Examining Oncolytic Poliovirus Bioactivity in Tumor Tissue After Intratumoral Administration of PVSRIPO in Women With Invasive Breast Cancer
Phase: EARLY_PHASE1
Conditions: Invasive Breast Cancer
Sponsor: Istari Oncology, Inc.

Pilot Study 
Examining Oncolytic Poliovirus Bioactivity in Tumor Tissue after 
Intratumoral Administration of PVSRIPO in Women with Invasive Breast 
Cancer 
 
 
 
 
Sponsor: PI and Istari Oncology, Inc. (Istari Oncology, 
Inc. is the IND Sponsor – Signatory, Darell 
Bigner, MD, PhD) 
Funding Source: DoD grant W81XWH-16-1-0354 
(Smita Nair, PI) 
Protocol Source: PI and Duke Cancer Institute 
Duke IRB#: Pro00085352 
IND#: 14,735 
 
Principal Investigator 
E. Shelley Hwang, MD, MPH 
 
Sub-Investigator(s) 
Jeremy Force, MD 
Lars Grimm, MD 
Smita Nair, PhD 
Matthias Gromeier, MD 
Darell D Bigner, MD, PhD (Istari signatory for IND) 
 
Pathologists 
Edgardo Castellar, MD, PhD 
Roger McLendon, MD 
 
Statistician 
James Herndon II, PhD 
 
Regulatory 
Alli Bosquet 
 
Original version: 
09/08/17 
Updated: 
10/09/17 
12/19/17 
01/29/18 
03/12/18 
06/29/18 
08/27/18 
07/15/19 
09/16/19 
08/12/21 
Change primary regulatory contact on title 
 
 
 
PT-PhIDEE v04.30.14 

page. Clarification that any subjects who do 
not have evaluable tumor tissue from both 
the pre-treatment biopsy and post-treatment 
surgery will be replaced. (Sections 5.3, 5.4, 
5.6, 15) This change is meant to allow 
replacement of subjects who have an active 
viral infection while on study, which would 
impact treatment related analysis and the 
primary objective of the study. 

 
 
 
1 TABLE OF CONTENTS 
1 
TABLE OF CONTENTS ...................................................................................................... 2 
2 
LIST OF FIGURES ............................................................................................................ 4 
3 
LIST OF TABLES .............................................................................................................. 4 
4 
LIST OF ABBREVIATIONS ............................................................................................... 5 
5 
PROTOCOL SYNOPSIS AND RESEARCH SUMMARY ................................................... 7 
Purpose ....................................................................................................................... 7 
Background and Significance ...................................................................................... 7 
Design and Procedure .................................................................................................. 8 
Selection of Subjects ................................................................................................... 9 
Duration of Study ....................................................................................................... 10 
Data Analysis and Statistical Considerations ............................................................. 11 
6 
STUDY SCHEMA ............................................................................................................ 11 
7 
BACKGROUND AND SIGNIFICANCE ............................................................................. 12 
Study Disease ........................................................................................................... 12 
Study Agent ............................................................................................................... 13 
7.2.1 
Pre-Clinical Experience ....................................................................................... 13 
7.2.2 
Clinical Experience .............................................................................................. 17 
Study Purpose and Rationale ..................................................................................... 18 
8 
OBJECTIVES AND ENDPOINTS .................................................................................... 19 
9 
INVESTIGATIONAL PLAN .............................................................................................. 19 
Study Design ............................................................................................................. 19 
Safety Considerations ................................................................................................ 20 
9.2.1 
Surgical Complications ......................................................................................... 20 
9.2.2 
Anesthesia .......................................................................................................... 20 
9.2.3 
Poliomyelitis ........................................................................................................ 20 
9.2.4 
Virus Recombination and Mutation ...................................................................... 20 
9.2.5 
Long-Term Sequelae of Virus Injection ............................................................... 21 
9.2.6 
Risks of Phlebotomy............................................................................................ 21 
9.2.7 
Risks to Household Contacts of Study Subjects .................................................. 21 
9.2.8 
Unacceptable Toxicities ....................................................................................... 22 
Rationale for Selection of Dose, Regimen, and Treatment Duration .......................... 22 
Rationale for Correlative Studies ............................................................................... 23 

 
 
Early Study Termination............................................................................................. 23 
10 
STUDY DRUG .............................................................................................................. 23 
Names, Classification, and Mechanism of Action .................................................... 23 
Supply, Receipt, and Storage .................................................................................. 23 
Dispensing and Preparation ................................................................................... 23 
Compliance and Accountability ............................................................................... 24 
Disposal and Destruction ......................................................................................... 25 
11 
SUBJECT ELIGIBILITY ................................................................................................. 25 
Inclusion and Exclusion Criteria .............................................................................. 25 
12 
SCREENING AND ON-STUDY TESTS AND PROCEDURES ..................................... 25 
Pre-treatment Period .............................................................................................. 27 
Treatment Period .................................................................................................... 28 
End of Treatment .................................................................................................... 28 
Follow-up Period ..................................................................................................... 28 
End of Study ........................................................................................................... 28 
Early Withdrawal of Subject(s) ................................................................................ 28 
12.6.1 
Criteria for Early Withdrawal ............................................................................ 28 
13 
SAFETY MONITORING AND REPORTING ................................................................. 29 
Adverse Events ...................................................................................................... 29 
Reporting of Serious Adverse Events ..................................................................... 29 
Safety Oversight Committee ................................................................................... 30 
External Data and Safety Monitoring ...................................................................... 30 
14 
QUALITY CONTROL AND QUALITY ASSURANCE .................................................... 31 
Monitoring ............................................................................................................... 31 
Audits ..................................................................................................................... 32 
Data Management and Processing ........................................................................ 32 
14.3.1 
Case Report Forms (CRFs) .............................................................................. 32 
14.3.2 
Data Management Procedures and Data Verification ...................................... 32 
14.3.3 
Study Closure .................................................................................................. 33 
15 
STATISTICAL METHODS AND DATA ANALYSIS ....................................................... 33 
16 
ADMINISTRATIVE AND ETHICAL CONSIDERATIONS ................................................ 34 
Regulatory and Ethical Compliance ......................................................................... 34 
DUHS Institutional Review Board and DCI Cancer Protocol Committee ................ 34 
Informed Consent ................................................................................................... 34 

 
 
Study Documentation ............................................................................................. 35 
Privacy, Confidentiality, and Data Storage ............................................................. 35 
Data and Safety Monitoring .................................................................................... 36 
Protocol Amendments ............................................................................................ 36 
Records Retention .................................................................................................. 36 
17 
REFERENCES ............................................................................................................. 36 
 
 
2 LIST OF FIGURES 
Figure 1. Study Schema PVSRIPO for Invasive Breast Cancer ............................................. 11 
Figure 2. Breast Cancer Susceptibility to PVSRIPO .............................................................. 14 
Figure 3. Immunohistochemistry for the PV Receptor (CD155) in 19 Triple-Negative Breast 
Cancer Cases ........................................................................................................................ 14 
Figure 4. PVSRIPO Cytotoxicity in Breast Cancer Cell Lines ................................................ 15 
Figure 5. Infection and Activation of Dendritic Cells (DCs) by PVSRIPO ............................... 16 
Figure 6. PVSRIPO-Induced Oncolysate Causes DC Activation in a Virus Dependent 
Fashion. ................................................................................................................................. 17 
3 LIST OF TABLES 
Table 1. Screening and On-Study Tests and Procedures ...................................................... 26 

 
 
4 LIST OF ABBREVIATIONS 
 
AE; adverse event 
ANC; absolute neutrophil count 
APC; antigen present cells 
BCRP; Breast Cancer Research Program 
CBC; complete blood count 
CBER; Center for Biologics Evaluation and Research 
CEA; carcinoembryonic antigen 
CMP; complete metabolic panel 
CNS; central nervous system 
CPC; Cancer Protocol Committee 
CRF; case report form 
CTC; Common Toxicity Criteria 
DAMP; damage-associated molecular pattern 
DC; dendritic cell 
DCI; Duke Cancer Institute 
DoD; Department of Defense 
DUHS; Duke University Health System 
ECG; electrocardiogram 
eCTD; electronic common technical document 
eIF; eukaryotic initiation factorELISA; enzyme-linked immunosorbent assay 
ER; estrogen receptor 
E:T; effector to target ratio 
FDA; Food and Drug Administration 
FFPE; Formalin-fixed, Paraffin-embedded 
H&E; hematoxylin and eosin 
HER2; human epidermal growth factor receptor 
HIV; human immunodeficiency virus 
HRPO; Human Research Protections Office 
HRV2; human rhinovirus type 2 
HSA; human serum albumin 
ICS; Investigational Chemotherapy Services 
IHC; immunohistochemistry 
IND; investigational new drug 
IRB; Institutional Review Board 
IRES; internal ribosomal entry site 
IT; information technology 
IV; intravenous 
MMG; mammogram 
MRI; magnetic resonance imaging 
MOI; multiplicity of Infection 
NCI; National Cancer Institute 
NHP; non-human primate 
OARC; Office of Audit, Risk, and Compliance 
OncPV; Oncolytic Poliovirus 

 
 
OR; operating roomPAMP; pathogen-associated molecular pattern 
PD-L1; programmed death ligand 1 
PET; positron emission tomography 
PFS; progression free survival 
PO; by mouth (latin term per os) 
PR; progesterone receptor 
PRTBTC; Preston Robert Tisch Brain Tumor Center 
PT; prothrombin time 
PTT; partial thromboplastin time 
PV1S; poliovirus serotype 1 (SABIN) 
PVSRIPO; Polio/Rhinovirus Recombinant 
RIO; Research Integrity Office 
RNA; ribonucleic acid 
SAE; serious adverse event 
SGOT; serum glutamic-oxaloacetic transaminase 
SOC; standard of care 
SOP; standard operating procedure 
TCID; tissue culture infectious dose 
TIL; tumor infiltrating lymphocytes 
TNBC; triple negative breast cancer 
ULN: upper limit of normal 
WHO; World Health Organization 

 
 
5 PROTOCOL SYNOPSIS AND RESEARCH SUMMARY 
Purpose 
To examine oncolytic poliovirus (OncPV) bioactivity in tumor tissue after intratumoral 
administration of PVSRIPO (Polio/Rhinovirus Recombinant) in women with invasive breast 
cancer. 
 
