Clinical trial NCT03267680
Conditions: Cervical Squamous Cell Carcinoma In Situ Vulvar High Grade Squamous Intraepithelial Lesion
Sponsor: University of Southern California
Start date: 2017-11-08; Completion date: 2024-03-13
Enrollment: 10 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This randomized phase II trial studies how well an IRX-2 Regimen works in treating women with cervical squamous intraepithelial neoplasia 3 or squamous vulvar intraepithelial neoplasia 3. The IRX-2 Regimen consists of a single dose of cyclophosphamide, followed by 21 days of indomethacin, zinc-containing multivitamins, and omeprazole. IRX-2, a human cell-derived biologic with multiple active cytokine components, may act as an immune booster to stimulate the immune system. Giving cyclophosphamide and IRX-2 may work better at treating cervical squamous intraepithelial neoplasia or squamous vulvar intraepithelial neoplasia.

Detailed description:
PRIMARY OBJECTIVES:

I. To compare the proportion of subjects who achieve a pathologic complete response (CR) or partial response (PR) in regimen 1 versus regimen 2 at week 25, based on the resected surgical specimen.

SECONDARY OBJECTIVES:

I. To evaluate the toxicity and feasibility of administration of IRX-2 in subjects with confirmed cervical intraepithelial neoplasia (CIN) 3 or vulvar intraepithelial neoplasia (VIN) 3.

II. To evaluate multiple parameters to assess the activity of the IRX-2 regimen for the treatment of CIN 3 or VIN 3: the occurrence of clinical CRs or PRs at weeks 6, 13 and 25.

III. To evaluate multiple parameters to assess the activity of the IRX-2 regimen for the treatment of CIN 3 or VIN 3: frequency of elimination of human papillomavirus (HPV) in cervical or vulvar tissue using a commercial HPV genotyping assay and viral load determination by quantitative polymerase chain reaction (PCR).

IV. To evaluate multiple parameters to assess the activity of the IRX-2 regimen for the treatment of CIN 3 or VIN 3: analysis of the immune infiltrates in the resected surgical specimens.

V. To evaluate multiple parameters to assess the activity of the IRX-2 regimen for the treatment of CIN 3 or VIN 3: immunophenotypic analysis of peripheral blood lymphocytes.

VI. To evaluate multiple parameters to assess the activity of the IRX-2 regimen for the treatment of CIN 3 or VIN 3: frequency of serum antibodies to HPV E6, E7 and L1 proteins by enzyme-linked immunosorbent assay (ELISA).

VII. To evaluate multiple parameters to assess the activity of the IRX-2 regimen for the treatment of CIN 3 or VIN 3: ribonucleic acid (RNA) expression profiling of immune-inflammatory markers from post-treatment resected surgical specimens.

OUTLINE: Patients are randomized to 1 of 2 arms.

Arm I: Patients receive cyclophosphamide intravenously (IV) on day 1 and IRX-2 via submucosal injections in the cervix or subcutaneously (SC) for vulvar lesions on days 4-7. Patients also receive indomethacin orally (PO) three times daily (TID), zinc-containing multivitamins (PO) once daily (QD) and omeprazole orally (PO) on days 1-21.

Arm II: Patients receive cyclophosphamide IV on day 1 and placebo via submucosal injections in the cervix or SC for vulvar lesions on days 4-7. Patients also receive indomethacin PO TID, zinc-containing multivitamins PO QD and omeprazole PO on days 1-21.

In both arms, treatment repeats every 6 weeks for up to 2 courses in the absence of disease progression or unacceptable toxicity. Beginning week 25, patients undergo surgical resection.

After completion of study treatment, patients are followed up at 1-8 weeks after surgery.

Arms: arm 1: Patients receive cyclophosphamide IV on day 1 and IRX-2 via submucosal injections in the cervix or SC for vulvar lesions on days 4-7. Patients also receive indomethacin PO TID, zinc-containing multivitamins PO QD and omeprazole PO on days 1-21. Treatment repeats every 6 weeks for up to 2 courses in the absence of disease progression or unacceptable toxicity. Beginning week 25, patients undergo surgical resection. arm 2: Patients receive cyclophosphamide IV on day 1 and placebo via submucosal injections in the cervix or SC for vulvar lesions on days 4-7. Patients also receive indomethacin PO TID, zinc-containing multivitamins PO QD and omeprazole PO on days 1-21. Treatment repeats every 6 weeks for up to 2 courses in the absence of disease progression or unacceptable toxicity. Beginning week 25, patients undergo surgical resection.
Interventions: intervention 1: Cyclophosphamide intervention 2: Indomethacin intervention 3: IRX-2 intervention 4: Laboratory Biomarker Analysis intervention 5: Multivitamin intervention 6: Omeprazole intervention 7: Placebo intervention 8: Therapeutic Conventional Surgery
Intervention descriptions: intervention 1: Given IV intervention 2: Given PO intervention 3: Given via submucosal injection or SC intervention 4: Correlative studies intervention 5: Given zinc-containing multivitamin PO intervention 6: Given PO intervention 7: Given via submucosal injections or SC intervention 8: Undergo surgical resection

Protocol documents:
Prot_SAP_000.pdf: 
 
 
Local Protocol #: 5GYN-16-2 
Protocol Title: A Two-Cohort, Open Label, Phase 2 Trial of the IRX 
2 Regimen in Women with Squamous Cervical Intraepithelial 
Neoplasia 3 (CIN 3) or Vulvar Intraepithelial Neoplasia 3 (VIN 3) 
Protocol version date: 08August2020 
 
ClinicalTrialsgov #: NCT03267680 
 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
1 
Title: 
A Two-Cohort, Open-label, Phase 2 Trial of the IRX-2 
Regimen in Women with Squamous Cervical Intraepithelial 
Neoplasia 3 (CIN 3) or Vulvar Intraepithelial Neoplasia 3 
(VIN 3) 
Protocol: 5GYN-16-2   
BB IND #: 
133686 
Date: 
August 08, 2020 
Sponsor: 
USC Norris Comprehensive Cancer Center 
Keck Hospital of USC, and LAC+USC Hospital 
Clinical Investigation Support Office 
1441 Eastlake Avenue, Suite 7310 
Los Angeles, CA 90033 
Phone: (323) 865-0451 
Fax: (323) 865-0089 
Phone: (212) 582-5352 
Email: CoreCISO@med.usc.edu 
Principal Investigator: 
Lynda Roman, MD 
Division of Gynecologic Oncology 
Department of Obstetrics & Gynecology 
USC Norris Comprehensive Cancer Center,  
Keck Hospital of USC, and LAC+USC Hospital 
1441 Eastlake Avenue, NTT 3411 
Los Angeles, CA 90033 
Phone: (323) 865-3922 
Co-Principal Investigators: 
Koji Matsuo, MD, PhD (Clinical) 
Diane Da Silva, PhD (Laboratory) 
W. Martin Kast, PhD (Laboratory) 
Co-Investigators: 
Laila Muderspach, MD     Annie Yessaian, MD 
Huyen Pham, MD             Laurie Brunette, MD 
Susan Groshen, PhD        Saloni Walia, MD 
Marcia Ciccone, MD 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
2 
Protocol Synopsis 
Study Title 
A Two-Cohort, Open-label, Phase 2 Trial of the IRX-2 Regimen in Women with 
Squamous Cervical Intraepithelial Neoplasia 3 (CIN 3) or Squamous Vulvar 
Intraepithelial Neoplasia 3 (VIN 3) 
Study Number 
5GYN-16-2 
Study Phase 
2 
IRX-2 Regimen 
IRX-2 is a primary cell-derived biologic with multiple active cytokine components, 
produced under pharmaceutical standards from phytohemagglutinin (PHA) and 
ciprofloxacin stimulated donor mononuclear cells.  
In this open label trial, a modified IRX-2 Regimen (as shown in the table below) will 
include cyclophosphamide on Day 1 (three days before the start of IRX-2), IRX-2 
daily x 4 days, and indomethacin, zinc with multivitamins, and a proton pump 
inhibitor (omeprazole) for 21 days. 
All treatments will be repeated at 6 weeks, for a total of two cycles.  
Agent 
Dose 
Route of 
Administration 
Treatment Days 
(each cycle) 
Cycle = 6 weeks 
Cyclophosphamide 
300 mg/m2 
IV 
Day 1 
IRX-2  
 
230 units daily 
(in 2 ml) 
in 4 divided doses: 
57.5 units (1/2 ml)  
in each quadrant of 
the cervix or affected 
areas of the vulva 
Submucosal 
injection in the 
cervix; 
subcutaneous for 
vulvar lesions 
4 consecutive days 
between D4 and 
D7 
Indomethacin 
25 mg TID 
Oral 
Days 1-21 
Zinc with multivitamins 
1 tab daily 
Oral 
Days 1-21 
Omeprazole 
20 mg daily 
Oral 
Days 1-21 
Route of 
Administration of 
IRX-2  
 
For cervical dysplasia: IRX-2 will be administered submucosally in the cervix in four 
divided doses, one into each quadrant of the cervix, irrespective of location of the 
CIN 3 lesion. 
For vulvar dysplasia:  IRX-2 will be administered into the subcutaneous tissue 
beneath the vulvar lesion. Doses may be divided at clinician discretion based on the 
geometry and size of the dysplastic lesion.  
Reference 
Therapy 
There will be no reference therapy. 
Study Design 
This trial was originally designed as a double-blinded randomized, 
placebo-controlled study of the IRX-2 Regimen in subjects with CIN 3 or VIN 3. 
After conversion to an open label, single-arm trial, this study will use a Simon two-
stage design for both the CIN 3 Cohort and VIN 3 Cohort.  
• 
CIN Cohort: N = 10+12 
o If 2 or fewer pathological responses are observed among the first 10 
patients, the enrollment will be stopped. If 3 or more responses are 
observed, the trial will continue to a total of 22 CIN patients.  
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
3 
• 
VIN Cohort: N = 5+5 
o If there is no pathological response observed among the first 5 
patients, the enrollment will be stopped.   If 1 or more responses are 
observed the trial will continue to a total of 10 VIN patients. 
The primary end-point is pathologic objective response at week 25. CIN subjects will 
be assessed with colposcopy and photography at baseline. The first six-week cycle 
treatment will be administered, after which the colposcopy and photography 
assessment will be repeated, and then a second cycle of treatment will be 
administered. Colposcopy and photography assessment will be performed again after 
the second six-week cycle at approximately Week 13 prior to LEEP or wide local 
excision of CIN lesion at week 25. HPV status will be determined at 4-months post-
surgical excision. 
VIN subjects will be assessed with colposcopy and photography of the vulvar lesion 
at screening and subsequently on Weeks 6 and 13, followed by wide local excision at 
week 25.HPV status will be determined at 4-months post-surgical excision. 
Secondary endpoints include toxicity, feasibility and exploratory immune changes.  
The protocol Study Team (PI, Co-PI’s, Study Monitor, and Study Statistician) will 
meet monthly and additionally as needed to review the status of subjects and the 
toxicities.  The USC/Norris Comprehensive Cancer Center DSMB will review the 
progress and safety of the trial every 6 months.   
All specimens will be analyzed for VIN or CIN (present/absent), grade of dysplasia, 
HPV 
virus 
(present/absent), 
and 
nature 
of 
the 
immune 
infiltrate 
by 
immunohistochemistry and gene expression profiling. 
Peripheral blood will be analyzed by ELISPOT and/or intracellular cytokine analysis 
for HPV T cell and other cellular responses as well as by flow cytometry and by TCR 
clonality studies.  
Projected 
Enrollment 
22 subjects with CIN 3 and 10 subjects with VIN 3 will be enrolled. 
Primary 
Objective 
 
Primary 
Endpoint  
To compare the proportion of subjects who achieve a pathologic complete response 
(CR) and/or a partial response (PR) at weeks 25 based on LEEP or vulvar surgical 
excision. Each cohort (CIN and VIN) will be assessed independently. 
The primary endpoint will be occurrence of a pathologic CR or PR of CIN 3 or VIN 
3 lesions in the Week 25 excisional specimen. CR is defined as absence of CIN. PR 
is a lower grade of dysplasia than present at baseline, i.e. grade 3 decreasing to grade 
1.  
Secondary 
Objectives  
1. To evaluate the toxicity and feasibility of administration of IRX-2 in 
subjects with confirmed CIN 3 or VIN 3. 
2. To evaluate multiple parameters to assess the clinical and immune activity 
of the IRX-2 Regimen for the treatment of CIN 3 or VIN 3. 
a. The occurrence of clinical CRs or PRs at weeks 6, 13 and 25 
b. Frequency of elimination of HPV in cervical or vulvar tissue using a 
commercial HPV genotyping assay and viral load determination by 
quantitative PCR. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
4 
c. Analysis of the immune infiltrates in the resected surgical specimens. 
Parameters to be evaluated include quantitation of lymphocyte subset 
infiltration including CD8 and CD4 FOXP3 cells, evaluation of 
PDL1 expression, evaluation of state of activation and location of 
infiltrating regions with respect to dysplasia and adjacent normal 
tissue. A commercial multiplex IHC assay will assess markers of 
Langerhans cells (CD1a, E-cadherin) and antigen presenting cell 
(APC) activation (CD80 and CD86). TCR clonality studies may also 
be performed. 
d. Immunophenotypic analysis of peripheral blood lymphocytes. 
Peripheral blood lymphocytes will be evaluated by direct ex-vivo 
ELISPOT analysis for evidence of induction of γ-Interferon 
expressing T cells in response to HPV antigens compared to baseline. 
Changes in naïve, memory, effector and regulatory T cell subset will 
be analyzed using immunophenotypic flow cytometry. 
e. Frequency of serum antibodies to HPV E6, E7 and L1 proteins by 
ELISA. Serum cytokine profile will be analyzed by multiplex ELISA 
at baseline and week 13 (pending availability of the antibodies).  
f. RNA expression profiling of immune-inflammatory markers from 
post-treatment resected surgical specimens. 
g. Frequency of long term HPV clearance following surgical excision. 
Subject 
Population 
Subjects with histologically confirmed squamous CIN 3 or VIN 3. 
Participating 
Center(s)  
University of Southern California (Clinical PI, Dr. Lynda Roman; Clinical Co-PI, 
Dr. Koji Matsuo; Laboratory Co-PIs, Dr. Diane Da Silva and Dr. Martin Kast) 
 
University of Oklahoma Health Science Center, Stephenson Cancer Center (PI:  
Kathleen Moore MD, Co-I’s:  Joan Walker MD, Katie Smith MD) 
Inclusion 
Criteria: 
1. Histologically confirmed squamous CIN 3, or VIN 3 (usual type only).  
2. Age ≥25 years.  
3. Surgically sterile, postmenopausal, or agrees to practice an effective method of 
birth control as determined by the investigator (to be continued for one year 
following last dose of study medication), except that subjects with CIN 3 are not 
permitted to use a cervical cap or diaphragm for contraception. 
4. In good health based upon the results of a medical history, physical 
examination, vital signs, and laboratory profile including: 
Hematology:  
• White blood cell >2,500/ mcL (>2.5 x 109/L)  
• Absolute neutrophil count >1,000/ mcL (>1 x 109/L) 
• Platelet count >75,000/ mcL (>75 x 109/L) 
• Hemoglobin ≥8 g/dL (≥80 g/L) (subjects who have received a 
transfusion or erythropoietin up to one week prior to receiving the first 
dose of cyclophosphamide are eligible for the study) 
Clotting Time: 
• International normalized ration (INR) or prothrombin time (PT) ≤1.5 x 
ULN (upper limit of normal) 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
5 
• Activated partial thromboplastin time (aPTT) ≤1.5 x ULN 
Renal Function: 
• Serum creatinine ≤1.5 x ULN  
Hepatic function: 
• Total bilirubin ≤2.0 x ULN unless thought to be related to inherited 
bilirubin conjugation disorder (ie Gilbert’s disease) 
• ALT and AST ≤2.0 x ULN 
5. Geographically accessible for ongoing follow-up and committed to comply with 
the designated visits. 
6. Capable of understanding and complying with the protocol and has given 
written informed consent. 
Exclusion 
Criteria: 
 
1. For subjects with cervical dysplasia: evidence of atypical glandular cells or 
adenocarcinoma in situ (ACIS) based on cervical cytology, colposcopy or biopsy. 
2. For subjects with either cervical or vulvar squamous dysplasia: evidence of 
microinvasive squamous carcinoma based on cytology, colposcopy or biopsy. 
3. For VIN subjects, those with >2 vulvar lesions. 
4. Pregnancy or lactation. 
5. Allergy to ciprofloxacin or other quinolones (because ciprofloxacin is used in 
preparation of IRX-2.) 
6. Allergy to indomethacin (a component of the IRX-2 Regimen) or to 
acetylsalicylic acid (aspirin) due to possible cross reaction. 
7. Imiquimod for the topical treatment of lower genital tract warts or dysplasia 
within 3 months of study enrollment. 
8. Known positive for human immunodeficiency virus-1 antibody, human 
immunodeficiency virus-2 antibody, hepatitis B surface antigen, or hepatitis C 
virus. 
9. Known to have other immunodeficiency diseases, including cellular 
immunodeficiencies, hypogammaglobulinemia, or dysgammaglobulinemia. 
10. Immunotherapy (eg, interferons, tumor necrosis factor, interleukins, or biological 
response 
modifiers 
[granulocyte-macrophage 
colony-stimulating 
factor, 
granulocyte colony-stimulating factor, macrophage colony-stimulating factor) or 
any investigational drug within 3 months of study enrollment. 
11. Concurrent treatment with systemic corticosteroids at a dose of ≥5 mg/day of 
prednisone (or equivalent). An infectious process or any other significant illness 
such as an autoimmune disease, comorbidities or advanced age that in the opinion 
of the investigator would compromise the subject’s ability to mount an immune 
response. 
12. Subjects should not take aspirin (except for low-dose aspirin as prescribed for 
vascular disease) or other non-prescribed, non-steroidal anti-inflammatory agents 
from start of treatment to surgery. 
13. An infectious process or any other significant illness such as an autoimmune 
disease or advanced age that in the opinion of the investigator would compromise 
the subject’s ability to mount an immune response. 
14. Active SARS-CoV2 infection or COVID-19 disease 
15. Impaired hepatic, renal or hematological function, evidenced by: 
a. Alanine aminotransferase, aspartate aminotransferase, or total bilirubin 
(in the absence of Gilbert’s syndrome) >2 times upper limit of normal, 
b. Serum creatinine >1.5 times upper limit of normal, or 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
6 
16. Clinically significant active cardiovascular disease, including a history of 
myocardial infarction within the past 6 months, heart failure as defined by New 
York Heart Association classes III or IV, and/or blood pressure greater than 
160/100 mm. 
17. History of severe allergic reaction to insect bites or stings, or to any biologic 
pharmaceutical product, including compounds similar to the test article. 
18. Any medical contraindications, allergies or previous therapy that would preclude 
treatment with the components of the IRX-2 Regimen, i.e., cyclophosphamide, 
indomethacin, zinc-containing multivitamins or omeprazole. 
19. Donation or loss of >450 mL of blood or plasma within 30 days of start of 
treatment 
Immunologic and 
Virologic 
Assessments 
Presence or absence of cervical HPV (using cervical swabs) for all subjects will be 
determined at Screening/Baseline, at Week 25, and 4 months post excision (Week 
41). 
HPV genotype will be determined by analyzing the Screening/Baseline biopsy and 
the Week 25 surgical excision. 
Immunophenotypic analysis of peripheral blood lymphocytes will be performed at 
Screening/Baseline; at Week 6, 13 and 25 by ELISPOT analyses using HPV peptide 
pools and assessment of cytokine release from HPV–specific T cells  
Enzyme-linked immunosorbent measurements of serum to determine the HPV 
antibody response will be performed at Screening/Baseline; Week 6, 13 and 25 
pending the availability of appropriate reagents. 
The Screening/Baseline biopsy and the subsequent cervical LEEP or vulvar 
excisional specimen will be compared to evaluate alterations in the tissue immune 
environment including T cell effector and T-regulatory and APC subsets looking at 
differentiation markers and markers of activation will be assessed dependent on 
obtaining adequate size tissue. Multiplex flow cytometry and nanostring gene 
expression profiling will be performed.  
Statistical 
Considerations 
The CIN and VIN cohorts will be analyzed separately.  Within each cohort, all 
patients who received any amount of treatment will be included in the summaries for 
primary and secondary endpoints with standard descriptive methods.   
For CIN Cohort, with a false-positive error rate of 5% when the true objective 
response rate is 20% and a false-negative error rate of 10% when the true CR + PR 
response rate is 50%, a total of 10 patients will be enrolled in the 1st stage. If 2 or 
fewer responses are recorded among the first 10 patients to receive the IRX Regimen, 
the enrollment will be stopped. If 3 or more responses are observed, the trial will 
continue to a total of 22 CIN patients.  
For the VIN Cohort, with a false-positive error rate of 10% when the true objective 
response rate is 5% and a false-negative error rate of 10% when the true CR + PR 
response rate is 40%, a total of 5 patients will be enrolled in the 1st stage.  If there is 
no response observed among the first 5 patients, the enrollment will be stopped. If 1 
or more responses are observed the trial will continue to a total of 10 VIN patients.     
The primary endpoint is the response, CR or PR of CIN 3 or VIN 3 lesions to the 
IRX-2 Regimen as determined by histologic examination of the Week 25 surgical 
excision specimen.   
Safety assessments will be summarized using descriptive statistics for continuous 
measures and frequency distributions for categorical responses. Differences between 
groups will be compared using standard statistical tests for small samples. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
7 
 
Immune assessment of peripheral blood lymphocytes will include determination of 
the percentage of γ-IFN secreting T cells by ELISPOT in response to various HPV 
epitopes. Responses will be considered positive if there is a 2x increase in the number 
of γ-IFN secreting cells over Baseline. Differences between groups will be compared 
using standard student’s T test. 
Immune infiltrates in cervical and vulvar biopsy specimens will be summarized and 
differences between groups will also be compared using standard student’s T test. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
8 
Table 1:  Schedule of Events  
Study Procedure 
Screening 
(Baseline) 
Treatment Cycles 
 
 
Surgical 
Resection 
(LEEP or 
wide local 
excision) 
Post-Op 
Checki 
 
Cycle 1: 1st 6-weeks 
Cycle 2: 2nd 6-weeks 
 
Pre-op 
procedure 
visit 
4-
month 
post-op 
Days 
−30 to 0 
Day(s) of Cycle 1 
Days(s) of Cycle 2 
 
Week  
21 
Week 
 25 
Weeks 
26+ 
 
1 
2 
3 
4 
5 
6 
7 
>8 
1 
2 
3 
4 
5 
6 
7 
>8 
Week 13 
safety 
visit  
Week 
41 
Day(s) of Protocol 
 
1 
2 
3 
4 
5 
6 
7 
8-
21 
22-
42 
43 44 
45 
46 
47 
48 
49 
50-
63 
64-
84 
91 
Day 147 
± 14 days 
Day 175 
 ± 7 daysg 
 
Day 290 
± 14 
days 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study Drug Administration 
 
Cyclophosphamide IVa 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Indomethacin, omeprazole, zinc 
with multivitaminsb 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
IRX-2 injectionb 
 
 
 
 
X 
X 
X 
X 
 
 
 
 
X 
X 
X 
X 
 
 
 
 
 
 
 
Procedures & Evaluations 
 
History & Complete Physical 
Exam 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
X 
 
 
 
HIV-1, HIV-2, HBV, HCV 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SARS-CoV2 
 
Xc 
 
 
 
 
 
 
 
Xc 
 
 
 
 
 
 
 
 
 
 
 
 
Focused Pelvic exam 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Xj 
X 
Injection Site Check 
 
 
 
 
X 
 
 
X 
 
 
 
 
X 
 
 
X 
 
X 
X 
 
 
 
ECOG Performance Status 
X 
X 
 
 
 
 
 
X 
 
X 
 
 
 
 
 
X 
 
X 
X 
 
X 
X 
AEs and SAEsd 
 
X 
 
 
X 
X 
X 
X 
 
X 
 
 
X 
X 
X 
X 
 
X 
X 
 
X 
X 
Concomitant Medications 
X 
X 
 
 
X 
X 
X 
X 
 
X 
 
 
X 
X 
X 
X 
 
X 
X 
 
X 
X 
Chemistry, CBC, PT/INR 
X 
Xe 
 
 
 
 
 
 
 
Xe 
 
 
 
 
 
 
 
Xe 
Xe 
 
 
 
Blood work for investigational 
studiesf 
Xe 
Xe 
 
 
 
 
 
 
 
Xe 
 
 
 
 
 
 
 
Xe 
Xe 
 
 
 
Blood for PK studiesh 
 
 
 
 
X 
X 
X 
X 
 
 
 
 
X 
X 
X 
X 
 
 
 
 
 
 
Pregnancy Testf 
X 
Xf 
 
 
 
 
 
 
 
Xf 
 
 
 
 
 
 
 
Xf 
Xf 
 
 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
9 
Colposcopy & cervix 
photography (CIN patients) 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
X 
X 
 
 
 
Cervical inspection & 
photography (CIN patients) 
 
 
 
 
XL 
 
 
XL 
 
 
 
 
XL 
 
 
XL 
 
 
 
 
 
 
Cervical cytology (pap 
smear/ECC) (CIN patients) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
 
 
X 
Vulvar inspection & photography 
(VIN patients) 
X 
 
 
 
XL 
 
 
XL 
 
X 
 
 
XL 
 
 
XL 
 
X 
X 
 
 
 
Cervical or vulvar swab for HPV 
assay (research) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
 
 
X 
Cervical or vulvar biopsy 
Xk 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Cervical LEEP excision or 
cone biopsy or vulvar excision 
or biopsy 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Footnotes:  
a. 
Dexamethasone or other steroids should not be used as an anti-emetic with the cyclophosphamide. Steroid administration may inhibit IRX-2 Regimen 
stimulation of an immune response. The prescribed dose is significantly less than used for conventional chemotherapy and thus steroids will not be needed. 
Suggested antiemetic regimen would include a serotonin (5-HT3) antagonist and lorazepam. 
b. Subjects will receive injection of study agent IRX-2 daily on Days 4, 5, 6, 7 or Days 5, 6, 7, 8. The indomethacin, omeprazole, zinc with multivitamins are 
administered for the first 3 weeks per cycle, on Days 1-21. For cycle 2 [Week 7] the comparable “Days” are shown in the table. 
c. 
SARS-CoV2 virus test required within 2-4 days prior to start of Cycle 1 and Cycle 2. If positive, treatment will be held. 
d. Adverse events (AEs) and serious adverse events (SAEs) are measured from day of Cycle 1 Day 1 until Week 25 of the study. Ongoing AE/SAEs will be 
reported and monitored until resolution. 
e. 
Ten ACD yellow top blood tubes (85 mL) and one red top serum tube should be drawn for investigational studies. Blood work can be done up to seven 
days prior to the specified day. Blood draws do not need to be repeated on indicated day if drawn within the previous 7 days.  
f. 
Pregnancy must be ruled out, or post-menopausal status verified, at study enrollment. Fertile subjects will need effective contraception during the study and 
for 1 year following last dose of study medication; investigator to repeat pregnancy tests during the study if needed for verification.  A blood pregnancy test 
should be used at screening, week 7 and week 21. A urine pregnancy test can be performed on Cycle 1 day 1 visit, and at week 13 safety visit. 
g. The “Week 25” lesion excision is to be scheduled for Day 175, +/- 7 days.  
h. One red top serum tube (5 mL) required at each of the following time points: Day 4 pre-injection, and then 30 minutes, 1 hour, 2 hours post-injection; Day 
5 pre-injection; Day 6 pre-injection; Day 7 pre-injection, and then 30 minutes, 1 hour, 2 hours post-injection. All time points allow +/- 5 minutes. 
i. 
Post-op check to be performed 1 to 8 weeks post-surgery; can occur by phone or office visit. 
j. 
If seen for office visit. 
k. If previous biopsy not done within past 60 days, or if slides/block from this are not available, or at discretion of investigator based on clinical impression. 
l. 
Photography to be performed prior to injection of study drug. 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
10 
Table 2:  Schedule of Virologic and Immune Monitoring (All Subjects) Based on Blood, Swab or Tissue Samples 
Study Procedure 
Screening 
(Baseline) 
Treatment Cycles 
Safety visit 
Surgical 
Excision 
4-month 
follow-up 
Cycle 1 
Cycle 2 
Week 13 
Week 25 
Week 41 
Days 
-30 to 0 
Day 1 
Day 43 
(Cycle 2, Day 1) 
Day 91 
Day 175 
 ± 7 days 
Day 290   
± 14 days 
Presence or Absence of HPVc 
X 
 
