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Cancer Therapy: Clinical

Clinical and Immunologic Biomarkers forHistologic Regression of High-Grade CervicalDysplasia and Clearance of HPV16 and HPV18after ImmunotherapyMatthew P. Morrow1, Kimberly A. Kraynyak1, Albert J. Sylvester1, Michael Dallas1,Dawson Knoblock1, Jean D. Boyer1, Jian Yan1, Russell Vang2, Amir S. Khan1,Laurent Humeau1, Niranjan Y. Sardesai1, J. Joseph Kim1, Stanley Plotkin3,4,David B.Weiner5, Cornelia L. Trimble2, and Mark L. Bagarazzi1

Abstract

Purpose: As previously reported, treatment of high-grade cer-vical dysplasia with VGX-3100 resulted in complete histopatho-logic regression (CR) concomitant with elimination of HPV16/18infection in 40.0% of VGX-3100–treated patients compared withonly 14.3% inplacebo recipients in a randomizedphase IIb study.Here, we identify clinical and immunologic characteristics thateither 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 cervi-cal swabs, whole blood, and tissue biopsies/resections to deter-mine correlates and predictors of treatment success.

Results: At study entry, the presence of preexisting immuno-suppressive factors such as FoxP3 and PD-L1 in cervical lesionsshowed no association with treatment outcome. The combina-tion of HPV typing and cervical cytology following dosing was

predictive for both histologic regression and elimination ofdetectable virus at the efficacy assessment 22weeks later (negativepredictive value 94%). Patients treated with VGX-3100 who hadlesion regression had a statistically significant >2-fold increase inCD137þperforinþCD8þ T cells specific for the HPV genotypecausing disease. Increases in cervical mucosal CD137þ andCD103þ infiltrates were observed only in treated patients.Perforinþ cell infiltrates were significantly increased >2-fold incervical tissue only in treated patients who had histologic CR.

Conclusions: Quantitative measures associated with an effec-tor immune response to VGX-3100 antigens were associated withlesion regression. Consequently, these analyses indicate thatcertain immunologic responses associate with successful resolu-tion of HPV-induced premalignancy, with particular emphasis onthe upregulation of perforin in the immunotherapy-inducedimmune response. Clin Cancer Res; 24(2); 276–94. �2017 AACR.

IntroductionDisease caused by mucosatrophic human papillomavirus

(HPV) remains common, despite preventive vaccines and screen-ing strategies. Globally, persistent HPV infections cause one-thirdof infection-associated cancers (1). All squamous cancers of thecervix (SCC) are thought to arise from untreated intraepithelial

disease, with particular emphasis on cervical intraepithelial neo-plasia 2/3 (CIN2/3). However, while all squamous cancers of thecervix arise from untreated CIN2/3, not all CIN2/3 progress tocancer. We and others have reported rates of spontaneous regres-sion in a relatively short, prospective observational protocol priorto planned therapeutic resection (2, 3), suggesting that in somepeople, endogenous immune responses can eliminate CIN2/3.Indeed, a previous stZudy of patients who spontaneouslyregressed, low-grade CIN1 lesions suggested higher granzyme Bþ

infiltrates into cervical tissue than those who did not regress (46),although this has not been reported for the regression of high-grade CIN2/3 disease. Patients who do not exhibit spontaneouslesion regression are indicated for ablative or surgical treatmentinterventions including cryotherapy, cold-knife conization, orloop electrical excision procedure (LEEP; refs. 4, 5). While effec-tive, such interventions have a number of undesirable possibleside effects including pain and cervical incompetence leading topreterm birth and low infant birthweight (6, 7). Moreover, evensuccessful procedures of this nature may not clear infection byhigh-risk HPV (HR-HPV), thus leaving the patient at risk forrecurrence of disease (8–10).

CIN2/3 is associated with expression of the viral early proteinsE6 and E7. Expression of both E6 and E7 are functionally required

1Inovio Pharmaceuticals, Plymouth Meeting, Pennsylvania. 2Johns Hopkins Uni-versity, School of Medicine, Baltimore, Maryland. 3The University of Pennsylva-nia, Philadelphia, Pennsylvania. 4Vaxconsult, Doylestown, Pennsylvania. 5TheWistar Institute, Philadelphia, Pennsylvania.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

M.P. Morrow, K.A. Kraynyak, C.L. Trimble, andM.L. Bagarazzi contributed equallyto this article.

Clinical Trial Registration ID: Clinicaltrials.gov NCT01304524

Corresponding Author: Matthew P. Morrow, Inovio Pharmaceuticals, Inc., 660West Germantown Pike, Suite 110, Plymouth Meeting, PA 19462. Phone: 267-440-4236; Fax: 267-440-4242; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-2335

�2017 American Association for Cancer Research.

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to initiate and maintain transformation (11, 12). T-cell effectorresponses to these viral, nonself oncoproteins, which are consti-tutively expressed by transformed cells, are likely to play a role inmediating lesion regression. Thus, immune therapies that targetadvanced dysplasia or cancer for which the goal is to elicit HPV-specific effector responses frequently focus on these two antigens.

We have previously reported efficacy results from our phaseIIb double blind, placebo-controlled trial of VGX-3100; aplasmid-based immunotherapy for high-grade dysplasiacaused by infection with HPV16/18 (13). In that trial, patientswith confirmed HPV16 and/or HPV18 infection and high-gradecervical dysplasia (CIN2/3) were randomized to receive eitherVGX-3100 (which encodes the E6 and E7 antigens of HPV16and HPV18) or blinded placebo via intramuscular immuniza-tion followed by in vivo electroporation using the CELLECTRAdevice at weeks 0, 4, and 12 to. VGX-3100 was efficacious asdefined by the primary endpoint of CIN2/3 lesion regressionand the secondary endpoint of lesion regression with concom-itant elimination of HPV16/18 infection (ref. 13; Supplemen-tary Fig S1). This report focuses on identifying and understand-ing patterns that predict treatment success. Specifically, weexplore whether or not cytologic, virologic, and immunologicassessments performed at study entry or in-study, as early astwo weeks following the completion of VGX-3100 dosing(week 14) had predictive value for treatment success prior toa formal histologic efficacy assessment 22 weeks later (week36). In addition, we also analyzed samples taken at the efficacyassessment to evaluate whether immunologic or virologicmarkers had correlative value with regards to histologic and/or virologic success or failure in the treatment of CIN2/3.

Materials and MethodsPatient sample selection

All patients designated as per protocol (i.e., those withoutprotocol violations) with sufficient sample were used foranalysis.

In situ hybridizationIn situ hybridization was performed using formalin-fixed, par-

affin-embedded tissue sections. Biotin-labeled HPV probe solu-tions (Dako Corporation) were applied to individual sections.These included separate type-specific probes for HPV16 andHPV18. Detection of hybridized probe was performed by tyra-mide-catalyzed signal amplification utilizing the Dako GenpointKit (Dako) as per the manufacturer's instructions. Chromogenicdetection was performed with DAB/H2O2. Controls includedtissue sections positive for the HeLa cell line for HPV18 and theSiHa cell line for HPV16. Biotin-labeled plasmid probes served asa negative control in each case. Cases with a discrete punctatereaction product specifically in tumor cell nuclei were interpretedas positive.