Objectives (Exploratory): 
To investigate PVSRIPO-mediated inflammation and immunity in women with stage II-IV 
invasive breast cancer. 
1. Primary Exploratory Objective: To describe the change in the amount of tumor 
infiltrating immune cells in tumor tissue pre- and post-injection of PVSRIPO by H&E 
(Hemotoxylin and Eosin). 
2. Other Exploratory Objectives: 
a. To examine tumor tissue pre- and post-injection of PVSRIPO for inflammatory 
and 
immune 
signatures 
using 
arrays, 
CD155 
expression 
by 
immunohistochemistry (IHC), immune cell infiltrate by IHC and tumor 
infiltrating immune cells using flow cytometry (post-injection only). 
b. To examine blood for inflammatory and immune signature using arrays, 
immune cell composition (antigen presenting cells, B cells and T cells), T cell 
activation by flow cytometry and B cell activation by ELISA and peptide arrays. 
Blood will be collected on day -7 (range day -7 to -10, before polio vaccine 
booster), day 0 (before PVSRIPO injection), day 2 (range day 1 to 2 after 
PVSRIPO), day 14 (range day 10 to 20, after PVSRIPO before surgery) and 
in follow-up at months 1 (± 10 days) and 6 (± 1 month) post-PVSRIPO. 
 
Hypothesis 
Administration of oncolytic poliovirus, PVSRIPO, in the tumor causes inflammation, which 
stimulates innate and adaptive immune activation, in invasive breast cancer. 
 
Background and Significance 
Breast cancer is the most common cancer in women in the United States, with an estimated 
246,660 new diagnoses in 2016, and is the second leading cause of cancer death in 
women, with an estimated 40,450 women in the United States dying from the disease in 
2016 (1). 
Breast cancer is a heterogeneous disease comprising several molecular 
subtypes, based on receptor status, specifically the estrogen receptor (ER), progesterone 
receptor (PR), and human epidermal growth factor 2-neu (HER2) receptor. These receptors 
are both prognostic but also predictive of response to targeted therapy. 
 
Triple Negative Breast Cancer or TNBC (negative for ER, PR, and HER2) represents 15- 
20% of all breast tumors and has the poorest prognosis, with an earlier age of diagnosis, 
higher rate of local recurrence and distant metastases, and an elevated 5-yr mortality rate 
compared to ER positive cancers (Breast Cancer Facts and Figures, 2015-2016, American 
Cancer Society). Therapies that stimulate durable systemic immunity and selectively 
eliminate malignant cells will be critical for extending survival and reducing risk of 
recurrence in women with breast cancer. In several phase I trials targeting the immune 

 
 
checkpoint PD-1/PD-L1, cancer immunotherapy has been shown to demonstrate some 
promise in TNBC by showing objective response rates in the metastatic setting. 
 
PVSRIPO is a recombinant polio:rhinovirus chimera, engineered to eliminate neuronal 
competence and is devoid of neuropathogenicity upon inoculation in primates and humans. 
PVSRIPO specifically targets and destroys tumor cells and initiates innate and adaptive 
immune events in the host. PVSRIPO has demonstrated promise in a phase I study at Duke 
University in patients with recurrent WHO Grade 4 malignant glioma and is currently being 
investigated in a Phase 2 trial in recurrent WHO Grade 4 malignant glioma. A single, 
intratumoral infusion of PVSRIPO has been shown to result in durable tumor regression in 
select patients with malignant glioma even in the presence of neutralizing antibodies and 
an innate antiviral response. 
 
This is a pilot study designed to study PVSRIPO bioactivity in invasive breast cancer 
following intratumoral injection. The endpoints are exploratory and will guide design of a 
phase I/II study of PVSRIPO and combinations of PVSRIPO with immune checkpoint 
blockade (for example PD-1/PD-L1) in women with breast cancer. This pilot study is funded 
by a grant from the Department of Defense (DoD) entitled “Regional oncolytic poliovirus 
immunotherapy for breast cancer” (Principal Investigator, Smita Nair, Ph.D.). 
 
Although we plan to target the TNBC population as the intended population for this novel 
therapeutic, for this feasibility study we will broaden the eligibility criteria to include all tumor 
subtypes, as there is no biologic rationale to support differing toxicities from different 
invasive breast cancer subtypes. After safety is confirmed in this feasibility study, we will 
focus our efforts on the intended TNBC population. 
 
 
Design and Procedure 
This pilot study is designed to assess PVSRIPO-mediated intratumoral bioactivity, 
inflammation, and immune events in women with invasive breast cancer. Enrollment target 
will include six women with invasive breast cancer. Women with stage II-IV invasive breast 
cancer with at least 1 cm of residual tumor after chemotherapy and scheduled for standard 
of care surgery will be eligible. 
 
Polio vaccine booster will be administered 1 week, or up to 10 days, prior to PVSRIPO 
injection. On day 0, after pretreatment biopsy is obtained, 1x108 TCID50 (tissue culture 
infectious dose) of PVSRIPO will be injected into the tumor mass. On day 14 (range 10-20 
days), women will undergo standard-of-care (SOC) surgical resection of PVSRIPO-treated 
tumor. Women will have follow-up at 1-2 days after PVSRIPO injection, 1 month (± 10 days) 
and 6 months (± 1 month) after PVSRIPO for adverse events, disease status, and whole 
blood draw for research purposes. 
 
Women will undergo a pre-treatment core needle biopsy with collection of research cores 
of tumor tissue prior to intratumoral injection of PVSRIPO. Ten to twenty days after 
PVSRIPO injection, tumor tissue will be collected at the time of SOC surgical tumor 
resection. The surgically resected tissue will be collected and processed as fresh and 
formalin-fixed paraffin-embedded (FFPE) tissue. The tissue will be used to evaluate tumor 

 
 
infiltrating lymphocytes (TILs), CD155 expression, and inflammatory and immune signature 
using arrays. Any subjects who do not have evaluable tumor tissue from both the pre- 
treatment biopsy and post-treatment surgery will be replaced. 
 
Additionally, inflammatory and immune analysis will be conducted using blood obtained on 
day -7 (range day -7 to -10, before polio vaccine booster), day 0 (before PVSRIPO 
injection), day 2 to 5 (range day 1-2 after PVSRIPO), day 14 (range day 10 to 20 after 
PVSRIPO, which is before surgery) and in follow-up at months 1 (± 10 days) and 6 (± 1 
month) after PVSRIPO. Blood will be analyzed for inflammatory and immune signature 
using arrays, immune cell composition (antigen presenting cells, B cells and T cells), T cell 
activation by flow cytometry and B cell activation by ELISA and peptide arrays. This pilot 
study will enable a phase I/II study of PVSRIPO and combinations of PVSRIPO with 
immune checkpoint blockade (for example PD-1/PD-L1) in women with advanced breast 
cancer. 
 
Selection of Subjects 
Eligibility Criteria: This pilot study will include women with stage II-IV invasive breast 
cancer scheduled to undergo surgical resection. Target enrollment is six women with 
invasive breast cancer over 24 months. Patients will be recruited among patients receiving 
their treatment for breast cancer at Duke Cancer Institute (DCI). Any subjects who do not 
have evaluable tumor tissue from both the pre-treatment biopsy and post-treatment surgery 
will be replaced. 
 
Inclusion Criteria 
• Age > 18 years 
• Confirmation of invasive breast cancer including any of the following: 
o Triple-negative breast cancer defined as receptor status being estrogen receptor 
expression ≤ 10%, progesterone receptor expression ≤ 10%, and HER2/Neu 
expression by IHC 0 or 1+, or 2+ with fluorescence in situ hybridization confirming 
no amplification of HER2 on a pretreatment tumor sample. 
o Hormone positive breast cancer defined as receptor status being estrogen receptor 
expression > 10%, progesterone receptor expression > 10% prior to initiation of 
chemotherapy. 
o HER2+ breast cancer defined as HER2/Neu expression by IHC 3+ or fluorescence 
in situ hybridization confirming amplification of HER2 on a pretreatment tumor 
sample prior to initiation of chemotherapy. HER2+ and hormone positive (ie triple 
positive) breast cancers are included in this study. 
• Stage II-III invasive breast cancer with ≥ 1 cm of residual tumor based on MRI, 
mammogram, ultrasound, or breast clinical exam as SOC after completion of neoadjuvant 
chemotherapy, OR Stage IV BC with ≥ 1 cm locally recurrent disease (i.e. chest wall 
recurrence only) 
• ECOG ≤ 1 
• Positive serum anti-PV titer prior to biopsy 
• Received a boost immunization with trivalent inactivated IPOL™ (Sanofi-Pasteur) at least 
1 week prior to administration of the study agent 
• Hemoglobin ≥ 9.0 g/dl, ANC ≥ 1,000 cells/µl, platelets ≥ 100,000 cells/µl 

 
 
• Women must have had last dose of chemotherapy at least 2 weeks prior to treatment with 
PVSRIPO 
• Women must have at least 2 weeks minimum (ideal 3-4 weeks) of a wash-out period after 
any steroid administration (IV, PO, or intraocular) 
• Serum creatinine ≤ 2.0 mg/dl, serum SGOT and bilirubin ≤ 2 times ULN (upper limit of 
normal) 
• Women must provide written informed consent prior to enrollment on study, prior to conduct 
of screening procedures and enrollment on study 
• Women of childbearing potential will have a negative serum pregnancy test at screening 
• Women of childbearing potential must be willing to avoid pregnancy utilizing approved forms 
of contraception for the course of the study through 120 days after PVSRIPO injection 
• Surgical resection of the tumor is planned and patient is willing to undergo surgical resection 
of the cancer 
 
Exclusion Criteria 
• T1 N0 invasive breast cancer 
• Breast cancer with skin necrosis 
• Concurrent immune therapy, chemotherapy, or steroid therapy 
• Is currently participating in or has participated in a study of an investigational agent or using 
an investigational device within 4 weeks prior to polio vaccine booster 
• Has a known diagnosis of immunodeficiency 
• Has a known additional malignancy that is progressing or requires active treatment 
• Has known active central nervous system metastases and/or carcinomatous meningitis 
• Has an active autoimmune disease requiring systemic treatment within the past 3 months 
or a documented history of clinically severe autoimmune disease or a syndrome that 
requires systemic steroids or immunosuppressive agents 
• Has an active infection requiring systemic therapy 
• Has known psychiatric or substance abuse disorders that would interfere with the 
requirements of the trial 
• Is pregnant, breastfeeding, or expecting to conceive children within the projected duration 
of the trial, starting with the screening visit through 120 days after trial treatment 
• Has received prior therapy with an anti-PD-1, anti-PDL-1, anti-PDL-2, anti-CD137, or anti- 
CTLA-4 (or any other antibody or drug specifically targeting T cell co-stimulation or 
checkpoint pathways) 
• Has a known history of Human Immunodeficiency Virus (HIV) 
• Has known active Hepatitis B or Hepatitis C 
• Active liver disease with elevated transaminases > 2x ULN 
• Has received a live vaccine within 30 days prior to PVSRIPO treatment 
o Inactivated vaccines are acceptable and are not an exclusion criterion 
Duration of Study 
This pilot study will accrue 6 women with invasive breast cancer. Follow-up will include 
immunological assessments at 1-2 days post-PVSRIPO injection, 1 month and 6 months 
post-PVSRIPO for blood draws. 