 
 
X 
X 
HPV Genotypingc 
X 
 
 
 
X 
X 
Flow Cytometryc 
X 
Xa,b 
Xb 
Xb 
Xb 
 
ELISPOTc 
X 
Xa,b 
Xb 
Xb 
Xb 
 
ELISAc 
X 
Xa,b 
Xb 
Xb 
Xb 
 
Phenotypic analysis of lymphocytic 
infiltrated 
X 
 
 
 
X 
 
a. 
Cycle 1, Day 1 labs do not need to be repeated if the Screening (Baseline) labs were drawn within seven days. For subject convenience, investigator should 
synchronize these phlebotomies with the blood draws specified in Table 1. 
b. Blood work for ELISPOT, ELISA and flow cytometry can be collected up to seven days prior to the specified day. 
c. HPV genotyping, flow cytometry, ELISPOT, and ELISA assays will be performed in the Norris Comprehensive Cancer Center Immune Monitoring Core 
laboratory. Immunogenicity assays will be performed by Brooklyn ImmunoTherapeutics on stored serum. 
d. Tissue immunohistochemistry and PK studies will be done by Brooklyn ImmunoTherapeutics via a third party. 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
11 
TABLE OF CONTENTS 
PROTOCOL SYNOPSIS ..............................................................................................................2 
1 
INTRODUCTION ...........................................................................................................200 
1.1 
BACKGROUND 
200 
1.1.1 Female Lower Genital Tract Dysplasia ...................................................200 
1.1.2 Nomenclature: Dysplasia, Intraepithelial Neoplasia, and Squamous 
Intraepithelial Lesion ...............................................................................211 
1.1.2.1 
Bethesda System for Grading Cervical Dysplasia .................211 
1.1.2.2 
Glandular Dysplasia, Adenocarcinoma-in-situ, and 
Adenocarcinoma of the Uterine Cervix .................................222 
1.1.2.3 
Vulvar Dysplasia ...................................................................233 
1.1.2.4 
Lower Anogenital Squamous Terminology (LAST) Project .233 
1.1.3 The Management of CIN 3, and its Long Term Sequelae .......................244 
1.1.3.1 
Management of CIN 3 ...........................................................244 
1.1.3.2 
Complications and Long-Term Sequelae from the Surgical 
Management of CIN 3 ...........................................................255 
1.1.4 Management of VIN ................................................................................277 
1.1.4.1 
Surgical Excision .....................................................................27 
1.1.4.2 
Efficacy of Current VIN Treatments .......................................27 
1.1.4.3 
Topical Therapy: Imiquimod Cream (Aldara®) .....................278 
1.1.4.4 
Investigational Therapeutic Vaccination for VIN 3 ................28 
1.1.5 Immune Deficiency and Immunotherapy in HPV-Mediated 
Premalignant and Malignant Conditions of the Lower Genital Tract ......288 
1.1.6 The IRX-2 Regimen for Treatment of CIN and VIN ..............................299 
1.1.7 Manufacture and Composition of IRX-2 .................................................299 
1.1.8 Mechanism of Action of IRX-2 ...............................................................299 
1.1.9 Rationale for the Components of the IRX-2 Regimen .............................300 
1.1.10 Delivery of IRX-2 ....................................................................................311 
1.2 
Clinical Experience with the IRX-2 Regimen 
311 
1.2.1 Phase 1 Trials ...........................................................................................311 
1.2.2 Phase 2 Trials in Head & Neck Squamous Cell Carcinoma ....................322 
1.2.2.1 
Study IRX-2 2005-A: Design and Clinical Results ...............322 
1.2.2.2 
Study IRX-2 2005-A: Safety Results and Adverse Events ...323 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
12 
1.2.2.3 
Study IRX-2 2005-A: Immunologic Results .........................333 
1.2.3 Phase 1/2 Cervical Cancer Study .............................................................344 
1.2.3.1 
Clinical Response ..................................................................344 
1.2.3.2 
Pathological Response ...........................................................344 
2 
STUDY OBJECTIVES ....................................................................................................355 
2.1 
Primary Objective 
355 
2.2 
Secondary Objectives 
355 
3 
INVESTIGATIONAL PLAN ..........................................................................................366 
3.1 
Study Design 
366 
3.2 
Selection of Study Population 
37 
3.2.1 Inclusion Criteria .......................................................................................37 
3.2.2 Exclusion Criteria ......................................................................................38 
3.3 
Assessment of Dysplastic Lesion and Surgical Resectability 
39 
3.4 
Termination of Study Treatment 
39 
3.5 
Withdrawal of Consent 
400 
3.6 
Medical Monitoring 
40 
3.6.1 Medical Monitors .......................................................................................40 
3.6.2 Principal Investigator(s) .............................................................................40 
3.7 
Data Safety Monitoring 
401 
4 
STUDY TREATMENTS .................................................................................................422 
4.1 
Study Design 
422 
4.2 
IRX-2 and Control Regimen 
422 
4.3 
Route of Administration 
422 
4.4 
Regimen Medications 
433 
4.4.1 IRX-2 .......................................................................................................433 
4.4.2 Other Medications ....................................................................................433 
4.5 
Method of Assigning Subject Numbers 
433 
4.6 
Treatment Compliance 
433 
4.7 
Prohibited, Prior and Concomitant Treatments 
433 
5 
STUDY PROCEDURES AND SCHEDULE ..................................................................434 
5.1 
Study Schedule 
435 
5.2 
Study Procedures 
48 
5.2.1 Screening....................................................................................................48 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
13 
5.2.2 Cycle 1, Day 1............................................................................................49 
5.2.3 Cycle 1, Days 4 to 7 or Days 5 to 8 ...........................................................50 
5.2.4 Cycle 1, Days 8 to 21 ...............................................................................511 
5.2.5 Cycle 2, Day 1 (Beginning of Week 7; Day 43 of Protocol) ...................511 
5.2.6 Cycle 2, Days 4 to 7 (Continuation of Week 7, or days 46 to 49 of 
Protocol) ...................................................................................................522 
5.2.7 Cycle 2, Days 8 to 21 (Days 50 to 63 of Protocol) ..................................522 
5.2.8 Management of Toxicity Related to IRX-2 Regimen. .............................522 
5.2.9 Week 13: Safety Assessment ...................................................................533 
5.2.10 Pre-Surgical Assessment ..........................................................................534 
5.2.11 Surgical Excision of the Dysplastic Lesion: Week 25 (Day 175 of 
Protocol) ...................................................................................................544 
5.2.12 Short-Term Follow-Up Schedule ...............................................................55 
5.2.13 Four-month follow-up surveillance……………………………………...55 
5.2.14 Longer-Term Follow-Up .........................................................................555 
6 
STUDY ASSESSMENTS .................................................................................................57 
6.1 
Endpoint Assessments 
57 
6.1.1 Primary Endpoint – Pathologic Response Rate .........................................57 
6.1.2 Secondary Endpoints .................................................................................57 
6.1.2.1 
Toxicity and Feasibility ...........................................................57 
6.1.2.2 
Clinical CR or PR at Weeks 6, 13 and 25 ................................58 
6.1.2.3 
HPV Status ...............................................................................58 
6.1.2.4 
Immunologic Studies ...............................................................58 
6.1.2.5 
Characterization of the Immune Infiltrate within the 
Surgical Specimens ..................................................................58 
6.1.2.6 
Relation Between HPV Status and Response to Therapy .......59 
6.1.2.7 
Activation, Clonality and Immunophenotypic Analyses of 
Peripheral Blood Lymphocytes ...............................................59 
7 
SURGICAL MANAGEMENT OF CIN 3 OR VIN 3 .......................................................60 
7.1 
Initial Diagnostic Biopsies 
60 
7.2 
Therapeutic Excision of Dysplastic Lesion: Week 25 
600 
7.2.1 CIN 3 Cohort: Cervical Excision ...............................................................60 
7.2.2 VIN 3 Cohort: Wide Local Excision of Dysplastic Lesion .......................60 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
14 
7.2.3 Multifocal Lower Genital Tract Dysplasia, and/or Concurrent Genital 
Tract Condylomata ...................................................................................611 
8 
STATISTICAL CONSIDERATIONS.............................................................................622 
8.1 
Summary of Design 
622 
8.2 
Major Endpoints 
643 
8.2.1 Primary Endpoint:  CR or PR in Surgical Specimen (≈week 25) ............643 
8.2.2 Secondary Endpoints of Toxicity and Feasibility ....................................643 
8.3 
Secondary Endpoint of HPV status 
654 
8.4 
Secondary Endpoints-Other 
654 
8.5 
Sample Size 
654 
8.6 
Power Calculations 
65 
8.7 
Accrual 
6Error! Bookmark not defined. 
8.8 
Endpoint Analyses 
665 
8.8.1 Primary Endpoint:  Complete Clearance or Major Regression (CR or 
PR) of Dysplasia Based on Surgical Excision Specimen ........................666 
8.8.2 Secondary Endpoints:  Toxicity and Feasibility ........................................68 
8.8.3 Additional Secondary Endpoints:  Clinical CR or PR at Weeks 6, 13, 
and 25 .........................................................................................................68 
8.8.4 Additional Secondary Endpoints:  HPV Status .........................................69 
8.8.5 Secondary/Exploratory Endpoints:  Immunologic Studies ........................69 
9 
ADVERSE EVENTS, REPORTING REQUIREMENTS AND STOPPING RULES ...680 
9.1 
Adverse Event Definitions 
680 
9.1.1 Definition of Serious and Life Threatening Adverse Events ...................691 
9.1.2 Definition of Relationship to Study Drug ................................................691 
9.1.3 Definition of Severity ..............................................................................691 
9.1.4 Definition of Action Taken ......................................................................702 
9.1.5 Definition of Outcome to Date ................................................................702 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
15 
9.2 
Notification of Serious or Unexpected Adverse Events 
70 
10 
ETHICAL AND REGULATORY CONSIDERATIONS ...............................................724 
10.1 
Protection of Human Subjects 
724 
10.2 
Informed Consent 
724 
10.3 
Institutional Review Board or Independent Ethics Committee 
724 
11 
STUDY ADMINISTRATION ..........................................................................................75 
11.1 
Monitoring Plan 
75 
11.1.1 Pre-study Site Evaluation ...........................................................................75 
11.1.2 Site Initiation ..............................................................................................75 
11.1.3 Interim Monitoring .....................................................................................75 
11.1.4 Study Close-out ..........................................................................................76 
11.1.5 Site Responsibility .....................................................................................76 
11.2 
Compliance Statement 
76 
11.3 
Investigational Product Accountability 
76 
11.4 
Study Documents and Access to Records 
76 
11.5 
Study Closure/Study Termination 
77 
11.6 
Quality Assurance 
77 
11.7 
Confidentiality and Publication Policy 
78 
12 
STUDY MANAGEMENT ................................................................................................79 
12.1 
Conflict of Interest 
79 
12.2 
Institutional Review Board (IRB) Approval and Consent Process 
79 
12.3 
Required Documentation (for multi-site studies) 
79 
12.4 
Registration Procedures 
78 
12.5 
RECORDS AND DATA SUBMISSION 
791 
12.6 
Data Management and Monitoring/Auditing 
802 
12.6.1 Active Monitoring Program Details ...........................................................80 
12.6.2 Quality Assurance Monitoring Committee (QAMC) Oversight ...............82 
12.6.2.1 
QAMC Annual Patient Audits ...............................................824 
12.6.2.2 
QAMC Annual Protocol Review ...........................................835 
12.6.3 Data and Safety Monitoring Committee (DSMC) Oversight ....................83 
12.7 
Adherence to the Protocol 
83 
12.7.1 Emergency Modifications ........................................................................835 
12.7.2 Non-Emergency departures from protocol ..............................................835 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
16 
12.7.3 Amendments to the Protocol ....................................................................846 
12.8 
Record Retention 
857 
12.9 
Obligations of Investigators 
85 
13 
REFERENCES ..................................................................................................................86 
 
LIST OF FIGURES 
Figure 1 
Lymphocyte Infiltration (Study IRX-2 2005-A) ................................................... 33 
Figure 2 
Flow Chart of Study Design ................................................................................. 37 
 
LIST OF TABLES 
Table 1 
IRX-2 Regimen ..................................................................................................... 42 
Table 2  
Schedule of Events ................................................................................................ 45 
Table 3: 
Schedule of Virologic and Immune Monitoring (All Subjects) ............................ 47 
 
