Clinical trial NCT03918499
Conditions: Clinical Stage IV Gastric Cancer AJCC v8 Clinical Stage IV Gastroesophageal Junction Adenocarcinoma AJCC v8 Clinical Stage IVA Gastric Cancer AJCC v8 Clinical Stage IVA Gastroesophageal Junction Adenocarcinoma AJCC v8 Clinical Stage IVB Gastric Cancer AJCC v8 Clinical Stage IVB Gastroesophageal Junction Adenocarcinoma AJCC v8 Metastatic Gastric Adenocarcinoma Metastatic Gastroesophageal Junction Adenocarcinoma Pathologic Stage IV Gastric Cancer AJCC v8 Pathologic Stage IV Gastroesophageal Junction Adenocarcinoma AJCC v8 Pathologic Stage IVA Gastroesophageal Junction Adenocarcinoma AJCC v8 Pathologic Stage IVB Gastroesophageal Junction Adenocarcinoma AJCC v8 Postneoadjuvant Therapy Stage IV Gastric Cancer AJCC v8 Postneoadjuvant Therapy Stage IV Gastroesophageal Junction Adenocarcinoma AJCC v8 Postneoadjuvant Therapy Stage IVA Gastroesophageal Junction Adenocarcinoma AJCC v8 Postneoadjuvant Therapy Stage IVB Gastroesophageal Junction Adenocarcinoma AJCC v8 Recurrent Gastric Adenocarcinoma Recurrent Gastroesophageal Junction Adenocarcinoma
Sponsor: City of Hope Medical Center
Start date: 2019-04-19; Completion date: 2023-02-09
Enrollment: 9 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This phase Ib/II trial studies the side effects of IRX-2, cyclophosphamide, and pembrolizumab work in treating participants with gastric or gastroesophageal junction cancer that has come back or that has spread to other places in the body. Interleukins, such as those found in IRX-2, are proteins made by white blood cells and other cells in the body and may help regulate immune response. Drugs used in chemotherapy, such as cyclophosphamide, work in different ways to stop the growth of tumor cells, either by killing the cells, by stopping them from dividing, or by stopping them from spreading. Immunotherapy with monoclonal antibodies, such as pembrolizumab, may help the body's immune system attack the cancer, and may interfere with the ability of tumor cells to grow and spread. Giving RX-2, cyclophosphamide, and pembrolizumab may work better in treating participants with gastric or gastroesophageal junction cancer.

Detailed description:
PRIMARY OBJECTIVES:

I. To determine the safety profile of combination IRX-2 regimen and pembrolizumab.

SECONDARY OBJECTIVES:

I. To evaluate the overall response rate of IRX-2 regimen combined with pembrolizumab using Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 and immune modified RECIST criteria.

II. To evaluate initial median progression-free and overall survival in these patients treated with combination IRX-2 regimen and pembrolizumab.

EXPLORATORY OBJECTIVES:

I. To evaluate the circulating T cell profiles in patients before and after therapy with combination IRX-2 regimen and pembrolizumab.

II. To evaluate the baseline and post-treatment tumor tissue immune gene expression profiling using the Nanostring platform.

III. To explore identification of tumor tissue neoantigens through a multiplex proteomic assay (MHC-PepSeq) paired with tumor genomic and transcriptomic sequencing.

IV. To explore putative biomarkers (including circulating tumor deoxyribonucleic acid \[DNA\] and immune cell profiles) in peripheral blood to generate hypotheses for response to treatment with combination IRX-2 regimen and pembrolizumab.

OUTLINE:

Participants receive pembrolizumab intravenously (IV) over 30 minutes on day 1. Participants also receive cyclophosphamide IV on day 1 and IRX-2 subcutaneously (SC) for 10 days starting on day 4 during cycles 1, 5, 9, 13, 17, 21, 25, 29, and 33. Treatment repeats every 3 weeks for up to 35 cycles in the absence of disease or unacceptable toxicity.

After completion of study treatment, participants are followed up for 30 days and then up to 1 year.

Arms: arm 1: Participants receive pembrolizumab IV over 30 minutes on day 1. Participants also receive cyclophosphamide IV on day 1 and IRX-2 SC for 10 days starting on day 4 during cycles 1, 5, 9, 13, 17, 21, 25, 29, and 33. Treatment repeats every 3 weeks for up to 35 cycles in the absence of disease or unacceptable toxicity.
Interventions: intervention 1: Cyclophosphamide intervention 2: Cytokine-based Biologic Agent IRX-2 intervention 3: Pembrolizumab
Intervention descriptions: intervention 1: Given IV intervention 2: Given SC intervention 3: Given IV

Protocol documents:
Prot_SAP_000.pdf: 
IRB#: 18069 
 
07/06/2020 
Packet: 06 
 
CITY OF HOPE  
1500 E. DUARTE ROAD 
DUARTE, CA 91010 
DEPARTMENT OF MEDICAL ONCOLOGY AND THERAPEUTICS RESEARCH 
TITLE: A Phase 1b/2 Trial of the IRX-2 Regimen and Pembrolizumab in Patients with Advanced Gastric 
and Gastroesophageal Junction (GEJ) Adenocarcinoma 
 
CITY OF HOPE PROTOCOL NUMBER: 
18069 
Protocol 
Version Date: 
07/06/2020 
 
 
Initial Approval           
Protocol dated 09/19/18 
 
 
Version: 00 
Amendment 01 
Protocol dated 01/11/19 
 
 
Version: 01 
Amendment 02 
Title Page dated 05/23/19 
 
 
Version: 02 
Amendment 03 
Protocol dated 08/23/19 
 
 
Version: 03 
Amendment 04 
Protocol dated 04/22/2020 
 
 
Version: 04 
Amendment 05 
Protocol dated 07/06/2020 
 
 
Version: 05 
Amendment 06 
Protocol dated 07/06/2020 (tp)  
 
Packet: 06 
 
 
 
DISEASE SITE: 
 
 
 
Gastric and Gastroesophageal Junction Adenocarcinoma 
 
STAGE (if applicable):  
 
 
 
 
 
MODALITY: 
 
 
 
 
Immunotherapy 
 
PHASE/TYPE: 
 
 
 
 
Phase Ib/II 
PRINCIPAL INVESTIGATOR: 
Joseph Chao, M.D. 
CO INVESTIGATOR: 
 
PARTICIPATING CLINICIANS: 
 
Timothy Synold, PharmD 
 
Vincent Chung, M.D., Marwan Fakih, M.D., 
Daneng Li, M.D., Dean Lim, M.D. 
 
BIOSTATISTICIAN:  
 
 
 
Paul Frankel 
 
PARTICIPATING SITES: 
 
 
 
City of Hope Duarte, CA 
Texas Oncology-Baylor Charles A. Sammons Cancer 
Center (Dallas, TX) 


 
 
1 
 
 
 
City of Hope National Medical Center 
1500 E. Duarte Road 
Duarte, CA 91010 
Clinical Trial Protocol 
A Phase 1b/2 Trial of the IRX-2 Regimen and Pembrolizumab in Patients with 
Advanced Gastric and Gastroesophageal Junction (GEJ) Adenocarcinoma 
 
Version Date: July 6, 2020 
Protocol Version: 04  
 
City of Hope #: 18069 
Agents: IRX-2, pembrolizumab   
IND Number:  141722 
Sponsor/IND Holder: City of Hope 
Funding Support: Brooklyn ImmunoTherapeutics, Inc.  
Industry Partner: Brooklyn ImmunoTherapeutics, Inc. 
Industry Partner Protocol ID: Pending 
NCT Number: Pending 
Participating Sites: City of Hope (Duarte)  
Honor Health Research Institute (Scottsdale, 
AZ) 
Texas Oncology-Baylor Charles A. Sammons 
Cancer Center (Dallas, TX)  
 
 
Principal Investigator 
Joseph Chao, MD 
Medical Oncology and Therapeutics Research 
City of Hope National Medical Center 
T: 626-218-3494 
Email: jchao@coh.org  
 
Coordinating Center 
Data Coordinating Center 
City of Hope National Medical Center 
T: (626)-256-4673 x 83968 
Email: DCC@coh.org 
 
 


 
 
2 
 
 
 
PROTOCOL TEAM 
COH Co-Investigators 
Co-Investigator 
Timothy Synold, PharmD 
Dept. of Cancer Biology 
T: 626-256-2110 
Email: tsynold@coh.org 
 
 
 
Non-COH Co-Investigators 
Co-Principal Investigator 
Sunil Sharma, MD 
TGen/HonorHealth Research Institute 
T: 602-343-8402 
Email: ssharma@tgen.org 
 
Biostatistician/Co-Investigator 
Kevin P. Gosselin, PhD 
HonorHealth Research Institute 
T: 480-323-3251 
Email: 
kevin.gosselin@honorhealth.com 
Co-Investigator 
Scott Paulson, MD 
Texas Oncology-Baylor Charles A. 
Sammons Cancer Center 
T: 214-370-1000 
Email: 
ascott.paulson@usoncology.com 
 
Co-Investigator 
Greg Caporaso, PhD 
TGen/Northern Arizona University 
T: 928-523-5854 
Email: gregcaporaso@gmail.com 
 
 
 
 
 


 
 
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Protocol Synopsis  
Protocol Title: 
A Phase 1b/2 Trial of the IRX-2 regimen and Pembrolizumab in Patients with Advanced Gastric 
and Gastroesophageal Junction (GEJ) Adenocarcinoma 
Brief Protocol Title for the Lay Public (if applicable): 
IRX-2 and Pembrolizumab Treatment in Gastric Cancer 
Study Phase: 
Phase 1b/2 
Participating Sites: 
City of Hope (Duarte, CA) 
Honor Health Research Institute (Scottsdale, AZ) 
Texas Oncology-Baylor Charles A. Sammons Cancer Center (Dallas, TX) 
Rationale for this Study: 
Advanced gastric cancer continues to account for a large proportion of worldwide causes of 
cancer-related mortality. Systemic and targeted therapies remain limited, though the immune 
checkpoint inhibitor pembrolizumab became FDA-approved in September of 2017 as third-line 
and beyond therapy based on results of the KEYNOTE-059 trial.1 However, durable objective 
responses remains limited to a small proportion of patients. The IRX-2 Regimen is an 
immunomodulatory regimen in development by Brooklyn ImmunoTherapeutics that includes a 
novel primary cell-derived biologic (IRX-2) that contains several active cytokines and 
cyclophosphamide. This treatment regimen has been demonstrated to bolster several facets of 
immune response, including tumor infiltration by lymphocytes.2-7 Therefore, it is possible that 
the combination of the IRX-2 regimen with pembrolizumab will improve treatment outcomes.  
This phase 1b/2 study will seek to investigate the safety and tolerability of the combination of 
the IRX-2 regimen with pembrolizumab.   It will be used to describe the safety profile of 
combination IRX-2 regimen and pembrolizumab and determine the maximum tolerable dose of 
combination IRX-2 regimen with pembrolizumab in patients with gastric or GEJ adenocarcinoma 
who have progressed on or are intolerant to two lines of systemic therapy. Clinical efficacy will 
also be evaluated with assessment of the objective clinical response rate of IRX-2 regimen 
combined with pembrolizumab using RECIST 1.1 and immune modified RECIST criteria, and 
resultant median progression-free and overall survival. Exploratory analyses will seek to 
evaluate the following: circulating T cell profile in patients before and after treatment with the 
IRX-2 regimen; the baseline and post-treatment tumor tissue for Nanostring gene expression 
profiling and correlation of these profiles to putative response; multiplex assays (MHC-PepSeq) 
in combination with genomic and transcriptomic sequencing to identify mutations associated 
with tumor neo-antigens; and other circulating biomarkers (other immune cell populations and 
circulating tumor DNA) in peripheral blood to develop hypotheses for response to these 
therapies.  
Objectives: 
Primary Objectives 


 
 
4 
 
 
• 
To determine the safety profile of the combination IRX-2 regimen and pembrolizumab 
Secondary Objectives 
• 
To evaluate the overall response rate of IRX-2 regimen combined with pembrolizumab 
using RECIST 1.1 and immune modified RECIST criteria. 
• 
To evaluate initial median progression-free and overall survival in these patients treated 
with combination IRX-2 regimen and pembrolizumab. 
Exploratory Objectives 
• 
To evaluate the circulating T cell profiles in patients before and after therapy with the 
combination IRX-2 regimen and pembrolizumab 
• 
To evaluate the baseline and post-treatment tumor tissue immune gene expression 
profiling using the Nanostring platform 
• 
To explore identification of tumor tissue neoantigens through a multiplex proteomic 
assay (MHC-PepSeq) paired with tumor genomic and transcriptomic sequencing 
• 
To explore putative biomarkers (including circulating tumor DNA and immune cell 
profiles) in peripheral blood to generate hypotheses for response to treatment with 
combination IRX-2 regimen and pembrolizumab.  
Study Design: 
The study will consist of two phases – a safety phase and a dose expansion phase: 
 
• 
Safety phase (phase 1b): A standard 3+3 dose finding rule will be employed with two dose 
levels.  Patients will start at dose level 1 [IRX-2 regimen at dose of 2 mL (230 units/day)]. 
Patients will be enrolled in cohorts of 3, separated by at least 1 week.  Initially, 3 patients 
will be treated with expansion to 3 more patients in the event of 1/3 dose limiting 
toxicities (DLTs) as observed during the first 3 weeks of treatment, escalation if 0/3 or 1/6 
DLTs, and termination of escalation if ≥ 2 DLTs.  Once ≥ 2 patients experience a DLT at a 
dose level, the next lower dose level will be expanded to 6 evaluable patients, if fewer 
than 6 evaluable patients have been treated at the dose level. The maximum tolerated 
dose (MTD) is defined as the highest dose tested in which only 0 or 1 out of 6 evaluable 
patients experience a DLT. The Recommended Phase 2 Dosing (RP2D) will not exceed the 
MTD but may be lower based on cumulative toxicities and dose modification patterns.  If 
the rules suggest escalation at the highest proposed dose, that dose will accrue to 6 
patients where <2 DLTs will determine that as the MTD, and 2 or more DLTs will require 
de-escalation. These rules will be used to determine the IRX-2 regimen dosing for the 
expansion phase.  
• 
Dose expansion phase (phase 2): 8-14 patients will be enrolled at the recommended dose 
level from the safety phase, for a total enrollment of 20 patients.  The 6-12 patients 
treated in the safety lead-in portion of the study will be counted as part of the dose 
expansion patient population.  
Endpoints: 
Primary Endpoint 
• 
Occurrence and severity of adverse events including dose limiting toxicities, with severity 
determined according to NCI Common Terminology Criteria for Adverse Events version 5.0. 
Secondary Endpoints 


 
 
5 
 
 
• 
Progression Free Survival (PFS), Overall Survival (OS), and Overall Response Rate (ORR) as 
assessed using RECIST version 1.1 and irRECIST. 
Exploratory Endpoints 
• 
Description of circulating T cell profiles in patients before and after therapy with 
combination IRX-2 regimen and pembrolizumab.  
• 
Correlation between baseline and post-treatment tumor tissue Nanostring gene expression 
profiling and clinical response.  
• 
Identification of tumor neoantigens using the MHC-PepSeq assay. 
• 
Development of hypotheses for response based on circulating immune cell profiles 
circulating tumor DNA from peripheral blood.  
Sample Size:  
Phase I:  6-12 patients 
Phase II: 20 patients (11 + 9 or 8 + 12)* 
* Patients treated at all dose levels in the phase I portion of the study will be counted as a part 
of the phase II patient population. 
Estimated Duration of the Study: 
42 Months 
Summary of Subject Eligibility Criteria: 
Inclusion Criteria: 
1. Patients with histologically or cytologically confirmed recurrent or metastatic gastric or 
gastroesophageal junction (GEJ) adenocarcinoma progressed or intolerant to ≥ 2 lines of 
systemic therapy. 
2. Patients must have recurrent or metastatic gastric/GEJ adenocarcinoma that are not 
amenable to local therapy with curative intent (surgery or radiation therapy with or 
without chemotherapy). 
3. Willing and able to give informed consent and adhere to protocol therapy; written 
informed consent and any locally required authorization must be obtained from the 
patient prior to performing any protocol-related procedures, including screening 
evaluations. 
4. At least 18 years of age. 
5. No prior exposure to PD-1/PD-L1 inhibitor therapy.  
6. Patients are deemed eligible for pembrolizumab therapy with tumors demonstrating 
PD-L1 expression by the Combined Positive Score (CPS) being ≥ 1 as per the FDA-
approved Dako PD-L1 IHC 22C3 PharmDx assay. 
7. ECOG 0-1 
8. Body weight must be >30 Kg. 
9. Adequate normal organ and marrow function as defined below: 
10. Hemoglobin >8 g/dL 
11. Absolute neutrophil count (ANC) >1,200 x 109/mL 
12. Platelet count >75 x 109/mL 
13. Serum bilirubin ≤1.5 × institutional upper limit of normal (ULN) OR Direct bilirubin ≤ ULN 
if total bilirubin levels > 1.5 x ULN 


 
 
6 
 
 
14. Aspartate aminotransferase (AST/SGOT) and alanine aminotransferase (ALT/SGPT) ≤ 5 x 
ULN  
15. Prothrombin time (PT) and partial thromboplastin time (PTT) < 1.5x the ULN. 
16. Serum creatinine ≤ 1.5 x ULN OR Measured creatinine clearance (CL) >40 mL/min or 
calculated creatinine clearance CL>40 mL/min by the Cockcroft-Gault formula 8 or by 24-
hour urine collection for determination of creatinine clearance: 
 
Males: 
 
Creatinine CL 
(mL/min) 
= 
Weight (kg) x (140 – Age)   
72 x serum creatinine (mg/dL) 
Females: 
 
Creatinine CL 
(mL/min) 
= 
Weight (kg) x (140 – Age) x 0.85 
72 x serum creatinine (mg/dL) 
17. Palliative radiation therapy is allowed to non-target lesions at the discretion of the 
treating physician.   
18. Patients must have measurable disease, defined as at least one lesion that can be 
accurately measured in at least one dimension (longest diameter to be recorded) or 
evaluable disease as outlined in RECIST version 1.1.  
19. Life expectancy of greater than 3 months in the opinion of the treating physician.  
20. Female patients of childbearing potential must have a negative serum pregnancy test 
within 7 days prior to enrollment. 
Exclusion Criteria: 
1. Prior exposure to the IRX-2 regimen and/or PD-1/PD-L1 inhibitors are excluded. 
2. Radiation therapy with a curable intent within 30 days of first dose of study treatment is 
excluded. However, radiation therapy with a palliative intent is allowed as long as 
treatment with study medication occurs ≥14 days after last dose of radiation.  
3. Any medical contraindications or previous therapy that would preclude treatment with 
the IRX-2 Regimen or pembrolizumab.  
4. Known allergies to ciprofloxacin or phytohemagglutin given trace amount of these 
agents are contained in IRX-2. 
5. Patients with ongoing chronic myelosuppression, myelodysplasia, or hemorrhagic 
cystitis which would contraindicate receipt of cyclophosphamide. 
6. Any unresolved toxicity NCI CTCAE Grade ≥ 2 from previous anticancer therapy with the 
exception of alopecia, vitiligo, and the laboratory values defined in the inclusion criteria. 
7. Patients with Grade ≥ 2 neuropathy will be evaluated on a case-by-case basis after 
consultation with the study physician. 
8. Patients with irreversible toxicity not reasonably expected to be exacerbated by 
treatment with IRX-2, pembrolizumab may be included only after consultation with the 
study physician. 
9. Active or prior documented autoimmune or inflammatory disorders (including 
inflammatory bowel disease [e.g., colitis or Crohn's disease], diverticulitis [with the 
exception of diverticulosis], systemic lupus erythematosus, Sarcoidosis syndrome, or 
Wegener syndrome [granulomatosis with polyangiitis, Graves' disease, rheumatoid 


 
 
