Dose-finding and Safety Study of an Oncolytic Polio/Rhinovirus Recombinant Against Recurrent WHO Grade IV Malignant Glioma
Phase: PHASE1
Conditions: GBM, Glioblastoma, Glioma, Malignant Glioma
Sponsor: Istari Oncology, Inc.

 
 
 
 
 
 
 
DOSE-FINDING AND SAFETY STUDY OF AN ONCOLYTIC 
POLIO/RHINOVIRUS RECOMBINANT AGAINST RECURRENT WHO 
GRADE IV MALIGNANT GLIOMA 
 
 
 
 
Duke Institutional Review Board #Pro00031169 
 
 
 
Food and Drug Administration Investigational New Drug (IND) # 14,735 
 
 
 
Duke University Medical Center Investigators 
 
Dina Randazzo, D.O., Principal Investigator 
 
IND Sponsor 
Darell D. Bigner, M.D., Ph.D. on behalf of Istari Oncology, Inc. 
 
Sub-Investigators 
David Ashley, M.B.B.S, F.R.A.C.P, Ph.D. 
Annick Desjardins, M.D., F.R.C.P.C. 
Allan Friedman, M.D. 
Henry Friedman, M.D. 
Matthias Gromeier, M.D. 
Katherine Peters, M.D., Ph.D. 
John Sampson, M.D., Ph.D. 
 
Statistician 
James Herndon, Ph.D. 
 
 

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1. TABLE OF CONTENTS 
 
1. 
Table of Contents ............................................................................................................... 2 
2. 
List of Tables ...................................................................................................................... 4 
3. 
List of Figures ..................................................................................................................... 4 
4. 
List of Abbreviations............................................................................................................ 5 
5. 
Study Synopsis ................................................................................................................... 6 
6. 
Abstract, Hypothesis and Objectives ..................................................................................14 
6.1. 
Abstract ......................................................................................................................14 
6.2. 
Hypothesis ..................................................................................................................14 
6.3. 
Objectives ...................................................................................................................14 
7. 
Study Schema ...................................................................................................................15 
8. 
Background and Rationale .................................................................................................16 
8.1. 
Disease and Current Therapy .....................................................................................16 
8.2. 
Viral Oncolysis ............................................................................................................16 
8.3. 
PVSRIPO Tumor Tropism ...........................................................................................16 
8.4. 
PSVRIPO Tumor Cell Killing .......................................................................................16 
8.5. 
Generating Tumor Specificity of PVSRIPO .................................................................17 
8.6. 
The Molecular Mechanisms of PVSRIPO Tumor Specificity ........................................17 
8.7. 
PVSRIPO Anti-Neoplastic Effects in Animal Tumor Models ........................................17 
8.8. 
PVSRIPO Non-Human Primate Toxicology .................................................................18 
9. 
Study Population ................................................................................................................18 
9.1. 
Inclusion Criteria .........................................................................................................18 
9.2. 
Exclusion Criteria ........................................................................................................19 
10. 
Investigational Product ...................................................................................................20 
10.1. 
Preparation of PVSRIPO .........................................................................................21 
11. 
Investigational Plan ........................................................................................................21 
11.1. 
Initial PVSRIPO Treatment Plan ..............................................................................21 
11.2. 
Retreatment with PVSRIPO.....................................................................................26 
11.3. 
Historical Modifications to the Protocol ....................................................................27 
11.4. 
Dose-Limiting Toxicity .............................................................................................31 
11.5. 
Toxicity Monitoring after dose escalation .................................................................32 
11.6. 
Toxicity Monitoring of PVSRIPO Retreatment .........................................................33 
11.7. 
Discharge, Follow-Up and Duration of Study ...........................................................33 
11.8. 
Criteria for Subject Discontinuation of Protocol Treatment .......................................33 
12. 
Data and Specimen Collection .......................................................................................34 
12.1. 
Data and Specimens to be Accessioned Prior to Treatment ....................................34 
12.2. 
Data and Specimens to be Accessioned During and After Treatment ......................34 

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13. 
Clinical Response Evaluations .......................................................................................35 
13.1. 
Radiographic Tumor Response Criteria ...................................................................36 
13.2. 
Progression Free Survival (PFS) .............................................................................36 
13.3. 
Survival ...................................................................................................................36 
14. 
Statistical Considerations ...............................................................................................36 
14.1. 
Safety and Dose-Finding Component ......................................................................36 
14.2. 
Analytical Methods ..................................................................................................36 
14.3. 
Dose Expansion Cohorts .........................................................................................36 
14.4. 
Retreatment with PVSRIPO.....................................................................................39 
15. 
Potential Benefits and Risks ...........................................................................................39 
15.1. 
Potential Benefits ....................................................................................................39 
15.2. 
Potential Risks.........................................................................................................39 
15.3. 
Treatment Alternatives and Financial Reimbursement.............................................43 
16. 
Ethical and Regulatory Issues ........................................................................................43 
16.1. 
Institutional Review Board (IRB) ..............................................................................43 
16.2. 
Recruitment and Informed Consent .........................................................................43 
16.3. 
Registration of Subjects ...........................................................................................44 
16.4. 
Data Collection and Maintenance ............................................................................44 
16.5. 
Documentation and Reporting of Adverse Events ...................................................44 
16.6. 
Data Safety and Monitoring .....................................................................................45 
16.7. 
Subject Privacy and Confidentiality..........................................................................47 
16.8. 
Inclusion of Women, Minorities, and Children ..........................................................47 
17. 
Study Financing ..............................................................................................................47 
18. 
Appendices ....................................................................................................................48 
18.1. 
Study Test Schema for Initial PVSRIPO Treatment .................................................48 
18.2. 
Study Test Schema For Retreatment with PVSRIPO...............................................50 
18.3. 
Investigational Product Handling Plan (Version 10.4.11) .........................................52 
18.4. 
Selection and Description of the Historical Control Cohort .......................................57 
19. 
References .....................................................................................................................61 
 
 
 

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2. LIST OF TABLES 
 
Table 1. Dose range-finding, toxicology, biodistribution and shedding study (BATTELLE Study 
916-G663301) of PVSRIPO in Cynomolgus macaques ...............................................24 
Table 2. Definitive, IND-directed toxicology, biodistribution, shedding and neutralizing antibody 
study (BATTELLE Study 1098-G663323) of PVSRIPO in Cynomolgus macaques ........24 
Table 3. Dose escalation schedule for PVSRIPO ......................................................................26 
Table 4. Power of the survival comparison to detect the noted hazard ratios ............................38 
Table 5. Study Test Schema .....................................................................................................48 
Table 6. Schedule of Study Tests and Procedures Associated with PVSRIPO Retreatment .....50 
 
3. LIST OF FIGURES 
 
Figure 1. Translation Initiation. ..................................................................................................17 
Figure 2. Genetic structure of PVSRIPO ...................................................................................21 
 

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4. LIST OF ABBREVIATIONS 
 
 
 
 
 
 
 
 
BDP; Biopharmaceutical Developmental Program  
 
CBER; Center for Biologics Evaluation & Research 
CED; convection-enhanced delivery  
CNS; central nervous system 
CPC; Cancer Protocol Committee 
CRM; continual reassessment method 
CSF; cerebrospinal fluid 
CTC; Common Toxicity Criteria 
DLT; dose-limiting toxicity 
DMEM; Dulbecco’s minimal essential medium 
DNA: deoxyribonucleic acid 
DVT; deep vein thrombosis 
EGFRvIII; epidermal growth factor receptor (variant III) 
eCTD; electronic common technical document 
eIF; eukaryotic initiation factor 
FDA; Food and Drug Administration 
FLAIR; fluid-attenuated inversion recovery 
GBM; glioblastoma 
GCS; Glasgow coma scale 
Gd-DTPA; Gadolinium diethylene triamine pentaacetic acid 
HRV2; human rhinovirus type 2 
  
 
HAS; human serum albumin 
IND; investigational new drug 
IRB; Institutional Review Board 
IRES; internal ribosomal entry site 
KPS; Karnofsky performance score  
 
 
MMSE; Mini-mental Status Examination 
MTD; maximally tolerated dose 
MVB; master virus bank 
NCI; National Cancer Institute 
Necl-5; nectin-like molecule 5 
NHP; non-human primate 
NINDS; National Institute of Neurological Disorders and Stroke 
NSICU; Neuro-Surgical Intensive Care Unit 
OS; overall survival 
PET; positron emission tomography 
PFS; progression free survival 
PKC; protein kinase C 
PV1S; poliovirus serotype 1 (SABIN) 
RIO; research integrity office 
SAE; serious adverse event 
SIADH; syndrome of inappropriate antidiuretic hormone 
TCID; tissue culture infectious dose 
UTR; untranslated region 
WHO; World Health Organization 

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5. STUDY SYNOPSIS 
 
Title: 
DOSE-FINDING 
AND SAFETY STUDY 
OF 
AN ONCOLYTIC POLIO/RHINOVIRUS 
RECOMBINANT AGAINST RECURRENT WHO GRADE IV MALIGNANT GLIOMA 
Study 
Agent: 
PVSRIPO administered intratumorally. PVSRIPO is the live attenuated, oral (SABIN) 
serotype 1 poliovirus vaccine containing a heterologous internal ribosomal entry site 
(IRES) stemming from human rhinovirus type 2 (HRV2). PVSRIPO recognizes 
nectin-like molecule-5 (Necl-5), an oncofetal cell adhesion molecule and tumor 
antigen widely expressed ectopically in malignancy, e.g. glioblastoma (GBM), as host 
cell receptor. Tumor specificity is mediated by selective expression of Necl-5 in GBM, 
translation repression at the heterologous HRV2 IRES in the normal central nervous 
system (CNS) and a supportive environment for PVSRIPO translation, growth and 
cytotoxicity mediated by universally active protein kinase C (PKC)-Ras-Erk signaling 
to translation machinery in GBM.  
Primary 
Objective: 
To determine the MTD and the Phase II dose of PVSRIPO when delivered 
intracerebrally by convection-enhanced delivery. 
Secondary 
Objectives: 
To obtain correlative mechanistic evidence for PVSRIPO’s effects on infected WHO 
grade IV malignant glioma tumors and to estimate PFS and OS in recurrent WHO 
grade IV malignant glioma patients.   
To estimate the efficacy of PVSRIPO administered at the optimal dose. 
Exploratory 
Objectives: 
The primary exploratory objectives focus on outcomes associated with the initial 
PVSRIPO treatment: 
• 
Describe changes visualized on imaging due to intratumoral inoculation of 
PVSRIPO.  
• 
Evaluate parameters of general immune activation: frequency of immune cell 
subsets in peripheral blood and changes in serum levels of cytokines. 
• 
Evaluate CD8 and CD4 immunologic response against polio specific and 
GBM specific antigens to be determined. 
• 
Identify genetic predictors of response or failure of response to treatment with 
PVSRIPO. 
Additional exploratory objectives focus on retreatment with PVSRIPO: 
• 
Describe the toxicity of retreatment with PVSRIPO in combination with a 
single dose of lomustine. 
• 
Describe the PFS and OS of patients after retreatment with PVSRIPO and a 
single dose of lomustine. 
• 
Describe changes visualized on imaging after retreatment with PVSRIPO in 
combination with a single dose of lomustine. 
• 
Evaluate parameters of general immune activation, including frequency of 
immune cell subsets in peripheral blood, changes in serum levels of 
cytokines, and CD8 and CD4 immunologic response against polio-specific 
and GBM- specific antigens. 
Inclusion 
Criteria: 
Please see Section 9 for further details regarding eligibility. 
• 
Patients must have a recurrent supratentorial WHO grade IV malignant 
glioma based on imaging studies with measurable disease (≥ 1 cm and ≤ 5.5 
cm of contrast-enhancing tumor).  Prior histopathology consistent with a 
World Health Organization (WHO) grade IV malignant glioma confirmed by 
the study pathologist, Roger McLendon, or his designate. 
• 
Age ≥ 18 years of age at the time of entry into the study. 
• 
Karnofsky Performance Score ≥ 70%. 
• 
Prothrombin and Partial Thromboplastin Times ≤ 1.2 x normal prior to biopsy. 

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• 
Total bilirubin, SGOT, SGPT, alkaline phosphatase ≤ 2.5 x normal prior to 
biopsy. 
• 
Neutrophil count ≥ 1000 prior to biopsy. 
• 
Hemoglobin ≥ 9 prior to biopsy. 
• 
Platelet count ≥ 125,000/µl prior to biopsy; Platelet count ≥ 100,000/µl prior 
to infusion. 
• 
Creatinine ≤ 1.2 x normal range prior to biopsy. 
• 
Positive serum anti-poliovirus titer prior to biopsy (except for retreatment). 
• 
The patient must have received a boost immunization with trivalent 
inactivated IPOL™ (Sanofi-Pasteur) at least 1 week prior to administration of 
the study agent. 
• At the time of biopsy, prior to administration of virus, the presence of recurrent 
tumor must be confirmed by histopathological analysis. 
• A signed informed consent form approved by the Duke University Institutional 
Review Board (IRB) will be required for patient enrollment into the study. 
Patients must be able to read and understand the informed consent document 
and must sign the informed consent indicating that they are aware of the 
investigational nature of this study. 
• Able to undergo brain MRI with and without contrast. 
Exclusion 
Criteria: 
• Because of the unknown risk of virus administration potentially affecting a 
developing fetus or growing infant, females who are pregnant or breast-
feeding will be excluded. Adults of reproductive potential not employing an 
effective method of birth control will be excluded. Sexually active women of 
child bearing potential, whose partner is male, must use medically accepted 
birth control. Sexually active men, whose partner is a female of child bearing 
potential, must use a medically accepted birth control. 
• Patients with an impending, life-threatening cerebral herniation syndrome, 
based on the assessment of the study neurosurgeons or their designate, will 
be excluded. 
• Because the potential toxicities from the agent being studied in this protocol 
may be similar to some known diseases or may be more dangerous in the 
context of certain known diseases, the following patients will be excluded: 
o Patients with an active infection requiring intravenous treatment or having 
an unexplained febrile illness (Tmax > 99.5 F/37.5 C),  
o Patients with known immunosuppressive disease or known human 
immunodeficiency virus infection,  
o Patients with unstable or severe intercurrent medical conditions such as 
severe heart (New York Heart Association Class 3 or 4) or known lung 
(FEV1 < 50%) disease, uncontrolled diabetes mellitus,  
o Patients with albumin allergy,  
o Patients with Gadolinium allergy. 
• 
Patients with a previous history of neurological complications due to poliovirus 
infection will be excluded.  
• 
Patients who have not recovered from the toxic effects of prior chemo- and/or 
radiation therapy will be excluded. Guidelines for this recovery period are 
dependent upon the specific therapeutic agent being used.  
• Patients may not have received chemotherapy or bevacizumab ≤ 4 weeks 
[except for nitrosourea (6 weeks) or metronomic dosed chemotherapy such 
as daily etoposide or cyclophosphamide (1 week)] prior to starting the study 
drug unless patients have recovered from side effects of such therapy.  

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• Patients may not have received immunotherapy ≤ 4 weeks prior to starting the 
study drug unless patients have recovered from side effects of such therapy.  
• Patients may not be less than 12 weeks from radiation therapy, unless 
progressive disease outside of the radiation field or 2 progressive scans at 
least 4 weeks apart or histopathologic confirmation. 
• Patients must have completed all standard of care treatments, including 
surgical procedure and radiation therapy (at least 59Gy)  
o If the MGMT promoter in their tumor is known to be unmethylated, 
patients are not mandated to have received chemotherapy prior to 
participating in this trial.  
o If the MGMT promoter in their tumor is known to be methylated or the 
MGMT promotor methylation status is unknown at the time of screening, 
patients must have received at least one chemotherapy regimen prior to 
participating in this trial. 
• Because of the potential toxicities from the agent, patients with neoplastic 
lesions in the brainstem, cerebellum, or spinal cord, radiological evidence of 
active (growing) multifocal disease, or leptomeningeal disease will be 
excluded.   Patients with evidence of diffuse subependymal disease or tumor 
in the brainstem, cerebellum, spinal cord, or CSF will also be excluded. 
 
Patients with the following will be excluded: 
• Undetectable anti-tetanus toxoid IgG (except for retreatment).   
• Known history of agammaglobulinemia. 
• Patients on greater than 4mg per day of dexamethasone within the 2 weeks 
prior to admission for PVSRIPO infusion.  
• Patients with worsening steroid myopathy (history of gradual progression of 
bilateral proximal muscle weakness, and atrophy of proximal muscle groups). 
• Patients with prior, unrelated malignancy requiring current active treatment 
with the exception of cervical carcinoma in situ and adequately treated basal 
cell or squamous cell carcinoma of the skin. 
Study 
Design: 
This protocol is designed primarily to determine the MTD and DLT of a novel oncolytic 
viral recombinant with GBM-specific viral translation, replication and cytotoxicity. 
PVSRIPO will be delivered intratumorally by convection-enhanced delivery using an 
intracerebral catheter placed within the enhancing portion of the tumor. To enable 
tracking of the inoculum, 1 mM Gadolinium will be co-infused. The contrast agent 
(Gd-DTPA) will be added to the virus-containing infusate.  Based on pre-clinical 
toxicity studies in non-human primates, the starting amount of the agent to be 
delivered will be 1 x 108 tissue culture infectious dose (TCID50). Due to limitations 
imposed by manufacture and dosing volume in Cynomolgus macaques, the MTD was 
not reached in non-human primates. Intra-thalamic inoculation of 5 x 109 TCID in 
Cynomolgus macaques did not cause any toxicity. A total of 3 mLs of the agent in 
physiologic saline stabilized with 0.2% human serum albumin will be delivered over 
approximately 6 hrs 30 minutes, corresponding to a flow-rate of 0.5 mL/hr. The agent 
is stable at room temperature during the intended instillation period and the intended 
delivery apparatus is not associated with adsorptive loss of PVSRIPO. PVSRIPO 
dose escalation will be accomplished by increasing agent concentration allowing 
flow-rate and infusion volume to remain constant. 
 
A two-step continual reassessment method design will be used for dose escalation. 
Starting at dose level 1, one patient will be treated at each dose level. Decisions 
concerning dose escalation for subsequent patients will be based upon the 
occurrence of DLT during the first 4 weeks after treatment administration.  Initially, 

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each patient will be observed for ≥ 4 weeks before the next patient is treated. If no 
patients experience a DLT on the first 4 dose levels, all subsequent patients will be 
treated at dose level 5. Escalation will be interrupted if any of the patients assigned 
to one of the first 4 dose levels or ≥ 20% of patients treated at dose level 5 experience 
a DLT. In that case, further dose escalation or de-escalation will be guided by the 
likelihood-based implementation of the continual reassessment method.  Greater 
detail is provided in section 11.1.5.1. 
 
Historical Modifications to the Protocol: 
As of 11/15/2013 (AMD025), 4 patients have been treated on dose level 5.  One 
patient experienced a DLT upon removal of the treatment catheter.  The remaining 3 
patients have not experienced a DLT; however, all 3 have had difficulties tapering off 
steroids.  To address this concern, the study is amended to reduce the dose for the 
next cohort of patients to dose level 2.  Up to 6 patients will be accrued at dose level 
2.  If 2 of these patients experience a DLT as defined in section 11.4, accrual will be 
suspended.  
 