Lytic granule loading assayThe lytic granule loading assay was performed as described

previously (44) with the exception that CD137 was used in placeof HLA-DR and CD38. Peripheral blood mononuclear cells(PBMC) were recovered from cryopreservation overnight in cellculture medium and spun, washed, and resuspended the follow-ing day. After counting, 1� 106 PBMCswere plated into a 96-wellplate in R10 medium. For antigen-specific responses, cells werestimulated 5 days with a combination of 15-mer peptides over-lapping by 8 amino acid residues corresponding toHPV16 E6 andE7orHPV18E6 andE7 that had beenpooled at a concentration of2 mg/mL, while an irrelevant peptide was used as a negativecontrol (OVA) and concanavalin A was used as a positive control(Sigma-Aldrich). All peptides were resuspended usingDMSO. Nocostimulatory antibodies or cytokines were added to cell culturesat anypoint. At the endof the 5-day incubationperiod, plateswerespun to pellet cells and all samples were washed with PBS andsubjected to staining for CD3-APCH7, CD4-PerCPCy5.5, CD14-Pacific Blue, CD-16 Pacific Blue, CD137-APC, Granulysin-FITC(BD Biosciences), CD8-BV605, Granzyme A-AF700 (BioLegend),CD-19 Pacific Blue, granzyme B-PETR (Invitrogen), and perforin-PE (Abcam). Staining for extracellular markers (CD4, CD8,CD137) occurred first, followed by permeabilization to stain forthe remainingmarkers. CD3was stained intracellularly to accountfor downregulation of the marker following cellular activation.Prepared cells were acquired using an LSRII flow cytometerequippedwith BDFACSDiva software (BDBiosciences). Acquireddata were analyzed using the FlowJo software version X.0.7 orlater (Tree Star).

IHCIHC was performed as described previously (44). In patients

with visible residual disease, an excisional procedure wasperformed. In patients with no obvious residual disease, abiopsy was obtained at the site of the original lesion. All IHCassays employed a polymer/multimer–based secondary detec-tion system. In the case of labvision assays, Vector ImmPRESSHRP appropriate for the species in which the primary antibodywas raised was used for detection. For the Roche Ventanaassays, Roche UltraView (a Universal kit using anti-mouse andanti-rabbit IgG/HRP conjugate cocktail) was used. For each IHCstaining run, irrespective of which platform was used, positivecontrol tissues were included that were treated with the primaryantibody and a buffer negative where the primary antibody wasomitted and acted as a negative control. Samples were initiallyallocated first for staining CD8 and Foxp3. Remaining tissue

Translational Relevance

Infection with human papillomavirus (HPV) continues tocause cervical cancer and the high-grade dysplasia that pre-cedes it despite effective preventive vaccines and screeningstrategies. Women with high-grade cervical dysplasia are trea-ted surgically by lesion ablation or excision. Although effec-tive, surgery is associated with pain and cervical incompetenceleading to preterm birth and low infant birthweight, and HPVmaypersist even after a "successful" treatment, thus leaving thepatient at risk for recurrence of disease, including cervicalsquamous cell carcinoma. VGX-3100 has been shown toregress HPV16/18–related high-grade cervical dysplasia andclear the HPV infection by targeting HPV directly in a blinded,placebo-controlled study. This report focuses on identifyingand understanding patterns of cytologic, virologic, and immu-nologic assessments that predicted treatment success with thismedical intervention. The identification of preexisting or in-treatment biomarkers could significantly improve the utility ofthis nonsurgical treatment for HPV-related high-grade cervicaldysplasia.

Biomarkers for VGX-3100–Induced Regression of CIN2/3

www.aacrjournals.org Clin Cancer Res; 24(2) January 15, 2018 277




was then allocated for staining CD137, CD103, granulysin, PD-L1, and perforin. Differences in number of patients stained foreach marker are due to sample availability. Whole-slide imagecapture was performed by Histologix (Biocity) at 20� magni-fication with a Hamamatsu Nanozoomer 1.0-HT digital slidescanner. Normal and dysplastic epithelium and subjacent stro-ma morphologic regions of interest (ROI) were digitally anno-tated, where present, onto each section image by the studypathologist. Quantitative image analysis of IHC staining withinthe annotated ROI was performed by OracleBio (Biocity) usingDefiniens Tissue Studio software. An analysis algorithm wasdeveloped to detect positive cellular staining across each tissueimage. Within the algorithm, image colors were initially sep-arated into respective stain components, for example, brownand blue. Cells were defined and generated on the basis of thepresence of a blue (hematoxylin) stained nucleus. A thresholdlevel based on identified positive and negative staining incontrol tissues was then applied to the positive brown colourintensity parameter within each cell, above which a cell wasdefined as positive. The number or area of positive and negativestained cells was then quantified within specific regions ofinterest across each tissue image.

Statistical analysisAll analyses are post hoc. Two-tailed Mann–Whitney U tests are

used to compare data between groups and two-tailed Wilcoxonsigned rank tests to compare data within a group, as indicated.Statistical analyses were carried out using Prism program 6.0(GraphPad Software Inc.) and SPSS Stats 22 with the exact Pvalue andbootstrappingmodules (IBMCorp. Released2013; IBMSPSS Statistics for Windows, Version 22.0).

ResultsPreexisting immune cell infiltration in cervical lesions at studyentry did not predict histologic regression

We have previously reported that treatment with VGX-3100resulted in histologic regression of high-grade cervical dysplasia(CIN2/3) and clearance of HPV16 and/or HPV18 in a phase IIbdouble blind, placebo-controlled trial (13). In this analysis, weevaluated cervical tissue obtained from these patients prior todosing with VGX-3100 to determine whether any preexistingfactors were predictive of the subsequent success or failure intreating CIN2/3. In patients withCIN2/3 observed over a 15-weekwindowprior to planned resection, the intensity of intraepithelialCD8þ T-cell infiltrates in lesional mucosa has been previouslyreported to be associated with the likelihood of subsequenthistologic regression (14). In this study, in patients treated withVGX-3100, we found no statistical association between the inten-sity of pretreated CD8þ immune infiltrates and subsequent treat-ment success in lesional epithelium and stroma (Fig. 1A, left andright graphs, respectively). Indeed, study entry CD8þ tissue infil-trates did not associate with lesion regression in either VGX-3100or placebo recipients (Supplementary Fig. S2).

We next assessed biopsies from study entry for the presence ofimmunosuppressive factors that might blunt immune responsesgenerated by VGX-3100 and thus possibly influence treatmentoutcomes.Weperformed IHC staining for the Foxp3 transcriptionfactor, whichmaybe indicative of regulatory T cells (Treg; Fig. 1B).Prior to treatment, the highest range of cells that were Foxp3þ indysplastic tissue were found in treated patients who did notachieve the primary or secondary endpoint compared with thosewho did, although the median Foxp3þ infiltration was higher in

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IHC analysis of CD8, Foxp3 infiltration, and PD-L1 expression in dysplastic cervical lesions of patients treated with VGX-3100 at study entry. A, Representativestaining of CD8 in dysplastic cervical epithelium (top) and number of CD8þ cells in dysplastic cervical tissue in VGX-3100–treated patients broken out byregression status (bottom left) or regression status concomitantwith HPV clearance (bottom right). Number of patients is listed under each group.B,Representativestaining of Foxp3 in immune infiltrates in dysplastic cervical epithelium (top) and number of Foxp3þ cells in dysplastic cervical tissue in VGX-3100–treated patientsbroken out by regression status (bottom left) or regression status concomitant with HPV clearance (bottom right). (Continued on the following page.)