LIST OF APPENDICES 
Appendix 1: 
Cervical Intraepithelial Neoplasia Nomenclature ............................................... 935 
Appendix 2: 
Vulvar Intraepithelial Neoplasia Nomenclature ................................................. 946 
Appendix 3: 
ECOG Performance Status ................................................................................... 95 
Appendix 4: 
Optimal Schedule for USC Investigators, Based on GYN Clinic Schedule ......... 96 
Appendix 5: 
Multi-site Registration Form ............................................................................... 939 
Appendix 6: 
Pill Diary ............................................................................................................. 100 
 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
17 
List of Definitions and Abbreviations  
ACIS 
adenocarcinoma in situ (of the cervix, in the context of this protocol) 
ACOG 
American College of Obstetricians and Gynecologists 
AE 
adverse event 
Akt 
protein kinase B 
ALT/SGPT 
alanine aminotransferase/serum glutamic pyruvic transaminase 
ASCCP 
American Society for Colposcopy and Cervical Pathology 
AST/SGOT 
aspartate aminotransferase/ serum glutamic oxaloacetic transaminase 
BUN 
blood urea nitrogen 
CAP 
College of American Pathology 
CBC 
complete blood count 
CD 
cluster of differentiation  
CFR 
Code of Federal Regulations 
CIN 3 
cervical intraepithelial neoplasia 3; comparable nomenclature is severe 
cervical dysplasia and cervical carcinoma in situ. 
CIS 
carcinoma in situ; see also CIN 3, VIN 3. Usually refers to squamous cell 
histology, in contrast to adenocarcinoma in situ (ACIS) 
COVID-19 
Coronavirus disease 2019 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
DSMB 
Data and Safety Monitoring Board 
ECC 
endocervical curettage 
ECOG 
Eastern Cooperative Oncology Group 
FDA 
Food and Drug Administration 
HBV 
hepatitis B virus 
HCV 
hepatitis C virus 
HIV 
human immunodeficiency virus 
HNSCC 
head and neck squamous cell carcinoma 
HPV 
human papillomavirus 
HSIL 
high-grade squamous intraepithelial lesion 
IB 
Investigator’s Brochure 
IEC 
Independent Ethics Committee 
IFN-α 
interferon alpha 
IFN-γ 
interferon gamma 
IL-1β 
interleukin 1 beta 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
18 
IL-2 
interleukin 2 
IL-7 
interleukin 7 
IL-10 
interleukin 10 
IL-12 
interleukin 12 
IL-15 
interleukin 15 
IRB 
Institutional Review Board 
IRX-2 
primary cell-derived biologic with multiple active cytokine components 
IRX-2 Regimen 
for CIN or VIN 
Sublesional injections of 230 U of IRX-2 for 4 days, preceded by 
cyclophosphamide with a 21-day course of indomethacin and zinc 
containing multivitamins; repeat cycle once in 6 weeks 
IV 
intravenous 
IxRS 
Interactive Recognition System 
LAST 
Lower Anogenital Squamous Terminology 
LC 
Langerhans cell(s) 
LDH 
lactate dehydrogenase 
LEEP 
large loop excision procedure (for excision of cervical dysplasia) 
LLETZ 
large loop excision of the transformation zone 
LSIL 
low-grade squamous intraepithelial lesion 
NCI-CTCAE 
National Cancer Institute Common Terminology Criteria for Adverse 
Events 
NK or NKT 
natural killer T cells 
PI 
Principal Investigator 
PI3-K 
phosphoinositide 3 kinase 
PK 
pharmacokinetic 
PS 
performance status  
PT 
prothrombin time 
PTT 
partial thromboplastin time 
SAE 
serious adverse event 
SAP 
statistical analysis plan 
SARS-CoV-2 
Severe acute respiratory syndrome coronavirus 2 
SIL 
squamous intraepithelial lesion 
T cells 
cellular immune lymphocytes; derivation – thymus lymphocytes 
T-bet 
T box transcription factor 
TCR 
T cell receptor  
Teff 
T effector cell 
TGF-β 
Transforming growth factor beta 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
19 
Treg 
T regulatory cell; suppressor T c ell 
TH 
T-helper 
TH1 
T-helper 1 (cell mediated) 
TH2 
T-helper 2 (humoral) 
TID 
three times a day 
TNF-α 
tumor necrosis factor alpha 
U 
unit(s) 
ULN 
upper limit of normal 
US 
United States 
VIN 3 
Vulvar intraepithelial neoplasia 3; comparable nomenclature is severe 
vulvar dysplasia and vulvar carcinoma in situ. 
WLE 
wide local excision 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
20 
1 
INTRODUCTION 
1.1 
BACKGROUND  
1.1.1 Female Lower Genital Tract Dysplasia 
Lower genital tract dysplasia in women encompasses premalignant conditions of the uterine 
cervix, vagina, vulva and adjacent perineal skin. Dysplasia of perianal tissues and anus are now 
recognized as part of the spectrum of this disorder due to common anatomic origin from the 
embryonic cloaca and shared etiology of antecedent infection with an oncogenic strain of human 
papilloma virus (HPV) [Schiffman, 1993; Darragh, 2013]. Lower anogenital dysplasia and lower 
genital tract dysplasia are used interchangeably in this introductory section.  
Untreated lower genital tract dysplastic lesions can progress to invasive cancers. Almost all 
cervical and vaginal cancers develop from pre-existing dysplasia, as do the majority of vulvar and 
anal cancers. Current Pap smear screening programs (for cervical dysplasia) and better recognition 
of visible or symptomatic lesions (for vulvar dysplasia) have improved the diagnosis of these 
premalignant lesions, at least in developed countries with the resources needed for this clinical 
care. Clinicians also now understand that infection with an oncogenic type of HPV is almost 
always the first step towards malignant transformation of anogenital epithelium. Unfortunately, 
HPV is endemic among sexually experienced individuals. While the risk does correlate with 
number of lifetime partners, it remains relatively high (4 to 20 %) even in those with one partner. 
Overall, at least 75 to 80% of sexually active women will have acquired a genital HPV infection 
by age 50 [Workowski, 2015]. Vaccines directed against HPV 16 and HPV 18 (2 of the more 
oncogenic HPV types) were introduced over 10 years ago, and a vaccine that also covers HPV 
types 31, 33, 45, 52 and 58 is now available [FDA, 2014]. Vaccination against these oncogenic 
types of HPV could prevent up to 90% of cervical cancers, with comparable reduction in the 
incidence of dysplasia and invasive cancer of other lower genital tract sites. However, current HPV 
vaccination programs will only benefit the generation of young woman eligible for vaccination. 
And to date the vaccination rate in the USA is only about 30%, which is below the level needed 
for protective herd immunity [Widgren, 2011; CDC, 2013]. Thus there remains an ongoing need 
for effective treatment to prevent the morbidity and mortality of HPV-associated lower genital 
tract dysplasia and cancer. 
Despite substantial gains in understanding the molecular biology of HPV-associated 
carcinogenesis [Schiffman, 1993; Alani, 1998; Darragh, 2013], the treatment of lower genital tract 
dysplasia has not improved. Clinicians use variations of the same crude ablative or excisional 
techniques dating to the 1950’s to eradicate dysplastic lesions [Montz, 2000; Martin-Hirsch, 2013; 
ACOG, 2011]. As detailed below, existing treatments of cervical and vulvar dysplasia cause 
substantial short term morbidity as well as long term risks to fertility, pregnancy outcomes, and 
sexual function. Furthermore, failure rates of these procedures are high, in part due to the 
multifocal nature of lower anogenital tract dysplasia; thus even women with apparent eradication 
of a dysplastic lesion remain at risk for recurrent dysplasia and cancer decades into the future 
[ACOG, 2012]. This is a particular risk for women with a history of VIN 3, of whom one-third 
will experience a recurrence of dysplasia, and 4 to 8% will eventually develop invasive cancer 
[van Seters, 2005]. 
The limitations of existing treatments for cervical and vulvar dysplasia led us to develop this Phase 
2 research protocol to evaluate the novel immunotherapy regimen, IRX-2, for this patient 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
21 
population. Women with severe cervical or vulvar dysplasia (CIN 3, VIN 3) will be invited to 
participate. All subjects will undergo the medically indicated surgical excision of their dysplastic 
lesion; subjects in the experimental arm will also receive sublesional injections of IRX-2. While 
this initial exploratory trial will of necessity have limited objectives, our hope is that our research 
can eventually achieve three goals: 
• 
Avoid large and potentially dangerous or disfiguring excisions currently required for 
conventional treatment of lower genital tract dysplasia. 
• 
Restore the subject’s immune system to prevent recurrent dysplasia and decrease 
future risk of secondary lower genital tract cancers. Multiple lines of experimental and 
clinical evidence have demonstrated the influence of the immune system upon relapse 
rates for lower genital tract dysplasia [Freiser, 2013; Gildener-Leapman, 2013]. Thus 
enhancing immunologic control of the pre-invasive lesions can confer benefits well 
beyond the initial excision of the dysplastic lesion. 
• 
Finally, if this IRX-2 Regimen is proven to be effective for eradicating HPV-related 
dysplasia, then it may also have a role in the adjuvant treatment of anogenital tract 
cancers. 
In the following sections, the diagnosis and management of cervical and vulvar dysplasia will be 
further reviewed, followed by a discussion of the interplay between HPV infection and local 
cellular immune-deficiencies. 
The IRX-2 Regimen has also been studied in a pilot study of 10 subjects with early stage cervical 
cancer as neoadjuvant, pre-operative therapy [Dueñas-Gonzalez, 2002]. This trial provides 
important data demonstrating the safety and feasibility of the IRX Regimen treatment of cervical 
disease. The treatment was well tolerated except for mild pain and minor bleeding during injections 
and gastric intolerance to indomethacin. Clinical response was seen in 50% of subjects (three 
partial responses, two minor responses). In addition, IRX-2 had been shown in vitro to induce 
significant upregulation of the immune response directed against human papilloma virus (HPV)-
exposed Langerhans cells, thereby potentially preventing HPV-induced cancers [Da Silva, 2016]. 
1.1.2 Nomenclature: Dysplasia, Intraepithelial Neoplasia, and Squamous Intraepithelial 
Lesion 
Historically, premalignant squamous lesions of the uterine cervix were described as mild, 
moderate or severe cervical dysplasia. Carcinoma-in-situ was used by most pathologists to denote 
full thickness replacement of the epithelium with severe dysplasia. Lesions of the vagina, vulva, 
perineum, and anus were similarly categorized. Several terminology systems have been introduced 
over the decades to allow better inter-observer diagnostic accuracy and more uniform reporting, 
clarify the distinction between cytology reports for Pap smears and histology reports for biopsies, 
and address the difficulties associated with the diagnosis of glandular neoplasia of the cervix 
[Muntz, 2004].  
1.1.2.1 Bethesda System for Grading Cervical Dysplasia 
For cervical dysplasia, the Bethesda system is most commonly used; its current iteration was 
released in 2001 [Solomon, 2002]. Pap smear cytology is described using the term “squamous 
intraepithelial lesion” (SIL) and histologic changes are described using the term “cervical 
intraepithelial neoplasia” (CIN). There are three degrees of severity for CIN: 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
22 
1. 
CIN 1 is a low grade lesion. A biopsy reveals mildly atypical cellular changes in the 
lower third of the epithelium. Koilocytotic atypia due to the cytopathic effect of HPV 
infection is usually present. 
2. 
CIN 2 is a higher grade lesion. A biopsy reveals moderately atypical cellular changes 
confined to the lower two-thirds of the epithelium with preservation of epithelial 
maturation. CIN 2 is analogous to moderate dysplasia. There is considerable 
diagnostic variability in this category. Clinical guidelines [Massad, 2013] used for 
treatment assume the cancer risk posed by CIN 2, while lower than the risk for CIN 3, 
is high enough to warrant treatment; nonetheless, many CIN 2 lesions have a low risk 
of malignant progression and will regress without treatment.  
3. 
CIN 3 is a high grade lesion with a substantial risk of progression to cancer if 
untreated. Histology reveals severely atypical cellular changes encompassing more 
than two-thirds of the epithelial thickness and up to full-thickness involvement. CIN 
3 is analogous to severe dysplasia or carcinoma in situ. 
1.1.2.2 Glandular Dysplasia, Adenocarcinoma-in-situ, and Adenocarcinoma of the 
Uterine Cervix 
The ectocervix, or the portion of the cervix that is visualized with a vaginal speculum examination, 
is covered by squamous epithelium. The cervical canal (endocervix) is covered with glandular 
epithelium. While dysplasia of both squamous and glandular tissues share the same HPV-mediated 
pathogenesis, recognizing and categorizing the severity of glandular dysplasia is very difficult. For 
that reason the Bethesda system specifically excludes glandular dysplasia from the CIN 
nomenclature; e.g. CIN refers only to squamous abnormalities [Solomon, 2002].  
The Bethesda system does provide guidance for reporting abnormal glandular cells detected by 
screening cytology Pap smears as well as biopsies showing glandular abnormalities. The preferred 
nomenclature is “atypical glandular cells” (AGC) for reporting abnormal glandular cytology, with 
“AGC favor neoplasia” used for more worrisome findings. But AGC interpretations are poorly 
reproducible, even by expert cytopathologists [Lee, 2002]. The underlying histologic abnormality 
may often be a squamous lesion. The glandular lesions associated with AGC cytology can range 
from benign polyps to low grade abnormalities to adenocarcinoma-in-situ to invasive 
adenocarcinoma of the uterine cervix; furthermore, an AGC pap smear can be caused by occult 
adenocarcinomas of the endometrium, fallopian tube, ovary, and other sites [Zhao, 2009]. Careful 
diagnostic evaluation is required for any patient with an AGC pap smear report, including 
extensive biopsies since glandular dysplasia is difficult to detect by colposcopy. There is also a 
small but important risk of occult adenocarcinoma; for example, in a large population data base, 
AGC pap smears in women over the age of 30 were due to occult invasive adenocarcinoma in 3% 
of cases [Katki, 2013]. Therefore it is prudent to excise a dysplastic glandular lesion without undue 
delay. Furthermore, if histology establishes adenocarcinoma-in-situ as the correct diagnosis, then 
hysterectomy remains the treatment of choice for women who have completed childbearing 
[Muntz, 1992; Muntz, 1996; Massad, 2013]. 
For all of these reasons, women with severe glandular dysplasia or adenocarcinoma in situ of the 
uterine cervix – even though this is the glandular corollary to squamous CIN 3 – are NOT eligible 
for participation in this clinical trial. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
23 
1.1.2.3 Vulvar Dysplasia 
In parallel with the descriptions used for cervical dysplasia, vulvar dysplasia has been historically 
divided into three grades: mild, moderate and severe (with the latter including carcinoma in situ). 
Since the late 1980’s the Bethesda nomenclature of intraepithelial neoplasia has been preferred, 
e.g. VIN 1, VIN 2 and VIN 3. As with CIN, all VIN lesions are by definition derived from 
squamous tissue, and are the most common precursors for vulvar squamous cell carcinoma. Unlike 
cervical cancers, of which a substantial proportion is adenocarcinoma, over 90% of vulvar cancers 
are primary squamous cell carcinomas. The few non-squamous categories include rare Bartholin 
gland cancers, adenocarcinomas arising from vulvar Paget’s disease, primary vulvar melanoma, 
or secondary malignancies from other sites [Fox, 2003]. 
In 2004 the International Society for the Study of Vulvar Diseases (ISVVD) made important 
updates to the VIN nomenclature [Sideri, 2005], including the following: 
1. 
There is now substantial evidence that what had been termed VIN 1 is not a cancer 
precursor requiring treatment [ACOG, 2011]. Thus, VIN 1 was dropped from the 
nomenclature, and those lesions are referred to as condyloma acuminatum. The term 
VIN is now limited to high grade squamous lesions (VIN 2 and VIN 3). 
2. 
VIN was classified into two main categories: 
a. 
VIN, usual type: These are the lesions associated with HPV infection, and are the 
most common in the general population. 
b. 
VIN, differentiated type: These lesions comprise less than 5% of VIN and 
typically occur in postmenopausal women. Differentiated VIN lesions are not 
associated with HPV infection. The lesions arise from areas of chronic vulvar 
irritation, most commonly due to vulvar dermatoses such as lichen sclerosus. The 
clinical, molecular, and immunologic characteristics of the differentiated type of 
VIN are distinct from those of the usual HPV-mediated type of VIN. For this 
reason, subjects with the differentiated type of VIN are excluded from this 
research study. 
1.1.2.4 Lower Anogenital Squamous Terminology (LAST) Project 
In 2012 the Lower Anogenital Squamous Terminology (LAST) project of the College of American 
Pathology (CAP) and the American Society for Colposcopy and Cervical Pathology (ASCCP) 
proposed uniform changes in terminology to describe all HPV-associated squamous lesions of the 
anogenital tract [Darragh, 2013]. The LAST project dropped the CIN and VIN nomenclature 
favored by the Bethesda and ISSVD systems for reporting cervical and vulvar histology (as well 
as VaIN, PAIN and AIN for vaginal, perianal and anal histology, respectively). Instead, the SIL 
nomenclature is used to report both cytologic and histologic abnormalities. Moderate grades of 
dysplasia are stratified according to p16 immunostaining to better identify precancerous lesions. 
Specimens that are p16-negative are categorized as LSIL and those that are p16-positive are 
categorized as HSIL. For vulvar dysplasia, VIN 2 and VIN 3 are combined into one category: 
vulvar HSIL. 
While there are cogent reasons for the changes proposed by the LAST project, at this time the new 
terminology is not widely used. Furthermore, clinicians principally rely on the Bethesda system’s 
CIN terminology when using ASCCP guidelines for the evaluation and management of abnormal 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
24 
cervical cytology and histology [Massad, 2013]. For these reasons the CIN terminology will be 
used for this research protocol. 
Appendix 1 and Appendix 2 provide a comparison between the different nomenclatures used to 
describe cervical and vulvar dysplasia. 
1.1.3 The Management of CIN 3, and its Long Term Sequelae 
1.1.3.1 Management of CIN 3 
Because of the risk CIN 3 lesions can progress to cancer, treatment requires complete destruction 
or excision of the dysplastic lesion with adequate margins. Lesion destruction can be done with 
cryotherapy or CO2 laser vaporization. But in general an excisional procedure is now favored 
because it is usually better tolerated by the patient and yields a histology specimen for 
determination of margin status and to rule out the presence of occult invasive carcinoma [Massad, 
2013]. 
Cervical excisions are accomplished with a superficial large loop excision of the transformation 
zone (LLETZ), also known as a loop electrosurgical excision procedure (LEEP). A deeper 
electrosurgical excision can also be performed that includes more of the endocervical canal and is 
commonly referred to as a LEEP conization procedure. These procedures are normally performed 
in a clinic or office setting; some patients, for instance who cannot tolerate an office procedure, 
are best managed in an operating room under sedation or anesthesia. Some patients have lesions 
that require a scalpel cervical conization for an adequate excision; these procedures are performed 
in an operating room with anesthesia [Montz, 2000; Kyrgiou, 2006; Martin-Hirsch, 2013] 
Success rates for excisional procedures for the management of CIN 3 are 90-95% with most 
failures diagnosed within two years [Paraskevaidis, 2004]. Long-term follow-up is also needed 
due to the risk that the patient will develop new dysplastic lesions elsewhere within the lower 
genital tract [Jakobsson, 2009]. With respect to future risk of cervical cancer, women with a history 
of CIN have a 10-fold increased risk during the next 20-25 years when compared to the general 
population [Melnikow, 2009; Rebolj, 2012].  
A small proportion of patients diagnosed with CIN 3 will have no histologic evidence of dysplasia 
in the subsequent excisional specimen performed for treatment. Whether this represents complete 
removal of a small lesion with the initial diagnostic biopsy, the effect of post-biopsy inflammatory 
changes, or spontaneous resolution of the dysplastic lesion is impossible to ascertain for any one 
patient. Based on observational studies and clinical trials, estimates of this rate range widely, from 
less than 5% up to 40%. The variability is best explained by several factors, including: 
• 
The population studied. 
o 
Women under age 25 have a greater likelihood of clearing a dysplastic lesion 
without treatment [Moscicki, 2001; Moscicki, 2010; Bosch, 2006];  
o 
Smokers have a greater risk of persistent disease [Deacon, 2000]. 
• 
Inclusion of subjects with varying degrees of dysplasia, for instance CIN 2, 3 vs. CIN 
3 only. CIN 2 lesions diagnosed by conventional histology have a higher rate of 
regression because many are best categorized as low grade SIL. As discussed above, 
the LAST classification system addresses this by requiring stratification of CIN 2 
lesions with p16 staining [Darragh, 2013]. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
25 
• 
Different definitions for resolution, such as complete absence of dysplasia in the 
surgical specimen vs. decrease in the extent or severity of the dysplasia. 
For these reasons this study is limited to women age 25 years or older, smoking history is a 
variable, and both complete and partial responses will be measured. Finally, the statistical model 
for our study used a modest spontaneous regression rate of 20% when calculating sample size to 
ensure an adequate number of subjects would be recruited. The spontaneous regression rate will 
not be as much of a confounding factor in VIN3 disease given the rate is much lower.  
1.1.3.2 Complications and Long-Term Sequelae from the Surgical Management of CIN 3 
The discomfort and psychological distress caused by the surgical treatment of CIN 3 should not 
be minimized [Lerner, 2010]. Nonetheless, immediate adverse events such as pain, hemorrhage 
and infection are uncommon, usually of mild to moderate severity, and manageable with routine 
clinical care [Martin-Hirsch, 2013]. Of greater concern is that CIN 3 most often affects 
reproductive-age women and damage to their cervix may impact normal menstruation, fertility and 
future pregnancy outcomes. 
Any surgical procedure can lead to scarring; for cervical excisions, this can result in cervical 
stenosis. This complication has been reported in up to 8% of women undergoing excisional 
treatment for CIN [Baldauf, 1996]. Cervical stenosis is linked to a number of complications, 
including: 
• 
Obstruction of menstrual flow, leading to painful menstruation (dysmenorrhea). In 
severe cases blood and debris accumulate inside the uterine cavity (hematometra), 
which can become secondarily infected (pyometra). Surgical management is required 
(ranging from cervical dilation to hysterectomy). 
• 
Severe cervical stenosis can prevent cervical dilation during labor. Ordinarily this is 
managed with a cesarean section. In other cases labor proceeds but only after 
disrupting the scar tissue with deep cervical lacerations followed by massive 
postpartum hemorrhage. 
Removal of cervical tissue from around the cervical os and into the endocervical canal can cause 
two other effects: structural weakness of the cervix and destruction of the glands that form the 
protective endocervical mucous plug. Complications linked to these alterations in normal cervical 
anatomy are summarized below: 
• 
The risk of second-trimester pregnancy loss is significantly increased, presumably due 
to loss of structural integrity of the cervix following an excisional procedure. This 
devastating complication usually occurs between 16 and 24 weeks gestational age – 
long after the mother has bonded with her infant but a few weeks too early for any 
hope of delivering a healthy live neonate. The baseline population risk of second 
trimester pregnancy loss is only 0.4%. Based on population studies [Albrechtsen, 
2008] and a large meta-analysis [Kyrgiou, 2014] this risk increases significantly to 
1.5-1.6% (RR approximately 2.60, 95% CI 1.45-4.67). 
• 
The risk of preterm premature rupture of membranes (PPROM) is significantly 
increased in women who have undergone a LEEP, rising from 2% to 5% [Kyrgiou, 
2006]. This increased risk has been attributed to a short, scarred and inelastic cervix 
transmitting shearing forces to the fragile amniotic sac. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
26 
• 
There is an increased risk for ascending bacterial invasion of the amniotic sac, leading 
to preterm delivery and both neonatal and maternal sepsis [Svare, 1992]. 
• 
Preterm delivery and perinatal mortality are both increased in most long-term studies 
evaluating the effects of CIN treatments on pregnancy outcomes. Preterm delivery and 
perinatal mortality are closely linked, with most perinatal deaths in these studies due 
to complications of prematurity. The relative risk of preterm delivery seems to vary 
widely based on the type of excision performed, with perinatal mortality also tracking 
with gestational age at delivery. Overall, the best risk estimates are derived from a 
large 20-study meta-analysis [Arbyn, 2008] and a large Danish population-based study 
[Noehr, 2009]. For example: 
o 
The meta-analysis linked cold knife conization to an increase in perinatal 
mortality, which rose from 0.5% to 4.3% (RR 2.9, 95% CI 1.4-5.8]. The increased 
perinatal mortality rate paralleled an increase in preterm deliveries between 28-
34 weeks. 
o 
The Danish population-based study found an association between LEEP and 
preterm deliveries throughout the third trimester. This included an increase in 
deliveries at 32 to 36 weeks (baseline rate 2.9%, increasing to 5.0%). Inclusion 
of these “late preterm infants” in the Danish study is important. Even though these 
neonates rarely die (and thus are not reported in mortality statistics), their 
increased morbidity is still substantially greater than full term infants. 
After reviewing these potentially catastrophic complications, is it imperative to remember that the 
main priority when managing a woman with CIN is to eliminate her risk of dying from cervical 
cancer. Yet a prudent clinician can mitigate the long term reproductive risks with common sense 
and by following current guidelines [Massad, 2013], such as: 
• 
Adolescents and young women (< age 25) have a very low risk of cancer. 
Furthermore, transient HPV infection is common (which even for “high risk” types 
is not linked to future cancer risk) and dysplastic lesions, if they develop, often 
regress [Moscicki, 2001; Moscicki, 2010]. For these reasons close observation can 
be chosen over either ablation or excision for carefully selected patients. 
• 
Ablation techniques (rather than excision) can be used for other young women – 
loosely defined based on number of children already delivered and future childbearing 
plans. This is most appropriate for women with more moderate degrees of dysplasia 
and other favorable risk factors in whom the likelihood of occult invasive cancer is 
negligible. 
• 
And for women with CIN 3, who will usually need an excisional procedure, the 
gynecologic surgeon should limit the depth of the excision, and if possible avoid 
multiple excisions. 
Another way to avoid long-term complication is to develop a non-surgical treatment for CIN. If 
IRX-2 can successfully eradicate CIN 3 then it could become a new treatment option for CIN. This 
could substantially benefit not only the many women facing a CIN diagnosis but also improve 
future pregnancy outcomes and neonatal health. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
27 
1.1.4 Management of VIN 
1.1.4.1 Surgical Excision 
Surgical management of VIN 3, similar to the management of CIN 3, requires either destruction 
(laser vaporization) or excision of the dysplastic lesion; and as with CIN 3, excisional procedures 
are generally preferred because those allow histologic assessment. Unlike CIN, spontaneous 
regression of vulvar dysplasia is very uncommon (under 1.5%) and treatment is required for almost 
all women [van Seters, 2005], excepting only the occasional very young patient with an ambiguous 
diagnosis favoring benign condyloma accuminata. Furthermore, VIN is often multifocal. The 
interlabial folds, clitoris, posterior fourchette, perineum and perianal tissues are frequently affected 
by widely dispersed lesions. More extensive disease can have confluent involvement of the labia 
majora, labia minora, perineal skin and anus. Multifocal or confluent high grade lesions are present 
in up to two-thirds of women with VIN, and these are often admixed with lower grade dysplastic 
lesions and condylomata [Friedrich, 1980]. 
Each patient therefore will have a unique distribution of abnormal tissue spread over a potentially 
wide area, and eradicating the dysplastic tissue while preserving normal vulvar anatomy and 
function can be very difficult [ACOG, 2011]. Small lesions can be managed with a wide local 
excision followed by reapproximation of the resulting surgical defect. Removing the epidermis 
with a small amount of underlying dermis is sufficient. Larger lesions require a partial vulvectomy. 
Extensive multifocal or confluent lesions are managed with a skinning vulvectomy. This procedure 
is done by removing all of the vulvar skin along the relatively avascular plane between the 
epidermis and dermis with the large defect covered by a split thickness skin graft [Rutledge, 1968]. 
Concurrent lower grade lesions and condylomata can be managed with ablative therapy to decrease 
the amount of vulvar tissue surgically excised. CO2 laser vaporization, argon beam coagulation, 
and cavitational ultrasonic surgical aspiration (CUSA) can be used for this purpose, although laser 
vaporization is currently preferred by most gynecologic oncologists [ACOG, 2011]. 
1.1.4.2 Efficacy of Current VIN Treatments 
Regardless of whether excision or ablation (or both) are used, many women require several painful 
and potentially disfiguring treatments for initial eradication of VIN 3. Even then, approximately 
1/3 will develop recurrent VIN [van Seters, 2005]. Risk factors for recurrence include 
immunosuppression, cigarette smoking, multifocal or multicentric disease, larger lesion size and 
involved margins. Long-term follow-up is required for decades and must include surveillance of 
the entire anogenital tract for appearance of new dysplastic lesions. Finally, approximately 3-8% 
of patients develop invasive squamous cell cancer following treatment for VIN 3 [van Seters, 
2005]. So while the overall population risk for vulvar cancer is lower than the risk of cervical 
cancer (approx. 5,000 vs. 13,000 cases/year in the US in 2015 [Siegel, 2015]), an individual 
woman’s risk of developing invasive cancer after treatment for VIN 3 is significant. 
Thus a more effective and less disfiguring treatment with better long term control of dysplasia and 
prevention of cancer is clearly needed for women diagnosed with VIN 3. 
1.1.4.3 Topical Therapy: Imiquimod Cream (Aldara®) 
Topical therapies have been tried in an attempt to preserve vulvar anatomy, especially in younger 
women. Imiquimod cream (Aldara®) is approved for the treatment of vulvar and perianal 
condyloma accuminata [Aldara Prescribing Information, 2010]. Imiquimod is a topical immune 
response modifier with antiviral and antitumor effects mediated through the stimulation of local 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
28 
cytokine production and cell-mediated immunity. A course of imiquimod treatment lasts 16 weeks, 
during which time the cream is topically applied to the wart. All patients experience blistering and 
desquamation of vulvar skin to some degree; most are severe enough to require dose modifications 
and interruption of treatment. “Off-label use” for VIN should be considered investigational but has 
been somewhat successful, with up to 50% complete response rate reported in a collection of 
patients (n = 162) pooled from ten small studies [Mahto, 2010]. Indeed the suggestion that the 
weak immunostimulatory properties of imiquimod can be effective supports our investigation of 
the cytokine-based IRX-2 Regimen for the treatment of VIN 3. 
1.1.4.4 Investigational Therapeutic Vaccination for VIN 3 
Preliminary investigations of therapeutic HPV vaccination for VIN 3 have been reported and are 
ongoing [Baldwin, 2003; Davidson, 2003; Smyth, 2004]. Distinct from the commercially available 
preventative vaccines (which only cause a humoral antibody response), the therapeutic vaccines 
elicit a cellular immune response. The most promising results were reported from a small trial of 
women with HPV-16-positive VIN 3. Subjects were vaccinated with a mixture of synthetic long-
peptides derived from the HPV-16 viral oncoproteins E6 and E7 [Kenter, 2009]. After 12 months 
there were 6 partial and 9 complete responses in the 19 evaluable subjects, for an overall response 
rate of 79% (95% CI, 54 to 94%).  
Like the imiquimod studies cited above, even partial success with vaccine therapy lends credence 
to our plan to use the IRX-2 Regimen to treat VIN 3, particularly since the IRX-2 Regimen is 
devised to overcome the inhibition of local cellular immunity that probably hampers the 
effectiveness of a systemically administered vaccine therapy. 
1.1.5 Immune Deficiency and Immunotherapy in HPV-Mediated Premalignant and 
Malignant Conditions of the Lower Genital Tract 
The rationale for the pursuit of immunotherapy of carcinoma in situ of the lower genital tract (such 
as CIN 3 and VIN 3) with the IRX-2 Regimen stems from a considerable body of evidence which 
indicates that immunologically competent cells are important host defense mechanisms. Many 
human cancers, including HPV-related cancers like cervical cancer, are associated with cellular 
immunodeficiency. Persistent high-risk human papillomavirus (HPV) infection leads to the 
development of numerous human cancers including cervical, vaginal, vulvar, anal, and an 
increasing proportion of head and neck cancers that cause significant morbidity and mortality 
worldwide [Forman, 2012; Tota, 2011]. While genital HPV infections are very common, more 
than one out of seven HPV-infected women cannot initiate an effective immune response against 
the virus, and among those that do, clearance is slow and generally takes more than one year 
[Frazer, 1996; Giuliano, 2002; Stanley, 2007]. To persist in this manner, HPV has evolved 
mechanisms to escape immune detection, and this persistence is the major risk factor for 
developing HPV-induced cancers. Previous studies have demonstrated that HPV-mediated 
suppression of Langerhans cell (LC) immune function is a central mechanism by which HPV 
evades immune detection [Fahey, 2009; Da Silva, 2015; Fausch, 2005; Woodham, 2015]. This 
effect is specific to LC, as dendritic cells (DC) are differentially targeted, and thus are activated 
by HPV. The impact of IRX-2 upon this HPV mediated suppression of LC was investigated after 
human LC activation by exposure to HPV16 followed by treatment with IRX-2 in vitro [Da Silva, 
2016]. The subsequent ability of the LC to induce HPV16-specific T cells was studied. HPV16 
alone is not sufficient to induce phenotypic and functional activation of LC, but IRX-2 induces a 
significant upregulation of antigen presentation and costimulatory molecules, Th1-associated 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
29 
cytokine release, and chemokine-directed migration of LC pre-exposed to HPV16. Furthermore, 
LC treated with IRX-2 after HPV16 exposure induced CD8+ T cell responses against specific 
HLA-A*0201-binding HPV16 T cell epitopes [Da Silva, 2016]. These results clearly suggest that 
IRX-2 is an attractive immunomodulator for assisting the immune response in eradication of HPV 
infected cells.  
1.1.6 The IRX-2 Regimen for Treatment of CIN and VIN 
The concept that subjects with squamous cell cancers and associated dysplastic lesions might 
benefit from immune stimulation led to the development of the IRX-2 Regimen. The regimen 
consists of the cell-derived biologic IRX-2 that contains multiple cytokines, plus 
cyclophosphamide, indomethacin, and zinc with multivitamins. For the treatment of pre-invasive 
squamous cell neoplasias such as CIN 3 and VIN 3, these four components are administered using 
an abbreviated schedule, when compared with the IRX-2 Regimen previously studied in subjects 
with invasive head and neck squamous cell cancers (HNSCC). The Regimen for this protocol 
consists of an intravenous (IV) infusion of low-dose cyclophosphamide on Day 1, followed by oral 
indomethacin and oral zinc with multivitamins daily for 21 days, and IRX-2 administered as daily 
sublesional injections per day for 4 days, as outlined in Section 5. Omeprazole, a proton pump 
inhibitor, is given concurrently to decrease the likelihood of intolerance of indomethacin.  
1.1.7 Manufacture and Composition of IRX-2 
IRX-2 is a primary cell-derived biologic with multiple active cytokine components produced under 
pharmaceutical standards as discussed in more detail in the Investigator’s Brochure. Briefly, 
human leukocytes (“buffy coats”) pooled from multiple donors are stimulated with 
phytohemagglutinin and ciprofloxacin. Subsequently, the phytohemagglutinin, ciprofloxacin and 
all cellular elements are removed or significantly reduced, and the cell-free supernatant is filter 
sterilized, nanofiltered to clear viral particles, vialed, and frozen as IRX-2. 
IL-2 is the major cytokine in IRX-2, followed by IFN-γ, TNF-α, and interleukin 1 beta (IL-1β). 
These cytokines when studied individually enhance cell-mediated immunity via several different 
mechanisms discussed below and in the IB.  
1.1.8 Mechanism of Action of IRX-2 
Recent in vitro studies have elucidated several potential mechanisms of action of IRX-2, and these 
various mechanisms of action need not be exclusive. IRX-2 treatment of human monocyte-derived 
dendritic cells results in changes consistent with the development of mature activated dendritic 
cells. Specifically, IRX-2 increased the percentage of cells expressing CD83 and CCR7, markers 
for dendritic cell maturation and migration and increased the expression of multiple markers that 
are critical mediators of T-cell activation [Egan, 2007]. Of relevance to the treatment of lower 
genital tract squamous cell carcinoma in situ, similar results were obtained in a later study in cells 
obtained from subjects with squamous cell cancer of the head and neck (HNSCC) [Schilling, 
2013]. Also, in an in vitro study of peripheral blood mononuclear cells obtained from subjects with 
HNSCC, IRX-2 up-regulated cytotoxicity of NK cells and did so more effectively than IL-2 
[Schilling, 2012]. 
IRX-2 can also protect T cells from activation induced cell death by reversing microvesicle 
induced inhibition of the PI3K/Akt pathway and correcting the imbalance of pro- versus anti-
apoptotic proteins induced by tumor-derived microvesicles [Czystowska, 2009; Czystowska 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
30 
2011]. IRX-2 was superior to recombinant IL-7 and IL-15 in protecting T cells from tumor-induced 
apoptosis. The presence of IRX-2 in a tumor microenvironment model promoted the induction and 
expansion of IFN-γ+T-bet+ Teff and significantly decreased the induction of inducible IL-10+TGF-
β+ Treg. The responsible mechanism involved IFN-γ+-driven T cell polarization towards Teff and 
suppression of Treg differentiation [Schilling, 2012]. 
The clinical utility of IRX-2 has been validated in a Phase IIa study in subjects with HNSSC 
(described below in Section 1.2.2). IRX-2 mediated reductions in circulating B and NKT cell 
numbers, suggesting redistribution of these cells to tissues [Whiteside, 2012]. A decrease in naïve 
T cells was also noted, suggesting their upregulation to memory T cells while unchanged numbers 
of Tregs (suppressor T cells) after IRX-2 therapy indicated that IRX-2 does not expand this 
compartment, potentially benefiting anti-tumor immune responses [Whiteside, 2012].  
Finally, IRX-2 has been shown to induce enhanced T cell responses when administered with tumor 
antigen vaccines, raising the possibility that IRX-2 treatment in subjects with carcinoma in situ or 
cancer can enhance endogenous antigen-specific T-cell responses to the tumor [Naylor, 2010]. 
1.1.9 Rationale for the Components of the IRX-2 Regimen 
The rationale for the components of the IRX-2 Regimen is outlined below. 
Cyclophosphamide: One mechanism for reversal of anergy and reversal of suppression of immune 
responses in subjects with malignancy by adoptive immunotherapy may be related to inhibition of 
suppressor T cell function [North, 1982; North, 1984]. Evidence indicates that cyclophosphamide 
inhibits Treg number and/or function [Emens, 2005]. Thus many clinical trials that involve 
immunotherapy or attempt to stimulate immune response to tumor antigens have employed low 
dose cyclophosphamide (300 mg/m2) as a component of the treatment regimen. This 
immunomodulatory dose is less than one-third of a typical anti-cancer dose and is intended to 
enhance the development of cell-mediated immunity by providing contrasuppression of tumor-
associated immune suppression (to reduce the number and function of suppressor T cells, (i.e. Treg) 
[Berd, 1982; Machiels, 2001]. 
Pathways of local immune tolerance, escape mechanisms active within the tumor 
microenvironment and superimposed potent systemic mechanisms of immune tolerance have been 
reviewed [Emens, 2005] and are discussed in more detail in the Investigator’s Brochure. The use 
of cytotoxic chemotherapy in doses and schedules designed to abrogate specific mechanisms of 
immune tolerance in order to release the full potential of an antitumor immune response is 
discussed. Specifically, cyclophosphamide may be used to prime the immune system by promoting 
the differentiation of CD4+ T helper cells and by abrogating the suppressive influence of 
CD4+CD25+ T regulatory (Treg) cells. In the absence of Treg influence, high-avidity CD8+ T cells 
are recruited to an antigen-specific immune response. Cyclophosphamide also facilitates the 
establishment of memory CD8+ T cells. Thus inclusion of cyclophosphamide in combination trials 
with other immune-modulatory agents is supported by both pre-clinical and clinical data as 
reviewed in more detail by Emens (2005) and in the Investigator’s Brochure. 
Indomethacin: Indomethacin, a nonselective COX-1/COX-2 inhibitor, is a potent inhibitor of 
prostaglandin synthesis and may reverse the immunosuppression induced by prostaglandin 
[Lapointe, 1992; Hadden, 1994]. Numerous experimental, epidemiologic, and clinical studies 
suggest that non-steroidal anti-inflammatory drugs, including indomethacin, suppress 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
31 
cyclooxygenase and have promise as anticancer agents, particularly for chemoprevention of and 
as adjuvant therapy in patients with cancer [Thun, 2002; Investigator’s Brochure]. 
Zinc-containing multivitamins: The importance of zinc in cellular immunity has been described 
and several reviews are available [Good, 1979; Keen, 1990; Blewett, 2012; Haase, 2014]. Other 
nutritional deficiencies can also result in impaired immune response [Słotwińska, 2014; Bianchini, 
2012; Wintergerst, 2007]. Thus, based on these observations and the lack of any contraindication 
to their use, zinc-containing multivitamins have been added to the IRX-2 Regimen. 
Omeprazole: Omeprazole, a proton pump inhibitor, is active at preventing indomethacin-induced 
gastritis. It is administered with the IRX-2 Regimen to decrease the likelihood of indomethacin-
induced gastritis. 
1.1.10 Delivery of IRX-2 
The sublesional route of administration of IRX-2 takes advantage of the normal afferent and 
efferent pathways of lymphatic drainage. Typically, lymphatics drain from an area of disease, such 
as a tumor bed, and antigens and other factors associated with disease migrate in the lymphatics to 
the regional nodes. By presenting the cytokine-containing biologic in the area of the CIN 3 and 
VIN 3, rather than systemically, there is an opportunity to directly mobilize and enhance the 
function of antigen presenting cells (APCs). The most important APCs in general are the dendritic 
cells. For the treatment of cervical neoplasia the Langerhans cells (LCs) are the members of the 
dendritic cell family that are most prominent in the mucosa of the urogenital tract; a variety of 
dendritic cell types may be important for the treatment of vulvar dysplasia, since those lesions are 
anatomically at a transition area between vaginal mucosa and skin. Sublesional injection of IRX-2 
may also directly activate T cells to proliferate and become cytotoxic lymphocytes. Additionally, 
sublesional administration may be less toxic since the systemic cytokine drug concentration is 
much lower. 
1.2 
Clinical Experience with the IRX-2 Regimen  
1.2.1 Phase 1 Trials 
The IRX-2 Regimen has been evaluated in two Phase I trials, one in patients with advanced 
HNSCC and one in patients with early stage cervical cancer.  
A Phase 1 trial in subjects with head and neck squamous cell carcinoma (HNSCC) who had 
progressed after surgery was undertaken to evaluate the clinical and laboratory safety and 
tolerability of the IRX-2 Regimen. Results of the study, which are presented in the IB and have 
been published [Freeman, 2010], indicated that the IRX-2 Regimen was well tolerated in these 
subjects. The reported toxicities were acceptable overall and did not preclude proceeding to 
additional trials. 
An earlier, pilot study of the IRX-2 Regimen was undertaken as neoadjuvant treatment in 10 
patients with untreated, resectable cervical cancer [Dueñas-Gonzalez, 2002]. IRX-2 was 
administered peri-lymphaticaly. All patients were scheduled for hysterectomy on day 21, although 
three patients opted not to proceed with surgery. Treatment was well tolerated with minor injection 
related pain and/or bleeding. Gastric symptoms were common, however, leading to substitution of 
ibuprofen for indomethacin in this study and the addition of omeprazole to subsequent studies of 
the IRX-2 Regimen. Clinical responses were seen in 5 of the 10 patients (80, 60, 50, 30 and 30% 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
32 
decreases in the product of longest lesion diameters). Pathological evaluation of the seven 
resection specimens showed tumor reduction and fragmentation in 5 patients and variable tumor 
infiltration of lymphocytes, plasma cells, neutrophils, macrophages and eosinophils. 
Immunohistochemical analysis of the surgical specimens showed an increase in tumor infiltrating 
CD8+ cells.  
1.2.2 Phase 2 Trials in Head & Neck Squamous Cell Carcinoma 
Two Phase 2 trials of neoadjuvant IRX-2 have been performed or are underway for the assessment 
of the IRX-2 Regimen in subjects with locally advanced HNSCC. 
The first study (IRX-2 2005 A) was a multicenter trial entitled “A Phase 2, Open-label Trial of the 
Safety and Biological Effect of Pre-operative Subcutaneous IRX-2 (with Cyclophosphamide, 
Indomethacin, and Zinc) in Subjects with Resectable Cancer of the Head and Neck.” Results of 
the study are presented in the IB, have been published [Wolf, 2011; Berinstein, 2012; Whiteside, 
2012], and are briefly summarized here. 
The second study is a multicenter, international trial entitled “A Randomized Phase 2 Trial of 
Neoadjuvant and Adjuvant Therapy with the IRX-2 Regimen in Patients with Newly Diagnosed 
Stage II, III or IVA Squamous Cell Carcinoma of the Oral Cavity” (NCT02609386). This trial 
designed to include 200 subjects in approximately 50 clinical sites opened for accrual in December, 
2015. 
1.2.2.1 Study IRX-2 2005-A: Design and Clinical Results 
The study objectives were to determine the safety of the IRX-2 Regimen when used as neoadjuvant 
(preoperative) therapy in a multi-center trial and to evaluate clinical, pathological, and 
radiographic tumor response and disease-free survival (DFS) and overall survival (OS). Of the 27 
subjects, 15 had oral cavity cancer, 8 had oropharyngeal cancer, 1 had hypopharyngeal cancer, and 
3 had laryngeal cancer.  
The IRX-2 Regimen used for this trial included cyclophosphamide, 300 mg/m2 on Day 1, 10 days 
of regional IRX-2 injections between days 4 and 15, and 21 days of oral indomethacin, zinc 
containing multivitamins and omeprazole. 
After 5 years of follow-up, only 7 of the 27 subjects had relapsed. DFS at 1, 2 and 3 years 
respectively was 72%, 64% and 62%; median DFS has not been reached. OS at 1, 2 and 3 years 
respectively was 92%, 73%, and 69%; median OS has not been reached. These results for both 
DFS and OS appeared to be slightly superior to those observed in a comparable group of 81 
historical controls, treated at the University of Michigan and matched for baseline characteristics 
[G. Wolf, personal data]. 
1.2.2.2 Study IRX-2 2005-A: Safety Results and Adverse Events 
The IRX-2 Regimen was tolerated with minimal toxicity. All subjects completed the regimen and 
there were no unplanned delays in surgery or difficulties with the planned resections and 
reconstructions as a result of the immunotherapy regimen.  
The most common adverse events (AEs) of the IRX-2 regimen (IRX-2, cyclophosphamide, 
indomethacin, omeprazole, zinc with multivitamins) were headache (30%), injection site pain 
(22%), nausea (22%), constipation (15%), dizziness (15%), fatigue (11%), aspiration pneumonia 
(11%), anemia (11%) and myalgia (7%). All were Grade 1-2 except for the aspiration pneumonias 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
33 
(one Grade 3, one Grade 4); and the aspiration pneumonias were related to the patient’s underlying 
head & neck malignancy. All AEs resolved without sequelae. There were only minor (Grade 1) 
alterations in post-treatment laboratory values. Of the 8 serious adverse events (SAEs) in 7 subjects 
reported during treatment and the 30-day post-operative period, none were ascribed to the IRX-2 
treatment except for a postoperative wound infection that was possibly related to the study 
treatment [Wolf, 2011]. 
1.2.2.3 Study IRX-2 2005-A: Immunologic Results 
Pretreatment tumor biopsies and the tumor surgical specimens were characterized for lymphocyte 
infiltration, 
necrosis 
and 
fibrosis 
using 
both 
hematoxylin 
and 
eosin 
stains 
and 
immunohistochemistry [Berinstein, 2012]. When lymphocyte infiltration in the pretreatment 
biopsies was compared to that in the resected surgical specimen, increases in lymphocyte 
infiltration were seen as shown in Figure 1 (change in mean lymphocyte infiltration from the 
biopsy to the surgical specimen is shown on the y-axis). 
 