7 
 
 
arthritis, hypophysitis, uveitis, etc]). The following are exceptions to this criterion: 
• 
Patients with vitiligo or alopecia. 
• 
Patients with hypothyroidism (e.g., following Hashimoto syndrome) stable on 
hormone replacement.  
• 
Any chronic skin condition that does not require systemic therapy. 
• 
Patients without active disease in the last 2 years may be included but only after 
consultation with the study physician. 
• 
Patients with celiac disease controlled by diet alone. 
10. Current or prior use of immunosuppressive medication within 14 days prior to Cycle 1, 
Day 1. The following are exceptions to this criterion: 
• 
Intranasal, inhaled, topical steroid or local steroid injections (e.g., intra articular 
injection). 
• 
Systemic corticosteroids at physiologic doses not to exceed 10 mg/day of 
prednisone or its equivalent. 
• 
Steroids as premedication for hypersensitivity reactions (e.g., CT scan 
premedication). 
11. Major surgical procedure (as defined by the Investigator) within 28 days prior to Cycle1, 
Day 1. Note: Local surgery of isolated lesions for palliative intent is acceptable. 
12. History of allogeneic organ transplantation. 
13. Symptomatic cardiopulmonary disease (including congestive heart failure and 
hypertension), coronary artery disease, serious arrhythmia or chronic lung disease. 
Patients with these conditions who are stable with relatively minor symptoms and who 
are appropriate candidates for systemic treatments need not be excluded. 
14. Myocardial infarction within the last 3 months. 
15. Has a known history of human immunodeficiency virus (HIV) (HIV 1/2 antibodies). 
16. Has a history of active Hepatitis B requiring ongoing antiviral therapy or a history of 
untreated Hepatitis C. 
17. Has received a live vaccine within 4 months of planned start of study therapy (Cycle 1, 
Day 1).  
Note: The killed virus vaccines used for seasonal influenza vaccines for injection are 
allowed provided not within 4 weeks of planned start of study therapy (Cycle 1, Day 1); 
however intranasal influenza vaccines (e.g., FluMist®) are live attenuated vaccines and 
are not allowed. 
18. Signs or symptoms of systemic infection (use of antibiotics to treat superficial infection 
or contamination of tumor shall not, by itself, be considered evidence of infection). 
19. Stroke or other symptoms of cerebral vascular insufficiency within the last 3 months. 
20. Previous diagnosis of invasive cancer from which the individual is not disease-free AND 
that has required treatment within the past 3 years, except for superficial skin, cervical 
cancer in-situ, or early stage prostate or bladder cancer (i.e. treatment with curative 
intent and long term disease-free expectations).  
21. History of leptomeningeal carcinomatosis 
22. Known allergy or hypersensitivity to any of the study drugs or any of the study drug 
excipients. 
23. Female patients who are pregnant or breastfeeding or male or female patients of 
reproductive potential who are not willing to employ effective birth control from 


 
 
8 
 
 
screening to one year after last dose of cyclophosphamide or 180 days after the last 
dose of pembrolizumab therapy, whichever is longer.  
Investigational Product and Reference Therapy: 
IRX-2 is supplied as a pale yellow, sterile liquid for subcutaneous injection in a 2.0 ml single dose 
vial.  
Pembrolizumab as a 200 mg IV Q3weeks as a 30 minute infusion 
 
Cycle 1 through 35 
Pembrolizumab as a 200 mg IV Q3weeks as a 30 minute infusion (every Cycle) 
IRX-2 regimen every 12 weeks as below starting on Cycle 1: 
Agent 
Dose 
Route of Administration 
Cyclophosphamide (Day 1) 
300 mg/m2 
IV 
IRX-2: For 10 days starting on 
Day 4; the 10 days are not 
required to be consecutive 
(e.g. dosing not mandatory on 
weekends/holidays) provided 
dosing is completed by Day 15 
Dose Level 1: 
230 units total daily 
(2 
injections 
daily 
comprised of 115 units in 
each injection) 
 
Dose Level 2: 
460 units total daily 
(4 
injections 
daily 
comprised of 115 units in 
each injection) 
Dose Level 1: Subcutaneous at or 
near the supraclavicular lymph 
node 
regions 
bilaterally 
(substitute 
axillary 
if 
supraclavicular nodes have been 
removed). 
 
Dose level 2: Subcutaneous at or 
near the supraclavicular and 
axillary node regions bilaterally 
(if any of these nodes have been 
removed, can inject at or near 
mastoid 
insertion 
of 
both 
sternocleidomastoid muscles). 
 
 
 Statistical Considerations: 
The safety phase of the study will enroll 3 to 6 patients per dose level. The dose expansion phase 
will enroll 8 to 12 patients at the recommended dose level from the safety phase for a total 
enrollment of 20 patients. The 6 to 12 patients treated in the safety phase of the study will be 
counted as a part of the dose expansion patient population. 
 
Historical data with single agent PD-1 inhibitors in gastroesophageal cancer indicate median PFS 
at 1.5 months. With an alpha of .05, hypothesized median PFS improved to 3.0 months with the 
combination IRX-2 regimen and pembrolizumab, study accrual of 30 months, and 12-month 
follow-up evaluation timeframe, a sample size of 20 patients will have 80% power to detect 
median differences in PFS.  Assuming that dose level 1 will be used for the dose expansion phase, 
these individuals will be included in phase II to reach the needed sample of 20 patients evaluable 
for immune activity. 
  
Sponsor/Licensee: 
City of Hope/Brooklyn ImmunoTherapeutics, Inc. 


 
 
9 
 
 
 
 
Study Schema  
  
 
 
 
 
 
 
*IRX-2 Regimen: IRX-2 x 10 days, Cyclophosphamide x 1 dose on Day 1; given every 12 weeks  
Pembrolizumab: 200 mg; IV administration; given every 3 weeks 
**Cycle 1 only 
***Imaging scans performed every 6 weeks for the first 18 weeks, then every 9 weeks afterwards 
Week 
1
Week 
2
Week 
3
Week 
4
Week 
5 
Week 
6
Week 
7
Week 
8
Week 
9 
Week 
10
Week 
11  
Week 
12 
IRX-2 Regimen* + 
Pembrolizumab  
Pembrolizumab  
Pembrolizumab administration , DLT 
Assessment Completed**  
Post-Treatment 
Biopsy* 
Additional 
12 Week 
Sequences 
Imaging for RECIST 
assessment*** 


 
 
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Table of Contents 
 
PROTOCOL SYNOPSIS ............................................................................................................................................. 3 
STUDY SCHEMA ..................................................................................................................................................... 9 
TABLE OF CONTENTS ............................................................................................................................................ 10 
1.0 
GOALS AND OBJECTIVES (SCIENTIFIC AIMS) ............................................................................................ 13 
1.1 
PRIMARY OBJECTIVES ....................................................................................................................................... 13 
1.2 
SECONDARY OBJECTIVES ................................................................................................................................... 13 
1.3 
EXPLORATORY OBJECTIVES ................................................................................................................................ 13 
2.0 
BACKGROUND ......................................................................................................................................... 13 
2.1 
GASTRIC CANCER ............................................................................................................................................ 13 
2.1.1 
Epidemiology ......................................................................................................................................... 13 
2.1.2 
Current Treatment Options .................................................................................................................... 13 
2.2 
IMMUNOTHERAPY ........................................................................................................................................... 14 
2.2.1 
PD-1 and Cancer .................................................................................................................................... 14 
2.2.2 
The IRX-2 Regimen ................................................................................................................................. 15 
2.2.3 
Manufacture and Composition of the IRX-2 Biologic ............................................................................. 15 
2.2.4 
Modifying the Lymph Node Microenvironment (IRX-2 Biologic) ............................................................ 15 
2.2.5 
Tumor-Induced Immune Suppression .................................................................................................... 16 
2.3 
RATIONALE FOR COMBINING THE IRX-2 REGIMEN AND PEMBROLIZUMAB IN GASTRIC/GEJ ADENOCARCINOMA ............... 16 
2.4 
OVERVIEW OF STUDY ....................................................................................................................................... 16 
3.0 
PATIENT ELIGIBILITY ................................................................................................................................ 17 
4.0 
SCREENING AND REGISTRATION PROCEDURES ....................................................................................... 20 
4.1 
SCREENING PROCEDURES .................................................................................................................................. 20 
4.2 
INFORMED CONSENT ....................................................................................................................................... 20 
4.3 
REGISTRATION REQUIREMENTS/PROCESS ............................................................................................................ 20 
4.4 
SCREEN FAILURES AND REGISTERED PARTICIPANTS WHO DO NOT BEGIN STUDY TREATMENT ........................................ 21 
5.0 
TREATMENT PROGRAM .......................................................................................................................... 21 
5.1 
TREATMENT OVERVIEW .................................................................................................................................... 21 
5.2 
TREATMENT CYCLE DEFINITION ........................................................................................................................... 21 
5.3 
TREATMENT PLAN ........................................................................................................................................... 22 
5.4 
DEFINITION OF DOSE LIMITING TOXICITY ............................................................................................................. 22 
5.5 
AGENTS ADMINISTRATION ................................................................................................................................ 24 
5.5.1 
IRX-2 Regimen ........................................................................................................................................ 24 
5.5.2 
Pembrolizumab ...................................................................................................................................... 24 
5.6 
STUDY PROCEDURES ........................................................................................................................................ 24 
5.7 
DURATION OF THERAPY AND CRITERIA FOR REMOVAL FROM STUDY TREATMENT ......................................................... 24 
5.8 
END OF STUDY FOLLOW UP .............................................................................................................................. 25 
5.9 
CRITERIA FOR COMPLETION OF STUDY PARTICIPATION ............................................................................................ 25 
5.10 
SUPPORTIVE CARE AND OTHER CONCOMITANT THERAPY ........................................................................................ 25 
5.10.1 
Included concomitant medications .................................................................................................... 25 
5.10.2 
Excluded concomitant medications ................................................................................................... 26 
6.0 
ANTICIPATED TOXICITIES AND DOSE DELAYS/MODIFICATIONS FOR ADVERSE EVENTS ........................... 27 


 
 
11 
 
 
6.1 
ANTICIPATED TOXICITIES ................................................................................................................................... 27 
6.2 
DOSE MODIFICATIONS AND DELAYS ........................................................................ ERROR! BOOKMARK NOT DEFINED. 
6.2.1 
General Information .............................................................................................................................. 27 
6.2.2 
Dose Modifications ................................................................................................................................ 28 
7.0 
DATA AND SAFETY MONITORING AND ADVERSE EVENT REPORTING ...................................................... 28 
7.1 
Adverse Events and Unanticipated Problems ............................................................................................ 28 
7.2 
PROTOCOL DEVIATIONS AND SINGLE SUBJECT EXCEPTIONS ...................................................................................... 33 
7.3 
STUDY OVERSIGHT, QUALITY ASSURANCE, AND DATA & SAFETY MONITORING ........................................................... 34 
 
8.0 
AGENT INFORMATION ............................................................................................................................ 36 
8.1 
DRUG INFORMATION FOR IRX-2 ........................................................................................................................ 37 
8.1.1 
Risks and Contraindications ................................................................................................................... 37 
8.1.2 
Supplier .................................................................................................................................................. 38 
8.1.3 
Dose and Administration ....................................................................................................................... 38 
8.1.4 
Formulation/Agent Preparation ............................................................................................................ 39 
8.1.5 
Storage ................................................................................................................................................... 39 
8.2 
DRUG INFORMATION FOR PEMBROLIZUMAB (KEYTRUDA®) ...................................................................................... 39 
8.2.1 
Risks and Contraindications ................................................................................................................... 39 
8.2.2 
Supplier .................................................................................................................................................. 39 
8.2.3 
Dose and Administration ....................................................................................................................... 40 
8.2.4 
Formulation/Agent Preparation ............................................................................................................ 40 
8.2.5 
Storage ................................................................................................................................................... 40 
9.0 
CORRELATIVE/SPECIAL STUDIES .............................................................................................................. 39 
9.1 
TUMOR TISSUE STUDIES ................................................................................................................................... 41 
9.2 
PERIPHERAL BLOOD STUDIES ............................................................................................................................. 41 
9.2.1 
Timepoints of Collection and Tube Type ................................................................................................ 40 
9.2.2 
APCF Notification, Blood Collection and Labeling .................................................................................. 40 
9.2.3 
Processing of samples at APCF............................................................................................................... 42 
9.2.4 
Sample maintenance and distribution/shipping to laboratories ........................................................... 42 
9.3 
STOOL SAMPLES ............................................................................................................................................. 43 
9.4 
TGEN DOWNSTREAM CORRELATIVE ANALYSES ..................................................................................................... 44 
10.0 
STUDY CALENDAR ................................................................................................................................... 46 
11.0 
EVALUATION CRITERIA/MEASUREMENT OF EFFECT ................................................................................ 48 
11.1 
PRIMARY ENDPOINT: TOXICITY .......................................................................................................................... 51 
11.2 
SECONDARY ENDPOINT: RESPONSE ..................................................................................................................... 51 
11.3 
RESPONSE CRITERIA ......................................................................................................................................... 50 
12.0 
DATA REPORTING/PROTOCOL DEVIATIONS ............................................................................................ 52 
12.1 
DATA REPORTING............................................................................................................................................ 52 
12.2 
PROTOCOL DEVIATIONS .................................................................................................................................... 53 
13.0 
STATISTICAL CONSIDERATIONS ............................................................................................................... 53 
13.1 
EVALUABLE PARTICIPANTS AND PARTICIPANT REPLACEMENT ................................................................................... 53 
13.2 
SAMPLE SIZE AND ACCRUAL RATE....................................................................................................................... 54 
13.3 
STATISTICAL ANALYSIS PLAN .............................................................................................................................. 54 


 
 
12 
 
 
14.0 
HUMAN SUBJECT ISSUES ......................................................................................................................... 55 
15.0 
REFERENCES ............................................................................................................................................ 61 
16.0 
APPENDIX I .............................................................................................................................................. 61 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
13 
 
 
1.0 Goals and Objectives (Scientific Aims) 
Primary Objectives 
• 
To determine the safety profile of combination IRX-2 regimen and pembrolizumab 
Secondary Objectives 
• 
To evaluate the overall response rate of IRX-2 regimen combined with pembrolizumab using 
RECIST 1.1 and immune modified RECIST criteria. 
• 
To evaluate initial median progression-free and overall survival in these patients treated with 
combination IRX-2 regimen and pembrolizumab. 
Exploratory Objectives 
• 
To evaluate the circulating T cell profiles in patients before and after therapy with combination 
IRX-2 regimen and pembrolizumab 
• 
To evaluate the baseline and post-treatment tumor tissue immune gene expression profiling 
using the Nanostring platform 
• 
To explore identification of tumor tissue neoantigens through a multiplex proteomic assay 
(MHC-PepSeq) paired with tumor genomic and transcriptomic sequencing  
• 
To explore putative biomarkers (including circulating tumor DNA and immune cell profiles) in 
peripheral blood to generate hypotheses for response to treatment with combination IRX-2 
regimen and pembrolizumab.  
2.0 Background  
Gastric Cancer 
2.1.1 Epidemiology  
In 2012 nearly 1 million new cases of gastric cancer were diagnosed globally with an estimated 723,100 
deaths, reflective of the high mortality rate associated with this disease.9  
2.1.2 Current Treatment Options  
A number of chemotherapy agents have demonstrated activity in advanced gastric and 
gastroesophageal junction (GEJ) cancer. These include platinum drugs, such as cisplatin and oxaliplatin, 
and fluoropyrimidines, such as intravenous 5-fluorouracil and oral capecitabine. Multi-agent 
chemotherapy regimens are able to improve by nearly 7 months the approximately 4 months median 
survival seen with best supportive care alone.10 In terms of predictive biomarkers to date that guide 
treatment decisions in front-line therapy, evidence of HER2 overexpression and/or gene amplification 
supports addition of HER2 blockade with trastuzumab to combination platinum and fluoropyrimidine 
chemotherapy based on the phase III ToGA trial.11 Despite initial activity seen with multi-agent 
chemotherapy, development of drug resistance and invariable disease progression still accounts for high 
mortality rates associated with advanced disease. 
Second-line chemotherapy with taxanes or irinotecan after platinum and fluoropyrimidine 
chemotherapy have demonstrated modest activity in randomized trials.12-14 More recently, evidence 
that angiogenesis inhibition can induce tumor responses and/or delay disease progression was 
demonstrated with delivery of the VEGFR2-inhibitory antibody ramucirumab in the phase III REGARD 
and RAINBOW trials, when utilized as a single agent or added to paclitaxel, respectively.15, 16 Median 
overall survival improved from 7.4 to 9.6 months (p=0.017) when ramucirumab was added to paclitaxel, 


 
 
14 
 
 
representing a stepwise improvement for this patient population, but still indicating more options are 
needed for this diagnosis. 
Immune checkpoint inhibitors have recently become an option for treatment-refractory advanced 
gastric cancer.  KEYNOTE 059 was a multi-cohort phase 2 trial, in which in its largest cohort, Cohort 1, 
examined pembrolizumab in 259 gastric and GEJ adenocarcinoma patients who had failed at least 2 
prior systemic regimens.1 Pembrolizumab was continued until completion of 35 cycles (approximately 2 
years) in patients without demonstration of progressive disease, intolerable toxicity, withdrawal of 
consent, or investigator decision to stop therapy. Fuchs et al. reported those with tumor PD-L1 
expression had an overall RR of 15.5% with 2.0% complete responses, and 13.5% partial responses by 
RECIST1.1.  Evidence of PD-L1 expression by a combined positive score (CPS) was defined as a cutoff ≥ 1 
tumor cells and/or immune cells out of 100 viable tumor cells with PD-L1 expression by 
immunohistochemistry using the 22C3 pharmDx assay. Microsatellite instability was also tested as a 
biomarker, and 4% of the study population harbored tumors with high microsatellite instability (MSI-H).  
ORR was more encouraging at 57.1% in the MSI-H subset vs. 9.0% in tumors defined to be non-MSI-H. 
Ultimately 13.3% of patients with non-MSI-H or undetermined MSI, PD-L1 CPS positive tumors 
demonstrated objective tumor responses which were durable with median durations of response 
between 2.3+ to 19.4+ months. While the durability of responses are very encouraging for a disease 
historically with dismal prognosis, the low proportion of patients having such clinical benefit speaks to 
the need to augment immunotherapy responses.  This was further highlighted by recent reporting of the 
phase III KEYNOTE-061 trial, in which 395 metastatic gastric or GEJ cancer patients with PD-L1 CPS 
positive tumors were randomized to single agent pembrolizumab or paclitaxel.17  In this trial, a median 
PFS of 1.5 months was observed with pembrolizumab versus 4.1 months with paclitaxel, indicating the 
vast majority of patients demonstrate disease progression early despite pembrolizumab therapy. 
Immunotherapy  
2.1.3 PD-1 and Cancer 
Developing immunity to a particular cancer can be viewed as a cyclic process in which immune-
stimulatory and inhibitory factors compete against each other to modulate the immune response.18 This 
relationship is further complicated by the ability of the tumor to down-regulate stimulatory signals and 
up-regulate inhibitory signals. Some tumors express Programmed death receptor ligand 1 (PD-L1) the 
ligand for programmed death-1 (PD-1), a cell surface molecule that regulates the adaptive immune 
response.19 PD-1 is found on the surface of T cells and sends inhibitory signals that inactivate T cell 
receptor (TCR) signaling upon the binding of PD-L1.20 The interaction of PD-L1 with PD-1 has been shown 
to inhibit the killing activity of cytotoxic T cells as well as interfere with the role of regulatory T cells.21 
Tumors take advantage of immune checkpoints such as the PD-1/PD-L1 pathway to evade immune 
destruction, resulting in disease progression. It is necessary to overcome multiple immunosuppressive 
mechanisms in order to establish an effective antitumor immune response. 22  Anti-PD1 and Anti-PD-L1 
are designed to block immune checkpoints thereby decreasing immune inhibitory pathways and 
increasing the antitumor effect. 23, 24 In targeting tumors that express PD-L1, immunotherapy has been 
used to produce an antibody blockade to the PD-1 receptor on T cells.25 
Pembrolizumab is a fully human, IgG4 (kappa) monoclonal antibody that binds PD-1, which is primarily 
expressed on activated T cells, B cells and myeloid cells.26-29  The PD-1 receptor-ligand interaction is a 
major pathway utilized by tumors to evade the surveillance and clearance by the immune system. 
Atezolizumab works in similar fashion, except it is an IgG1 isotype antibody which blocks the interaction 
of PD-L1 with PD-1 and CD80.30, 31  A variety of cancers have demonstrated the ability to express PD-L1 