As of 6/5/14 (AMD041), 5 subjects have been treated on dose level 2.  Four subjects 
have completed the two-week follow-up period and have not had a DLT. One subject 
remains in the 14 day follow up period. Once one last patient has been treated, the 
study per the November 2013 amendment is complete.  We are amending the study 
to allow continued patient accrual to garner more information about patient safety 
while the single-institution phase 2 study is being developed and reviewed by various 
regulatory bodies.  We intend to continue accruing subjects for treatment at dose level 
2 until such time that the phase 2 study has been activated. After each patient has 
been followed for a one-week observation period, during which the patient will have 
been evaluated in clinic on day 7 (± 2 days), a patient summary will be prepared and 
submitted to the DSMB chair for approval. Once approval from the DSMB chair has 
been obtained, treatment of the next patient will be allowed. We will treat up to four 
subjects within a 30-day period as allowed by DSMB chair, and there will be only one 
PVSRIPO treatment per patient without specific approval for a second or more 
treatments from the FDA and the IRB. 
As of 10/06/2014 (AMD048), 7 subjects have been treated on protocol at dose level 
2.  One additional subject has been treated under a Single Subject Emergency Use 
Request at dose level 2.  Of these eight subjects, six subjects had difficulty or 
remained unable to be tapered off steroids more than three months post infusion 
(three subjects required the addition of bevacizumab to facilitate the taper down or 
off steroids). We are amending the study to reduce the dose to dose level -1 (5.0 X 
107 TCID50) with the goal of limiting the occurrence of known possible side effects of 
prolonged steroid use in patients who otherwise benefit from this investigational 
therapy. This dose reduction will allow continued patient accrual and garner more 
information about patient safety and optimal dosing while the single-institution phase 
2 study is being developed and reviewed by various regulatory bodies. Our last 
amendment allowed the enrollment of up to 18 patients, one patient was enrolled 
under that amendment and thus, we are planning to enroll up to 17 patients on dose 
level -1.  After each patient has been followed for a one-week observation period, 
during which the patient will have been evaluated in clinic on day 7 (± 2 days), a 
patient summary will be prepared and submitted to the DSMB chair for approval. 
Once approval from the DSMB chair has been obtained, treatment of the next patient 
will be allowed. We will treat up to four subjects within a 30-day period as allowed by 

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DSMB chair, and there will be only one PVSRIPO treatment per patient without 
specific approval for a second or more treatments from the FDA and the IRB. 
Study Design Modification (1/13/2015 AMD054)). Once the optimal dose level of 
PVSRIPO is determined, a total of 26 patients will be treated at that dose level on a 
dose expansion cohort.  The survival associated with these 26 patients will be 
compared to that of a historical cohort (see section 14.3). The study is being amended 
to allow accrual and treatment of a total of 26 patients at the optimal dose level of 
PVSRIPO and, as needed, radiation-necrosis dose levels and schedules of 
bevacizumab to estimate the efficacy of PVSRIPO relative to a historical cohort. 
Sample Size Modification (6/24/2015 AMD071). As of 6/24/15, we have treated 13 
patients on dose level -1. None of the patients have had prolonged steroid needs, two 
patients died at 3 and 6 months respectively, and five patients total (including the two 
patients who died) were initiated on bevacizumab. It is now felt that dose level -1 is 
the optimal dose, as none of the patients have experienced undue side effects due to 
PVSRIPO or the management of the cerebral inflammation secondary to PVSRIPO. 
We are now amending the protocol to enroll a total of 50 patients on dose level -1.  
Study Design Modification (11/10/2015 AMD078).  Imaging response criteria were 
previously developed based on imaging observations following chemoradiation 
treatment (Macdonald criteria) and subsequently revised based on new observations 
from anti-angiogenic trials (RANO criteria). As imaging is dramatically different 
following treatment with immunotherapy, new imaging criteria are being developed 
(iRANO). This process is likely to take several years to validate. For that reason, we 
are removing response rate as a secondary objective and adding, as an exploratory 
objective, a description of changes visualized on imaging due to intratumoral 
inoculation of PVSRIPO. 
Study Design Modification (2/1/2016 AMD081).  The protocol has been re-formatted 
in an electronic Common Technical Document (eCTD) format for electronic 
submission to the FDA for the associated IND.  The statistical section has been 
revised and an appendix (section 18.4) has been added to define and describe the 
historical control group being used as a statistical comparison for the subject 
population.  We have also further described the types of subject follow-up activity that 
will occur in this study. 
Study Design Modification (6/15/2016 AMD096). As of 6/15/2016, 23 subjects have 
been treated on dose level -1, including 12 subjects that were treated more than 12 
months ago.  Three of these 12 subjects demonstrated tumor reduction without 
significant inflammation necessitating prolonged use of bevacizumab and/or 
additional chemotherapy.  In contrast, the remaining 9 patients had inflammation that 
was burdensome and required prolonged use of bevacizumab and/or additional 
chemotherapy.  Caregivers of some of these patients were overwhelmed by the 
experience.  The 3 patients without significant inflammation are alive at 20.2, 15.5, 
and 13.1 months after PVSRIPO infusion, and 7 of the 9 patients with burdensome 
inflammation have died, including 5 that died within 12 months of PVSRIPO 
treatment. 
Despite the fact that subjects on dose level -1 have been able to remain off significant 
doses of steroids, we believe that the subjects benefiting the most from PVSRIPO 
have been those who have experienced minimal or easily controllable inflammation.   

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As such, we are amending this study to reduce the PVSRIPO dose to dose level -2 
(1.0 X 107 TCID50) with the goal of limiting the occurrence of undesirable burden from 
the inflammation and its treatment on as many subjects (including caregivers) as 
possible. Based upon additional animal studies, we are confident that dose level -2 
(1.0 X 107 TCID50) will be a therapeutic dose. This amendment is not due to concerns 
for the safety of the patients on dose level -1, but due to the observation that less 
inflammatory reaction is a predictor of better survival and treatment response.  
We plan to treat 27 patients at dose level -2.  After each patient has been followed for 
a one-week observation period, during which the patient will have been evaluated in 
clinic on day 7 (± 2 days), a patient summary will be prepared and submitted to the 
DSMB chair for approval. Once approval from the DSMB chair has been obtained, 
treatment of the next patient will be allowed. We will treat up to four subjects within a 
30-day period as allowed by DSMB chair, and there will be only one PVSRIPO 
treatment per patient without specific approval for a second or more treatments from 
the FDA and the IRB.  
Furthermore, patients previously treated on trial received high dose steroids prior to 
catheter insertion and throughout the infusion, as part of undergoing biopsy. Given 
concerns for negative impact of high dose steroids on the efficacy of PVSRIPO, we 
will limit steroid usage to the minimal dose necessary, if even needed.  
Study Design Modification (6/29/2016 AMD101).  This amendment includes several 
revisions to tests and procedures, which we believe can be safely implemented with 
the aim of reducing the time between consenting to participate in the study, screening 
to determine eligibility, and study treatment.  Previously, patients had to demonstrate 
a serum total IgG level of ≥ 400 mg/dL to be eligible for the study.  All patients who 
have undergone this testing thus far have met this eligibility criterion; therefore, it is 
no longer considered necessary to test serum IgG in patients going forward prior to 
their enrollment.  We are also changing the timing of the boost immunization with 
trivalent inactivated IPOL™ (Sanofi-Pasteur) from between 6 months and 2 weeks 
prior to PVSRIPO infusion to between 6 months and 1 week prior to PVSRIPO 
infusion.  Also, for previously enrolled patients, a patient had to be followed for a one-
week observation period post-infusion, before the next patient could be treated.  Now, 
once a subject has been observed through successful completion of the PVSRIPO 
infusion, the next subject can be treated.  We are updating pharmacy instructions for 
the preparation and administration of PVSRIPO in this amendment as well.  Lastly, 
per Duke’s Research Integrity Office (RIO), a study-specific DSMB may no longer be 
necessary for this study given the oversight provided by the BTC’s DSMB-Plus.  If the 
FDA concurs, the study-specific DSMB may be dissolved per its charter.   
Study Design Modification (9/5/2016 AMD104).  This amendment includes the 
addition of an exploratory objective to identify genetic predictors of response or failure 
of response to treatment with PVSRIPO.  Molecular genetic tests, such as, but not 
limited to, DNA sequencing, gene amplification, and gene expression, will be 
performed on the tissue obtained at the protocol-specified biopsy prior to PVSRIPO 
infusion.  Tissue may be obtained for testing as either fresh, frozen, or fixed tissue, 
or as slides, after the diagnostic pathologist has retained all tissue needed for 
diagnosis. The amount of tissue required for this testing should be sufficient to yield 
a minimum of one microgram of DNA.   

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Study Design Modification (3/1/2017 AMD124).  Based upon recent pre-clinical data 
and clinical data collected on dose levels -1 and -2, we have concluded that there is 
no evidence of pre-clinically or clinically important differences between these dose 
levels. The initial need for treatment with the radiation necrosis dose levels and 
schedules of bevacizumab for cerebral inflammation secondary to PVSRIPO or tumor 
growth has been comparable thus far.  In addition, the toxicity profiles for the two 
dose levels are also similar.  Therefore, we have decided to proceed with the 
development of PVSRIPO dose level -1 (5.0 X 107 TCID50) within the phase II study 
that is now in development.  We are amending this phase I study to treat all future 
patients at the ­1 dose level.  We intend to continue accruing patients and treating 
patients at this dose level while the phase II study is being finalized and reviewed by 
various regulatory bodies.   
The protocol is also being modified to allow multiple patients to be infused with study 
drug simultaneously if the neuro-surgical schedule allows.  There are currently 3 
neurosurgeons listed as sub-investigators who are trained on the surgical procedures 
for this study.  In the study modification dated 6/29/2016, the study was modified so 
that the next patient could be treated immediately following the completed infusion of 
the previous patient.  However, we would like to increase our enrollment potential by 
allowing multiple patients to be treated simultaneously.  As of 1/20/2017, no patients 
have experienced complications during the infusion of PVSRIPO.  Only 1 out of 51 
treated patients has experienced severe complications (Grade III or higher) during 
catheter removal following the infusion of the study drug, and therefore we are 
confident that treating multiple patients at a time would be safe for the patients and 
would allow for increased data collection. 
This amendment also includes updates to the program used for planning catheter 
placement, the timing of catheter removal, the infusion pump information, and to 
clarify the history of DLT monitoring in the study.  The statement related to sending 
MRIs to Therataxis was modified to state that the MRIs may be sent to Therataxis.  
The blood test for anti-epileptics has been removed.  Lastly, the exclusion criterion 
related to completion of standard of care treatments has been clarified based on 
whether or not the MGMT promotor methylation status is known at the time of 
screening.  There are other minor corrections throughout.  
Following the FDA’s review of this amendment, an additional section (see section 
11.5) describing guidelines and criteria for study pause as a result of toxicity 
monitoring has been added.  Specifically, the study is being monitored for the 
occurrence of unacceptable toxicities within dose levels -1 and -2 at any time after 
the initiation of protocol treatment. 
Study Design Modification (6/22/2017 AMD142).  It has been recently observed that 
patients who originally benefitted from the infusion of PVSRIPO can demonstrate 
tumor recurrence years later. Given the limited life expectancy of recurrent 
glioblastoma patients, it has been hypothesized that retreatment of long-term 
survivors previously treated with PVSRIPO could trigger an immune recall effect and 
thus further extend the survival of patients. As such, in the current amendment, we 
will allow selected patients who have previously benefited from PVSRIPO infusion to 
be retreated with PVSRIPO in the event of tumor recurrence.  A patient is considered 
to have benefited from the initial PVSRIPO infusion if they survived 12 or more 
months after PVSRIPO treatment. All patients retreated will be treated at dose level -
1 as per the same procedure previously used. Furthermore, all retreated patients will 

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receive a single dose of lomustine 8 weeks post infusion of PVSRIPO. As lomustine 
temporarily reduces the number of regulatory T-cells, it is hypothesized that it could 
potentially increase the efficacy of PVSRIPO and of the immune recall.  Further 
details regarding this retreatment plan are in Section 11.2. 
Study Design Modification (9/25/2017 AMD149).  Based upon the Final Clinical 
Shedding Study for PVSRIPO (IND 14,735) dated 8/6/2017, the IBC reviewed the 
requirement for stool sampling for patients receiving investigational treatment with 
PVSRIPO.  The purpose of the stool sampling was to test for presence of viral 
shedding in stool samples of patients receiving PVSRIPO via intracerebral delivery.  
The Final Clinical Shedding Report indicates no presence of PVSRIPO in stool 
collected from 59 subjects who received 61 treatments with PVSRIPO (2 patients 
were re-treated).  Per the report, “we feel that the serologic evidence indicates a 
formidable protective shield against shedding in patients with pre-existing and 
boosted anti-polio immunity.”  On 8/17/2017, the IBC determined that the requirement 
for viral shedding studies may be discontinued on the basis of the report. Therefore, 
we have removed stool sampling from Section 12.2 and from Table 6.  In addition, 
we have added 3 additional biopsy core samples to be taken at the time of biopsy, 
prior to catheter placement, if possible.  The additional biopsy core samples will be 
used for genetic analysis, including full genome or full exome sequencing, as well as 
other molecular genetic testing. 

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6. ABSTRACT, HYPOTHESIS AND OBJECTIVES 
6.1. 
ABSTRACT 
Despite aggressive surgery, prolonged radiation therapy, and multi-agent chemotherapy, survival 
after diagnosis with a malignant brain tumor is usually < 16 mo. and patients with recurrent tumors 
usually survive < 3 mo. Current therapies for malignant brain tumors are limited by ineffective 
delivery beyond the blood-brain barrier, limited diffusion of regionally-delivered macromolecules, 
and a lack of tumor specificity, which leads to dose-limiting toxicity.  
 
We and others demonstrated that sustained direct intracerebral infusion at slow flow rates can 
overcome delivery barriers. This innovative technique, called convection-enhanced delivery 
(CED), may induce a convective interstitial fluid current that has the potential to homogeneously 
distribute even large molecules great distances within the brain by displacing interstitial fluid.  
 
PVSRIPO is a genetically recombinant, non-pathogenic poliovirus:rhinovirus chimera with a 
tumor-specific conditional replication phenotype. It consists of the genome of the live attenuated 
poliovirus serotype 1 (SABIN) vaccine (PV1S) with its cognate IRES element replaced with that of 
HRV2. PVSRIPO tumor tropism is mediated by the poliovirus receptor, Necl-5. Necl-5, an onco-
fetal cell adhesion molecule ectopically upregulated in all ectodermal/neuroectodermal cancers, 
is broadly expressed on cancerous cells, cancer ‘stem-cell-like cells’ and tumor-associated 
proliferating vasculature. Infection with PVSRIPO results in swift destruction of tumor cells. 
Poliovirus’ inherent neuropathogenicity was removed by IRES exchange. This ablated the virus’ 
ability to propagate in cells of neuronal lineage and to cause poliomyelitis in non-human primates. 
However, PVSRIPO replicates efficiently in cancerous cells and exhibits potent anti-neoplastic 
effects in animal tumor models. Tumor cell-specificity mediated by the foreign IRES in PVSRIPO 
relies (i) on deficient anti-viral defenses centered on nuclear factor associated with dsRNA 
(NFAR) proteins in tumor cells; (ii) constitutive signal transduction via Ras-Erk to translation 
machinery, which stimulates viral, cap-independent translation via the HRV2 IRES in cancerous 
cells. The anti-neoplastic effects of PVSRIPO are due to direct, virus-mediated tumor cell killing 
and secondary, host-mediated inflammatory effects directed against infected and/or lysed tumor. 
 
In this protocol, we will determine the largest feasible and phase II dose of PVSRIPO given directly 
into intracerebral tumors.  
6.2. 
HYPOTHESIS 
Intratumoral treatment with PVSRIPO will be feasible and safe in patients with recurrent WHO 
grade IV malignant glioma. 
6.3. 
OBJECTIVES 
6.3.1. Primary Objective 
• 
To determine the MTD and phase II dose of PVSRIPO when delivered 
intracerebrally by convection-enhanced delivery. 
6.3.2. Secondary Objectives 
• 
To obtain correlative mechanistic evidence for PVSRIPO’s effects on 
infected WHO grade IV malignant glioma tumors and to estimate PFS and 
OS in recurrent WHO grade IV malignant glioma patients.  
• 
To estimate the efficacy of PVSRIPO administered at the optimal dose. 
6.3.3. Exploratory Objectives   

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The primary exploratory objectives focus on outcomes associated with the 
initial PVSRIPO treatment: 
• 
Describe changes visualized on imaging due to intratumoral inoculation of 
PVSRIPO. 
• 
Evaluate parameters of general immune activation: frequency of immune 
cell subsets in peripheral blood and changes in serum levels of cytokines. 
• 
Evaluate CD8 and CD4 immunologic response against polio-specific and 
GBM-specific antigens to be determined. 
• 
Identify genetic predictors of response or failure of response to treatment 
with PVSRIPO 
 
Additional exploratory objectives focus on retreatment with PVSRIPO: 
• 
Describe the toxicity of retreatment with PVSRIPO in combination with a 
single dose of lomustine. 
• 
Describe the PFS and OS of patients after retreatment with PVSRIPO and 
a single dose of lomustine. 
• 
Describe changes visualized on imaging after retreatment with PVSRIPO 
in combination with a single dose of lomustine. 
• 
Evaluate parameters of general immune activation, including frequency of 
immune cell subsets in peripheral blood, changes in serum levels of 
cytokines, and CD8 and CD4 immunologic response against polio-specific 
and GBM- specific antigens  
7. STUDY SCHEMA  
 
HGG Recurrence 
Consent 
Biopsy 
Confirm Tumor 
Catheter Placement 
Confirm Catheter Placement 
Start Infusion 
Catheter Removal 
Stop Infusion 
~30 min 
~390 min 

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8. BACKGROUND AND RATIONALE 
8.1. 
DISEASE AND CURRENT THERAPY 
The brain is the most frequent site of crippling and incurable disease. Brain tumors account for 
more than 100,000 deaths each year in the US (1). Malignant primary brain tumors are more 
common than Hodgkin’s disease and cause more deaths than cancer of the bladder or kidney, 
leukemia, or melanoma. Despite aggressive surgery, prolonged radiation therapy and toxic 
chemotherapy, most patients with such tumors live only one year from the time of diagnosis and 
patients with recurrent tumors usually survive less than 12 weeks (2-6). The estimated cost of 
treatment for each patient with a malignant brain tumor is between $30,000 and several hundred 
thousand dollars annually. Therefore, the annual treatment cost alone for these patients, not 
mentioning the lost earning potential of afflicted individuals, is greater than the entire annual 
budget of the National Institute of Neurological Disorders and Stroke (NINDS). In fact, 
conventional therapy for a malignant brain tumor is the most expensive medical therapy per 
quality-adjusted life-year saved currently provided in the US (7, 8). Moreover, the non-specific 
nature of conventional therapy for brain tumors often results in incapacitating damage to 
surrounding normal brain (9, 10). The inherent selectivity of approaches based on biological 
agents with tumor specificity offers the prospect of more precise and effective therapy. 
8.2. 
VIRAL ONCOLYSIS 
After being recognized for their anti-neoplastic properties in the early 1900’s, viruses are again 
being considered for use as therapeutic agents against cancer. ‘Oncolytic’ viruses carry the 
promise to efficiently target cancer cells for destruction and spread throughout tumor tissue to 
reach distant neoplastic loci without causing collateral damage to healthy tissues. Lytic, viral 
destruction of tumors elicits host immune responses directed against the infected cancer. To 
unfold their anti-neoplastic activity, oncolytic viruses must engage in intricate interactions with 
their host. Unraveling the molecular basis for specific targeting of-, translation and replication in- 
and killing of tumor cells is pivotal to properly support clinical investigations. PVSRIPO oncolysis 
is due to an unusual confluence of factors that mediate specific tropism for tumor cells and tumor-
specific viral translation and cytotoxicity. The principal elements determining PVSRIPO tumor 
tropism, tumor-specific cell killing and safety are understood. 
8.3. 
PVSRIPO TUMOR TROPISM 
Poliovirus host cell tropism categorically depends on its sole cellular receptor, Necl-5 (11). In the 
normal human organism, narrowly restricted Necl-5 expression occurs at sites of poliovirus 
replication in the gastrointestinal tract and associated lymphatic structures and in the spinal cord. 
Necl-5 expression is low in normal adult tissues, but is rapidly induced upon acute injury, 
malignant transformation, or other events disrupting tissue architecture (12). Necl-5 is an onco-
fetal cell adhesion molecule with physiological roles in morphogenesis (during embryonic 
development) and tissue regeneration and repair (in adult age) (12). Necl-5 is universally 
expressed in ectodermal/neuroectodermal malignancies, but its association with GBM is 
particularly compelling. Necl-5 is abundant in all high-grade GBMs, is present on GBM ‘stem cell-
like’ cells, occurs in proliferating tumor vessels and is implicated in GBM cell dispersal and 
dissemination in the brain (13-16). 
8.4. 
PSVRIPO TUMOR CELL KILLING 
Infection of tumor cells with PVSRIPO results in their swift destruction. This is due to a viral 
strategy that relies on early biosynthesis of highly cytotoxic viral proteins engaging in the shut-off 
of host cell protein synthesis. PVSRIPO achieves this through an unorthodox mechanism of 
translation initiation (Figure 1). At eukaryotic mRNAs, protein synthesis occurs upon recruitment 

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of a multi-partite protein complex at the canonical 5’ ‘cap’ structure (Figure 1A). Poliovirus RNAs 
are un-capped and, hence, rely on cap-independent translation for viral protein synthesis. This 
involves direct recruitment of ribosomal subunits via binding of the eukaryotic initiation factor (eIF) 
4G to viral RNA (Figure 1B). eIF4G binding occurs via the IRES, a cis-acting genetic element 
located in the 5’ untranslated region of the viral RNA (17). 
8.5. 
GENERATING TUMOR SPECIFICITY OF PVSRIPO 
Due to its key role in viral translation, the IRES element determines virus:host interactions and, 
hence, pathogenesis. We generated tumor-specificity by replacing the cognate poliovirus IRES 
with its counterpart of HRV2 (18). To enhance safety, the chimera was based on PV1S (19), 
generating PVSRIPO. PVSRIPO exhibits profound translation deficits in primate neuron-like cell 
lines, and lacks neuropathogenicity in mice transgenic for the human Necl-5 gene and in non-
human primates (18-20). Yet, the virus exhibits potent cytotoxic effects in malignant cells, e.g. 
GBM (21). 
 