 
 
Data Analysis and Statistical Considerations 
Six women with invasive breast cancer will participate in this clinical pilot study, in which we 
will explore PVSRIPO bioactivity. Any subjects who do not have evaluable tumor tissue 
from both the pre-treatment biopsy and post-treatment surgery will be replaced. Descriptive 
statistics will be used to summarize inflammatory and immune signatures in tumor and 
blood, tumor infiltrating immune cell analysis, changes in immune cell composition in blood, 
and changes in T cell activation in blood. As such, the correlative studies and the pilot 
clinical study design are part of Aim 1 of the funded DoD grant application entitled “Regional 
oncolytic poliovirus immunotherapy for breast cancer” (Principal Investigator, Smita Nair, 
Ph.D.). 
 
 
6 STUDY SCHEMA 
 
 
 
 
 
 
Figure 1. Study Schema PVSRIPO for Invasive Breast Cancer. 

 
 
7 BACKGROUND AND SIGNIFICANCE 
Study Disease 
Breast cancer is the most common cancer in women, with an estimated 246,660 new 
diagnoses in 2016, and is the second leading cause of cancer death in women, with an 
estimated 40,450 women in the United States dying from the disease in 2016 (1). Breast 
cancer is a heterogeneous disease comprising several molecular subtypes, based on 
receptor status, specifically the estrogen receptor (ER), progesterone receptor (PR), and 
human epidermal growth factor 2-neu (HER2) receptor. These receptors are both 
prognostic but also predictive of response to targeted therapy. Although often curable when 
localized to the breast and lymph nodes, on the whole, breast cancer remains incurable for 
most women in the metastatic setting (2). An estimated 20% to 30% of women diagnosed 
with invasive breast cancer will have a recurrence and may eventually die of their disease 
(Department of Defense (DoD) Breast Cancer Research Program (BCRP), “The Breast 
Cancer Landscape”). 
 
Triple Negative Breast Cancer (TNBC) refers to tumors lacking the expression of estrogen 
receptor (ER), progesterone receptor (PR), and the human epidermal growth factor receptor 
2 (HER2) and portends a poor prognosis compared with tumors expressing these receptors. 
These tumors account for about 15% of all breast cancers and have been shown to be more 
aggressive than other tumor subtypes. These women are diagnosed at an earlier age 
and have a higher risk of local and distant recurrence (3). The higher risk of distant 
metastases results in an elevated 5-year mortality rate compared to ER positive cancers 
(4). In addition, the paucity of targeted treatment available for TNBC likely contributes to 
the lower survival rate. Despite the risk of increased local recurrence and distant 
metastases, standard criteria are used to select the approach to local therapy as more 
extensive surgeries have not been shown to improve survival (3). At the time of distant 
recurrence, there are no standard of care approaches for systemic chemotherapy. 
Common chemotherapies utilized in the neoadjuvant, adjuvant, and metastatic 
setting 
for 
invasive 
breast 
cancers 
include anthracyclines, 
cyclophosphamide, 
taxanes, capecitabine, eribulin, vinorelbine, and platinums. In addition to these 
chemotherapies, common anti-HER2 treatments used in HER2+ breast cancer include 
trastuzumab, pertuzumab, T-DM1, and lapatinib. 
 
Immunotherapy has been demonstrated to mediate regression of large bulky tumors in 
the stage IV setting in multiple clinical settings, resulting in improved overall and disease 
free survival in melanoma and non-small cell lung cancer, as well as promising 
objective response rates in head and neck, gastric, and urothelial cancers (5-10). The 
presence of tumor infiltrating lymphocytes (TILs) correlates with a favorable 
prognosis in various malignancies including TNBC (11). Several phase I/II clinical 
trials targeting the immune checkpoint PD-1/PD-L1 pathways in metastatic TNBC have 
shown promising results with overall response rates of 18-20% (9, 12). 
 
This is a pilot study designed to examine PVSRIPO bioactivity in breast cancer 
following intratumoral injection. The endpoints are exploratory and will guide a phase I/ 
II study of PVSRIPO and combinations of PVSRIPO with immune checkpoint blockade in 
women with breast cancer. Although we plan to target the TNBC population as the 
intended population 

 
 
for this novel therapeutic, for this feasibility study we will broaden the eligibility criteria to 
include all tumor subtypes, as there is no biologic rationale to support differing toxicities 
from different invasive breast cancer subtypes. After safety is confirmed in this feasibility 
study we will focus our efforts on the intended TNBC population. 
 
Study Agent 
PVSRIPO is the live attenuated, oral (Sabin) serotype 1 poliovirus vaccine containing a 
heterologous internal ribosomal entry site (IRES) derived from the human rhinovirus type 2 
(HRV2). Poliovirus receptor is mediated by the poliovirus receptor, CD155, an oncofetal cell 
adhesion molecule, which is widely expressed ectopically in malignancy, e.g. invasive 
breast cancer. Tumor specificity is mediated by selective expression of CD155 in invasive 
breast cancer. Translation is repressed at the heterologous HRV2 IRES in normal breast 
tissue and a supportive environment for PVSRIPO translation, growth, and cytotoxicity is 
mediated by universally active protein kinase C (PKC)-Ras-Erk signaling to translation 
machinery in cancers. PVSRIPO is administered intratumorally. Since PVSRIPO is a 
version of the serotype 1 live-attenuated (Sabin) poliovirus vaccine (PV1), its immunogenic 
properties and potential for long-term sequelae are expected to be similar. Poliovirus 
serotype 1 (SABIN) (PV1S) has been safely administered to >10 billion individuals 
worldwide without untoward long-term immunogenic sequelae. The pre-clinical data 
described below was included in the funded DoD grant application to provide rationale for 
this pilot study. 
7.2.1 
Pre-Clinical Experience 
For details about the pre-clinical experience with PVSRIPO, please refer to the 
Investigator’s Brochure for PVSRIPO. 
 
PVSRIPO tumor tropism: PVSRIPO has natural cancer tropism, due to widespread 
ectopic expression of the poliovirus receptor (PVR) CD155 in solid cancers, where it 
may serve as a tumor immune evasion strategy (13-17). Despite neuronal 
incompetence, PVSRIPO is highly cytotoxic in neoplastic cells (17). PVSRIPO’s oncolytic 
efficacy depends on ectopic CD155 expression and unhinged MAPK-signals to 
translation machinery. Both properties define most malignancies, including breast 
cancer. Efficient tumor tropism and rapid, violent cytotoxicity distinguish PVSRIPO 
from other oncolytic viruses. PVSRIPO uniquely has oncolytic efficacy despite pre- 
existing neutralizing antibodies and viral cell killing proceeds in the presence of an 
active innate antiviral response (18, 19). 
 
Figure 2A depicts CD155 expression on breast cancer cell lines and primary breast 
tumor tissue (20) (de-identified primary tissue was used and the sub-type of primary 
tumor tissue was not determined). Note that CD155 is not expressed on normal breast 
epithelial cultures (S2N). PVSRIPO exhibits anti-tumor activity in breast cancer models 
of brain metastases and leptomeningeal disease, which are notoriously refractory to 
therapy (20) (Figure 2B). Figure 3 depicts CD155 expression on primary triple negative 
breast tumor tissue. 

 
 
 
 
Figure 2. Breast Cancer Susceptibility to PVSRIPO. A. CD155 expression in breast cancer cell 
lines and primary breast cancer tissues by western blot. B. PVSRIPO efficacy in vivo. Rats (n=10) with 
intrathecal xenografts were treated once with PVSRIPO at 1x107 pfu (open triangles) or 1x109 pfu 
(closed squares), 3 days after tumor implant. UV-inactivated PVSRIPO was used as control (closed 
diamonds). 
 
 
Figure 3. Immunohistochemistry for the PV Receptor (CD155) in 19 Triple-Negative 
Breast 
Cancer 
Cases. 
For methods and detailed information on the assay, 
expression scoring, etc. see Chandramohan et al. (21). The table summarizes expression 
scores in the 19 cases. The numbered panels show IHC results for the corresponding cases 
in the table. Size bars represent 1 mm (7, 14) or 100 mm (4, 8, 10, 17). 
 
