specific CD8+ T cells to load granzymes and perforin in response to treatment (Total n = 8, n=2 from Cohort I, n=6 from Cohort 2). To that end, we analyzed the CD8+ T cell compartment for immune activation via antigen-specific expression of cell surface markers such as CD38, CD69 and CD137 as well as for lytic potential

as determined by the presence of granzyme A (GrzA), granzyme B (GrzB) and perforin (Prf) (Fig 2A and Supplementary Fig S1) after in vitro stimulation with cognate antigens. When assessing for activation based on expression of CD137, three of the eight patients showed immune activity to HPV16 antigens prior to MEDI0457 and three patientsshowed baseline reactivity to HPV18 (Supplementary Tables 3 and 4). Following MEDI0457 administration, three patients showed clear elevations in activated HPV16 specific CD8+CD137+ T cell populations expressing GrzA, GrzB and Prf, in a range of 0.24% to 0.52% of total CD8+ T cells over baseline (Fig 3B top left panel and Supplementary Tables 3 and 4) after stimulation in this assay, one of whom had limited activity at baseline. Five patients showed an elevation in this population of CD8+ T cells specific for HPV18 antigens in a range of 0.04% to 0.35% (Fig 3B, top right panel), including three patients that also showed HPV16 reactivity post MEDI0457. We performed an additional assessment of CD8+ T cell activation based on the expression of CD69 and found that, at baseline two of eight patients showed immune activity to HPV16 antigens and two non-overlapping patients showed reactivity to HPV18. Following treatment, four patients showed clear elevations in activated HPV16 specific CD8+CD69+ T cell populations expressing GrzA, GrzB and Prf, in a range of 0.28% to 1.16% of total CD8+ T cells over baseline after stimulation in this assay (Fig 3B, middle left panel). Three of those four patients also showed an elevation in this population of CD8+ T cells specific for HPV18 antigens in a range of 0.40% to 1.21% (Fig 3B, middle right panel). Finally, we assessed CD38 regulation based on antigen specific cellular activation and found that at baseline, three of eight patients showed immune activity to HPV16 antigens with two of those three patients also showing reactivity to HPV18 at baseline. Following MEDI0457, four patients showed clear elevations in activated HPV16 specific CD8+CD38+ T cell populations expressing GrzA, GrzB and Prf, in a range of 0.50% to 1.38% of total CD8+ T cells over baseline after stimulation in this assay (Fig 3B, bottom left panel). All four of these patients and an additional fifth patient showed an elevation in this population of CD8+ T cells specific for HPV18 antigens in a range of 0.06% to 1.33% (Fig 3B, bottom right panel). In total, five of the eight patients tested showed both HPV16 and HPV18 specific elevations in at least one of the three parameters tested by flow cytometry after treatment with MEDI0457 (Supplementary Tables 3 and 4). These results are not statistically significant, likely due to the small sample size. Nevertheless, the results suggest that MEDI0457 drove the induction of CD8+ T cells capable of activation in the

context of antigenic exposure and that these cells were capable of granzyme and perforin synthesis, thus exhibiting a clear CTL phenotype.

MEDI0457 alters the composition of Tumor Infiltrating Lymphocytes

Paired tumor samples obtained from baseline (prior to treatment with MEDI0457) and at definitive surgery (after a single dose of MEDI0457) were available on five Cohort I patients. These paired samples were analyzed for the presence of tumor infiltrating lymphocytes (TILs) and stained for CD8 (Fig 4A), and FoxP3 (Fig 4B) by IHC. After MEDI0457, increases in CD8+ infiltrates per mm2 of neoplastic tissue were noted in two patients (Fig 4C), and decreases in FoxP3+ infiltrates were noted in three patients (Fig 4C). We calculated the CD8/FoxP3 ratio within this tissue to assess the possibility of a shift towards a pro-inflammatory response and showed a positive change in four of five patients, including one patient with a greater than 3-fold rise in this measure (Fig 4C). The fifth patient showed a CD8/FoxP3 ratio shift in the negative direction (decrease in both CD8 and FoxP3 infiltration, with the former being greater), suggesting for this patient the possibility of a dampened inflammatory response. We further assessed these surgical specimens for the presence of immune infiltrates that were perforin positive in order to determine cytolytic capacity of the infiltrates. Interestingly, all five patients showed increases in the number of perforin positive immune infiltrates in neoplastic tissue (Fig 4C and 4D). The presence of perforin positive infiltrates in tissue samples from patient 21 (with the negative shift in the CD8/FoxP3 ratio) suggests the presence of a mixed immune state (increases in both pro- and anti-inflammatory markers). PD-L1 assessment by IHC staining was also performed on these paired samples, with a “positive” score being assigned to total percent of tumor cells with membrane staining of any intensity of greater than or equal to 25% (Fig 4E). Interestingly, all five patients assessed showed negative PD-L1 expression at study entry, with one patient showing up-regulation of PD-L1 expression after treatment with MEDI0457 (Fig 4C). Since there was no intervening therapy between the paired biopsies other than MEDI0457, the results of CD8, FoxP3 and perforin staining provide evidence that MEDI0457 directly altered the composition of TILs in tumor tissue.