Morrow et al.

Clin Cancer Res; 24(2) January 15, 2018 Clinical Cancer Research278




patients who did meet those endpoints (Fig. 1B, left and rightgraphs, respectively). Statistical analysis revealed that there wasno significant difference between the groups. As the ratio ofCD8 to Foxp3 has been shown to change in response toimmunotherapy and may be informative for clinical prognosisin some diseases (15, 16), we undertook this approach toanalysis as well. Finally, the CD8þ:Foxp3þ ratio at study entrydid not associate with either the primary or secondary end-points (Fig. 1C), suggesting this assessment did not havepredictive power for clinical outcome.

The immunosuppressive ligand PD-L1 is expressed by avariety of cell types including dysplastic, neoplastic, andimmune cells in various disease states including cervical dys-

plasia. The pattern of PD-L1 staining in dysplastic tissue at studyentry was restricted to dysplastic epithelium, and was predom-inantly cytoplasmic (Fig. 1D), consistent with other reports ofPD-L1 expression profiles in cervical dysplasia (17, 18). Thehighest range of cells that were PD-L1þ in dysplastic tissue werefound in treated patients who did not achieve the primary orsecondary endpoint compared with those who did, althoughthere was no significant difference between the groups (Fig. 1D,left and right graphs, respectively), suggesting that similar to theintensity of CD8þ or Treg cellular infiltration, the expression ofPD-L1 in pretreatment cervical lesions was not predictive of thesuccess or failure of VGX-3100 to treat high-grade cervicaldysplasia. These data and those presented above suggest

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(Continued. ) C, The ratio of CD8þ/Foxp3þ cells in dysplastic cervical tissueinVGX-3100–treated patients broken outby regression status (left) or regressionstatus concomitant with HPV clearance(right). D, Representative staining ofPD-L1 in immune infiltrates in dysplasticcervical epithelium (top) and frequencyof PD-L1 staining in dysplastic cervicaltissue in VGX-3100–treated patientsbroken out by regression status (bottomleft) or regression status concomitantwith HPV clearance (bottom right). Forall analyses, the number of patientsassessed is listed under each group.Hatched boxes in pictures indicate areabeing more closely displayed within theinset. Arrows indicate positive staining(see Materials and Methods fordetermination of positive stain). In allgraphs, each dot represents one patient.The groups were compared with two-tailed Mann–Whitney tests for allanalyses.






baseline CD8, Foxp3, and PD-L1 assessments were not predic-tive for treatment response to thisHPV-specific immunotherapy.

Real-time examination of cytology and virology were clinicalpredictors of treatment outcome

In addition to analyzing preexisting factors thatmight influencetreatment outcomes, we assessed whether longitudinal cytologicand/or virologic testing performed during the studymight predictthe ultimate treatment outcomes. Specifically, we assessed thetime period following the completion of VGX-3100 dosing butprior to the efficacy assessment.We assessed the utility of cytologyand HPV typing data collected from cervical swabs for use aspredictors of treatment success or failure. The diagnosis of "Nointraepithelial lesion" at week 14 was compared with any abnor-mal diagnosis, at that timepoint, including "high-grade squa-mous intraepithelial lesion (HGSIL)," "atypical squamous cells,cannot rule out HGSIL (ASC-H)," and calculated sensitivity,specificity, and positive and negative predictive values. The pre-dictive value of detecting HPV16 and/or 18 at the same timepointwas also assessed alone or in combination with the cytologydata. Figure 2Apresents the results of these analyses from cytologyandvirologyperformed atweek14,which is 2weeks following thefinal dose of VGX-3100. Normal cytology (NIL) alone was not astrong predictor of ultimate histologic outcome, only 68% ofpatients with this cytology ultimately had histologic regression byweek 36 (Fig. 2A).However, in patientswith persistent high-gradecytology (HSIL/ASC-H) at week 14, only 17% of lesions wouldultimately regress histologically in the remaining 22 weeks.Eighty-four percent (84%) of patients whose HPV16/18 wasundetectable at week 14 were likely to regress by week 36. Whilethe sensitivity and specificity of these individual measures werenot uniformly high, the combination of week 14 cytology andvirology was a much stronger predictor of ultimate histologic

regression results. The combined findings of normal (NIL) cytol-ogy andHPV16/18 clearance at week 14weremuchmore likely topredict histologic regression at week 36 than a finding of HGSIL/ASC-H and persistence of HPV16/18 (negative predictive value94%; sensitivity 96%). Taken together, these data suggest that thecombination of clinical cytologic and virologic assessments fromcervical swabs at week 14 have high predictive value for lesionregression status at week 36.

Genotype-specific peripheral bloodCD8þ T-cell responses afterthe third dose of VGX-3100 predict treatment success

While only the combination cytologic and virologic assess-ments had strongnegative andpositive predictive values for lesionregression status, the strength of these assessments applied toapproximately half of the patients in the trial receiving VGX-3100(49 of 101 evaluable patients) who exhibited two positive indi-cators or two negative indicators. Cytology and virology resultswere not particularly predictive in theother half of patients, whoseresults were divergent, prompting us to pursue other indicators oftreatment success. We assessed peripheral blood immuneresponses to VGX-3100 antigens at week 14 (twoweeks followingthe third and final dose of VGX-3100). Postimmunization sig-natures at week 14 were analyzed for their ability to predicthistologic regression status prior to the definitive determinationat week 36.

Usingmultiparametric flow cytometry, we first assessed wheth-er or not quantification of the frequency of CD8þ T cells that werespecific for any of the VGX-3100 antigens would be sufficient todiscriminate between patients who met primary or secondaryendpoints and those who did not. We employed CD137 stainingfor this purpose, a marker that has been shown by our group andothers to identify CD8þ T cells activated by their cognate antigen(Fig. 3A; Supplementary Fig. S3; refs. 19–21). While treatment

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NIL – No Intraepithelial Lesion, HGSIL – High Grade Squamous Intraepthelial Lesion, ASC-H – Atypical Squamous Cells, cannot rule out High GradeNega�ve Predic�ve Value is the probability of regression given a nega�ve Week 14 result, which are the following: NIL, Not HGSIL/ASCH, HPV 16/18 Clearance, and NIL and HPV 16/18 Clearance Posi�ve Predic�ve Value is the probability of non-regression given a posi�ve Week 14 result, which are the following: Not NIL, HGSIL/ASCH, No HPV 16/18 Clearance, and HGSIL/ASCH and No HPV 16/18 Clearance

Figure 2.

Cytologic and virologic data collected at week 14 represented in tabular form for positive and negative predictive value, sensitivity and specificity forregression and nonregression of advanced dysplastic lesions in VGX-3100–treated patients at week 36.