Molecular mechanisms of PVSRIPO tumor specificity: PVSRIPO targets constitutively 
active Raf-Erk1/2 signal transduction to translation machinery via Erk1/2 to downstream 

 
 
MAPK-interacting kinases and to eIF4G to generate conditions that favor 5’ 
methyl-7- guanidine cap-independent, IRES-mediated translation (22-24). The Raf-Erk1/2 
pathway is a central gene regulatory node in malignancy that is recalcitrant to small- 
molecule inhibitors, due to drug resistance mechanisms that restore Erk1/2 activity, for 
example to trastuzumab in breast cancer (25). PVSRIPO oncolysis depends on active 
Raf-Erk1/2 signals thus exploiting an essential mitogenic mechanism in cancer. 
PVSRIPO causes potent cytotoxicity, with irreversible, lethal effects on infected host 
cells as early as 90 min post PVSRIPO infection (26). The TNBC cell line MDA- 
MB231 and the triple-negative inflammatory breast cancer (IBC) cell line SUM149, are 
susceptible to PVSRIPO-mediated lysis and express CD155 (Figure 4). Lethal 
PVSRIPO-mediated cytotoxicity results in the loss of intact eIF4G and the generation of 
the eIF4G cleavage product (Figure 4A) (27). 
 
Figure 4. PVSRIPO Cytotoxicity in Breast Cancer Cell Lines. Breast cancer cell lines, MDA- 
MB231 and SUM149, were infected at multiplicity of Infection (MOIs) of 0.1 or 10 with 
PVSRIPO. A. Lysates were collected at the denoted intervals and tested by immunoblot for 
markers of viral cytotoxicity (eIF4G cleavage), host cell demise (PARP cleavage), viral 
replication (viral proteins P2/2BC/2C), and antiviral responses (p-eIF2α(S51)/IFIT1/ISG15). 
Loss of cellular proteins (e.g. tubulin) and viral protein at later time points correlates with lysis 
of infected cells. B. Cells were infected at an MOI of 0.1 and harvested at 48 h post infection 
(hpi) for analysis of MHC class I and CD155 expression by flow cytometry (light grey, uninfected 
cells stained with isotype control; dark grey, uninfected cells stained for MHC or CD155; and 
red, infected cells stained for MHC or CD155). 
 
PVSRIPO activates dendritic cells: PVSRIPO oncolysis releases tumor antigens and 
generates DAMPs and PAMPs (damage-associated and pathogen-associated molecular 
patterns) that stimulates immune cells (28). Poliovirus RNA is a classic PAMP that 
engages cytoplasmic pattern recognition receptors and downstream responses (Mda5- 
Stat1-IFN/) in immune cells. Poliovirus naturally targets antigen presenting cells 
(APCs), such as macrophages, monocytes, and dendritic cells (DCs), via CD155. To 
delineate the effects of PVSRIPO on DCs, we examined markers of cytotoxicity and 
activation status on antigen presentation. DCs infected with PVSRIPO at MOI of 1, 10, 
and 100 did not exhibit lytic 

 
 
cytoxicity as compared to lytic cytotoxicity in cancer cells infected with PVSRIPO (Figure 
5A). Viral propagation was detected, however, in sharp contrast to cancer cells, did 
not yield progeny beyond background levels (Figure 5A) (29). An increase in 
Stat1 phosphorylation (which leads to increased Stat1 total protein) followed by the 
upregulation of interferon (IFN)-stimulated genes ISG15, IFIT1, and MDA5 protein, signify 
the activation of type-I IFN responses (Figure 5B) (27). Inefficient cleavage of eIF4G 
and PARP and decreased 2C expression following infection, suggests severe limitations 
on viral replication and cytotoxicity. This is in sharp contrast to what we observe in 
tumor cells treated with PVSRIPO (Figure 4). In animal tumor models, there is 
histologic evidence for virus-mediated tumor cell killing and indirect host-mediated 
inflammatory responses focused against tumor (24, 30-33). 
 
Figure 5. Infection and Activation of Dendritic Cells (DCs) by PVSRIPO. Human monocyte- 
derived DCs were infected with PVSRIPO at MOI of 1, 10, and 100. A. LDH-release assay 
(top) and viral progeny (bottom) analysis was conducted using infected DCs and compared to 
infected DM6/MDA-MB231 cells. B. Western blot analysis was conducted at the designated 
time points post infection (PI). PVSRIPO viral protein (2C) detection signifies productive 
infection. An increase in Stat1 phosphorylation (which leads to increased Stat1 total protein) 
followed by the upregulation of IFN-stimulated genes ISG15, IFIT1, and MDA5 protein, signify 
activation of IFN responses. Inefficient cleavage of eIF4G and PARP and decreased 2C 
expression following infection, suggests severe limitations on viral replication and cytotoxicity. 
 
To examine if presence of PAMPs and DAMPs in PVSRIPO oncolysate activates DCs, 
tumor lysates were collected after exposing tumor cells to mock- or PVSRIPO-infection 
(MOI = 0.1; 48h) and treating human monocyte-derived DCs with the resulting 
oncolysate (Figure 6) (27). To control for the effects of infectious PVSRIPO particles 
present in the oncolysate on DCs (~5x108 pfu/mL), virus was removed using a 100kD 
filter. This approach removes all infectious virus but does not remove IFN/dsRNA/HMGB1 
or other conceivable PAMPs/DAMPs. Exposure to oncolysate increased DC co- 
stimulatory molecule expression 

 
 
and other markers of DC activation (Figure 6B). This effect was mitigated by 
100kD filtration, suggesting that PVSRIPO infection may actively participate in DC 
activation (27). 
 
Figure 6. PVSRIPO-Induced Oncolysate Causes DC Activation in a Virus Dependent 
Fashion. A. Outline of in vitro human DC activation experiment. B. PVSRIPO-induced tumor 
lysate (PVSRIPO oncolysate) activates human DCs. DCs were generated (34) from HLA-A2+ 
donor cells. Supernatant from untreated or PVSRIPO-infected (MOI = 0.1; 48hpi) tumor cells 
were either unfiltered or filtered through a 100 kD filter. DCs were treated with the resulting 
supernatants for 24 hours. Standard immature DCs (iDCs) or DCs matured in cytokine cocktail 
(mDCs) were used as controls. Flow cytometry was used to assess DC activation/maturation 
phenotype and viability. This experiment was repeated twice and representative data is shown. 
7.2.2 
Clinical Experience 
Clinical efficacy of OncPV therapy against recurrent WHO Grade 4 malignant glioma 
PVSRIPO has been tested in a phase I study in subjects with recurrent WHO Grade 4 
malignant glioma and has generated a durable clinical response in approximately 20% of 
patients. These responses occurred despite prior disease progression following surgical 
resection, cranial radiation therapy, chemotherapy (temozolomide, TMZ), and (sometimes) 
bevacizumab. Fifteen patients were enrolled into the PVSRIPO dose-escalation portion of 
this phase I study. Dose was rapidly escalated from 1x108 TCID50 (dose level 1) to 1x1010 
TCID50 (dose level 5) after which dose was reduced due to long-term steroid or 
bevacizumab therapy to control inflammatory reaction secondary to PVSRIPO-mediated 
immune response. For more information about the clinical findings of PVSRIPO, refer to the 
Investigator’s Brochure for PVSRIPO. Based on the original dose finding and safety study 
in patients with recurrent malignant glioma (ClinicalTrials.gov Identifier: NCT01491893; 
Duke IRB Pro00031169), we will use 1x108 TCID50 (dose level 1) in women with invasive 
breast cancer. 
We have chosen to use 1x108 TCID50 (dose level 1 from the phase 1 dose-escalation study 
in patients with recurrent WHO Grade 4 malignant glioma) for the following reasons. 
1. In the dose escalation study conducted in patients with malignant glioma, patient 1 
received dose level 1, 1x108 TCID50. This patient responded favorably to PVSRIPO 
therapy and was disease-free 60+ months after receiving a single intratumoral injection 

 
 
of PVSRIPO. 
2. Brain inflammation in the area of the tumor or immediately adjacent was the main toxicity 
associated with intratumoral infusion of PVSRIPO and the primary reason for dose 
reduction in the phase I brain tumor study and use of dose level -1 (5 x 107 TCID50) in 
the Phase 2 glioma trial. There were no signs of encephalitis, systemic toxicity or 
systemic inflammatory processes. Given that the breast is not in a physiologically 
confined space (like brain in the cranium) the effects of inflammation due to PVSRIPO 
are not of significant clinical impact. Additionally, the breast tumor will be resected 10- 
20 days following PVSRIPO infusion therefore limiting the time in which the patient may 
experience discomfort from tumor site hyper-inflammation. 
3. A higher dose is warranted in this setting given that there may be some systemic leakage 
upon injection, therefore requiring an overall higher dose to ensure that a substantial 
amount of the PVSRIPO remains in the tumor. We will therefore start with dose level 1 
(1x108 TCID50) from the phase 1 recurrent WHO Grade 4 malignant glioma study, to 
ensure that we elicit a response in tumor (both tumor killing and immune consequences) 
post-PVSRIPO in this pilot study. 
 
Study Purpose and Rationale 
PVSRIPO, a recombinant oncolytic poliovirus, has shown promising results in recurrent 
malignant glioma when administered intracerebrally. Once administered, PVSRIPO enters 
tumor cells through the poliovirus receptor CD155, which is overexpressed in solid 
malignancies. As such, it both specifically targets and destroys tumor cells that it enters and 
initiates innate and adaptive immune events in the host. 
 
A phase I clinical trial of PVSRIPO in recurrent malignant glioma patients has yielded 
durable complete radiographic and clinical responses with a proportion of patients achieving 
prolonged disease survival up to 61+ months. The first subject treated with PVSRIPO is 
alive and well 5 years after PVSRIPO administration. PVSRIPO was granted Breakthrough 
Therapy Designation by FDA/CBER on May 16th, 2016. 
 
As CD155 is also overexpressed in all subtypes of invasive breast cancer, a pilot study to 
examine the effects of intratumoral injection of PVSRIPO into invasive breast cancer tumors 
in 6 women is of interest. Importantly, if effective, this will lead to a phase I/II trial of 
PVSRIPO and combinations of PVSRIPO with immune checkpoint blockade (for example 
PD-1/PD-L1) in women with triple negative breast cancer. Of note, PD-1/PD-L1 blockade 
has already demonstrated some clinical efficacy in women with TNBC. 
 