Figure 1 
Lymphocyte Infiltration (Study IRX-2 2005-A) 
 
 
In addition, subjects in whom the greatest increase in tumor lymphocyte infiltration from biopsy 
to surgery (n=14) was observed had a trend toward superior survival compared to subjects in whom 
no or more limited change was observed (n=11) (p = 0.10) [Brooklyn ImmunoTherapeutics, 
unpublished observations]. 
Peripheral blood lymphocyte subsets also were monitored pre- and post-treatment with the IRX-2 
Regimen to evaluate changes induced by the IRX-2 Regimen (summarized in Section 1.1.8 above 
and in the IB). The IRX-2 Regimen-mediated reductions in B and NKT cell numbers in the blood 
suggested a redistribution of these cells to tissues while the unchanged numbers of Tregs after IRX-2 
therapy indicated that IRX-2 does not expand this compartment, potentially benefiting anti-tumor 
immune responses [Whiteside, 2012].  


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
34 
1.2.3 Phase 1/2 Cervical Cancer Study 
1.2.3.1 Clinical Response 
Of the 10 patients treated with perilymphatic, subcutaneous injections of IRX-2 and IRX-2 
Regimen, 3 were reported to have a partial response (>50% reduction in the product of the longest 
perpendicular tumor diameters) and 2 to have minor responses (<50% but >25% reduction in 
product of tumor diameters) [Dueñas-Gonzalez, 2002]. Regardless of the response observed by 
these criteria, tumors showed flattening with less bleeding and return of normal pain sensation and 
epithelial-like appearance. A relationship between clinical response and surgical or pathological 
findings was not evident.  
1.2.3.2 Pathological Response 
Five of the seven patients who underwent hysterectomy showed tumor fragmentation with variable 
patterns of leucocyte infiltration. Immunohistochemical studies showed an increase percentage of 
CD8+ T cells, and these cells appeared larger and more granular that before treatment. Peripheral 
blood T cell counts in patients with normal lymphocyte counts pretreatment did not change, but in 
two patients who had lymphopenia pretreatment, an increase in peripheral blood T cells was noted 
[Dueñas-Gonzalez, 2002]. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
35 
2 
STUDY OBJECTIVES  
2.1 
Primary Objective 
The primary objective is to compare the proportion of subjects who achieve a pathologic CR or 
PR at week 25, based on the resected surgical specimen.  
Each cohort (CIN and VIN) will be assessed independently. 
2.2 
Secondary Objectives 
1. To evaluate the toxicity and feasibility of administration of IRX-2 in subjects with 
confirmed CIN 3 or VIN 3. 
2. To evaluate multiple parameters to assess the activity of the IRX-2 Regimen for the 
treatment of CIN 3 or VIN 3: 
a. The occurrence of clinical CRs or PRs at weeks 6, 13 and 25 
b. Frequency of elimination of HPV in cervical or vulvar tissue using a commercial HPV 
genotyping assay and viral load determination by quantitative PCR. 
c. Analysis of the immune infiltrates in the resected surgical specimens. Parameters 
to be evaluated include quantitation of lymphocyte subset infiltration including 
CD8 and CD4, FOXP3+ cells, evaluation of PDL1 expression, evaluation of state 
of activation and location of infiltrating regions with respect to dysplasia and 
adjacent normal tissue. A commercial multiplex IHC assay will assess markers of 
Langerhans cells (CD1a, E-cadherin) and antigen presenting cell (APC) activation 
(CD80 and CD86). TCR clonality studies may also be performed. 
d. Immunophenotypic analysis of peripheral blood lymphocytes. Peripheral blood 
lymphocytes will be evaluated by direct ex-vivo ELISPOT analysis for evidence of 
induction of γ-Interferon expressing T cells in response to HPV antigens compared 
to baseline. Changes in naïve, memory, effector and regulatory T cell subset will 
be analyzed using immunophenotypic flow cytometry. 
e. Frequency of serum antibodies to HPV E6, E7 and L1 proteins by ELISA. Serum 
cytokine profile will be analyzed by multiplex ELISA at baseline and week 13 
(pending availability of the antibodies).  
f. RNA expression profiling of immune-inflammatory markers from post-treatment 
resected surgical specimens. 
g. Frequency of long term HPV clearance following surgical excision. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
36 
3 
INVESTIGATIONAL PLAN 
3.1 
Study Design  
This is a Phase 2, open-label study of the IRX-2 Regimen in subjects with CIN 3 or VIN 3. Subjects 
with CIN 3 will constitute one cohort (“CIN Cohort”) and subjects with VIN 3 will constitute a 
second cohort (“VIN Cohort”).  
All subjects are to receive standard of care evaluation of lower genital tract dysplasia, including 
comprehensive colposcopy of the lower genital tract with diagnostic biopsies as clinically 
indicated. Only women with CIN 3 or VIN 3 who would ordinarily be then managed with an 
excisional procedure are eligible for this trial (see Section 3.2 for complete inclusion and exclusion 
criteria). Following the 12 weeks (two cycles) of study treatment all subjects will receive a standard 
of care excision of the dysplastic lesion involving the cervix or vulva. 
For women with cervical dysplasia (the CIN 3 cohort) this usually requires a loop electrosurgical 
excision procedure (LEEP) of the cervix, with the depth and breadth of the excised tissue based 
on the geographic extent of dysplasia as determined by the treating physician. A cervical 
conization procedure (cone biopsy) can also be performed, again at the discretion of the treating 
physician. 
For women in the VIN 3 cohort a wide local excision (partial vulvectomy) will be performed, with 
the extent of the surgical resection determined by the treating physician based on the extent of the 
dysplastic lesion. If no lesion is visible on exam, a biopsy will be taken of the original lesion site 
to confirm complete pathological response. 
The excisional procedures for subjects in either the CIN or VIN cohort can be done in an office 
setting, an ambulatory surgery center, or a hospital operating room based on subject and physician 
preference. 
These non-investigational, standard of care colposcopies, biopsies, excisions and related 
evaluations and supportive care are reasonable and medically necessary for the clinical 
management of the subject.  
Adverse events (AEs) will be assessed daily beginning with the first dose of cyclophosphamide 
until the week 25 surgical excision. Ongoing AE/SAEs will be reported and monitored until 
resolution (See Table 2 in Section 5) and classified according to the Common Terminology Criteria 
for Adverse Events (CTCAE) v4. AEs and SAEs will be tabulated by investigators as definitely, 
possibly or not related to the study regimen. For further details on AE and SAE data collection, 
see Section 9. 
The flow chart presented in Figure 2 illustrates the study design for both the CIN and VIN cohorts. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
37 
Figure 2 
Flow Chart of Study Design 
  
3.2 
Selection of Study Population 
The subject population will consist of 32 subjects with histologically confirmed squamous CIN 3 
or VIN 3 who meet the following inclusion and exclusion criteria. Up to 40 subjects may be 
enrolled to ensure 32 evaluable subjects. 
3.2.1 Inclusion Criteria 
To be enrolled in the study, subjects must meet the following inclusion criteria: 
1. Histologically confirmed squamous CIN 3, or VIN 3 (usual type only).  
2. Female aged ≥25 years. 
3. The subject is either surgically sterile, postmenopausal, or agrees to practice an effective 
method of birth control as determined by the investigator (to be continued for one year 
following last dose of study medication), except that subjects with CIN 3 are not permitted 
to use a cervical cap or diaphragm for contraception. 
4. The subject is judged to be in good health based upon the results of a medical history, 
physical examination, vital signs, and laboratory profile including: 
Hematology:  
• White blood cell >2,500/ mcL (>2.5 x 109/L)  
• Absolute neutrophil count >1,000/ mcL (>1 x 109/L) 
• Platelet count >75,000/ mcL (>75 x 109/L) 
• Hemoglobin ≥8 g/dL (≥80 g/L) (subjects who have received a transfusion or 
erythropoietin up to one week prior to receiving the first dose of cyclophosphamide 
are eligible for the study) 
Clotting Time: 
• International normalized ration (INR) or prothrombin time (PT) ≤1.5 x ULN (upper 
limit of normal) 
• Activated partial thromboplastin time (aPTT) ≤1.5 x ULN 
Renal Function: 
• Serum creatinine ≤1.5 x ULN  
Hepatic function: 
• Total bilirubin ≤2.0 x ULN unless thought to be related to inherited bilirubin 
conjugation disorder (ie Gilbert’s disease) 
• ALT and AST ≤2.0 x ULN 
5. The subject is geographically accessible for ongoing follow-up and is committed to comply 
with the designated visits. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
38 
6. The subject is capable of understanding and complying with the protocol and has signed 
the enrollment informed consent form at screening. 
3.2.2 Exclusion Criteria 
Subjects who meet any of the following criteria will be excluded from the study: 
1. For subjects with cervical dysplasia: evidence of atypical glandular cells or 
adenocarcinoma in situ (ACIS) based on cervical cytology, colposcopy or biopsy. 
2. For subjects with either cervical or vulvar squamous dysplasia: evidence of microinvasive 
squamous carcinoma based on cytology, colposcopy or biopsy. 
3. For VIN subjects, those with >2 vulvar lesions. 
4. Pregnancy or lactation. 
5. Allergy to ciprofloxacin or other quinolones (because ciprofloxacin is used in preparation 
of IRX-2.) 
6. Allergy to indomethacin (a necessary component of the regimen) or to acetylsalicylic acid 
(aspirin) due to likely allergy cross-reaction. 
7. Aldara (imiquimod) for the topical treatment of lower genital tract warts or dysplasia within 
3 months of study enrollment. 
8. Known to be positive for human immunodeficiency virus-1 (HIV-1) antibody, human 
immunodeficiency virus-2 (HIV-2) antibody, hepatitis B surface antigen, or hepatitis C 
virus antibody. 
9. Known to have other immunodeficiency diseases, including cellular immunodeficiencies, 
hypogammaglobulinemia, or dysgammaglobulinemia. 
10. Immunotherapy (eg, interferons, tumor necrosis factor, interleukins) or biological response 
modifiers (granulocyte-macrophage colony-stimulating factor, granulocyte colony-
stimulating factor, macrophage colony-stimulating factor) or any investigational drug 
within 3 months of study enrollment. 
11. Concurrent treatment with systemic corticosteroids at a dose of ≥5 mg/day of prednisone 
(or equivalent). 
12. Subjects should not take aspirin (except for low-dose aspirin as prescribed for vascular 
disease) or other non-prescribed, non-steroidal anti-inflammatory agents from start of 
treatment to surgery. 
13. An infectious process or any other significant illness such as an autoimmune disease or 
advanced age that in the opinion of the investigator would compromise the subject’s ability 
to mount an immune response. 
14. Active SARS-CoV2 infection or COVID-19 disease 
15. Impaired hepatic, renal or hematological function, evidenced by: 
a. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), or total 
bilirubin >2 times upper limit of normal (ULN), 
b. Serum creatinine >1.5 times ULN, or 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
39 
16. Clinically significant active cardiovascular disease, including a history of myocardial 
infarction within the past 6 months, heart failure as defined by New York Heart Association 
classes III or IV, and/or blood pressure greater than 160/90 mm Hg (1 repeat measure 
allowed no more than 5 minutes after the first measurement). 
17. History of severe allergic reaction to insect bites or stings, or to any biologic 
pharmaceutical product, including compounds similar to the test article. 
18. Any medical contraindications, allergies or previous therapy that would preclude treatment 
with the components of the IRX-2 Regimen, i.e., cyclophosphamide, indomethacin, zinc-
containing multivitamins or omeprazole. 
19. Donation or loss of >450 mL of blood or plasma within 30 days of start of treatment. 
3.3 
Assessment of Dysplastic Lesion and Surgical Resectability  
For women with CIN, a colposcopic examination will be performed after application of 5% acetic 
acid to the cervix. The site (based on a 1-12 o’clock system) and appearance of each suspicious 
lesion (colpsocopic features suggestive of CIN 3 include acetowhitening and the presence of 
specific vascular patterns such as punctate vessels or mosaicism) will be documented. A rebiopsy 
will not be performed unless the visualized lesions have features suggestive of invasive cancer, 
such as presence of a gross lesion. A determination will be made as to the “adequacy” of the 
colpsocopic examination (whether the entirety of each lesion can be seen and as to whether the 
entirety of the squamocolumnar junction can be seen). A photograph will be taken with the 
attached camera at each colposcopic evaluation. At the prescribed time, an excisional procedure 
(either a large loop excision of the transformation zone for women who have had an adequate 
colposcopy and negative ECC) or a conization biopsy (to be performed either via LEEP technique 
or via the cold knife technique, depending on the judgment of the surgeon as to whether the patient 
requires general anesthesia for the procedure for women with a positive ECC or in whom the 
colposcopy is not adequate) will be performed. 
For women with VIN 3, the site of all lesions based on which anatomical portion of the vulva the 
lesion is involving, as well as the size of each lesion, measured with a ruler will be recorded. The 
vulva will be photographed at each vulvar examination. At the proscribed time, either a wide local 
excision or biopsy of each residual lesion followed by laser ablation will be performed, depending 
on the number and sites of residual lesions. If a lesion is no longer visible at a site where a lesion 
was seen at study entry, a punch biopsy will be taken of the area to assess for pathologic complete 
response.  
3.4 
Termination of Study Treatment 
The investigator can stop the study treatment regimen due to an allergic or hypersensitivity 
reaction to the study drug, an SAE or clinically significant AE or laboratory abnormality or for 
protocol noncompliance. If a subject is discontinued from the study due to an AE, the investigator 
should notify Brooklyn ImmunoTherapeutics within 24 hours of the event. Regardless of the 
reason for study treatment discontinuation, the investigator must complete the Study Completion 
Form in the Case Report Form (CRF), specifying the reason. Subjects should continue follow-up 
visits and be monitored for recurrence and survival, unless the subject withdraws consent for 
follow-up.  


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
40 
NOTE: Subjects may experience changes in injection sites or regional lymph nodes during 
treatment with the IRX-2 Regimen, including inflammation, necrosis, or change in 
consistency. These changes should not be mistaken for malignant transformation or 
progression. 
3.5 
Withdrawal of Consent 
Any subject may terminate participation in the study at any time but every effort should be made 
to continue with study treatment and evaluations. If a subject elects to discontinue study 
participation at any time for safety, medical or personal reasons, the investigator should make a 
reasonable effort to determine the reason for the subject’s withdrawal and document the reason on 
the CRF. 
An attempt will be made, unless consent for follow-up is specifically withdrawn, to follow all 
subjects per study protocol, with emphasis on ensuring the subject does undergo the necessary 
surgical excision of the dysplastic lesion (e.g. cervical LEEP or cold knife conization or vulvar 
WLE). 
Any clinically significant AEs or SAEs leading to premature withdrawal are to be followed until 
resolution. 
3.6 
Medical Monitoring 
3.6.1 Medical Monitors 
The Medical Monitors are physicians trained in the conduct of clinical trials who are available to 
the clinical investigators for discussion of questions regarding the inclusion/exclusion criteria and 
any other medical issues that may arise.  
3.6.2 Principal Investigator(s) 
The Study Principal Investigator or his designee should be contacted to discuss questions related 
to enrollment, subject assessment, surgical resectability, and optimal technique for the cervical 
LEEP or vulvar WLE, and any other details of the study. 
Principal Investigator: 
Lynda Roman, MD 
Contact information: 
(213) 919-0209 (Pager) 
 
  
3.7 
Data Safety Monitoring 
 
The Study Team (PI, Co-PI’s, and Study Statistician) will meet at least monthly and as needed by 
teleconference to review the progress of the trial as well as all AE’s.  Minutes will be taken at each 
meeting and circulated to the Study Team.  
Should a decision be made that toxicities and adverse events due to the IRX-2 regimen are too 
frequent or unexpectedly severe, the Study Team will propose modifications to the regimen 
beyond those specified in Section 5.2.9 and if appropriate, the protocol will be formally amended.   
Section 8.1 describes the technical aspects of the proposed safety monitoring. 
The toxicities, study compliance and progress, will be reviewed every 6 months by the USC/Norris 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
41 
Comprehensive Cancer Center DSMB.  This DSMB will review the actions and modifications 
taken by the Study Team in response to issues that have arisen during the conduct of the trial. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
42 
4 
STUDY TREATMENTS 
4.1 
Study Design  
Subjects who meet all eligibility criteria will receive the IRX Regimen.  
4.2 
IRX-2 Regimen 
In this trial, the IRX-2 Regimen (as shown in Table 1, below) will include cyclophosphamide 3 
days before the start of IRX-2, IRX-2 daily x 4 consecutive days, and indomethacin, zinc with 
multivitamins, and a proton pump inhibitor for the first 21 days of each six week cycle. 
All treatments will be repeated beginning on Week 7, for a total of two cycles. 
 
Table 1 
IRX-2 Regimen 
Agent 
Dose 
Route of 
Administration 
Treatment Days 
(each cycle) 
Cycle = 6 weeks 
Cyclophosphamide 
300 mg/m2 
IV 
Day 1 
IRX-2 
230 units daily 
(in 2 ml) 
in 4 divided doses: 
57.5 units (1/2 ml)  
in each quadrant of the 
cervix or affected areas 
of the vulva 
Submucosal injection 
in the cervix; 
subcutaneous for 
vulvar lesions 
Days 4-7 
Indomethacin 
25 mg TID 
Oral 
Days 1-21 
Zinc with multivitamins  
1 tab daily 
Oral 
Days 1-21 
Omeprazole 
20 mg daily 
Oral 
Days 1-21 
   
All IRX-2 preparations will be prepared by the pharmacist or designated staff.  The IRX-2 will be 
transferred from their sterile vials to syringes provided by Brooklyn ImmunoTherapeutics.  
4.3 
Route of Administration 
For cervical dysplasia: IRX-2 will be administered submucosally in the cervix with four divided 
doses into each quadrant of the cervix, irrespective of location of the CIN 3 lesion. 
In women with CIN 3, 2.0 cc of study drug will be drawn into one 3 cc syringe,  which will be 
attached to a 22 gauge spinal needle (9cm length). 0.5 cc of study drug will be injected into the 
submucosa of each of the 4 quadrants of the cervix.  
For vulvar dysplasia:  In women with VIN 3, 2.0 cc of study drug will be drawn into a 3 cc syringe 
which will then be attached to a 25 gauge needle. 0.5 cc of study drug will be injected at 12, 3, 6 
and 9 o'clock at the borders of a single lesion, into the subcutaneous tissue beneath the lesion. For 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
43 
>1 lesion, doses shall be divided at clinician discretion based on the geometry and size of the 
dysplastic lesions.  
4.4 
Regimen Medications 
4.4.1 IRX-2 
IRX-2 is supplied as a pale yellow, sterile liquid for subcutaneous injection in 2.0 mL single-dose 
vials. IRX-2 must be stored in a secure area and maintained under labeled storage conditions at a 
range of −15o C to –50o C. All study supply containers will be appropriately labeled to identify 
study number, kit numbers, lot number, and product identity. Study syringes for subcutaneous 
study drug administration will be disposed of in accordance with biosafety procedures. Empty 
study drug vials will be retained for accountability and may be disposed of on-site following drug 
reconciliation. 
4.4.2 Other Medications 
Cyclophosphamide, indomethacin, zinc with multivitamins and omeprazole shall be sourced 
locally for each study subject.  
4.5 
Method of Assigning Subject Numbers 
After a subject has signed the informed consent form and has been deemed eligible for enrollment, 
the subject will be assigned a 3-digit enrollment numbering (starting with 001) such that all 
subjects are given consecutive identification numbers in successive order of enrollment.   
4.6 
Treatment Compliance 
The investigator or other study personnel will administer study drug injections to ensure treatment 
compliance. Subjects should be asked each day that they are seen about compliance with oral 
medications and compliance (or lack thereof) documented in the clinical record. 
4.7 
Prohibited, Prior and Concomitant Treatments 
Subjects, from start of treatment to completion of Cycle 2, should not take aspirin (except for low-
dose aspirin as prescribed for vascular disease) or other non-prescribed, non-steroidal anti-
inflammatory agents.  
All other concomitant prescribed medications, recognized over-the-counter medications, or any 
changes in medications during the study from start of treatment to the 25th week excisional 
procedure will be recorded. Medications given to treat a reported AE or SAE will continue to be 
recorded until the AE/SAE resolves. 
5 
STUDY PROCEDURES AND SCHEDULE 
5.1 
Study Schedule 
The Schedule of Study Procedures is outlined in Table 2 and Table 3. 
Tests and procedures should be performed on days indicated. Variation of ±3 days for tests 
indicated in “days”, 1 week for tests indicated in “weeks” and 2 weeks for tests indicated in months 
is permitted. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
44 
See also Appendix 4 for the optimal schedule for investigators at USC. For subjects at that 
institution, the “Day 1” cyclophosphamide is best infused on a Thursday or Friday; the subsequent 
IRX-2 injections are performed on the subsequent Monday through Thursday or Tuesday to Friday 
(Days 4, 5, 6, 7 or Days 5, 6, 7, 8). 
 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
45 
Table 2  
Schedule of Events 
Study Procedure 
Screening 
(Baseline) 
Treatment Cycles 
 
 
Surgical 
Resection 
(LEEP or 
wide local 
excision) 
Post-Op 
Checki 
 
Cycle 1: 1st 6-weeks 
Cycle 2: 2nd 6-weeks 
 
Pre-
procedure 
visit 
4-
month 
post-op 
Days 
−30 to 0 
Day(s) of Cycle 1 
Days(s) of Cycle 2 
 
 
Week 
 25 
Weeks 
26+ 
 
1 
2 
3 
4 
5 
6 
7 
>8 
1 
2 
3 
4 
5 
6 
7 
>8 
Week 13 
safety 
visit  
Week 
21 
Week 
41 
Day(s) of Protocol 
 
1 
2 
3 
4 
5 
6 
7 
8-
21 
22-
42 
43 44 
45 
46 
47 
48 
49 
50-
63 
64-
84 
91 
Day 147 
± 14 days 
Day 175 
 ± 7 daysg 
 
Day 290 
± 14 
days 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Study Drug Administration 
 
Cyclophosphamide IVa 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Indomethacin, omeprazole, zinc 
with multivitaminsb 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
IRX-2 injectionb 
 
 
 
 
X 
X 
X 
X 
 
 
 
 
X 
X 
X 
X 
 
 
 
 
 
 
 
Procedures & Evaluations 
 
Complete History & Physical 
Exam 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
X 
 
 
 
HIV-1, HIV-2, HBV, HCV 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SARS-CoV2 
 
Xc 
 
 
 
 
 
 
 
Xc 
 
 
 
 
 
 
 
 
 
 
 
 
Focused pelvic exam 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Xj 
X 
Injection Site Check 
 
 
 
 
X 
 
 
X 
 
 
 
 
X 
 
 
X 
 
X 
X 
 
 
 
ECOG Performance Status 
X 
X 
 
 
 
 
 
X 
 
X 
 
 
 
 
 
X 
 
X 
X 
 
X 
X 
AEs and SAEsd 
 
X 
 
 
X 
X 
X 
X 
 
X 
 
 
X 
X 
X 
X 
 
X 
X 
 
X 
X 
Concomitant Medications 
X 
X 
 
 
X 
X 
X 
X 
 
X 
 
 
X 
X 
X 
X 
 
X 
X 
 
X 
X 
Chemistry, CBC, PT/INR 
X 
Xe 
 
 
 
 
 
 
 
Xe 
 
 
 
 
 
 
 
Xe 
Xe 
 
 
 
Blood work for investigational 
studies 
Xe 
Xe 
 
 
 
 
 
 
 
Xe 
 
 
 
 
 
 
 
Xe 
Xe 
 
 
 
Blood for PK analysish 
 
 
 
 
X 
X 
X 
X 
 
 
 
 
X 
X 
X 
X 
 
 
 
 
 
 
Pregnancy Testf 
X 
Xf 
 
 
 
 
 
 
 
Xf 
 
 
 
 
 
 
 
Xf 
Xf 
 
 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
46 
Colposcopy & cervix photography 
(CIN patients) 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
X 
X 
 