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Flow cytometric analysis of CD137,granulysin, perforin, granzyme A, andgranzymeB fromperipheral CD8þT cells.A,Representative staining patterns at week 0and week 14 for CD137 in placebo patientsand VGX-3100–treated patients (top),granulysin, perforin, granzyme A, andgranzyme B (bottom) within CD8þ T cells.(Continued on the following page.)






with VGX-3100 significantly increased the frequency of HPV16/18–specific CD8þ T cells (Fig. 3B, top), there was no significantdifference in themagnitude of response at this timepoint betweentreated patients who would go on to meet the primary or sec-ondary endpoints from those who did not. In contrast, weobserved a significant posttreatment increase in the frequency ofCD8þ/CD137þ cells in patients treated with VGX-3100 irrespec-tive of histologic regression outcome (Fig. 3B, bottom), suggest-ing this measure spoke to general immune activation by VGX-3100 but not an immune signature specific for treatment success.

We reasoned that further characterizing the functionality oftheseCD8þ T cellsmight aid in identifying an immune phenotypethat discriminates between treated patients whose lesions do ordonot regress or clearHPV.We assessed antigen-activatedCD8þ Tcells (CD137þ) for markers associated with lytic function: gran-zyme A, granzyme B, perforin, and granulysin (Fig. 3A; refs. 22–28). Expression of these lytic markers in the CD8þCD137þ

population significantly increased in both treated patients whomet primary and secondary endpoints as well as in treatedpatients who did not, in all but one analysis (Fig. 3C, bottomleft; analysis of granulysin for regression endpoint), suggestingthat the inclusion of lytic proteins with respect to responses to anyVGX-3100 antigen was informative for immune activation

induced in the treated arm of the study, but was not informativefor lesion regression (Fig. 3C).

VGX-3100 targets four HPV antigens: E6 and E7 from HPV16and E6 and E7 from HPV18. The majority of patients included inthe efficacy analyses were infected with HPV16 alone (89%),while a minority were infected with either HPV18 alone (6%),or coinfected with both HPV genotypes (5%; Supplementary Fig.S4). We reasoned that immune responses directed against someantigens may have confounded our analysis to this point due tothe fact that they were being weighed against clinical benefitwithout being relevant to the infecting HR-HPV type of anindividual patient. Therefore, to perform the most focused andrelevant evaluation, we undertook additional analyses whichexcluded immune responses against HPV16 antigens or HPV18antigens if the patient did not have an active infection with thatHR-HPV type. Immune assessment therefore focused onresponses relevant only to the HR-HPV type causing the high-grade dysplasia in each patient. Results of these analyses showedthat the frequency of lytic protein expression within this infectingHPV type-specific CD8þCD137þ T-cell population were signifi-cantly increased only in treated patients who met primary orsecondary endpoints (Fig. 3D). The discriminatory potential ofthis analysis was noted for perforin (increase in mean of 5.17%

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(Continued. ) B, Frequency of CD137 expression on VGX-3100–specific CD8þ T cells broken out by treatment allocation (top), primary efficacy endpoint(bottom left) and secondary efficacy endpoint (bottom right). (Continued on the following page.)

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and 5.54% after immunization for patients meeting primary andsecondary endpoints) and granulysin expression (increase inmean of 4.80% and 4.75% after immunization for patientsmeeting primary and secondary endpoints) as well as coexpres-sion of granzyme A and perforin (increase in mean after immu-nization of 5.27% and 5.60% for patients meeting primary and

secondary endpoints), and coexpression of granzyme B withperforin (increase in mean after immunization of 4.79% for bothprimary and secondary endpoints, Fig. 3D). This observationconfirms that lytic potential in peripheral CD8þ T cells specificto the infecting HPV type of the treated subjects is predictive oftreatment success with VGX-3100.

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ta

re

Pr

f+(%

)

P la c e b o


V G X - 3 1 0 0 – T r e a t e d r e g r e s s o r s

P = 0 .0 0 7

P < 0 .0 0 0 1

n = 3 0 n = 4 3 n = 3 9

W k 0 W k 1 4 W k 0 W k 1 4 W k 0 W k 1 40

1 0

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Gn

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a l l o t h e r V G X - 3 1 0 0 t r e a t e d


P < 0 .0 0 0 1ns

n = 3 0 n = 4 3 n = 3 9

W k 0 W k 1 4 W k 0 W k 1 4 W k 0 W k 1 40

1 0

2 0

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cif

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7+

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sth

at

ar

eP

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(%)

P la c e b o


V G X - 3 1 0 0 r e g r e s s o r s


P = 0 .0 0 8

P < 0 .0 0 0 1

n = 3 0 n = 4 8 n = 3 4

W k 0 W k 1 4 W k 0 W k 1 4 W k 0 W k 1 40

1 0

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pec

ific

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cel l

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at

are

Gn

ly+

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P la c e b o


V G X - 3 1 0 0 r e g r e s s o r s


P = 0 .0 2 7P < 0 .0 0 0 1

n = 3 0 n = 4 8 n = 3 4

W k 0 W k 1 4 W k 0 W k 1 4 W k 0 W k 1 40

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zA

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P la c e b o



P = 0 .0 0 5

P < 0 .0 0 0 1

n = 3 0 n = 4 3 n = 3 9

W k 0 W k 1 4 W k 0 W k 1 4 W k 0 W k 1 40

1 0

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Pr

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V G X - 3 1 0 0 r e g r e s s o r s


P = 0 .0 0 6

P < 0 .0 0 0 1

n = 3 0 n = 4 8 n = 3 4

W k 0 W k 1 4 W k 0 W k 1 4 W k 0 W k 1 40

1 0

2 0

3 0

5 0

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HP

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6/1

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cif

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lls

tha

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re

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P la c e b o


V G X - 3 1 0 0 r e g r e s s o r s


P = 0 .0 0 5

P < 0 .0 0 0 1

n = 3 0 n = 4 8 n = 3 4

W k 0 W k 1 4 W k 0 W k 1 4 W k 0 W k 1 40

1 0

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HP

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re

Gr

zB

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(%)

P la c e b o



p = 0 .0 0 5

p < 0 .0 0 0 1

n = 3 0 n = 4 3 n = 3 9

Figure 3.

(Continued. ) C, Expression of granulysin, perforin,granzyme A, and granzyme B within CD8þ/CD137þ Tcells responding to HPV16 and HPV18 E6 and E7 brokenout by primary efficacy endpoint (left column) orsecondary efficacy endpoint (right column).(Continued on the following page.)






D

W k 0 W k 1 4 W k 0 W k 1 4 W k 0 W k 1 40

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Infe

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1 0

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Infe

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w it h H P V cle a r a n c e

P = 0 .0 0 3

n = 3 3 n = 5 0 n = 3 4

W k 0 W k 1 4 W k 0 W k 1 4 W k 0 W k 1 40

1 0

2 0

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5 0

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V G X - 3 1 0 0 r e g r e s s o r s


P = 0 .0 0 5

n = 3 3 n = 5 0 n = 3 4

W k 0 W k 1 4 W k 0 W k 1 4 W k 0 W k 1 40

1 0

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P = 0 .0 2 1

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W k 0 W k 1 4 W k 0 W k 1 4 W k 0 W k 1 40

1 0

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V G X - 3 1 0 0 T r e a t e d r e g r e s s o r s

P = 0 .0 0 2

n = 3 3 n = 4 5 n = 3 9

W k 0 W k 1 4 W k 0 W k 1 4 W k 0 W k 1 40

1 0

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Infe

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P la c e b o


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P = 0 .0 2 1

n = 3 3 n = 4 5 n = 3 9

Figure 3.