Response to anti PD-1 therapy after MEDI0457

Three patients exhibited disease progression while on study. One patient in Cohort I, patient 21, had disease recurrence 7 months after completion of adjuvant chemoradiation therapy, including hemorrhagic dermal and lymph node metastases. Palliative radiation therapy was instituted for control of bleeding metastases followed by therapy with nivolumab. The patient had a complete radiographic response to treatment following just 4 cycles of nivolumab (Fig 5A-B). The patient’s tonsil HNSCCa was confirmed to be HPV16 associated by genotyping. In light of an unusually rapid and complete response to anti-PD-1 antibody therapy we analyzed the immune responses in detail in this patient. Assessment of peripheral immune responses induced by MEDI0457 in this patient revealed the induction of humoral responses (endpoint titers of 1:450 and 50 for HPV16 E6 and E7, respectively) but only limited cellular responses as gauged by IFN ELISpot (peak ELISpot responses above baseline of 7 SFU/106 PBMC for HPV16 E6 and 7 SFU/106 PBMC for HPV16 E7 as compared to median responses of 30 and 10 SFU/106 PBMC for all of Cohort I patients, Fig 5C). Patient 21 was also the only patient in Cohort I who did not show a positive shift in the CD8/FoxP3 ratio in resected tumor (Fig 4C). This suggests the possible absence of an anti-tumor inflammatory state and may partly explain the disease progression that occurred. While this non-inflamed tumor profile appears to fit with the patient’s early disease progression, the unusually rapid achievement of CR after PD-1 blockade was surprising. In order to investigate this paradox further, we performed additional assessments of peripheral blood samples taken from this patient prior to MEDI0457 dosing as well as 8 months following the completion of dosing (15 days prior to the first infusion of Nivolumab). We specifically assessed MEDI0457-driven CD8+ T cell activity and the expression of PD-1 after in vitro stimulation with HPV16 antigens. Interestingly, HPV16 antigen specific PD-1+ CD8+ T cells were not found prior to MEDI0457 but were found at robust levels after MEDI0457 dosing (0% vs. 1.8%, Fig 5D left panel). In order to determine if such cells might harbor the potential for a desirable effector response, we assessed the expression of granzyme A, granzyme B and perforin. Results of this analysis indicate that nearly half of the HPV16-specific PD-1+ CD8 T cells observed in the assay also expressed these lytic proteins (0.70%, Fig 5D right panel), suggesting the potential for HPV16 specific lytic activity in this T cell subset. We hypothesize that the expression of PD-1 on peripheral CD8+ T cells may have allowed them to be inhibited

when bound to tumor cells expressing PD-L1. Nivolumab may have relieved the inhibition of these MEDI0457-induced HPV16 specific CTLs, allowing their outgrowth and leading to the durable complete response. At the time of this publication, this patient remains in CR, 24 months post initiation of anti-PD-1 therapy.

Discussion

Here we report results of a phase I/IIa immunological proof of principle clinical trial of a HPV16 and HPV18 E6/E7 DNA immunotherapy in patients with HPV associated locally advanced HNSCCa that included a novel IL-12 DNA adjuvant (MEDI0457), both delivered by electroporation with the CELLECTRA® device. We demonstrated that administration of the immunotherapy was safe and well-tolerated both in the perioperative setting (cohort I), and after multimodality therapy with chemotherapy and radiation (cohort II). There were no treatment- related serious adverse events. The immunotherapy could be safely delivered as part of clinical standard of care, and in the Cohort I patients did not result in delays in time to surgery. We observed induction of strong humoral and cellular responses even after a single dose of MEDI0457. All patients showed induction of humoral responses against at least one HPV- specific antigen, with persistence of humoral responses for up to 23 months. That all patients responded to the immunotherapy is especially striking in Cohort II. Despite receiving cisplatin and radiation therapy, which cause lymphopenia that might blunt an immune response, patients in Cohort II were nonetheless able to mount a potent immune response to the immunotherapy. While the humoral responses induced against the E6 or E7 oncoproteins may not play a major role against progression of HPV-associated HNSCCa, E6- or E7-specific antibodies do reflect immunotherapy potency and as shown in our study were accompanied by the parallel induction of antigen-specific cellular immune responses. Why there are differences in the induction of immune reactivity to HPV 16/ 18 E6- and E7- antigens is currently unclear but may be due to a number of factors, including intrinsic immunogenic potential of these antigens as well as their relative ordering on the plasmid constructs, which in turn, may affect the expression, processing, or presentation of the antigens.

We have previously reported the results of a phase IIb randomized, blinded, placebo-controlled study, testing VGX-3100 (HPV-16 and HPV-18 E6 and E7 antigens without IL-12) in women with high-grade cervical dysplasia (CIN2/3) who were positive for HPV-16 and/or HPV-18(23). Immune responses observed in the phase IIb study in patients with CIN2/3 were similar or greater when compared to the magnitude of the responses observed in the current study of patients with HNSCCa. Relative immune competency of the two patient populations may be a factor accounting for this difference as patients with an untreated locally advanced cancer or who have just completed chemoradiation may be immune suppressed by the tumor itself and/or treatment with chemotherapy or radiation (35). That said, we were encouraged by the robustness of the immune response seen in patients on the current clinical trial.

In the current study, we demonstrated that immunotherapy with MEDI0457 induced antigen- specific cytotoxic T cells that were functionally capable of granzyme and perforin synthesis. Cells with this phenotype were also noted in the randomized controlled trial in patients with CIN and on that study, were shown to be significantly associated with clinical response to treatment (23, 32). Pre- and post-immunotherapy tissue analyses from the current study revealed that MEDI0457 altered the composition of TILs in tumor tissue tilting the cellular immune profile in most of the patients towards a pro-inflammatory state. These findings confirm our hypothesis that HPV-specific immunotherapy would induce potentially beneficial anti-tumor immune responses and are consistent with our previous finding that HPV-specific immunotherapy is able to generate tissue infiltrating immune responses that correlate with complete resolution of high grade CIN (23, 32).