 
 
15 
 
 
and binding of PD-1 to PD-L1 or PD-L2 has been shown to down-regulate T-cell activation.32-35 
Interactions between PD-1/PD-L1 may also indirectly modulate the response to tumor antigens through 
T-cell/APC interactions. PD-1 blockade by nivolumab, pembrolizumab and PD-L1 blockade by 
atezolizumab has been shown to reverse immune tolerance and enhance T cell effector function in 
several tumor types including melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), 
urothelial carcinoma, hepatocellular carcinoma (HCC), microsatellite instability-high (MSI-H) colorectal 
cancer, and head and neck squamous cell carcinoma (HNSCC).28, 36-42 As discussed earlier, for the FDA 
approval of pembrolizumab in the third-line treatment of advanced gastric/GEJ adenocarcinoma, the 
observed response rate was 15.5%, indicating the possibility for improvement of this therapy through 
combination with an additional immunomodulatory regimen.   
2.1.4 The IRX-2 Regimen 
The IRX-2 Regimen is an immunomodulatory regimen in development by Brooklyn ImmunoTherapeutics 
that includes a novel primary cell-derived biologic (IRX-2) and cyclophosphamide. Preclinical studies 
showed that the IRX-2 biologic promotes upregulation of antigen presenting machinery, improved 
antigen presentation, dendritic cell (DC) maturation, improved T cell activation, and T cell protection 
from extrinsic and intrinsic forms of tumor-induced apoptosis.2-7 Clinical studies evaluating a 3-week 
cycle of the IRX-2 Regimen in therapy-naïve subjects with squamous cell carcinoma of the head and neck 
(HNSCC) intended for curative surgery demonstrated modest tumor regression with increased 
lymphocyte infiltration into tumors when surgical specimens were compared to pre-treatment biopsies. 
Increase in tumor lymphocyte infiltration appeared to be associated with better overall survival.43-46 
These findings suggest that the IRX 2 Regimen may mobilize tumor-infiltrating effector lymphocytes 
resulting in a clinically significant anti-tumor immune response. 
2.1.5 Manufacture and Composition of the IRX-2 Biologic 
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. Multiple healthy donor leukocytes (“buffy coats”) are pooled and then stimulated 
with PHA (to mimic a cellular immune challenge), and then locked in pre-s phase by ciprofloxacin. 
Subsequently, the PHA, ciprofloxacin and all cellular elements are removed and the cell-free 
supernatant is filter-sterilized, nanofiltered to clear viral particles, vialed and frozen as IRX-2. IRX-2 
contains nanogram quantities (physiologic) of IL-1β, IL-2, IFN-γ, and TNFα, and also includes IL-6, IL-8, 
GM-CSF, and G-CSF. Each of these human cytokines when studied individually enhances cell-mediated 
immunity via different mechanisms.  
2.1.6 Modifying the Lymph Node Microenvironment (IRX-2 Biologic) 
The IRX-2 biologic is administered by subcutaneous injection in the peri-lymphatic area adjacent to 
lymph nodes draining the tumor bed to take advantage of the normal afferent and efferent pathways of 
lymph node activation. The immunosuppressive microenvironment contributes to incomplete or 
ineffective tumor antigen processing in regional lymph nodes. Administering IRX-2 near lymphatic 
capillaries and vessels enables the immunostimulatory cytokines to be drawn up into and potentially 
restore the lymph node microenvironment to facilitate DC maturation, antigen processing, and effector 
T cell activation. 


 
 
16 
 
 
2.1.7 Tumor-Induced Immune Suppression 
Tumor-induced immune suppression ranges from tumors utilizing immune checkpoints (CTLA-4, PD-
1/PD-L1) to production of soluble factors such as IL-10, TGF-β, prostaglandins, vascular endothelial 
growth factor, and Fas ligand which contribute to depression of cellular immunity by suppressing 
lymphocyte and monocyte function.47-49 The production of these factors by the tumor results in the 
induction of host suppressor cells of both lymphoid and myeloid origin including regulatory T cells, 
myeloid suppressor cells, suppressor macrophages and DCs.50-52 
Rationale for Combining the IRX-2 Regimen and Pembrolizumab in Gastric/GEJ Adenocarcinoma  
Recently it has been shown that the degree of lymphocyte infiltration is an important prognostic factor 
for treatment with anti-PD-1 monoclonal antibodies in melanoma.53 Additionally, it has been well 
established that the level of T cell infiltration in primary tumor biopsies is a strong predictive factor of 
improved outcome in multivariate analyses for many cancers.54-56 This provides a compelling basis for 
incorporating treatment with the IRX-2 Regimen to increase the level of lymphocyte infiltration in 
tumors with anti-PD-1 treatments. In a Phase 2 trial, IRX-2 was shown to increase T cell infiltration into 
the tumor and this correlated with overall survival in HNSCC.44, 45 In the Phase 2a study in patients with 
HNSCC, IRX-2 facilitated reductions in circulating B and NKT cell numbers, suggesting redistribution of 
these cells to tissues.44 
In pre-clinical models, the IRX-2 Regimen was shown to be superior to recombinant IL-7 and IL-15 in 
protecting T cells from tumor- induced apoptosis and to be involved in IFN-γ+-driven T cell polarization 
towards Teff and suppression of Treg differentiation in the tumor microenvironment.4 IRX-2 was 
subsequently shown to restore the function of dendritic cells from head and neck cancer patients.5 A 
decrease in naïve T cells and unchanged numbers of Tregs (suppressor T cells) after IRX-2 therapy 
indicated that IRX-2 does not expand suppressor T cells thus potentially benefiting anti-tumor immune 
responses.44  
As the IRX-2 regimen has been shown to work in an upstream immune-activating manner and PD-1 
inhibitors work downstream by reducing tumor-induced immune suppression, a combination of such 
therapies would likely be synergistic. Pembrolizumab, a fully human monoclonal immunoglobulin (Ig) G4 
antibody that binds to the PD-1 cell surface membrane receptor has been approved by FDA for the 
third-line treatment of gastroesophageal cancer although only a 13% objective response rate was 
observed in non-MSI-H tumors with positive PD-L1 expression by the CPS score (Fuchs ref). Therefore, it 
is likely that patients with evidence of immune checkpoint expression in the tumor and tumor 
microenvironment would benefit from additional immune system activation in conjunction with 
immune checkpoint inhibition.  With employment of an immune checkpoint inhibitor, indomethacin and 
the need for omeprazole prophylaxis against NSAID-induced gastritis has been deemed unnecessary and 
will not be utilized in the trial.  Zinc deficiency and supplementation in the HNSCC trials were unique to 
that patient population, and will also not be utilized in this trial for patients with metastatic 
gastroesophageal adenocarcinomas. 
Overview of Study  
The IRX-2 Regimen is an immunomodulatory regimen in development by Brooklyn ImmunoTherapeutics 
that includes a novel primary cell-derived biologic (IRX-2) that contains several active cytokines and 
cyclophosphamide. This treatment regimen has been demonstrated to bolster several facets of immune 


 
 
17 
 
 
response, including tumor infiltration by lymphocytes.2-7 Therefore, it is possible that the combination of 
the IRX-2 regimen with pembrolizumab will improve treatment outcomes in advanced, treatment-
refractory gastric and GEJ adenocarcinoma.  
This phase 1b/2 study will seek to investigate the safety and tolerability of the combination of the IRX-2 
regimen with pembrolizumab in this patient population.   It will be used to describe the safety profile of 
combination IRX-2 regimen and pembrolizumab and determine the maximum tolerable dose of 
combination IRX-2 regimen with pembrolizumab in patients with gastric and gastroesophageal junction 
adenocarcinoma who have progressed on or are intolerant to ≥ 2lines of prior systemic therapy. The 
objective clinical response rate of IRX-2 regimen combined with pembrolizumab using RECIST 1.1 and 
immune modified RECIST criteria and progression free survival will also be evaluated. Exploratory 
analyses will seek to evaluate the following: circulating T cell profiles in patients before and after 
treatment with the IRX-2 regimen, the baseline and post-treatment tumor tissue for Nanostring gene 
expression profiling and correlation of these profiles to clinical response and evaluate additional 
biomarkers to develop hypotheses for response to these therapies.  
3.0 Patient Eligibility 
Inclusion Criteria: 
1. Patients with histologically or cytologically confirmed recurrent or metastatic gastric or 
gastroesophageal junction (GEJ) adenocarcinoma progressed or intolerant to ≥ 2 lines of 
systemic therapy. 
2. Patients must have recurrent or metastatic gastric/GEJ adenocarcinoma that are not 
amenable to local therapy with curative intent (surgery or radiation therapy with or 
without chemotherapy). 
3. Willing and able to give informed consent and adhere to protocol therapy; written 
informed consent and any locally required authorization must be obtained from the 
patient prior to performing any protocol-related procedures, including screening 
evaluations 
4. At least 18 years of age. 
5. No prior exposure to PD-1/PD-L1 inhibitor therapy. 
6. Patients are deemed eligible for pembrolizumab therapy with tumors demonstrating 
PD-L1 expression by the Combined Positive Score (CPS) being ≥ 1 as per the FDA-
approved Dako PD-L1 IHC 22C3 PharmDx assay . 
7. ECOG 0-1 
8. Body weight must be >30 Kg. 
9. Adequate normal organ and marrow function as defined below: 
10. Hemoglobin >8 g/dL 
11. Absolute neutrophil count (ANC) >1,200 x 109/mL 
12. Platelet count >75 x 109/mL 
13. Serum bilirubin ≤1.5 × institutional upper limit of normal (ULN) OR Direct bilirubin ≤ ULN 
if total bilirubin levels > 1.5 x ULN 
14. Aspartate aminotransferase (AST/SGOT) and alanine aminotransferase (ALT/SGPT) ≤ 5 x 
ULN  
15. Prothrombin time (PT) and partial thromboplastin time (PTT) < 1.5x the ULN. 
16. Serum creatinine ≤ 1.5 x ULN OR Measured creatinine clearance (CL) >40 mL/min or 


 
 
18 
 
 
calculated creatinine clearance CL>40 mL/min by the Cockcroft-Gault formula 8 or by 24-
hour urine collection for determination of creatinine clearance: 
 
Males: 
 
Creatinine CL 
(mL/min) 
= 
Weight (kg) x (140 – Age)   
72 x serum creatinine (mg/dL) 
Females: 
 
Creatinine CL 
(mL/min) 
= 
Weight (kg) x (140 – Age) x 0.85 
72 x serum creatinine (mg/dL) 
17. Palliative radiation therapy is allowed to non-target lesions at the discretion of the 
treating physician.   
18. Patients must have measurable disease, defined as at least one lesion that can be 
accurately measured in at least one dimension (longest diameter to be recorded) or 
evaluable disease as outlined in RECIST version 1.1.  
19. Life expectancy of greater than 3 months in the opinion of the treating physician.  
20. Female patients of childbearing potential must have a negative serum pregnancy test 
within 7 days prior to enrollment. 
Exclusion Criteria: 
1. Prior exposure to the IRX-2 regimen and/or PD-1/PD-L1 inhibitors are excluded. 
2. Radiation therapy with a curable intent within 30 days of first dose of study treatment is 
excluded. However, radiation therapy with a palliative intent is allowed as long as 
treatment with study medication occurs ≥14 days after the last dose of radiation.  
3. Any medical contraindications or previous therapy that would preclude treatment with 
the IRX-2 Regimen or pembrolizumab.  
4. Known allergies to ciprofloxacin or phytohemagglutin given trace amount of these 
agents are contained in IRX-2. 
5. Patients with ongoing chronic myelosuppression, myelodysplasia, or hemorrhagic 
cystitis which would contraindicate receipt of cyclophosphamide. 
6. Any unresolved toxicity NCI CTCAE Grade ≥ 2 from previous anticancer therapy with the 
exception of alopecia, vitiligo, and the laboratory values defined in the inclusion criteria. 
7. Patients with Grade ≥ 2 neuropathy will be evaluated on a case-by-case basis after 
consultation with the study physician 
8. Patients with irreversible toxicity not reasonably expected to be exacerbated by 
treatment with IRX-2, pembrolizumab may be included only after consultation with the 
study physician. 
9. Active or prior documented autoimmune or inflammatory disorders (including 
inflammatory bowel disease [e.g., colitis or Crohn's disease], diverticulitis [with the 
exception of diverticulosis], systemic lupus erythematosus, Sarcoidosis syndrome, or 
Wegener syndrome [granulomatosis with polyangiitis, Graves' disease, rheumatoid 
arthritis, hypophysitis, uveitis, etc]). The following are exceptions to this criterion: 
• 
Patients with vitiligo or alopecia. 
• 
Patients with hypothyroidism (e.g., following Hashimoto syndrome) stable on 
hormone replacement.  


 
 
19 
 
 
• 
Any chronic skin condition that does not require systemic therapy. 
• 
Patients without active disease in the last 2 years may be included but only after 
consultation with the study physician. 
• 
Patients with celiac disease controlled by diet alone. 
10. Current or prior use of immunosuppressive medication within 14 days prior to Cycle 1, 
Day 1. The following are exceptions to this criterion: 
• 
Intranasal, inhaled, topical steroid or local steroid injections (e.g., intra articular 
injection). 
• 
Systemic corticosteroids at physiologic doses not to exceed 10 mg/day of 
prednisone or its equivalent. 
• 
Steroids as premedication for hypersensitivity reactions (e.g., CT scan 
premedication). 
11. Major surgical procedure (as defined by the Investigator) within 28 days prior to Cycle 1, 
Day 1. Note: Local surgery of isolated lesions for palliative intent is acceptable. 
12. History of allogeneic organ transplantation. 
13. Symptomatic cardiopulmonary disease (including congestive heart failure and 
hypertension), coronary artery disease, serious arrhythmia or chronic lung disease. 
Patients with these conditions who are stable with relatively minor symptoms and who 
are appropriate candidates for systemic treatments need not be excluded. 
14. Myocardial infarction within the last 3 months. 
15. Has a known history of human immunodeficiency virus (HIV) (HIV 1/2 antibodies). 
16. Has a history of active Hepatitis B requiring ongoing antiviral therapy or a history of 
untreated Hepatitis C. 
17. Has received a live vaccine within 4 months of planned start of study therapy (Cycle 1, 
Day 1).  
Note: The killed virus vaccines used for seasonal influenza vaccines for injection are 
allowed provided not within 4 weeks of planned start of study therapy (Cycle 1, Day 1); 
however intranasal influenza vaccines (e.g., FluMist®) are live attenuated vaccines and 
are not allowed. 
18. Signs or symptoms of systemic infection (use of antibiotics to treat superficial infection 
or contamination of tumor shall not, by itself, be considered evidence of infection). 
19. Stroke or other symptoms of cerebral vascular insufficiency within the last 3 months. 
20. Previous diagnosis of invasive cancer from which the individual is not disease-free AND 
that has required treatment within the past 3 years, except for superficial skin, cervical 
cancer in-situ, or early stage prostate or bladder cancer (i.e. treatment with curative 
intent and long term disease-free expectations).  
21. History of leptomeningeal carcinomatosis 
22. Known allergy or hypersensitivity to any of the study drugs or any of the study drug 
excipients. 
23. Female patients who are pregnant or breastfeeding or male or female patients of 
reproductive potential who are not willing to employ effective birth control from 
screening to one year after last dose of cyclophosphamide or 180 days after the last 
dose of pembrolizumab therapy, whichever is longer.  
 


 
 
20 
 
 
4.0 Screening and Registration Procedures 
Screening Procedures 
Diagnostic or laboratory studies performed exclusively to determine eligibility for this trial will be done 
only after obtaining written informed consent. Studies or procedures that were for clinical indications 
(not exclusively to determine study eligibility) may be used for baseline values, even if the studies were 
done before informed consent was obtained.  All screening procedures and their respective windows 
are detailed in the Study Calendar.  
Informed Consent  
The investigational nature and objectives of the trial, the procedures and treatments involved and their 
attendant risks and discomforts, and potential alternative therapies will be carefully explained to the 
subject and a signed informed consent will be obtained.  Documentation of informed consent for 
screening will be maintained in the subject’s research chart and medical record and the patient will 
receive a copy of the signed inform consent document. All institutional, Federal, and State regulations 
concerning Informed Consent will be fulfilled.   
COH Data Coordinating Center 
Eligible participants will be registered on the study centrally by the Data Coordinating Center (DCC) at 
City of Hope.   
DCC staff are available between the hours of 8.00 am and 5.00 pm PST, Monday through Friday (except 
holidays).  
o E-mail: DCC@coh.org 
Registration Requirements/Process 
4.1.1 Confirming, reserving a slot and dose level assignment  
Staff (including physicians, protocol nurses and/or CRCs) should contact the DCC to verify the current 
dose level and slot availability, and to reserve a slot for a specific prospective subject. Slots can only be 
held for a limited time.  
Eligible subjects must be registered prior to the start of protocol therapy. Issues that would cause 
treatment delays should be discussed with the Principal Investigator. If a subject does not receive 
protocol therapy following registration, the subject’s registration on the study may be canceled after 
discussion with the PI. The Data Coordinating Center should be notified of cancellations as soon as 
possible.  
4.1.2 Registration Process 
Once a slot at a dose level has been reserved, the signed informed consent has been obtained, all 
pretreatment evaluations have been performed, and subject’s eligibility has been confirmed by the Data 
Coordinating Center (DCC), a subject will be registered on study.  
To register a subject, the treating physician should contact the protocol nurse or the responsible clinical 
Research Coordinator (CRC) in the Clinical Trial Office (CTO) to complete the eligibility checklist.  
Allow up to 24 hours for the DCC to review eligibility. To register a participant the subsequent procedure 
is to be followed: 


 
 
21 
 
 
1. The study team should contact the DCC via email to provide notification regarding the pending 
registration and communicate desired timeline of the registration, especially if it must be 
completed promptly to meet the registration window.  
2. The study team will email a Complete Eligibility Packet to the DCC, which consists a copy of the 
following documents: 
o Registration Cover Sheet (Appendix II) 
o Completed eligibility checklist (printed from Section 3.0 of the protocol) with required 
signature(s)  
o Source documents that support all eligibility criteria listed in the checklist  
o Signed Informed Consent  
o Signed HIPAA authorization form (if separate from informed consent) 
o Signed subject’s bill of Rights (California only) 
3. When all source documents are received, the DCC will review to verify eligibility, working with the 
study team to resolve any missing required source elements. Any missing documents may delay 
review and registration. A participant failing to meet all protocol eligibility requirements will not 
be registered and the study team will be immediately notified.  
4. Once eligibility is confirmed, the DCC will send a Confirmation of Registration Form and signed 
Eligibility Checklist, including the Subject Study Number and cohort assignment to:  
o The study team: Site Lead Investigator, treating physician/sub-investigator, protocol 
nurse, CRC and pharmacy (as needed). 
o The COH Study PI and sponsor team designees (including but not limited to study 
monitor(s) and statistician(s)).  
5. Upon receipt of the Confirmation of Registration Form, COH study team will register the patient 
in OnCore. The DCC will register non-COH patients in OnCore. 
 