(Continued. ) D, Expression ofgranulysin, perforin, granzymeA, and granzyme B withinCD8þ/CD137þT cells responding tothe E6 and E7 antigens only of theHPV type the patient is infectedwith broken out by primary efficacyendpoint (left column) orsecondary efficacy endpoint (rightcolumn). For all graphs containingsubject data, all visible symbolsrepresent a single subject, withother subjects being representedwithin the box andwhiskers (whererelevant). Changes in responsemagnitude between study weekswere compared with two-tailedWilcoxon signed-rank tests for allcomparisons. P values for theplacebo cohort are >0.05 for allassessments presented in thefigure.

Morrow et al.





Contribution of HPV genotypes other than HPV16 and HPV18to treatment failures: retrospective analysis

After establishing that HPV type specificity was a key com-ponent of a peripheral correlate of successful treatment withVGX-3100, we performed direct in situ HPV typing of dysplasticlesions that remained present in treated patients at the time ofthe efficacy assessment (week 36) to determine whether unre-solved lesions were HPV16 or HPV18 positive (Fig. 4A). In thistrial, 56% of patients had mixed infections at study entry.However, assessment of HPV was performed on exfoliated cellsamples. We reasoned that as VGX-3100 was designed to treathigh-grade dysplasia driven by HPV16 or HPV18, but not otherHR-HPV types, persistent lesions that were not positive forHPV16 or HPV18 might be due to non HPV16/18 types thatwere also present at diagnosis and may account for a portion oftreatment failures noted in the trial. Forty-two of the 54 patientstreated with VGX-3100 whose lesions had not regressed hadsample evaluable for in situ analysis. Of these 42 patients,approximately one quarter (10) were found to have persistenthigh-grade lesions that were not HPV16 or HPV18 positive by insitu hybridization (Fig. 4B), and 6 of those patients were alsonegative by PCR from a cervical swab (Fig. 4B). Further analysisof the PCR data of the cervical swab samples revealed that 4 ofthese patients were positive for other HR-HPV types (Fig. 4B).Agreement of these two independent assays and the presence ofother HR-HPV types by PCR suggests that there is a subset ofpatients who were formally classified as VGX-3100–treatednonregressors, but may not have been true treatment failuresas HPV16/18 was undetectable, suggesting that in thesepatients, persistent dysplasia was likely due to infection withanother high-risk HPV type. However, this observation accountsfor only 11% of treatment failures in the VGX-3100–treated

cohort, and thus other analyses of samples taken after theefficacy assessment were performed.

Retrospective assessment of tissue-infiltrating immune cellsubsets correlated with treatment outcome

While our assessment of cervical tissue in this study demon-strates that pretreatment CD8þ infiltrates were not predictive ofhistologic response (Fig. 1A), we have previously reported thatposttreatment assessment of cervical tissue revealed a statisticallysignificant increase in CD8þ immune infiltration in VGX-3100–treated patients whose lesions regressed histologically and alsocleared virus (13).We further assessed the quality of this responseby performing quantitative digital image post hoc analyses ofmarkers associated with antigen-induced activation, and differ-entiation into tissue-residentmemory T cells. We assessed cervicalepithelial tissue obtained prior to treatment with VGX-3100 andobtained at the week 36 efficacy assessment for the presence ofcells expressing CD137. Virologic and cytologic data suggest thatthe majority of treatment successes may have exhibited lesionregression and viral clearance as early as week 14 and thusevidence of an active immune response might be difficult todetect in cervical tissue at week 36. However, we hypothesizedthat some residual activated cellsmight still be detectedwithin thecervical epithelium.No significant elevation inCD137þ infiltratesfrom week 0 to week 36 was observed in patients with eitherregression alone or concurrent lesion regression and viral clear-ance (Fig. 5A). This observation may indicate that enough timehad indeed passed after resolution of infection and associatedpathology for an active immune response to wane and tissuehomeostasis to be reestablished. However, VGX-3100-treatedpatients with persistent lesions had significant increases in theintensity of CD137þ infiltrates in residual dysplastic cervical

H&E of lesion (CIN3) HPV16/18 ISH of lesion

Neg

a�ve

HPV1

6+

H&E of lesion (CIN3)

H&E of lesion (CIN2)

HPV1

8+

HPV16/18 ISH of lesion

A

HPV16/18 ISH of lesion

Pa�ent ID of pa�ents in intersec�on

Linear array result from cervix at week 36

In situ HPV16/HPV18 result of lesion at week 36

600-002 33, 52 Neg

015-005 45, CP6108 Neg

301-009 35, 52 Neg

012-007 42, 52, CP6108 Neg

017-002 Neg Neg

004-010 Neg Neg

B VGX-3100�Treated Nonregressors3 Pa�ents HPV16/18nega�ve by RLA Only

6 Pa�ents HPV16/18nega�ve by ISH and RLA

4 Pa�ents HPV16/18nega�ve by ISH Only

Figure 4.

Results from in situ hybridization and PCR assays for HPV type. A, Left, H&E staining identifying the presence of cervical dysplasia. Right, representativepositive staining patterns for presence of HPV16 or HPV18 in cervical lesions present at week 36. Black arrows denote positive signal. B, Pie chart (top) showing 13patients treated with VGX-3100 who were negative for HPV16 or HPV18 by at least one assay (ISH or PCR) at week 36. Six patients were negative in bothassays. PCR confirmed the presence of other HR-HPV types present in 4 of 6 patients (bottom).






A

0

5

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CD

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2

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ith

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H is to lo g ic r e g re s s io n N o h is to lo g ic r e g re s s io n

W k 0 W k 0 W k 0 W k 0W k 3 6 W k 3 6 W k 3 6 W k 3 6

P = 0 .0 0 0 4

P la c e b o V G X -3 1 0 0

n = 6 n = 8 n = 1 5 n = 2 6

Placebo VGX-3100Histologicregression

week 0


week 36


week 0


week 36


week 0


week 36


week 0


week 36

Median 0.50 0.50 0.50 3.00 0.00 1.00 0.50 3.00

Mean 1.83 0.50 4.75 11.63 3.67 2.73 1.19 17.54

SD 2.48 0.55 9.02 22.75 11.75 7.87 2.00 33.57

0

5

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a n d vira l cle a ra n c eA ll o th e rs

W k 0 W k 0 W k 0 W k 0W k 3 6 W k 3 6 W k 3 6 W k 3 6

P = 0 .0 0 0 4

n = 3 n = 1 1 n = 1 3 n = 2 8

Placebo VGX-3100Histologic

regression and viral clearance

week 0

Histologicregression and viral clearance

week 36

All othersweek 0

All othersweek 36


week 0


week 36

All othersweek 0

All othersweek 36

Median 1.00 1.00 0.00 1.00 0.00 1.00 0.50 3.00

Mean 2.00 0.67 3.91 8.55 4.08 3.08 1.18 16.32

SD 2.65 0.58 7.79 19.75 12.63 8.44 1.94 32.61

Figure 5.

IHC analysis of immune infiltration andimmunosuppressive factors in cervical tissue at week 36.A, CD137 staining on immune infiltrates in cervicalepithelium at week 36 (top panel with zoomed inset)broken out by primary efficacy endpoint (middle) andsecondary efficacy endpoint (bottom). Hatched boxesindicate area being more closely displayed within theinset. (Continued on the following page.)