The observation of a sustained complete clinical response after just 4 doses of nivolumab (ongoing for > 18 months) in a patient with progressive metastatic disease is remarkable. Complete responses after treatment with PD-1 inhibitors have been observed, but are uncommon, and usually occur later in the treatment course (9, 10, 13, 14). Our data showing expansion of antigen specific PD-1+ CD8+ cells with cytolytic potential in this patient is evidence of the immunogenicity of MEDI0457. One hypothesis is that subsequent therapy with nivolumab facilitated the outgrowth of functional HPV16-specific CTLs that had been induced by the MEDI0457 immunotherapy, but were impeded from anti-tumor effector activity by co- expression of PD-1. A combination approach, using immunotherapy to “prime” the immune

response by activation of antigen specific T cells followed by therapeutic PD-1 blockade, may be one way to increase response rates to immunotherapy in HPV associated HNSCCa.

In murine tumor models, several experimental HPV immunotherapy strategies, including vector-, peptide-, protein-, and nucleic-acid-based, as well as dendritic immunotherapies and RNA replicon approaches have been shown to enhance HPV-specific immune cell activity and anti- tumor responses (36-41). Many of these strategies have been tested in humans in the setting of CIN or HPV-associated cervical cancer, but evaluation as treatment for advanced HNSCCa remains understudied, despite a large unmet medical need in the face of an “epidemic” of HPV associated HNSCCa. Axalimogene filolisbac (AXAL or ADXS11-001) is a novel immunotherapeutic based on live irreversibly attenuated Listeria monocytogenes (Lm) fused to the nonhemolytic fragment of listeriolysin O (Lm-LLO) and secretes the Lm-LLO-HPV E7 fusion protein targeting HPV-positive tumors(42). ADXS11-001 is currently being evaluated in Phase 2 clinical trials for cervical cancer, and HPV-positive head and neck cancer (43). Preliminary data suggest robust induction of immune responses by ADXS11-001; side effects include pyrexia, anemia, vomiting, chills and muscle pain (36, 44) and clinical responses have not yet been reported. We did not observe systemic effects such as fever, myalgia, arthralgia, or malaise with MEDI0457. Another phase I dose escalation trial is evaluating the safety and immunologic responses to peptide immunomodulatory immunotherapy GL-0810 (HPV16) and GL-0817 (MAGE-A3) in HPV16 and MAGE-A3-positive metastatic or recurrent HNSCCa patients, respectively. The immunotherapy was safe, and no significant adverse events were noted. In patients who received all four doses, 80% of HPV16 positive and 67% of MAGE-A3- positive patients developed both a T cell and antibody response. In both groups, there was a significant correlation between T cell response and subsequent antibody response, but these immune responses did not translate into any clinical responses as measured by RECIST (45).

Finally, ISA 101, a synthetic long peptide immunotherapy directed against HPV 16, is being studied in combination with nivolumab in patients with recurrent/metastatic HPV 16-associated oropharyngeal cancer. In a recent report, an overall response rate of 36% (8/22 patients) was reported with the use of this combination (46). Immunological biocorrelative data have not yet been reported, and it is unclear how much of the clinical benefit is attributable to the immunotherapy vs. PD-1 blockade alone in this PD-1 treatment- naïve population. Nevertheless,

this report provides early signals of efficacy with a therapeutic vaccine combined with PD-1 blockade in patients with metastatic HPV-associated HNSCCa.

Our study has important limitations that must be acknowledged. First, in the initial iteration of the clinical protocol, patients were not selected for HPV16 and 18 genotypes based on HPV genotyping, but were enrolled solely on the basis of p16 positivity. HPV genotyping was performed retrospectively, and consequently, three patients with non-HPV 16 and 18 tumors were enrolled. The protocol was then amended to require demonstration of HPV16 and/ or HPV18 positivity. Second, none of the patients on this study were positive for the HPV 18 genotype. Third, this was a relatively small, single institution study and our results need to be confirmed in a larger population of patients. Finally, our study was not designed to answer questions related to optimal dosing schedule, timing and duration of immunotherapy or the contribution of the IL-12 DNA adjuvant. Instead, these aspects of dosing were based on the prior experience in patients with CIN and warrant further investigation.

Our observations provide novel insights into advancing therapy for HNSCCa. Indeed, this is the first report of detailed immune responses following therapeutic HPV specific DNA-based immunotherapy in patients with locally advanced HNSCCa. There have been very few, if any, novel approaches in the management of HPV-associated HNSCCa since the HPV epidemic was first described (47). There are currently no FDA-approved therapies specific to this population. Instead, treatments developed in patients with HPV negative HNSCCa - a biologically different disease are typically used. Approximately a third of patients with HPV-associated HNSCCa will go on to develop fatal metastatic disease. Based on our data, one can envision using therapeutic HPV-specific immunotherapy across the various settings that are relevant in this disease, including the pre-invasive stage where immunotherapy could be used to slow progression to carcinoma or in conjunction with existing therapies (surgery, chemotherapy and radiation) for locally advanced high risk HPV-associated HNSCC to reduce local recurrence and metastasis. Additionally, as noted in our discussion of an exceptional responder, HPV immunotherapies may be useful to increase the activity of checkpoint inhibitors by inducing the requisite pro- inflammatory state. A Phase 1b/2a, open-label study of MEDI0457 in combination with durvalumab (anti PD-L1 antibody) is underway to determine whether this combined approach will improve the response rate in patients with recurrent/ metastatic HPV-associated HNSCCa

(NCT03162224). The consistent induction of antigen-specific humoral and functional cellular immune responses in the current study should encourage the use of this “targeted immunotherapeutic” approach in the management of HPV-associated HNSCCa.