A subject failing to meet all protocol requirements will not be registered.  
Patients must begin protocol treatment within 1 week following registration date.  
Screen Failures and Registered Participants Who Do Not Begin Study Treatment  
Notify the DCC immediately if the participant screen fails after registration or if the participant does not 
start treatment. For non-COH sites, the reason for screen failure will be documented in the registration 
coversheet (Appendix II) and submitted to the DCC. 
5.0 Treatment Program 
Treatment Overview 
For a detailed tabular view of the treatment, monitoring, and follow-up schedule, see the Study 
Calendar (Section 10) 
Treatment cycle definition 
Each cycle will consist of 3 weeks coinciding with timing of pembrolizumab infusions.  Cycle 1, Day 1 will 
start with the 1st day of IRX-2 regimen and a dose of pembrolizumab. 


 
 
22 
 
 
Treatment Plan 
Treatment on study will continue up to 24 months or until confirmed disease progression, patient 
refusal, patient lost to follow up, unacceptable toxicity, whichever occurs first, or the study is 
terminated by the Sponsor. 
The study will consist of two phases – a safety phase and a dose expansion phase: 
 
Safety phase: A standard 3+3 dose finding rule will be employed with two dose levels.  Patients will start 
at dose level 1 [IRX-2 regimen at dose of 2 mL (230 units/day)]. Patients will be enrolled in cohorts of 3, 
separated by at least 1 week.  Initially, 3 patients will be treated with expansion to 3 more patients in 
the event of 1/3 dose limiting toxicities (DLTs) as observed during the first 3 weeks of treatment, 
escalation if 0/3 or 1/6 DLTs, and termination of escalation if ≥ 2 DLTs.  Once ≥ 2 patients experience a 
DLT at a dose level, the next lower dose level will be expanded to 6 evaluable patients, if fewer than 6 
evaluable patients have been treated at the dose level. The maximum tolerated dose (MTD) is defined 
as the highest dose tested in which only 0 or 1 out of 6 evaluable patients experience a DLT. The 
Recommended Phase 2 Dosing (RP2D) will not exceed the MTD but may be lower based on cumulative 
toxicities and dose modification patterns.  If the rules suggest escalation at the highest proposed dose, 
that dose will accrue to 6 patients where <2 DLTs will determine that as the MTD, and 2 or more DLTs 
will require de-escalation. These rules will be used to determine the IRX-2 regimen dosing for the 
expansion phase.   
 
Dose expansion phase: 8-14 patients will be enrolled at the recommended dose level from the safety 
phase, for a total enrollment of 20 patients. The 6-12 patients treated in the dose escalation portion of 
the study will be counted as part of the dose expansion patient population. Two doses of IRX-2 will be 
considered (Table 5-1). There will be no intra-patient dose escalation; participants will undergo dose 
modification due to toxicity (Section 6).  
Table 5-1: Dose Levels of IRX-2 
Dose Level 
IRX-2 Dose  
Starting 
Dose Level: 
1 
230 units (2 mL) total daily for 10 days: 2 injections daily comprised of 115 units in each 
1 mL injection; 1 injection administered into the left and 1 injection into the right 
supraclavicular lymph node regions (substitute axillary regions if supraclavicular nodes 
have been removed)  
2 
460 units (4 mL) total daily for 10 days: 4 injections daily comprised of 115 units in each 
1 mL injection; 1 injection each into both supraclavicular and 1 injection each into both 
axillary lymph node regions (substitute mastoid insertion if supraclavicular or axillary 
nodes have been removed)  
 
Definition of Dose Limiting Toxicity 
A DLT will be defined as any Grade 3 or higher toxicity that occurs during the DLT evaluation period of 3 
weeks (between Cycle 1 Day 1 and Cycle 1 Day 21) and are considered related to study treatment. Toxicity 
that is clearly and directly related to the primary disease or to another etiology is excluded from this 
definition. A patient is evaluable for DLT if he/she receives the cycle 1 dose of pembrolizumab (100%) and 


 
 
23 
 
 
80% of the planned doses of IRX-2 during cycle 1 OR he/she experiences a DLT.  If this minimum amount 
of treatment is not reached due to treatment related AEs, this is considered a DLT.  However, patients 
who fail to achieve this minimum amount of treatment due to reasons that are clearly unrelated to 
treatment (e.g. error on part of patient or healthcare provider) will be replaced.  The following will be 
DLTs: 
• 
Any Grade 4 adverse event deemed to be immune-related 
• 
Any ≥Grade 3 colitis 
• 
Any Grade 3 or 4 non-infectious pneumonitis irrespective of duration 
• 
Any Grade 2 pneumonitis that does not resolve to ≤Grade 1 within 3 days of the initiation of 
maximal supportive care 
• 
Any Grade 3 irAE, excluding colitis or pneumonitis, that does not downgrade to Grade 2 within 3 
days after onset of the event despite optimal medical management including systemic 
corticosteroids or does not downgrade to ≤Grade 1 or baseline within 14 days 
• 
Liver transaminase elevation > 8 × ULN or total bilirubin > 5 × ULN 
• 
Any unresolved toxicity necessitating a delay in the delivery of cycle 2, day 1 of pembrolizumab 
by > 3 weeks 
• 
Any ≥Grade 3 non-irAE, except for the exclusions listed below 
 
The definition excludes the following conditions:  
• 
Grade 3 fatigue lasting ≤7 days 
• 
Grade 3 endocrine disorder (thyroid, pituitary, and/or adrenal insufficiency) that is managed with 
or without systemic corticosteroid therapy and/or hormone replacement therapy and the patient 
is asymptomatic 
• 
Grade 3 inflammatory reaction attributed to a local antitumor response (e.g., inflammatory 
reaction at sites of metastatic disease, lymph nodes, etc.) 
• 
Concurrent vitiligo or alopecia of any AE grade 
• 
Grade 3 infusion-related reaction (first occurrence and in the absence of steroid prophylaxis) that 
resolves within 6 hours with appropriate clinical management 
• 
Grade 3 or 4 neutropenia that is not associated with fever or systemic infection that improves by 
at least 1 grade within 3 days. Grade 3 or Grade 4 febrile neutropenia will be a DLT regardless of 
duration or reversibility 
• 
Grade 3 or 4 lymphopenia 
• 
Grade 3 thrombocytopenia that is not associated with clinically significant bleeding that requires 
medical intervention, and improves by at least 1 grade within 3 days 


 
 
24 
 
 
• 
Isolated Grade 3 electrolyte abnormalities that are not associated with clinical signs or symptoms 
and are reversed with appropriate maximal medical intervention within 3 days 
Agents Administration 
5.1.1 IRX-2 Regimen  
The IRX-2 regimen will be given every 12 weeks starting with Cycle 1.  Cyclophosphamide will be 
administered as an intravenous infusion on Day 1 of each IRX-2 regimen at a dose of 300 mg/m2.  
IRX-2 will be administered subcutaneously into a maximum of 4 lymph node-bearing regions, i.e. the 
bilateral supraclavicular and axillary regions.  For dose level 1, 1 ml (115 units/ml) will be injected into 
each of the 2 lymph node regions (total 2 ml (230 units/day)) corresponding to the bilateral supraclavicular 
areas only.  If dose level 2 is reached, 1 ml (115 units/ml) will be injected into each of the 4 lymph node 
regions (total 4 ml (460 units/day)).  
IRX-2 Regimen includes one dose of cyclophosphamide on Day 1 of each designated cycle, and 
subcutaneous IRX-2 injections in bilateral supraclavicular (and axillary regions if applicable) for 10 days 
between Days 4 and 15.  The 10 days are not required to be consecutive (e.g. dosing not mandatory on 
weekends/holidays) provided dosing is completed by day 15. 
 
5.1.2 Pembrolizumab  
Pembrolizumab will be given at the approved dose of 200 mg every 3 weeks beginning with Day 1 of 
Cycle 1.  
Pembrolizumab will be administered as an intravenous infusion over 30 minutes every 21 days (+/- 3 days). 
Study Procedures 
For a detailed list of all study procedures including timing and windows, see the Study Calendar.  
Duration of Therapy and Criteria for Removal from Study Treatment 
Patients will receive study treatment until disease progression, unacceptable toxicity, or other criteria 
for removal from study treatment are satisfied. Participants may be removed from treatment for any of 
the following reasons:  
• 
Evidence of disease progression 
• 
Patient is deemed intolerant to study treatment because of toxicity, despite dose 
modification/delay.  
• 
Intercurrent illness that prevents further administration of treatment 
• 
Participant withdraws from the treatment phase of the study 
• 
General or specific changes in the participant’s condition, including non-compliance, which 
renders the participant unacceptable for further treatment in the opinion of the treating 
investigator.  
Documentation of the reason for discontinuing therapy and the date effective should be made in the 
medical record and appropriate eCRF. The participant should then proceed to off-treatment procedures 
for safety monitoring, and follow-up procedures. The participant’s status is to be modified in the MIDAS 
system once the off-treatment period is completed. Alternative care options will be discussed with the 
participant.  


 
 
25 
 
 
End of Study Follow Up 
In End of Study Follow-Up participants will be followed for survival information which may be obtained 
by reviewing the medical record, contacting the participant and/or a review of outside medical records 
(note: this is not an all-inclusive list).  
The schedule and windows for assessments and data collection points are further detailed in the Study 
Calendar.  
Criteria for Completion of Study Participation 
Participants may be removed from the study as a whole at any time at their own request, or they may 
be withdrawn at the discretion of the investigator for safety, behavioral or administrative reasons. The 
reasons for discontinuing study participation should be documented and may include: 
• 
Participant completes all study procedures including all study follow-up procedures. 
• 
Participant withdraws consent for follow-up. 
• 
Participant is determined to be lost to follow-up. All attempts to contact the subject must be 
documented.  
The reason for study removal and the date the participant was removed must be documented in the 
source documentation and the study-specific case report form (CRF). The participant’s status is to be 
modified in the MIDAS system once the participant completes the study.  
Supportive Care and Other Concomitant Therapy 
All concomitant medications taken during the study treatment will be recorded in the Case Report Form. 
The minimum requirements are that drug name and the dates of administration are to be recorded. All 
prescription and over-the-counter medications that have been taken within 1 month prior to the first 
dose of investigational treatment should also be reported in the Case Report Form. 
Patients should receive full supportive care during the study, including treatment with antibiotics, 
antiemetics, blood transfusions, antidiarrheals, and analgesics as appropriate. 
5.1.3 Included concomitant medications  
Supportive medications: 
Supportive medication/class of drug: 
Usage: 
Concomitant 
medications 
or 
treatments 
(e.g., acetaminophen or diphenhydramine) 
deemed necessary to provide adequate 
prophylactic or supportive care, except for 
those medications identified as “prohibited”. 
To be administered as prescribed by the Investigator 
Best supportive care (including antibiotics, 
nutritional support, correction of metabolic 
disorders, optimal symptom control, and pain 
management [including palliative radiotherapy 
to non-target lesions, etc.]) 
Should be used, when necessary, for all patients 
 


 
 
26 
 
 
5.1.4 Excluded concomitant medications 
 Prohibited concomitant medications 
Prohibited medication/class of drug: 
Usage: 
Any investigational anticancer therapy other 
than those under investigation in this study 
Should not be given concomitantly whilst the patient 
is on study treatment 
mAbs against CTLA-4, PD-1, or PD-L1 other 
than pembrolizumab 
Should not be given concomitantly whilst the patient 
is on study treatment 
Any concurrent chemotherapy, radiotherapy, 
immunotherapy, or biologic or hormonal 
therapy for cancer treatment other than those 
under investigation in this study 
Should not be given concomitantly whilst the patient 
is on study treatment. (Concurrent use of hormones 
for non-cancer-related conditions [e.g., insulin for 
diabetes and hormone replacement therapy] is 
acceptable. Local treatment of isolated lesions, 
excluding target lesions, for palliative intent is 
acceptable [e.g., by local surgery or radiotherapy]) 
Immunosuppressive medications including, but 
not limited to, systemic corticosteroids at 
doses exceeding 10 mg/day of prednisone or 
equivalent, methotrexate, azathioprine, and 
tumor necrosis factor-α blockers 
Should not be given concomitantly, or used for 
premedication prior to the immunotherapy infusions. 
The following are allowed exceptions: 
• 
Use of immunosuppressive medications for 
the management of immune-related AEs,  
• 
Short-term premedication for patients 
receiving combination agents where the 
prescribing information for the agent 
requires the use of steroids for documented 
hypersensitivity reactions,  
• 
Use in patients with contrast allergies.  
• 
In addition, use of inhaled, topical, and 
intranasal corticosteroids is permitted. 
  
A temporary period of steroids will be allowed if 
clinically indicated and considered to be essential for 
the management of non-immunotherapy related 
events experienced by the patient (e.g., chronic 
obstructive pulmonary disease, radiation, nausea, 
etc.). 
 


 
 
27 
 
 
6.0 Anticipated Toxicities and Dose Delays/Modifications for Adverse Events 
 
Anticipated Toxicities 
Immune checkpoint inhibitors are known to cause autoimmune toxicities including pneumonitis, colitis, 
hepatitis, endocrinopathies, nephritis, rash, encephalitis.  
In prior Phase 2 studies the IRX-2 Regimen was tolerated with acceptable toxicity. The most common 
adverse events (AEs) 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 (one Grade 3, one Grade 4) and all resolved without 
sequelae. There were only minor (grade 1) alterations in post-treatment laboratory values. Eight serious 
adverse events (SAEs) in 7 subjects were reported during treatment and the 30-day post-operative 
period: aspiration pneumonia (n = 3), respiratory tract infection, asthma exacerbation, wound infection, 
neck abscess and alcohol withdrawal (n = 1 each); only the postoperative wound infection was 
considered related to the study treatment (42). During treatment, several subjects noted decreased pain 
or improved swallowing and no significant progressive symptoms were noted. 
6.1.1 Potential Overlapping Toxicities 
BOTH: diarrhea, nausea, abdominal pain, decreased appetite, fever, headache, infusion related 
reaction (injection site reaction), arthralgia, shortness of breath, asthenia 
Pembrolizumab only: fatigue, itching rash, increased creatinine, increased blood sugar changes, 
low sodium, increased lipase, alterations in thyroid hormones, increased alkaline phosphatase, 
ALT, amylase, and AST, chills, constipation, cough, dizziness, dry mouth, dry skin, inflammation 
of color, or mouth, loss of color pigmentation, lung inflammation, musculoskeletal pain, nausea, 
redness, swelling, vomiting 
IRX-2 only: lymphopenia, anemia, difficult swallowing, myalgia, rhinorrhea, neutrophilia, 
hypoalbuminemia, dehydration, hypotension, painWith elimination of indomethacin, 
omeprazole, and zinc supplementation, we aim to see fewer toxicities that may be associated 
with indomethacin-induced gastritis.  To still carefully assess overlapping toxicities, dose 
escalation will start with dose level 1 as per Section 5.0. 
 
 
6.1.2 General Information 
The study will use the NCI Common Terminology for Adverse Events (CTCAE) Version 5.0 to grade 
toxicities.  
Intra-patient dose escalation is never permitted in this study. Rules for dose modification are found in 
Appendix I.   
Baseline values are from the last values obtained prior to treatment. 
For holds due to toxicities related to study agent, if the participant does not meet the criteria to resume 
treatment within 14 days, the participant must permanently discontinue study treatment.  


 
 
28 
 
 
6.1.3 Dose Modifications  
Guidelines for the management of immune-mediated reactions for pembrolizumab are provided in the 
Dosing Modification and Toxicity Management Guidelines in Appendix I. Patients should be thoroughly 
evaluated and appropriate efforts should be made to rule out neoplastic, infectious, metabolic, toxin, or 
other etiologic causes of the immune related AE. Serologic, immunologic, and histologic (biopsy) data, as 
appropriate, should be used to support an immune related AE diagnosis. In the absence of a clear 
alternative etiology, events should be considered potentially immune related. 
In addition, there are certain circumstances in which pembrolizumab should be permanently 
discontinued (see the Dosing Modification and Toxicity Management Guidelines in Appendix I).  
Dose reductions for pembrolizumab are not permitted. In case of doubt, the local investigator should 
consult with the study principal investigator and Sponsor. 
Patients should be thoroughly evaluated and appropriate efforts should be made to rule out neoplastic, 
infectious, metabolic, toxin, or other etiologic causes of the immune related AE. Serologic, immunologic, 
and histologic (biopsy) data, as appropriate, should be used to support an immune related AE diagnosis. 
In the absence of a clear alternative etiology, events should be considered potentially immune related. 
All toxicities will be graded according to NCI CTCAE, Version 5. 
Dose reductions for IRX-2 injections are not permitted. In case of doubt, the local investigator should 
consult with the study principal investigator. 
7.0 Data and Safety Monitoring and Adverse Event Reporting  
7.1.0  Adverse Events and Unanticipated Problems  
7.1.1  Definitions  
7.1.1.1 Adverse Event (AE) 
An adverse event is any untoward medical experience or change of an existing condition that occurs 
during or after treatment, whether or not it is considered to be related to the protocol intervention. 
 
 
7.1.1.2 Serious Adverse Event (SAE) 
A serious adverse event is any expected or unexpected adverse events that result in any of the following 
outcomes: 
• 
Death 
• 
Is life-threatening experience (places the subject at immediate risk of death from the event as it 
occurred) 
• 
Unplanned hospitalization (equal to or greater than 24 hours) or prolongation of existing 
hospitalization 
• 
A persistent or significant disability/incapacity 
• 
A congenital anomaly/birth defect 
• 
Secondary malignancy* 


 
 
29 
 
 
• 
Any other adverse event that, based upon appropriate medical judgment, may jeopardize the 
subject’s health and may require medical or surgical intervention to prevent one of the 
outcomes listed above (examples of such events include allergic bronchospasm requiring 
intensive treatment in the emergency room or at home, blood dyscrasias of convulsions that do 
not result in inpatient hospitalization, or the development of drug dependency or drug abuse). 
*Modified from 21 CFR 312.32 
 
7.1.1.3 Unanticipated Problems Involving Risks to Subjects or Others 
An unanticipated problem is any incident, experience, or outcome that meets all three of the following 
criteria: 
1. Unexpected (in terms of nature, severity, or frequency) given the following: a) the research 
procedures described in the protocol-related documents such as the IRB approved research 
protocol, informed consent document or Investigator Brochure (IB); and b) the characteristics of 
the subject population being studied; AND 
2. Related or possibly related to participation in the research (possibly related means there is a 
reasonable possibility that the incident, experience, or outcomes may have been caused by the 
drugs, devices or procedures involved in the research); AND 
3. Suggests that the research places subjects or others at greater risk of harm (including physical, 
psychological, economic, or social harm) than previously known or recognized. 
 
7.1.1.4 Adverse Events of Special Interest (AESI) 
Specific adverse events, or groups of adverse events, will be followed as part of standard safety 
monitoring activities. These events, regardless of seriousness, will be reported. 
  
 
 
7.1.2  Assessment of Adverse Events 
The site Investigator will be responsible for determining the event name, assessing the severity (i.e. 
grade), expectedness, and attribution of all adverse events.  
 
7.1.2.1 Assessment of Adverse Event Name and Grade 
Adverse events will be characterized using the descriptions and grading scales found in the most recent 
CTCAE v 5.0.  A copy of the scale can be found at NCI/ CTEP web site. The determination of severity for 
all other events not listed in the CTCAE v 5.0 should be made by the investigator based on medical 
judgment and the severity categories of Grade 1 to 5 as defined below: 
• 
Grade 1 (mild) – An event that is usually transient and may require only minimal treatment or 
therapeutic intervention. The event does not generally interfere with usual activities of daily 
living. 
• 
Grade 2 (moderate) – An event that is usually alleviated with additional specific therapeutic 
intervention. The event interferes with usual activities of daily living, causing discomfort but 
poses no significant or permanent risk of harm to the subject. 