Figure 1. Translation Initiation. A. Conventional, cap-dependent initiation occurs via eIF4E, mediating 
assembly of the initiation complex at the m7G-cap. B. Cap-independent translation initiation at poliovirus 
RNA occurs by direct binding of eIF4G to the IRES (22). 
8.6. 
THE MOLECULAR MECHANISMS OF PVSRIPO TUMOR SPECIFICITY 
PVSRIPO exhibits tumor specific viral translation and cell killing because: 
• 
The foreign HRV2 IRES associates with NFAR proteins in the normal CNS. NFAR proteins 
are predominantly cytoplasmic, associate with translation machinery and readily bind 
PVSRIPO RNA in neuronal cells. Association with NFAR proteins causes translation 
repression of PVSRIPO RNA in normal neuronal cells (23-25). In GBM, NFAR proteins are 
inactive, due to sequestration in the nucleus, expression of distinct protein isoforms and 
altered RNA-binding capacity (24-26). Thus, PSVRIPO translation is unimpeded in GBM; 
• 
Mitogenic signal transduction to translation machinery benefits PVSRIPO cap-independent 
translation. Signaling via the PKC-Ras-Erk axis to the translation apparatus stimulates cap-
independent viral translation (27). This may be due to enhanced affinity of eIF4G for the viral 
IRES element and may involve (i) direct phosphorylation of eIF4G affecting protein 
conformation and altering RNA-binding capacity (28); (ii) altered association of eIF4G with its 
binding partner, Mnk1 (29); and (iii) eIF4E phosphorylation by Mnk1, resulting in a shift from 
cap-dependent to IRES-mediated translation (30). 
8.7. 
PVSRIPO ANTI-NEOPLASTIC EFFECTS IN ANIMAL TUMOR MODELS 
In animal tumor models, oncolytic polioviruses elicit efficient anti-neoplastic effects resulting in 
tumor regression and, eventually, destruction in a dose-dependent manner. There is histologic 
evidence for direct, virus-mediated tumor cell killing and indirect, host-mediated inflammatory 
responses directed against tumor (21, 27, 31-35). 

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8.8. 
PVSRIPO NON-HUMAN PRIMATE TOXICOLOGY 
PSVRIPO was subjected to dose-range finding, toxicology, biodistribution, shedding and 
neutralizing antibody tests with intrathalamic inoculation of up to 5 x 10e9 TCID50 of PVSRIPO 
in Cynomolgus macaques (36). These revealed: 
• 
The absence of morbidity and mortality; 
• 
The absence of neuropathological signs consistent with virus-induced CNS damage; 
• 
The absence of virus dissemination form the brain or viremia; 
• 
The absence of extraneural replication; 
• 
The absence of shedding with saliva, urine or stool; 
• 
The presence of a neutralizing antibody response. 
9. STUDY POPULATION  
9.1. 
INCLUSION CRITERIA 
9.1.1. Disease Status 
Patients must have a recurrent supratentorial WHO grade IV malignant glioma based on 
imaging studies with measurable disease (≥ 1 cm and ≤ 5.5 cm of contrast-enhancing 
tumor). Prior histopathology consistent with a World Health Organization (WHO) Grade IV 
malignant glioma confirmed by the study pathologist, Roger McLendon, or his designate.  
9.1.2. Age 
Due to the potential implications of the treatment on the developing CNS, all patients must 
be ≥ 18 years of age at the time of entry into the study. 
9.1.3. Performance Status 
The patient must have a Karnofsky Performance Score (KPS) of ≥ 70% at the time of 
entry. 
9.1.4. Laboratory Studies 
• 
Platelet count ≥ 125,000/µl prior to biopsy.  Platelets ≥ 100,000/µl prior to infusion; 
• 
Prothrombin and Partial Thromboplastin Times ≤ 1.2 x normal prior to biopsy; 
• 
Positive serum anti-poliovirus titer prior to biopsy (except for retreatment); 
• 
Creatinine ≤ 1.2 x normal prior to biopsy; 
• 
Total bilirubin, SGOT, SGPT, alkaline phosphatase ≤ 2.5 x normal prior to biopsy; 
• 
Neutrophil count ≥ 1000 prior to biopsy; 
• 
Hemoglobin ≥ 9 prior to biopsy. 
9.1.5. Poliovirus Immunization Booster 
The patient must have received a boost immunization with trivalent inactivated IPOL™ 
(Sanofi-Pasteur) at least 1 week prior to administration of the study agent. 
9.1.6. Disease Confirmation 
At the time of biopsy, prior to administration of virus, the presence of recurrent tumor must 
be confirmed by histopathological analysis. 
9.1.7. Informed Consent 
A signed informed consent form approved by the Duke University Institutional Review 
Board (IRB) will be required for patient enrollment into the study. Patients must be able to 

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read and understand the informed consent document and must sign the informed consent 
indicating that they are aware of the investigational nature of this study. 
9.1.8. Brain MRI 
Able to undergo brain MRI with and without contrast. 
9.2. 
EXCLUSION CRITERIA 
9.2.1. Pregnancy 
Because of the unknown risk of virus administration potentially affecting a developing fetus 
or growing infant, females who are pregnant or breast-feeding during the study period will 
be excluded. Adults of reproductive potential not employing an effective method of birth 
control will be excluded. Sexually active women of child bearing potential, whose partner 
is male, must use medically accepted birth control. 
 
Sexually active men, whose partner is a female of child bearing potential, must use a 
medically accepted birth control. 
9.2.2. Disease Status 
Because patients will receive drug intracerebrally, patients with an impending, life-
threatening cerebral herniation syndrome, based on the assessment of the study 
neurosurgeons, Allan Friedman, John Sampson, or Peter Fecci, or their designate, will be 
excluded. 
9.2.3. Medical Conditions 
Because the potential toxicities from the agent being studied in this protocol may be similar 
to some known diseases or may be more dangerous in the context of certain known 
diseases, if the patients meet any of the following they will be excluded: 
 
• 
Active infection requiring intravenous treatment or having an unexplained febrile 
illness (Tmax > 99.5 F/37.5 C). 
• 
Known immunosuppressive disease or known human immunodeficiency virus 
infection. 
• 
Unstable or severe intercurrent medical conditions such as severe heart (New 
York Heart Association Class 3 or 4) or known lung (FEV1 < 50%) disease, 
uncontrolled diabetes mellitus. 
• 
Albumin allergy. Albumin is added to the agent as a stabilizer. Patients with a 
known allergy will be excluded. 
• 
Gadolinium allergy. Gadolinium is used as contrast for the MRI. 
9.2.4. Previous Poliomyelitis 
A history of neurological complications due to poliovirus infection would imply previous 
virus replication in the CNS. Based on animal studies, previous exposure to poliovirus 
administered intracerebrally can reduce subsequent virus replication in the CNS. 
9.2.5. Prior Therapy 
Patients who have not recovered from the toxic effects of prior chemotherapy and/or 
radiation therapy will be excluded. Guidelines for this recovery period are dependent upon 
the specific therapeutic agent being used.  
 
• 
Patients may not have received chemotherapy or bevacizumab ≤ 4 weeks [except 
for nitrosourea (6 weeks) or metronomic dosed chemotherapy such as daily 

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etoposide or cyclophosphamide (1 week)] prior to starting the study drug unless 
patients have recovered from side effects of such therapy.  
• 
Patients may not have received immunotherapy ≤ 4 weeks prior to starting the 
study drug unless patients have recovered from side effects of such therapy.  
• 
Patients may not be less than 12 weeks from radiation therapy, unless progressive 
disease outside of the radiation field or 2 progressive scans at least 4 weeks apart 
or histopathologic confirmation. 
• 
Patients must have completed all standard of care treatments, including surgical 
procedure and radiation therapy (at least 59Gy) 
o If the MGMT promoter in their tumor is known to be unmethylated, patients 
are not mandated to have received chemotherapy prior to participating in 
this trial. 
o If the MGMT promoter in their tumor is known to be methylated or the 
MGMT promoter status is unknown at the time of screening, patients must 
have received at least one chemotherapy regimen prior to participating in 
this trial. 
9.2.6. Location and Extent of Tumor 
Because of the potential toxicities from the agent, patients with neoplastic lesions in the 
brainstem, cerebellum or spinal cord, radiological evidence of active (growing) multifocal 
disease, or leptomeningeal disease will be excluded. Patients with evidence of diffuse 
subependymal disease or tumor in the brainstem, cerebellum, spinal cord, or CSF will also 
be excluded. 
 
Since the study agent is a local treatment, patients with radiological evidence of active 
(growing) multifocal disease, tumors extending into or crossing the corpus callosum or 
leptomeningeal disease, will be excluded.   
9.2.7. Patients must not have diagnosis of agammaglobulinemia 
Patients with the following will be excluded: 
• 
Undetectable anti-tetanus toxoid IgG (except for retreatment)   
• 
Known history of agammaglobulinemia 
9.2.8. Patients on greater than 4mg per day of dexamethasone within the 2 weeks 
prior to admission for PVSRIPO infusion 
9.2.9. Patients with worsening steroid myopathy (history of gradual progression 
of bilateral proximal muscle weakness, and atrophy of proximal muscle 
groups) 
9.2.10. Patients with prior, unrelated malignancy requiring current active treatment 
with the exception of cervical carcinoma in situ and adequately treated basal 
cell or squamous cell carcinoma of the skin 
10. 
INVESTIGATIONAL PRODUCT 
PVSRIPO is a version of the serotype 1 life-attenuated (Sabin) poliovirus vaccine (PV1), its 
immunogenic properties and potential for long-term sequelae are expected to be similar. PV1S 
has been safely administered to >10 billion individuals worldwide without untoward long-term 
immunogenic sequelae. The main immunogenic effect of administration of PV1S to human 
subjects is neutralizing immunity to poliovirus. 

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10.1. 
PREPARATION OF PVSRIPO 
PVSRIPO is PV1S containing a heterologous IRES of HRV2 (Figure 2). The IRES is a cis-acting, 
non-coding genetic element within the 5’ untranslated region (UTR) of all enteroviruses and is 
essential for translation of the viral genome. PVSRIPO is a non-enveloped, positive-sense ssRNA 
virus with a genome of ~7300 nucleotides (nt) in length (Figure 2). PVSRIPO particles consist of 
a proteinaceous capsid composed of 60 copies of each of 4 capsid proteins (VP1-VP4) arranged 
in icosahedral geometry. Since the coding regions for the viral polyprotein (giving rise to all viral 
polypeptides) of PVSRIPO and PV1S are the same, the physical structure of the viral capsid and 
all non-structural viral polypeptides of PVSRIPO and PV1S are identical. 
 
Figure 2. Genetic structure of PVSRIPO 
PVSRIPO is formulated in 50 mM sodium phosphate in 0.9% sodium chloride, pH 7.4 with 0.2% 
human serum albumin. It is provided in sterile, single use containers. PVSRIPO must be stored 
at less than or equal to -70°C. Once thawed, it is a clear colorless liquid with no evidence of 
particulates or foreign matter. PVSRIPO was manufactured at the Biopharmaceutical 
Development Program/SAIC-Frederick at NCI-Frederick. For tracking intratumoral/intracerebral 
distribution of the inoculum, the study agent suspension will be supplemented with gadolinium-
DTPA diamide (Magnevist) at a final concentration of 1 mM. Magnevist is stored at room 
temperature. 
11. 
INVESTIGATIONAL PLAN 
11.1. 
INITIAL PVSRIPO TREATMENT PLAN 
11.1.1. Catheter Implantation 
Systemic delivery of high molecular weight therapeutic agents to brain tumors is limited 
by the blood-brain barrier and increased interstitial pressure within the tumor. Poliovirus 
CNS invasion after intravascular administration via trans-endothelial passage is 
exceedingly inefficient (37). Intratumoral delivery bypasses these physiologic barriers and 
concentrates the therapeutic agent at the tumor site while minimizing systemic exposure. 
Therefore, PVSRIPO will be delivered directly into the tumor. A stereotactic biopsy will be 
performed prior to virus administration for frozen section confirmation of viable tumor and 
further analysis (see below). The biopsy needle will be placed with stereotactic guidance 
by a Cosman-Robert-Wells, MRI-compatible, stereotactic head frame or similar frameless 
device. Collection of biopsy tissue for clinical pathologic diagnosis will be performed under 
traditionally accepted conditions according to standard of care.  Up to three additional core 
biopsies will be obtained for molecular genetic testing.  Molecular genetic tests, such as, 
but not limited to, DNA sequencing, gene amplification, and gene expression, will be 
performed on the tissue obtained from the three additional core biopsies.  The goal of 
these molecular genetic tests is to identify genetic predictors of response or failure of 
response to treatment with PVSRIPO.     

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Immediately following the stereotactically-guided tumor biopsy, a catheter [Vygon PIC-030 
(Sophysa, Inc.; Crown Point, IN)] will be implanted in the operating room at a site the same 
or different from that used for the biopsy using sterile techniques under general anesthesia 
with monitoring of cardiac rhythm and oxygen saturation. Implantation will occur at a co-
ordinate selected by the operating surgeon with the assistance of the clinically-approved 
iPlan Flow (BrainLAB). The catheter will be implanted at least 1 cm away from the 
ventricles. Based on our experience, a tumor ≤ 1cm from the ventricles can safely and 
feasibly have a catheter placed ≥ 1cm from the ventricles and would absolutely minimize 
the possibility of infusates going into the ventricles. The catheter will be tunneled beneath 
the scalp for a distance of at least 5 cm to aid in the prevention of infection. The catheter 
will be pre-filled with preservative-free saline. A CT scan may be used to confirm catheter 
placement post-operatively.  
11.1.2. Agent Infusion 
The entire volume of the agent to be delivered will be pre-loaded into a syringe by the 
investigational pharmacist and connected to the catheter under sterile conditions in the 
Neuro-Surgical Intensive Care Unit (NSICU) or neuro step down unit just prior to beginning 
of infusion. Due to the complexity of scheduling all of the necessary components for the 
infusion (operating room time, pharmacy time, and radiology appointments), a +1 day 
window has been built in to the study for the study drug infusion.  This means that the 
infusion is allowed to start the following day after the biopsy/catheter placement.  This will 
still be considered “day 0” in regards to the protocol and the timing of the subsequent 
events. At the time of virus injection, emergency drugs, including epinephrine and 
diphenhydramine will be available and the neurologic status, oxygen saturation, and 
cardiac rhythm will be monitored. Drug infusion will occur in the Neuro-Surgical Intensive 
Care Unit (NSICU) or neuro step down unit so that all other emergency facilities will be 
available. Patients will be treated with appropriate antibiotic prior to biopsy and catheter 
insertion per DUHS Neuro-surgical standard practice.  
 
Based on our own experience, previously published reports (38) and  IRB- and FDA-
approved trials using similar infusion techniques (IRB# 4774-03-4R0), patients will be 
infused at a rate of 500 µL/hr. A Medfusion 3500 or 3010 infusion pump will be pre-
programmed to a delivery rate of 500 µL/hr. The agent (which will be in a total volume of 
8 mL to account for ‘dead-space’ of 3.3723 mL in the infusion system) will be loaded in a 
20 mL syringe into the syringe pump at the initial onset to avoid any interruptions in the 
infusion. The total amount of the inoculum delivered to the patient will be 3 mL. The 
catheter itself (30 cm length, 1 mm interior diameter) cannot be preloaded with virus 
suspension. The infusion pump will be programmed for delivery of 500 µL/hr per hour over 
approximately 6.5 hours.  To account for the dead space, the pump will be stopped when 
the delivered amount is between 3.125 to 3.250 mL. The infusion will be performed using 
a Medfusion 3500 or 3010 (Smiths Medical ASD, Inc, Minneapolis, MN) syringe infusion 
pump. The virus injection procedure will be completed within approximately 6.5 hrs. The 
catheter will be removed following the post-PVSRIPO infusion MRI. 
 
The infusion catheter (PIC 030) and infusion tubing (PIT 400) will be supplied by Sophysa, 
Inc. (Crown Point, IN). The Infusion Catheter Kit is a 30 cm clear, open-ended catheter 
(1.0 mm ID/2.0 mm OD) with 1 cm markings for 20 cm. The catheter comes with a 30 cm 
stainless steel stylet, a barbed female luer lock with cap and a stainless steel trocar. The 
Infusion Tubing Kit consists of a 3-way stopcock connector with air filter, 4 m of microbore 
tubing with antisiphon valve, a red, vented cap and a white luer lock cap. The catheter 

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products are packaged sterile and non-pyrogenic and are intended for single (one-time) 
use only.  
 
Acute Reaction. Any acute reaction symptoms determined to be an acute reaction to the 
study drug will be manage by the Neuro-Surgical Intensive Care Unit (NSICU) or the neuro 
step down unit. 
11.1.3. Gadolinium Distribution Quantitation 
MRI imaging, within 4 hours of the completion of infusion, will be registered to define the 
shape and position of the contrast agent distribution relative to the patient’s brain anatomy. 
While gadolinium–diethylene triamine pentaacetic acid (Gd-DTPA) is a widely available 
MRI contrast agent, there has been speculation that its small molecular weight (938 
Dalton) could limit its ability to predict the distribution of the larger molecules typically 
infused therapeutically with CED [reviewed in (39)]. Development of large molecule tracers 
labeled with gadolinium (Gd) has been problematic, but it has been stipulated that infusion 
of low molecular weight Gd-DTPA can predict the distribution of larger molecules by 
systematic post-infusion manipulation of the images based on theoretical differences in 
the predicted distribution of the Gd-DTPA and the therapeutic drug being infused (39). 
This has been confirmed by simultaneously infusing a patient with a supratentorial 
recurrent malignant glioma with an epidermal growth factor receptor (variant III) 
(EGFRvIII)-targeted immunotoxin in combination with 124I-human serum albumin (HSA) [to 
permit positron emission tomography (PET) imaging] and Gd-DTPA. Gd-DTPA co-
infusion provided direct information about the distribution of large molecules with high 
resolution (39). In combination with fluid-attenuated inversion recovery (FLAIR) imaging, 
Gd-DTPA co-infusion provides additional information about leak into cerebrospinal fluid 
spaces and resection cavities (39). The MRI obtained prior to CED (planning MRI) of the 
study drug and the post infusion MRI may be sent to Therataxis for analysis and simulation 
of the intraparenchymal infusions. If sent, these MRIs will be sent de-identified to 
Therataxis per Duke radiology department de-identification processes. They will be 
labelled with the patient study ID and will include a limited data set that is described in the 
Data Transfer and Analysis Agreement with Therataxis. These MRIs will be shipped via 
Fed-Ex to Therataxis, Attention Raghu Raghavan, 1101E 33rd Street, Suite 8305, 
Baltimore, MD, 21218. Standard IRB language will be placed in the informed consent form 
to inform study participants. 
11.1.4. Biopsy Sampling and Analyses 
Biopsy material will be obtained from tumor tissue prior to virus administration. This tissue 
material will be subjected to routine histology to confirm tumor recurrence by the study 
neuropathologist, Dr. R. McLendon or his designate.  Molecular genetic tests, as 
described in section 11.1.1, will also be conducted on extracts of tumor cells from the 
protocol-specified biopsy prior to PVSRIPO infusion.  After acquiring sufficient tissue for 
standard clinical pathologic testing, up to three additional core biopsies will be obtained, if 
possible.  These additional core biopsies will be frozen in optimal cutting temperature 
(OCT) fixative and kept at liquid nitrogen temperature.  They will be used for genetic 
analysis, including full genome or full exome sequencing, as well as other molecular 
genetic testing. This testing will include, but is not limited to, DNA sequencing, gene 
amplification, and gene expression.    Additional pathology tests on the protocol-specified 
biopsy tissue may include, but are not limited to, EGFRvIII and EGFRwt status, TERT, 
IDH 1 and 2, MGMT IHC and MGMT promoter methylation, and anti-poliovirus receptor, 
if a sufficient amount of tissue remains after standard clinical pathologic testing.   
 