Rationale: The rationale for doing this pilot clinical study in six women with invasive breast 
cancer is as follows: 
1. To characterize intratumoral PVSRIPO injection in a non-CNS cancer 
2. To investigate inflammation and immunity in tumor tissue post-PVSRIPO treatment. 
PVSRIPO treatment is showing promise in malignant glioma patients but the tumor 
tissue is not accessible to examine PVSRIPO bioactivity. This pilot study in women 
with accessible invasive breast cancer would enable investigation of PVSRIPO 
bioactivity in tumor tissue. 
3. To demonstrate that post-transcriptional gene regulatory principles that mediate 
PVSRIPO tumor cytotoxicity apply to breast cancer. 

 
 
8 OBJECTIVES AND ENDPOINTS 
To examine oncolytic poliovirus (OncPV) bioactivity in tumor tissue after intratumoral 
administration of PVSRIPO in women with invasive breast cancer. 
 
Objectives (Exploratory): 
To investigate PVSRIPO-mediated inflammation and immunity in women with stage II-IV 
invasive breast cancer. 
1. Primary Exploratory Objective: To describe the change in the amount of tumor 
infiltrating immune cells in tumor tissue pre- and post-injection of PVSRIPO by H&E 
(Haemotoxylin and Eosin). 
2. Other Exploratory Objectives: 
a. To examine tumor tissue pre- and post-injection of PVSRIPO for inflammatory 
and 
immune 
signature 
using 
arrays, 
CD155 
expression 
by 
immunohistochemistry (IHC), immune cell infiltrate by IHC and tumor 
infiltrating immune cells using flow cytometry (post-injection only). 
b. To examine blood for inflammatory and immune signature using arrays, 
immune cell composition (antigen presenting cells, B cells and T cells), T cell 
activation by flow cytometry and B cell activation by ELISA and peptide arrays. 
Blood will be collected on day -7 (range day -7 to -10, before polio vaccine 
booster), day 0 (before PVSRIPO injection), day 2 (range day 1-2 after 
PVSRIPO), day 14 (range day 10 to 20, after PVSRIPO before surgery) and 
in follow-up at months 1 (± 10 days, after PVSRIPO) and 6 (± 1 month, after 
PVSRIPO). 
 
Hypothesis 
Administration of oncolytic poliovirus, PVSRIPO, in the tumor causes inflammation, which 
stimulates innate and adaptive immune activation, in invasive breast cancer. 
 
9 INVESTIGATIONAL PLAN 
Study Design 
This is a single institution pilot study designed to assess PVSRIPO-mediated intratumoral 
bioactivity, inflammation and immune cell infiltration in 6 patients with invasive breast 
cancer. To maximize immunological stimulation, we will wait at least 2 weeks (ideal 3-4 
weeks) after the last dose of chemotherapy or steroids to administer the polio vaccine 
booster. PVSRIPO will be injected into the tumor mass at 1x108 TCID50 7-10 days after polio 
vaccine booster is administered. 
 
Biopsy material will be obtained from tumor tissue immediately prior to virus administration 
and resected tumor tissue will be obtained at 10-20 days (ideally 12-16) after PVSRIPO at 
the time of surgical resection. The PVSRIPO-treated tumor tissue will be transported to 
Surgical Pathology by the Brain Tumor BioRepository (BTBR) Team, as directed by Dr. 
Roger McLendon, MD or per clinical protocol, for gross examination and research tissue 
procurement, thereby safe-guarding the diagnostic integrity of the specimen. Additional 
expertise in breast pathology will be provided by Dr. Edgardo Parilla Castella, MD, PhD. 

 
 
Tissue deemed appropriate for collection (i.e. wasteful excess and of no diagnostic value) 
will be collected by the Brain Tumor BioRepository (BTBR) Team, as directed by Dr. Smita 
Nair, PhD, and transported to her lab for correlative molecular and immune analyses. Our 
primary objective is to determine change in the amount of tumor infiltrating immune cells in 
tumor tissue pre- and post-injection of PVSRIPO by H&E. We will also evaluate 
inflammatory and immune signatures using inflammatory and innate/adaptive arrays, 
CD155 expression and immune cell infiltrate by IHC and tumor infiltrating immune cells by 
flow cytometry (post-injection only). 
 
Additionally, inflammatory and immune analysis will be conducted using blood obtained on 
day -7 (range day -7 to -10, before polio vaccine booster), day 0 (before PVSRIPO 
injection), day 1-2 (after PVSRIPO), day 14 (range day 10 to 20 after PVSRIPO, which is 
before surgery), and in follow-up at months 1 (± 10 days) and 6 (± 1 month) after PVSRIPO. 
Blood will be analyzed for inflammatory and immune signature using arrays, immune cell 
composition (antigen presenting cells, B cells and T cells), T cell activation by flow cytometry 
and B cell activation by ELISA and peptide arrays. Immune cell composition and T cell 
activation in blood will be done using DuraClone IM panels from Beckman Coulter. 
 
Safety Considerations 
9.2.1 Surgical Complications 
Patients undergoing biopsy and excision of breast tumors may develop infection at site 
of injection or breast tissue removal, cellulitis, or abscess formation. Seromas or 
hematomas may also be a complication in this setting. The risk of perioperative infection 
will be the same as encountered routinely for breast surgery. 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 
and/or undergo incision and drainage procedure. 
9.2.2 
Anesthesia 
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. 
9.2.3 
Poliomyelitis 
PVSRIPO has been tested in non-human primates (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. In addition, the use of intracerebral 
PVSRIPO in human subjects with recurrent malignant glioma in phase I trials have not 
induced symptoms of poliomyelitis. 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. 
9.2.4 
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 malignant glioma cells does not select for 
genetic changes in viral progeny. However, the occurrence of such events cannot be 
categorically excluded in patients receiving PVSRIPO. So far, the use of intracerebral 
PVSRIPO in human subjects with malignant glioma in phase I trials have not 
demonstrated these phenomena. 
9.2.5 
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. In addition, the use of PVSRIPO in human subjects with 
recurrent malignant glioma in phase I have not demonstrated this phenomena. 
9.2.6 
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). 
9.2.7 
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. In addition, this also has not been observed with the use of PVSRIPO in 
human subjects with recurrent malignant glioma in phase I trials. 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. 

 
 
9.2.8 
Unacceptable Toxicities 
Clinical trials using PVSRIPO in breast tumor patients have not been performed. Since 
the overall risk classification of this research is unknown we plan to take the following 
approaches to ensure patient safety: 
I. 
We will stagger enrollment of each patient by at least 4 weeks, which will be 
sufficient time to implement dose reduction or other safety measures based on 
adverse events observed. 
II. 
The study will only treat 6 patients total. However, the trial will be paused for 
enrollment and undergo reevaluation if two patients have any Grade 3 or any 
Grade 4 toxicity that is not reversible within 2 weeks after surgery (~24-34 days 
after PVSRIPO, depending on when the surgery is performed), or any life- 
threatening event, or treatment-related death will be considered an unacceptable 
toxicity. Any grade 2 or higher serious autoimmune toxicities particularly those 
affecting vital organs (e.g. cardiac, renal, CNS) will be considered an 
unacceptable toxicity. Also, the study will be paused to enrollment if a patient 
death occurs within a month after PVSRIPO injection possibly or likely related to 
PVSRIPO. 
 
Rationale for Selection of Dose, Regimen, and Treatment Duration 
The dose of PVSRIPO for this trial was selected based on IND-directed toxicity studies 
and on experience from a phase I study in adults with recurrent WHO grade IV malignant 
glioma. For more information on the results from the dose-finding study, please refer to 
the Investigator’s Brochure for PVSRIPO. 
 
We have chosen to use 1x108 TCID50 (dose level 1 from phase 1 dose-finding malignant 
glioma trial) for the following reasons. 
1. In the dose escalation study conducted in patients with recurrent malignant glioma, 
patient 1 received dose level 1, 1x108 TCID50. This patient responded favorably to 
PVSRIPO therapy and was disease-free 60+ months after receiving a single 
intratumoral injection of PVSRIPO. 
2. Brain inflammation in the area of the tumor or immediately adjacent was the main toxicity 
associated with intratumoral infusion of PVSRIPO and the primary reason for dose 
reduction in the phase I brain tumor study. Because of this, dose level -1 (5 x 107 TCID50) 
is being investigated in the phase 2 recurrent malignant glioma study in adults and phase 
1b study in pediatric patients. There were no signs of encephalitis, systemic toxicity or 
systemic inflammatory processes. Given that the breast is not in a physiologically 
confined space (like brain in the cranium) the effects of inflammation due to PVSRIPO 
are not of significant clinical impact. Additionally, the breast tumor will be resected 10- 
20 days following PVSRIPO infusion therefore limiting the time in which the patient may 
experience discomfort from tumor site hyper-inflammation. 
3. A higher dose is warranted in the breast cancer setting given that there may be some 
systemic leakage upon injection, therefore requiring an overall higher dose to ensure 
that a substantial amount of the PVSRIPO remains in the tumor. We will therefore start 
with dose level 1 (1x108 TCID50) from the phase 1 malignant glioma study, to ensure 
that we elicit a response in tumor (both tumor killing and immune consequences) post- 
PVSRIPO in this pilot study. 

 
 
4. If a study pause occurs due to adverse events observed according to Section 9.2.8 and 
these adverse events are concluded to be possibly or likely related to PVSRIPO injection 
and it is deemed safe to continue the study, then we will continue to enroll patients at a 
lower dose of 5x107 TCID50, the dose currently being investigated in the phase 2 
recurrent malignant glioma study in adults and a phase 1b study in recurrent malignant 
glioma in pediatric patients. 
 
Rationale for Correlative Studies 
As this is a pilot study with exploratory endpoints only, correlative studies will be used to 
assess PVSRIPO tumor bioactivity. Specifically, the study will investigate PVSRIPO- 
mediated inflammation and immunity in women with stage II-IV invasive breast 
cancer. Greater detail is provided in Section 8. 
 
Early Study Termination 
This study can be terminated at any time for any reason by the PI or Sponsor. If this occurs, 
all subjects on study will be notified as soon as possible. Additional procedures and/or 
follow up should occur in accordance with Section 12.6, which describes criteria 
for Early Withdrawal of Subject(s). 
 