Acknowledgments

We would like to thank the patients who participated and their families as well as Hoyin Mok, Jiping Zha and Lily Cheng for their work on the ACD and IHC data. We would also like to thank Histologix and OracleBio for assistance with additional IHC staining and digital image analysis. We would like to thank Alison Berry and Kristine Mykulowycz at the University of Pennsylvania for clinical research support.

References

1. Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA: a cancer journal for clinicians. 2017;67(1):7-30. Epub 2017/01/06. 2. Varilla V, Atienza J, Dasanu CA. Immune alterations and immunotherapy prospects in head and neck cancer. Expert opinion on biological therapy. 2013;13(9):1241-56. Epub 2013/06/25. 3. Sun W, Li WJ, Wu CY, Zhong H, Wen WP. CD45RA-Foxp3high but not CD45RA+Foxp3low suppressive T regulatory cells increased in the peripheral circulation of patients with head and neck squamous cell carcinoma and correlated with tumor progression. Journal of experimental & clinical cancer research : CR. 2014;33:35. Epub 2014/04/26. 4. Mandal R, Senbabaoglu Y, Desrichard A, Havel JJ, Dalin MG, Riaz N, et al. The head and neck cancer immune landscape and its immunotherapeutic implications. JCI insight. 2016;1(17):e89829. Epub 2016/10/26. 5. Chaturvedi AK, Engels EA, Pfeiffer RM, Hernandez BY, Xiao W, Kim E, et al. Human papillomavirus and rising oropharyngeal cancer incidence in the United States. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2011;29(32):4294-301. Epub 2011/10/05. 6. Gillison ML, D'Souza G, Westra W, Sugar E, Xiao W, Begum S, et al. Distinct risk factor profiles for human papillomavirus type 16-positive and human papillomavirus type 16-negative head and neck cancers. Journal of the National Cancer Institute. 2008;100(6):407-20. Epub 2008/03/13. 7. Lyford-Pike S, Peng S, Young GD, Taube JM, Westra WH, Akpeng B, et al. Evidence for a role of the PD-1:PD-L1 pathway in immune resistance of HPV-associated head and neck squamous cell carcinoma. Cancer research. 2013;73(6):1733-41. Epub 2013/01/05. 8. Schoenfeld JD. Immunity in head and neck cancer. Cancer immunology research. 2015;3(1):12-7. Epub 2015/01/09. 9. Bauml J, Seiwert TY, Pfister DG, Worden F, Liu SV, Gilbert J, et al. Pembrolizumab for Platinum- and Cetuximab-Refractory Head and Neck Cancer: Results From a Single-Arm, Phase II Study. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2017;35(14):1542-9. Epub 2017/03/23.

10. Ferris RL, Blumenschein G, Jr., Fayette J, Guigay J, Colevas AD, Licitra L, et al. Nivolumab for Recurrent Squamous-Cell Carcinoma of the Head and Neck. The New England journal of medicine. 2016;375(19):1856-67. Epub 2016/10/11. 11. Harrington KJ, Ferris RL, Blumenschein G, Jr., Colevas AD, Fayette J, Licitra L, et al. Nivolumab versus standard, single-agent therapy of investigator's choice in recurrent or metastatic squamous cell carcinoma of the head and neck (CheckMate 141): health-related quality-of-life results from a randomised, phase 3 trial. The Lancet Oncology. 2017;18(8):1104-15. Epub 2017/06/28. 12. Segal NH AS, Brahmer JR, Maio M, Blake-Haskins A, Li X, et al. Preliminary data from a multi-arm expansion study of MEDI4736, an anti-PD-L1 antibody. J Clin Oncol 32:5s, 2014 (suppl; abstr 3002). 2014. 13. Chow LQ, Haddad R, Gupta S, Mahipal A, Mehra R, Tahara M, et al. Antitumor Activity of Pembrolizumab in Biomarker-Unselected Patients With Recurrent and/or Metastatic Head and Neck Squamous Cell Carcinoma: Results From the Phase Ib KEYNOTE-012 Expansion Cohort. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2016. Epub 2016/09/21. 14. Seiwert TY, Burtness B, Mehra R, Weiss J, Berger R, Eder JP, et al. Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial. The Lancet Oncology. 2016;17(7):956-65. Epub 2016/06/02. 15. Chen DS, Mellman I. Elements of cancer immunity and the cancer-immune set point. Nature. 2017;541(7637):321-30. Epub 2017/01/20. 16. Economopoulou P, Perisanidis C, Giotakis EI, Psyrri A. The emerging role of immunotherapy in head and neck squamous cell carcinoma (HNSCC): anti-tumor immunity and clinical applications. Annals of translational medicine. 2016;4(9):173. Epub 2016/06/09. 17. Wieking BG, Vermeer DW, Spanos WC, Lee KM, Vermeer P, Lee WT, et al. A non-oncogenic HPV 16 E6/E7 vaccine enhances treatment of HPV expressing tumors. Cancer gene therapy. 2012;19(10):667- 74. Epub 2012/08/25. 18. Sarkar AK, Tortolero-Luna G, Follen M, Sastry KJ. Inverse correlation of cellular immune responses specific to synthetic peptides from the E6 and E7 oncoproteins of HPV-16 with recurrence of cervical intraepithelial neoplasia in a cross-sectional study. Gynecologic oncology. 2005;99(3 Suppl 1):S251-61. Epub 2005/09/29. 19. Peng S, Trimble C, Wu L, Pardoll D, Roden R, Hung CF, et al. HLA-DQB1*02-restricted HPV-16 E7 peptide-specific CD4+ T-cell immune responses correlate with regression of HPV-16-associated high- grade squamous intraepithelial lesions. Clinical cancer research : an official journal of the American Association for Cancer Research. 2007;13(8):2479-87. Epub 2007/04/18. 20. Kadish AS, Timmins P, Wang Y, Ho GY, Burk RD, Ketz J, et al. Regression of cervical intraepithelial neoplasia and loss of human papillomavirus (HPV) infection is associated with cell-mediated immune responses to an HPV type 16 E7 peptide. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2002;11(5):483-8. Epub 2002/05/16. 21. Farhat S, Nakagawa M, Moscicki AB. Cell-mediated immune responses to human papillomavirus 16 E6 and E7 antigens as measured by interferon gamma enzyme-linked immunospot in women with cleared or persistent human papillomavirus infection. International journal of gynecological cancer : official journal of the International Gynecological Cancer Society. 2009;19(4):508-12. Epub 2009/06/11. 22. Bagarazzi M, Yan J, Morrow M. Immunotherapy Against HPV16/18 Generates Potent TH1 and Cytotoxic Cellular Immune Responses. Sci Transl Med. 2012;4(155). 23. Trimble CL, Morrow MP, Kraynyak KA, Shen X, Dallas M, Yan J, et al. 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. Lancet. 2015;386(10008):2078-88. Epub 2015/09/21.