Patients may undergo tumor resection or biopsy following PVSRIPO administration and 
outside the context of this protocol. Resection or biopsy may be medically indicated for 

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reasons not part of the investigative protocol. Should subjects have a resection or biopsy 
at a later time, we will request samples of this resected tissue for tissue analysis. If 
subjects consent, portions of resected tissue will be delivered to the study 
neuropathologist, Dr. R. McLendon or his designate, for histopathological analyses and to 
Dr. M. Gromeier or his designate for correlative molecular analyses. 
11.1.5. Dose 
The anti-neoplastic effects of PVSRIPO in animal tumor models are dose-dependent. 
PVSRIPO produced complete tumor regress in all treated animals at a ‘mouse-adjusted’ 
dose equivalent to 5 x 108 TCID50 [1/10th of the non-toxic dose in NHPs in the dose-range 
finding toxicology study (Table 1)]. ‘Mouse-adjusted’ means that the dose was 1/70th of 5 
x 108 TCID50, since the starting tumor volume in athymic mice was 1/70th of the maximum 
tumor size in patients. Reducing this dose 100-fold significantly diminished the anti-
neoplastic efficacy of PVSRIPO in animal tumor models. 
 
Since no prior experience with intracerebral inoculation of PVSRIPO in humans exists, 
consideration of dose-escalation is based on the results of neurovirulence assessments 
in dose-range finding and biodistribution studies in non-human primates. These 
assessments evaluated the clinical outcome and toxicity after intrathalamic administration 
of PVSRIPO in Cynomolgus macaques (Table 1 & Table 2). 
 
The highest dose in the definitive, IND-directed study in NHPs (1 x 109 TCID50; Table 2) 
was limited by the highest feasible concentration in an acceptable inoculation volume 
(limited to 250 µL in Cynomolgus). In this NHP study, DLTs were not observed and the 
MTD was not reached. Therefore, in this trial, based on pre-clinical NHP toxicity studies, 
the proposed starting dose is 1 x 108 TCID50, which is 1/10th of the highest non-toxic dose 
in NHPs in the definitive, IND-directed toxicology study (Table 2) and 1/50th of the highest 
non-toxic dose in NHPs in the dose-range finding toxicology study (Table 1).  
 
Table 1. Dose range-finding, toxicology, biodistribution and shedding study (BATTELLE Study 916-
G663301) of PVSRIPO in Cynomolgus macaques
PVSRIPO 
Dose 
Equiv. Human 
Dose 
No. of 
Animals 
Route 
Day of 
Sacrifice 
Clinical Outcome 
Vehicle 
N/A 
1 
intrathalamic 
3 
no toxicity/neurologic 
symptoms 
Vehicle 
N/A 
1 
intrathalamic 
21 
1 x 107 TCID50 
1 x 108 TCID50 
1 
intrathalamic 
3 
1 x 107 TCID50 
1 x 108 TCID50 
1 
intrathalamic 
21 
1 x 109 TCID50 
1 x 1010 TCID50 
1 
intrathalamic 
3 
1 x 109 TCID50 
1 x 1010 TCID50 
1 
intrathalamic 
21 
5 x 109 TCID50 
5 x 1010 TCID50 
1 
intrathalamic 
3 
5 x 109 TCID50 
5 x 1010 TCID50 
2 
intrathalamic 
21 
 
Table 2. Definitive, IND-directed toxicology, biodistribution, shedding and neutralizing antibody 
study (BATTELLE Study 1098-G663323) of PVSRIPO in Cynomolgus macaques
PVSRIPO 
Dose 
Equiv. Human 
Dose 
No. of 
Animals 
Route 
Day of 
Sacrifice 
Clinical Outcome 
Vehicle 
N/A 
2 
intrathalamic 
3 
no toxicity/neurologic 
symptoms 
Vehicle 
N/A 
2 
intrathalamic 
10 
Vehicle 
N/A 
2 
intrathalamic 
56 
5 x 107 TCID50 
5 x 108 TCID50 
4 
intrathalamic 
3 
5 x 107 TCID50 
5 x 108 TCID50 
4 
intrathalamic 
10 
5 x 107 TCID50 
5 x 108 TCID50 
4 
intrathalamic 
56 

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1 x 109 TCID50 
1 x 1010 TCID50 
4 
intrathalamic 
3 
1 x 109 TCID50 
1 x 1010 TCID50 
4 
intrathalamic 
10 
1 x 109 TCID50 
1 x 1010 TCID50 
4 
intrathalamic 
56 
 
 
11.1.5.1. Rules for Escalation 
We propose adopting a phase I escalation strategy that avoids well-publicized 
shortcomings of standard ‘3+3’ designs, in particular the problem of ‘memory loss’ with 
such designs (40). To minimize the number of patients treated at low-dose levels, 
minimize the number of patients needed to complete the study and efficiently use 
accumulated data to evaluate the appropriateness of dose escalation (40, 41), we plan to 
use a two-step continual reassessment method (CRM) design that includes an escalation 
step and a model-guided step (42, 43). In the escalation step, dose levels will be rapidly 
escalated as preclinical data suggest that dose-limiting toxicity (DLT) will not occur at any 
of the five dose levels that will be evaluated (44). Decisions concerning dose escalation 
for subsequent patients will be based upon the occurrence of DLT during the first 4 weeks 
after treatment administration.  Initially, each patient will be observed for ≥4 weeks before 
the next patient is treated.  Starting at dose level 1, one patient will be treated at each 
higher dose level (Table 3).  If no DLT is observed within 4 weeks of administration, then 
the next patient will be treated at the next higher dose. If no DLTs are observed among 
patients treated on the first four dose levels, subsequent patients will be treated at dose 
level 5. The escalation step will be interrupted if a patient assigned to one of the first 4 
dose levels or more than 20% of patients treated at dose level 5 experiences a DLT. In 
that case, further dose escalation or de-escalation will be guided by the likelihood-based 
implementation of the CRM, in which the one-parameter hyperbolic tangent model will be 
used to estimate the probability of DLT at each of the 5 dose levels based upon available 
data (45). The highest dose level for which this estimated probability is less than 20% will 
be identified. If this optimal dose is less than or the same as the current dose, subsequent 
patients will be treated at that dose level. If greater than the current dose, subsequent 
patients will be treated at the one dose level higher than the current dose. The optimal 
dose will be recomputed whenever the status of DLT has been determined for a patient. 
 
As more patients are treated with PVSRIPO the impact of a DLT on subsequent patient 
treatment assignments by the model described above will decrease with the expectation 
that treatment assignments will stabilize over time at a particular dose level.  If the outcome 
(i.e. DLT or not DLT) of a particular patient has no impact upon treatment assignment for 
the subsequent patient or patients, a 4-week observation period will not be required before 
accruing that additional patient or patients.   
 
For example, consider the situation where one patient has already been treated at each 
of the first 4 dose levels without DLT and 4 patients have been treated at dose level 5 
without DLT.  Under the model described above, a 5th patient would be treated on dose 
level 5.  If this 5th patient experiences a DLT, the treatment assignment for the subsequent 
patient would be dose level 5 according to the rules described above.  If this 5th patient 
does not experience a DLT, the subsequent treatment would also be dose level 5.  Hence, 
a 4-week observation period for the 5th patient on dose level 5 is unnecessary to determine 
the dose treatment assignment for the following patient.  In this case, a 4-week observation 
period for the 5th patient will not be required as it has no impact upon treatment assignment 
for the subsequent patient. 
 
The clinical team will keep the statistical team apprised of the current status of all patients.  
The statistical team will determine and inform clinical investigators as to whether a 4-week 
observation period is required for some or all patients before another patient is accrued.  
More specifically, based upon the rules described above, the statistical team will inform 

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clinical investigators as to how many patients can be accrued while other patients are 
within their 4-week post-treatment observation period. 
Table 3. Dose escalation schedule for PVSRIPO
Dose Level 
PVSRIPO dose 
Anticipated No. of 
patients treated in 
original design*  
Administration route 
1 
1.0 x 108 TCID50 
1 
intratumoral 
2 
3.3 x 108 TCID50 
1 
intratumoral 
3 
1.0 x 109 TCID50 
1 
intratumoral 
4 
3.3 x 109 TCID50 
1 
intratumoral 
5 
1.0 x 1010 TCID50 
21 
intratumoral 
 
*Please refer to study design modifications below. 
11.2. 
RETREATMENT WITH PVSRIPO 
Selected patients who benefited from the initial infusion of PVSRIPO within this protocol will be 
eligible for a second infusion.  The criteria for a patient to be considered for a second infusion is 
provided in section 11.2.1.  Details about that treatment plan are provided in section 11.2. 
11.2.1. Eligibility Criteria for Retreatment 
• 
Subjects can be considered for PVSRIPO retreatment if they meet the pertinent 
inclusion and exclusion criteria for study retreatment.  Specifically, the subject must 
satisfy inclusion criteria provided in Sections 9.1.2 - 9.1.4 and the exclusion criteria in 
Sections 9.2.1 - 9.2.10.  In addition, the subject must also satisfy the following criteria: 
Patient must have benefited from initial treatment with PVSRIPO (i.e. be at least 12 
months following their initial PVSRIPO infusion). 
• 
Patients must have a recurrence of their supratentorial WHO grade IV malignant 
glioma based on imaging studies with measurable disease (≥ 1 cm of contrast-
enhancing tumor).   
• 
At the time of biopsy, prior to administration of virus, the presence of recurrent tumor 
must be confirmed by histopathological analysis, unless already proven within the last 
3 months via tissue sampling (biopsy or resection). 
• 
The patient must have received a new boost immunization with trivalent inactivated 
IPOL™ (Sanofi-Pasteur) at least 1 week and no more than 4 weeks prior to re-
administration of the study agent.  The boost immunization may be obtained locally, 
prior to signing consent, as long as the patient has been informed about this study 
procedure via phone script. 
• 
A new signed informed consent form for retreatment approved by the Duke University 
Institutional Review Board (IRB) will be required for retreatment. Patients must be able 
to read and understand the informed consent document and must sign the informed 
consent indicating that they are aware of the investigational nature of this study. 
11.2.2. Retreatment Plan 
Catheter implantation (section 11.1.1), PVSRIPO infusion at dose level -1 (section 11.1.2), 
gadolinium distribution quantitation (section 11.1.3), and biopsy sampling (section 11.1.4) 
will occur according to the procedures previously described in this protocol.  In addition to 
the PVSRIPO retreat infusion, the patients will also receive a single dose of lomustine at 
approximately 8 weeks post-infusion of PVSRIPO.  All procedures previously described 
will be followed; however, the addition of the single dose of lomustine will alter the 
schedule of events that was previously described.  All retreated patients will follow the 
schedule of events shown in Table 6 and described in section 12. 

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11.3. 
HISTORICAL MODIFICATIONS TO THE PROTOCOL 
11.3.1. Study Design Modifications (11/15/2013 AMD025) 
As of 11/15/2013, 4 patients have been treated on dose level 5.  One patient experienced 
a DLT upon removal of the treatment catheter.  The remaining 3 patients have not 
experienced a DLT; however, all 3 have had difficulties tapering off steroids.  To address 
this concern, the study is amended to reduce the dose for the next cohort of patients to 
dose level 2.  Up to 6 patients will be accrued at dose level 2.  If 2 of these patients 
experience a DLT as defined in section 11.4, accrual will be suspended. Following a two-
week observation period, during which the patient will be evaluated in clinic on day 7 (± 2 
days) and day 14 (± 2 days), a patient summary will be prepared and submitted to the 
DSMB chair for approval. Once approval from the DSMB chair has been obtained, 
treatment of the next patient will be allowed. Only one patient will be treated every 2 weeks 
and there will be only one PVSRIPO treatment per patient without specific approval for a 
second or more treatments from the FDA and the IRB. 
11.3.2. Study Design Modifications (6/05/2014 AMD041) 
As of 06/05/2014, 5 subjects have been treated on dose level 2.  Four subjects have 
completed the two-week follow-up period and have not had a DLT. One subject remains 
in the 14 day follow up period. Once one last patient has been treated, the study per the 
November 2013 amendment is complete.  We are amending the study to allow continued 
patient accrual to garner more information about patient safety while the single-institution 
phase 2 study is being developed and reviewed by various regulatory bodies.  We intend 
to continue accruing subjects for treatment at dose level 2 until such time that the phase 
2 study has been activated. After each patient has been followed for a one-week 
observation period, during which the patient will have been evaluated in clinic on day 7 (± 
2 days), a patient summary will be prepared and submitted to the DSMB chair for approval. 
Once approval from the DSMB chair has been obtained, treatment of the next patient will 
be allowed. We will treat up to four subjects within a 30-day period as allowed by DSMB 
chair, and there will be only one PVSRIPO treatment per patient without specific approval 
for a second or more treatments from the FDA and the IRB. 
11.3.3. Study Design Modifications (10/06/2014 AMD048) 
As of 10/06/2014, 7 subjects have been treated on protocol at dose level 2.  One additional 
subject has been treated under a Single Subject Emergency Use Request at dose level 
2.  Of these eight subjects, six subjects had difficulty or remained unable to be tapered off 
steroids more than three months post infusion (three subjects required the addition of 
bevacizumab to facilitate the taper down or off steroids). We are amending the study to 
reduce the dose to dose level -1 (5.0 X 107 TCID50) with the goal of limiting the occurrence 
of known possible side effects of prolonged steroid use in patients who otherwise benefit 
from this investigational therapy. This dose reduction will allow continued patient accrual 
and garner more information about patient safety and optimal dosing while the single-
institution phase 2 study is being developed and reviewed by various regulatory bodies. 
Our last amendment allowed the enrollment of up to 18 patients, one patient was enrolled 
under that amendment and thus, we are planning to enroll up to 17 patients on dose level 
-1.  After each patient has been followed for a one-week observation period, during which 
the patient will have been evaluated in clinic on day 7 (± 2 days), a patient summary will 
be prepared and submitted to the DSMB chair for approval. Once approval from the DSMB 
chair has been obtained, treatment of the next patient will be allowed. We will treat up to 
four subjects within a 30-day period as allowed by DSMB chair, and there will be only one 
PVSRIPO treatment per patient without specific approval for a second or more treatments 
from the FDA and the IRB. 

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11.3.4. Study Design Modifications (1/13/2015 AMD054) 
Once the optimal dose level of PVSRIPO is determined, a total of 26 patients will be 
treated at that dose level, on a dose expansion cohort.  The survival associated with these 
26 patients will be compared to that of a historical cohort (see section 14.3). The study is 
being amended to allow accrual and treatment of a total of 26 patients at the optimal dose 
level of PVSRIPO and, as needed, radiation-necrosis dose levels and schedules of 
bevacizumab to estimate the efficacy of PVSRIPO relative to a historical cohort. 
11.3.5. Sample Size Modification (6/24/2015 AMD071) 
As of 6/24/15, we have treated 13 patients on dose level -1. None of the patients have 
had prolonged steroid needs, two patients died at 3 and 6 months respectively, and five 
patients total (including the two patients who died) were initiated on bevacizumab. It is 
now felt that dose level -1 is the optimal dose, as none of the patients have experienced 
undue side effects due to PVSRIPO or the management of the cerebral inflammation 
secondary to PVSRIPO. We are now amending the protocol to enroll a total of 50 patients 
on dose level -1. 
11.3.6. Study Design Modification (11/10/2015 AMD078) 
Imaging response criteria were previously developed based on imaging observations 
following chemoradiation treatment (Macdonald criteria) and subsequently revised based 
on new observations from anti-angiogenic trials (RANO criteria). As imaging is 
dramatically different following treatment with immunotherapy, new imaging criteria are 
being developed (iRANO). This process is likely to take several years to validate. For that 
reason, we are removing response rate as a secondary objective and adding, as an 
exploratory objective, a description of changes visualized on imaging due to intratumoral 
inoculation of PVSRIPO. 
11.3.7. Study Design Modification (2/1/2016 AMD081) 
The protocol has been re-formatted in an electronic Common Technical Document (eCTD) 
format for electronic submission to the FDA for the associated IND.  The statistical section 
has been revised and an appendix (section 18.4) has been added to define and describe 
the historical control group being used as a statistical comparison for the subject 
population.  We have also further described the types of subject follow-up activity that will 
occur in this study. 
11.3.8. Study Design Modification (6/15/2016 AMD096) 
As of 6/15/2016, 23 subjects have been treated on dose level -1, including 12 subjects 
that were treated more than 12 months ago.  Three of these 12 subjects demonstrated 
tumor reduction without significant inflammation necessitating prolonged use of 
bevacizumab and/or additional chemotherapy.  In contrast, the remaining 9 patients had 
inflammation that was burdensome and required prolonged use of bevacizumab and/or 
additional chemotherapy.  Caregivers of some of these patients were overwhelmed by the 
experience.  The 3 patients without significant inflammation are alive at 20.2, 15.5, and 
13.1 months after PVSRIPO infusion, and 7 of the 9 patients with burdensome 
inflammation have died, including 5 that died within 12 months of PVSRIPO treatment. 
 
Despite the fact that subjects on dose level -1 have been able to remain off significant 
doses of steroids, we believe that the subjects benefiting the most from PVSRIPO have 
been those who have experienced minimal or easily controllable inflammation.   
 
As such, we amend this study to reduce the PVSRIPO dose to dose level -2 (1.0 X 107 
TCID50) with the goal of limiting the occurrence of undesirable burden from the 
inflammation and its treatment on as many subjects (including caregivers) as possible. 

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Based upon additional animal studies, we are confident that dose level -2 (1.0 X 107 
TCID50) will be a therapeutic dose. This amendment is not due to concerns for the safety 
of the patients on dose level -1, but due to the observation that less inflammatory reaction 
is a predictor of better survival and treatment response.  
 
We plan to treat 27 patients at dose level -2.  After each patient has been followed for a 
one-week observation period, during which the patient will have been evaluated in clinic 
on day 7 (± 2 days), a patient summary will be prepared and submitted to the DSMB chair 
for approval. Once approval from the DSMB chair has been obtained, treatment of the 
next patient will be allowed. We will treat up to four subjects within a 30-day period as 
allowed by DSMB chair, and there will be only one PVSRIPO treatment per patient without 
specific approval for a second or more treatments from the FDA and the IRB.  
 
Furthermore, patients previously treated on trial received high dose steroids prior to 
catheter insertion and throughout the infusion, as part of undergoing biopsy. Given 
concerns for negative impact of high dose steroids on the efficacy of PVSRIPO, we will 
limit steroid usage to the minimal dose necessary, if even needed.  
11.3.9. Study Design Modification (6/29/2016 AMD101) 
This amendment includes several revisions to tests and procedures, which we believe can 
be safely implemented with the aim of reducing the time between consenting to participate 
in the study, screening to determine eligibility, and study treatment.  Previously, patients 
had to demonstrate a serum total IgG level of ≥ 400 mg/dL to be eligible for the study.  All 
patients who have undergone this testing thus far have met this eligibility criterion; 
therefore, it is no longer considered necessary to test serum IgG in patients going forward 
prior to their enrollment.  We are also changing the timing of the boost immunization with 
trivalent inactivated IPOL™ (Sanofi-Pasteur) from between 6 months and 2 weeks prior 
to PVSRIPO infusion to between 6 months and 1 week prior to PVSRIPO infusion.  Also, 
for previously enrolled patients, a patient had to be followed for a one-week observation 
period post-infusion, before the next patient could be treated.  Now, once a subject has 
been observed through successful completion of the PVSRIPO infusion, the next subject 
can be treated.  We are updating pharmacy instructions for the preparation and 
administration of PVSRIPO in this amendment as well.  Lastly, per Duke’s Research 
Integrity Office (RIO), a study-specific DSMB may no longer be necessary for this study 
given the oversight provided by the BTC’s DSMB-Plus.  If the FDA concurs, the study-
specific DSMB may be dissolved per its charter.  
11.3.10. 
Study Design Modification (9/5/2016 AMD104)   
This amendment includes the addition of an exploratory objective to identify genetic 
predictors of response or failure of response to treatment with PVSRIPO.  Molecular 
genetic tests, such as, but not limited to, DNA sequencing, gene amplification, and gene 
expression, will be performed on the tissue obtained at the protocol-specified biopsy prior 
to PVSRIPO infusion.  Tissue may be obtained for testing as either fresh, frozen, or fixed 
tissue, or as slides, after the diagnostic pathologist has retained all tissue needed for 
diagnosis. The amount of tissue required for this testing should be sufficient to yield a 
minimum of one microgram of DNA. 
11.3.11. 
Study Design Modification (3/1/2017 AMD124) 
Based upon recent pre-clinical data and clinical data collected on dose levels -1 and -2, 
we have concluded that there is no evidence of pre-clinically or clinically important 
differences between these dose levels. The initial need for treatment with the radiation 
necrosis dose levels and schedules of bevacizumab for cerebral inflammation secondary 
to PVSRIPO or tumor growth has been comparable thus far.  In addition, the toxicity 

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profiles for the two dose levels are also similar.  Therefore, we have decided to proceed 
with the development of PVSRIPO dose level -1 (5.0 X 107 TCID50) within the phase II 
study that is now in development.  We are amending this phase I study to treat all future 
patients at the ­1 dose level.  We intend to continue accruing patients and treating patients 
at this dose level while the phase II study is being finalized and reviewed by various 
regulatory bodies. 
 