10 STUDY DRUG 
Names, Classification, and Mechanism of Action 
PVSRIPO is the live attenuated poliovirus (PV) serotype 1 (SABIN) vaccine with its internal 
ribosomal entry site (IRES) element replaced with that of human rhinovirus type 2. IRES 
exchange causes an inability of PVSRIPO to drive translation of its genome in normal 
cells (35, 36). Thus, PVSRIPO is devoid of neuropathogenicity after high-dose 
intracerebral inoculation in non-human primates (13) and in humans. Because PVSRIPO 
is a version of the serotype 1 live-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. 
 
Supply, Receipt, and Storage 
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. The PVSRIPO being utilized in this study was 
manufactured at the Biopharmaceutical Development Program/National Cancer Institute 
(NCI)-Frederick. 
 
Dispensing and Preparation 
The study agent and vehicle will be supplied to the Investigational Pharmacy by the Sponsor 
or their representative. 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 not compromise biological and biophysical properties for at least 5 years. 

 
 
The agent will be stored long-term in an ultra-low temperature freezer (e.g., less than or 
equal to -80°C freezer). 
 
For aliquot preparation, the agent will be thawed slowly on ice (~4C) and kept at that 
temperature. Thawed vials of PVSRIPO are stable at 4°C for 48 hours. The supplier will 
provide the study agent’s potency (as tissue culture infectious doses [TCID]) 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. 
 
The study agent will be in a disposable, 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 site of patient injection in a ‘ziplock’ bag placed in a portable cooler while maintaining a 
temperature of 4°C. 
 
Each patient will be injected with a total intratumoral dose of 1x108 TCID50 PVSRIPO in 
physiologic saline stabilized with 0.2% human serum albumin. The injection surface will be 
cleaned with Chloraprep or betadine and allowed to dry. A local anesthetic agent will be 
applied to the injection area. There will be a single entry point into the breast. The study 
agent in 0.6 mL will be loaded into a 1 cc syringe connected to the injection needle. Under 
direct ultrasound visualization, a 20 or 21-gauge needle will be inserted into the tumor. 
Following visualization of the needle tip within the tumor, study agent will be injected over 
10-15 seconds by hand injection. The needle tip will then be repositioned to another site 
within the tumor and the injection repeated. A total of 3-6 sites will be injected with the total 
study dose evenly divided among sites. The location of sites will be selected by the 
performing radiologist with the aim of evenly distributing the study agent over the volume of 
a typically irregularly shaped tumor. All injections will be performed under direct real-time 
ultrasound visualization. The needle will then be removed very slowly. 
 
Post-Injection pressure will be applied to the injection site(s) with sterile gauze for at least 
30 seconds. The injection site and surrounding area will be swabbed with alcohol. After a 
change of gloves, the injected lesion(s) will be covered with an absorbent pad and dry 
occlusive dressing. Exterior of the occlusive dressing will be wiped with alcohol. Following 
injection and bandaging, the patients will be transferred to clinic 2.2. Vital signs will be 
monitored every 10 minutes for an hour prior to patient discharge. Patients will be advised 
to keep the injection site(s) covered. Bandage will be replaced when the patient returns to 
the clinic 1-2 days after PVSRIPO injection. Patients will be advised to leave the dressing 
on for 2-3 more days. If the bandage falls off at home, the patients will be asked to replace 
the bandage. They will receive instructions on how to replace bandage and will be provided 
with gloves and plastic Ziploc disposal bags. The old bandage and gloves will be placed in 
the plastic Ziploc bag, the bag closed and then put in regular plastic trash bag for disposal. 
 
Compliance and Accountability 
Compliance and accountability will be monitored by the Investigational Chemotherapy 

 
 
Service (ICS) according to their standards of practice (SOPs). 
 
Disposal and Destruction 
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. Disposal of any unused 
or partially used study agent will be the responsibility of the Investigational Pharmacy. All 
surgical materials used in the procedure and potentially coming into contact with the study 
agent will be disposable. Used sharps with be disposed in a biohazard sharps container 
and incinerated in final disposal. 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. 
 
Spill procedure. PVSRIPO can 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 the 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. 
 
11 SUBJECT ELIGIBILITY 
Inclusion and Exclusion Criteria 
Please refer to Section 5.4. 
 
12 SCREENING AND ON-STUDY TESTS AND PROCEDURES 
Informed consent will occur after confirming tumor size by imaging per SOC between days 
-28 and -7 After obtaining informed consent and during screening (days -28 to -7), women 
with invasive breast cancer will undergo complete history and physical exam, blood work 
including CBC, CMP, and pregnancy test (if applicable). An ultrasound, mammogram, or 
breast MRI showing a residual breast tumor at least 1 cm in size before PVSRIPO 
injection is required. 
 
After completing informed consent for the protocol, the patient will receive a polio vaccine 
booster (day -7). On day 0, an ultrasound guided core biopsy will be performed at radiology 
prior to injection of PVSRIPO into the tumor. Injection will be performed in Duke Breast 
Radiology and the patients will be transferred to Duke Cancer Center clinic 2-2. Vital signs 
will be monitored every 15 minutes for an hour prior to patient discharge. On day 10-20 

 
 
(ideal 12-16 days), the patient will undergo SOC surgery. Part of the excised tumor will be 
transported to Surgical Pathology by the Brain Tumor BioRepository (BTBR) Team, as 
directed by Dr. Roger McLendon, MD or per clinical protocol, for gross examination and 
research tissue procurement. Additional expertise in breast pathology will be provided by 
Dr. Edgardo Parrilla Castellar, MD, PhD. 
 
Tissue deemed appropriate for collection (i.e. wasteful excess and of no diagnostic value) 
will be collected by the Brain Tumor BioRepository (BTBR) Team, as directed by Dr. Smita 
Nair, PhD, and transported to her lab for correlative molecular and immune analyses. 
Surgically resected tissue will be used fresh and/or processed into formalin-fixed paraffin- 
embedded (FFPE) blocks, required for immunohistochemistry and immunofluorescence. 
Additionally, inflammatory and immune analysis will be conducted using blood obtained on 
day -7 (range day -7 to -10, before polio vaccine booster), day 0 (before PVSRIPO 
injection), day 1-2 (range day 1-2 after PVSRIPO), day 14 (range day 10 to 20 after 
PVSRIPO, which is before surgery) and in follow-up at months 1 (± 10 days) and 6 (± 1 
month) after PVSRIPO. Blood will be analyzed for inflammatory and immune signature 
using arrays, immune cell composition (antigen presenting cells, B cells and T cells), T cell 
activation by flow cytometry and B cell activation by ELISA and peptide arrays. Whole blood 
will be collected for immunological correlates on day -7 (range -7 to -10 just before polio 
vaccine booster), day 0 (before PVSRIPO injection), day 1-2 and day 14 (range day 10 to 
20 after PVSRIPO, just before surgery) and in follow-up at months 1 and 6 after PVSRIPO. 
 
Table 1. Screening and On-Study Tests and Procedures. 
 
Anticipated Time Frame 
Screening 
Day -28 to 
-7 
Day -7 
Day 0 
Day 
1-2 
Day 10- 
20 
Follow- 
up 
Scheduling Window 
(Days) 
-28 to -7 
-7 to - 
10 
0 
1-2 
10 to 
20 
1, 6 
months 
Informed consent 
X 
Medical history 
X 
Physical Exam, vitals 
X 
X 
X 
X 
Wound/injection site 
evaluation 
X 
X 
Concomitant medications 
Continuous 
12-Lead ECG 
X 
Pregnancy test (whole 
blood - only if 
premenopausal prior to 
chemotherapy) 
X 
CBC w/diff 
X 
X 
X 
X 
X 
CMP 
X 
X 
X 
Urinalysis 
X 

 
 
 
Anticipated Time Frame 
Screening 
Day -28 to 
-7 
Day -7 
Day 0 
Day 
1-2 
Day 10- 
20 
Follow- 
up 
SOC MRI, MMG, or 
ultrasound of tumor 
X 
X 
X1 
X2 
Polio vaccine booster 
X 
PVSRIPO injection 
X 
Tumor biopsy 
X 
Tumor resection 
X 
Whole blood for immune 
analysis (35 mL) 
X 
X 
X 
X 
X 
X 
Blood for polio titer (5 mL) 
X 
X 
X 
X 
X3 
SAEs / AEs 
Continuous 
Stool sample* 
X4 
Days 
7,14,21,56 
 
Screening and On-Study Tests and Procedures 
• History: Complete history, ECOG status, and physical exam including vital signs, and 
weight, must be obtained within 7-28 days prior to completion of informed consent. 
• Blood work: CBC with differential, CMP, and pregnancy test. Pre-study laboratory tests 
must be obtained within 7-28 days prior to completion of informed consent. 
• Imaging: All participants must have a MRI, MMG or ultrasound after completion of 
chemotherapy (days -28 to -7) showing a breast tumor at least 1 cm in size. 
• Pathology: Pathological report confirming diagnosis of invasive breast cancer prior 
to start of chemotherapy will be required. 
• Systemic imaging: CT chest/abdomen/pelvis, PET scans, ultrasounds, and bone 
scans during the trial are at the discretion of the PI and will be ordered as indicated to 
assess for extent of disease or disease relapse per SOC. 
• *Stool samples: Stool samples will be collected on days 7, 14, 21, and 56 after 
PVSRIPO injection to comply with Duke Biosafety Protocol. This is not a clinic visit. 
Patients will be given a kit to collect stool that includes the following items: a toilet hat 
specimen collector, tongue depressor(s), a urine sample container, 2 plastic bags, and a 
brown paper bag. Samples will be stored frozen until the next clinic visit. 
Pre-treatment Period 
Women with stage II-IV invasive breast cancer with a residual tumor mass of at least 1 cm 
by imaging, after completion of neoadjuvant chemotherapy will be included in this study. A 
polio vaccine booster will be given 7 to 10 days before PVSRIPO injection. Thirty-five ml 
whole blood will be drawn for immunological profiling before polio vaccine booster. 
 
 
1 At the discretion of the physician, MMG or ultrasound may be done the day prior to surgery 
2 MMG or ultrasound only done at the 6 (± 1) month follow-up visit. 
3 At 1 month follow-up visit only. 
4 This is a take-home kit and does not involve a clinic/hospital visit. Days refer to days after PVSRIPO. 