24. Morrow MP, Tebas P, Yan J, Ramirez L, Slager A, Kraynyak K, et al. Synthetic consensus HIV-1 DNA induces potent cellular immune responses and synthesis of granzyme B, perforin in HIV infected individuals. Molecular therapy : the journal of the American Society of Gene Therapy. 2015;23(3):591- 601. Epub 2014/12/23. 25. Diehl MC, Lee JC, Daniels SE, Tebas P, Khan AS, Giffear M, et al. Tolerability of intramuscular and intradermal delivery by CELLECTRA((R)) adaptive constant current electroporation device in healthy volunteers. Human vaccines & immunotherapeutics. 2013;9(10):2246-52. Epub 2013/09/21. 26. Ake JA, Schuetz A, Pegu P, Wieczorek L, Eller MA, Kibuuka H, et al. Safety and Immunogenicity of PENNVAX(R)-G DNA Prime Administered by Biojector(R) 2000 or CELLECTRA(R) Electroporation Device with Modified Vaccinia Ankara-CMDR Boost. The Journal of infectious diseases. 2017. Epub 2017/10/03. 27. Boyer JD, Robinson TM, Kutzler MA, Parkinson R, Calarota SA, Sidhu MK, et al. SIV DNA vaccine co-administered with IL-12 expression plasmid enhances CD8 SIV cellular immune responses in cynomolgus macaques. Journal of medical primatology. 2005;34(5-6):262-70. Epub 2005/09/01. 28. Kim JJ, Ayyavoo V, Bagarazzi ML, Chattergoon MA, Dang K, Wang B, et al. In vivo engineering of a cellular immune response by coadministration of IL-12 expression vector with a DNA immunogen. J Immunol. 1997;158(2):816-26. Epub 1997/01/15. 29. Morrow MP, Yan J, Pankhong P, Ferraro B, Lewis MG, Khan AS, et al. Unique Th1/Th2 phenotypes induced during priming and memory phases by use of interleukin-12 (IL-12) or IL-28B vaccine adjuvants in rhesus macaques. Clinical and vaccine immunology : CVI. 2010;17(10):1493-9. Epub 2010/08/06. 30. Hsieh CS, Macatonia SE, Tripp CS, Wolf SF, O'Garra A, Murphy KM. Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science. 1993;260(5107):547-9. Epub 1993/04/23. 31. Jacobson JM, Zheng L, Wilson CC, Tebas P, Matining RM, Egan MA, et al. The Safety and Immunogenicity of an Interleukin-12-Enhanced Multiantigen DNA Vaccine Delivered by Electroporation for the Treatment of HIV-1 Infection. J Acquir Immune Defic Syndr. 2016;71(2):163-71. Epub 2016/01/14. 32. Morrow MP, Kraynyak K, Sylvester AJea. Clinical and Immunological Biomarkers for Histologic Regression of High Grade Cervical Dysplasia and Clearance of HPV16 and