 
 
30 
 
 
• 
Grade 3 (severe) – An event that requires intensive therapeutic intervention. The event 
interrupts usual activities of daily living, or significantly affects the clinical status of the subject. 
• 
Grade 4 (life threatening) – An event, and/or its immediate sequelae, that is associated with an 
imminent risk of death or with physical or mental disabilities that affect or limit the ability of the 
subject to perform activities of daily living (eating, ambulation, toileting, etc). 
• 
Grade 5 (fatal) – Death (loss of life) as a result of an event. 
 
7.1.2.2 Assessment of Attribution 
The following definitions will be used to determine the causality (attribution) of the event to the study 
agent or study procedure. 
• 
Unrelated – The event is clearly related to other factors such as the participant’s clinical state, 
other therapeutic interventions, or concomitant medications administered to the participant. 
• 
Unlikely – The event is doubtfully related to the investigational agent(s). The event was most 
likely related to other factors such as the participant’s clinical state, other therapeutic 
interventions, or concomitant drugs. 
• 
Possible – The event follows a reasonable temporal sequence from the time of drug 
administration, but could have been produced by other factors such as the participant’s clinical 
state, other therapeutic interventions, or concomitant drugs.  
• 
Probable – The event follows a reasonable temporal sequence from the time of drug 
administration, and follows a known response pattern to the study drug. The event cannot be 
reasonably explained by other factors such as the participant’s clinical state, therapeutic 
interventions, or concomitant drugs.  
• 
Definite – The event follows a reasonable temporal sequence from the time of drug 
administration, follows a known response pattern to the study drug, cannot be reasonably 
explained by other factors such as the participant’s condition, therapeutic interventions, or 
concomitant drugs, AND occurs immediately following study drug administration, improves 
upon stopping the drug, or reappears on re-exposure.  
 
7.1.2.3 Assessment of Expectedness 
The following definitions will be used to determine the expectedness of the event: 
 
• 
Unexpected– An adverse event is unexpected if it is not listed in the investigator’s brochure 
and/or package insert; is not listed at the specificity or severity that has been observed; is not 
consistent with the risk information described in the protocol and/or consent; is not an 
expected natural progression of any underlying disease, disorder, condition, or predisposed risk 
factor of the research participant experiencing the adverse event. *Modified from 21 CFR 
312.32 (a) 
• 
Expected – An adverse event is expected if it does not meet the criteria for an unexpected 
event, OR is an expected natural progression of any underlying disease, disorder, condition, or 
predisposed risk factor of the research participant experiencing the adverse event. 
 
 


 
 
31 
 
 
7.1.3   Reporting of Adverse Events 
 
7.1.3.1 Routine Reporting of Non-Serious Adverse Events by Site Investigators  
Routine AE recording will occur via data entry into the study eCRF. Recording of adverse events will 
begin once the patient is consented and will continue until 30 days after the last dose of treatment. 
Adverse events will be monitored by the Protocol Management Team (PMT). Adverse events that do not 
meet the criteria of serious OR are not unanticipated problems do not require expedited reporting.  AEs 
reported through expedited processes (i.e. reported to the IRB, DSMC, FDA, etc.) must also be reported 
in routine study data submissions.  
 
7.1.3.2  Expediting Reporting Requirements of SAEs and UPs by Site Investigators 
7.1.3.2.1 
Criteria for Reporting SAEs/UPs to the Coordinating Center 
Serious Adverse Events meeting the criteria specified below will be reported to the Coordinating Center 
within 24 hours of notification that the event occurred.  
 
Adverse events that require expedited reporting include: 
• 
AEs or SAEs that meet the definition of an unanticipated problem 
• 
All deaths that occur within 30 days of active treatment 
• 
All deaths that occur after 30 days of active treatment that are unexpected and possibly, 
probably, or definitely related to the study agent or procedure 
• 
All serious adverse events, regardless of relationship to study agent or study procedure, that 
occur within 30 days of the last day of treatment 
• 
All serious adverse events that occurred after 30 days of active treatment/therapy that are 
considered possibly, probably, or definitely related to the study agent or procedure  
 
Note: Follow-up reports must be submitted for all events that require expedited reporting when the 
status of the event changes and until the resolution or stabilization of the event. 
 
Reportable serious adverse events must be followed until the event is resolved, stabilized, or 
determined to be irreversible by the participating investigator; for ongoing reportable adverse events 
that are unrelated to study agent, the follow-up period may end at the 30-days post study-drug 
assessment. The Coordinating Center should be consulted prior to ending the follow-up of events that 
have stabilized. 
 
7.1.3.2.2 
Non-COH Sites: Procedure for Reporting SAEs/UPs to the COH Data Coordinating 
Center  
1. Sites are to report to their local IRB per their site’s specific institutional and IRB 
guidelines. As soon as possible, non-COH sites will provide to the COH Data 
Coordinating Center copies of the IRB submission and corresponding IRB 
response.  
2. Document/describe the SAE/UP on each of the following:  


 
 
32 
 
 
a. MedWatch 3500A   
i. Downloadable form at http://www.fda.gov/medwatch/getforms.htm  
b. UP/SAE Coversheet 
i. SAE Coversheet is found in Appendix III.  A modifiable Microsoft Word 
document is also available from the Data Coordinating Center.  An 
electronic signature on the document will be accepted. 
3. Scan and email above documents to DCC@coh.org with the subject title as “IRB 
# SAE”.   
a. All SAE reports received at this account are forwarded immediately to 
study Principal Investigator, and to Coordinating Center personnel.  
b. While not required, if available and applicable, please also include the 
local IRB submission for this event in the submission.  
4. If an email receipt from Coordinating Center personnel is not received within 
one working day, please call 626-256-4673 x 83968 and/or email DCC@COH.org. 
 
7.1.3.2.3 
COH Investigative Sites: Procedure for Reporting SAEs/UPs to the Coordinating 
Center 
1. Email the following information to DCC@coh.org and jchao@coh.org:   
a. Participant ID, date the event met criteria for reporting, whether 
the event meets the definition of serious, whether the event is an 
unanticipated problem, grade of event, attribution of event, 
whether the event is a known expected toxicity to study agent. 
2. Complete the iRIS AE/UP reporting form per COH reporting timeline. 
 
 
7.1.3.3 Additional Reporting Requirements of the Study Principal Investigator  
7.1.3.3.1 
Reporting to COH IRB and DSMC 
The study PI (or designee) will report to COH IRB and DSMC via iRIS all reportable serious adverse events 
that occur at COH and non-COH sites and meet COH IRB and DSMC expedited reporting criteria 
according to City of Hope’s Institutional policy. The study PI will also submit a Protocol Management 
Team (PMT) report to the COH DSMC at the frequency outlined in Section 3.6. This report will include a 
review of aggregate adverse event data.   
 
7.1.3.3.2 
Reporting to the FDA  
The study PI (or designee) will be responsible for contacting the Office of IND Development and 
Regulatory Affairs (OIDRA) at COH to ensure prompt reporting of safety reports to the FDA.  OIDRA will 
assist the PI with the preparation of the report and submit the report to the FDA in accordance with the 
approved City of Hope’s Institutional policy.  
 


 
 
33 
 
 
Serious Adverse Events meeting the requirements for expedited reporting to the Food and Drug 
Administration (FDA), as defined in 21 CFR 312.32, will be reported as an IND safety report using the 
MedWatch Form FDA 3500A for Mandatory Reporting. 
 
The criteria that require reporting using the Medwatch 3500A are: 
• 
Any unexpected fatal or life threatening adverse experience associated with use of the drug 
must be reported to the FDA no later than 7 calendar days after initial receipt of the 
information [21 CFR 312.32(c)(2)] 
• 
Any adverse experience associated with use of the drug that is both serious and unexpected 
must be submitted no later than 15 calendar days after initial receipt of the information [21 
CFR 312.32(c)(1)] 
• 
Any follow-up information to a study report shall be reported as soon as the relevant 
information becomes available. [21 CFR 312.32(d)(3)] 
 
In addition, the study PI will submit annually within 60 days (via COH OIDRA) of the anniversary date of 
when the IND went into effect, an annual report to the FDA which is to include a narrative summary and 
analysis of the information of all FDA reports within the reporting interval, a summary report of adverse 
drug experiences, and history of actions taken since the last report because of adverse drug experiences. 
 
7.1.3.3.3 
Reporting to Participating Investigators 
The study PI (or designee) will report all reportable serious adverse events to participating investigators 
as an IND Safety Report occurring within 30 calendar days of receipt of sponsor (lead site) notification, 
and indicate whether or not a protocol and/or consent form change is required.  A cover letter will 
indicate the protocol title, the IND#, whether the FDA was informed, and, for non-COH sites, a 
statement that the report should be submitted to their local IRB for review as an IND safety report if 
applicable per local IRB policy. 
 
The study PI will also forward to participating sites all IND safety reports received from Brooklyn 
ImmunoTherapeutics (formerly IRX Therapeutics), indicating whether a consent form or protocol change 
is required within 30 days of notification to study PI. 
 
7.1.3.3.4 
Reporting to Brooklyn ImmunoTherapeutics 
All serious adverse events and AESIs (initial and follow-up information) will be reported by the study PI 
to Brooklyn ImmunoTherapeutics (formerly IRX Therapeutics) within the same timeframes as required 
by reporting to the FDA. A copy of Medwatch Form FDA 3500A submitted to the FDA will be submitted 
to Brooklyn ImmunoTherapeutics.   
 
 
7.2.0  Protocol Deviations and Single Subject Exceptions 
It is understood that deviations from the protocol should be avoided, except when necessary to 
eliminate an immediate hazard to a research participant. Brief interruptions and delays may occasionally 
be required because of travel delays, airport closures, inclement weather, family responsibilities, 
security alerts, government holidays, and so forth. Delays can also extend to complications of disease or 
unrelated medical illnesses not related to disease progression. The PI has the discretion to deviate from 


 
 
34 
 
 
the protocol when necessary so long as such a deviation does not threaten patient safety or protocol 
scientific integrity. As a result of deviations, corrective actions are to be developed by the study staff 
and implemented promptly. 
 
7.2.1  Definitions 
7.2.1.1 Deviation 
A deviation is a divergence from a specific element of a protocol that occurred without prior IRB 
approval.  Investigators may deviate from the protocol to eliminate immediate hazard(s) for the 
protection, safety, and well-being of the study subjects without prior IRB approval.  
Examples include, but are not limited to: a) dose adjustments based on excessive patient weight; b) 
alteration in treatment schedule due to non-availability of the research participant for treatment; and c) 
laboratory test results which are slightly outside the protocol requirements but at levels that do not 
affect participant safety. 
 
 
7.2.1.2 
Single Subject Exceptions (SSE) 
An SSE is a planned deviation, meaning that it involves circumstances in which the specific procedures 
called for in a protocol are not in the best interests of a specific patient.  It is a deviation that is 
anticipated and receives prior approval by the PI and the IRB.  
  
7.2.2  Reporting of Deviations and SSEs 
 
7.2.2.1 
Reporting Deviations 
For any deviation, the Investigator will notify the COH DSMC and IRB within 5 calendar days of its 
occurrence via iRIS in accordance with the Clinical Research Protocol Deviation policy. A list of deviations 
from all participating sites will be submitted along with the Protocol Management Team (PMT) progress 
report to the COH DSMC. 
 
For non-COH sites:  
o The local IRB and/or DSMC must be notified according to local institutional policies.  
o The study Principal Investigator must be notified as soon as practical (within 24 hours of 
notification of the event) via email to jchao@coh.org and dcc@coh.org.  This email 
should provide input on the following: 
▪ 
Description of the event 
▪ 
Impact on participant safety or the safety to others 
▪ 
Impact on the study design 
▪ 
A corrective and preventative action plan 
 
7.2.2.2 Reporting Single Subject Exceptions 


 
 
35 
 
 
The SSE must be submitted as a “Single Subject Exception Amendment Request” via iRIS in accordance 
with IRB guidelines and the Clinical Research Protocol Deviation policy.  An IRB approved SSE does not 
need to be submitted as a deviation to the DSMC. 
 
All non-emergency planned deviations from the protocol must have prior approval by the Study 
Principal Investigator, the Site Principal Investigator, COH IRB, and when applicable, the local IRB.  In 
addition, if contractually obligated, the sponsor must also approve the deviation.  
 
7.3.0  Study Oversight, Quality Assurance, and Data & Safety Monitoring 
7.3.1  All Investigator Responsibilities 
 
An investigator is responsible for ensuring that an investigation is conducted according to the signed 
investigator statement, the investigational plan, and applicable regulations; for protecting the rights, 
safety, and welfare of subjects under the investigator's care; and for the control of drugs under 
investigation.  
 
All Investigators agree to: 
• 
Conduct the study in accordance with the protocol and only make changes after notifying the 
Sponsor (or designee), except when necessary to protect the safety, rights or welfare of 
subjects. 
• 
Personally conduct or supervise the study (or investigation).  
• 
Ensure that the requirements relating to obtaining informed consent and IRB review and 
approval meet federal guidelines, as stated in § 21 CFR, parts 50 and 56.  
• 
Report to the Sponsor or designee any AEs that occur in the course of the study, in accordance 
with §21 CFR 312.64.  
• 
Ensure that all associates, colleagues and employees assisting in the conduct of the study are 
informed about their obligations in meeting the above commitments.  
• 
Maintain adequate and accurate records in accordance with §21 CFR 312.62 and to make those 
records available for inspection with the Sponsor (or designee).  
• 
Ensure that an IRB that complies with the requirements of §21 CFR part 56 will be responsible 
for initial and continuing review and approval of the clinical study.  
• 
Promptly report to the IRB and the Sponsor all changes in the research activity and all 
unanticipated problems involving risks to subjects or others (to include amendments and IND 
safety reports).  
• 
Seek IRB and Sponsor approval before any changes are made in the research study, except when 
necessary to eliminate hazards to the patients/subjects.  
• 
Comply with all other requirements regarding the obligations of clinical investigators and all 
other pertinent requirements listed in § 21 CFR part 312. 
 


 
 
36 
 
 
7.3.2  Study Principal Investigator Responsibilities 
The Study Principal Investigator is responsible for the conduct of the clinical trial, including overseeing 
that sponsor responsibilities as defined in § 21 CFR 312. Subpart D are executed in accordance with 
federal regulations.  
 
7.3.3  Protocol Management Team (PMT) 
The Protocol Management Team (PMT), minimally consisting of the study PI, site investigators, 
collaborating investigators, research nurse, clinical research associate/coordinator, and the study 
biostatistician, is responsible for ongoing monitoring of the data and safety of this study, including 
implementation of the stopping rules for safety/toxicity. 
 
The PMT is recommended to meet (in person or via teleconference) at least monthly to review study 
status. This review will include, but not be limited to, reportable AEs and UPs, and an update of the 
ongoing study summary that describes study progress in terms of the study schema. The meeting will be 
a forum to discuss study related issues including accrual, SAE/AEs experienced, study response, 
deviations/violations and study management issues. The appropriateness of further subject enrollment 
and the specific intervention for subsequent subject enrollment are addressed. It is recommended that 
minutes of these discussions be taken to document the date of these meetings, attendees and the 
issues that were discussed (in a general format).   
 
7.3.4  Monitoring  
Clinical site monitoring is conducted to ensure that the rights of human subjects are protected, that the 
study is implemented in accordance with the protocol and regulatory requirements, and that the quality 
and integrity of study data and data collection methods are maintained.  Monitoring for this study will 
be performed by the City of Hope Office of Clinical Trials Auditing and Monitoring (OCTAM).  
 
The site Investigator/Institution will permit the study monitors and appropriate regulatory authorities 
direct access to the study data and to the corresponding source data and documents to verify the 
accuracy of this data. The Investigator will allocate adequate time for such monitoring activities.  The 
Investigator will also ensure that the monitor or other compliance or quality assurance reviewer is given 
access to all the above noted study-related documents and study related facilities (e.g. pharmacy, 
diagnostic laboratory, etc.), and has adequate space to conduct the monitoring visit. 
 
Details of clinical site monitoring are documented in the OCTAM SOP that is provided as a supplement 
to this document. This document specifies the frequency of monitoring, monitoring procedures, the 
level of clinical site monitoring activities (e.g., the percentage of subject data to be reviewed), and the 
distribution of monitoring reports.  Staff from OCTAM will conduct monitoring activities and provide 
reports of the findings and associated action items in accordance with the details described in the SOP.  
Documentation of monitoring activities and findings will be provided to the site study team, the site PI, 
study PI, and the COH DSMC.  
 
7.3.5  Quality Assurance  
The City of Hope Clinical Research Information Support will provide support for this multi-center trial as 
detailed in the COH DCC Operations Plan provided as a supplement to this document.  
 


 
 
37 
 
 
7.3.6  City of Hope Data and Safety Monitoring Committee 
This is a risk level 4 study as defined in the City of Hope Institutional Data and Safety Monitoring Plan. 
This determination was made because the study involves COH as the IND holder and escalates the study 
agent to a dose above that indicated by the FDA for the agent’s currently approved indication. 
 
The DSMC is a multidisciplinary committee charged with overseeing the monitoring of safety of 
participants in clinical trials, and the conduct, progress, validity, and integrity of the data for all clinical 
trials that are sponsored by City of Hope. The committee is composed of clinical specialists with 
experience in oncology and who have no direct relationship with the study. The committee reviews the 
progress and safety of all active research protocols that are not monitored by another safety and data 
monitoring committee or board.  
 
The Study Principal Investigator is required to submit periodic status reports (the PMT report) according 
to the guidelines outlined in the City of Hope Institutional Data and Safety Monitoring Plan.  The PMT 
report will be submitted to the COH DSMC quarterly from the date of activation.   
 
The COH Data and Safety Monitoring Committee (DSMC) will review and monitor toxicity and accrual 
data from this trial. The DSMC will review up-to-date participant accrual; summary of all adverse events 
captured via routine and expedited reporting; a summary of deviations; any response information; 
monitoring reports, and summary comments provided by the study team. Other information (e.g. scans, 
laboratory values) will be provided upon request. For Phase I studies, a Phase I Tracking Log will be 
utilized and reviewed by the DSMC to monitor data and safety for dose escalation.  A review of outcome 
results (response, toxicity and adverse events) and factors external to the study (such as scientific or 
therapeutic developments) is discussed, and the Committee votes on the status of each study. 
Information that raises any questions about participant safety will be addressed with the Principal 
Investigator, statistician and study team. The PMT report and DSMC recommendations will be circulated 
to all participating sites for submission to their IRBs, in accordance with NIH guidance. 
 
8.0 Agent Information 
Drug Information for IRX-2 
Mode of Action:  IRX-2 is a primary cell-derived biologic containing multiple active cytokine 
components produced under pharmaceutical standards from phytohemagglutinin (PHA) and 
ciprofloxacin stimulated donor mononuclear cells. Route of administration of IRX-2 takes advantage 
of the normal afferent and efferent pathways of lymph node activation. By presenting the cytokine-
containing biologic in the area of lymph nodes rather than systemically, there is an opportunity to 
facilitate the mobilization of antigen presenting cells and enhance dendritic cell function as well as 
directly activate T cells to proliferate and become cytotoxic lymphocytes. T cells may become 
activated to common tumor antigens or mutated autologous neoantigens that are found in the local 
tumor microenvironment. Additionally, subcutaneous administration may be less toxic since the 
systemic cytokine drug concentration is much lower. 
8.1.1 Risks and Contraindications 
Ciprofloxacin and PHA are used in the manufacture of IRX-2. Ciprofloxacin and PHA concentrations are 
significantly reduced by centrifugation and dilution during manufacture and only very small amounts 


 
 
38 
 
 
remain in the final product. Nevertheless, individuals who are allergic to ciprofloxacin or PHA should not 
be treated with IRX-2. 
Cyclophosphamide is contraindicated in individuals with evidence of pre-existing myelosuppression, 
myelodysplasia or hemorrhagic cystitis. 
 