This protocol is also being modified to allow multiple patients to be infused with study drug 
simultaneously if the neuro-surgical schedule allows. There are currently 3 neurosurgeons 
listed as sub-investigators who are trained on the surgical procedures for this study.  In 
the study modification dated 6/29/2016, the study was modified so that the next patient 
could be treated immediately following the completed infusion of the previous patient.  
However, we would like to increase our enrollment potential by allowing multiple patients 
to be treated simultaneously.  As of 1/20/2017, no patients have experienced 
complications during the infusion of PVSRIPO.  Only 1 out of 51 treated patients has 
experienced severe complications (Grade III or higher) during catheter removal following 
the infusion of the study drug, and therefore we are confident that treating multiple patients 
would be safe for the patients and would allow for increased data collection.   
 
This amendment also includes updates to the program used for planning catheter 
placement, the timing of catheter removal, the infusion pump information, and to clarify 
the history of DLT monitoring in the study.  The statement related to sending MRIs to 
Therataxis was modified to state that the MRIs may be sent to Therataxis.  A clarification 
has been made regarding the blood test for anti-epileptics.  Lastly, the exclusion criterion 
related to completion of standard of care treatments has been clarified based on whether 
or not the MGMT promotor methylation status is known at the time of screening.  There 
are other minor corrections throughout. 
 
Following the FDA’s review of this amendment, an additional section (see section 11.5) 
describing guidelines and criteria for study pause as a result of toxicity monitoring has 
been added.  Specifically, the study is being monitored for the occurrence of unacceptable 
toxicities within dose levels -1 and -2 at any time after the initiation of protocol treatment. 
11.3.12. 
Study Design Modification (6/22/2017 AMD142) 
It has been recently observed that patients who originally benefitted from the infusion of 
PVSRIPO can demonstrate tumor recurrence years later. Given the limited life expectancy 
of recurrent glioblastoma patients, it has been hypothesized that retreatment of long-term 
survivors previously treated with PVSRIPO could trigger an immune recall effect and thus 
further extend the survival of patients. As such, in the current amendment, we will allow 
selected patients who have previously benefited from PVSRIPO infusion to be retreated 
with PVSRIPO in the event of tumor recurrence. A patient is considered to have benefited 
from the initial PVSRIPO infusion if they survived 12 or more months after PVSRIPO 
treatment.  All patients retreated will be treated at dose level -1 as per the same procedure 
previously used. Furthermore, all retreated patients will receive a single dose of lomustine 
8 weeks post infusion of PVSRIPO. As lomustine temporarily reduces the number of 
regulatory T-cells, it is hypothesized that it could potentially increase the efficacy of 
PVSRIPO and of the immune recall.  Further details regarding this retreatment plan are in 
Section 11.2. 
11.3.13. 
Study Design Modification (9/25/2017 AMD149) 
Based upon the Final Clinical Shedding Study for PVSRIPO (IND 14,735) dated 8/6/2017, 
the IBC reviewed the requirement for stool sampling for patients receiving investigational 
treatment with PVSRIPO.  The purpose of the stool sampling was to test for presence of 

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viral shedding in stool samples of patients receiving PVSRIPO via intracerebral delivery.  
The Final Clinical Shedding Report indicates no presence of PVSRIPO in stool collected 
from 59 subjects who received 61 treatments with PVSRIPO (2 patients were re-treated).  
Per the report, “we feel that the serologic evidence indicates a formidable protective shield 
against shedding in patients with pre-existing and boosted anti-polio immunity.”  On 
8/17/2017, the IBC determined that the requirement for viral shedding studies may be 
discontinued on the basis of the report. Therefore, we have removed stool sampling from 
Section 12.2 and from Table 6.  In addition, we have added 3 additional biopsy core 
samples to be taken at the time of biopsy, prior to catheter placement, if possible.  The 
additional biopsy core samples will be used for genetic analysis, including full genome or 
full exome sequencing, as well as other molecular genetic testing. 
11.4. 
DOSE-LIMITING TOXICITY 
Patients will be monitored for toxicity indefinitely or until confirmation of recurrent disease or the 
patient prematurely discontinues from the study at Duke University Medical Center.  Adverse 
events will be categorized and graded in accordance with the NCI CTC (Version 4).   
 
During the dose escalation phase of this study, patients were also monitored for dose-limiting 
toxicity (DLT).   As no patient experienced a DLT on dose level 2, dose level 2 was determined to 
be the MTD. On 10/06/2014, the protocol was amended to reduce the dose to dose level -1 (see 
section 11.3.3). Since dose level 2 was determined to be the MTD, DLT monitoring was 
discontinued at that time. 
 
With the removal of oversight by the study-specific DSMB, a patient summary with toxicity review 
will no longer need to be generated after each patient (see section 11.3.9).   
11.4.1. Definition of Dose-limiting Toxicity 
Dose-limiting toxicity (DLT) will be defined using the NCI Common Toxicity Criteria (CTC) 
(Version 4).  Any Grade 3 or any Grade 4 toxicity that is not reversible within 2 weeks, or 
any life-threatening event, or treatment-related death will be considered a DLT. Any grade 
2 or higher serious autoimmune toxicities particularly those affecting vital organs (e.g. 
cardiac, renal, CNS) will be considered a DLT if it occurs within 2 weeks with or without 
treatment. Exceptions to these DLT’s are as follows: 
• 
Events associated with the biopsy procedure/catheter placement: seizures or 
hemorrhages occurring during anesthesia or the biopsy/catheter insertion proper 
prior to administration of the agent if Grade 2 or lower (Grade 3 or higher surgical 
complication from insertion of catheter is considered DLT). 
• 
Seizures: Due to the nature of the disease under investigation in this protocol, 
patients may have pre-existing seizures or be susceptible to new seizures as a 
result of the underlying disease process.  Although seizures may be defined as 
Grade 3 or 4 toxicities under NCI CTC, and will be reported as such in this protocol, 
seizures will not be considered DLT if, in the opinion of the Principal Investigator 
they have not increased in frequency or can be attributed to another recognized 
cause of increasing seizure frequency such as sub-therapeutic anti-convulsant 
levels or biopsy proven tumor progression. 
• 
New neurologic deficits: Due to the nature of the disease under investigation in this 
protocol, patients may develop new neurologic deficits as a result of tumor 
invasion. A new neurologic deficit, which resolves within 2 weeks after initiation of 
medical therapy, will not be considered a DLT. Similarly, late (≥ 4 weeks after 
completion of infusion) DLTs thought to be drug or procedure related, which 
resolves within 2 weeks after initiation of medical therapy, will not be considered a 
DLT. New neurological symptoms will not be a DLT if they can be ascribed to tumor 

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progression (e.g. documented with histopathologic analyses of biopsy tissue; or; 
they respond to treatment (e.g. oral steroids, within 2 weeks)). 
• 
Thromboembolism:  Due to the high incidence of deep vein thrombosis (DVT) in 
this patient population, patients may have undiagnosed pre-existing DVTs or be 
susceptible to the development of DVTs due to the underlying disease process.  
Although DVT may be defined as Grade 3 or 4 toxicities under NCI CTC, and will 
be reported as such in this protocol, DVT will not be considered DLT in this 
protocol. 
• 
Syndrome of Inappropriate Antidiuretic Hormone (SIADH): Due to the high 
incidence of SIADH in this patient population, patients may be susceptible to the 
development of SIADH due to the underlying disease process.  Although SIADH 
may be defined as Grade 3 toxicity under NCI CTC, and will be reported as such 
in this protocol, SIADH will not be considered DLT in this protocol unless it is 
refractory to medical management. 
• 
Muscle Weakness and Weight Gain:  Due to the high incidence of muscle 
weakness and weight gain in patients taking steroids in this patient population, 
patients may be susceptible to the development of muscle weakness or weight 
gain which is due to steroids alone.  Although muscle weakness may be defined 
as Grade 3 or Grade 4 toxicity and weight gain ≥ 20% may be defined as Grade 3 
toxicity under NCI CTC, and will be reported as such in this protocol, muscle 
weakness or weight gain will not be considered DLT in this protocol if the patient 
has required steroids greater than physiologic doses in the interval between the 
immunization and the development of the toxicity. 
• 
Tumor Progression:  Due to the nature of the disease under investigation in this 
protocol, patients may have an increase in pre-existing neurologic deficits or have 
an onset of new neurologic deficits due to tumor progression.  Although such 
neurologic deficits may be defined as DLTs under NCI CTC, and will be reported 
as such in this protocol, these clinical changes are not an unexpected 
phenomenon in this disease in the setting of tumor growth. As a result, neurologic 
deficits will not be considered a DLT if unequivocal tumor progression can be 
documented radiographically or histologically. 
11.4.2. Management of Toxicities 
If a Grade 3 or 4 NCI CTC toxicity is observed, the patient will be monitored until the NCI 
CTC toxicity improves to a Grade 1. If this resolves within 4 weeks with or without 
treatment, these toxicities will not be considered dose limiting. If a new Grade 3 or 4 NCI 
CTC toxicity is seen again in the same subject after initial resolution within 4 weeks of the 
initial infusion, a DLT will be declared.  
 
If Grade 2 or higher serious autoimmune NCI CTC toxicity is observed the patient will be 
monitored until the NCI CTC toxicity improves to a Grade 1. If resolution to Grade 1 occurs 
within 4 weeks with or without treatment, these toxicities will not be considered dose 
limiting.  
11.5. 
TOXICITY MONITORING AFTER DOSE ESCALATION 
Though DLT monitoring has been discontinued due to the declaration of dose level 2 as the MTD, 
toxicity monitoring continues.  Specifically, the study is being monitored for the occurrence of 
unacceptable toxicities within dose levels -1 and -2 at any time after the initiation of protocol 
treatment.  We will use the definition of DLT provided in section 11.4.2 as the definition of an 
unacceptable toxicity.  
 

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Given that both long- and short-term toxicities are of interest, it is not feasible to suspend accrual 
while toxicity is assessed as was done within the dose-escalation portion of this study.  If 25% or 
more of patients have experienced an unacceptable adverse event or there are other reasons for 
concern about the safety of patient treatment (e.g. treatment-related toxic death), accrual will be 
suspended and data will be carefully reviewed to determine if accrual should be permanently 
terminated or the protocol modified.  These guidelines have not been adjusted for differential 
length of follow-up of accrued patients.  
 
Every 6 months, the toxicity experienced by patients accrued to this study will be summarized 
and reviewed, regardless of the number of patients accrued, to determine whether the overall 
toxicity profile of treatment is unacceptable or not. 
11.6. 
TOXICITY MONITORING OF PVSRIPO RETREATMENT 
Though there is growing knowledge about the long- and short-term toxicities associated with an 
initial PVSRIPO infusion, there is a possibility that the toxicities associated with a second infusion 
might differ in some manner from the initial infusion.  Hence, the toxicity monitoring that has 
occurred with the initial PVSRIPO infusion will continue with the second PVSRIPO infusion.  The 
guidelines provided in section 11.5 will also be implemented in the monitoring of toxicity 
associated with the second infusion of PVSRIPO. 
11.7. 
DISCHARGE, FOLLOW-UP AND DURATION OF STUDY 
 
Patients will be followed at a minimum of 2, 4, 8, 16, 24, 32, 40 and 48 week intervals after infusion 
as outlined in the Study Test Schema (see section 18.1). 
 
Patients may not be treated with any other modality (other than bevacizumab per the ‘special 
considerations’ in section 15.2.7) unless progressive tumor is noted or they are otherwise 
removed from the study. Patients will be considered off study upon tumor progression or upon 
treatment of the tumor with another modality.  When subjects are considered off study, this 
indicates that subjects will no longer be obligated to undergo study-related tests and procedures, 
but the data described below will still be collected from these subjects as feasible.    Subjects will 
be followed for serious adverse events for 30 days after coming off study. Collection of the 
following additional data from off study subjects will be performed, if possible, but is not mandatory 
and will not be considered a deviation if the data cannot be obtained.  Subject’s medical records 
will be reviewed for the remainder of their life, in order to follow survival, as will subjects’ MRIs.  
Subjects will also be followed for progression and subsequent treatments.  Follow-up activity will 
be at the discretion of the treating physician. 
11.8. 
CRITERIA FOR SUBJECT DISCONTINUATION OF PROTOCOL TREATMENT 
Protocol treatment for a subject can be discontinued for any of the following reasons listed below, 
although patients will continue to be followed as described above in section 11.7, if possible. An 
explanation will be recorded for each patient taken off treatment and the off treatment summary 
completed. 
• 
Progressive or recurrent disease as documented by MRI or physical examination 
for which the patient’s tumor is treated with another modality, with the exception of 
bevacizumab as described in section 15.2.7. 
• 
Development of DLT during the dose escalation phase (please refer to sections 
11.1.5.1 and 11.4). 
• 
The occurrence of two or more grade 4 events or any death in the study that are 
possibly related to PVSRIPO. 

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• 
Upon request of the patient or the patient’s legal representative. 
• 
Administrative reasons, such as a major violation of the clinical trial protocol. 
• 
Non-compliance of the patient. 
12. 
DATA AND SPECIMEN COLLECTION 
12.1. 
DATA AND SPECIMENS TO BE ACCESSIONED PRIOR TO TREATMENT 
• 
Registered in the Velos e-Research software system. 
• 
Complete physical and neurologic examination with KPS rating. 
• 
MRI (with and without gadolinium enhancement) of the brain. 
• 
PT/PTT, CBC with diff, CMP, within 14 days of treatment  
• 
β-HCG serum pregnancy test within 48 hours of treatment for female patients. 
• 
Receive a boost immunization with trivalent inactivated IPOL™ (Sanofi-Pasteur) 
poliovirus vaccine at least 1 week prior to administration of the study agent.  For 
retreatments, this may be obtained locally and prior to signing consent, as long as 
the patient has agreed via the phone script consent. 
• 
Anti-tetanus toxoid IgG, LSQ, anti-poliovirus antibody, 76.5 mL of whole blood for 
tests of anti-tumor immune response, and 10 mL of whole blood for tests of 
immunologic assays within 6 months prior to treatment, prior to the boost 
immunization with trivalent inactivated IPOL™ (Sanofi-Pasteur).  Among patients 
who are retreated with PVSRIPO, the anti-tetanus toxoid IgG, LSQ, anti-poliovirus 
antibodies, 76.5 mL of whole blood for tests of anti-tumor immune response, and 
10 mL of whole blood for tests of immunologic assays obtained prior to the boost 
immunization will not be collected. 
• 
76.5 mL of whole blood for tests of anti-tumor immune responses and 10 mL of 
whole blood for tests of immunologic assays within 14 days of treatment, at any 
time post boost immunization with trivalent inactivated IPOL™ (Sanofi-Pasteur), 
but before PVSRIPO infusion. 
 
Pathology Studies: (This will only be performed once the patient has been treated with PVSRIPO 
so that tissue is not being requested on ineligible patients).  Tissue from the patient’s original 
surgery or any other surgery will be requested in the form of either a block or enough unstained 
slides for the following tests:  Immunohistochemistry (IHC), including but not limited to EGFRvIII, 
TERT, IDH 1 and 2, MGMT,  anti-poliovirus receptor, and MGMT quantitative level.  Any other 
laboratory tests that may guide treatment or determine prognosis may also be performed with this 
tissue.  If slides are requested, they will be prepared on Fischer Plus glass or Histostix coated 
slides and will be unstained FFPE or otherwise appropriately prepared slides.  Also, if any of this 
testing requires de-identified tissue, the slides will be de-identified by the study team and sent to 
Dr. Gromeier or his designee for additional testing.   
12.2. 
DATA AND SPECIMENS TO BE ACCESSIONED DURING AND AFTER 
TREATMENT  
DURING INFUSION AND AFTER TREATMENT AT VISITS AT 1, 2, 4, 8, 12 (RETREATMENT ONLY), 
AND 16 WEEKS AND EVERY 8 OR 9 (RETREATMENT) WEEKS THEREAFTER (STARTING WITH DAY 
7 [WEEK 1] ALL TESTS AND PROCEDURES HAVE A SEVEN DAY PLUS OR MINUS WINDOW.) 

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• 
General physical examination and neurologic examination will be obtained daily 
after infusion until discharge from hospital and at each follow-up visit. 
• 
CT to confirm catheter placement ≥ 1cm from the ventricles prior to beginning 
infusion. 
• 
MRI (without gadolinium-enhancement) of the brain within 4 hours of completing 
the infusion to monitor distribution of infusion.  
• 
MRI (with gadolinium-enhancement) at follow-up visits (MRI only at weeks 4, 8, 16 
[retreatment] and every 8 or 9 [retreatment] weeks thereafter.) 
  
• 
76.5 mL of whole blood for tests of anti-tumor immune responses at Weeks 1, 4, 
8, 12 (retreatment), 16, and 24 or 25 (retreatment) after treatment. At the discretion 
of the PI, an additional blood sample at least 2 years post study drug infusion may 
be obtained.  31 mL collection of whole blood at Day 14. 
• 
10 mL of whole blood for tests of immunologic assays and anti-poliovirus antibody 
titer at Weeks 1, 4, 8, 12 (retreatment), 16, 24 or 25 (retreatment), and 34 
(retreatment). Five mL collection of whole blood at Day 14. At the discretion of the 
PI, an additional blood sample at least 2 years post study drug infusion may be 
obtained.   
• 
Biopsy material will be obtained from tumor tissue prior to virus administration.  
This tissue material will be subjected to routine histology to confirm tumor 
recurrence by the study neuropathologist, Dr. R. McLendon or his designate.  
Molecular genetic tests will also be conducted on extracts of tumor cells from the 
protocol-specified biopsy prior to PVSRIPO infusion.  After acquiring sufficient 
tissue for standard clinical pathologic testing, up to three additional core biopsies 
will be obtained, if possible.  These additional core biopsies will be frozen in optimal 
cutting temperature (OCT) fixative and kept at liquid nitrogen temperature.  They 
will be used for genetic analysis, including full genome or full exome sequencing 
as well as other molecular genetic testing (please refer to section 11.1.4 for 
details). 
• 
Tumor material obtained from biopsy or resection at any time after PVSRIPO 
administration will be tested for parameters of virus tropism, virus translation and 
replication, virus: host interaction, host response to infection and host response to 
viral tumor lysis.  1 sq cm of tumor.  1/3 stored as OCT block for future staining, 
and 1/3 saved as either dissociated tumor or snap frozen.   
• 
CBCs and CMPs will be drawn day 1 post infusion and at each follow-up visit. 
• 
Treatment data may be collected for the remainder of subject’s lives. 
Subjects will have approximately 465 mL’s of blood drawn during the first 8 weeks of this 
study. 
 
Immune monitoring samples may be shipped to the NIH for analysis per Materials Transfer 
Agreement.  
13. 
CLINICAL RESPONSE EVALUATIONS 

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13.1. 
RADIOGRAPHIC TUMOR RESPONSE CRITERIA 
Given that imaging following immunotherapy differs greatly from what is typically seen following 
chemoradiation treatment or treatment with anti-angiogenic compounds, evaluation of response 
using with Macdonald criteria or RANO criteria is not appropriate in this trial.  Immunotherapy can 
trigger an inflammatory immune response that is observed on imaging.  Distinguishing between 
the inflammatory immune response and progressive disease is difficult.  Therefore, an exploratory 
objective of this study is to describe radiographic imaging post-PVSRIPO treatment. 
13.2. 
PROGRESSION FREE SURVIVAL (PFS) 
Patients potentially eligible for this study will be followed from the time protocol treatment is 
initiated. The PFS is defined as the time between initiation of protocol treatment and the first 
occurrence of disease progression. The PFS will be defined radiologically, if possible; or 
alternatively defined by a significant overall change in clinical status as assessed by the study 
principal investigator, Dr. Dina Randazzo, or her designees. 
13.3. 
SURVIVAL 
Survival time is defined as the time between the initiation of protocol treatment and death, with 
the survival of live patients being censored at the last follow-up. Survival data in the context of a 
Phase I/II trial must be interpreted with great care.  There are a few studies in the literature that 
may be able to be compared to our study to some degree. For example, the carmustine polymer 
study (47), has a set of very similar inclusion, and eligibility criteria. However, two important 
differences do exist. First, this study required a gross total resection prior to implantation of 
carmustine polymers. Second, patients were randomized to treatment. This will allow us to 
compare the results of the current clinical trial to previously published trials of patients with similar 
prognostic features. 
14. 
STATISTICAL CONSIDERATIONS 
14.1. 
SAFETY AND DOSE-FINDING COMPONENT 
The dose-escalation criteria as outlined above in section 11.1.5 will be used to determine the 
MTD of PVSRIPO.   
14.2. 
ANALYTICAL METHODS 
Descriptive analyses will be conducted to assess secondary objectives. These analyses should 
be interpreted with caution in light of the relatively small number of patients and the pooling of 
data from all dose levels.  A specific statistical hypothesis will not be tested. The product limit 
estimator of Kaplan and Meier will be used to describe the distribution of survival time and time 
to progression. 
14.3. 
DOSE EXPANSION COHORTS  
As described in section 11.3, the protocol amendment dated 6/24/2015 (AMD071) allowed the 
treatment of up to 50 subjects at dose level -1 to assess efficacy.  The amendment dated 
6/15/2016 (AMD096) reduced the PVSRIPO dose for treatment to dose level -2.  Subsequently, 
amendment dated 2/9/2017 (AMD124) increased the treatment dose of PVSRIPO to dose level 
–1.  Section 11.3 provides the rationale for all these changes. 
14.3.1. Dose Expansion at Dose Level “-1” 
Accrual at dose level -1 will be extended in order that a sufficient number of patients will be treated 
at that dose level to estimate its efficacy relative to a historical cohort.   
 