 
 
Cervical inspection & 
photography (CIN patients) 
 
 
 
 
XL 
 
 
XL 
 
 
 
 
XL 
 
 
XL 
 
 
 
 
 
 
Cervical cytology (pap 
smear/ECC)(CIN patients) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
 
 
X 
Vulvar inspection & photography 
(VIN patients) 
X 
 
 
 
XL 
 
 
XL 
 
X 
 
 
XL 
 
 
XL 
 
X 
X 
 
 
 
Cervical or vulvar swab for HPV 
assay (research) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
 
 
X 
Cervical or vulvar biopsy 
Xk 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Cervical LEEP excision or cone 
biopsy or vulvar excision or 
biopsy 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Footnotes for Table 2:  
a. Dexamethasone or other steroids should not be used as an anti-emetic with the cyclophosphamide. Steroid administration may inhibit IRX-2 Regimen 
stimulation of an immune response. The prescribed dose is significantly less than used for conventional chemotherapy and thus steroids will not be needed. 
Suggested antiemetic regimen would include a serotonin (5-HT3) antagonist and lorazepam. 
b. Subjects will receive injection of either study agent IRX-2 daily on Days 4, 5, 6, 7 or Days 5, 6, 7, 8. The indomethacin, omeprazole, zinc with multivitamins 
are administered for the first 3 weeks per cycle, on Days 1-21. For cycle 2 [Week 7] the comparable “Days” are shown in the table. 
c. 
SARS-CoV2 virus test required within 2-4 days prior to start of Cycle 1 and Cycle 2. If positive, treatment will be held. 
d. Adverse events (AEs) and serious adverse events (SAEs) are measured from day of Cycle 1 Day 1 until Week 25 of the study. Ongoing AE/SAEs will be 
reported and monitored until resolution. 
e. 
Ten ACD yellow top blood tubes (85 mL) and one red top serum tube should be drawn for investigational studies. Blood work can be done up to seven 
days prior to the specified day. Blood draws do not need to be repeated on indicated day if drawn within the previous 7 days. 
f. 
Pregnancy must be ruled out, or post-menopausal status verified, at study enrollment. Fertile subjects will need effective contraception during the study and 
for 1 year following last dose of study medication; investigator to repeat pregnancy tests during the study if needed for verification. A blood pregnancy test 
should be used at screening, week 7 and week 25. A urine pregnancy test can be performed on Cycle 1 day 1 visit, and at week 13 safety visit.  
g. The “Week 25” lesion excision is to be scheduled for Day 175, +/- 7 days.  
h. One red top serum tube (5 mL) required at each of the following time points: Day 4 pre-injection, 30 minutes, 1 hour, 2 hours post-injection; Day 5 pre-
injection; Day 6 pre-injection; Day 7 pre-injection, and then 30 minutes, 1 hour, 2 hours post-injection. All time points allow +/- 5 minutes. 
i. Post-op check to be performed 1 to 8 weeks post-surgery; can occur by phone or office visit. 
j. If seen for office visit. 
k. If previous biopsy not done within past 60 days, or if slides/block from this are not available, or at discretion of investigator based on clinical impression.  
l. 
Photography to be performed prior to injection of study drug. 
 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
47 
Table 3:  Schedule of Virologic and Immune Monitoring (All Subjects) Based on Blood, Swab or Tissue Samples 
Study Procedure 
Screening 
(Baseline) 
Treatment Cycles 
Safety 
visit 
Surgical 
Excision 
4-month 
follow-up 
Cycle 1 
Cycle 2 
Week 13 
Week 25 
Week 41 
Days 
-30 to 0 
Day 1 
Day 43 
(Cycle 2, Day 1) 
Day 91 
Day 175 
 ± 7 days 
Day 290 
 ± 14 days 
Presence or Absence of HPVc 
X 
 
 
 
X 
X 
HPV Genotypingc 
X 
 
 
 
X 
X 
Flow Cytometryc 
X 
Xa,b 
Xb 
Xb 
Xb 
 
ELISPOTc 
X 
Xa,b 
Xb 
Xb 
Xb 
 
ELISAc 
X 
Xa,b 
Xb 
Xb 
Xb 
 
Phenotypic analysis of lymphocytic infiltrated 
X 
 
 
 
X 
 
a. 
Cycle 1, Day 1 labs do not need to be repeated if the Screening (Baseline) labs were drawn within four days. For subject convenience, investigator should 
synchronize these phlebotomies with the blood draws specified in Table 1. 
b. Blood work for ELISpot, ELISA and flow cytometry can be collected up to four days prior to the specified day (e.g. labs can be obtained on M-T-W-Th 
before a Friday treatment day). 
c. HPV genotyping, flow cytometry, ELISPOT, and ELISA assays will be performed in the Norris Comprehensive Cancer Center Immune Monitoring Core 
laboratory. 
d. Tissue immunohistochemistry and PK studies will be done by Brooklyn ImmunoTherapeutics via a third party. 
 
 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
48 
 
5.2 
Study Procedures  
5.2.1 Screening  
The purpose and procedures of the study will be fully explained to participants. Those wishing to 
enroll in the study will sign a written informed consent prior to initiating any protocol specific 
evaluations or procedures. 
The following screening evaluations are to be performed within 30 days prior to beginning 
treatment: 
• History of present illness, including recording of prior cytology and histology results, and 
collection of corresponding laboratory reports with subject identifiers and accession numbers 
to allow retrieval of archived specimens for comparative studies.  
• Medical history, including assessment of all entry and exclusionary criteria, demographics 
(age, sex, race, menstrual status, weight, height), history of hypersensitivity (drug allergies), 
general medical history, and assessment of all current symptoms including severity. 
• History and current use of tobacco and/or alcohol. 
• Determination of ECOG performance status (Appendix 3). 
• Concomitant medication review. 
• General physical examination, including vital signs (blood pressure and pulse). 
• Detailed pelvic examination, including: 
o Cervical swab for HPV status and genotyping for investigational studies; 
o Colposcopy of the lower genital tract and detailed description of the dysplastic lesion(s); 
o Photography of the dysplastic lesion(s) 
o Biopsy of dysplastic lesion(s), if not done at time of initial diagnosis; if a biopsy has been 
done previously within the past 60 days, it is not necessary, nor desirable, to repeat it. 
• Clinical laboratory assessments: 
o Hematology: complete blood count (CBC), differential, platelet count, PT and PTT. 
o Serum chemistry: serum albumin, total protein, serum bilirubin, lactate dehydrogenase 
(LDH), ALT/SGPT, AST/SGOT, alkaline phosphatase, creatinine, blood urea nitrogen 
(BUN), glucose, and electrolytes. 
• Pregnancy test (serum) <OR> confirmation subject is post-menopausal (absence of menses for 
> six months and LH or FSH in menopausal range). 
• Collect additional blood samples for investigational correlative studies, including assessment 
of the immunogenicity of IRX-2. 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
49 
All of the above screening evaluations, except for the collection of blood samples for 
investigational correlative studies, are reasonable and medically necessary for the direct clinical 
management of the subject, and thus should be appropriate for reimbursement by insurance 
providers [Martin, 2014]. 
5.2.2 Cycle 1, Day 1     
The following procedures will be completed at this visit: 
• Review documentation of history, physical exam, and laboratory information obtained during 
screening assessment. 
• Review results of SARS-CoV2 viral RNA test administered within the prior 2-4 days. A 
positive test will result in delay of study drug administration until infection clears. 
• Interval history and assessment of all current symptoms (AEs) including severity. 
• ECOG performance status. 
• Record concomitant medications. 
• Repeat the following laboratory assessments if more than 3 weeks since screening values 
obtained. 
o Hematology: CBC, differential, platelet count, PT and PTT. 
o Serum chemistry: serum albumin, total protein, serum bilirubin, LDH, ALT/SGPT, 
AST/SGOT, creatinine, BUN, glucose, and electrolytes. 
• Verification premenopausal subjects are not pregnant (by history and/or laboratory studies at 
discretion of investigator. Urine pregnancy test is acceptable). 
• Collect blood samples for investigational correlative studies, including assessment of the 
immunogenicity of IRX-2 (if not collected at screening visit). 
• Administer the following medications: 
o Infusion of cyclophosphamide, 300 mg/m2, over a one hour period. 
o Steroids must not be administered as anti-emetic agents since these might interfere with 
immunomodulation by IRX-2. Antiemetic regimen should include a serotonin (5-HT3) 
antagonist and lorazepam or equivalent medications. 
o Begin indomethacin (25 mg, approx. q 8 hours [TID]) to be taken orally after meals from 
Day 1 through Day 21. 
o Begin zinc (24 mg, once daily) with multivitamins to be taken orally from Day 1 through 
Day 21. 
o Begin omeprazole (20 mg, once daily) to be taken orally after meals from Day 1 through 
Day 21. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
50 
5.2.3 Cycle 1, Days 4 to 7 or Days 5 to 8 
On Treatment Days 4 to 7 (or Days 5 to 8), the investigational pharmacist will supply the clinical 
investigators with the pre-filled syringes containing 2.0 ml of IRX-2 solution. The solution will be 
administered by injection, as follows:  
• Total daily dose of the IRX-2 is 230 units, contained in 2 ml of solution. 
• For the CIN 3 cohort: The solution should be administered in four divided doses into the 
cervical mucosa of each quadrant of the cervix (0.5 ml, or 57.5 U, per injection).  
• For the VIN 3 cohort: The solution should be administered into the subcutaneous tissue beneath 
the dysplastic lesion(s) in divided doses. The number and distribution of the injections is 
deferred to the investigator based on the size and geometry of the vulvar lesion. The 
investigator will record this information, aided by diagrams and/or photographs. 
• On each injection day, subjects will be monitored for at least 15 minutes after the injections 
for signs or symptoms of any reaction. 
  
On the Day 4 visit (the first day of solution injections), the following evaluations will be performed 
and documented: 
• Interval history and assessment of all current symptoms including severity (AEs); 
o 
Irrespective of scheduled days for AE assessment, any information regarding AEs 
received by clinicians caring for the subject will be documented and the study site 
personnel informed. 
• Record concomitant medications; 
• Evaluation of injection sites; 
• Photography of the lesion site obtained before the injection of IRX-2; 
• Blood will be drawn pre-injection, followed by 30 minutes, 1 hour, and 2 hours after 
injection for pharmacokinetic analysis. All time points allow +/- 5 minutes. 
 
 
On the Day 5 and Day 6 visits (the second and third day of solution injections), the following 
evaluations will be performed and documented: 
• Interval history and assessment of all current symptoms including severity (AEs); 
o 
Irrespective of scheduled days for AE assessment, any information regarding AEs 
received by clinicians caring for the subject will be documented and the study site 
personnel informed. 
• Record concomitant medications; 
• Evaluation of injection sites; 
• Blood will be drawn pre-injection for pharmacokinetic analysis 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
51 
On the Day 7 visit (the fourth day of solution injections), the following evaluations will be 
performed and documented: 
• Interval history and assessment of all current symptoms including severity (AEs); 
o 
Irrespective of scheduled days for AE assessment, any information regarding AEs 
received by clinicians caring for the subject will be documented and the study site 
personnel informed. 
• Record concomitant medications; 
• Evaluation of injection sites;  
• Photography of the lesion site obtained before the injection of IRX-2; 
• Blood will be drawn pre-injection, followed by 30 minutes, 1 hour, and 2 hours after 
injection for pharmacokinetic analysis. All time points allow +/- 5 minutes. 
• Any missed doses due to reasons other than toxicity should be administered as soon as 
possible within 10 days from the first injection, not to exceed one injection dose per day. If 
ten days has elapsed from initial IRX-2 injection, patient should continue being monitored 
until the next scheduled study visit. Missed doses should be documented on case report 
forms. 
5.2.4 Cycle 1, Days 8 to 21 
Subjects continue the indomethacin, omeprazole, zinc with multivitamins regimen. 
5.2.5 Cycle 2, Day 1 (Beginning of Week 7; Day 43 of Protocol) 
 
Subjects return at the start of Week 7 to begin the second of 2 cycles of treatment. Day 43 is almost 
identical to Day 1, as detailed below: 
• Review documentation of history, physical exam, and laboratory information obtained during 
screening assessment and during the first cycle of treatment. 
• Review results of SARS-CoV2 viral RNA test administered within the prior 2-4 days. A 
positive test will result in delay of study drug administration until infection clears. 
• Interval history and assessment of all current symptoms (AEs) including severity. 
• ECOG performance status. 
• Record concomitant medications. 
• Brief general physical examination including weight and vital signs (blood pressure and pulse). 
• Clinical laboratory assessments: 
o Hematology: CBC, differential, platelet count, PT and PTT. 
o Serum chemistry: serum albumin, total protein, serum bilirubin, LDH, ALT/SGPT, 
AST/SGOT, creatinine, BUN, glucose, and electrolytes. 
• Pregnancy test (serum or urine) <OR> confirmation subject is post-menopausal (absence of 
menses for > six months and LH or FSH in menopausal range). 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
52 
• Collect blood samples for investigational correlative studies, including assessment of the 
immunogenicity of IRX-2. 
• Administer the following medications: 
o Infusion of cyclophosphamide, 300 mg/m2, over a one hour period. 
o Steroids must not be administered as anti-emetic agents since these might interfere with 
immunomodulation by IRX-2. Antiemetic regimen should include a serotonin (5-HT3) 
antagonist and lorazepam or equivalent medications. 
o Begin indomethacin (25 mg, approx. q 8 hours (TID)) to be taken orally after meals from 
Day 43 through Day 49 (Cycle 2, days 1-21). 
o Begin zinc (24 mg, once daily) with multivitamins to be taken orally from Day 43 through 
Day 49 (Cycle 2, days 1-21). 
o Begin omeprazole (20 mg, once daily) to be taken orally after meals from Day 43 through 
Day 49 (Cycle 2, days 1-21). 
5.2.6 Cycle 2, Days 4 to 7 (Continuation of Week 7, or days 46 to 49 of Protocol) 
During these days the subjects receive the second series of solution injections.  
Procedures are identical to Cycle 1, Days 4-7 detailed in Section 5.2.3 (above). 
5.2.7 Cycle 2, Days 8 to 21 (Days 50 to 63 of Protocol) 
Subjects continue the indomethacin, omeprazole, zinc with multivitamins regimen. 
5.2.8 Management of Toxicity Related to IRX-2 Regimen. 
In the event of Grade 3 or higher toxicity, no further IRX-2 should be injected and indomethacin 
should be discontinued for that Cycle. Should the toxicity occur during Cycle 1 and if the subject 
has fully recovered, Cycle 2 may be given at full or reduced doses as specified below.   In the event 
of Grade 2 toxicity, treatment should be held until stable and then resumed per protocol, but if 
treatment must be delayed for more than 7 days that Cycle should be discontinued, except that 
omeprazole and zinc with multivitamins can be continued at the discretion of the investigator. 
For subjects with toxicity most likely related to indomethacin (e.g. gastrointestinal intolerance), 
the indomethacin may be discontinued or the dose reduced and the rest of the protocol treatment 
continued. 
For subjects with toxicity most likely related to cyclophosphamide, including myelosuppression 
or hematuria, the Cycle 2 dose should be reduced by 50%.  For cyclophosphamide related 
unacceptable nausea or for emesis, the antiemetic regimen should be enhanced by adding a 
neurokinin-1 antagonist or olanzapine, but corticosteroids should not be administered. 
For subjects with Grade 2 or greater local toxicity most likely related to the injections of IRX-2, 
the details of administration, as specified in Section 4.3, may be modified at the discretion of the 
investigator, e.g. by increasing or decreasing the number of injections or the volume per injection 
(not to exceed 2 mL), or the total dose of IRX-2 may be decreased by 50%. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
53 
5.2.9 Week 13: Safety Assessment 
Subjects will return on week 13 for a safety assessment. The purpose of this visit will be to 
assess the well-being of the subject including performance status, adverse event profile and 
injection site reactions. In addition, the dysplastic lesion will be assessed either by direct visual 
inspection and photography or by colposcopy. The status of the lesion will be determined and 
regression will be documented. If there is evidence of progression consideration will be given to 
performing the surgical excision at this time (see Sections 5.2.11 and 5.2.12). Exploratory assays 
from the blood will be performed at this timepoint. 
• Review documentation of history, and laboratory information obtained while on study. 
• Complete physical. 
• Assessment of all current symptoms (AEs) including severity. 
• ECOG performance status. 
• Record concomitant medications. 
• Pelvic examination with colposcopy (CIN patients) and photographs of the lesion (CIN and 
VIN patients). 
• Evaluation of injection sites. 
• Cervical cytology (pap smear/ECC) for CIN patients. 
• Clinical laboratory assessments: 
o Hematology: CBC, differential, platelet count, PT and PTT. 
o Serum chemistry: serum albumin, total protein, serum bilirubin, LDH, ALT/SGPT, 
AST/SGOT, creatinine, BUN, glucose, and electrolytes. 
• Pregnancy test (serum or urine) <OR> confirmation subject is post-menopausal (absence of 
menses for > six months and LH or FSH in menopausal range). 
• Collect blood samples for investigational correlative studies, including assessment of the 
immunogenicity of IRX-2. 
• Cervical or vulvar swab for HPV status and genotyping for investigational studies. 
 
5.2.10 Week 21-23: Pre-Surgical Assessment 
Subjects return between Week 21-23 for pre-surgical assessment, counseling and surgical 
scheduling for excision of the dysplastic lesion. As detailed in Section 7, for some subjects the 
excision will be an office procedure – either a cervical LLETZ for the CIN 3 cohort or a vulvar 
WLE for the VIN 3 cohort. Some procedures will require surgery in an outpatient surgical center 
or hospital-based operating room, depending on subject preference and clinical considerations 
deferred to the managing clinician; those subjects will likely have a separate “pre-operative visit” 
at week 21-23, or based on local institutional policies for outpatient surgery. 
Regardless, the assessments listed below are to be done no earlier than four weeks prior to the 
excisional procedure; e.g. from -28 to 0 days before excision of the dysplastic lesion. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
54 
• Review documentation of history, and laboratory information obtained while on study. 
• Assessment of all current symptoms (AEs) including severity. 
• ECOG performance status. 
• Record concomitant medications. 
• Complete physical. 
• Pelvic examination with colposcopy (CIN patients) and photographs of the lesion (CIN and 
VIN patients). 
• Evaluation of injection sites. 
• Cervical cytology (pap smear/ECC) for CIN patients. 
• Pregnancy test (serum or urine) <OR> confirmation subject is post-menopausal (absence of 
menses for > six months and LH or FSH in menopausal range). 
• Clinical laboratory assessments: 
o Hematology: CBC, differential, platelet count, PT and PTT 
o Serum chemistry: serum albumin, total protein, serum bilirubin, LDH, ALT/SGPT, 
AST/SGOT, creatinine, BUN, glucose, and electrolytes. 
• Collect blood samples for investigational correlative studies, including assessment of the 
immunogenicity of IRX-2. 
• Cervical or vulvar swab for HPV status and genotyping. 
All of the above pre-surgical evaluations, except for the collection of blood samples and cervical 
swab for investigational correlative studies, are reasonable and medically necessary for the direct 
clinical management of the subject, and thus should be appropriate for reimbursement by insurance 
providers [Martin, 2014]. 
5.2.11 Surgical Excision of the Dysplastic Lesion: Week 25 (Day 175 of Protocol) 
All subjects will undergo excision of their dysplastic lesion following the usual and customary 
care guidelines presented in Section 7.  
Surgery is to take place on or around Day 175 (Week 25) of the protocol, adjusted if needed for 
any treatment delays allowed for toxicity (See Section 5.2.9) or minor changes in schedule within 
the acceptable parameters (See Section 5.1). Regardless, surgical excision should occur no later 
than 7 months since starting treatment to avoid unacceptable delay in definitive surgical 
management of the subject’s dysplastic lesion. 
For CIN3 patients, in cases where no identifiable lesion is seen on colposcopy at the pre-procedure 
visit, the surgical approach (LLETZ, versus cone biopsy via either LEEP or cold knife technique) 
will be the same as that which the subject would have received at baseline to rule out any occult 
dysplasia, particularly in the endocervix.  
For VIN3 patients with no visible lesion at the end of the study period, the extent of surgical 
excision will be different from that of VIN3 at baseline. Instead of a WLE, a punch biopsy will be 
performed in the location of the original lesion. If the VIN lesion is smaller in diameter compared 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
55 
to baseline, these lesions will be removed with a surgical approach appropriate to the size and 
location of the lesion at week 25, at the discretion of the treating physician.  
The surgical specimen will be processed as detailed in the Laboratory Manual. Conventional 
histologic assessment will be done and a pathology report generated for the subject’s medical 
record following routine practice at the institution. Routine histologic assessment that guides 
clinical management of the subject will take priority over the investigational studies. 
The surgical excision of the dysplastic lesions and routine pathology assessment are reasonable 
and medically necessary for the direct clinical management of the subject, and thus should be 
appropriate for reimbursement by insurance providers [Martin, 2014]. Expenses for the 
investigational studies performed using the excised tissue will be borne by Brooklyn 
ImmunoTherapeutics. 
5.2.12 Short-Term Follow-Up Schedule  
Consistent with usual and customary care, all subjects will have at least a telephone contact from 
the clinical investigators 1 to 8 weeks after the excisional procedure to inquire about post-
procedure recovery, inform the subject of the final pathology report, and discuss plans for long-
term surveillance of the subject’s lower genital tract dysplasia. Based upon clinician or subject 
preference, this post-procedure assessment can be performed as part of an office visit. 
The post-procedure assessment, whether done by phone or during an office visit, will include: 
• Interval history and assessment of all current symptoms including severity (AEs)  
• Record concomitant medications. 
• ECOG performance status. 
• And IF subject is seen for an office visit: 
o 
Detailed pelvic examination, including: 
 Inspection of surgical site 
Subjects should be seen more frequently for management of post-procedure complications, other 
medical problems, or regimen-related toxicities at the discretion of the managing clinician. 
Information from these additional encounters will be collected on CRFs and reported to the study 
team. 
Routine post-procedure care, including office visits advised by the clinician, and care of any 
expected complications related to the surgical procedure itself, is appropriate for reimbursement 
by insurance providers [Martin, 2014]. Other study-related visits, such as photography at follow-
up office visits, as well as care required for the management of study-related AEs, will be borne 
by Brooklyn ImmunoTherapeutics.  
5.2.13 Four-month follow-up surveillance 
Subjects return four months post excisional procedure or biopsy to assess HPV status post 
treatment. The assessments listed below are to be done no earlier than 16 weeks from the excisional 
procedure. 
• Focused pelvic exam 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
56 
• Assessment of all current symptoms (AEs) including severity. 
• ECOG performance status. 
• Record concomitant medications. 
• Cervical cytology (pap smear) for CIN patients. 
• Cervical or vulvar swab for HPV status and genotyping. 
Routine follow-up care, including office visits advised by the clinician, and care of any expected 
complications related to the surgical procedure itself, is appropriate for reimbursement by 
insurance providers [Martin, 2014]. Study-related procedures, such as HPV swab at follow-up 
office visits, as well as care required for the management of study-related AEs, will be borne by 
Brooklyn ImmunoTherapeutics.  
 
5.2.14 Longer-Term Follow-Up 
Active surveillance of subjects will cease after a) all treatment-related toxicities have resolved and 
b) completion of the 4-month post-excision assessment detailed in Section 5.2.13. 
If all toxicities related to participation in the study have not resolved by the final office visit post 
surgical excision, investigators may obtain longer-term follow-up information from the subjects’ 
medical records related to these delayed complications or adverse events related to the study, as 
well as any history or laboratory evidence of lower genital tract dysplasias or cancers. This 
additional information will be collected in a HIPAA-compliant fashion with informed consent of 
the subject and will be narrowly focused for only this study of IRX-2 treatment of lower genital 
tract dysplasia. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
57 
6 
STUDY ASSESSMENTS  
6.1 
Endpoint Assessments 
6.1.1 Primary Endpoint – Pathologic Response Rate 
The primary endpoint is the response of CIN 3 or VIN 3 lesions to the IRX-2 regimen as 
determined by histologic examination of the Week 25 surgical excision specimen. Complete 
response is defined as a reduction to no CIN or VIN. The number of subjects with lesions that 
change to a lower grade of dysplasia (CIN/VIN 1) will also be quantified, as well as subjects with 
evidence of persistent CIN/VIN 3 or progression to invasive cancer. 
Pathologic Complete response 
Disappearance of any dysplasia 
Pathologic Partial response 
Regression of CIN3 to CIN1 in cervical cases; 
and VIN3 to VIN1 I in vulvar cases  
 
Pathologic Non-Responder 
Stable high grade cervical dysplasia (CIN2 or 
CIN3) or stable high grade vulvar dysplasia 
(VIN2 or VIN3) 
Pathologic Progression 
Evidence of invasive carcinoma 
 
Histologic evaluation of both the initial diagnostic biopsies and the subsequent surgical excisions 
(For CIN3: cervical LEEP or Conization; For VIN3: vulvar WLE or biopsy of original lesion site) 
will be performed and reported to the clinicians caring for each subject by the pathology laboratory. 
Clinical decisions will be based on this information. 
For the purpose of the study, a pathologist with specialty expertise in the assessment of lower 
genital tract dysplasia will also evaluate the specimen. In the event of a clinically significant 
diagnostic discrepancy between different pathologists, the principal investigator and the clinician 
caring for the subject will be notified, steps taken to resolve the discrepancy, the subject informed 
of the final diagnostic decision, and her clinical management altered if necessary. 
The gynecologic pathologist selected for this study at USC is Dr. Saloni Walia (USC Department 
of Pathology).  Gynecological pathologists from other participating sites will be selected by the 
institutions Principal Investigator. Entry biopsy slides and final excisional procedure pathology 
slides obtained at other clinical sites will be reviewed by USC’s pathologists for confirmation of 
final diagnosis. 
6.1.2 Secondary Endpoints 
6.1.2.1 Toxicity and Feasibility 
Toxicity of the IRX-2 Regimen overall, and specifically for the IRX-2 injection, will be assessed 
by the incidence and severity of AEs, SAE, as classified and graded according to the current 
version of the CTCAE v4, with one modification:  attribution of AEs will be coded as definitely, 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
58 
possibly, and not related to treatment (per Section 9.1.2). All subjects will be assessed for AEs as 
detailed on the Study Schedules (Section 5.1) and evaluated for severity. 
Feasibility for each patient will be determined if she (a) has received the 2 full cycles of treatment 
(specifically all 4 doses of IRX-2 in each cycle) and (b) can undergo surgical resection within the 
planned time window.  
6.1.2.2 Clinical CR or PR at Weeks 6, 13 and 25 
At the time of the planned colposcopies (and photographs) at weeks 6, 13, and 25, the lesions sizes 
will be evaluated. Clinical complete response is no evidence of CIN or VIN visually by colposcopy 
and documented by the photographs. Clinical partial response is defined as a reduction in diameter 
of the lesion by 50% or greater.  
6.1.2.3 HPV Status 
HPV status will be assessed at baseline, at week 13 (at the time of planned colposcopy), at week 
25 (at the time of surgical resection), and at 4 months post excision or biopsy. HPV status will be 
classified as positive if HPV virus is detected by PCR or negative if it is not detected. The log 
reduction in the viral load will be recorded. 
6.1.2.4 Immunologic Studies 
Multiple parameters will also be studied to assess the immunologic activity of the IRX-2 Regimen 
for the treatment of CIN 3 or VIN 3, as detailed below. These studies are investigational and costs 
will be borne by Brooklyn ImmunoTherapeutics. These will include assessments of serologic 
response to HPV by ELISA, evidence of an antigen-specific T cell response by ELISPOT and 
changes in peripheral blood lymphocyte subsets and state of activation by flow cytometry. In 
addition, the development of anti-drug antibody (ADA) immune responses to the active 
components of IRX-2 will be measured in blood samples collected at pre-dose of 1st cycle (day 1), 
week 1-2, pre-dose of 2nd cycle (week 6), week 13, and week 25. Blood samples collected at the 
time of injections (days 4-7 of each cycle) will be used for pharmacokinetic analysis.  
6.1.2.5 Characterization of the Immune Infiltrate within the Surgical Specimens 
Analysis of the immune infiltrates in the pre-treatment tumor biopsies and the post-treatment 
excisional specimens will be determined and compared where sufficient biopsy/excisional material 
is available. Parameters to be evaluated include: 
• Lymphocyte infiltration within the center of the tumor and at the tumor edge or margins. 
• Lymphocyte subsets will be evaluated including total T cells and naïve, cytotoxic and 
regulatory T cells. 
• The analyses may include quantifications of cellular infiltrates and degree of activation by 
immunohistochemistry, including (but not limited to): CD3, CD8, CD68 (myeloid derived 
suppressor cells), CD45RO and Fox P3 on lymphocytes and MHC class I and II and PDL1 on 
tumor cells and APCs. IHC assay will also assess markers of Langerhans cells (CD1a, e-
cadherin) and antigen presenting cell (APC) activation (CD80 and CD86). Other immune 
subsets and activation markers may be evaluated. Multiplex IHC assays may be used.  
• Gene expression signatures within the dysplastic tissue may also be performed. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
59 
• Lymphocyte infiltrates and changes after treatment will be evaluated using T cell receptor 
(TCR) repertoire analyses performed on extracted RNA. 
Methodology is detailed in the accompanying Laboratory Manual and/or the Investigator’s 
Brochure.  
6.1.2.6 Relation Between HPV Status and Response to Therapy 
• For both the CIN 3 and the VIN 3 cohorts, cervical HPV status including genotyping will be 
determined based on cervical or vulvar swabs obtained at baseline, during, and after treatment 
with the IRX-2 Regimen at time-points specified in the study protocol using a commercially 
available assay, Roche LINEAR ARRAY® HPV Genotyping Assay, or equivalent. Clearance 
of HPV from the cervix or vulva as determined by these assessments will be measured as a 
secondary endpoint of the study. 
• The tissue specimens obtained during the study, including both the cervical and vulvar 
biopsies and excisions, will be evaluated for a) the presence or absence of different HPV 
genotypes; and b) expression of p16, using immunohistochemistry (IHC). The IHC will be 
performed using the paraffin embedded tissue blocks prepared by the clinical pathology lab. 
Because some of the tissue biopsies may be of limited size, investigational use of specimens will 
always be secondary to the conventional histology needed for clinical management. 
 