8.1.2 Supplier  
Brooklyn ImmunoTherapeutics  
8.1.3 Dose and Administration 
IRX-2 will be administered subcutaneously into a maximum of 4 lymph node-bearing regions: bilateral 
supraclavicular and bilateral axillary regions are preferred. IRX-2 can be administered in the bilateral 
mastoid lymph node-bearing region if supraclavicular or axillary nodes have been removed. Two dose 
levels will be considered. For dose level 1, one ml (115 units/ml) will be injected into 2 lymph node regions 
(total 2 ml (230 units/day)) preferentially into the supraclavicular node regions with substitution of axillary 
node regions if the supraclavicular lymph nodes have been removed. For dose level 2, one ml (115 
units/ml) will be injected into 4 lymph node regions (total 4 ml (460 units/day)).  
Other IRX-2 Regimen Medications:    
Cyclophosphamide will generally be purchased by the pharmacy of the study site and will be supplied to 
study personnel by the study site investigational pharmacist.  
 
IRX-2 regimen 
 
 
Agent 
Dose 
Route of Administration 
Cyclophosphamide 
(Day 1) 
300 mg/m2 
IV 
IRX-2: For 10 days 
starting on Day 4; the 
10 days are not 
required to be 
consecutive (e.g. dosing 
not mandatory on 
weekends/holidays) 
provided dosing is 
completed by Day 15  
Dose Level 1: 
230 units total daily 
(2 
injections 
daily 
comprised of 115 units in 
each injection) 
 
Dose Level 2: 
460 units total daily 
(4 injections daily 
comprised of 115 units in 
each injection) 
Dose Level 1: Subcutaneous at or near the 
supraclavicular lymph node regions bilaterally 
(substitute axillary if supraclavicular nodes have 
been removed). 
 
Dose level 2: Subcutaneous at or near the 
supraclavicular and axillary node regions 
bilaterally (if any of these nodes have been 
removed, can inject at or near mastoid 
insertion of both sternocleidomastoid muscles). 
 
The study personnel or other trained individuals will administer IRX-2 injections to ensure treatment 
compliance. IRX-2 can be given as a subcutaneous injection through a 25-guage needle. 
Any non-compliance with the study treatment needs to be documented in the clinical record. 


 
 
39 
 
 
8.1.4 Formulation/Agent Preparation 
IRX-2 is supplied as a pale yellow, sterile liquid for subcutaneous injection in 2.0 mL single-dose 
vials. All immediate study supply containers will be appropriately labeled to identify study 
number, lot number, and product identity. Study syringes for subcutaneous IRX-2 administration 
will be disposed of in accordance with biosafety procedures. Prepared drug must be used within 
24 hours of removing the vial from frozen storage. 
  
8.1.5 Storage 
IRX-2 must be stored in a secure area and maintained under labeled storage conditions at a range 
of −15oC to –50o C.   
Drug Information for Pembrolizumab (Keytruda®) 
Other Names: Keytruda® 
Mode of Action:  Pembrolizumab is a fully human immunoglobulin (Ig) G4 monoclonal antibody 
which binds to and blocks the activation of PD-1, an Ig superfamily transmembrane protein.  This 
prevents the interaction between PD-1 with its ligands programmed cell death ligand 1 (PD-L1), 
overexpressed on certain cancer cells, and programmed cell death ligand 2 (PD-L2), which is primary 
expressed on antigen presenting cells (APCs).  This results in the activation of T-cells and cell-
mediated immune responses against tumor cells or pathogens. 
8.1.6 Risks and Contraindications 
No contraindications or drug interactions 
Warnings and Precautions include development of: 
• 
Immune-Mediated Pneumonitis 
• 
Immune-Mediated Colitis 
• 
Immune-Mediated Hepatitis 
• 
Immune-Mediated Endocrinopathies 
• 
Immune-Mediated Nephritis and Renal Dysfunction 
• 
Immune-Mediated Skin Adverse Reactions  
• 
Immune-Mediated Encephalitis 
• 
Other Immune-Mediated Adverse Reactions 
• 
Infusion Reactions 
• 
Complications of Allogeneic HSCT  
• 
Embryo-Fetal Toxicity 
Adverse Reactions: (please refer to package insert for full details) 
Most common adverse reactions (>20%) in patients treated with pembrolizumab include: 
• 
fatigue, musculoskeletal pain, decreased appetite, pruritus, diarrhea, nausea, rash, 
pyrexia, cough, dyspnea, and constipation  
Dose delays or discontinuations as a result of treatment expected adverse events are listed in 
Section 6.0 and in the package insert. 
8.1.7 Supplier 
Routine commercial supply of medication based on FDA approved indication for advanced 
gastric/GEJ adenocarcinoma. 


 
 
40 
 
 
8.1.8 Dose and Administration 
Dosage Forms/Strengths: 100mg/4mL (25 mg/mL) solution in a single vial. 
Administer as an intravenous infusion over 30 minutes. 
• 
Gastric and GEJ adenocarcinoma 
o Pembrolizumab 200 mg every 3 weeks. 
8.1.9 Formulation/Agent Preparation 
Preparation for Intravenous Infusion 
• 
Visually inspect the solution for particulate matter and discoloration prior to administration. 
The solution is clear to slightly opalescent, colorless to slightly yellow. Discard the vial if 
visible particles are observed. 
• 
Dilute KEYTRUDA injection (solution) or reconstituted lyophilized powder prior to 
intravenous administration. 
• 
Withdraw the required volume from the vial(s) of KEYTRUDA and transfer into an 
intravenous (IV) bag containing 0.9% Sodium Chloride Injection, USP or 5% Dextrose 
Injection, USP. Mix diluted solution by gentle inversion. The final concentration of the 
diluted solution should be between 1 mg/mL to 10 mg/mL. 
• 
Discard any unused portion left in the vial. 
 
8.1.10 
Storage 
Store non-diluted vials under refrigeration at 2°C to 8°C (36°F to 46°F) in original carton to protect 
from light. Do not freeze. Do not shake. 
Store the diluted solution from the pembrolizumab 100 mg/4 mL vial either: 
• 
At room temperature for no more than 6 hours from the time of dilution.  This includes 
room temperature storage of the infusion in the IV bag, and the duration of the infusion. 
• 
Under refrigeration at 2°C to 8°C (36°F to 46°F) for no more than 24 hours from the time of 
dilution. If refrigerated, allow the diluted solution to come to room temperature prior to 
administration. 
• 
Do not freeze. 
 
Use of IRX-2 and COVID-19 
As the extent of the exposure and infection with COVID-19 grows, Brooklyn ImmunoTherapeutics (BITX) 
has reviewed the available literature and is providing the following as guidance for use of IRX-2 in 
patients at this time. 
Published literature suggests that some of the deaths from COVID-19 have been linked to cytokine 
release syndrome, or “Cytokine storm”. It is important to note that during the development of IRX-2, we 
have not had any reports of Cytokine Release Syndrome to date, but, given the components, and 
mechanism of action of the drug, BITX feels there could be a small, but theoretically possible risk in the 
administration of IRX-2 to a patient who may be co-infected with COVID-19, by creating an increased 
risk of Cytokine Release Syndrome. 
Given this, we recommend the following: 
• 
All subjects should have a negative COVID-19 screening test within 5 days of 


 
 
41 
 
 
administration of first dose of a cycle of IRX-2. 
• 
Subjects should be instructed to take all precautions possible to limit their exposure to 
COVID-19 during their treatment with IRX-2. 
• 
Any subjects that present with symptoms of COVID-19 infection should immediately 
discontinue treatment with IRX-2. Once symptoms have fully resolved and the subject has 
demonstrated a negative COVID-19 test, resumption of IRX-2 dosing can occur at the 
Investigator’s discretion. 
9.0 Correlative/Special Studies 
 Tumor Tissue Studies  
Radiology-guided biopsies will utilize 18 to 20-gauge core biopsy needles.  Endoscopic biopsies will utilize 
standard 3-6 mm biopsy forceps. As feasible, the same region of the tumor will be sampled. 
An overview of collection, processing, and analysis details are shown in Table 9.1. 
Table 9.1 Tumor Tissue Studies Overview 
Tissue Type 
Timepoint of Collection 
Receiving Lab 
Downstream Analysis & Lab 
Performing Analysis  
Formalin 
fixed (FFPE)  
 
 Screening 
 Week 7 
 
COH 
Pathology 
Core 
 
TGen 
 
FFPE: 
MHC-PepSeq 
 
 
Molecular Pathology Core 
 
FFPE: 
Nanostring Gene Expression 
Profiling 
9.1.1 
Distribution to laboratories for analysis 
The COH Pathology core will distribute tissue to internal collaborators for downstream analysis. 
Peripheral Blood Studies  
Blood samples will be collected from an indwelling venous catheter or by venipuncture for the below 
stated analyses (see Table 9.2).  


 
 
42 
 
 
9.1.2 
Timepoints of Collection and Tube Type  
Timepoint of collection 
Volume 
per 
Timepoint 
Tube Type 
Receiving & 
Processing Lab 
Downstream Analysis & Lab 
Performing Analysis 
 Week 1 
 Week 4 
 Week 7 
 Week 10 
 End of Treatment 
20 mL 
Green-top 
COH APCF 
(Dr. Synold) 
 ctDNA analysis – TGen Tardis 
assay 
 Cytokine profile on plasma - Dr. 
Synold 
 PBMC- immuneprofiling- COH 
Immuno-oncology Core 
 Anti-Drug Antibodies Testing – 
Dr. Synold 
 
6 mL 
Purple-top 
6 mL 
Red-top 
9.1.3 
APCF Notification, Blood Collection and Labeling 
 
Notification to COH APCF of 
Pending Collection 
Tube Type 
Labeling and Collection Details 
Post-collection Instructions 
 Notify at least one day in 
advance) 
 Send calendar invite via 
e-mail: Leslie Smith-
Powell (Lsmith-
Powell@coh.org) or 
Dauhlian Chi 
(dchi@coh.org) 
Green-top 
Purple-top 
Red-top 
1. Label tubes with COH protocol #, 
subject ID, actual collection time in 
24-hour format, institution (for CP 
sites), and timepoint of collection 
(e.g. pre-chemo, pre-immuno, 
post-immuno, F/U). 
2. Timepoints of collection are stated 
in Section 9.1.2. 
3. Blood samples will be collected 
from an indwelling venous catheter 
or by venipuncture  
4. Invert tubes eight times after 
collection.  
5. Green-top and Purple-top tube(s): 
Immediately place the tubes on 
ice. 
6. Red-top tube(s): Keep red-top 
tubes at room temperature. 
 
 
 Promptly deliver the blood 
samples to the COH APCF, 
Shapiro room 1042 (Duarte 
Campus) for processing within 
1-2 hours (± 30 minutes) of 
collection.  
 Delivery temperature: 
− 
Green-top/purple-top 
tubes on ice or 4oC. 
− 
Red-top tube at room 
temperature. 
 For non-Duarte sites process 
samples and store until future 
batched shipments. 
 


 
 
43 
 
 
9.1.4 
Processing of samples 
Keep blood samples on a rocker set at low speed to mimic circulation and avoid clot formation until 
processing.  
Efforts should be made to process the samples within 4 hours of collection. 
Tube Type  
Processing Details  
Green-top 
(x2) 10mL 
NaHep tubes 
(cat# BD 
367874) 
 
 
 
 
Plasma  
1. For plasma preparation use 20 mL whole blood from green-top tubes. 
2. Centrifuge for 10 minutes at 1800 x g at 4 oC.  
3. The resulting upper plasma layer from each tube will be drawn up sequentially 
into a sterile 5 mL syringe and pushed through a sterile 0.2/0.8 micron disposable 
filter (Acrodisc Filter .8/.2ucron filter. Thermo Fisher:  Andwin Scientific 28139-
702).  
a. Save the plasma-depleted portion for isolation of PBMC (see below). 
4. The filtered plasma will then be transferred in 500 µL aliquots into multiple 
appropriately-labeled Starstedt microfuge tubes (Sarstedt micro tube PP 1.5ml 
screw cap tubes ThermoFisher: 50-809-238).  
5. To one aliquot, add 0.5 mL glycerol/0.02% sodium azide solution to dilute the 
plasma 50/50 v/v. Keep the diluted plasma sample at -20˚C and do not freeze.  
6. All the remaining plasma aliquots will be stored frozen at -80˚C until at City of 
Hope or Shipment to City of Hope for Non-COH sites. 
Peripheral 
blood 
mononuclear 
cells (PBMC) 
 
1. Any blood remaining in the two green-top tubes used to prepare plasma above 
will be diluted 1:1 with Hank’s Balanced Salt Solution (or equivalent)  and 
combined with the whole blood from the unused green-top tube in a sterile 50 
ml conical centrifuge tube. 
2. PBMC will then be isolated per COH APCF procedures at City of Hope. Non COH 
sites will process PBMC per site standard process. 
3. Isolated PBMC will be aliquoted(Thermo: 377627. Thermo Scientific™ Nunc™ 
Biobanking and Cell Culture Cryogenic Tubes 1.8mL) and stored at -80˚C/ liquid 
nitrogen until at City of Hope or Shipment to City of Hope for Non-COH sites.   
Purple-top 
(x1) 6mL K2-
EDTA (Cat# 
BD 367863) 
 
Plasma 
1. City of Hope subjects will process per APCF Procedures. Non-COH sites:   
Centrifuge the 6ml PT tube at 1000x g, 10min at 4deg. 
2. Tubes should be de-identified prior to aliquoting and freezing (i.e. tubes should 
only contain protocol ID, study subject ID and timepoint of collection) 
3. Plasma aliquots (~500 µL aliquots) (Sarstedt micro tube PP 1.5ml screw cap tubes 
ThermoFisher: 50-809-238). )will be stored frozen at -80˚C until shipment to 
TGen.  
Red-top 
(x1) 6mL red 
top (Cat# BD 
367815) 
Serum 
1. If not already done so, allow the blood to clot for at least 1 hour at room 
temperature. 
2. Keep the red-top tubes upright and at room temperature during transport and 
prior to processing. 
3. Centrifuge at 1000 x g at 4˚C for 15 minutes, max brakes.  
4. Transfer as many 0.5 mL aliquots as possible to labeled 1.5 mL Sarstedt tubes. 
4. Serum aliquots will be stored frozen at -80˚C until use at City of Hope or 
Shipment to City of Hope for Non-COH sites 


 
 
44 
 
 
9.1.5 
Sample maintenance and distribution/shipping to laboratories 
A sample manifest will be maintained by the PI or designee. Samples will be maintained at APCF until 
distribution to internal collaborators/external vendors. Samples will be batch shipped to non-COH 
vendors. 
Stool Samples 
9.1.6 
Timepoints 
o Screening 
 
o Week 7 
9.1.7 
Collection, labeling, storage 
1. Provide participant with a biospecimen bag and 
sterile swab (BD BBLTM CultureSwabTM Sterile, 
Media-free Swabs). 
2. Instruct participant on proper collection from a used toilet tissue paper. 
3. Stool will be collected either just before the scheduled clinic visit or during the clinic visit. 
4. Remind the participant at least 1-2 days in advance of the protocol visit to perform stool collection 
and store the sample at room temperature until the day of the scheduled clinic visit. 
a. Optional: Participant may instead provide a stool sample at the clinic during the protocol visit 
provided they are reporting regular bowel movements. 
5. Labeling: Label samples with COH protocol #, subject ID, institution (for CP sites), date of collection, 
and timepoint of collection (e.g. pre-chemo, pre-immuno, post-immuno, F/U). 
6. Promptly store samples are at -20 OC until analysis. 
7. Community Practice Sites:  
a. Ship on ice at time of blood shipment to the COH Duarte campus  
b. COH APCF will notify the study team to retrieve the sample 
9.1.8 
Shipping to TGen/Northern Arizona University 
Samples will be batch shipped on dry ice via overnight courier by the study team designee to Dr. Caporaso. 
Dr. J. Gregory Caporaso, 
Northern Arizona University 
1350 S Knoles Drive 
Flagstaff, AZ 86011, USA 
 
 
 


 
 
45 
 
 
 
 
 
 
 
 
 
TGen Downstream Correlative Analyses 
An overall low abundance of somatic 
mutations with antigenic potential is a 
second feature of Gastroesophageal cancers 
that may limit its responsiveness to 
immunotherapy. T cell-mediated immunity 
to neo-antigens (novel peptide sequences 
created by tumor mutations) shapes the 
genomic landscape of emergent tumors and 
mediates the efficacy of successful 
immunotherapies57. However, predicting 
which tumor mutations have immunogenic 
potential is a multi-factorial and highly-personalized inquiry, 
dependent upon: (i) the set of particular mutant sequences in the 
tumor, (ii) the expression of proteins containing those sequences, 
(iii) the ability of mutant sequences to be presented by autologous 
HLA proteins, and (iv) the activation and expansion of appropriate T 
cells that can eliminate the tumor. We estimate a median ~40 
somatic coding mutations per GE tumor. Based on published 
estimates, 5-20% of these might be expected to have antigenic 
potential. As a consequence, even if the tumor microenvironment is 
modulated to become more immunogenic, the potential for a 
clinical response to combination immunotherapy is likely to depend 
upon an intrinsic capacity for T cell recognition that varies from 
tumor to tumor58. 
Both MHC class I-restricted CD8+ and MHC class II-restricted CD4+ T 
cells have been implicated in successful anti-tumor immunity59, 60. 
Athough in silico methods for predicting class I binding sequences 
have shown some utility, models of HLA class II binding are generally of limited predictive value60. To 
address this gap, we developed a novel multiplexed proteomics technology ‘MHC-PepSeq’ that enables 
large (10,000s), programmable sets of DNA-encoded peptides to be assayed for binding against a panel of 
human HLA class II proteins, using a digital sequencing readout. This technology will be deployed in 
association with comprehensive genomic and transcriptomic sequencing (Figure 1). Importantly, our 
recent validation experiments, indicate this system has proven to be highly effective at predicting the 
Figure 1. An integrated approach to 
predict and monitor neo-antigen-specific 
immunity. (A) Comprehensive genomic 
tumor analysis (exome, transcriptome 
sequencing, in silico neoantigen prediction) 
will be combined with the MHC-PepSeq 
assay to identify tumor mutations that 
result in variant peptides capable of binding 
host HLA. The landscape of such mutations 
may be predictive of the response to 
immunotherapy. (B) Non-invasive (ctDNA) 
assays measuring the abundance of 
different 
classes 
of 
the 
mutations 
characterized in (A) will be developed for 
serial study of each individual and used to 
monitor 
the 
response 
over 
time. 
Disproportionate 
changes 
in 
the 
abundance 
of 
immunogenic 
v 
non-
immunogenic mutations may provide a 
sensitive measure of immune pressure in 
response to immunotherapy. 


 
 
46 
 
 
epitope targets of naturally-arising T cell responses to influenza virus, substantially outperforming in silico 
methods. 
To look for immunological predictors and correlates of therapeutic effect within the trial, we will assay 
tumor sections for tumor infiltrating lymphocytes (TILs). While TIL density/localization has previously been 
correlated with therapeutic anti-tumor immunity, it can be challenging to obtain serial samples of tumor 
tissue after treatment and, even when such biopsies are obtained, traditional tissue analysis affords only 
a fairly narrow view of the response. Therefore, in parallel, we will develop a richer view using an 
integrated comprehensive genomics approach to predict and monitor neo-antigen-specific immunity 
(outlined in Figure 1). This approach combines 2 novel multiplexed assays (“MHC-PepSeq” and “TARDIS”) 
that both provide personalized information about the immunogenicity of mutations within individual 
tumors. 
(i) MHC-PepSeq – identifying mutations with 
immunogenic potential 
We will use a new multiplexed assay (MHC-
PepSeq), together with in silico prediction, to assay 
all tumor mutations expressed in each individual 
(identified by both DNA and mRNA sequencing) 
against autologous HLA II proteins. Each mutation 
will be represented, alongside its wild-type 
counterpart, at each position of a 15mer peptide - 
resulting in ~30 peptides per SNV/indel.  
 