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As of 6/15/2015, 28 patients have been treated on this protocol, including 13 patients treated at  
dose level -1.  The median survival is 15.2 months (95% confidence interval:  7, ∞).  Median 
follow-up among patients treated at dose level -1 is 3.5 months.   
 
Informal comparisons of survival associated with PVSRIPO (including all patients at all dose 
levels) relative to that observed in other recurrent WHO grade IV malignant glioma patient groups 
suggest a hazard ratio in the ballpark of 0.5 to 0.65.  
 
The historical cohort that will be used for this comparison will include recurrent WHO grade IV 
malignant glioma patients in the PRoGREss registry (IRB# Pro00027120; Primary and Recurrent 
Glioma Registry) who would have been eligible to receive PVSRIPO if the treatment had been 
available.  Details about the construction of this comparison group, as well as a summary of 
control group patient characteristics are provided in Appendix 18.4.  Two control groups were 
generated, with one group having no limit to pre-treatment steroid usage, and the other group 
having a steroid limit of ≤ 4 mg per day of dexamethasone within the 2 weeks prior to being eligible 
for PVSRIPO infusion.  The first control group has eligibility criteria similar to that which was in 
existence for dose levels 1-5 and the first 5 patients on dose level “-1”.  The second control group 
has eligibility criteria similar to those currently being used for dose level “-1”.  The first historical 
control group includes 124 patients of which 123 are dead; whereas, the second historical control 
group includes 104 patients of which 103 are dead. 
 
Korn and Friedlin (48) recommend the use of an unconditional power estimator for the 
determination of sample size in single arm studies that compare time-to-event outcomes such as 
survival with that observed in a historical control group.  That estimator resembles an estimator 
for a 2-arm randomized study.  The power of such a comparison is dependent upon the total 
number of observed deaths (49). 
 
The ultimate goal of this accrual expansion and survival comparison is to determine whether 
PVSRIPO administered at dose level -1 and, as needed, with a radiation-necrosis dose level and 
schedule of bevacizumab in the manner described within this protocol has merit for further 
evaluation in additional clinical studies.  This goal is analogous to that of a randomized phase II 
screening study, except for the fact that the two groups are not created by randomization.  The 
second historical control group will be considered in this comparison. Typically, with a randomized 
phase II study, there is a need to constrain the sample size requirements at the expense of either 
an increased false negative or false positive rate.  It has been recommended that a false-positive 
rate of 0.2 be used to test a hypothesis comparing arms while maintaining a high power level (e.g. 
90%). 
 
A total of 50 subjects will be accrued and treated at  dose level -1.  Assuming survival is 
exponentially distributed and the median survival associated with the -1 dose level is 15.2 months, 
we anticipate that approximately 15 of the 50 patients will be deceased by the time accrual is 
complete in June 2016.  Without additional follow-up, the power of a logrank test conducted at 
the 0.2 level of significance (one-tailed) to detect a hazard ratio of 0.5, 0.55, 0.6, and 0.65 are 
0.95, 0.91, 0.84, and 0.76, respectively.  Tabulated below is the power to detect these hazard 
ratios as a function of the number of additional months of follow-up after accrual completion (Table 
4). 

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Table 4. Power of the survival comparison to detect the noted hazard ratios 
Follow-
up 
(Months) 
Expected # 
Dead if Median 
OS = 15.2 
months 
Power of Comparison to Detect Noted Hazard 
Ratio 
Hazard 
Ratio = 
0.5 
Hazard 
Ratio = 
0.55 
Hazard 
Ratio = 
0.6 
Hazard 
Ratio = 
0.65 
0 
15 
0.95 
0.91 
0.84 
0.76 
1 
16 
0.96 
0.92 
0.85 
0.78 
2 
18 
0.97 
0.93 
0.88 
0.80 
3 
19 
0.97 
0.94 
0.89 
0.81 
4 
21 
0.98 
0.95 
0.90 
0.83 
5 
22 
0.98 
0.96 
0.91 
0.84 
6 
23 
0.98 
0.96 
0.91 
0.85 
7 
24 
0.99 
0.96 
0.92 
0.85 
8 
26 
0.99 
0.97 
0.93 
0.87 
9 
27 
0.99 
0.9 
0.94 
0.87 
10 
28 
0.99 
0.97 
0.94 
0.88 
11 
29 
0.99 
0.98 
0.94 
0.89 
12 
30 
0.99 
0.98 
0.95 
0.89 
 
Given that the study is not a randomized study, there is a need to statistically adjust survival 
comparisons for confounding factors.  Hence, survival comparisons will be conducted using the 
Cox model.  Predictors within the Cox model will include treatment, age, extent of resection, prior 
bevacizumab treatment, Karnofsky performance status at the time of PVSRIPO treatment, 
number of prior recurrences, and an indicator of secondary GBM. 
 
It should be noted that the calculations and analyses described above assume that survival with 
PVSRIPO is exponentially distribution and that the proportional hazards assumption is satisfied.  
Recently reported studies suggest that such properties may not exist for immunotherapeutic 
agents due to (1) delayed antitumor effects and hence delayed separation of survival curves, and 
(2) a percentage of patients who are “cured” or survive for a relatively long period of time.  If that 
is the situation with PVSRIPO, the power calculations and planned analyses may not be 
appropriate.  It might be more appropriate to focus on long-term survivorship at a specific time 
point such as 18 and 24-month survival.   Within the second historical control group, the 18 and 
24-month survival probabilities are 23.1% and 13.5%, respectively.  A two-tailed chi-square test 
(α=0.2) conducted to compare the proportion of patients alive at 18 or 24 months at the 0.2 level 
has >84% power to detect an increase of 15% in survivorship.  Logistic regression will be used to 
adjust for confounding factors. 
14.3.2. Dose Expansion at Dose Level -2 
As described in section 11.3.8, this protocol is amended to reduce the PVSRIPO dose to dose 
level -2 (1.0 X 107 TCID50) with the goal of limiting the occurrence of undesirable burden from 
the inflammation and its treatment on as many subjects (including caregivers) as possible.   Based 
upon additional animal studies, we are confident that dose level -2 (1.0 X 107 TCID50) will be a 
therapeutic dose. This amendment is not due to concerns for the safety of the patients on dose 
level -1, but due to the observation that less inflammatory reaction is a predictor of better survival 
and treatment response.  
 

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Twenty-seven (27) patients will be accrued to the dose expansion at dose level -2.  The ultimate 
goal of this accrual expansion is to determine whether PVSRIPO administered at dose level -2 
has merit for further evaluation in additional clinical studies.  The second historical control group 
as described in Appendix 18.4 and section 14.3.1 will be to make this assessment. 
 
A false-positive rate of 0.2 will be used to test a hypothesis comparing dose level -2 with the 
historical control group while maintaining a high power level (e.g. 90%).  The rationale is provided 
in section 14.3.1. 
 
Given that recently reported studies of effective immunotherapeutic agents show (1) delayed 
antitumor effects and hence delayed separation of survival curves, and (2) a percentage of 
patients who are “cured” or survive for a relatively long period of time, we will focus on long-term 
survivorship at a specific time point (i.e. 24 months).   Within the second historical control group, 
the 24-month survival probability is 13.5%.  Assuming 27 patients in the historical group, 
simulation studies show that a two-tailed chi-square test (α=0.2) conducted to compare the 
proportion of patients alive at 24 months at the 0.2 level has 88.7% power to detect an increase 
to 35% in 24-month survival.  Logistic regression will be used to adjust for confounding factors.  
14.4. 
RETREATMENT WITH PVSRIPO 
The analysis of data collected in conjunction with PVSRIPO retreatment will be descriptive as the 
sample size is expected to be limited.  The rigor and approach to all the associated exploratory 
analyses will be dependent upon the number of patients who are retreated. 
 
The toxicity associated with the second infusion of PVSRIPO will be summarized separately from 
those observed with the first infusion, but in a similar manner.  A landmark survival analysis will 
describe PFS and OS starting at the time of the second infusion.  Changes over time after the 
first infusion of PVSRIPO in various immune function/response parameters will be described.  
Among patients who receive a second PVSRIPO treatment, changes observed in immune 
function/response associated with the second infusion will be compared to those observed with 
the first infusion.   
15. 
POTENTIAL BENEFITS AND RISKS 
15.1. 
POTENTIAL BENEFITS 
The potential benefits may include reduction and/or remission of the subject’s brain cancer.  
Because this procedure is experimental, it cannot be guaranteed that subjects will receive any 
benefit as a result of participating in this research study. The information collected in this research 
may help scientists better understand the mechanisms involved in viral oncolysis. If such an 
understanding emerges from this research, it may benefit society by furthering the development 
of improved treatment methods for WHO grade IV malignant glioma in the future. 
15.2. 
POTENTIAL RISKS 
15.2.1. Surgical Complications 
The stereotactic catheter implantation procedure carries a risk for loss of neurologic 
function, non-neurologic complications and death. These risks depend primarily on the 
preoperative condition of the patient, the size and location of the tumor and associated 
diseases. The potential risk for the patient will be discussed in detail with the patient and 
family. 
15.2.2. Anesthesia 

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Patients undergoing general anesthesia may be subjected to associated risks including 
pneumothorax, pneumonia, airway injury, hypotension, myocardial infarction, stroke, 
hepatic and renal injury and death. 
15.2.3. Poliomyelitis 
PVSRIPO has been tested in NHPs according to the WHO standardized monkey 
neurovirulence tests. These tests revealed the absence of neuropathogenic properties, 
evident as failure to induce symptoms of poliomyelitis in NHPs after intracerebral 
inoculation of virus. However, PVSRIPO are replication-competent viral agents that, in 
principle, retain the potential to cause motor neuron damage. Possible complications 
include transient or permanent mono- or paraparesis, paralysis, and life-threatening 
respiratory insufficiency. 
15.2.4. Virus Recombination and Mutation 
PVSRIPO retains the ability to alter its genome during replication upon administration to 
patients. Various mechanisms are known to lead to genetic adaptation, including 
spontaneous mutagenesis and recombination that may give rise to viral progeny with 
changed properties. Empirical studies in tissue culture and laboratory animals 
demonstrated that prolonged passaging in GBM cells does not select for genetic changes 
in viral progeny. However, the occurrence of such events cannot be categorically excluded 
in patients receiving intracerebral PVSRIPO. Genetic changes may cause an altered 
phenotype of PVSRIPO, including adaptation to improved virus replication in the normal 
CNS. 
15.2.5. Long-Term Sequelae of Virus Injection 
PVSRIPO does not encode foreign genetic material, polioviruses are unable to insert viral 
genetic material in the host genome and polioviruses are incapable of inducing latent or 
chronic CNS infection. Therefore, PVSRIPO is unable to induce long-term expression of 
foreign polypeptides or long-term persistence of virus replication. Thus, there are no long-
term neurologic consequences to intracerebral PVSRIPO administration in study subjects. 
For these reasons, no specific measures to analyze the potential for persistence of virus 
replication in the CNS of long-term survivors are indicated.  
 
Prolonged gastrointestinal propagation of polioviruses, associated with prolonged virus 
shedding with stool, has been reported in select individuals with acquired or rare inherited 
immunodeficiency disorders. Primate toxicology results indicate that extraneural 
dissemination and shedding of PVSRIPO with stool do not occur after intracerebral 
inoculation. However, if PVSRIPO shedding is detected in stool samples scheduled to be 
collected from study subjects, virus excretion will be analyzed and documented in study 
subjects at monthly intervals as long as it persists.  
15.2.6. Gastrointestinal Infection and Virus Excretion 
After oral uptake, poliovirus replicates in the gastrointestinal tract and is excreted by 
infected individuals with stool. Gastrointestinal viral replication usually is asymptomatic, 
but may cause mild symptoms of gastrointestinal discomfort. Tests of PVSRIPO in NHPs 
suggest that excretion of virus does not occur after intracerebral administration, implying 
the absence of gastrointestinal replication. However, PVSRIPO excretion with stool cannot 
be categorically excluded in patients enrolled in this study.   
15.2.7. Cerebral Edema and Increased Intracranial Pressure 
Cerebral edema may be secondary to the disease process itself, the surgical procedure, 
necrosis from previous radiation, or inflammation due to immune infiltration of the brain or 
destruction of tumor cells. Symptoms may include, but are not limited to, severe headache, 

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confusion, lethargy, unresponsiveness, coma, or focal neurological deficits. Patients will 
be monitored throughout the course of the study and upon any signs or symptoms of 
cerebral edema, may have their steroid doses increased or receive treatment with an 
osmotic diuretic, or surgical decompression. Edema that fails to respond to aggressive 
therapy may lead to permanent neurological impairment. The probability of this risk can 
be predicted to some degree based upon tumor size, location, pre-operative neurological 
impairment, and post-operative course prior to virus administration. 
 
Special consideration: In the event a patient demonstrates neurologic or radiographic 
signs suggestive of an inflammatory reaction secondary to the immune response expected 
from PVSRIPO for which it would be considered to increase dexamethasone above 4 mg 
a day after the first 2 weeks post PVSRIPO infusion, dexamethasone will not be increased 
any further.  Instead, patients will be initiated on bevacizumab 7.5 mg/kg IV every 3 weeks 
and imaged with an MRI after 3 doses (every 9 weeks +/- 2 weeks) to assess if continued 
utilization of bevacizumab to control the cerebral inflammation is needed. Bevacizumab 
will not be provided by the study. Every attempt should be made to discontinue 
dexamethasone.  Once patients start bevacizumab, the follow-up visit schedule will be 
adjusted to coincide with the timing of the bevacizumab instead of the prior PVSRIPO 
infusion.  Therefore, patients will return approximately every 9 weeks for an evaluation 
with MRI. 
 
If there are adverse events or other circumstances prohibiting the use of bevacizumab, 
we will use corticosteroids or surgery, or other interventions deemed more appropriate for 
the patient by the treating physician, if needed, to treat the inflammatory reaction 
secondary to PVSRIPO. 
15.2.8. Risk of Infection 
The intracerebral catheter placement and infusion may include the risk of infection. 
However, similar procedures including stereotactic biopsy and ventriculostomy placement 
are commonly used in the routine clinical care of patients with malignant brain tumors with 
a very low and acceptable rate of infection. In the most extreme situation, however, 
infection may lead to systemic bacterial/fungal sepsis and possibly death. The risk of 
infection will be minimized though by performing catheter implantations in the Operating 
Room. Patients will be monitored throughout the course of the study for any signs and 
symptoms of infection. If an active infection is suspected, patients will be cultured and 
treated with appropriate antibiotics. 
15.2.9. Risks of Phlebotomy 
Drawing blood or inserting an intravenous catheter into an arm vein may result in bruising 
or swelling in the area of the insertion, bleeding at the site of the needle puncture, light 
headedness, fainting and very rarely, local infection, which may be severe. These risks 
are reduced by the fact that the blood will be drawn by a qualified physician, nurse or 
phlebotomist (a professional trained to draw blood). 
15.2.10. 
Risks of MRI 
Risks and/or discomforts associated with MRI scans include anxiety produced from being 
in a tight, enclosed space (claustrophobia). In addition, the machine operates using a large 
and powerful magnet, which attracts certain metals. Therefore, people with these metals 
in their bodies (specifically pacemakers, infusion pumps, metal aneurysm clips, metal 
prostheses, joints, rods or plates) will be excluded from the study. Patients will also be 
checked to make sure that they do not bring any metal objects into the MRI facility.  Dental 
fillings are less affected by the magnetic fields generated and are therefore permitted.  It 

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will be asked that patients let the physicians conducting this study know of any metal in 
their bodies other than dental fillings.   
15.2.11. 
Allergic Reactions to Contrast Agents 
During the MRI, patients will be given a contrast agent. The agent is given routinely to 
obtain enhanced MRI scans of the brain. The agent is administered through the vein and 
requires the placement of an IV catheter.  The catheter placement is similar to drawing 
blood except that the catheter remains in the vein during the time the agent is actively 
delivered. The risks of a blood draw and insertion of a catheter are similar. There have 
been a few, rare cases of allergies to the agent used in MRI contrast enhanced scans. 
Patients with any known severe allergies to contrast agents will be excluded from the 
study. Patients with mild allergies (i.e., rash only) will be pretreated with Tylenol and 
Benadryl prior to injection of the contrast agent. In addition to the gadolinium (contrast 
agent) being given for MRI contrast, it will also be co-infused with the study drug to assess 
infusion distribution.  It is not FDA approved for gadolinium to be administered in this 
way.  The potential risks of intracerebral infusion of gadolinium contrast agents are not 
completely known but are believed to be small. Risks for infusion of gadolinium contrast 
agents intrathecally for procedures such as cistemography have been somewhat better 
studied, and were recently summarized by Selcuk et al (2009) (50). Encephalopathy, 
coma and seizures have been reported as side effects in case reports of accidental 
administration of large amounts of gadolinium contrast agents intrathecally in humans (51, 
52). When these contrast agents are used in appropriately low dose, however, the risk of 
intrathecal administration appears reasonably low. No neurological sequelae attributable 
to the procedure were detected in any patients in one year follow-up of series of 85 
patients (50) and 95 patients (53), or in any of 51 patients after over 4 years of mean 
follow-up in another (54). Although these results cannot be extrapolated to the procedure 
proposed in the current research, they indicate that direct exposure of the brain to small 
amounts of gadolinium contrast agent is generally well tolerated.  
 
A rare but serious adverse reaction has been observed in patients that received a 
gadolinium-based contrast material during MRI examinations, a reaction called 
nephrogenic systemic fibrosis (NSF). Patients with kidney disease are at increased risk of 
developing NSF. NSF may cause skin thickening, joint pain and/or swelling. In rare cases 
NSF can lead to lung and heart problems and cause death. 
15.2.12. 
Risks to household contacts of study subjects 
Primate toxicology studies showed that intracerebral infusion of PVSRIPO does not lead 
to extraneural dissemination or replication and, hence, is not associated with virus 
shedding. Therefore, and because poliovirus transmission occurs via the fecal-oral route, 
the likelihood of unintended exposure of patient household contacts is exceedingly low. If 
accidental exposure occurred, it would equal the risk of exposure to any type 1 Sabin 
vaccine virus or vaccine virus derivatives. Thus, in essence, exposure with PVSRIPO is 
equal to oral immunization with a safe version of type 1 Sabin. Since type 1 Sabin vaccine 
virus or vaccine virus derivatives have to be considered part of the human environment, 
exposure to PVSRIPO would not represent an added risk beyond the possibility for 
exposure that already exists. Study subjects will be advised of the risks of exposure to 
unvaccinated household contacts. 
15.2.13. 
Unknown Risks 
The overall risk classification of this research is unknown.  Clinical trials using PVSRIPO 
in brain tumor patients have not been performed. 