HPV18 after Immunotherapy Clinical cancer research : an official journal of the American Association for Cancer Research. 2017;DOI: 10.1158/1078-0432.CCR-17-2335. 33. Migueles SA, Rood JE, Berkley AM, Guo T, Mendoza D, Patamawenu A, et al. Trivalent adenovirus type 5 HIV recombinant vaccine primes for modest cytotoxic capacity that is greatest in humans with protective HLA class I alleles. PLoS pathogens. 2011;7(2):e1002002. Epub 2011/03/09. 34. Varadarajan N, Julg B, Yamanaka YJ, Chen H, Ogunniyi AO, McAndrew E, et al. A high-throughput single-cell analysis of human CD8(+) T cell functions reveals discordance for cytokine secretion and cytolysis. The Journal of clinical investigation. 2011;121(11):4322-31. Epub 2011/10/04. 35. Chang S, Kohrt H, Maecker HT. Monitoring the immune competence of cancer patients to predict outcome. Cancer immunology, immunotherapy : CII. 2014;63(7):713-9. Epub 2014/02/04. 36. Maciag PC, Radulovic S, Rothman J. The first clinical use of a live-attenuated Listeria monocytogenes vaccine: a Phase I safety study of Lm-LLO-E7 in patients with advanced carcinoma of the cervix. Vaccine. 2009;27(30):3975-83. Epub 2009/04/25. 37. Davidson EJ, Boswell CM, Sehr P, Pawlita M, Tomlinson AE, McVey RJ, et al. Immunological and clinical responses in women with vulval intraepithelial neoplasia vaccinated with a vaccinia virus encoding human papillomavirus 16/18 oncoproteins. Cancer research. 2003;63(18):6032-41. Epub 2003/10/03.

38. Kenter GG, Welters MJ, Valentijn AR, Lowik MJ, Berends-van der Meer DM, Vloon AP, et al. Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. The New England journal of medicine. 2009;361(19):1838-47. Epub 2009/11/06. 39. Muderspach L, Wilczynski S, Roman L, Bade L, Felix J, Small LA, et al. A phase I trial of a human papillomavirus (HPV) peptide vaccine for women with high-grade cervical and vulvar intraepithelial neoplasia who are HPV 16 positive. Clinical cancer research : an official journal of the American Association for Cancer Research. 2000;6(9):3406-16. Epub 2000/09/22. 40. Karanam B, Gambhira R, Peng S, Jagu S, Kim DJ, Ketner GW, et al. Vaccination with HPV16 L2E6E7 fusion protein in GPI-0100 adjuvant elicits protective humoral and cell-mediated immunity. Vaccine. 2009;27(7):1040-9. Epub 2008/12/20. 41. Barrios K, Celis E. TriVax-HPV: an improved peptide-based therapeutic vaccination strategy against human papillomavirus-induced cancers. Cancer immunology, immunotherapy : CII. 2012;61(8):1307-17. Epub 2012/04/25. 42. Miles B, Safran HP, Monk BJ. Therapeutic options for treatment of human papillomavirus- associated cancers - novel immunologic vaccines: ADXS11-001. Gynecologic oncology research and practice. 2017;4:10. Epub 2017/07/21. 43. Wallecha A, French C, Petit R, Singh R, Amin A, Rothman J. Lm-LLO-Based Immunotherapies and HPV-Associated Disease. Journal of oncology. 2012;2012:542851. Epub 2012/04/07. 44. Cohen EE MK, Slomovitz BM. Phase I/II study of ADXS11–001 or MEDI4736 immunotherapies alone and in combination, in patients with recurrent/metastatic cervical or human papillomavirus (HPV)- positive head and neck cancer. J Immunother Cancer. 2015;3(suppl 2, abstract P147). 45. Zandberg DP, Rollins S, Goloubeva O, Morales RE, Tan M, Taylor R, et al. A phase I dose escalation trial of MAGE-A3- and HPV16-specific peptide immunomodulatory vaccines in patients with recurrent/metastatic (RM) squamous cell carcinoma of the head and neck (SCCHN). Cancer immunology, immunotherapy : CII. 2015;64(3):367-79. Epub 2014/12/30. 46. Glisson B, Massarelli, E., William, W.N. et al. Nivolumab and ISA 101 HPV vaccine in incurable HPV-16+ cancer. Annals of Oncology (2017) 28 (suppl_5): v403-v427 101093/annonc/mdx376. 2017. 47. Gillison ML, Koch WM, Capone RB, Spafford M, Westra WH, Wu L, et al. Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. Journal of the National Cancer Institute. 2000;92(9):709-20. Epub 2000/05/04.

Table Legends

Table 1. Patient Demographics and Baseline Characteristics

Figure Legends

Figure 1: Trial Schema. Patients were enrolled in 2 cohorts: Cohort I: patients that underwent definitive surgical resection. Patients could receive 1-2 doses of MEDI0457 prior to surgery. About 4 weeks post-surgery, if adequate healing was documented by the surgeon, the patient

resumed immunotherapy dosing every 3 weeks, for a total of 4 doses. Patients then went on to receive standard adjuvant therapy based on pathologic features. Cohort II: patients started immunotherapy 2 months after completion of chemoradiation. Blue arrows denote the timing of peripheral immune analyses. Red arrows denote the timing of tissue analyses. V= visits for immunotherapy administration.

Fig 2A: Treatment with MEDI0457 induces the generation of HPV16 and HPV18 specific antibodies. Immunoglobulin G (IgG) responses at baseline, and peak response post immunotherapy with MEDI0457 (a to d) HPV16 E6 (a), HPV16 E7 (b), HPV18 E6 (c), and HPV18 E7 (d) measured by ELISA. Peak response is defined as the highest titer noted against the graphed antigen at any time point post treatment. Statistical significance is noted where confirmed for a given comparison.