For each patient, the assay will output a personalized 
immunogenicity landscape that represents the predicted 
interaction between tumor and host. We expect ~5-20% 
of the coding somatic mutations to have immunogenic 
potential restricted by at least one of the patient’s HLAs, 
resulting in a fairly limited and variable set of potential 
neo-antigens in each patient. We will assess whether 
these neo-antigen sets are predictive of the clinical 
response to combination therapy across the trial cohort. 
(ii) ctDNA-based monitoring tumor response to combination immunotherapy 
We have developed a targeted multiplexed digital sequencing assay for circulating tumor DNA (‘TARDIS’) 
that enables non-invasive high-dimensional tumor monitoring by quantifying a panel of personalized 
tumor variant sequences in circulating cell-free DNA. Changes in the allele fraction of these variants over 
time are indicative of changes in tumor activity, load and/or composition – and have been shown to 
provide early indicators of the response to therapy61,62. Here, we propose to extend this concept to the 
detection of immune-mediated selection. Using the immunogenicity-predicting assay described above, 
we will identify 2 categories of mutations for each individual’s tumor – putatively ‘immunogenic’ and 
‘non-immunogenic’. For each category/patient, a multiplexed panel of ~5-10 TARDIS assays will be 
designed and used to monitor serial patient plasma samples. Tumor-specific immunity is expected to 
manifest as the disproportionate loss of immunogenic mutations v non-immunogenic mutations over 
Figure 2. Accurate T cell epitope prediction using the MHC-
PepSeq assay. A library comprising 3431 DNA-encoded 
peptides representing the proteome of H5N1 influenza was 
synthesized and tested for binding against 10 HLA-DR proteins, 
resulting in ~34000 multiplexed peptide-MHC measurements. 
The subset (~1%) of data corresponding to HLA-DRB1*01:01 
binding of NP-derived peptides is shown (upper panel), and 
compared to in silico prediction data generated for the same 
peptides using the tool available at www.iedb.org (lower panel). 
Overlaid in vertical red / orange bars are the positions of all six 


 
 
47 
 
 
time (represented in Fig 1b), as has been observed in serial tissue biopsies of melanomas treated with 
nivolumab63. Tracking multiple mutations in each category will afford statistical power and help to 
compensate for any limitations in the immunogenicity prediction step. Overall, this strategy will offer a 
sensitive molecular correlate of anti-tumor immunity that will be viewed alongside – and used to enrich 
– the interpretation of standard clinical monitoring (i.e. imaging).   


 
 
48 
 
 
10.0 Study Calendar 
Study Activity Calendar  
Test 
Screening 
(Day -28 to 0) 
Cycles 1-35 (+/- 3 day window starting with Cycle 2) 
End of 
treatment (EOT) 
or disease 
progression 
Safety Follow-
up (30 days 
post-
discontinuation 
for toxicity) 
Survival 
Follow-
up (up to 
1 year 
after 
EOT) 
Weeks 
W1 
W4 
W7 
W10 
W13+ 
History and Physicala 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Weight and ECOG PS 
X 
X 
X 
X 
X 
X 
X 
X 
 
CBC and diff b 
X 
X 
X 
X 
X 
X 
X 
X 
 
Chemistries (Na, CL, CO2, K, 
BUN, Creatinine, Ca, Mg, Phos) 
X 
X 
X 
X 
X 
X 
X 
X 
 
Liver function panelc 
X 
X 
X 
X 
X 
X 
X 
X 
 
PT, PTT, INRd 
X 
X 
X 
X 
 
X 
 
 
 
TSH 
X 
X 
X 
X 
X 
X 
X 
X 
 
ECGe 
X 
 
 
 
 
 
 
 
 
Pembrolizumabf 
 
X 
X 
X 
X 
X 
 
 
 
IRX-2 regimeng 
 
X 
 
 
 
X 
 
 
 
Tissue for Research Purposes 
(paraffin embedded)h 
X 
 
 
X 
 
 
 
 
 
Stool Collection for Research 
Purposes 
X 
 
 
X 
 
 
 
 
 
Blood for Research Purposesi 
 
X 
X 
X 
X 
 
X 
 
 


 
 
49 
 
 
 
a Once the patient has fully recovered from the study drug-related toxicities or the patient enrolls in a hospice, the follow up can be done by a phone call if the 
patient is off the study drugs. 
b CBC and diff includes: WBC, ANC, HCT, HGB, PLT, % lymphocytes, % monocytes, % neutrophils, and other differential. 
c Liver function panel includes: Alk Phos, Total Bilirubin, SGOT (AST), SGPT (ALT), Total Protein, and albumin. 
d PT, PTT, and INR testing to occur week 1, week 4, and week 7 of each 12 week cycle. 
e Resting 12-lead ECGs will be recorded at screening, and then as clinically indicated throughout the study. 
f Pembrolizumab (200mg) administered IV Q3weeks as a 30 minute infusion for up to 2 years provided no intolerable toxicities and no progression of disease as 
per label of pembrolizumab (35 cycles) 
g IRX-2 Regimen administered every 12 weeks starting week 1 corresponding to cycles 1, 5, 9, 13, 17, 21, 25, 29, and 33.   
h If tumor paraffin blocks are not available, fresh biopsy will be obtained before the first treatment. If fresh biopsy is indicated, one core (or punch) will be 
collected in formalin and the second core (or punch) will be collected in liquid nitrogen.  
i Research blood will be collected at the beginning of cycle 1 prior to patient receiving week 1 of therapy and must be processed within 2 hours of collection in 
order to separate PBMC and plasma. Plasma will be utilized for Pepseq based ctDNA determination 
j Toxicity assessment: Common Toxicity Criteria (CTC) version 5.0 will be used. All toxicity grades (including grade 1) should be captured on the case report forms.  
All toxicities that occurred during treatment should be followed until resolution.     
     
k. Tumor measurement: The same type of scan should be used for repeat measurements. Scans should be obtained every 6 weeks for the first 18 weeks, then 
every 9 weeks afterwards. For patients with the first documentation of progressive disease, treatment can be continued if no clinical deterioration and no grade 
3-4 toxicities. Confirmatory scan will be obtained in ≥ 4 weeks. If the disease progression is confirmed, the patient will be taken off the study. If the disease 
progression is not confirmed and the patient does not have grade 3-4 toxicities, the patient will continue the treatment on the study.  For patients who complete 
35 cycles of therapy, scans can be spaced out to every 12-24 weeks at the discretion of the treating physician.  
l Pregnancy test should be done in woman of child bearing age who are sexually active and may potentially be pregnant. Prior to study enrollment, women of 
childbearing potential (WOCBP) must be advised of the importance of avoiding pregnancy during trial participation and the potential risk factors for an 
unintentional pregnancy. In addition, men enrolled on this study should understand the risks to any sexual partner of childbearing potential and should practice 
an effective method of birth control. All WOCBP MUST have a negative pregnancy test within 7 days prior to enrollment. If the pregnancy test is positive, the 
patient must not receive investigational product and must not be enrolled in the study. In addition, all WOCBP should be instructed to contact the Investigator 
Toxicity Assessmentj 
X 
X 
X 
X 
X 
X 
X 
X 
 
Radiology: CT-chest, abdomen, 
and pelvis for tumor 
measurementsk 
X 
 
 
X 
 
X 
X 
 
 
Serum B-HCG l 
X 
 
 
 
 
 
 
 
 


 
 
50 
 
 
immediately if they suspect they might be pregnant (e.g., missed or late menstrual period) at any time during study participation. The Investigator must 
immediately notify IRX in the event of a confirmed pregnancy in a patient participating in the study. 


 
 
51 
 
 
11.0 Evaluation Criteria/Measurement of Effect 
Primary Endpoint: Toxicity  
The primary endpoint is toxicity. Toxicity will be captured in a standardized manner using the NCI 
Common Toxicity Criteria for Adverse Events (v5). Dose limiting toxicity is defined in Section 5.4 of the 
protocol. The MTD will be based on the assessment of DLT. All patients who are not evaluable for dose 
limiting toxicity will be replaced.  A patient is evaluable for DLT if he/she receives the cycle 1 dose of 
pembrolizumab (100%) and 80% of the planned doses of IRX-2 during cycle 1 OR he/she experiences a 
DLT.  If this minimum amount of treatment is not reached due to treatment related AEs, this is 
considered a DLT.  However, patients who fail to achieve this minimum amount of treatment due to 
reasons that are clearly unrelated to treatment (e.g. error on part of patient or healthcare provider) will 
be replaced. 
Secondary Endpoint: Response 
Response is the secondary endpoint. Response and progression will be evaluated in this study using the 
new updated international criteria proposed by the Response Evaluation Criteria in Solid Tumors 
(RECIST) Committee version 1.1. 
Only those patients who have measurable disease present at baseline, have received at least one cycle 
of therapy, and have had their disease re-evaluated will be considered evaluable for response.  These 
patients will have their response classified according to the definitions stated below.  (Note:  Patients 
who exhibit objective disease progression prior to the end of cycle 1 will also be considered evaluable.) 
11.1.1 
Definitions 
• 
Evaluable for toxicity:  All patients will be evaluable for toxicity from the time of their first treatment 
with IRX-2 and/or pembrolizumab. 
• 
Evaluable for objective response:  Only those patients who have measurable disease present at 
baseline, have received at least one cycle of therapy, and have had their disease re-evaluated will be 
considered evaluable for response.  These patients will have their response classified according to 
the definitions stated below.  (Note:  Patients who exhibit objective disease progression prior to the 
end of cycle 1 will also be considered evaluable.) 
11.1.2 
Disease Parameters 
• 
Measurable disease:  Measurable lesions are defined as those that can be accurately measured in at 
least one dimension (longest diameter to be recorded) as >10mm with conventional techniques (CT, 
MRI, or caliper measurement) and as >20mm by chest X-ray (if clearly defined and surrounded by 
aerated lung.) Lymph nodes greater than 10mm on short axis are considered measureable as well. 
All tumor measurements must be recorded in millimeters (or decimal fractions of centimeters). 
• 
Non-measurable disease:  All other lesions (or sites of disease), including small lesions (longest 
diameter <20 mm by chest X-ray or <10 mm using CT, MRI or caliper measurement), are considered 
non-measurable disease.  Organomegaly, leptomeningeal disease, ascites, pleural/pericardial 
effusions, lymphangitis cutis/pulmonis, inflammatory breast disease, and abdominal masses (not 
followed by CT or MRI are all non-measurable. 
• 
Target lesions:  All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, 
representative of all involved organs, should be identified as target lesions and recorded and 
measured at baseline. Lymph nodes less than 15mm in the short axis cannot be used as target 


 
 
52 
 
 
lesions.  Target lesions should be selected on the basis of their size (lesions with the longest 
diameter) and their suitability for accurate repeated measurements (either by imaging techniques or 
clinically).  A sum of the longest diameter (LD) for all target lesions will be calculated and reported as 
the baseline sum LD.  The baseline sum LD will be used as reference by which to characterize the 
objective tumor response. 
• 
Non-target lesions:  All other lesions (or sites of disease) including any measurable lesions over and 
above the 10 target lesions should be identified as non-target lesions and should also be recorded 
at baseline.  Measurements of these lesions are not required, but the presence or absence of each 
should be noted throughout follow-up.  
11.1.3 
Methods for Evaluation of Measurable Disease 
All measurements should be taken and recorded in metric notation using a ruler or calipers. All 
baseline evaluations should be performed as closely as possible to the beginning of treatment and 
never more than 30 days before the beginning of the treatment. 
The same method of assessment and the same technique should be used to characterize each 
identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is 
preferred to evaluation by clinical examination when both methods have been used to assess the 
antitumor effect of a treatment. 
• 
Clinical lesions:  Clinical lesions will only be considered measurable when they are superficial (e.g., 
skin nodules and palpable lymph nodes).  In the case of skin lesions, documentation by color 
photography, including a ruler to estimate the size of the lesion, is recommended.  
• 
Chest x-ray:  Lesions on chest x-ray are acceptable as measurable lesions when they are clearly 
defined and surrounded by aerated lung.  However, CT is preferable.  
• 
Conventional CT and MRI:  These techniques should be performed with cuts of 10 mm or less in slice 
thickness contiguously.  Spiral CT should be performed using a 5 mm contiguous reconstruction 
algorithm.  This applies to tumors of the chest, abdomen, and pelvis.  Head and neck tumors and 
those of extremities usually require specific protocols. 
• 
Ultrasound (US):  When the primary endpoint of the study is objective response evaluation, US 
should not be used to measure tumor lesions.  It is, however, a possible alternative to clinical 
measurements of superficial palpable lymph nodes, subcutaneous lesions, and thyroid nodules.  US 
might also be useful to confirm the complete disappearance of superficial lesions usually assessed 
by clinical examination. 
• 
Endoscopy, Laparoscopy:  The utilization of these techniques for objective tumor evaluation has not 
yet been fully and widely validated.  Their uses in this specific context require sophisticated 
equipment and a high level of expertise that may only be available in some centers.  Therefore, the 
utilization of such techniques for objective tumor response should be restricted to validation 
purposes in reference centers. However, such techniques may be useful to confirm complete 
pathological response when biopsies are obtained. 
• 
Tumor markers:  Tumor markers alone cannot be used to assess response.  If markers are initially 
above the upper normal limit, they must normalize for a patient to be considered in complete 
clinical response.  
• 
Cytology, Histology:  These techniques can be used to differentiate between partial responses (PR) 
and complete responses (CR) in rare cases (e.g., residual lesions in tumor types, such as germ cell 
tumors, where known residual benign tumors can remain). 


 
 
53 
 
 
The cytological confirmation of the neoplastic origin of any effusion that appears or worsens 
during treatment when the measurable tumor has met criteria for response or stable disease is 
mandatory to differentiate between response or stable disease (an effusion may be a side effect 
of the treatment) and progressive disease. 
Response Criteria 
11.1.4 
Evaluation of Target Lesions 
Complete Response (CR): 
Disappearance of all target lesions. Lymph node CR is when the 
lymph node has decreased to less than 10mm in the short axis. 
Partial Response (PR):  
At least a 30% decrease in the sum of the longest diameter (LD) 
of target lesions, taking as reference the baseline sum LD 
Progressive Disease (PD): 
At least a 20% increase in the sum of the LD of target lesions, 
taking as reference the smallest sum LD recorded since the 
treatment started (including the baseline scan if that is the 
smallest), and at least a 5mm increase or the appearance of new 
lesions. 
Stable Disease (SD): 
 
Neither sufficient shrinkage to qualify for PR nor sufficient 
increase to qualify for PD, taking as reference the smallest sum 
LD since the treatment started 
11.1.5 
Evaluation of Non-Target Lesions 
Complete Response (CR): 
Disappearance of all non-target lesions and normalization of 
tumor marker level. 
Incomplete 
Response/Stable Disease 
(SD): 
Persistence of one or more non-target lesion(s) and/or 
maintenance of tumor marker level above the normal limits 
Progressive Disease (PD): 
Appearance of one or more new lesions and/or unequivocal 
progression of existing non-target lesions. However, unequivocal 
progression should not normally trump target disease status.  It 
must be representative of overall disease status change, not a 
single lesion increase. 
Note:  If tumor markers are initially above the upper normal limit, they must normalize for a 
patient to be considered in complete clinical response. 
Although a clear progression of “non-target” lesions only is exceptional, the opinion of the 
treating physician should prevail in such circumstances. 
11.1.6 
Evaluation of Best Overall Response 
The best overall response is the best response recorded from the start of the treatment until 
disease progression/recurrence (taking as reference for progressive disease the smallest 
measurements recorded since the treatment started).  The patient's best response assignment will 
depend on the achievement of measurement criteria, but confirmation is not necessary. 


 
 
54 
 
 
 
 
 
Table 10: Assessment of Best Overall Response Using Target and Non-Target Lesions 
Target Lesions 
Non-Target Lesions 
New Lesions 
Overall Response 
CR 
CR 
No 
CR 
CR 
Non-CR/Non-PD 
No 
PR 
PR 
Non-PD 
No 
PR 
SD 
Non-PD 
No 
SD 
PD 
Any 
Yes or No 
PD 
Any 
PD* 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
* 
In exceptional circumstances, unequivocal progression in non-target lesions 
may be accepted as disease progression. 
Note: Patients with a global deterioration of health status requiring 
discontinuation of treatment without objective evidence of disease progression 
at that time should be reported as “symptomatic deterioration”.  Every effort 
should be made to document the objective progression even after 
discontinuation of treatment. 
11.1.7 
Duration of Response 
Duration of overall response:  The duration of overall response is measured from the time 
measurement criteria are met for CR or PR (whichever is first recorded) until the first date that 
recurrent or progressive disease is objectively documented (taking as reference for progressive 
disease the smallest measurements recorded since the treatment started). 
The duration of overall CR is measured from the time measurement criteria are first met for CR 
until the first date that recurrent disease is objectively documented. 
Duration of stable disease:  Stable disease is measured from the start of the treatment until the 
criteria for progression are met, taking as reference the smallest measurements recorded since 
the treatment started.  
11.1.8 
Disease Control Rate (DCR) 
DCR is defined as the proportion of patients with the best overall response of CR, PR, or SD 
according to RECIST 1.1 criteria.  


 
 
55 
 
 
11.1.9 
Progression-Free Survival 
Progression-free survival is defined as the length of time between study enrollment and when 
objective evidence of disease progression is documented. 
11.1.10 
Overall Survival 
Overall survival is defined as the length of time between study enrollment and death. 
11.1.11 
Tumor Imaging on Study per Immune-related RECIST (irRECIST) 
RECIST 1.1 may not provide an accurate assessment of immunotherapeutic agents such as 
pembrolizumab.  Immune-related RECIST (irRECIST) is RECIST 1.1 adapted as described below to 
account for the unique tumor response seen with immunotherapeutics.   
Patients with 1st radiologic assessment of PD by RECIST 1.1 may continue treatment if clinically 
stable as per Table 11.  If the repeat scan confirms PD, patients will be discontinued from 
treatment.  Exception can be made if the Investigator feels the patient is deriving benefit from 
treatment and after discussion with the study PI and Sponsor.  Subsequent imaging should be 
pursued every 6 weeks until demonstration of confirmed PR or CR in which event imaging will 
then be conducted every 9 weeks if a patients has completed initial 18 weeks of protocol therapy.  
Imaging may be more frequent if clinically indicated. 
 