 
 
Treatment Period 
Patients will undergo a core biopsy of the tumor after which PVSRIPO will be administered 
via intratumoral injection using ultrasound guidance in Radiology. Core biopsy will be tested 
for CD155 expression, immune infiltrate by H&E and IHC, and inflammatory and immune 
signature by arrays. After injection, the patient will be transferred to clinic 2.2. Vital signs 
will be monitored every 10 minutes for an hour prior to patient discharge. Thirty-five ml of 
whole blood will be drawn for immunological profiling prior to PVSRIPO injection. Thirty-five 
ml of whole blood will be drawn for immunological profiling 1-2 days after PVSRIPO 
injection. 
 
End of Treatment 
End of treatment, defined as definitive standard-of-care surgery, will be performed on 
patients 10-20 days after the PVSRIPO injection. Tumor samples obtained from the surgical 
resection will be tested for CD155 expression, immune infiltrate by H&E and IHC, tumor 
infiltrating immune cell profile using flow cytometry, and inflammatory and immune 
signatures using arrays. The excised tumor will be transported to Surgical Pathology by 
the Brain Tumor BioRepository (BTBR) Team, as directed by Dr. Roger McLendon, MD or 
per clinical protocol. Dr. Edgardo Parilla Castellar will provide additional expertise in breast 
pathology. Tissue deemed appropriate for collection will be transported to Dr. Smita Nair, 
PhD, by the BTBR Team for correlative molecular and immune analyses. Surgically 
resected tissue will be used fresh and/or processed into FFPE blocks, required for 
immunohistochemistry and immunofluorescence. Thirty-five ml of whole blood will be drawn 
for immunological profiling. 
 
Follow-up Period 
Follow up will be at 1-2 days, 1 month, and 6 months after PVSRIPO. Thirty-five ml of whole 
blood will be drawn at each time point for immunological profiling. At 6 month SOC visit, 
the patient will undergo an MMG or ultrasound to monitor disease progression. 
 
End of Study 
End of study is defined as after the 6 month follow-up visit. Patient will be removed from 
the trial if they choose to withdraw from the trial or if unable to comply with the study 
requirements, please refer to Section 12.6.1 for more details on early withdrawal. The 
decision to remove a patient from trial due to failure to comply with study requirements 
is at the discretion of the PI. Patients will continue to be monitored indefinitely for 
progression, further treatment regimens, and survival. 
 
Early Withdrawal of Subject(s) 
12.6.1 
Criteria for Early Withdrawal 
Patients may voluntarily withdraw from the study at any time. The PI may also withdraw 
a patient from the study at any time based on his/her discretion. Reasons for PI- 
initiated withdrawal may include, but is not limited to the following: 
- 
Adverse events 
- 
Abnormal laboratory values 
- 
Abnormal test procedure results 
- 
Protocol deviation 

 
 
- 
Administrative issues 
- 
Pregnancy 
 
13 SAFETY MONITORING AND REPORTING 
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 the polio vaccine booster is given on day -7. 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 is fatal or life threatening, requires inpatient hospitalization or a prolongation 
of existing hospitalization, results in persistent or significant disability/incapacity, constitutes 
a congenital anomaly or a birth defect and/or 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 a 21 CFR Part 11 compliant database. 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. 
 
Reporting of Serious Adverse Events 
SAEs must be reported by the PI to the Sponsor or their designee within 24 hours of 
discovery if the event occurs during the clinical study or within 28 days of receiving the study 
drug, whether or not the SAE is considered to be related to the study drug. Applicable forms 
provided by the Sponsor or their designee must be transmitted via secure e-mail within 24 
hours of discovery to pv_istari@klserv.com. 
 
All suspected adverse events that are both serious and unexpected should be reported 
immediately to the PI, Dr. Shelley Hwang, or her designee. 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 or their designee followed by a complete report within 8 additional calendar 
days. Unexpected adverse events that are both serious and suspected, but 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 or their 
designee. 
 
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 Serious Adverse Event (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 days 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 report to the FDA, a summary of the overall toxicity experience will 
be provided. 
 
Safety Oversight Committee 
The Duke Cancer Institute Safety Oversight Committee is responsible for annual data and 
safety monitoring of DUHS sponsor-investigator phase I and II therapeutic interventional 
studies that do not have an independent Data Safety Monitoring Board (DSMB). The 
primary focus of the Safety Oversight Committee is the review of safety data, toxicities and 
new information that may affect subject safety or efficacy. Annual safety reviews include 
but may not be limited to review of safety data, enrollment status, stopping rules if 
applicable, accrual, toxicities, reference literature, and interim analyses as provided by the 
Principal Investigator. The Safety Oversight Committee in concert with the DCI 
Monitoring Team (Section 14.1 below) oversees the conduct of DUHS cancer-related, 
greater-than-minimal-risk intervention studies that do not have an external monitoring 
plan, ensuring subject safety and that the protocol is conducted, recorded and reported in 
accordance with the protocol, standing operating procedures (SOPs), Good Clinical 
Practice (GCP), and applicable regulatory requirements. 
 
External Data and Safety Monitoring 
Dr. Elizabeth A. Mittendorf will serve as the research monitor for this protocol. Per DoD 
3216.02, at a minimum, the research monitor may discuss the research protocol with the 
investigators, interview human subjects, and consult with others outside of the study about 
the research; shall have authority to stop a research protocol in progress, remove individual 
human subjects from a research protocol, and take whatever steps are necessary to protect 
the safety and well-being of human subjects until the IRB can assess the monitor's report; 
and shall have the responsibility to promptly report their observations and findings to the 
IRB or other designated official and the Human Research Protection Office (HRPO). IND 
safety reports and other notable safety information from all ongoing PVSRIPO clinical 
investigations will be provided to the PI by the Sponsor or their designee, who will then 
communicate relevant information with Dr. Mittendorf and the DoD, per their communication 
plans. 
 
If the PI or DCI has a potential conflict of interest with conduct of this protocol, the protocol 
may require additional oversight beyond or in place of the SOC in the form of an external 
DSMB. If a potential conflict of interest exists, the PI should contact the Duke Research 
Integrity Office (RIO) to develop a conflict of interest management plan for the protocol. The 
plan should describe who will comprise the external DSMB, how frequently it will meet, what 
it will review, and the criteria it will use to determine whether it is safe to proceed with the 
protocol. Due to a potential conflict of interest, an external DSMB-Plus was created for the 

 
 
original phase I clinical trial in adult patients with Grade IV malignant glioma performed by 
the Preston Robert Tisch Brain Tumor Center (PRTBTC) at Duke University Medical Center 
and was extended to all other studies with PVSRIPO conducted by the PRTBTC. As the 
current study is being supported by a grant from the DoD, which requires and provides 
independent medical monitoring, RIO has determined that no additional oversight is 
required by an external DSMB. 
 
14 QUALITY CONTROL AND QUALITY ASSURANCE 
Monitoring 
This clinical research study will be monitored both internally by the PI and institutionally by 
the Duke Cancer Institute (DCI) and possibly by the Sponsor or their designee. 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 occur, 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: 
• If stopping rules for toxicity are met, the study will be halted; 
• Risk/benefit ratio is not altered to the detriment of the subjects; 
• Appropriate internal monitoring of AEs 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 reasonable and timely manner. 
 
The DCI Monitoring Team will conduct monitoring visits to ensure subject safety and to 
ensure that the protocol is conducted, recorded, and reported in accordance with the 
protocol, standard operating procedures, good clinical practice, and applicable regulatory 
requirements. DCI review and monitoring of this protocol occurs in accordance with the 
NCI-approved Data and Safety Monitoring Plan. Briefly, protocol review begins with an 
initial review by the Cancer Protocol Committee (CPC), which assesses the ethics and 
safety of the protocol. Documentation of these assessments will be maintained. Formal, 
independent monitoring will be conducted by the DCI Monitoring Team after the first 3 
subjects are enrolled, followed by annual monitoring of 1-3 subjects until the study is closed 
to enrollment and subjects are no longer receiving study interventions that are more than 
minimal risk. 
 
DCI Monitoring Team reports and additional data/safety/toxicity reports submitted by the PI 
will be reviewed by the Safety Oversight Committee on an annual basis. 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, Safety Oversight Committee, a 
sponsor, an investigator, or the IRB. All study documents must be made available upon 
request to the DCI Monitoring Team and other authorized regulatory authorities, including, 
but not limited to the National Institute of Health, National Cancer Institute, and the FDA. 
Every reasonable effort will be made to maintain confidentiality during study monitoring. 

 
 
Audits 
The Duke School of Medicine Office of Audit, Risk, and Compliance (OARC) office and the 
Sponsor or their designee may conduct confidential audits to evaluate compliance with the 
protocol and the principles of GCP. The PI agrees to allow the auditor(s) direct access to 
all relevant documents and to allocate his/her time and the time of the study team to the 
auditor(s) in order to discuss findings and any relevant issues. 
 
Audits are designed to protect the rights and well-being of human research subjects. OARC 
audits may be routine or directed. Routine audits are selected based upon risk metrics 
generally geared towards high subject enrollment, studies with limited oversight or 
monitoring, investigator initiated investigational drugs or devices, federally-funded studies, 
high degree of risk (based upon adverse events, type of study, or vulnerable populations), 
phase I studies, or studies that involve Medicare populations. Directed audits occur at the 
directive of the IRB or an authorized institutional official. 
 
OARC audits examine research studies/clinical trial methodology, processes and systems 
to assess whether the research is conducted according to the protocol approved by the 
DUHS IRB. The primary purpose of the audit/review is to verify that the standards for safety 
of human subjects in clinical trials and the quality of data produced by the clinical trial 
research are met. The audit/review will serve as a quality assurance measure, internal to 
the institution. Additional goals of such audits are to detect both random and systemic errors 
occurring during the conduct of clinical research and to emphasize “best practices” in the 
research/clinical trials environment. 
 