6.1.2.7 Activation, Clonality and Immunophenotypic Analyses of Peripheral Blood 
Lymphocytes 
Peripheral blood mononuclear cells will be obtained by Ficol Hypaque centrifugation and 
cryopreservation of peripheral blood samples within 8 hours of collection. Peripheral blood 
lymphocytes will be evaluated by direct ex vivo ELISPOT analysis and TCR clonotypic analysis 
for evidence of induction of γ-interferon expressing T cells or T cell clones in response to HPV 
antigens. Baseline T cell reactivity and clonality will be compared to on-treatment and post-
treatment levels.  
Immunophenotypic flow cytometry will also be utilized to assess changes in naïve, memory, 
effector and regulatory T cell subsets. 
The methodology to be used has been previously described by Dr. Martin Kast and his colleagues 
[Da Silva, 2016; Da Silva, 2015; van Poelgeest, 2016].  
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
60 
7 
SURGICAL MANAGEMENT OF CIN 3 OR VIN 3 
Surgical management of CIN 3 or VIN 3 is within the scope of practice for a gynecologist and so 
will only be summarized. 
7.1 
Initial Diagnostic Biopsies 
The initial cervical or vulvar biopsies to establish the diagnosis of CIN 3 or VIN 3 will be done by 
the subject’s referring clinician in the course of normal clinical care [Massad, 2013]. Upon 
enrollment in the trial of IRX-2 therapy, one of the clinical investigators will perform a baseline 
assessment of the subject, including a comprehensive colposcopy of the lower genital tract. 
Baseline photographs should be obtained. Biopsies will only be obtained if a previous biopsy has 
not been obtained or if slides/block from this are not available. This is comparable to what is 
normally done for a patient who is referred to a Colposcopy Clinic. 
7.2 
Therapeutic Excision of Dysplastic Lesion: Week 25 
Following enrollment, each subject will receive two courses of therapy, the first administered 
during Week 1, and the second during Week 7. Six weeks later (Week 13), the dysplastic lesion is 
reevaluated by colposcopy, with photographic documentation, after which (week 25) it is 
surgically excised. In this trial the surgical excision will be performed but after a time period where 
immunotherapy will have had sufficient time to mediate a clinical effect (month 6 from start of 
treatment). Where progression at week 13 is suspected the surgical excision procedure would be 
moved earlier. 
7.2.1 CIN 3 Cohort: Cervical Excision 
The entire dysplastic lesion with a surrounding margin of transformation zone will be excised. For 
most subjects this can be accomplished with a loop electrosurgical excision procedure (LEEP); or 
for more superficial lesions, a large loop excision of the transformation zone (LLETZ). Subjects 
with more extensive lesions, including lesions extending into the endocervical canal, will require 
a deeper conization procedure (either a LEEP conization or a cone biopsy performed with 
conventional “scalpel” technique). If no lesions are visible at week 25, a LLETZ or cone biopsy 
via either LEEP or cold knife technique will still be performed for diagnostic purposes to rule out 
any occult dysplasia. 
An endocervical curettage (ECC) will be done for all subjects at the time of the cervical excision, 
in keeping with standard of care, to rule out occult endocervical involvement with either dysplasia 
or cancer. 
All decisions regarding the optimal type of excisional procedure to be performed (LLETZ, LEEP, 
scalpel conization); setting (office vs. operating room); and analgesia/anesthesia are to be made by 
the clinical investigator based on subject’s needs and preferences. 
7.2.2 VIN 3 Cohort: Wide Local Excision of Dysplastic Lesion 
The size, location, and even geometry of a subject’s dysplastic lesion or lesions will determine the 
optimal technique for surgical excision. Most VIN 3 lesions can be removed with a superficial 
wide local excision (WLE) as would be done with any cutaneous lesion. For larger lesions, an 
excision with adequate margins will require a superficial partial vulvectomy, and is usually done 
in the operating room for better subject comfort and safety. At week 25, if no VIN lesion is visible, 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
61 
a punch biopsy of the original lesion site will be performed instead of WLE to confirm complete 
pathological response. Punch biopsies can be performed in a clinic setting. 
7.2.3 Multifocal Lower Genital Tract Dysplasia, and/or Concurrent Genital Tract 
Condylomata 
Multifocal lower genital tract dysplasia, with or without concurrent condylomata, is common, 
especially in younger women. While this protocol requires that the CIN 3 or VIN 3 lesion be 
surgically excised, the clinical investigator caring for the subject may treat other lesions in any 
manner consistent with standard of care, for example: concurrent CO2 laser ablation of vaginal 
dysplasia or vulvar condylomata at the time of excision of VIN 3. 
Questions regarding permissible treatments in these circumstances should be referred to the PI. 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
62 
8 
STATISTICAL CONSIDERATIONS 
8.1 
Summary of Design (revised design – Amendment dated November 08, 2019) 
This trial was originally designed as a two-cohort randomized placebo-controlled Phase 2 trial in 
women with  squamous cervical intraepithelial neoplasia 3 (CIN 3) (Cohort 1) or  squamous vulvar 
intraepithelial neoplasia 3 (VIN 3) (Cohort 2). Eligible patients would be randomized 2:1 to 
Regimen 1 or Regimen 2. In the experimental Regimen 1, patients receive IRX-2 injections (20 
women in each cohort – 40 total) and in control Regimen 2 patients will receive placebo injections 
(10 women in each cohort – 20 total). Prior to randomization, patients were to be stratified by 
smoking history (current or recent smoking vs. not – see Section 5.2.2); after stratification, a 
permuted block design (with varying block sizes) was to be used to randomly assign the regimen. 
After randomization, patients were to receive two cycles of treatment followed by surgical 
resection of the CIN or VIN lesions.  
Current, Revised Design 
Based on slow accrual and in order to ensure completion in a timely fashion, the trial was 
redesigned to be a non-randomized, open-label trial.  There will still be 2 cohorts of patients: 
(Cohort 1) those with squamous cervical intraepithelial neoplasia 3 (CIN3) and (Cohort 2) those 
with squamous vulvar intraepithelial neoplasia 3 (VIN3).  This is still a Phase II/Screening trial.   
The primary endpoint will be the complete or partial disappearance of all dysplasia (CR or PR) as 
determined by histologic examination of the Week 25 surgical specimen obtained at the time of 
surgical excision (LEEP, wide local excision, etc. – see Section 8.2.1); any patient who does not 
experience a CR or PR will be classified as being a “non-responder”.   
For this specific regimen to proceed to further clinical testing, we require a very strong signal - 
with at least 50% of subjects with CIN3, and with at least 40% of subjects with VIN3, experiencing 
the clear benefit of a CR or PR.  A modest effect, while intriguing, would not be enough to justify 
further testing in otherwise healthy subjects.   
It is known that some, relatively small, proportion of subjects with CIN 3 or VIN 3 will have no 
evidence of dysplasia in a subsequent excisional specimen or just a minimal disease remaining 
(CR and PR is defined in Section 6.1). Whether this represents complete removal of a small lesion 
with the initial diagnostic biopsy, an effect of post-biopsy inflammatory changes, or spontaneous 
resolution of the dysplastic lesion is impossible to ascertain for any one subject. In addition, all 
subjects in this trial will receive immuno-modulatory doses of cyclophosphamide, indomethacin, 
and zinc with multivitamins – any one of which may by itself affect immunologic clearance of the 
dysplastic lesion. Furthermore, repetitive injections of any substance into or below the dysplastic 
lesion may influence the resolution rate. Because subjects with very small lesions (which could be 
excised with the initial diagnostic biopsy) are excluded from this trial, a baseline rate of lesion 
regression or resolution is expected to be no more than 20% at the time of the “Week 25” excisional 
procedure for CIN [Trimble, 2005; Munk, 2007] and no more than 1.5% for VIN [van Seters, 
2005]. 
After discussion with the team, it was determined that for the IRX-2 Regimen to be considered 
sufficiently promising for further study we would need to observe: 
• in the population of subjects with CIN 3, the IRX-2 Regimen should result in a response 
rate that was clearly greater than 20% and not significantly less than 50%, and 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
63 
• in the population of subjects with VIN 3, the IRX-2 injections should result in a response 
rate that was clearly greater than 5% and not significantly less than 40%. 
Study Design.   For each cohort, this trial will use a Simon two-stage design. 
 
Cohort 1 (CIN3): with a false-positive error rate of 5% when the true objective response rate is 
20% and a false-negative error rate of 10% when the true response rate is 50%.  A total of 10 
patients will be enrolled in the 1st stage.  The first stage stopping rule is defined as observing only 
2 or fewer responses among the first 10 patients. If 3 or more responses are observed, the trial will 
continue to a total of 22 patients.   If 8 or more patients of the 22 experience a CR or PR then this 
will be evidence that the true response rate of IRX-2 Regimen is greater than 20% and not 
significantly less than 50%. 
 
Cohort 2 (VIN3): with a false-positive error rate of 10% when the true objective response rate is 
5% and a false-negative error rate of 10% when the true response rate is 40%.  A total of 5 patients 
will be enrolled in the 1st stage.  If there is no response observed among the first 5 patients, the 
enrollment will be stopped. If 1 or more responses are observed the trial will continue to a total of 
10 patients.   If 1 or more patients of the 10 experience a CR or PR then this will be evidence that 
the true response rate of IRX-2 Regimen is greater than 5% and not significantly less than 40%. 
 
8.1.1 Toxicity Monitoring 
Toxicity will be monitored on an ongoing basis during the conduct of the trial by the Study Team. 
To assist in this review of the proposed regimen, monitoring boundaries have been developed.   
For these guidelines: 
• Significant Local Toxicity will be defined as (a) any Grade 3 or greater AE or reaction 
(per CTCAE v4) related to study drug injections, at or near the site of injection, or (b) 
withdrawal from study due to intolerance of cervical or vulvar injection. 
• Unacceptable Toxicity will be defined as any Grade 3 or greater AE, occurring during the 
observation period (from the time of the 1st injection until the Week 26 post resection 
evaluation) that cannot be clearly attributed to a non-IRX-2 treatment cause.   
• Each of these toxicities will be monitored separately using the boundaries described below.  
CIN and VIN subjects who are treated with the IRX-2 regimen, will be combined to 
monitor toxicity.    The toxicity monitoring boundaries are: 
- 
2 or more of the 1st 10 subjects experience unacceptable toxicity 
- 
3 or more of the 1st 15 subjects experience unacceptable toxicity  
- 
4 or more of the 1st 20 subjects experience unacceptable toxicity  


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
64 
If the boundary is crossed for either definition of toxicity, this will serve as a flag to the Study 
Team to review the tolerability of the regimen and consider modifying some aspect of the regimen 
and amending the protocol.   
 
8.1.1.1 Justification of the Monitoring Boundaries 
The rules (2+/10 or 3+/15 or 4+/20) were selected to ensure a reasonable chance of crossing the 
boundary if the regimen produced either significant local toxicity or unacceptable toxicity (each 
monitored separately) in 20-25% of subjects.  The table below summarizes the chance of crossing 
the boundary. 
Chance of Unacceptable Toxicity 
5% 
10% 
15% 
20% 
25% 
30% 
Probability of Crossing the Boundary 
(2+/10 or 3+/15 or 4+/20) 
10% 
31% 
55% 
74% 
87% 
94% 
 
8.1.1.2 Toxicities related to indomethacin, cyclophosphamide, omeprazole and zinc-
containing multivitamins 
AEs including gastrointestinal symptoms such as heartburn or dyspepsia which could possibly be 
related to the indomethacin or hematologic changes such as anemia, neutropenia or 
thrombocytopenia which may be associated with cyclophosphamide will be monitored. 
Modifications to the study regimen for AEs related to these study drugs are described in Section 
5.2.9. 
8.2 
Major Endpoints 
8.2.1 Primary Endpoint:  CR or PR in Surgical Specimen (≈week 25) 
The primary endpoint is the response of CIN3 or VIN3 lesions to the IRX-2 Regimen as 
determined by histologic examination of the Week 25 surgical excision specimen. Complete 
response is defined as complete resolution of all dysplasia with no evidence of any dysplasia or 
cancer on the end of study pap smear, excisional procedure, ECC or biopsy. A partial response is 
defined as regression of CIN3 to CIN1 in cervical cases or VIN3 to VIN1 in vulvar cases based on 
the end of study excisional procedure or biopsy. In cervical cases where the internal margins of 
the cone are involved with CIN1, the pre-conization pap smear and post conization ECC must 
reveal no pathology more severe than CIN1.) (see Section 6.1). 
8.2.2 Secondary Endpoints of Toxicity and Feasibility 
Toxicity of the IRX-2 Regimen will be assessed by the incidence and severity of AEs and/or SAEs, 
as classified and graded according to the current version of the CTCAE v4, with one modification:  
attribution will be coded as definitely, possibly, and not related to treatment (per Section 9.1.2). 
All subjects will be assessed for AEs as detailed on the Study Schedules (Section 5.1) and 
evaluated for severity. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
65 
Feasibility for each patient will be determined if she (a) has received the 2 full cycles of treatment 
(specifically all 4 doses of IRX-2 in each cycle) and (b) can undergo surgical resection within the 
planned time window.  
8.3 
Secondary Endpoint of HPV status 
An important endpoint in this trial will be the change in HPV status from before to after protocol 
treatment. All patients with CIN 3 on this trial will be “HPV positive” based on a commercial HPV 
genotyping assay which will be performed prior to treatment start; there is no such equivalent 
commercially available HPV genotyping assay for patients with VIN 3, and therefore patients with 
VIN 3 will be tested for HPV status after enrollment but before treatment using an assay in the 
USC Norris Immune Monitoring Core Laboratory, but this will not affect eligibility. Thus it is 
possible that 1 or 2 patients with VIN 3 will be “HPV negative” prior to start of treatment. 
Nonetheless, the HPV status, prior to start of protocol therapy, as well as at the time of surgical 
excision, will be known for all patients.  
8.4 
Secondary Endpoints-Other 
A number of other exploratory secondary endpoints will be performed including 
immunophenotypic analysis of peripheral blood lymphocytes, frequency of serum antibodies and 
RNA expression profiling. These will be primarily descriptive end-points.  
8.5 
Accrual  
Cervical dysplasia is more common than vulvar dysplasia, so study accrual will be different for 
each cohort. For the CIN 3 cohort, it is reasonable to expect enrollment of one subject per month, 
after an initial lag of two months when the study is first opened for recruitment. On average, there 
will be about a two-week interval between recruitment and Day 1 of treatment. With these 
considerations, all 22 CIN 3 subjects should be recruited by 22 months, and the surgical excision 
of the dysplastic lesion for the last recruited subject will occur around 28 months after opening the 
trial. 
The VIN 3 cohort will probably enroll at a rate of one subject per quarter, so estimated completion 
of enrollment is at 30 months and performance of the last excisional procedure is at 36 ½ months. 
An active outreach program is planned to accelerate recruitment of the vulvar dysplasia subject to 
shorten this timeline as much as possible. 
We expect approximately that 20% of patients may not complete therapy and/or surgical excision 
and therefore may not be fully evaluable. (Any such subjects will be contacted in keeping with 
clinic policies to ensure she receive appropriate care, irrespective of continued participation in the 
research study.)  Thus about 8 additional patients may need to be enrolled adding approximately 
3 months for completion of Cohort 1 and 6 months for completion Cohort 2.  
The accrual times listed above may be shortened with the addition of any additional clinical sites. 
8.6 
Endpoint Analyses 
All patients who received any amount of treatment will be accounted for; listings and summaries 
will include patients who receive at least one IRX-2 injection. For these patients, the following 
will be listed and summarized with standard descriptive methods:  basic demographic and clinical 
baseline data, smoking status, number of cycles begun, number of injections received in each 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
66 
cycle, reason off treatment, timing of surgical excision, amounts of cyclophosphamide, 
indomethacin, zinc-containing multivitamins, and omeprazole received, HPV levels, clinical 
response at each colposcopy, pathologic response, and toxicities (grade, type, cycle, and 
attribution) experienced.  
8.6.1 Primary Endpoint:  Complete Clearance or Major Regression (CR or PR) of 
Dysplasia Based on Surgical Excision Specimen 
For the primary endpoint, the primary analysis will include all eligible patients who receive at least 
one cycle (4 injections) of IRX-2 and who undergo surgical excision. In an important secondary 
analysis, all eligible patients who receive at least one injection, will be included. 
As stated above, the primary endpoint will be the complete or partial disappearance of all dysplasia 
(CR or PR) at the time of surgical excision (LEEP, wide local excision, etc.); any patient who does 
not experienced a CR or PR will be classified as having a “non-responder”. An exact logistic 
regression will be used to test for an association between treatment and experiencing a CR/PR with 
history of smoking (currently or recently vs. not) as the single covariate in logistic model. Initially, 
the two cohorts of patients will be analyzed separately.  
8.6.2 Secondary Endpoints:  Toxicity and Feasibility 
All patients who receive at least one injection of IRX-2 will be included in the analyses of toxicity 
and feasibility. All patients will be followed for adverse events as summarized in Section 6.1.  
AEs will be recorded and classified by system organ class and preferred term coded in accordance 
with the version of CTCAE/MedDRA that is most recent at the start of the study - with one 
modification:  attribution will be coded as definitely, possibly, and not related to treatment (per 
Section 9.1.2).  Adverse event definitions and reporting requirements are detailed in Section 9. 
Study participants will be summarized in terms of  
• each type of AE at worst grade of severity 
• any AE at worst grade of severity 
• SAEs 
• withdrawals (and reasons for them) 
• dose modifications (and reasons for them) 
• treatment interruptions (and reasons for them)  
• abnormal laboratory findings that are considered clinically significant by the investigator 
• any deaths occurring within 210 days (approx. 7 months) after Day 1 of treatment. 
For reporting results, toxicities will be tabulated and reported according to regimen, cycle, toxicity 
type, grade, and attribution. The proportion of patients for whom the regimen was feasible (i.e. 
completed 2 cycles (received all 8 injections) and underwent surgical excision within the planned 
time period) will be calculated with associated (exact) 95% confidence intervals. For these 
analyses, both cohorts will be combined.  


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
67 
8.6.3 Additional Secondary Endpoints:  Clinical CR or PR at Weeks 6, 13, and 25 
The initial analysis will include all eligible patients who complete 1 cycles of therapy and undergo 
the 1st colposcopy at 6 weeks. The clinical response at weeks 6, 13, and 25 will be tabulated by 
Cohort and Regimen. The proportion of patients who achieve a CR’s and PR’s at each time point 
will be calculated with associated (exact) 95% confidence intervals. If numbers permit, changes 
over time will be summarized using models that accommodate repeated measures; the two Cohorts 
will be analyzed separately. 
8.6.4 Additional Secondary Endpoints:  HPV Status 
HPV status will be assessed at baseline, at week 13 (at the time of planned colposcopy), at week 
25 (at the time of surgical resection), and at week 41 (four months post excision or biopsy). All 
eligible patients who complete at least 1 cycle of treatment and have the HPV status assessed at 
week 13, 25, or 41 will be included in the initial analysis. The results at baseline and weeks 13, 
25, and 41 will be tabulated by Cohort. Results will be reported as virus detected or not detected. 
Quantitative measurements of viral load will also be recorded.  
8.6.5 Secondary/Exploratory Endpoints:  Immunologic Studies 
It is also planned that exploratory endpoints will be assessed in all subjects who undergo lesion 
excision, limited only by availability of histologic material (as explained in Section 6.1.2.4) if the 
quantity of biopsy material is small then priority is given to conventional diagnostic purposes over 
the performance of investigational studies). Analyses of some or all of the exploratory endpoints 
will be conducted by designated researchers at intervals depending on the availability of sufficient 
specimens. The Study Team may alter or discontinue some or all of these exploratory endpoints.  


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
68 
9 
ADVERSE EVENTS, REPORTING REQUIREMENTS AND STOPPING RULES 
9.1 
Adverse Event Definitions 
Definitions of AEs will follow the International Conference on Harmonization E6: Good Clinical 
Practice Step 5, Consolidated Guideline 1.5.96, April 1998 edition, and are summarized below. 
For this study, the investigational product is the IRX-2 Regimen, including IRX-2, 
cyclophosphamide, indomethacin, omeprazole and zinc-containing multivitamins. 
An adverse drug experience (event) is any new undesirable medical occurrence or change 
(worsening) of an existing condition in a subject that occurs from the first day of treatment until 
the time of surgical excision of the dysplastic lesion (with the exception of an SAE, if it occurs 
within 30 days from the last dose of study medication), whether or not considered to be drug 
related. Therefore, AEs are treatment-emergent signs and symptoms. This includes those events 
occurring from drug overdose, whether accidental or intentional, from drug abuse or from drug 
withdrawal. In general, abnormal laboratory findings without clinical significance based on the 
investigator’s judgment are part of the database and should not be recorded as AEs. 
Adverse events reported by the subject or observed by the investigator will be individually listed 
on an AE CRF page. The signs and symptoms date of onset, duration, action taken, severity, 
outcome to date, and relationship to the study drug will be recorded. The severity of AEs will be 
graded according to the National Cancer Institute Common Terminology Criteria for Adverse 
Events (NCI-CTCAE), Version 4.03 toxicity criteria. Severity will be classified as described in 
Section 9.1.3. 
Adverse events of Grade 3 or 4 will be reported to the Study Team and Medical Monitors, who 
will assess with the Site Principal Investigator whether to modify or discontinue study treatment. 
Adverse events will be measured from Cycle 1 Day 1 until the week 25 surgical excision. Ongoing 
AE/SAEs will be reported and monitored until resolution. Evidence of toxicity after surgery will 
be assessed by tabulation of AEs felt by the investigators to be definitely or possibly related to the 
study regimen.  
Serious adverse events will be reported, measured and monitored from the first day of treatment 
until the SAE resolves or the trial is terminated by the Study Team. All SAEs be tabulated 
regardless of relatedness to study drugs. Ongoing AEs at the time of surgery will be followed for 
30 days post-surgery. 
At each visit, the investigator will be prompted to report AEs as “not”, “possibly,” or “definitely” 
related to the IRX-2 Regimen. 
Pregnancy. While pregnancy is not an adverse event per se, any pregnancy or suspected 
pregnancy occurring during the clinical study  or 1 year following the last dose of study 
medication should be reported to the Study Team and Medical Monitor immediately using the 
pregnancy report form. If the subject is still active in the study, the protocol drugs will be 
discontinued immediately and the subject removed from the trial.  The Investigator will follow 
the subject until completion of the pregnancy and report pregnancy outcomes, including adverse 
maternal and neonatal outcomes.  All neonatal deaths that occur within 28 days of birth should 
be reported, without regard to causality, as SAEs.   
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
69 
9.1.1 Definition of Serious and Life Threatening Adverse Events 
A serious adverse drug experience (event) is any AE occurring at any dose that results in any of 
the following outcomes: 
• Death 
• Is life-threatening  
- A life-threatening adverse drug experience is any AE that places the subject, in the view of 
the investigator, at immediate risk of death from the reaction as it occurred. This does not 
include a reaction that, had it occurred in a more severe form, might have caused death. 
• A persistent or significant disability/incapacity 
• Inpatient hospitalization or prolongation of existing hospitalization, except that planned 
hospitalization or hospitalization for the social reasons or the convenience of the subject or 
physician shall not be considered an SAE  
• A congenital anomaly/birth defect 
• Required intervention to prevent permanent impairment/damage 
9.1.2 Definition of Relationship to Study Drug 
Association or relatedness to the study drug will be graded as either “not”, “possibly,” or 
“definitely” related to the study regimen. Determination of relatedness includes: 
• Definitely, characterized as an AE that 
- Follows direct temporal sequence from drug administration. 
- Abates upon discontinuation of the drug. 
- Cannot be explained by the known characteristics of the subject’s clinical state or by other 
modes of therapy administered to the subject.  
• Possibly, characterized as an AE that 
- Follows a reasonable temporal sequence from drug administration. 
- Abates upon discontinuation of the drug. 
- Could have been produced by the subject’s clinical state or by other modes of therapy 
administered to the subject. 
• Not Related, characterized as an AE that 
- Does not follow any temporal sequence from drug administration.  
- Is explained by the subject’s clinical state or by other modes of therapy administered to the 
subject. 
9.1.3 Definition of Severity 
The severity of adverse changes in physical signs or symptoms will be graded according to the 
NCI-CTCAE v4.03. For AEs not listed in the table, the severity of adverse changes in physical 
signs or symptoms will be classified as follows: 
 
Grade 1: 
Mild (transient or mild discomfort; no limitation in activity; no medical 
intervention/therapy required) 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
70 
Grade 2: 
Moderate (mild to moderate limitation in activity, some assistance may be needed; 
no or minimal medical intervention/therapy required) 
Grade 3: 
Severe (marked limitation in activity, some assistance usually required;  
medical intervention/therapy required, hospitalizations possible) 
Grade 4: 
Life-threatening 
(extreme 
limitation 
in activity, 
significant 
assistance  
required; significant medical intervention/therapy required, hospitalization or 
hospice care probable) 
Grade 5: 
Death 
9.1.4 Definition of Action Taken 
None: 
No action taken with regard to the study drug 
Interrupted: 
The study drug was stopped but restarted after the subject’s symptom abated. 
The subject was rechallenged with the study drug 
Discontinued: The study drug was permanently stopped 
9.1.5 Definition of Outcome to Date 
Resolved with sequelae: 
The subject has recovered from the AE with observable  residual 
effects 
Resolved without sequelae: 
The subject has fully recovered from the AE with no  observable 
residual effects 
Unresolved: 
The AE is present and observable 
Death: 
The subject died as a result of the AE 
Lost to Follow-up: 
Source documentation confirms that repeated attempts to  
contact subject have failed 
9.2 
Notification of Serious or Unexpected Adverse Events 
• For all sites- the sponsor/USC Principal Investigator, and USC DSMC and Brooklyn 
ImmunoTherapeutics  must be notified within 24 hours of learning of any serious adverse 
events, regardless of attribution, occurring during the study or within 30 days of the last 
administration of the study drug. 
o USC Principal Investigator: Lynda Roman, MD 
Email: lroman@med.usc.edu 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
71 
o Brooklyn ImmunoTherapeutics: SAE@linical.accelovance.com with cc to 
safety@brooklynitx.com 
 
• The Institutional IRB must be notified of “any unanticipated problems involving risk to 
subjects or others” (UPR) in accordance with the Institutional policy. 
• The following events meet the definition of UPR: 
1. Any serious event (injuries, side effects, deaths or other problems), which in the 
opinion of the Principal Investigator was unanticipated, involved risk to subjects or 
others, and was possibly related to the research procedures. 
2. Any serious accidental or unintentional change to the IRB-approved protocol that 
alters the level of risk. 
3. Any deviation from the protocol taken without prior IRB review to eliminate 
apparent immediate hazard to a research subject. 
4. Any new information (e.g., publication, safety monitoring report, updated safety 
report), interim result or other finding that indicates an unexpected change to the 
risk/benefit ratio for the research. 
5. Any breach in confidentiality that may involve risk to the subject or others. 
6. Any complaint of a subject that indicates an unanticipated risk or that cannot be 
resolved by the Principal Investigator. 
 