Morrow et al.





B

‡

0013-XGVobecalPHistologicregression

week 0


week 36


week 0


week 36


week 0


week 36


week 0


week 36

Median 220.50 202.50 117.00 369.50 73.00 172.00 143.00 212.00

Mean 228.83 270.00 215.38 360.88 100.40 186.53 202.11 275.89

SD 128.99 275.40 236.24 246.18 87.89 132.22 209.22 222.52

CD

103+

Cel

ls/m

m2

cerv

ical

epi

thel

ium

CD

103+

Cel

ls/m

m2

cerv

ical

epi

thel

ium

0013-XGVobecalPHistologic


week 0


week 36

All othersweek 0

All othersweek 36


week 0


week 36

All othersweek 0

All othersweek 36

Median 131.00 264.00 194.00 237.00 73.00 140.00 143.00 199.00

Mean 147.67 392.33 241.18 302.73 99.15 186.92 195.66 269.55

SD 92.14 366.75 209.01 233.20 88.53 142.74 204.28 215.75

P = 0.027

P = 0.0391,000

800

600

400

200

0Wk 0 Wk 0 Wk 0 Wk 0

Placebo VGX-3100

All othersHistologic regressionand viral clearance

Wk 36 Wk 36Wk 36Wk 36n = 3 n = 11 n = 13 n = 29

P = 0.013

ns1,000

800

600

400

200

0Wk 0 Wk 0 Wk 0 Wk 0

Placebo VGX-3100

No histologic regressionHistologic regression

Wk 36 Wk 36Wk 36Wk 36n = 3 n = 11 n = 13 n = 29

Figure 5.

(Continued. ) B, CD103 staining on immune infiltrates incervical epithelium atweek 36 (top panel with zoomed inset)as broken out by the primary efficacy endpoint (middle) andsecondary efficacy endpoint (bottom). Hatched boxesindicate area being more closely displayed within the inset.z, P ¼ 0.055 (Continued on the following page.)






tissue at week 36 (Fig. 5A), while patients in the placebo cohortwhose lesions persisted to week 36 did not. This observationsuggests that VGX-3100 induced an immune response character-ized by CD137 expression in dysplastic epithelium that was stilldetectable in lesions that had not yet regressed by the week 36endpoint evaluation.

As a detectable immune response was no longer present insubjects whose lesions had regressed, we turned to analysis ofresidentmemory immune responses by evaluatingCD103 expres-sion, which is a hallmark of intraepithelial CD8þ T residentmemory (Trm) cells (29–31). We compared the intensity ofCD103 infiltration in cervical epithelium taken prior to VGX-3100 dosing and at week 36. VGX-3100–treated patients whoselesions regressed and HPV16/18 was cleared showed significantincreases in the frequency of intraepithelial CD103þ cells incervical epithelium (Fig. 5B). While the frequency of CD103þ

cells was also elevated in treated patients whose lesions did notregress, themagnitudeof these increases didnot reach significance(P¼0.055). Patientswhose lesions regressed, but failed to achievethe secondary endpoint of concomitant viral clearance also hadstatistically significant elevation ofmucosal CD103þ infiltrates (P¼ 0.039). Patients who received placebo did not have significantincreases in CD103þ tissue infiltrates (Fig. 5B). These data suggestthat the active immune response in treated patients whose lesionsregressed had converted to a residentmemory phenotype byweek36 and that the ongoing immune response noted in the treatednonregressors may also have been converting to a memoryresponse as well.

The increase in CD137þ and CD103þ immune infiltrationobserved in the cervical tissue of VGX-3100–treated nonregressorswas paradoxical, because one might predict that immune infil-tration of this nature would be a hallmark of patients who wouldbe likely to regress their lesions, not a hallmark of lesion persis-

tence (32–34). We reasoned that if an immune response waspresent in the cervical epithelium of patients with persistentdisease, there could be an underlying immunoregulatory orimmunosuppressive mechanism at work preventing efficienteffector function. Our assessment of Treg and PD-L1 expressionprior to treatment with VGX-3100 had indicated that neither werepredictive of treatment failure (Fig. 1A–D). However, it has beenreported that an infiltrating effector immune response can resultin increases in either Treg infiltrates or PD-L1 expression (35–39).Thus, we compared Foxp3 infiltration and PD-L1 expression indysplastic lesions before and after VGX-3100 treatment (week36). In the residual lesions of VGX-3100–treated nonregressors,Foxp3þ infiltrates did not increase significantly between week 0and week 36. This observation suggested that VGX-3100 did notelicit mucosal FoxP3þ infiltrates (Fig. 5C) and that FoxP3þ

infiltration is likely not to be a mechanism of treatment failure.Indeed, because of increases in CD8þ infiltrates, the CD8:Foxp3ratio increased in VGX-3100–treated regressors from week 0 toweek 36 (Fig. 5D).

In contrast, in VGX-3100–treated nonregressors, PD-L1 expres-sion in persistently dysplastic lesions was significantly increasedfrom week 0 to week 36, a finding not replicated in patientsreceiving placebo (Fig. 5E). PD-L1 expressionmaybe induced by avariety cytokines, including TNFa and IFNg (16, 37, 40). Thus, theincrease in PD-L1 expression in residual lesions suggested theongoing presence of an active infiltrating effector immuneresponse. In patients whose lesions did regress, PD-L1 expressiondid not change significantly (data not shown). These data suggestthat upregulation of PD-L1 in persistently dysplastic lesions ofVGX-3100–treated patients may have contributed to treatmentfailure, although the specific impact of PD-L1 in this context isspeculative as most evaluable patients displayed less than 5%staining of this immunosuppressive ligand and when viewed in

C

Placebo VGX-3100No histologic

regression and no viral clearance

week 0

No histologicregression and no

viral clearanceweek 36





Median 232.00 331.00 255.00 183.00

Mean 380.18 376.00 282.82 339.86

SD 442.39 162.34 172.46 371.88

0250500750

1,0001,2501,5001,750

0 kW0 kW 63 kW63 kW

Placebo

No regression and no clearanceNo regression

VGX-3100

ns

n = 11 n = 28

FoxP

3+ p

er m

m2

FoxP

3+ p

er m

m2


regressionweek 0


week 36


week 0


week 36

Median 268.00 413.00 255.00 183.00

Mean 434.67 405.78 282.82 339.86

SD 473.20 164.66 172.46 371.88

0250500750

1,0001,2501,5001,750

0 kW0 kW 63 kW63 kW

Placebo VGX-3100

ns

n = 9 n = 28

Figure 5.

(Continued. ) C, Comparison of increases in Foxp3 infiltration from week 0 to week 36 as broken out by primary efficacy endpoint (top) and secondary efficacyendpoint (bottom). (Continued on the following page.)

Morrow et al.





the context of variations of expression of PD-L1 in patientsreceiving placebo (Fig. 5E).