Data Management and Processing 
14.3.1 
Case Report Forms (CRFs) 
The electronic CRF (eCRF) will be the primary data collection document for the study and 
is developed in conjunction with statistical oversight. The CRFs will be updated in a timely 
manner following acquisition of new source data. Only the PI, the study coordinator, the 
data management team, and the clinical trials manager are permitted to make entries, 
changes, or corrections in the eCRF. 
 
An audit trail will be maintained automatically by the electronic CRF management system. 
All users of this system will complete user training, as required or appropriate per DCI 
requirements and other regulations. 
14.3.2 
Data Management Procedures and Data Verification 
Access to electronic databases will be managed by the DCI IT staff. 
 
Completeness of entered data will be checked automatically by the eCRF system and users 
will be alerted to the presence of data inconsistencies. Additionally, the data manager and 
the statistical team will cross-reference the data to verify accuracy. Missing or implausible 
data will be highlighted for the PI requiring appropriate responses (i.e. confirmation of data, 
correction of data, completion or confirmation that data is not available, etc.). 
 
The database will be reviewed and discussed prior to database closure, and will be closed 

 
 
only after resolution of all remaining queries. An audit trail will be kept of all subsequent 
changes to the data. 
14.3.3 
Study Closure 
Following completion of the studies, the PI will be responsible for ensuring the following 
activities: 
- 
Data clarification and/or resolution 
- 
Accounting, reconciliation, and destruction/return of used and unused study drugs 
- 
Review of site study records for completeness 
- 
Shipment of all remaining laboratory samples to the designated laboratories 
 
15 STATISTICAL METHODS AND DATA ANALYSIS 
Six women with breast cancer will participate in this pilot clinical study. The study is 
designed as an exploratory pilot study only to assess PVSRIPO bioactivity. Any subjects 
who do not have evaluable tumor tissue from both the pre-treatment biopsy and post- 
treatment surgery will be replaced. 
 
Our primary objective is to describe the change in the amount of tumor infiltrating immune 
cells in tumor pre- and post-injection of PVSRIPO by H&E. Given the small sample size of 
this study, descriptive statistics will be used to summarize inflammatory and immune 
signatures in tumor and blood, tumor infiltrating immune cell analysis, changes in immune 
cell composition in blood and changes in T cell activation in blood. Some statistical 
comparisons may be conducted in an exploratory manner. 
 
Tumor specimens collected pre and post injection of PVSRIPO will be examined for 
inflammatory and immune signature using arrays, tumor infiltrating cells by H&E and IHC, 
and CD155 expression by IHC. The level of the immune checkpoint PD-L1 expression in 
tumor tissue before and after PVSRIPO injection will also be examined. Mean changes in 
these outcomes will be computed, and a paired t-test or Wilcoxon signed-rank test may be 
conducted with appropriate adjustment for multiple comparison. 
 
Changes over time in inflammatory and immune signature using arrays, immune cell 
composition (antigen presenting cells, B cells and T cells) and T cell activation by flow 
cytometry will be described. Changes over time in B cell activation using ELISA and peptide 
arrays will be described. Measurements will be obtained on day -7 (range day -7 to -10, 
before polio vaccine booster), day 0 (before PVSRIPO injection), day 2 (range day 1-2 after 
PVSRIPO), day 14 (range day 10 to 20, after PVSRIPO before surgery) and in follow-up at 
months 1 and 6 (after PVSRIPO). 
 
Additional descriptive analyses besides those described here may be conducted using the 
samples collected. 

 
 
16 ADMINISTRATIVE AND ETHICAL CONSIDERATIONS 
Regulatory and Ethical Compliance 
This protocol was designed and will be conducted and reported in accordance with the 
International Conference on Harmonization (ICH) Harmonized Tripartite Guidelines for 
Good Clinical Practice, the Declaration of Helsinki, and applicable federal, state, and local 
regulations. 
 
DUHS Institutional Review Board and DCI Cancer Protocol 
Committee 
The protocol, informed consent form, advertising material, and additional protocol-related 
documents will be submitted to the DUHS Institutional Review Board (IRB) and DCI Cancer 
Protocol Committee (CPC) for review. The study will be initiated only after the Principal 
Investigator has received written and dated approval from the CPC, IRB, and Sponsor 
representative or their designee. 
 
The Principal Investigator will submit and obtain approval from the IRB for all subsequent 
protocol amendments and changes to the informed consent form. The CPC will be informed 
about any protocol amendments that potentially affect research design or data analysis (i.e. 
amendments affecting subject population, inclusion/exclusion criteria, agent administration, 
statistical analysis, etc.). 
 
The Principal Investigator will obtain protocol re-approval from the IRB within 1 year of the 
most recent IRB approval. The Principal Investigator must also obtain protocol re-approval 
from the CPC within 1 year of the most recent IRB approval, for as long as the protocol 
remains open to subject enrollment. 
 
Informed Consent 
The informed consent form will be written in a manner that is understandable to the subject 
population. Prior to its use, the informed consent form will be approved by the IRB. 
 
The Principal Investigator or authorized key personnel will discuss with the potential subject 
the purpose of the research, methods, potential risks and benefits, subject concerns, and 
other study-related matters. This discussion will occur in a location that ensures subject 
privacy and in a manner that minimizes the possibility of coercion. Appropriate 
accommodations will be made available for potential subjects who cannot read or 
understand English or are visually impaired. Potential subjects will have the opportunity to 
contact the Principal Investigator or authorized key personnel with questions, and will be 
given as much time as needed to make an informed decision about participation in the 
study. 
 
Before conducting any study-specific procedures, the Principal Investigator must obtain 
written informed consent from the subject or a legally acceptable representative. The 
original informed consent form will be stored with the subject’s study records, and a copy 
of the informed consent form will be provided to the subject. The Principal Investigator is 
responsible for asking the subject whether the subject wishes to notify his/her primary care 

 
 
physician about participation in the study. If the subject agrees to such notification, the 
Principal Investigator will inform the subject’s primary care physician about the subject’s 
participation in the clinical study. 
 
Study Documentation 
Study documentation includes, but is not limited to, source documents, case report forms 
(CRFs), monitoring logs, appointment schedules, study team correspondence with 
sponsors/their designees or regulatory bodies/committees, and regulatory documents that 
can be found in the DCI-mandated “Regulatory Binder,” which includes, but is not limited 
to, approved protocols, approved informed consent forms, FDA Forms 1572, and laboratory 
certifications. 
 
Source documents are original records that contain source data, which is all information in 
original records of clinical findings, observations, or other activities in a clinical trial 
necessary for the reconstruction and evaluation of the trial. Source documents include, but 
are not limited to, hospital records, clinical and office charts, laboratory notes, memoranda, 
subjects’ diaries or evaluation checklists, pharmacy dispensing records, recorded data from 
automated instruments, copies or transcriptions certified after verification as being accurate 
copies, microfiches, photographic negatives, microfilm or magnetic media, x-rays, subject 
files, and records kept at the pharmacy, at the laboratories and at medico-technical 
departments involved in the clinical trial. When possible, the original record should be 
retained as the source document. However, a photocopy is acceptable provided that it is a 
clear, legible, and an exact duplication of the original document. 
 
An electronic database, specifically RAVE, will be the primary documentation for case 
report forms (CRFs) in this study. The CRFs will be updated within two weeks of acquisition 
of new source data. Only approved staff as indicated in the in key personnel log, are 
permitted to make entries, changes, or corrections in the CRF. Changes or corrections will 
be dated, initialed, and explained (if necessary). The Principal Investigator or authorized 
key personnel will maintain a record of the changes and corrections. 
 
Privacy, Confidentiality, and Data Storage 
The Principal Investigator will ensure that subject privacy and confidentiality of the subject’s 
data will be maintained. Research Data Security Plans (RDSPs) will be approved by the 
appropriate institutional Site Based Research group. 
 
To protect privacy, every reasonable effort will be made to prevent undue access to subjects 
during the course of the study. Prospective participants will be consented in an exam room 
where it is just the research staff, the patient and his family, if desired. For all future visits, 
interactions with research staff (study doctor and study coordinators) regarding research 
activities will take place in a private exam room. All research related interactions with the 
participant will be conducted by qualified research staff who are directly involved in the 
conduct of the research study. 
 
To protect confidentiality, subject files in paper format will be stored in secure cabinets 
under lock and key accessible only by the research staff. Subjects will be identified only by 
a unique study number and subject initials. Electronic records of subject data will be 

 
 
maintained using a dedicated 21 CFR Part 11 compliant database (Medidata RAVE), which 
is housed in an encrypted and password-protected file on a secure network drive. Access 
to electronic databases will be limited to designated key personnel as indicated in the 
delegation log. Subject data may be stored temporarily on encrypted and password- 
protected portable memory devices such as flash drives and external hard drives, but only 
when absolutely necessary. Data stored on portable memory devices will be de-identified. 
Subject data will be deleted from the portable memory device at the earliest opportunity. 
The security and viability of the IT infrastructure will be managed by the DCI and/or Duke 
Medicine. 
 
Upon completion of the study, research records will be archived and handled per DUHS 
HRPP policy. Subject names or identifiers will not be used in reports, presentations at 
scientific meetings, or publications in scientific journals. 
 
Data and Safety Monitoring 
Data and Safety Monitoring will be performed in accordance with the DCI Data and 
Safety Monitoring Plan. For a more detailed description, please refer to Section 13.3 and 
14.1 in this protocol. 
 
Protocol Amendments 
All protocol amendments must be initiated by the Principal Investigator and approved by 
the IRB and Sponsor prior to implementation. IRB approval is not required for protocol 
changes that occur to protect the safety of a subject from an immediate hazard. However, 
the Principal Investigator must inform the IRB and all other applicable regulatory agencies 
of such action immediately. 
 
Though not yet required, the CPC should be informed about any protocol amendments that 
potentially affect research design or data analysis (i.e. amendments affecting subject 
population, inclusion/exclusion criteria, agent administration, etc.). 
 
Records Retention 
The Principal Investigator will maintain study-related records for a period of: 
- at least two years after the date on which a New Drug Application is approved by the FDA 
(if an IND is involved) 
- at least two years after formal withdrawal of the IND associated with this protocol (if an 
IND is involved) 
- at least six years after study completion (Duke policy) 
 
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