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15.3. 
TREATMENT ALTERNATIVES AND FINANCIAL REIMBURSEMENT 
Alternative treatments for recurrent malignant brain tumors include additional surgery, radiation, 
bevacizumab and/or chemotherapy. If a patient chooses not to participate in this trial, they may 
seek alternative treatment. If the patient fails treatment through this trial, these alternatives may 
still be available. There will be no financial reimbursement to subjects for study participation. 
16. 
ETHICAL AND REGULATORY ISSUES 
This study will be conducted in accordance with current US FDA regulations, ICH guidelines, GCP 
standards, the Declaration of Helsinki, and local ethical and legal requirements. 
16.1. 
INSTITUTIONAL REVIEW BOARD (IRB) 
Prior to patient accrual, this protocol and the protocol informed consent must be approved in 
writing by the Cancer Protocol Committee of the Duke Comprehensive Cancer Center (CPC) and 
the Duke University Medical Center IRB. The term of study approval will not exceed one year.  A 
progress report will be submitted annually to the IRB and CPC and re-approval obtained to 
continue the study. The IRB will also approve any significant changes to the protocol as well as a 
change of PI. Records of all study review and approval documents will be kept on file by the PI 
and are subject to FDA inspection during or after completion of the study. Adverse events will be 
reported to the IRB. The IRB will receive notification of the completion of the study and final report 
within three months of study completion or termination. The PI will maintain an accurate and 
complete record of all submissions made to the IRB, including a list of all reports and documents. 
The IND number assigned will be reported to the IRB. Study activities will not commence until 
there is full resolution of any question or concerns raised during the process of IND review by the 
FDA. Any changes to the protocol or informed consent will be submitted for approval to the FDA 
and will not be enacted until that approval is obtained. Finally, due to use of an infectious agent 
in this study, this protocol will also be reviewed and approved by the Duke University Medical 
Center Institutional Biosafety Committee (IBC) prior to patient accrual. 
16.2. 
RECRUITMENT AND INFORMED CONSENT 
Upon determination that a patient’s tumor histology and radiographic findings are compatible with 
the eligibility criteria of this protocol, the clinical study will be briefly explained to the patient by the 
principal investigator (PI) or colleague. If the patient indicates interest in study participation, 
patient education sheets and possibly the protocol consent form will be provided to the patient as 
these provide the most comprehensive explanation of the study in lay terms. If the patient shows 
continued interest, the PI or designee will thoroughly explain the required elements of informed 
consent and all aspects of the study to the subject including inclusion/exclusion criteria, risks, 
benefits, and alternatives to study participation. 
 
The PI or designee will fully explain the purpose and potential risks and benefits of the study to 
the subject prior to enrollment and address any questions posed by the subject. In accordance 
with guidelines in the 21CFR50.27, all subjects will sign a statement of informed consent, which 
has been approved by the IRB. The subject will receive a copy of the executed consent document. 
The signed consent will be retained at the investigative site for each subject. The informed 
consent document serves as authorization as defined under HIPPA and contains the appropriate 
statements regarding privacy and confidentiality of protected health information as well as 
information on withdrawal from the study. This phase I study involves research that presents risk, 
but holds the prospect of direct benefit to the individual subject. The PI will report to the IRB and 
FDA changes in the research protocol and all unanticipated problems involving risks to human 
subjects and others, and no changes will be made in the research activity without IRB approval. 

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16.3. 
REGISTRATION OF SUBJECTS 
All subjects enrolled into this clinical study must be registered by the PRTBTC staff into eResearch 
per Duke Oncology CRU SOP’s. 
16.4. 
DATA COLLECTION AND MAINTENANCE 
The PI will be responsible for accurate, consistent, timely, complete and reliable data collection. 
 
The study coordinator and PI are responsible for ensuring that the following forms are completed 
in a legible and timely manner for every patient enrolled on study. These forms are an integral 
part of the study data and will be maintained in the patient’s clinical chart at the Preston Robert 
Tisch Brain Tumor Center at Duke (BTC). Errors on these forms and any source documents 
should be lined through, but not obliterated with the correction inserted, initialed and dated by the 
study coordinator or PI. These forms and the source documents will be available at all times for 
inspection by the FDA and the DCI SOC.  
 
Eligibility Checklist 
Electronic DUHS Adverse Event Form 
Mandatory MedWatch Form (Form 3500A) when appropriate 
 
In addition to the patient data maintained on the forms listed above, patient data will also be 
entered primarily into an Oracle Clinical (OC) database. Ancillary information will be stored in 
Excel spreadsheets maintained on a secure DUHS server.  The data are backed up daily and 
stored on a secure medical center server. The PI, study coordinators, and data coordinators for 
the study are the only individuals who will have access to the password-protected OC data file.  
Regulatory coordinators may have access to data stored on the Excel spreadsheets.  
16.5. 
DOCUMENTATION AND REPORTING OF ADVERSE EVENTS 
An ‘Adverse Event’ will be defined as any adverse change from the subject’s pre-treatment 
baseline condition, including any clinical or laboratory test abnormality that occurs during the 
course of research after treatment has started. Adverse events will be categorized and graded in 
accordance with the NCI CTC (Version 4). 
 
A ‘Serious Adverse Event’ will be defined as an undesirable sign, symptom or medical condition 
which 1) is fatal or life threatening; 2) requires inpatient hospitalization or a prolongation of existing 
hospitalization; 3) results in persistent or significant disability/incapacity; 4) constitutes a 
congenital anomaly or a birth defect and/or; 5) medically significant impairment such that it may 
jeopardize the subject, and may require medical or surgical intervention to prevent one of the 
outcomes listed above. 
 
A summary of all adverse events (not just those considered related to the study drug) will be 
maintained in Oracle Clinical. The event will be categorized by organ system, relationship to 
treatment, its grade of severity, and resolution. The PI and study statistician will periodically review 
the collective adverse events with the intention of identifying any trends or patterns in toxicity. If 
any such trends are identified, depending on their severity and frequency, a protocol amendment 
will be considered. 
 
All suspected adverse events that are both serious and unexpected should be reported 
immediately to both the PI, Dr. Dina Randazzo (pager 970-9692) or her designee (919-684-8111), 
and to the FDA. Unexpected fatal or life-threatening suspected adverse events will be reported to 
the FDA by telephone, facsimile, or in writing as soon as possible, but no later than 7 calendar 
days after first knowledge by the sponsor followed by as complete a report as possible within 8 
additional calendar days. Unexpected adverse events that are both serious and suspected, but 

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not fatal or life-threatening, will be reported to the FDA by telephone, facsimile, or in writing as 
soon as possible, but no later than 15 calendar days after first knowledge by the sponsor. 
 
All adverse events that are considered serious, unanticipated or unexpected, and related or 
possibly related to the research (as defined by 21CRF312.32[a]) will be reported to the Duke 
University Medical Center IRB using the appropriate SAE report form within the following 
guidelines: 
• 
Report within 24 hours of learning about any subject’s death that was unanticipated and 
more likely related to the research than unrelated; 
• 
Report within 5 business days of learning about any serious, unanticipated, and related or 
possibly/probably related adverse event; 
• 
Report within 10 business day of learning about any other unanticipated problem or event 
that was more likely related to the research than unrelated. 
 
At the time of the annual progress report to the Duke University Medical Center IRB, a summary 
of the overall toxicity experience will be provided.  
16.6. 
DATA SAFETY AND MONITORING 
This clinical research study will be monitored by the PI and the Duke Cancer Institute (DCI) 
Protocol Review and Monitoring system in accord with the NCI-approved “Institutional Protocol 
Monitoring Procedures and Guidelines for NIH-sponsored Research Involving Human Subjects.” 
In terms of internal review, the PI will continuously monitor and tabulate adverse events. 
Appropriate reporting to the Duke University Medical Center IRB will be made. If an unexpected 
frequency of grade III or IV events occurs, depending on their nature, action appropriate to the 
nature and frequency of these adverse events will be taken.  This may require a protocol 
amendment, dose de-escalation, or potentially closure of the study. The PI of this study will also 
continuously monitor the conduct, data, and safety of this study to ensure that: 
• 
Interim analyses occur as scheduled when applicable; 
• 
Stopping rules for toxicity and/or response are met; 
• 
Risk/benefit ratio is not altered to the detriment of the subjects; 
• 
Appropriate internal monitoring of adverse events and outcomes is done; 
• 
Over-accrual does not occur; 
• 
Under-accrual is addressed with appropriate amendments or actions; 
• 
Data are being appropriately collected in a reasonably timely manner. 
 
DCI Protocol Review and Monitoring systems review of this protocol begins with an initial review 
by the Cancer Protocol Committee (CPC). CPC new protocol review focuses on scientific 
relevance, study design, adequacy of biostatistical input, protocol prioritization, feasibility of 
completing the study within a reasonable time frame and risk assessment of the trial. The PI will 
abide by CPC assessment of the level of risk, which will determine the intensity of subsequent 
DCI monitoring. CPC also conducts annual scientific progress reviews on protocols that are open 
to enrollment and focus on protocol prioritization, accrual and scientific progress. These reviews 
are conducted at the time of IRB annual renewals and documentation of all CPC reviews will be 
maintained in eIRB systems. 
 
A determination for the degree of monitoring conducted by the DCI monitoring team is made at 
the time of initial CPC approval to commensurate with the type and level of intervention, phase, 
endpoints, degree of risk, size and complexity of the protocol. Typically, a formal monitoring visit 
is conducted by the DCI monitoring team after the first 3 subjects have been enrolled, followed 
by annual monitoring of 1-3 subjects until closed to enrollment or subjects are no longer receiving 
study drug or other interventions that are more than minimal risk. In this study, beginning with the 
monitoring visit (MV) of September 2013, the DCI Monitoring Team has monitored the eligibility 

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of all subjects enrolled following completion of their 4-week follow-up.  This monitoring of eligibility 
is in conjunction with annual monitoring of accrual, regulatory, consenting, eligibility, conduct, 
safety, data quality, investigation product accountability, and biologic samples.  Additional 
monitoring may be prompted by findings from monitoring visits, unexpected frequency of serious 
and/or unexpected toxicities, or other concerns.  Monitoring visits may also be initiated upon 
request by DUHS and DCI Leadership, CPC, SOC, a sponsor, an investigator, or the IRB.  
Following closure of enrollment to new subjects in June 2017, DCI monitoring will revert to 
standard monitoring practices (i.e. individual subject monitoring will be discontinued, while annual 
monitoring will continue while selected patients are being retreated).   
 
The DCI monitoring team reviews the adequacy of informed consent, enrollment of appropriate 
patients, implementation of protocol-specified procedures and treatment, adequacy of data 
collection, and appropriateness of adverse event monitoring and reporting. The DCI monitoring 
team presents final monitoring reports to the DCI Safety Oversight Committee (SOC) highlighting 
safety concerns and unresolved issues.  The SOC chair assigns an overall rating of satisfactory 
(no major deviations), marginal (1 major deviation), or unsatisfactory (2 or more major deviations 
or an ineligible subject) to reflect overall quality of data, regulatory, consent, eligibility, conduct 
and AE reporting.  Corrective action plans (CAPs) are developed, implemented, and evaluated 
as indicated.  The SOC will notify the Sponsor and DUHS IRB when significant safety concerns 
are identified. 
 
The SOC, in concert with DCI monitoring team, conducts annual data and safety monitoring for 
DUHS sponsor-investigator phase 1 and 2, therapeutic interventional studies that do not have an 
independent DSMB.  While this study does utilize an independent DSMB (see below), it is also 
being monitored as described by the DCI monitoring team.  Annual safety reviews by the DCI 
monitoring team include review of safety data, enrollment status, stopping rules if applicable, 
accrual, toxicities, reference literature, and interim analyses as provided by the Sponsor. Studies 
are rated satisfactory when adequate accrual with lack of excessive toxicity is present.   
 
This phase 1 study is limited to Duke University Medical Center. There will be a team meeting 
with the study investigator and study team members that occurs weekly, on average, during which 
all adverse events will be reviewed and discussed (exceptions to the weekly schedule may be 
made to accommodate the investigator’s schedule or holidays). Monitoring of accrual, outcomes 
and compliance will be carried out on an ongoing basis.  This will include email correspondence 
at each occurrence of an SAE.  The principal investigator and IND holder, as well as any relevant 
key personnel, will participate in this correspondence.  The severity, relatedness and whether or 
not the event is expected will all be reviewed via email. In addition, the IND sponsor and research 
staff meet weekly and go over all study AE’s.  The IND sponsor and PI sign off each week on the 
listing of all AE’s and SAE’s.  If the meeting does not take place with the IND holder and Study 
PI, the PI may sign the AE and SAE summary as reviewed. 
 
DSMB-Plus 
The Duke School of Medicine Research Integrity Office (RIO) has issued an institutional conflict 
of interest management plan to the Preston Robert Tisch Brain Tumor Center requiring a DSMB-
Plus for this project. Because of Duke University’s potential for conflict of interest, there is a 
necessity for review of the study for bias in the protocol design, performance of the study, and 
assessment of endpoints.  
  
In addition to the usual roles of a DSMB, this committee will also consider conflict of interest 
questions related to this research.  As such, it will be referred to as a “DSMB-Plus.”  The DSMB-
Plus will evaluate whether financially-linked biases have affected the design, conduct, or reporting 
of the research related to PVSRIPO.  

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Policies of the DSMB–Plus will be described in the DSMB-Plus Charter, which will be signed by 
the DSMB members. 
16.7. 
SUBJECT PRIVACY AND CONFIDENTIALITY 
The information obtained during the conduct of this clinical study is confidential, and disclosure to 
third parties other than those noted below is strictly prohibited. 
 
All subject data will be identified by a subject identification number and subject initials only to 
protect the subject’s privacy. The data will be blinded accordingly in all data analyses. However, 
in compliance with federal guidelines regarding the monitoring of clinical studies, the investigator 
will permit a representative of the FDA to review that portion of the subject’s medical record that 
is directly related to the study. This will include all relevant study documentation including medical 
histories to verify eligibility, laboratory test results to verify transcription accuracy, X-ray reports, 
admission, discharge summaries for hospital/outpatient admissions while the subject is on-study 
and autopsy reports for deaths occurring during the study. As part of the required content of 
informed consent, the subject will be informed that his/her medical chart may be reviewed by a 
representative of the FDA. 
 
Information obtained during the conduct of this study will be used by the sponsor in connection 
with development of the study drug. This information may be disclosed to other physicians who 
are conducting similar studies and to the FDA as deemed necessary by the sponsor. Patient-
specific information may be provided to other appropriate medical personnel only with the 
patient’s permission. 
 
To ensure compliance with current Federal Regulations, data generated by this study must be 
available for inspection upon request by representatives of the FDA and national and local health 
authorities, the sponsor, and the IRB/IBC for each study site. 
 
Should access to the medical record require a separate waiver or authorization, it is the PI’s 
responsibility to obtain such permission from the patient in writing before the subject is entered 
into the study. 
 
Any publications resulting from this study will not use any patient identifying data.  
16.8. 
INCLUSION OF WOMEN, MINORITIES, AND CHILDREN 
The subject (with no gender or minority restrictions) population for the protocol will include patients 
meeting the eligibility criteria. Inclusion of women and minorities is encouraged. The Brain Tumor 
Center at Duke typically sees more than 500 new patients per year; all patients with recurrent 
malignant brain tumors encountered by the investigators will be considered for the study. 
17. 
STUDY FINANCING 
Charges related to the medical care of the patient will be the responsibility of the patient or their 
insurance carrier as outlined in the Informed Consent document. The study agent will be made 
available free of charge by the NCI. Charges related to testing of tumor biopsy material will be 
covered by research funds administered by M. Gromeier. 

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18. 
APPENDICES 
18.1. 
STUDY TEST SCHEMA FOR INITIAL PVSRIPO TREATMENT 
Note:  Patients that require the addition of bevacizumab to their treatment regimen will complete the same procedures at each visit per the schema below, 
however, they will follow a 9 week (+/- 2 week) schedule once bevacizumab is started.  If a patient starts bevacizumab prior to the 8 week post-PVSRIPO infusion 
visit, the patient will bring in the stool sample at their next MRI evaluation. 
Table 5. Study Test Schema 
 
-6 
months 
-14 days 
Screening 
and prior 
to catheter 
place- 
ment 
Day -2 
48 hours 
prior to 
PVSRIPO 
Day 0 
Catheter 
place- 
ment, 
biopsy, 
PVSRIPO 
infusion 
(infusion 
has a + 
one day 
window) 
 
Day 0 After 
Completion 
of Infusion 
Day 1 
Post 
PVSRIPO 
Day 7 
(week 1) 
Post 
PVSRIPO 
Day 14 
(week 2) 
Post 
PVSRIPO 
4 weeks 
post 
PVSRIPO 
8 weeks 
post 
PVSRIPO 
Weeks 
16, 24, 32, 
40, 48 
post 
PVSRIPO 
History and 
Physical, 
including 
KPS, 
neurologic 
evaluation 
 
X 
 
 
 
X a 
 
X c 
X c 
X c 
X c 
MRI 
 
X 
 
 
X b 
 
 
 
X c 
X c 
X c 
CT 
 
 
 
X 
 
 
 
 
 
 
 
CBC diff, CMP 
 
X 
 
 
 
X 
 
X c 
X c 
X c 
X c 
PT, PTT 
 
X 
 
 
 
 
 
 
 
 
 
Beta HCG 
 
X 
X 
 
 
 
 
 
 
 
 
Polio virus 
immunization 
booster 
X (must 
receive 
booster 
between 
6 mon 
and 1 wk 
prior to 
PVSRIP
O) 
 
 
 
 
 
 
 
 
 
 

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Serum for  
LSQ, anti-
tetanus 
toxoid IgG, 
and 
poliovirus 
titer 
X 
 
 
 
 
 
 
 
 
 
 
Whole Blood 
for 
immunologic 
analysis and 
poliovirus 
titer (10 mL 
except day 
14.  Day 14 is 
5 mL) 
X 
X (10 mL) 
 
 
 
 
X c (10 
mL) 
X c (5 mL) 
X c (10 
mL) 
X c (10 mL) 
X c,d (at 16 
and 24 
weeks) 
Whole Blood 
for 
immunologic 
analysis (76.5 
mL except 
day 14.  Day 
14 is 31 mL) 
(p.22) 
X c 
(76.5 
mL) 
(whole 
blood 
must be 
collecte
d before 
booster) 
X 
(76.5 mL) 
 
 
 
 
X c (76.5 
mL) 
X c (31 mL) 
X c (76.5 
mL) 
X c(76.5 mL) 
X c,d (at 16 
and 24 
weeks) 
Stool for 
PVSRIPO titer 
 
 
 
 
 
 
 
 
 
X c 
 
 
a Daily after infusion until discharged from hospital. 
b  Within 4 hours after completion of infusion.  
c Starting with Day 7 (Week 1) all tests and procedures have a seven day plus or minus window.  
d At the discretion of the PI, an additional blood sample at least 2 years post study drug infusion may be obtained. 
 

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18.2. 
STUDY TEST SCHEMA FOR RETREATMENT WITH PVSRIPO 
Table 6. Schedule of Study Tests and Procedures Associated with PVSRIPO Retreatment 
                                               
1 Starting with Day 7 (Week 1), all tests and procedures have a 7-day plus or minus window. 
2 Daily after infusion until discharged from hospital. 
3 Polio immunization booster and anti-tetanus toxoid IgG blood test may be obtained locally. 
4 At the discretion of the PI, an additional poliovirus titer blood sample may be obtained at least 2 years post study drug infusion. 
Description 
Within 6 
months 
Screening: 
Within14 days 
prior to 
catheter 
placement 
Screening: Within 2 
days prior to 
PVSRIPO 
Catheter 
Placement 
biopsy, 
PVSRIPO 
infusion 
Post infusion (follow-up period)1  
Week 
 
 
 
 
0 
1 
2 
4 
8 
12 
16 
+ every 9 
weeks until 
week 61 
Day 
 
Within 14 
days prior to 
catheter 
placement 
Within 2 days prior 
to PVSRIPO 
0 
1 
7 
14 
28 
56 
84 
112 
General 
Evaluations 
 
 
 
 
 
 
 
 
 
 
 
Informed Consent 
 
X 
 
 
 
 
 
 
 
 
 
Physical Exam 
 
X 
X 
 
X2 
X 
X 
X 
X 
X 
X 
Neurologic Exam 
 
X 
X 
 
X2 
X 
X 
X 
X 
X 
X 
Performance 
Status 
 
X 
X 
 
X2 
X 
X 
X 
X 
X 
X 
Adverse Events 
 
 
 
Continuous 
Laboratory 
Evaluations 
 
 
Poliovirus 
Immunization 
Booster 
X3 
 
 
 
 
 
 
 
 
 
 
CBC w/diff 
 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
CMP 
 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
PT, aPTT 
 
X 
 
 
 
 
 
 
 
 
 
Serum Pregnancy 
Test 
 
X 
X 
 
 
 
 
 
X 
 
 
Whole blood for 
immunologic 
analysis and 
poliovirus titer (10 
 
X 
 
 
 
X 
X 
X 
X 
X 
X (at  16, 25 
and 34 
weeks)4  

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5 Blood samples for immune analysis will continue to be collected at each MRI visit at the discretion of the PI. 
6 MRI should be obtained within 4 hours after completion of infusion.  This MRI after the infusion is without gadolinium enhancement.  All other MRIs in the study include gadolinium enhancement. 
7 One cycle of 110 mg/m2 oral lomustine at approximately 8 weeks post PVSRIPO infusion. 
mL except Day 14 
will be 5 mL) 
Whole blood for 
immunologic 
analysis  (76.5 mL 
except day 14 will 
be 31 mL) 
 
X 
 
 
 
X 
X 
X 
X 
X 
X (at 16 and 
25 weeks)5  
Disease 
Evaluations 
 
 
MRI 
 
 
X 
X6  
 
 
 
X 
X 
 
X 
CT Scan 
 
 
 
X 
 
 
 
 
 
 
 
Biopsy 
 
 
 
X 
 
 
 
 
 
 
 
Treatment 
 
 
 
 
 
 
 
 
 
 
 
PVSRIPO 
 
 
 
X 
 
 
 
 
 
 
 
Lomustine7 
 
 
 
 
 
 
 
 
X7  
 
 

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18.3. 
INVESTIGATIONAL PRODUCT HANDLING PLAN (VERSION 10.4.11) 
Study Agent. The study agent, PVSRIPO, is the live-attenuated, serotype 1 poliovirus (SABIN) 
vaccine (PV1S) containing a heterologous human rhinovirus type 2 internal ribosomal entry site 
(IRES). PVSRIPO will be administered by convection-enhanced delivery to intracerebral tumors 
of patients with recurrent glioblastoma. The procedure consists of approximately a 6.5 hr infusion 
via an implanted catheter connected to an infusion pump via 4m of coiled connector tubing. 
 