Finally, we evaluated cervical tissue for functional lytic markersfor correlation with treatment success. As the peripheral immune

analysis had revealed that changes in expression of granulysin orperforin were the strongest predictors of concomitant lesionregression and clearance of HPV16 or HPV18 (Fig. 3D), thesecytolytic effector molecules were targeted for further analysis in

D



week 0


week 36

All othersweek 0

All othersweek 36


week 0


week 36

All othersweek 0

All othersweek 36

Median 3.30 16.30 5.80 5.80 5.10 7.40 4.20 4.80

Mean 3.63 17.70 6.42 7.42 6.02 9.68 11.44 8.41

SD 2.02 9.38 4.95 5.85 5.68 6.92 28.05 13.91

Wk 0Wk 0n = 6 n = 10 n = 25 n = 31

No histologic regressionHistologic regression

Rat

io o

f CD

8+/F

oxP3

+ ce

llsce

rvic

al e

pith

eliu

mWk 0 Wk 0 Wk 36Wk 36 Wk 36Wk 36

Placebo VGX-3100


week 0


week 36


week 0


week 36


week 0


week 36


week 0


week 36

Median 4.55 10.75 5.60 5.15 5.10 7.60 4.20 4.80

Mean 5.05 12.77 6.41 7.29 11.69 12.24 7.57 6.17

SD 3.47 8.68 5.32 6.29 30.14 16.05 13.85 5.36

200P = 0.031 P = 0.022

10050

40

30

20

10

0

Wk 0 Wk 0 Wk 0 Wk 0

Placebo VGX-3100

Histologic regressionand viral clearance

All others

Wk 36 Wk 36Wk 36Wk 36

n = 3 n = 21

P = 0.011

20010050

40

30

20

10

0

Rat

io o

f C

D8+

/Fox

P3+

cel

lsce

rvic

al e

pit

hel

ium

n = 13 n = 35

Figure 5.

(Continued. ) D, Comparison ofCD8þ/Foxp3þ ratios in cervicalepithelium at week 36 as broken outby the primary efficacy endpoint (top)and secondary efficacy endpoint(bottom). (Continued on the followingpage.)






cervical tissue. We reasoned that confirmation of the presence of asimilar signature in cervical tissue would confirm that theresponses noted in the periphery were mechanistically relevantfor VGX-3100–driven efficacy. The intensity of granulysinþ

immune infiltration in cervical epithelium did not change signif-icantly between week 0 and week 36 in any group (data notshown). However, perforinþ immune infiltrates increased signif-icantly at week 36 compared with study entry, only in the VGX-3100–treated patients who met primary or secondary endpoints(Fig. 5E). This finding correlated directly with the immunesignature noted in the peripheral blood (Fig. 3D).

Taken together, these data confirm that treatment with VGX-3100 drove immune infiltration into cervical lesions and thatincreased infiltration of lymphocytes with a cytotoxic signaturemarked specifically by perforin expression was discriminatory infavor of histologic regression of cervical HSIL and clearance ofHPV16/18 infection. Expression of perforin, but not granulysin,was discriminatory, suggesting a possible specificity for the activ-ity of perforin in the context of lesion regression that is notapplicable to granulysin.

DiscussionA detailed understanding of the mechanisms contributing to

successful treatment of chronic disease with an immunotherapymay serve as a roadmap for continued development of theimmunotherapy platform as a whole. We have previouslyreported on the successful clearance of HPV16/18–associatedhigh-grade cervical dysplasia (CIN2/3) in a phase IIb double-blind, placebo-controlled trial of VGX-3100 (13). VGX-3100 notonly led to the regression of CIN2/3, but also eradicated theunderlying HPV16 or HPV18 infection in about 80% of the

responders, thereby potentially reducing the risk of recurrentdisease (41). However, although VGX-3100 induced detectableperipheral blood cellular immune responses in many patients,only about half of treated patients exhibited complete lesionregression during the study window. More in-depth analysespresented here show that cellular correlates of VGX-3100–induced regression of diseasewasmultifactorial (Fig. 6), requiringboth antigen specificity for the HPV type the patient was infectedwith as well as the expression of effector molecules stronglyassociated with lytic function. While all four lytic proteins ana-lyzed (granzymeA, granzymeB, granulysin, andperforin) showedstatistical association with lesion regression and eliminationof HPV infection, the expression of perforin in particular inHPV-infecting type-specific CD8þCD137þ cells had the strongestassociation with successful treatment with VGX-3100. Upregula-tion of PD-L1 in the lesions of VGX-3100–treated patients withpersistent disease was noted, although the relatively low frequen-cy of PD-L1 expression in most patients suggests that while thismay be a contributing factor, it may not represent themain driverof treatment failure. The pattern of PD-L1 staining was predom-inantly cytoplasmic as opposed tomembranous, andwhile this isin keeping with other published reports of PD-L1 staining incervical dysplasia (17, 18), it raises the question of what type ofimpact this localization has in immunosuppression.

The antibody responses elicited by the preventive HPV vaccinesare qualitatively and quantitatively different from those elicitedby natural infection in that vaccine-induced memory B cells havegreater avidity and breadth of specificity than naturally occurringresponses (43, 45). Generation of virus-specific T-cell responses inthe clinical setting of preinvasive HPV lesions may be analo-gous—in natural infection, virus-specific T cells are exceedinglyrare in the circulation. Natural infection is intraepithelial, and

E


regressionweek 0


week 36


week 0


week 36

Median 0.25 2.60 0.40 1.55

Mean 1.22 3.92 0.91 5.66

SD 2.45 4.81 1.72 9.23


regression and no viral clearance

week 0







Median 0.40 3.00 0.40 1.55

Mean 1.13 3.89 0.91 5.66

SD 2.25 4.39 1.72 9.23

0

5

10

15203040

0

5

PD-L

1+ C

ells

/mm

2

PD-L

1+ C

ells

/mm

2

10

15203040

Wk 0Wk 0 Wk 36Wk 36

PlaceboPlacebo VGX-3100VGX-3100

P = 0.027P = 0.027

n=7 n=12Wk 0Wk 0 Wk 36Wk 36n=6 n=12

No regression No regression and no clearance

Figure 5.

(Continued. )E,Comparison of increases in PD-L1 expression fromweek 0 toweek 36 broken out by primary efficacy endpoint (top) and secondary efficacy endpoint(bottom). (Continued on the following page.)

Morrow et al.





F


week 0


week 36


week 0


week 36


week 0


week 36


week 0


week 36

Median 2.00 2.00 0.50 5.00 1.00 3.00 3.00 5.00

Mean 2.50 2.67 2.88 8.38 1.73 4.13 4.70 5.93

SD 2.26 3.08 4.94 10.20 1.98 4.02 8.08 5.53



week 0


week 36

All othersweek 0

All othersweek 36


week 0


week 36

All othersweek 0

All othersweek 36

Median 4.00 4.00 1.00 4.00 1.00 3.00 3.00 5.00

Mean 3.67 4.00 2.45 6.45 1.69 3.77 4.52 5.97

SD 2.52 4.00 4.25 9.17 1.97 3.27 7.84 5.61

Figure 5.