• 
The USC NCCC Data and Safety Monitoring Committee (DSMC) must be notified within 
24 hours of submission of such reportable event to the IRB. The patient ID and the study 
number as well as identifier of the SAE report should be submitted to the DSMC 
Coordinator via email (Grace.Kim@med.usc.edu) or Fax to the attention of the DSMC 
Coordinator at 323-865-0089. 
• 
The FDA should be notified within 7 business days of any unexpected fatal or life-
threatening adverse event with possible relationship to study drug, and 15 business days of 
any event that is considered: 1) serious, 2) unexpected, and 3) at least possibly related to 
study participation. 
• 
USC: Study team to work with CISO QA to submit to the FDA using 
MedWatch3500A form in accordance within the FDA required timelines 
• 
Non-USC site: study team to notify USC PI, and USC DSMC Coordinator, 
within 24hrs of site becoming aware of the event by completing the 
MedWatch3500A form. USC will review and submit the applicable reports 
to the FDA and notify Brooklyn ImmunoTherapeutics of the submission. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
72 
10 ETHICAL AND REGULATORY CONSIDERATIONS 
10.1 Protection of Human Subjects 
This study has been designed and will be conducted in accordance with the principles outlined in 
the Declaration of Helsinki. 
10.2 Informed Consent 
Once a subject is referred for consideration in the study, that individual’s history and current status 
will be completely evaluated, and treatment recommendations will then be discussed thoroughly 
with the subject. The investigator will discuss with the subject any other treatments that may be 
available and their relative value compared to the possible use of IRX-2 therapy. The risks and 
hazards of the procedures will be explained to the subject. The investigator shall seek consent only 
under circumstances that provide the individual sufficient opportunity to consider whether or not 
to participate and that minimize the possibility of coercion or undue influence. The information 
that is given to the individual shall be in understandable language. No informed consent may 
include any exculpatory language through which the individual is made to waive or appear to 
waive any of their legal rights, or releases or appears to release the investigator, Brooklyn 
ImmunoTherapeutics, the institution, or its agents from liability for negligence. The subject, or 
legal guardian, must be able to comprehend the informed consent form and sign prior to 
enrollment. The subject will then receive a signed copy of the consent form. 
10.3 Institutional Review Board or Independent Ethics Committee 
The investigator must obtain approval for the study from the Institutional Review Board (IRB) or 
Independent Ethics Committee (IEC). All changes to the protocol must be reviewed and approved 
prior to implementation, except where purely administrative or necessary to eliminate apparent 
immediate hazards to subjects. The investigator is responsible for obtaining annual IRB/IEC 
renewal through the duration of the study. Copies of the investigator’s report and the IRB/IEC’s 
continuance of approval must be submitted as directed.   


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
73 
11 STUDY ADMINISTRATION 
11.1 Monitoring Plan 
11.1.1 Pre-study Site Evaluation 
The study monitor will visit each study center to confirm the site’s qualifications for implementing 
this protocol. The protocol and study procedures will be reviewed with the investigator and study 
personnel. This visit may be combined with the study initiation visit or by completion of site 
qualification form. 
11.1.2 
 Site Initiation 
The study monitor will visit the site or perform the visit as a teleconference/WebEx to train 
personnel on all protocol elements, study procedures, and safety, and to confirm site readiness 
prior to enrollment. There may be visits to clinical, diagnostic laboratory, and pathology study 
participants or completion of site qualification form. 
11.1.3 Interim Monitoring 
The NCCC Data and Safety Monitoring Committee (DSMC) will monitor all NCCC initiated 
multi-center studies. If external sites have local policies and procedures that require them to 
perform internal auditing or data and safety monitoring of their patients enrolled to USC trials, the 
local auditing and/or monitoring schedule must be provided to the MCC team and USC PI. In 
addition, auditing and/or monitoring reports must be provided to the MCC team and USC PI within 
2 weeks of issuance of the local report to the external site study team or local PI. Remote auditing 
will be conducted yearly or more frequently as needed, regardless of the external site’s monitoring 
policies. The USC Audit Team, may elect to do an independent review if major issues are found 
on the local reports. The first two patients registered on a protocol will be audited. USC will notify 
external site study teams via an email reminder of an upcoming audit at least 2 weeks prior to a 
scheduled audit. 
Auditing will be performed on multi-site protocols opened by the CISO-MCC. All reviews will be 
performed remotely, unless otherwise specified in a contractual agreement and agreed upon by 
both institutions. The MCC and external site study team will make available the required medical 
records, research files, regulatory documents and other documentation via a secure electronic 
method agreed upon by both parties. The Clinical Research Auditor will review these materials 
during the audit or monitoring review. Once the review for each subject selected is complete, a 
final report will be made available. Trends and other important findings will be reviewed during 
the standing trial and safety review teleconferences organized by the MCC. Specific findings for 
individual external sites will not be outlined during all-site teleconferences, but will rather be 
provided to the respective site during a dedicated teleconference with the site or via email, 
depending on the findings. 
The CISO-MCC may require a “for cause” audit; this audit is required when significant findings 
or data indicate potential issues on the part of the site. The USC PI of the trial reserves the right to 
call for this audit with all sites. 
 
If the audit reveals compliance issues, further follow up will include: 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
74 
 
• A re-audit of the protocol in question by the MCC 
• Suspension of an external site or the entire protocol to accrual or conduct at the 
discretion of the USC DSMC 
• Recommendation of permanent closure of the protocol to the IRB; this is a rare 
occurrence and has to be done in consultation with USC PI and CISO leadership. 
 
11.1.4 Study Close-out 
At the study’s conclusion, there will be a monitoring close-out visit at which time a final review 
and reconciliation of all study and regulatory documents and study drug will be performed. 
11.1.5 Site Responsibility 
The procedures defined in the protocol and in the CRFs will be carefully reviewed by the 
investigator and his/her staff, prior to the time of study initiation, to ensure appropriate 
interpretation and implementation. No deviations from the protocol may be made. Minor 
exceptions may be approved on a case-by-case basis.  Any changes to the protocol will originate 
from the Investigator in the form of an amendment. 
11.2 Compliance Statement 
This study will be conducted in accordance with the US CFR, Title 21, Parts 11, 50, 54, 56 and 
312; the Declaration of Helsinki (1964) including all amendments and revisions; the Good Clinical 
Practices: Consolidated Guideline (E6), International Conference on Harmonization, and the Food 
and Drug Administration Guidance for Industry: Computerized Systems Used in Clinical Trials. 
11.3 Investigational Product Accountability 
The principal investigator is responsible for investigational product accountability. The 
investigator or designee must maintain a current record of the receipt and disposition of 
investigational product, including dates, quantity, kit, lot, and subject numbers. The investigator 
or responsible designee will record the receipt, dispensation by subject number, kit number, lot 
number, and disposition of the product onto the drug accountability forms. The investigator will 
maintain a copy for the institutional records. Drug dispensing records must also be maintained. 
In addition, a quality assurance release form and a shipping form (if applicable) will be included 
with each shipment stating lot number and quantity of product shipped. The investigator or 
responsible designee will sign the forms in the designated area, which certifies the receipt of 
supplies, and retain a copy for the institutional records. At sites that are dispensing drug, the 
investigator, or designee, will also maintain a dispensing record of IRX-2. 
Used and unused drug vials will be destroyed by the responsible pharmacist at each clinical site 
after study monitor reconciliation procedures. Specific instructions will be provided in the 
Investigational Product Guidelines. Each site will be requested to use the procedures that are 
locally approved for destruction of biological products, and the details of such destruction will be 
captured on a form provided in the guidelines. These forms will be retained and available for 
inspection at the study site. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
75 
11.4 Study Documents and Access to Records 
It is the responsibility of the investigator and study staff to maintain a comprehensive and 
centralized filing system of all study-related documentation, which is suitable for inspection at any 
time by the study sponsor (USC), Brooklyn ImmunoTherapeutics or its designee, the FDA and 
other Regulatory Agencies. Elements should include, but are not limited to: 
• Financial disclosure information to allow Brooklyn ImmunoTherapeutics to submit complete 
and accurate certification or disclosure statements required under Part 54 of Title 21 of the 
CFR. In addition, the investigators must provide to Brooklyn ImmunoTherapeutics a 
commitment to promptly update this information if any relevant changes occur during the 
course of the investigation and for 1 year following the completion of the study 
• Study Files, containing the curriculum vitae of all investigators and his/her designees, FDA 
Form 1572, Financial Disclosure Forms, the protocol with all amendments, the IB, copies of 
all pre-study documentation, local lab certifications and lab normal ranges, and all 
correspondence to and from local Regulatory Authorities to Brooklyn ImmunoTherapeutics or 
its designee. 
• Subject Files, containing the completed original CRFs, supporting source documentation and 
the signed informed consent form(s). 
• Dispensing records of test agents. 
The FDA requires that an investigator retain records for a period of at least 2 years from the date 
of FDA approval to market the product for this indication, or 2 years from the date that the 
application for approval is withdrawn by Brooklyn ImmunoTherapeutics. Please contact Brooklyn 
ImmunoTherapeutics or its designee prior to discarding any study-related files. 
11.5 Study Closure/Study Termination 
The investigational site or study may be terminated at any time for any reason, including but not 
limited to the following: 
• Successful completion of the study 
• Failure of the investigator to comply with the protocol or Good Clinical Practice/ International 
Conference on Harmonization guidelines 
• Safety concerns 
• Inadequate recruitment of subjects at a clinical site 
• Decision to close the study for any reason 
Disclosure of study termination will be made immediately to the IRB/IEC by the investigator. 
11.6 Quality Assurance 
Brooklyn ImmunoTherapeutics and all designees will ensure the integrity of all data collected and 
calculations made during the conduct of the study according to their quality assurance standards 
of operations. Edit checks will be run on the data and queries issued. All data will undergo 100% 
review. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
76 
11.7 Confidentiality and Publication Policy 
This trial will be listed in clinical trial databases as appropriate, e.g., www.clintrials.gov. 
No written or oral reports or information about the trial, its progress, or results obtained for the 
duration of the trial will be provided by any investigator or anyone associated with this study, to 
anyone not involved in the trial other than to the Brooklyn ImmunoTherapeutics or its designees, 
or in confidence to the IRB/IEC, without the express, written permission of Brooklyn 
ImmunoTherapeutics. 
Following the completion of the study, the data will be reported at a scientific meeting and by 
publication in a scientific journal at the discretion of the participating investigators, voting in 
proportion to the number of subjects each has entered on the study. Since this research effort is a 
multicenter investigation, each investigator acknowledges that participation in the protocol 
involves a commitment to present and/or publish the data from this study in a cooperative manner 
prior to doing so on an individual basis. 
Any abstract or publication presenting results of this trial shall be given to Brooklyn 
ImmunoTherapeutics for review and approval at least 30 days prior to intended submission to a 
scientific journal for publication or to a scientific meeting for oral or poster presentation. Brooklyn 
ImmunoTherapeutics will not arbitrarily withhold or delay agreement to present the results of this 
study in appropriate scientific settings. 
Brooklyn ImmunoTherapeutics endorses published guidelines for the publication and presentation 
of clinical study results. Brooklyn ImmunoTherapeutics and the investigators acknowledge their 
intent to resolve in good faith any differences of opinion regarding presentation or publication of 
study results. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
77 
12 STUDY MANAGEMENT 
12.1 Conflict of Interest 
All investigators will follow the University conflict of interest policy appropriate to their 
institution. Any USC investigator who has a conflict of interest with this study (patent 
ownership, royalties, or financial gain greater than the minimum allowable by their 
institution, etc.) must complete a “Statement of Outside Interests Related to Research” 
Form. The application is reviewed and approved by the Conflict of Interest Review 
Committee (CIRC) USC conflict of interest policy is available at 
http://ooc.usc.edu/conflict-interest-research 
12.2 Institutional Review Board (IRB) Approval and Consent Process 
It is expected that the IRB will have the proper representation and function in accordance 
with federally mandated regulations. The IRB should approve the consent form and 
protocol and all study related documents used in the study (e.g. QOL questionnaire, pill 
diary, brochure, advertisement etc). 
In obtaining and documenting informed consent, the investigator should comply with the 
applicable regulatory requirement(s), and should adhere to Good Clinical Practice (GCP) 
and to ethical principles that have their origin in the Declaration of Helsinki. 
Before recruitment and enrollment onto this study, the patient will be given a full 
explanation of the study and will be given the opportunity to review the consent form. 
Each consent form must include all the relevant elements currently required by the FDA 
Regulations and local or state regulations. Once this essential information has been 
provided to the patient and the investigator is assured that the patient understands the 
implications of participating in the study, the patient will be asked to give consent to 
participate in the study by signing a dated IRB approved consent form. 
Prior to a patient’s participation in the trial, the written informed consent form should be 
signed and personally dated by the patient and by the person authorized to   obtain the 
informed consent  
12.3 Required Documentation (for multi-site studies) 
Before the study can be initiated at any site, the following documentation must be 
provided to the Clinical Investigation Support Office (CISO)  
• 
A copy of the official IRB approval letter for the protocol and informed consent 
• 
IRB membership list 
• 
CVs and medical licensure for the principal investigator and any associate 
investigators who will be involved in the study 
• 
Protocol signature page with Investigator signature  
• 
Form FDA 1572 appropriately filled out and signed with appropriate 
documentation (NOTE: this is required if {institution} holds the IND.  
• 
A copy of the IRB approved consent form 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
78 
• 
CAP and CLIA Laboratory certification numbers and institution lab normal 
values 
• 
Executed clinical research contract 
 
12.4 Registration Procedures 
Multi-Site Registration:  
All participants in the multi-site trial are subject to central registration, which is used for tracking 
study accrual, checking eligibility, and monitoring adequate participation of women and 
minorities. Subject registration will be conducted through the coordinating center at the NCCC-
CISO. External sites will identify eligible subjects and verify enrollment availability with the MCC 
prior to consenting patients. The external site is required to notify the MCC of a new signed 
informed consent within 48 business hours and note the basic consent information on the screening 
log. A copy of the consent will accompany the complete eligibility packet for verification. The 
MCC will enter the patient, demographic, and consent information in the applicable USC database. 
The MCC will assign a study patient sequence ID and communicate this to the external site. 
 
The Coordinating Center Program Hours are 8 am to 4 pm, Monday through Friday, based on the 
PST zone. The MCC will be closed on official government holidays unless otherwise indicated. 
The contact number for the MCC is 323-865-3122. A copy of the registration sheet is located in 
the Appendix. 
 
External sites will verify eligibility prior to submitting documents to the MCC for central 
registration. External sites must submit registration requests to the MCC at least one full business 
day prior to the planned treatment start date. Registration will require the external site to submit to 
the MCC all of the following: 
 
• A completed registration form with patient demographics: 
• 
Zip code 
• 
Payor Source 
• 
Age 
• 
Sex 
• 
Race 
• 
Ethnicity 
• 
Initials 
• 
Date of Birth (DOB) 
• A completed Eligibility Checklist signed by the investigator 
• A copy of the most recently IRB-approved, patient signed informed consent form 
• All required screening tests, within the time parameters specified by the protocol study 
calendar 
• All other de-identified source documents needed to verify all points of eligibility 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
79 
• Any On-Study forms for registration specified by protocol 
 
These documents must be securely emailed to the MCC staff. With advance notice documents will 
also be accepted faxed to 323-865-0457. The MCC will verify completeness of documents and 
confirm eligibility. The MCC will enter the registration information in the USC OnCore® database. 
The MCC will then fax or securely email the completed Registration Form with the assigned study 
sequence ID to the external site as confirmation of patient registration. 
An external site must maintain a log of all subjects who sign informed consents. The log must also 
document an explanation for exclusion due to screen failure. The MCC will provide sites with a 
Patient Tracking Log at the time of site activation. In the event of screen failure, external sites 
must submit the Screen Failure form to the MCC within one business day of determining screen 
failure. 
 
Participating sites are required to retain, in a confidential manner, sufficient information on each 
subject so that the subject may be contacted should the need arise. 
 
All documents, investigative reports, or information relating to the patient are strictly confidential. 
Any patient specific reports (i.e. Pathology reports, MRI reports, Operative reports, etc.) submitted 
to the CISO-MCC must have the patient’s full name and social security number redacted (blacked 
out) and the assigned CISO-MCC patient ID number, protocol number, and site number written 
in. Patient initials only may be included or retained for cross verification of identification. 
 
A registration verification letter will be emailed (preferred) or faxed to the registering site within 
one working day for patients registered to CISO-MCC multi-site trials. Treatment may not be 
initiated until the site receives this faxed or emailed verification.  
USC Registration:  
For patients enrolled at USC, the Research Coordinator must complete the protocol 
eligibility form to ensure that the patient is eligible. The PI will review the patient 
eligibility (with assistance from the Research Coordinator- who will assemble the 
required source documents, and do an initial review) prior to registering the patient on 
study. 
The Research Coordinator or data manager will then register the patient into the Cancer 
Center database, Café, by accessing the Registration forms. Likewise, after the patient 
has completed the study, the Off Study forms in cafe will need to be completed, for Off 
Treatment and Off Study. 
 
12.5 RECORDS AND DATA SUBMISSION 
A. 
Confidentiality of Records 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
80 
The original data collection forms will be kept in secure file cabinets, for USC 
patients forms will be kept in the Clinical Investigations Support Office (CISO). 
For additional clinical sites, original records may be stored locally in a secure 
fashion. Copies of data collection forms shall be sent to USC. 
 
B. 
 Patient Consent Form   
At the time of registration, signed and dated copies of the patient Informed 
Consent with the Human Rights and the HIPAA authorization must be given to 
the patient. Institutional policy regarding distribution and location of original 
consent documents should be followed.  When a study is opened at two or more 
institutions, a copy of the signed consent and HIPAA should be sent to USC 
CISO QA team as soon as possible, and not later than within 5 business days of 
obtaining consent.  For patients consented at USC/LAC, institutional policy 
should be followed: a copy of ICF and HIPAA should be uploaded through   True 
to USC CRO and to CISO QA Team. The original will be kept in the patient 
research chart maintained by the study assigned Data Manager. 
 
C. 
  Registration Eligibility Worksheet   
At the time of registration, the completed Eligibility Worksheet will be submitted 
to the QA Monitor at CISO for review of eligibility compliance. 
 
D. 
Data Collection Forms and Submission Schedule 
If a treatment trial, protocol data will be entered into eCRFs in Café. 
Within two weeks of registration, the data manager will complete the initial set of 
On Study forms and baseline Toxicities. 
Within two weeks of completion of each course of treatment, the data manager 
must complete the Course Assessment, Toxicities, and if appropriate Response 
data. 
After Off Treatment, within two weeks of each follow up, complete the Follow 
Up forms. 
 
12.6 Data Management and Monitoring/Auditing            
12.6.1 Active Monitoring Program Details 
a. 
Adherence to Protocol/Per Patient: It is the responsibility of the USC 
Principal Investigator (PI) to ensure that patient recruitment and enrollment, 
treatment, follow-up for toxicities and response, and documentation and 
reporting at USC are all performed as specified in the protocol. When a study is 
opened at two or more institutions, the PI at each institution will assume the 
responsibilities for the day-to-day monitoring of the trial, as described below. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
81 
b. 
Day-to-Day Monitoring – Eligibility:  At USC, the Study Coordinator will 
assist the Investigator in reviewing eligibility and will assemble the required 
source documents, and do a final review by completing an Eligibility 
Registration Worksheet. When a study is opened at two or more institutions, the 
PI at each institution will review the patient eligibility in accordance with that 
institution’s policy.  For all institutions, the Eligibility Registration Worksheet 
with a copy of Informed Consent and supporting source documents will be 
submitted to CISO QA via email or Fax for verification prior to registering the 
patient on study. 
c. 
Day-to-Day Monitoring – Informed Consent: Prior to registering the 
patient on study, the Study Coordinator will review the informed consent, to 
ensure that the patient has signed and dated the most current IRB-approved form, 
and that the form has been signed and dated by the person obtaining the consent 
as well as appropriate witnesses. A copy of the ICF will also be provided to 
CISO QA for review. CISO SOP 3.3 will be followed.  
d. 
Day-to-Day Monitoring – Treatment: The PI and co-investigators are 
responsible for ensuring that treatment is given per protocol. The Study 
Coordinator will review the treatment orders with the treating investigator. 
Regardless of who the treating physician is, there will be only one responsible 
Study Coordinator for each study at each of the hospitals affiliated with the USC 
Norris Cancer Center. The treating investigator will review the status of each 
patient on-study, with the Study Coordinator and treating physicians, on an on-
going basis.  When a study is opened at two or more institutions, CISO QA will 
periodically audit medical records for the subjects on study at other institutions 
to ensure compliance and adherence to the protocol. 
e. 
Data Management – Patient Charts: When a study is opened at two or 
more institutions, the policy in place at each institution will be followed for 
maintaining medical and research related records. Such policies will be provided 
to the CISO QA prior to enrolling 1st patient.  At USC, All written source 
documents not associated with the study research are maintained in the patient 
chart, which is stored in the Department of Medical Records at the appropriate 
hospital. At the Norris Hospital, the official medical record is the Electronic 
Patient File (EPF). Radiographical images are stored in the Department of 
Radiology and in an electronic system called Synapse. At Los Angeles County 
General Hospital the official medical record is called ORCHID. These are the 
permanent, official documents for each patient on-study. A copy of the signed 
informed consent, physician’s notes, orders, test results and pathology notes are 
maintained in the patients’ hospital charts. It is the responsibility of the research 
staff to ensure that the patient chart contains the required documents and work 
closely with treating investigators to ensure all protocol-related assessments are 
carefully documented. 
f. 
Data Management – Research Charts: When a study is opened at two or 
more institutions, the policy in place at each institution will be followed for 
maintaining medical and research related records. Such policies will be provided 
to the CISO QA prior to enrolling 1st patient.  At USC, to facilitate adherence to 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
82 
the protocol schedule and data management, research charts are created to collect 
copies of the relevant notes, orders and results, that are in the Patient Chart. In 
addition, all source documents related to the research, such as original informed 
consent forms, HIPAA Forms, AE assessment worksheets, disease response 
worksheets and NTFs are maintained in the Research Charts. Protocol calendars, 
worksheets, and checklists, are also kept in the research chart. These are 
maintained in the Clinical Investigation Support Office until the study is 
completed and the results are published and no further need is anticipated. These 
are then stored off-site. It is the responsibility of the Data Manager to ensure that 
the research chart contains all the required documents. 
g. 
Data Management – Case Report Forms: It is the responsibility of the Data 
Manager to complete the required case report forms. For in-house trials, case 
report forms are developed for each trial; these are used to finalize the data entry 
screens in the Cancer Center clinical trials database. It is the responsibility of the 
PI to review the Off-Study Summary form which summarizes pertinent toxicity, 
response and adherence information, once the patient has completed treatment. 
 
12.6.2 Quality Assurance Monitoring Committee (QAMC) Oversight 
The Quality Assurance and Monitoring Committee (QAMC) of the NCCC has the 
responsibility for study auditing and monitoring for protocol compliance, data 
accuracy, performance of audits and monitoring of accrual. QAMC procedures 
are detailed in the NCCC Data Safety and Monitoring Plan available on CISO 
Website. 
12.6.2.1 QAMC Annual Patient Audits 
The QAMC is responsible for conducting audits and providing the initial 
review of the audits, for all open institutional (i.e. USC initiated), CCCP-
sponsored trials, and any trials identified by the CIC.  These trials are 
audited by the QAMC once a year. Faculty and staff at the Cancer Center 
involved in clinical research – but not directly involved in the research 
under evaluation – are asked to serve as auditors. Twenty percent of 
patients accrued during the past 12 months – and a minimum of 2 patients 
– are selected at random; however, additional patients may be selected for 
audit if there is some indication that there might have been a problem or 
unusual circumstance (possibly related to compliance, toxicity, response 
or some indication of an irregularity). The audit involves a review of the 
research chart, hospital medical record (i.e., source documentation) and 
evaluates the following: documentation of eligibility (including failure to 
obtain appropriate informed consent) and baseline status of the patient; 
documentation of adherence to protocol-specified treatment and follow-
up; evaluation of toxicity; and evaluation of response or other outcome. In 
addition, for investigative agents, a drug audit is also performed for these 
patients by the Research Pharmacist. In addition, for Institutional, 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
83 
Investigator Initiated Trials, Data in the CAFÉ database are compared to 
the information in the medical record.   
12.6.2.2 QAMC Annual Protocol Review 
All open trials are reviewed at least once a year by the QAMC (or more 
often if stipulated by the CIC). This annual review includes the following: 
evaluation of the current accrual relative to the planned total accrual; 
examination of gender and minority accrual; examination of all reported 
violations; review of past audits and correspondence with the PI; review of 
results of current audit (by an outside agency or by the NCCC QAMC); 
review of previous correspondence between the PI and the QAMC/DSMC. 
The QAMC review process is detailed in USC NCCC DSM Plan available 
on the CISO website. 
12.6.3 Data and Safety Monitoring Committee (DSMC) Oversight 
The Data and Safety Monitoring Committee (DSMC) is an independent body responsible 
for the safety of study subjects through the review of new protocols to ensure an adequate 
adverse event assessment/reporting plan, study stopping rules and through the real-time 
and periodic monitoring of severe adverse events (SAEs) or those AEs that require 
expedited reporting. The DSMC performs quarterly and annual safety reviews as well as 
interim efficacy/futility analyses on institutional trials. DSMC procedures are detailed in 
USC NCCC DSM Plan available on the CISO website. 
12.6.4 Phase I Committee Oversight (for Phase I or I/II IIT only) 
The USC Norris Comprehensive Cancer Center Phase I DLT committee reviews all open 
institutional phase I studies at regularly scheduled intervals. The committee reviews the 
adverse events, serious adverse events, and treatment administration for each patient 
during the DLT observation period as specified per protocol. The committee will 
determine whether a patient is evaluable for DLT and whether an AE meets the DLT 
definition or not. Decisions regarding dose escalation, de-escalation and cohort expansion 
are made by the committee in coordination with the PI. 
12.7 Adherence to the Protocol 
Except for an emergency situation in which proper care for the protection, safety, and 
well-being of the study patient requires alternative treatment, the study shall be 
conducted exactly as described in the approved protocol.  
12.7.1 Emergency Modifications 
Investigators may implement a deviation from, or a change of, the protocol to 
eliminate an immediate hazard(s) to trial subjects without prior IRB approval.  
For any such emergency modification implemented, an IRB modification form 
must be completed within five (5) business days of making the change.  
12.7.2 Non-Emergency departures from protocol  
A protocol deviation is any variance from an IRB approved protocol.  


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
84 
If the deviation meets all of the following criteria, it is considered a minor 
protocol deviation that:  
• 
Is generally noted or recognized only after it occurs 
• 
Has no substantive effect on the risks to research participants 
• 
Has no substantive effect on the scientific integrity of the research plan or 
the value of the data collected  
• 
Did not result from willful or knowing misconduct on the part of the 
investigator(s).  
 