Fig 2B: IFN-γ ELISpot responses to HPV16 and HPV18 antigens are elevated after treatment with MEDI0457 when assessed irrespective of cohort. Number of antigen-specific IFN-γ– secreting cells in response to stimulation with HPV16 E6 and E7 pools (top panel) and HPV18 E6 and E7 pools (lower panel) at baseline and peak response measured by IFN-γ ELISpot assay for all patients in the trial irrespective of cohort. The dashed line represents a cutoff below which responses are considered “low”. Peak response is defined as the highest SFU reading noted against the graphed antigen at any time point post treatment. Statistical significance is noted where confirmed for a given comparison.

Figure 2C: IFN-γ ELISpot responses to HPV16 and HPV18 antigens are elevated after treatment with MEDI0457 when assessed by cohort. Number of antigen-specific IFN-γ– secreting cells in response to stimulation with HPV16 E6 and E7 pools in cohort I (upper left) and Cohort II (upper right) or HPV18 E6 and E7 pools in Cohort 1 (lower left) and Cohort II (lower right). Peak response is defined as the highest SFU reading noted against the graphed antigen at any time point post treatment. Statistical significance is noted where confirmed for a given comparison.

Figure 2D: Peak per-subject and median (plus range) IFN-γ ELISpot responses to HPV16 E6 and E7 antigens after treatment with MEDI0457. For Cohort I (left panel) and Cohort II (right panel) at the “peak” timepoint post treatment.

Figure 2E: Persistent per subject and median (plus range) IFN-γ ELISpot responses to HPV16 E6 and E7 antigens after treatment with MEDI0457. For Cohort I (top panels) and Cohort II (bottom panels) the time is three months post treatment. Where sample was unable to be recovered for testing “N/A” is listed.

Fig 3A: MEDI0457 induces HPV16 and HPV18 CD8+ T cells with phenotypic lytic markers as gauged by flow cytometry. A: Flow cytometric assessments of CD8+ T cell compartment for cell surface markers CD38, CD69, and CD137 and lytic markers granzyme A (GrzA), granzyme B (GrzB) and perforin (Prf). Representative gates are shown.

Figure 3B: Induction of antigen specific CD8+ T cell populations expressing GrzA, GrzB and Prf; HPV 16 specific (left column, black triangles) and HPV 18 specific (right column, red circles). Flow cytometry was performed on patients who had sufficient recoverable PBMCs (Total n = 8, n=2 from Cohort I, n=6 from Cohort 2). Each triangle or circle represents an individual patient. The line that connects triangles or circles shows the increase or decrease from the timepoint prior to dosing with MEDI0457 to the timepoint following dosing with MEDI0457. Although mean frequencies increase for all comparisons, the increases are not statistically significant.

Fig 4: Treatment with MEDI0457 modulates immune infiltration into tumor tissues. Immunohistochemical analysis of neoplastic tissue in paired samples pre- and post-MEDI0457, (A) CD8 and (B) FoxP3.

Figure 4C: Numbers for cells staining positive for CD8, FoxP3, Perforin and PD-L1 per mm2 neoplastic tissue are listed for each patient along with CD8/FoxP3 ratio.

Fig 4D: Immunohistochemical analysis of neoplastic tissue in paired samples pre- and post- MEDI0457 for Perforin and Fig 4E: PD-L1; PD-L1 status of positive is assigned to cases with total percent of tumor cells with membrane staining of any intensity of greater than or equal to 25%

Fig 5: Treatment of a patient with progressive disease with dermal and lymph node metastases (patient 21) with nivolumab resulted in radiographic complete response (A): CT neck with IV contrast and (B): PET CT scan images pre- and 6 weeks post- nivolumab. There is abnormal

tissue in the right oropharynx involving the tonsil, lateral oropharyngeal wall, right glossotonsillar sulcus (green arrows) and right base of tongue extending to the midline. At 6 weeks post nivolumab, there was interval reduction in this solid enhancing tissue component. PET images showed interval resolution of a hypermetabolic left supraclavicular lymph node (green arrow) seen on the prior exam.

Fig 5C: Assessment of peripheral immune responses in patient 21; ELISA antibody responses (left panel) and cellular responses by IFNGamma ELISpot (right panel).

Fig 5D: Analysis of CD8+T cells specific for HPV16 E6 and E7 peptides, HPV18 E6 and E7 peptides or both (MEDI0457) prior to and post dosing with MEDI0457. Frequencies of PD-1+ (left panel) and GrzA+GrzB+Prf+PD-1+ (right panel) of all peripheral CD8+ T cells specific for HPV 16 peptides (blue bars), HPV 18 peptides (red bars) or pooled HPV16 and HPV18 peptides (MEDI0457, black bars).

Fig 1 Cohort I Baseline 4-6 weeks 4 weeks 6-9 weeks Follow up x 6 months

p16+ HNSCCa Standard to undergo Visual Standard of Care definitive V V Surgery Inspection V V V of Care Adjuvant surgical by surgeon Follow up Therapy resection

Archival Surgical Biopsy Specimen Correlative studies Cohort II Baseline 2 months 12 weeks Follow up x 6 months

p16+ HNSCCa undergoing Baseline V V V V Standard concurrent Visit of Care chemoradiation Follow up

Archival Biopsy Correlative studies Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 21, 2018; DOI: 10.1158/1078-0432.CCR-18-1763 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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Fig 2C Cohort I Cohort II

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Fig 2D Peak and Median IFN ELISpot (SFU/106 PBMC) Cohort II Median Median HPV16 E6 HPV16 E7 Subject HPV16 E6 HPV16 E7 Peak Peak (range) (range)