(Continued. ) F, Perforin staining on immune infiltrates incervical epithelium at week 36 (top panel with zoomedinset) broken out by primary efficacy endpoint (middle),and secondary efficacy endpoint (bottom). Hatchedboxes in tissue photographs indicate area being moreclosely displayed within the inset. Arrows indicatepositive staining (see Materials and Methods fordetermination of positive stain). Changes in thefrequency of staining between study weeks werecomparedwith two-tailedWilcoxon signed-rank tests forcomparisons in which statistical analysis was performed.P values for the placebo cohort are >0.05 for allassessments presented in the figure unless otherwiseindicated.






noninflammatory, so the likelihood of generating a strong sys-temic response is relatively low. In contrast, peripheral adminis-tration of antigens through vaccination is inherently systemic. Theperipheral administration of VGX-3100 induces T cells expressingperforin that are detectable in the peripheral blood as well as incervical epithelium whose presence appears to play a role in theelimination of preinvasive HPV disease in the cervix. In addition,cervical cytology and HPV testing specific for the two genotypestargeted by VGX-3100 (HPV16, HPV18) are two simple, com-mercially available tests routinely used in clinical practice thatprovide prognostic information (Fig. 6).

The increase in tissue infiltrates expressing CD137, CD103, orperforin subsequent to treatmentwith VGX-3100provide indirectevidence of activation and differentiation mediated by recogni-

tion of cognate antigen. The success of peripheral therapeuticvaccination is predicated on the concept that peripheral immu-nization can drive an immune response that homes to diseasedtissues, such as the cervical mucosa in the context of HR-HPVinfection (42). One previous study of immune therapy for HR-HPV infection has reported immune changes in cervical mucosafollowing treatment, but did not assess activation status, memoryphenotype, or the presence of effector molecules (42). In theearlier study, it became apparent that the endpoint was effectivelybeing censored only three weeks after the final vaccine dose, asrobust immune responseswere observed in the residual dysplasticmucosa. Alternatively, in the current clinical study, where end-points were assessed 6 months after the final dose of VGX-3100tissue analyses reflected a shift toward homeostasis in patients

dessessA tniopemiTepyT elpmaSdetseT retemaraPPredic�ve of Clinical

Response?

Correla�on with Clinical

Response?CD8+ Infiltra�on

Cervical Epithelial TissuePrior to VGX-3100

dosingNo N/A

FoxP3+ Infiltra�onCervical Epithelial Tissue

Prior to VGX-3100 dosing

No N/A

CD8/FoxP3 Ra�oCervical Epithelial Tissue


No N/A

PD-L1 ExpressionCervical Epithelial Tissue


No N/A

Pap SmearThinPrep Collec�on Kit Study Week 14 No N/A

HPV TypingDigene Cervical Sampler Study Week 14 No N/A

Pap Smear and HPV TypingDigene Cervical Sampler and ThinPrep Collec�on

KitStudy Week 14 Yes N/A

CD137+CD8+ T cells specific for any VGX-3100 an�gen Peripheral Blood Study Week 14 No N/ACD137+CD8+ T cells expressing GrzA, GrzB, Prf and/or Gnly specific for any VGX-3100 an�gen Peripheral Blood Study Week 14 No N/A

CD137+CD8+ T cells expressing GrzA, GrzB, Prf and/or Gnly specific for HPV type causing dysplasia

Peripheral Blood Study Week 14 Yes N/A

PD-L1 ExpressionCervical Epithelial Tissue

Study Week 36 Efficacy Assessment

N/A Yes

FoxP3+ Infiltra�onCervical Epithelial Tissue


N/A No

CD137+ Infiltra�onCervical Epithelial Tissue


N/A No

CD103+ Infiltra�onCervical Epithelial Tissue


N/A No

CD8/FoxP3 Ra�oCervical Epithelial Tissue


N/A No

Perforin+ Infiltra�on Cervical Epithelial Tissue


N/A Yes

Predictors and Correlates of Efficacy of Treatment with VGX-3100

Figure 6.

Chart of parameters tested, sample type, timepoints assessed and predictive as well as correlative associations with clinical response to VGX-3100. Allassessments performed prior to the efficacy assessment at week 36 are considered to be gauging data for predictive ability, while assessments performed atweek 36 are considered to be exploring correlative associations.


Morrow et al.




whose lesions had regressed. We did not see these changes in theresection specimens in patients who had received placebo. Thefinding of increased CD103-expressing cells is encouraging in thatthe differentiation into tissue-resident memory cells could resultin lower rates of recurrence in treated patients compared withwomen whose lesions regressed spontaneously. The HPV type–specific expression and persistence of perforin by cells in theperiphery as well as cervical mucosa identify a mechanism bywhich these cellsmaymediate this effect. Toour knowledge, this isthe first report of immunologic findings in target tissues that arecongruent with vaccine-induced changes in peripheral blood andthe first time that changes in a peripheral cytolytic CD8þ T-cellresponse have been statistically linked with successful treatmentof a neoplastic process by an active immunotherapy (Fig. 6). Thesefindings will be further investigated in phase III, and if confirmed,would provide impetus to carry out more granular analyses todetermine what constitutes an effective immune response, as wellas identify barriers that prevent immune-mediated regression.

Disclosure of Potential Conflicts of InterestM. Dallas and J.J. Kim hold ownership interest (including patents) in Inovio

Pharmaceuticals. S. Plotkin reports receiving speakers bureau honoraria fromand is a consultant/advisory board member for Dynavax and Inovio Pharma-ceuticals. D.B. Weiner is an employee of, reports receiving commercial researchgrants from, holds ownership interest (including patents) in, and is a consul-tant/advisory boardmember for Inovio Pharmaceuticals; reports receiving othercommercial research support from GeneOne; and reports receiving speakersbureau honoraria from BMGF, Medimmune, and Merck. C.L. Trimble is aconsultant/advisory board member for Inovio Pharmaceuticals. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: M.P. Morrow, J. Yan, N.Y. Sardesai, J.J. Kim,D.B. Weiner, C.L. Trimble, M.L. BagarazziDevelopment of methodology: A.S. Khan, D. Weiner, M.L. BagarazziAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.P. Morrow, J.D. Boyer, R. Vang, C.L. Trimble,M.L. BagarazziAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):M.P.Morrow, K.A. Kraynyak, A.J. Sylvester,M.Dallas,D. Knoblock, N.Y. Sardesai, J.J. Kim, C.L. Trimble, M.L. BagarazziWriting, review, and/or revision of the manuscript: M.P. Morrow,K.A. Kraynyak, A.J. Sylvester, M. Dallas, D. Knoblock, J. Yan, L. Humeau,N.Y. Sardesai, S. Plotkin, D.B. Weiner, C.L. Trimble, M.L. BagarazziAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.J. Sylvester, K.A. Kraynyak, L. HumeauStudy supervision: A.S. Khan, L. Humeau, N.Y. Sardesai, M.L. Bagarazzi

AcknowledgmentsThe authors thank the patients who participated in this study and the entire

HPV-003 clinical team from the participating study sites and Histologix andOracleBio for assistance with IHC staining and digital image analysis andFlowMetric for aid in flow cytometry. This work was supported by InovioPharmaceuticals Inc.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 29, 2017; revised October 4, 2017; accepted October 24,2017; published OnlineFirst October 30, 2017.

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Morrow et al.




2018;24:276-294. Published OnlineFirst October 30, 2017.Clin Cancer Res Matthew P. Morrow, Kimberly A. Kraynyak, Albert J. Sylvester, et al. after ImmunotherapyHigh-Grade Cervical Dysplasia and Clearance of HPV16 and HPV18 Clinical and Immunologic Biomarkers for Histologic Regression of

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