If the deviation meets any of the following criteria, it is considered a protocol 
violation: 
• 
Has harmed or increased the risk of harm to one or more research 
participants. 
• 
Has damaged the scientific integrity of the data collected for the study. 
• 
Results from willful or knowing misconduct on the part of the 
investigator(s). 
• 
Demonstrates serious noncompliance with federal regulations, State laws, 
or University policies. 
Protocol Deviations: personnel will report to any sponsor or data and safety 
monitoring committee in accordance with their policies.  
Protocol Violations: All protocol violations will be entered in the clinical trial 
database by the Research Coordinator. In addition, Research Coordinator and 
Investigator should report all protocol violations within one (1) week of the 
knowledge of the event using iStar. 
 
12.7.3 Amendments to the Protocol    
Should amendments to the protocol be required, the amendments will be 
originated and documented by the Principal Investigator. It should also be noted 
that when an amendment to the protocol substantially alters the study design or 
the potential risk to the patient, a revised consent form might be required.  
The written amendment, and if required the amended consent form, must be sent 
to the IRB as well as to all the sponsoring agencies (FDA, NCI, etc.) for review 
and for approval prior to implementation. It is the responsibility of the study PI to 
ensure that the appropriate agencies have been informed of the proposed 
amendments and that these have been reviewed and approved.   
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
85 
12.8 Record Retention   
Study documentation includes all Case Report Forms, data correction forms or queries, 
source documents, Brooklyn ImmunoTherapeutics-Investigator correspondence, 
monitoring logs/letters, and regulatory documents (e.g., protocol and amendments, IRB 
correspondence and approval, signed patient consent forms). 
Source documents include all recordings of observations or notations of clinical activities 
and all reports and records necessary for the evaluation and reconstruction of the clinical 
research study. 
Government agency regulations and directives require that the study investigator must 
retain all study documentation pertaining to the conduct of a clinical trial. In the case of a 
study with a drug seeking regulatory approval and marketing, these documents shall be 
retained for at least two years after the last approval of marketing application in an 
International Conference on Harmonization (ICH) region. In all other cases, study 
documents should be kept on file until three years after the completion and final study 
report of this investigational study. 
12.9 Obligations of Investigators 
The Principal Investigator is responsible for the conduct of the clinical trial at the site in 
accordance with Title 21 of the Code of Federal Regulations and/or the Declaration of 
Helsinki. The Principal Investigator is responsible for personally overseeing the treatment 
of all study patients. The Principal Investigator must assure that all study site personnel, 
including sub-investigators and other study staff members, adhere to the study protocol 
and all FDA/GCP/NCI regulations and guidelines regarding clinical trials both during 
and after study completion. 
The Principal Investigator at each institution or site will be responsible for assuring that 
all the required data will be collected and entered onto the Case Report Forms. 
Periodically, monitoring visits will be conducted and the Principal Investigator will 
provide access to his/her original records to permit verification of proper entry of data. At 
the completion of the study, all case report forms will be reviewed by the Principal 
Investigator and will require his/her final signature to verify the accuracy of the data. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
86 
13 REFERENCES 
Committee on Gynecologic Practice of American College of Obstetricians and Gynecologist. 
ACOG Committee Opinion no. 509: management of vulvar intraepithelial neoplasia. Obstet 
Gynecol 2011;118:1192. 
Committee on Practice Bulletins – Gynecology. ACOG Practice Bulletin Number 131: Screening 
for cervical cancer. Obstet Gynecol 2012; 120:1222 
Alani RM, Munger K. Human papillomaviruses and associated malignancies. J Clin Oncol 
1998;16:330. 
Albrechtsen S, Rasmussen S, Thoresen S, et al. Pregnancy outcome in women before and after 
cervical conisation: population based cohort study. BMJ 2008;337:a1343. 
Aldara Prescribing Information, 2010; 
http://www.accessdata.fda.gov/drugsatfda_docs/label/2010/020723s022lbl.pdf 
Arbyn M, Kyrgiou M, Simeons C, et al. Perinatal mortality and other severe adverse pregnancy 
outcomes associated with treatment of cervical intraepithelial neoplasia: meta-analysis. BMJ 
2008;337:a1284. 
Bachaud JM, Cohen-Jonathan E, Alzieu C, et al. Combined postoperative radiotherapy and 
weekly cisplatin infusion for locally advanced head and neck carcinoma: final report of a 
randomized trial. Int J Radiat Oncol Biol Phys. 1996;36:999-1004. 
Baldauf JJ, Dreyfus M, Ritter J, Meyer P, Philippe E. Risk of cervical stenosis after large loop 
excision or laser conization. Obstet Gynecol 1996;88:933 
Baldwin PJ, van der Burg SH, Boswell CM, et al. Vaccinia-expressed human papillomavirus 16 
and 18 E6 and E7 as a therapeutic vaccination for vulval and vaginal intraepithelial neoplasia. 
Clin Cancer Res 2003;9:5205-13. 
Berd D, Mastrangelo MJ, Engstrom PF, et al. Augmentation of the human immune response by 
cyclophosphamide. Cancer Res 1982;42:4862-4866. 
Berinstein NL, Wolf GT, Naylor PH, et al. Increased lymphocyte infiltration in patients with 
head and neck cancer treated with the IRX-2 immunotherapy regimen. Cancer Immunol 
Immunother 2012;61:771-782. 
Bernier J. Cetuximab in the treatment of head and neck cancer. Expert Rev Anticancer Ther 
2006;11:1539-52. 
Bernier J, Domenge C, Ozsahin M, et al. Postoperative irradiation with or without conconmitant 
chemotherapy for locally advanced head and neck cancer. NEJM 2004;19:1945-52. 
Bianchini C, Ciorba A, Stomeo F, et al. Immunonutrition in head and neck cancer: have a look 
before surgery! Eur Arch Otorhinolaryngol. 2012;Jan;269(1):5-8 
Blewett HJ, Taylor CG. Dietary zinc deficiency in rodents: effects on T-cell development, 
maturation and phenotypes. Nutrients 2012;4:449-66 
Bosch X, Harper D. Prevention strategies of cervical cancer in the HPV vaccine era. Gynecol 
Oncol 2006;103:21. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
87 
Brookes GB, Clifford P. Nutritional status and general immune competence in patients with head 
and neck cancer. J Royal Soc Med 1981;74:132-139. 
Bruinsma FJ, Quinn MA. The risk of preterm birth following treatment for precancerous changes 
in the cervix: a systematic review and meta-analysis. BJOG 2011;118:1031-41. 
Centers for Disease Control and Prevention (CDC). Human Papillomavirus Vaccination 
Coverage Among Adolescent Girls, 2007–2012, and Postlicensure Vaccine Safety Monitoring, 
2006–2013—United States. MMWR Recomm Rep. 2013;62:591 
Chung CH, Zhang Q, Kong CS, et al. p16 protein expression and human papillomavirus status as 
prognostic biomarkers of nonoropharyngeal head and neck squamous cell carcinoma. J Clin 
Oncol. 2014;32:3930-8. 
Cooper JS, Pajak TF, Forastiere AA, et al. Postoperative concurrent radiotherapy and 
chemotherapy for high-risk squamous-cell carcinoma of the head and neck. NEJM 
2004;19:1937-44. 
Czystowska M, Han J, Szczepanski MJ, et al. IRX-2, a novel immunotherapeutic, protects 
human T cells from tumor-induced cell death. Cell Death Differ. 2009;16:708-718. 
Czystowska M, Szczepanski MJ, Szajnik M, et al. Mechanisms of T-cell protection from death 
by IRX-2: a new immunotherapeutic. Cancer Immunol Immunother. 2011;60:495-506. 
Darragh TM, Colgan TJ, Thomas Cox J, et al. The Lower Anogenital Squamous Terminology 
Standardization prject for HPV-associated lesions: background and consensus recommendations 
from the College of American Pathologists and the American Society for Colposcopy and 
Cervical Pathology. Int J Gynecol Pathol 2013;32:76. 
Da Silva DM, Woodham AW, Naylor PH, Egan JE, Berinstein NL, Kast, WM. 
Immunostimulatory activity of the cytokine-based biologic, IRX-2, on human papillomavirus-
exposed Langerhans cells. J Interferon Cytokine Res. 2016;36(5):291-301. 
doi:10.1089/jir.2015.0115 
Da Silva DM, Woodham AW, Skeate JG, et al. Langerhans cells from women with cervical 
precancerous lesions become functionally responsive against human papillomavirus after 
activation with stabilized Poly-I:C. 2015;161(2):197-208. 
Davidson EJ, Boswell CM, Sehr P, et al. Immunological and clinical responses in women with 
vulval intraepithelial neoplasia vaccinated with a vaccinia virus encoding human papillomavirus 
16/18 oncoproteins. Cancer Res 2003;63:6032-41. 
Deacon JM, Evans CD, Yule R, et al. Sexual behavior and smoking as determinants of cervical 
HPV infection and of CIN 3 among those infected: a case-control study nested within the 
Manchester cohort. Br J Cancer 2000;83:1565. 
Dueñas-Gonzalez A, Verastegui E, Lopez-Graniel C, Gonzalez A, Mota A, Barrera-Franco JL, et 
al. A pilot study of perilymphatic leukocyte cytokine mixture (IRX-2) as neoadjuvant treatment 
for early stage cervical carcinoma. International Immunopharmacol 2002;2:1007-1016. 
Egan JE, Quadrini KJ, Santiago-Schwarz F, et al. IRX-2, a novel in vivo immunotherapeutic, 
induces maturation and activation of human dendritic cells in vitro. J Immunother 2007;30:624-
633. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
88 
Emens LA, Jaffee EM. Leveraging the activity of tumor vaccines with cytotoxic chemotherapy. 
Cancer Res 2005;65:8059-8064. 
Fahey LM, Raff AB, Da Silva DM, Kast WM. Reversal of human papillomavirus-specific T cell 
immune suppression through TLR agonist treatment of Langerhans cells exposed to human 
papillomavirus type 16. J Immunol. 2009;182:2919-28.  
Fausch S, Fahey LM, Da Silva DM, Kast WM. Human papillomavirus can escape immune 
recognition through Langerhans cell phosphoinositide 3-kinase activation. J Immunol 
2005;174:7172-7178. 
FDA News Release, December 10, 2014. FDA approves Gardasil 9 for prevention of certain 
cancers caused by five additional types of HPV. 
http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm426485.htm  
Forman D, de Martel C, Lacey CJ. Global burden of human papillomavirus and related diseases. 
Vaccine 2012;30:Supplement 5. 
Fox H, Wells M. Recent advances in pathology of the vulva. Histopathology 2003;42:209. 
Frazer IH. The role of vaccines in the control of STDs: HPV vaccines. Genitourin Med 
1996;72:398-403. 
Freeman SM, Franco JLB, Kenady DE, Baltzer L, Roth Z, Brandwein HJ, Hadden JW. A Phase 
1 safety study of an IRX-2 regimen in patients with squamous cell carcinoma of the head and 
neck. Am J Clin Oncol 2010;34:173-8. 
Freiser ME, Serafini P, Weed DT. The immune system and head and neck squamous cell 
carcinoma: from carcinogenesis to new therapeutic opportunities. Immunol Res. 2013;57:52-59. 
Friedrich ER Jr, Wilkinson EJ, Fu YS. Carcinoma in situ of the vulva: a continuing challenge. 
Am J Obstet Gynecol 1980;136:830. 
Gildener-Leapman N, Ferris RL, Bauman JE. Promising systemic immunotherapies in head and 
neck squamous cell carcinoma. Oral Oncol. 2013;49:1089-1096. 
Giuliano AG, Sedjo RL, Roe, DJ. Clearance of oncogenic human papillomavirus (HPV) 
infection: effect of smoking (United States). Cancer Causes and Control. 2002;13:839–846. 
Good RA, Fernandes G. Nutrition, immunity, and cancer-a review. Part I: Influence of protein or 
protein-calorie malnutrition and zinc deficiency on immunity. Clin Bull 1979;1:3-12. 
Haase H, Rink L. Zinc signals and immune function. Biofactors 2014;40:27-40. 
Hadden JW, Endicott J, Baekey P, et al. Interleukins and contrasuppression induce immune 
regression of head and neck cancer. Arch Otolaryngol Head Neck Surg. 1994;120:395-403. 
Jakobsson M, Gissler M, Paavonen J, Tapper AM. Long-term mortality in women treated for 
cervical intraepithelial neoplasia. BJOG 2009a;116:838-44 
Jakobsson M, Gissler M, Paavonen J, Tapper AM. Loop electrosurgical excision procedure and 
the risk for preterm birth. Obstet Gynecol 2009b;114:504-10 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
89 
Katki HA, Schiffman M, Castle PE, Fetterman B, Poitras NE, Lorey T, et al. Five-year risk of 
cervical cancer and CIN3 for HPV-positive and HPV-negative high-grade Pap results. J Low 
Genit Tract Dis 2013;17:S50-S55 
Keen CL, Gershwin ME. Zinc deficiency and immune function. Ann Rev Nutr 1990;10:413-415. 
Kenter GG, Welters MJP, Valentijn ARPM, Lowik MJG, Berends-van der Meer DMA, Vloon 
APG, et al. Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N 
Engl J Med 009;361:1838-47. 
Khalid S, Dimitriou E, Conroy R, Paraskevaidis E, Kyrgiou M, Harrity C, et al. The thickness 
and volume of LLETZ specimens can predict the relative risk of pregnancy related morbidity. 
BJOG 2012;119;685-91. 
Kyrgiou M, Koliopoulos G, Martin-Hirsch P, et al. Obstetric outcomes after conservative 
treatment for intraepithelial or early invasive cervical lesions: systemic review and meta-
analysis. Lancet 2006;367:489. 
Kyrgiou M, Mitra A, Arbyn M, et al. Fertility and early pregnancy outcomes after treatment for 
cervical intraepithelial neoplasia: systematic review and meta-analysis. BMJ 2014;349:g6192. 
Lapointe H, Lampe H, Banerjee D. Head and neck squamous cell carcinoma cell line-induced 
suppression of in vitro lymphocyte proliferative responses. Otolaryngol Head Neck Surg 
1992;106:149-158. 
Lee KR, Darragh TM, Joste NE, Krane JF, Sherman ME, Hurley LB, et al. Atypical glandular 
cells of undetermined significance (AGUS): interobserver reproducibility in cervical smears and 
corresponding thin-layer preparations. Am J Clin Pathol 2002;117:96-102 
Lerner D, Parsons SK, Justicia-Linde F, Chelmow D, Chang H, Rogers WH, et al. The impact of 
pre-cancerous cervical lesions on functioning at work and work productivity. J Occup Environ 
Med. 2010;52:926-33. 
Machiels J-PH, Reilly RT, Emens LA, et al. Cyclophosphamide, doxorubicin, and paclitaxel 
enhance the antitumor immune response of granulocyte/macrophage-colony stimulating factor-
secreting whole-cell vaccines in HER-2/neu tolerized mice. Cancer Research 2001;61:3689-
3697. 
Mahto M, Nathan M, O’Mahony C. More than a decade on: review of the use of imiquimod in 
lower anogenital intraepithelial neoplasia. Int J STD AIDS 2010;21:8. 
Martin PJ, Davenport-Ennis N, Petrelli NJ, et al. Responsibility for costs associated with clinical 
trials. J Clin Oncol. 2014;32:3357-9. 
Martin-Hirsch PPL, Paraskevaidis E, Bryant A, Dickinson HO. Surgery for cervical 
intraepithelial neoplasia. Cochrane Database Syst Rev 2013;12: CD001318. doi: 
10.1002/14651858.CD001318.pub3 
Massad LS, Einstein, MH, Huh WK, et al. 2012 Updated consensus guidelines for the 
management of abnormal cervical cancer screening tests and cancer precursors. J Low Genit 
Tract Dis. 2013;17:S1. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
90 
Melnikow J, McGahan C, Sawaya GF, et al. Cervical intraepithelial neoplasia outcomes after 
treatment long-term follow-up from the British Columbia Cohort Study. J Natl Cancer Inst 
2009;101:721. 
Montz FJ. Management of high-grade cervical intraepithelial neoplasia and low-grade squamous 
intraepithelial lesion and potential complications. Clin Obstet Gynecol 2000; 43:394. 
Moscicki AB, Hills N, Shiboski S, Powell K, Jay N, Hanson E, et al. Risks for incident human 
papillomavirus infection and low-grade squamous intraepithelial lesion development in young 
females. JAMA. 2001;285:2995-3002. 
Moscicki AB, Ma Y, Wibbelsman C, et al. Rate of and risks for regression of cervical 
intraepithelial neoplasia 2 in adolescents and young women. Obstet Gynecol 2010;116:1373. 
Munk AC, Kruse AJ, van Diermen B, et al. Cervical intraepithelial neoplasia grade 3 lesions can 
regress. APMIS 2007;115(12):1409-14. 
Muntz HG. Cervical cancer screening and management of the abnormal Papanicolaou smear. In: 
Current Care of Women: Diagnosis & Treatment. 2nd Edition. Lemcke DP, Pattison J, Marshall 
LA, Cowley DS (eds). McGraw-Hill/Appleton & Lange, New York, NY, 2004, pp. 520-531. 
Muntz HG. Can cervical adenocarcinoma in situ be safely managed by conization alone?  
Gynecol Oncol 1996;61:301-303 
Muntz HG, Bell DA, Lage JM, Goff BA, Feldman S, Rice LW. Adenocarcinoma in-situ of the 
uterine cervix. Obstet Gynecol 1992;80:935-939 
National Cancer Institute. Common Terminology Criteria for Adverse Events (CTCAE) Version 
4.03, June 14, 2010. 
Naylor PH, Hernandez KE, Nixon AE, et al. IRX-2 increases the T cell-specific immune 
response to protein/peptide vaccines. Vaccine 2010;28:7054-7062. 
Noehr B, Jensen A, Frederiksen K, et al. Loop electrosurgical excision of the cervix and 
subsequent risk for spontaneous preterm delivery: a population-based study of singleton 
deliveries during a 9-year period. Am J Obstet Gynecol 2009;201:33.e1. 
North RJ. Cyclophosphamide-facilitated adoptive immunotherapy of an established tumor 
depends on elimination of tumor-induced suppressor T cells. J Exp Med 1982;55:1063-1074. 
North RJ. The murine antitumor immune response and its therapeutic manipulation. Adv 
Immunol 1984;35:89-155. 
Oken M, Creech R, Tormey D, et al. Toxicity and response criteria of the Eastern Cooperative 
Oncology Group. Am J Clin Oncol. 1982;5:649-655. 
Paraskevaidis E, Arbyn M, Sotiriadis A, Diakomanolis E, Martin-Hirsch P, Koliopoulos G, et al. 
The role of HPV DNA testing in the follow-up period after treatment for CIN: a systematic 
review of the literature. Cancer Treat Rev 2004;30:205-11. 
Rebolj M, Helmerhorst T, Habbema D, et al. Risk of cervical cancer after completed post-
treatment follow-up of cervical intraepithelial neoplasia: population based cohort study. BMJ 
2012;345:e6855. 
Reyes MC, Cooper K. An update on vulvar intraepithelial neoplasia: terminology and a practical 
approach to diagnosis. J Clin Pathol 2014;67:290. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
91 
Rutledge F, Sinclair M. Treatment of intraepithelial carcinoma of the vulva by skin excision and 
graft. Am J Obstet Gynecol 1968;102:807. 
Sadler L, Saftlas A, Wang W, Exeter M, Whittaker J, McCowan L. Treatment for cervical 
intraepithelial neoplasia and risk of preterm delivery. JAMA 2004;29:2100-6. 
Schiffman MH, Bauer HM, Hoover RN, et al. Epidemiologic evidence showing that human 
papillomavirus infection causes most cervical intraepithelial neoplasia. J Natl Cancer Inst 
1993;85:958. 
Schilling B, Harasymczuk M, Schuler P, et al. IRX-2, a novel biologic, favors the expansion of T 
effector over T regulatory cells in a human tumor microenvironment model. J Mol Med (Berl) 
2012;90:139-147. 
Schilling B, Harasymczuk M, Schuler P, et al. IRX-2, a novel immunotherapeutic, enhances 
functions of human dendritic cells. PLoS One 2013;8:e47234. 
Sideri M, Jones RW, Wilkinson EJ, et al. Squamous vulvar intraepithelial neoplasia: 2004 
modified terminology, ISSVD Vulvar Oncology Subcommittee. J Reprod Med 2005;50:807. 
Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2015. CA Cancer J Clin 2015;65:5-29 
Słotwińska SM, Słotwiński R. Host response, malnutrition and oral diseases. Part 1. Cent Eur J 
Immunol. 2014;39(4):518-21. 
Smyth LJ, van Poelgeest MI, Davidson EJ, et al. Immunological responses in women with 
human papillomavirus type 16 (HPV-16)-associated anogenital intraepithelial neoplasia induced 
by heterologous prime-boost HPV-16 oncogene vaccination. Clin Cancer Res 2004;10:2954-61. 
Solomon D, Davey D, Kuman R, et al. The 2001 Bethesda System: terminology for reporting 
results of cervical cytology. JAMA 2002;287:2114. 
Stanley MA, Pett MR, Coleman N. HPV: from infection to cancer. Biochemical Society 
Transactions 2007;35,part 6:1456-60. 
Svare JA, Andersen LF, Langhoff-Roos J, et al. The relationship between prior cervical 
conization, cervical microbial colonization and preterm premature rupture of the membranes. Eur 
J Obstet Gynecol Reprod Biol. 1992;47:41. 
Thun MJ, Henley SJ, Patrono C. Nonsteroidal anti-inflammatory drugs as anticancer agents: 
mechanistic, pharmacologic, and clinical issues. J Natl Cancer Inst. 2002;94:252-66. 
Tota JE, Chevarie-Davis M, Lyndsay A. Richardson LA. Epidemiology and burden of HPV 
infection and related diseases: Implications for prevention strategies. Preventive Medicine 
2011;53:S12–S21. 
Trimble CL, Piantadose S, Gravitt P, et al. Spontaneous regression of high-grade cervical 
dysplasia: effects of human papillomavirus type and HLA phenotype. Clin Cancer Res. 
2005;11(13):4717-23. 
van Poelgeest MI, Welters MJ, Vermeij R, et al. Vaccination against oncoproteins of HPV16 for 
noninvasive vulvar/vaginal lesions: lesion clearance is related to the strength of the T-cell 
response. Clin Cancer Res. 2016;22(10):2342-50. 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
92 
van Seters M, van Beurden M, de Craen AJ. Is the assumed natural history of vulvar 
intraepithelial neoplasia III based on enough evidence?  A systematic review of 3322 published 
patients. Gynecol Oncol 2005; 97:645 
Whiteside TL, Butterfield LH, Naylor PH, et al. A short course of neoadjuvant IRX-2 induces 
changes in peripheral blood lymphocyte subsets of patients with head and neck squamous cell 
carcinoma. Cancer Immunol Immunother 2012;61:783-788. 
Widgren K, Simonsen J, Valentiner-Branth P, Mølbak K. Uptake of the human papillomavirus-
vaccination within the free-of-charge childhood vaccination programme in Denmark. Vaccine. 
2011 Dec;29(52):9663-7. Epub 2011 Oct 18 
Wintergerst ES, Maggini S, Hornig DH. Contribution of selected vitamins and trace elements to 
immune function. Ann Nutr Metab. 2007;51(4):301-23.  
Wolf GT, Fee WE, Dolan RW, et al. Novel neoadjuvant immunotherapy regimen safety and 
survival in head and neck squamous cell cancer. Head Neck 2011;33:1666-1674. 
Woodham AW, Raff AB, Da Silva DM, Kast WM. Molecular analysis of human papillomavirus 
virus-like particle activated Langerhans cells in vitro. Methods Mol Biol. 2015;1249:135-49. 
Workowski KA, Bolan GA. Centers for Disease Control and Prevention. Sexually transmitted 
diseases treatment guidelines, 2015. MMWR Recomm Rep 2015;64:1. 
Zhao C, Florea A, Onisko A, Austin RM. Histologic follow-up results in 662 patients with Pap 
test findings of atypical glandular cells: results from a large academic women’s hospital 
laboratory employing sensitive screening methods. Gynecol Oncol 2009;114:383-9 
 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
93 
Appendix 1: 
Cervical Intraepithelial Neoplasia Nomenclature 
Descriptive Terminology 
Mild 
Dysplasia 
Moderate 
Dysplasia 
Severe 
Dysplasia 
Carcinoma 
in situ 
Bethesda 
Classification 
Systema 
Cytology 
LSIL 
HSIL 
Histology 
CIN 1 
CIN 2 
CIN 3 
LAST Systemb 
Cytology 
LSIL 
HSIL 
Histology 
LSIL 
Perform p16 
staining: 
negative is LSIL 
positive is HSIL 
HSIL 
a. 
Solomon, 2002 
b. 
Darragh, 2013 
 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
94 
Appendix 2: 
Vulvar Intraepithelial Neoplasia Nomenclature 
Descriptive Terminology 
Mild 
Dysplasia 
Moderate 
Dysplasia 
Severe 
Dysplasia 
Carcinoma 
in situ 
VIN Terminology 
(Derived from Bethesda System)a 
VIN 1 
VIN 2 
VIN 3 
ISSVD Systemb 
HPV Associated 
Condyloma 
Acuminatum 
VIN, usual type 
Antecedent 
Vulvar 
Dystrophy 
N/A  
(e.g., Lichen 
Sclerosus) 
VIN, differentiated type* 
LAST Systemc 
Condyloma 
Acuminatum 
HSIL 
a. Reyes, 2014 
b. Sideri, 2005 
c. Darragh, 2013 
*Subjects with the non-HPV associated, or differentiated type of VIN, are excluded from 
enrollment in this trial. 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
95 
Appendix 3: ECOG Performance Status 
Score 
Performance 
0 
Fully active, able to carry on all pre-disease performance without restriction 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out 
work of a light or sedentary nature, e.g., light house work, office work 
2 
Ambulatory and capable of all self-care but unable to carry out any work activities; 
up and about more than 50% of waking hours  
3 
Capable of only limited self-care; confined to bed or chair more than 50% of 
waking hours 
4 
Completely disabled; cannot carry on any self-care; totally confined to bed or chair 
5 
Dead 
 
Oken M, Creech R, Tormey D, et al. Toxicity and response criteria of the Eastern Cooperative 
Oncology Group. Am J Clin Oncol. 1982;5:649-655. 
 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
96 
Appendix 4: Optimal Schedule for USC Investigators, Based on GYN Clinic Schedule 
 
IV Cytoxan given on Day 1 Friday of each cycle. 
GYN Clinic days on Monday through Thursday, Days 4-7 each cycle, for the 4 daily  
IRX-2 sub-lesional injections 
 
Fri 
Sat 
Sun 
Mon 
Tues 
Wed 
Thurs 
Week 
------ Cycle 1 ------ 
1 
1 
2 
3 
4 
5 
6 
7 
2 
8 
9 
10 
11 
12 
13 
14 
3 
15 
16 
17 
18 
19 
20 
21 
4 
22 
23 
24 
25 
26 
27 
28 
5 
29 
30 
31 
32 
33 
34 
35 
6 
36 
37 
38 
39 
40 
41 
42 
 
Week 
------ Cycle 2 ------ 
7 
(1) 
43 (1) 
44 (2) 
45 (3) 
46 (4) 
47 (5) 
48 (6) 
49 (7) 
8 
(2) 
50 (8) 
51 (9) 
52 (10) 
53 (11) 
53 (12) 
55 (13) 
56 (14) 
9 
(3) 
57 (15) 
58 (16) 
59 (17) 
60 (18) 
61 (19) 
62(20) 
63 (21) 
10 
(4) 
64 (22) 
65 (23) 
66 (24) 
67 (25) 
68 (26) 
69 (27) 
70 (28) 
11 
(5) 
71 (29) 
72 (30) 
73 (31) 
74 (32) 
75 (33) 
76 (34) 
77 (35) 
12 
(6) 
78 (36) 
 79 (37) 
80 (38) 
81 (39) 
82 (40) 
83 (41) 
84 (42) 
 
Week 
Week 13 Safety Assessment (Day 91) 
13 
85 
86 
87 
88 
89 
90 
91 
 
21 
Pre-surgical assessment Week 21 (Day 147 ±14 days) 
 
25 
Surgical Resection Week 25 (Day 175 ±7 days) 
 
26+ 
Post-Op Check Week 26+ (1 to 8 weeks after surgery) 
 
 
 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
97 
 
 
 


 
Clinical Study Protocol 5GYN-16-2 
Version date: 08/08/2020 
CONFIDENTIAL 
98