01 50.0 35.8

03 20.0 11.7

Peak and Median IFN ELISpot (SFU/106 PBMC) 04 7.0 1.0 Cohort I Median Median 05 17.0 20.0 HPV16 E6 HPV16 E7 Subject HPV16 E6 HPV16 E7 Peak Peak (range) (range) 06 22.0 182.5

02 233.3 26.7 10 54.0 267.5

09 133.3 43.3 11 8.0 25.0

30.0 10.0 20 10.0 10.0 13 154.0 19.2 (6.7 – 233.3) (0.0 – 26.7) 31.7 19.6 (5.0 – 1700.0) (0.0 – 267.5) 21 6.7 6.7 15 19.2 0.0

16 226.7 50.0 24 30.0 0.0 17 50.0 77.5

18 38.3 15.0

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27 1700.0 94.2 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 21, 2018; DOI: 10.1158/1078-0432.CCR-18-1763 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Fig 2E Cohort I IFN ELISpot (SFU/106 PBMC) for Cohort I IFN ELISpot (SFU/106 PBMC) for HPV16E6 Above Baseline Three Months Post HPV16E7 Above Baseline Three Months Post Final dose of MEDI0457 Final dose of MEDI0457 Median Spot # Median Spot # Subject Spot # Subject Spot # (range) (range)

02 131.7 02 6.7

09 133.3 09 43.3 66.7 3.3 20 N/A 20 N/A (1.7-133.3) (0.0-43.3)

21 1.7 21 0.0

24 1.7 24 0.0

Cohort II IFN ELISpot (SFU/106 PBMC) for HPV16E6 Cohort II IFN ELISpot (SFU/106 PBMC) for HPV16E7 Above Baseline Three Months Post Final dose of Above Baseline Three Months Post Final dose of MEDI0457 MEDI0457 Median Spot # Median Spot # Subject Spot # (range) Subject Spot # (range) 01 20.0 01 10.8 03 N/A 03 NA 04 0.0 04 1.0 05 0.0 05 0.0 06 11.7 06 81.7 10 N/A 10 NA 11 N/A 11 NA 13.3 5.0 13 113.5 13 5.0 (0.0-113.5) (0.0-81.7) 15 N/A 15 NA

16 106.7 16 20.0 17 25.0 17 77.5 18 13.3 18 0.0 19 40.0 19 10.0 22 0.0 22 5.0 25 0.0 25 0.0 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer 27 N/A Research. 27 NA Author Manuscript Published OnlineFirst on September 21, 2018; DOI: 10.1158/1078-0432.CCR-18-1763 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Fig 3A

CD69 CD38 CD137

CD3 CD3 CD3 Granzyme B Granzyme Granzyme A Granzyme

Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Perforin Research. Perforin Author Manuscript Published OnlineFirst on September 21, 2018; DOI: 10.1158/1078-0432.CCR-18-1763 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Fig 3B

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Fig 4A

CD8 staining prior to MEDI0457 CD8 staining after MEDI0457

Fig 4B

FoxP3 staining prior to MEDI0457 FoxP3 staining after MEDI0457

Fig 4C

Immunohistochemical Analysis of Neoplastic Tissue CD8 FoxP3 CD8/FoxP3 Ratio Perforin PD-L1 Patient ID Pre Post Pre Post Pre Post Pre Post Pre Post MEDI0457 MEDI0457 Delta MEDI0457 MEDI0457 Delta MEDI0457 MEDI0457 Delta MEDI0457 MEDI0457 Delta MEDI0457 MEDI0457 045-509 933 1509 576 725 1145 420 1.29 1.32 0.03 4 17 13 Negative Positive 045-514 1659 1185 -474 1235 689 -546 1.34 1.72 0.38 6 20 14 Negative Negative 045-520 935 445 -490 678 317 -361 1.38 1.4 0.02 0 1 1 Negative Negative 045-521 1185 396 -789 1253 663 -590 0.95 0.6 -0.35 1 10 9 Negative Negative 045-524 685 2462 1777 1263 1475 212 0.54 1.67 1.13 1 16 15 Negative Negative

Fig 4D

Perforin staining prior to MEDI0457 Perforin staining after MEDI0457

Fig 4E

PD-L1 staining prior to MEDI0457 PD-L1 staining after MEDI0457

Fig 5A

Fig 5B

Fig 5C

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Table 1. Patient Demographics and Baseline Characteristics n % Sex Male 20 91% Female 2 9% Age Median (Range) 57.5 years (32-76)

Performance Status ECOG 0 18 82% ECOG 1 4 18% Race African American 3 14% Caucasian 19 86% Smoking Status Current Smoker 1 4% Former Smoker 12 54% Never Smoker 9 41% Site of Primary Tonsil 12 54% Base of Tongue 10 45% HPV Genotype HPV 16 19 86% HPV 18 0 0% Other 3 (HPV- 33, 35, 26) 14%

Cohort I: Treatment Post Surgery RT alone 2 33% Concurrent CRT 1 17%

Cohort II: Chemotherapy Received Cisplatin based therapy 15 94%

Weekly cisplatin 5 33% High dose cisplatin 10 67%

RT: Radiation Therapy, CRT: Concurrent chemoradiation therapy Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 21, 2018; DOI: 10.1158/1078-0432.CCR-18-1763 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Immunotherapy targeting HPV 16/18 generates potent immune responses in HPV-Associated Head and Neck Cancer

Charu Aggarwal, Roger B. Cohen, Matthew P Morrow, et al.

Clin Cancer Res Published OnlineFirst September 21, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-1763

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