Risk assessment. The study agent PVSRIPO is based on the serotype 1 poliovirus vaccine 
(PV1S). The oral polio vaccine (OPV) was administered by the oral route to millions of Americans 
before it was replaced by the inactivated polio vaccine. OPV includes a combination of three 
attenuated polio strains, corresponding to serotypes 1, 2, and 3.  
 
OPV was administered to millions of American children until 2000. Currently only the inactivated 
polio vaccine (IPV) is used. The primary risk associated with OPV is vaccine-associated paralytic 
polio (VAPP). VAPP is similar in presentation to paralytic polio produced by natural infection with 
the wild type virus. The rate of paralytic polio after OPV is about one case in 2.4 million 
vaccinations. The risk is higher for the first OPV dose (about 1 in 750,000 first-dose recipients) 
than for subsequent doses.  
 
VAPP risk is increased among vaccine recipients with specific congenital and acquired 
immunodeficiency disease, generally those conditions associated with agammaglobulinemia (no 
antibodies), or hypogammaglobulinemia (low levels of antibodies). There does not appear to be 
an increased risk of VAPP associated with HIV infection or with the receipt of immunsuppressive 
drugs (e.g., steroids, cancer chemotherapy). 
 
PVSRIPO has not been previously administered to humans, so there is no direct human safety 
experience. On the basis of primate toxicology studies, PVSRIPO appears to have lower 
neurovirulence than the PV1S vaccine strain from which it was derived. The risks of VAPP in 
employees from this PVSRIPO are predicted to be lower than exposure to the live attenuated 
polio vaccine. These risks are reduced by the following factors: 
• 
Poliovirus is normally acquired by ingestion. This route of exposure is relatively unlikely 
for clinicians. Other routes of transmission such as aerosol exposure or needlestick are 
unlikely to lead to infection. 
• 
PVSRIPO will be injected intracerebrally, and is unlikely to lead to viral shedding by the 
subjects. 
• 
Most Americans have received a complete polio vaccine series. Childhood polio 
vaccination is predicted to provide a substantial level of protection from VAPP due to 
PVSRIPO. 
 
PVSRIPO has been handled by hundreds of research personnel for routine research purposes in 
various laboratories, in NCI production facilities, laboratory animal facilities and NCI-appointed 
primate testing facilities, using BSL-2/ABSL-2. 
 
Employee screening. Work practices will be designed to minimize the number of employees 
who come into contact with the viral preparation of materials that are in contact with the viral 
preparation. These employees will include personnel responsible for aliquot preparation and 
administration, and those who dispose of materials that in contact with the virus. Prior to work 
with PVSRIPO, these employees will be screened by Duke Employee Occupational Health and 
Wellness (EOHW) using an EOHW screening form designed to identify individuals with 
immunodeficiency conditions. Those who screen positive on the form will be further evaluated by 
EOHW to determine if they can safely handle PVSRIPO. 

PVSRIPO Clinical Protocol version 9/25/2017 
  
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Most American adults have received a polio vaccine series. Inactivated polio vaccine is available 
for adults, but there is no recommendation for booster vaccination for employees participating in 
this research. Employees with uncertainty about their vaccination status or with any questions 
related to vaccination may contact EOHW for personalized advice. 
 
Receipt and Storage. The study agent and vehicle will be supplied directly to the Investigational 
Pharmacy by the manufacturer, the National Cancer Institute, Developmental Therapeutics 
Program, Biological Resources Branch, Ft. Detrick, MD. The study agent will be shipped via 
approved methods in the appropriate packaging on dry ice. Storage of the study agent at -70°C 
has been empirically determined to occur without compromise in biological or biophysical 
properties for at least 5 years. The agent should be stored long-term at -80°C.  Thawed vials 
should be kept at 4°C whenever possible. Thawed vials of PVSRIPO are stable at 4°C for 48 hrs. 
 
Aliquot Preparation. Preparation of PVSRIPO will occur in a biosafety cabinet designated for 
viral vector agents.  For thawing, vials containing PVSRIPO should be removed from the -80°C 
freezer and kept at room temperature. PVSRIPO contained in the clinically intended delivery 
apparatus is stable at room temperature for 18 hrs.  The vials contain a clear, aqueous solution, 
which does not require reconstitution.  Do not thaw with heating devices (waterbath, heat-
block) as a loss of potency may result.  Do not expose vials to UV-light (in biosafety 
cabinets), as a loss of potency will result.  Thawed vials should not be re-frozen/thawed for 
later use, because potency may be lost.  Thawed vials/study syringes containing PVSRIPO, which 
were kept for >48 hrs at 4°C prior to the infusion procedure, should be discarded, as a loss of 
potency may have occurred.  Thawed vials/study syringes containing PVSRIPO, which were 
exposed for >12 hrs at room temperature prior to the infusion procedure, shall be discarded as a 
loss of potency may have occurred.  For aliquot preparation, the agent will be thawed slowly on 
ice (4°C) and kept at that temperature. The study agent is stable at 20°C for at least 5 days, but 
as a precaution, the cold chain should be maintained. The manufacturer will provide the study 
agent’s potency (as tissue culture infectious doses) and will also supply the appropriate vehicle 
for aliquot preparation. Aliquot preparation will occur in the Investigational Pharmacy. All handling 
of the study agent will occur in a biosafety cabinet or a similarly contained environment.  
 
Any materials in contact with the study agent, e.g. pipettes, vials, etc., will be disposed of as 
biological waste. The final desired aliquot of the study agent will be prepared at the intended 
volume (8 mL) and drawn into the intended delivery device (a 20 mL syringe) using a needle. The 
disposable needle will be a safer hypodermic needle with a safety sheath to cover the needle 
using a one-handed technique after the agent is drawn into the syringe.  The sheathed needle is 
then removed from the syringe containing the study agent aliquoted at the desired dose, and the 
syringe will be capped. The capped syringe will be transported to the study site in a ‘ziplock’ bag 
placed in a portable cooler while maintaining a temperature of 4°C. 
 
For dose levels -1 and -2, the stock of PVSRIPO must be diluted according to the tables below 
respectively. 
 
 
 
 
 
 
 
 
 

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Preparation for Dose Levels -2 through 5 
Dose 
level 
PVSRIPO 
dose 
(In 3 mL) 
PVSRIPO 
dose 
(in 8 mL 
total 
syringe 
load) 
PVSRIPO 
 
Previously 
Diluted (1:10) 
gadopentetate 
dimeglumine 
(Magnevist®) 
Vehicle 
(phosphate-
buffered 
saline, 0.2% 
human serum 
albumin) 
Total Volume 
in 20 ml 
syringe 
-2 
1 x 107 
TCID50 
2.67 x 
107 
TCID50 
See Preparation for Dose Level -2 table 
-1 
5 x 107 
TCID50 
1.33 x 
108 
TCID50 
See Preparation for Dose Level -1 table 
1 
1.0 x 108 
TCID50 
2.67 x 
108 
TCID50 
0.07 mL 
0.16 mL 
7.77 mL 
8 mL 
2 
3.3 x 108 
TCID50 
8.8 x 108 
TCID50 
0.22 mL 
0.16 mL 
7.62 mL 
8 mL 
3 
1.0 x 109 
TCID50 
2.67 x 
109 
TCID50 
0.67 mL 
0.16 mL 
7.17 mL 
8 mL 
4 
3.3 x 109 
TCID50 
8.8 x 109 
TCID50 
2.2 mL 
0.16 mL 
5.64 mL 
8 mL 
5 
1.0 x 1010 
TCID50 
2.67 x 
1010 
TCID50 
6.7 mL 
0.16 mL 
1.14 mL 
8 mL 
 
 
Preparation for Dose Level -1 
PVSRIPO Dilution for Dose Level -1: 
• 
To measure to PVSRIPO dose, we must make a 1:10 dilution. 
• 
Dilute 0.5 ml (1 vial) PVSRIPO with 4.5 ml of Vehicle (phosphate-buffered saline, 0.2% 
human serum albumin) in a syringe.  Refer to table 2 for remainder of steps needed for 
preparation. 
Dose 
level 
PVSRIPO 
dose 
(In 3 mL) 
PVSRIPO 
(in 8 mL 
total 
syringe 
load) 
Diluted 
1:10 (see 
above) 
PVSRIPO 
 
Previously 
Diluted (1:10) 
gadopentetate 
dimeglumine 
(Magnevist®) 
Vehicle 
(phosphate-
buffered saline, 
0.2% human 
serum albumin) 
Total Volume in 
20 ml syringe 
-1 
5 x 107 
TCID50 
1.33 x 108 
TCID50 
0.33 mL 
0.16 mL 
7.51 mL 
8 mL 
 
 
 

PVSRIPO Clinical Protocol version 9/25/2017 
  
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Preparation for Dose Level -2 
PVSRIPO Dilution for Dose Level -2: 
• 
To measure to PVSRIPO dose, we must make a 1:100 dilution. 
• 
Dilute 0.5 ml (1 vial) PVSRIPO with 49.5 ml of Vehicle (phosphate-buffered saline, 0.2% 
human serum albumin) in a syringe.  Refer to table 3 for remainder of steps needed for 
preparation.  
Dose 
level 
PVSRIPO 
dose 
(In 3 mL) 
PVSRIPO 
(in 8 mL 
total 
syringe 
load) 
Diluted 
1:100 
(see 
above) 
PVSRIPO 
 
Previously 
Diluted (1:10) 
gadopentetate 
dimeglumine 
(Magnevist®) 
Vehicle 
(phosphate-
buffered saline, 
0.2% human 
serum albumin) 
Total Volume in 
20 ml syringe 
-2 
1 x 107 
TCID50 
2.67 x 107 
TCID50 
0.67 mL 
0.16 mL 
7.17 mL 
8 mL 
 
Administration. The study agent will be administered to patients via intracerebral delivery using 
a previously implanted tube radiographically determined to be placed correctly within the tumor 
bed. The syringe containing the study agent (upon delivery from the Investigational Pharmacy) 
will be connected to the tube equipped with the appropriate adaptor. This occurs by removing the 
cap and fastening the syringe to the Luer-Lok adaptor and does not involve needles. The infusion 
procedure is driven by an infusion pump at a set pace measured to last ~6 hrs. The infusion 
procedure will take place in dedicated space in the Neurosurgical ICU or the Neuro Step-Down 
Unit, where patients will remain overnight for observation. 
 
The infusion procedure in the Neurosurgical ICU or the Neuro Step-Down Unit follows established 
principles, e.g. those previously used for delivery of radiochemicals by a similar infusion method 
and under similar containment precautions. All procedures are restricted to a single room where 
the patient remains throughout the duration of the infusion. Access to the room is restricted to 
staff caring for the patient.  
 
For reasons pointed out above, persons involved with the care of patients enrolled in the study 
and who received the standard recommended poliovirus vaccine schedule as a child, are not 
required to be re-vaccinated.  
 
All materials coming in contact with the study agent, the syringe delivered from the Investigational 
Pharmacy, tubing, dressings or coverings used to protect the trepanation site, will be disposed of 
as biological waste in the treatment room. All personnel caring for the patient will be wearing 
appropriate safety devices, e.g. disposable gloves, gown, and face protection, which will be 
disposed of as biological waste in the treatment room. Appropriate hand hygiene is required 
before and after any handling of the study agent  
 
Disposal. All surgical materials used in the procedure and (potentially) coming in contact with the 
study agent will be disposable. These materials will be disposed of as biological waste using 
established procedures. Used sharps will be disposed in biohazard sharps container and 
incinerated for final disposal.  
 
List of locations. 
• 
Investigational Pharmacy (receipt, storage, aliquot preparation) 
• 
Closed transport container; at 4°C (for transport of the sealed delivery device containing 
the study agent) 
• 
Study site in the Neurosurgical ICU or the Neuro Step-Down Unit (administration) 

PVSRIPO Clinical Protocol version 9/25/2017 
  
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• 
Investigational Pharmacy (disposal of unused or partially used study agent) 
 
Spill procedure. PVSRIPO can all be easily and completely inactivated with household bleach. 
In the event of a spill, the liquid will be absorbed with gauze (then treated as biohazardous waste 
as above). The spill area will then be liberally wiped down with a 20% household bleach solution 
for chemical disinfection. The disinfectant will be left in contact with contaminated area for at least 
30 minutes, then wiped away with wet towels, which will be disposed as biohazard waste as well. 
 
Exposure follow-up. Any virus exposures will be reported to the Employee Health Exposure 
Hotline (115), followed by submission of the Report of Occupational Injury or Illness form. There 
is no specific preventative treatment. Based on the circumstances of exposure, employee health 
may choose to monitor for infection by submitting stool for enterovirus culture at 7, 14, and 21 
days after an exposure event. 
 
 

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18.4. 
SELECTION AND DESCRIPTION OF THE HISTORICAL CONTROL COHORT 
The historical cohort that will be used for the evaluation of PVSRIPO efficacy among WHO grade 
IV malignant glioma patients was derived from the PRoGREss registry (IRB# Pro00027120; 
Primary and Recurrent Glioma Registry). 
 
Description of PRoGREss registry:  The PRoGREss registry study is both a retrospective and 
prospective chart review study of all patients diagnosed after 12/31/2004 with a primary CNS 
tumor who were seen at the PRTBTC.  Specifically, that database includes: (1) data from all 
patients that were deceased as of 11/3/2011, and (2) data collected retrospectively and 
prospectively from patients who were alive as of 11/3/2011 and provided registry consent.  The 
latter group includes patients who were diagnosed after 12/31/2004 but before 11/3/2011, as well 
as patients diagnosed after 11/3/2011.  Both demographic and clinical information was extracted 
from clinical and research records, which included such information as diagnosis, 
gender/race/ethnicity, medical history, medications, and types of treatment.  Details about the 
data available from the PRoGREss registry can be found in the PRoGREss protocol.   
 
Criteria Used to Select Historical Control Cohort:  To create the historical cohort, data available 
from the PRoGREss registry as of 12/15/2014 were reviewed to determine which patients would 
have been eligible to participate in the PVSRIPO phase I study at the time their disease recurred 
in 2007 or later.  All radiologic records were reviewed by Dr. Annick Desjardins without knowledge 
of the patient’s survival outcome in making this determination. 
 
During the conduct of the PVSRIPO WHO grade IV malignant glioma Phase I study, most 
modifications to the patient eligibility criteria were minor.  As such, the eligibility criteria used for 
identifying the historical control cohort from the PRoGREss registry were standardized.  The 
following relevant criteria were used: 
 
1. Patients must have a diagnosis of recurrent supratentorial WHO Grade IV MG based on 
imaging studies with measurable disease (≥ 1 cm and ≤ 5.5 cm of contrast-enhancing tumor) 
2. Age ≥ 18 years of age 
3. Patients must not have taken part in the PVSRIPO study 
4. KPS ≥70 at the time patient could have been enrolled on the PVSRIPO study 
5. Absence of rapid clinical decline 
 
After the first 5 patients were treated at dose level “-1,” the PVSRIPO WHO grade IV malignant 
glioma Phase I study was amended to institute a limit on pre-treatment steroid usage to ≤ 4 mg 
per day of dexamethasone within the 2 weeks prior to admission for PVSRIPO infusion.  There 
were no steroid limitations for all patients treated on dose levels 1-5 and for the first 5 patients 
treated on dose level “-1”.  To address this eligibility amendment, two control groups have been 
generated, with one group having no limitations on pre-treatment steroid usage, and the other 
group having a steroid limit of ≤ 4 mg per day of dexamethasone (or equivalent) within the 2 
weeks prior to being eligible for PVSRIPO infusion. 
 
Within these historical control cohorts, survival time was computed as the time between the date 
of the first MRI at which time the patient would have been eligible to receive PVSRIPO and the 
date of death.  If the patient remained alive at the date of last follow-up, survival time was censored 
at that date. 
 
Characteristics of the Patients within the Historical Control Group:  A summary of selected patient 
characteristics in these two historical control groups is provided below. 
 

PVSRIPO Clinical Protocol version 9/25/2017 
  
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Patient Characteristics 
Group 
Historical 
controls      
(with steroid 
expanded pts) 
Historical 
controls    
(without 
steroid 
expanded pts) 
N 
% 
N 
% 
All 
124 
100.0 
104 
100.0 
Gender 
44 
35.5 
39 
37.5 
Female 
Male 
80 
64.5 
65 
62.5 
Histologic Diagnosis 
124 
100.0 
104 
100.0 
GBM 
Age at GBM Diagnosis, mean (SD) 
55.3 (10.9) 
54.7 (11.2) 
Age at GBM Diagnosis, median (range) 
55 (20 – 76) 
54 (20 – 76) 
Primary or Secondary GBM 
110 
88.7 
92 
88.5 
Primary 
Secondary 
13 
10.5 
11 
10.6 
Unknown 
1 
0.8 
1 
1.0 
Karnofsky Performance Status 
8 
6.5 
8 
7.7 
100 
90 
69 
55.6 
64 
61.5 
85 
2 
1.6 
2 
1.9 
80 
36 
29.0 
27 
26.0 
70 
7 
5.6 
2 
1.9 
Undetermined but ≥70 
2 
1.6 
1 
1.0 
Resection Type at diagnosis* 
73 
58.9 
60 
57.7 
GTR 
STR 
29 
23.4 
25 
24.0 
Biopsy 
22 
17.7 
19 
18.3 
Further Surgery prior to Treatment** 
8 
6.5 
8 
7.7 
GTR 
STR 
6 
4.8 
4 
3.8 
None 
110 
88.7 
92 
88.5 
PDs prior to being eligible 
102 
82.3 
85 
81.7 

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Patient Characteristics 
Group 
Historical 
controls      
(with steroid 
expanded pts) 
Historical 
controls    
(without 
steroid 
expanded pts) 
N 
% 
N 
% 
1 
2 
14 
11.3 
12 
11.5 
3 
8 
6.5 
7 
6.7 
Total Daily Dex (or other steroid) dose 
at MRI 
89 
71.8 
89 
85.6 
0 
0.125 
1 
0.8 
1 
1.0 
0.5 
1 
0.8 
1 
1.0 
1 
1 
0.8 
1 
1.0 
2 
3 
2.4 
3 
2.9 
4 
8 
6.5 
8 
7.7 
6 
2 
1.6 
0 
0 
8 
4 
3.2 
0 
0 
12 
4 
3.2 
0 
0 
14 
1 
0.8 
0 
0 
16 
3 
2.4 
0 
0 
30 mg Hydrocortisone 
1 
0.8 
1 
1.0 
Unknown 
6 
4.8 
0 
0 
Avastin prior to eligible PD 
61 
49.2 
49 
47.1 
No 
Yes 
63 
50.8 
55 
52.9 
Avastin Failure prior to eligible PD 
75 
60.5 
61 
58.7 
No 
Yes 
49 
39.5 
43 
41.3 
Vital Status 
1 
0.8 
1 
1.0 
Alive 
Dead 
123 
99.2 
103 
99.0 
 

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*In both control groups, this includes 1 patient with a questionable STR/GTR, and 1 patient with 
a GTR on the primary tumor and a STR on a second tumor. 
**In both control groups, this includes 1 patient with a questionable STR/GTR. 
 
 
 

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