Clinically stable is defined by the following criteria: 
• 
Absence of signs and symptoms indicating disease progression 
• 
No decline in ECOG performance status 
• 
Absence of rapid progression of disease 
• 
Absence of progressive tumor at critical anatomical sites (e.g., cord compression) requiring 
urgent alternative medical intervention 
 
Table 11.  Imaging and Treatment after 1st radiologic evidence of progressive disease (PD)  
 
 
Clinically Stable 
Clinically Unstable 
Imaging 
Treatment 
Imaging 
Treatment 
1st radiologic 
evidence of PD 
per RECIST 1.1 
Repeat imaging at 
6 weeks to 
confirm PD 
May continue 
study treatment 
at the 
Investigator’s 
discretion while 
awaiting 
confirmatory scan 
Repeat imaging at 
≥ 4 weeks to 
confirm PD if 
possible 
Discontinue 
treatment 


 
 
56 
 
 
Repeat scan 
confirms PD 
No additional 
imaging required 
Discontinue 
treatment 
(exception is 
possible after 
discussion with 
the PI and 
Sponsor) 
No additional 
imaging required 
N/A 
Repeat scan 
shows SD, PR, or 
CR 
Continue 
regularly 
scheduled 
imaging 
assessments 
every 6-9 weeks 
dependent on 
cycle of therapy 
Continue study 
treatment at the 
Investigator’s 
discretion 
Continue 
regularly 
scheduled 
imaging 
assessments 
every 6-9 weeks 
dependent on 
cycle of therapy 
May restart study 
treatment if 
condition has 
improved and/or 
clinically stable 
per Investigator’s 
discretion 
 
 
12.0 Data Reporting/Protocol Deviations 
Data Reporting 
12.1.1 
Confidentiality and Storage of Records  
Electronic Data Collection will be used for this protocol. The data will be stored in encrypted, password 
protected, secure computers that meet all HIPPA requirements. When the results of this study are 
reported in medical journals or at meetings, identification of those taking part will not be disclosed. 
Medical records of subjects will be securely maintained in the strictest of confidence, according to 
current legal requirements. They will be made available for review, as required by the FDA, HHS, or 
other authorized users such as the NCI, under the guidelines established by the Federal Privacy ACT and 
rules for the protection of human subjects.  
12.1.2 
Subject Consent Form 
At the time of registration, the original signed and dated Informed Consent for, HIPPA research 
authorization form, and the California Experimental Subject’s Bill of Rights (for the medical record) and 
three copies (for the subject, the research record, and the Coordinating Center) must be available. All 
Institutional, NCI, Federal, and State of California requirements will be fulfilled.  
12.1.3 
Data Collection Forms and Submission Schedule 
All data will be collected using electronic data collection, stored as indicated above, and will be 
submitted according to the timelines presented in the table below.  
Table 12-1: Data Submission Schedule 
Form 
Submission Timeline 
Eligibility Checklist 
Complete prior to registration 


 
 
57 
 
 
On Study Forms 
Within 14 calendar days of registration 
Baseline Assessment Forms 
Within 14 calendar days of registration 
Treatment Forms 
Within 10 calendar days of treatment administration 
Adverse Event Reporting 
Forms 
For cycle 1: with 7 calendar days of AE assessment/notification 
All other cycles: within 10 calendar days of AE 
assessment/notification 
Response Assessment Forms 
With 10 calendar days of response assessment 
Other Assessment Forms 
(Concomitant Medications) 
Within 10 calendar days of the assessment 
Off Treatment/Off Study 
Forms 
Within 10 calendar days of end of treatment/study 
Follow-up/Survival Forms 
Within 14 calendar days of the protocol defined follow-up visit or 
call 
 
 
Eligibility checklist 
The eligibility checklist must be completed by a protocol nurse or clinical research coordinator and 
signed by an authorized investigator prior to registering the subject. See Section 4.3 for the registration 
procedure.  
Protocol Deviations 
12.1.4 
Deviation Policy 
This protocol will be conducted in accordance with COH’s “Clinical Research Protocol Deviation Policy” 
Deviations from the written protocol that could increase patient risk or alter the protocol integrity 
require prior IRB approval of a single subject exception (SSE) request. IRB pre-approved SSE protocol 
modifications are considered an amendment to the protocol and not a deviation. The submission of a 
deviation report is not required.  
Brief interruptions and delays may occasionally be required due to travel delays, airport closure, 
inclement weather, family responsibilities, security alerts, government holidays, etc. This can also 
extend to complications of disease or unrelated medical illnesses not related to disease progression. The 
PI has the discretion to deviate from the protocol when necessary so long as such deviation does not 
threaten patient safety or protocol scientific integrity. Example include, but are not limited to: dose 
adjustments based on excessive patient weight, alteration in treatment schedule due to non-availability 
of the research participant for treatment, laboratory test results which are slightly outside the protocol 
requirements but at levels that do not affect participant safety. These instances are considered 
deviations from the protocol. A deviation report will be submitted to the DSMC/IRM within five days.  


 
 
58 
 
 
12.1.5 
Reporting of Deviations 
All deviations will be reported to the COH DSMC within five days. The DSMC will forward the report to 
the IRM following review.  
12.1.6 
Resolving Disputes 
The COH Investigational Drug Service (IDS) cannot release a research agent that would cause a protocol 
deviation without approval by the PI. Whenever the protocol is ambiguous on a key point, the IDS 
should rely on the PI to clarify the issue.  
In situations where there is misperception or dispute regarding a protocol deviation among the persons 
involved in implementing the protocol it is the responsibility of the PI to resolve the dispute and the PI 
may consult with the DSMC chair or designee to arrive at resolution.  
13.0 Statistical Considerations 
Evaluable Participants and Participant Replacement 
All participants will be evaluable for toxicity from the time of their first treatment with IRX-2 regimen 
and pembrolizumab. 
.  
13.1.1 
Design  
Phase I of the investigation will comprise a safety lead-in phase: A standard 3+3 dose finding rule will be 
employed with two dose levels.  Patients will start at dose level 1 [IRX-2 regimen at dose of 2 mL (230 
units/day)]. Patients will be enrolled in cohorts of 3, separated by at least 1 week.  Initially, 3 patients 
will be treated with expansion to 3 more patients in the event of 1/3 dose limiting toxicities (DLTs) as 
observed during the first 3 weeks of treatment, escalation if 0/3 or 1/6 DLTs, and termination of 
escalation if ≥ 2 DLTs.  Once ≥ 2 patients experience a DLT at a dose level, the next lower dose level will 
be expanded to 6 evaluable patients, if fewer than 6 evaluable patients have been treated at the dose 
level. The maximum tolerated dose (MTD) is defined as the highest dose tested in which only 0 or 1 out 
of 6 evaluable patients experience a DLT. The Recommended Phase 2 Dosing (RP2D) will not exceed the 
MTD but may be lower based on cumulative toxicities and dose modification patterns.  If the rules 
suggest escalation at the highest proposed dose, that dose will accrue to 6 patients where <2 DLTs will 
determine that as the MTD, and 2 or more DLTs will require de-escalation. These rules will be used to 
determine the IRX-2 regimen dosing for the expansion phase. Occurrence and severity of adverse events 
including dose limiting toxicities, with severity determined according to NCI Common Terminology 
Criteria for Adverse Events version 5. Phase II is a single arm, non-randomized study to investigate 
secondary endpoints that include median progression free survival (PFS) and overall response rate (ORR) 
by RECIST version 1.1 criteria. Exploratory endpoints include descriptive statistics on circulating T cell 
profiles in patients pre and post therapeutic intervention with the combination IRX-2 regimen and 
pembrolizumab.  
 
Sample Size and Accrual Rate 
The safety phase of the study will enroll 3 to 6 patients per dose level. The dose expansion phase will 
enroll 8 to 14 patients at the recommended dose level from the safety phase for a total enrollment of 20 


 
 
59 
 
 
patients. The 6 to 12 patients treated at all dose levels in the safety phase of the study will be included as 
a part of the phase II dose expansion patient population. 
 
Historical data with single agent PD-1 inhibitors in gastroesophageal cancer indicate median PFS at 1.5 
months. With an alpha of .05, hypothesized median PFS improved to 3.0 months with the combination 
IRX-2 regimen and pembrolizumab, study accrual of 30 months, and 12-month follow-up evaluation 
timeframe, a sample size of 20 patients will have 84% power to detect median differences in PFS.   
 
Statistical Analysis Plan 
13.1.2 
Primary Objective 
A standard 3+3 dose finding rule will be employed with two dose levels.  Patients will start at dose level 1 
[IRX-2 regimen at dose of 2 mL (230 units/day)]. Patients will be enrolled in cohorts of 3, separated by at 
least 1 week.  Initially, 3 patients will be treated with expansion to 3 more patients in the event of 1/3 
dose limiting toxicities (DLTs) as observed during the first 3 weeks of treatment, escalation if 0/3 or 1/6 
DLTs, and termination of escalation if ≥ 2 DLTs.  Once ≥ 2 patients experience a DLT at a dose level, the 
next lower dose level will be expanded to 6 evaluable patients, if fewer than 6 evaluable patients have 
been treated at the dose level. The maximum tolerated dose (MTD) is defined as the highest dose tested 
in which only 0 or 1 out of 6 evaluable patients experience a DLT. The Recommended Phase 2 Dosing 
(RP2D) will not exceed the MTD but may be lower based on cumulative toxicities and dose modification 
patterns.  If the rules suggest escalation at the highest proposed dose, that dose will accrue to 6 patients 
where <2 DLTs will determine that as the MTD, and 2 or more DLTs will require de-escalation. These rules 
will be used to determine the IRX-2 regimen dosing for the expansion phase.  
 
13.1.3 
 Secondary Objectives 
Progression free survival is defined as the time from the first day of study drug administration to disease 
progression or death due to any cause. For PFS, the Kaplan-Meier estimates and corresponding 95% 
confidence intervals for the median and quartiles will be provided.  
 
13.1.4 
Correlative Exploratory Objectives 
Descriptive statistics will be compiled on circulating T cell profiles for each patient at pre- and post-
therapeutic intervention with the combination IRX-2 Regimen and Pembrolizumab. Repeated measures 
t-tests will be conducted to examine pre and post-intervention differences between baseline and post-
treatment tumor tissue Nanostring expression profiling. Data will be screened for parametric statistical 
assumption and the alpha level for all statistical tests will be set at .05. The Bonferroni correction will be 
applied to adjust for potentially inflated family wise error rates for multiple comparisons with the same 
sample.  
14.0 Human Subject Issues 
Institutional Review Board 
In accordance with City of Hope policies, as Institutional Review Board (IRB) that complies with the 
federal regulations at 45 CFR 46 and 21 CFR50, 56 and State of California Health and Safety code, Title 


 
 
60 
 
 
17, must review and approve this protocol and the informed consent form prior to initiation of the 
study. All institutional, NCI, Federal, and State of California regulations must be fulfilled.  
Recruitment of Subjects 
The HCC subjects will be recruited from patients undergoing treatment for HCC at the City of Hope 
Cancer Center and Honor Health Research Center. Any recruitment materials will be reviewed and 
approved by the IRB prior to their use to recruit potential study subjects.  
Advertisements  
Advertisements to include print, media (radio, television, and billboards), telephone scripts, etc., will be 
reviewed and approved by the IRB prior to their use to recruit potential study subjects.   
 Study Location and Performance Sites 
This study will be performed at City of Hope National Medical Center in Duarte, CA and Honor Health 
Research Institute (Scottsdale, AZ).  
Confidentiality  
This research will be conducted in compliance with federal and state of California requirements relating 
to protected health information (PHI).  The study will record individual response to the study drugs and 
any side effects, and this will be linked to the subject’s identity using a coded study number.  The 
principal investigator, co-investigators, and laboratory technicians will have access to this information, 
but all information will be treated confidentially.  No identifiers will be used in any subsequent 
publication of these results. 
Financial Obligations and Compensation 
The investigational drug, IRX-2 regimen will be provided free of charge by City of Hope for the duration 
of the trial.  
The standard of care drug(s), pembrolizumab and standard of care procedures provided will be the 
responsibility of the research participant and/or insurance carrier. The research participant will be 
responsible for all copayments, deductibles, and other costs of treatment and diagnostic procedures as 
set forth by the insurance carrier. The research participant and/or the insurance carrier will be billed for 
the costs of treatment and diagnostic procedures in the same way as if the research participant were 
not in a research study. However, neither the research participant nor the insurance carrier will be 
responsible for the research procedures related to this study.  
In the event of physical injury to a research participant, resulting from research procedures, appropriate 
medical treatment will be available at each participating institution in the injured research participant; 
however, financial compensation will not be available.  
The research participant will not be paid for taking part in this study.  
Informed Consent Processes  
The Principal Investigator or IRB approved named designate will explain the nature, duration, purpose of 
the study, potential risks, alternatives and potential benefits, and all other information contained in the 
informed consent document.  In addition, they will review the experimental subject’s bill of rights and 
the HIPAA research authorization form.  Research subjects will be informed that they may withdraw 
from the study at any time and for any reason without prejudice, including as applicable, their current or 
future care or employment at their hospital or any relationship they have with their hospital.   


 
 
61 
 
 
Should sufficient doubt be raised regarding the adequacy of comprehension, further clarifications will be 
made and the questionnaire repeated until a satisfactory result is obtained.  Prospective research 
subjects who cannot adequately comprehend the fundamental aspects of the research study with a 
reasonable amount of discussion, education and proctoring will be ineligible for enrollment.  For those 
subjects who do comprehend the fundamental aspects of the study, the research team will review the 
results of eligibility testing and determine if the subject is a candidate for study enrollment.  
 
 
 
 
 
 
 
 
 
15.0 References  
1. Fuchs CS, Doi T, Jang RW-J, et al. KEYNOTE-059 cohort 1: Efficacy and safety of pembrolizumab 
(pembro) monotherapy in patients with previously treated advanced gastric cancer. Journal of Clinical 
Oncology. 2017;35: 4003-4003. 
2. Egan JE, Quadrini KJ, Santiago-Schwarz F, Hadden JW, Brandwein HJ, Signorelli KL. IRX-2, a novel in 
vivo immunotherapeutic, induces maturation and activation of human dendritic cells in vitro. J 
Immunother. 2007;30: 624-633. 
3. Schilling B, Halstead ES, Schuler P, Harasymczuk M, Egan JE, Whiteside TL. IRX-2, a novel 
immunotherapeutic, enhances and protects NK-cell functions in cancer patients. Cancer Immunol 
Immunother. 2012;61: 1395-1405. 
4. Schilling B, Harasymczuk M, Schuler P, Egan JE, Whiteside TL. 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. 
5. Schilling B, Harasymczuk M, Schuler P, Egan J, Ferrone S, Whiteside TL. IRX-2, a novel 
immunotherapeutic, enhances functions of human dendritic cells. PLoS ONE. 2013;8: e47234. 
6. Czystowska M, Han J, Szczepanski MJ, et al. IRX-2, a novel immunotherapeutic, protects human T cells 
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16.0 Appendix I 
Treatment modifications (e.g. dose delay or discontinuation) will be based on the specific laboratory and 
immune-related adverse event criteria per the package insert as shown in the table below: 
Adverse 
Reaction 
Severity* 
Dose Modification 
Diarrhea/Colitis 
Grade 2 
Withhold dosea 
Grade 3 
Withhold dosea 
Grade 4 
Permanently discontinue 
Pneumonitis 
Grade 2 
Withhold dosea 
Grade 3 or 4 or recurrent Grade 2 pneumonitis 
Permanently discontinue 
Hepatitis (non-
HCC) 
Aspartate aminotransferase (AST) or alanine 
aminotransferase (ALT) more than 3 and up to 5 
times the upper limit of normal (ULN) or total 
bilirubin more than 1.5 and up to 3 times ULN 
Withhold dosea 
AST or ALT >5x ULN or total bilirubin >3x ULN 
Permanently discontinue 
Hepatitis (HCC) 
• 
If AST/ALT is within normal limits at baseline 
and increases to more than 3 and up to 5 
times the ULN 
• 
If AST/ALT is more than 1 and up to 3 times 
ULN at baseline and increases to more than 5 
and up to 10 times the ULN 
• 
If AST/ALT is more than 3 and up to 5 times 
ULN at baseline and increases to more than 8 
and up to 10 times the ULN 
Withhold doseb 
If AST or ALT increases to more than 10 times the ULN 
or total bilirubin increases to more than 3 times the 
ULN 
Permanently discontinue 
Hypophysitis 
Grade 2 or 3 
Withhold dosea 
Grade 4 
Permanently discontinue 
Adrenal 
insufficiency 
Grade 2 
Withhold dosea 
Grade 3 or 4 
Permanently discontinue 
Type 1 Diabetes 
Mellitus 
Grade 3 hyperglycemia 
Withhold dosea 
Grade 4 hyperglycemia 
Permanently discontinue 
Nephritis and 
Renal 
Dysfunction 
Serum creatinine more than 1.5 and up to 6 times 
ULN 
Withhold dosea 
Serum creatinine more than 6 times ULN 
Permanently discontinue 
Skin 
Grade 3 rash or suspected Stevens-Johnson syndrome 
(SJS) or toxic epidermal necrolysis (TEN) 
Withhold dosea 
Grade 4 
Permanently discontinue 
Encephalitis 
New-onset moderate or severe neurologic signs or 
symptoms 
Withhold dosea 
Immune-medicated encephalitis 
Permanently discontinue 
Other 
Other Grade 3 immune-related adverse reaction: 
 


 
 
67 
 
 
First occurrence 
Recurrence of same grade 3 adverse reactions 
Withhold dosea 
Permanently discontinue 
Life-threatening or Grade 4 immune-related adverse 
reaction 
Permanently discontinue 
Requirement for 10mg per day or greater prednisone 
or equivalent for more than 12 weeks 
Permanently discontinue 
Persistent Grade 2 or 3 immune-related adverse 
reactions lasting 12 weeks or longer after last dose of 
pembrolizumab (excluding endocrinopathies 
controlled with hormone replacement therapy) 
Permanently discontinue 
*Grading according to CTCAE Version 5.0.  
CTCAE=Common Terminology Criteria for Adverse Events. 
aTreatment can be resumed once adverse reaction returns to Grade 0 or 1. 
bTreatment can be resumed once AST/ALT returns to baseline 
 
In all cases where the subject is withdrawn due to unusual or unusually severe adverse event considered 
related to pembrolizumab, the investigator must report the withdrawal as a Serious Adverse Event. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
68 
 
 
APPENDIX II: REGISTRATION COVERSHEET  
COH IRB# 18069: A Phase 1b/2 Trial of the IRX-2 Regimen and Pembrolizumab in Patients with 
Advanced Gastric and Gastroesophageal Junction (GEJ) Adenocarcinoma 
Data Coordinating Center:   
City of Hope 
1500 Duarte Road 
Duarte, CA 91010  
Tel: (626)-218-7904 
Email: DCC@coh.org (use #secure# in subject line) 
Site Principal Investigator  
Name: 
 
CRA/Study Coordinator:     
Contact Number:   
Patient’s Initials: (F M L): 
Institution: 
Medical Record No: 
PI/ Sub-Investigator: 
Patient’s DOB: 
IRB approval valid until (date): 
Sex: 
______Male 
______Female 
Date Informed Consent Signed: 
Projected start date of treatment: 
Race 
Ethnicity 
Method of 
Payment:_______________________ 
 
Black 
 Hispanic 
Codes: 
 
Caucasian 
 Non-Hispanic 
01 Private 
06 Military or Veterans 
Adm. sponsored 
 
Asian 
 Other_________________ 
02 Medicare 
07 Self-pay (no 
insurance) 
 
American Indian 
 
03 Medicare & 
private ins. 
08 No means of 
payment  (no insurance) 
 
Native Hawaiian/Pacific 
Islander 
04 Medicaid 
09 Unknown 
 
Other_______________
____________________ 
05 Medicaid & Medicare 
Reason for Screen Failure:  
 
Reason for Failing to Initiate Protocol Therapy: 
 
 
 
 
 
 


 
 
69 
 
 
APPENDIX III: EXPEDITED REPORTING COVERSHEET 
NOTIFICATION OF UNANTICIPATED PROBLEM/SERIOUS ADVERSE EVENT 
For Use by Participating Institutions Only 
THIS FORM ALONG WITH A COPY OF THE MEDWATCH 3500 OR IRB REPORTING FORM MUST BE EMAILED TO DCC@COH.ORG WITHIN 24 
HOURS OF KNOWLEDGE OF ONSET OF SERIOUS ADVERSE EVENT OR UNANCTICIPATED PROBLEM  
COH IRB #18069_- Participating Site IRB # ___________ 
From: 
Date: 
Phone No.: 
Email: 
Reporting Investigator: 
Event:  
Participant ID: 
Institution: 
Date Event Met Reporting Criteria (as defined in protocol):   
 
Type of Report: 
 Initial    Follow-up 
CTCAE Grade:   
 G1/mild    G2/moderate     G3/severe    G4/life threatening   
 G5 
Attribution to IRX-2: 
 Not Applicable*   Unrelated   Unlikely   Possible    Probable   Definite 
Attribution to pembrolizumab: 
 Not Applicable*   Unrelated   Unlikely   Possible    Probable   Definite 
Historical/Known Correlation to 
IRX-2: 
 Expected    Unexpected 
Historical/Known Correlation to 
pembrolizumab: 
 Expected    Unexpected   
Meets Definition of Serious AE:   
   Serious      Non-serious 
Meets Definition of 
Unanticipated Problem: 
   UP      Not a UP 
Has the event been reported to 
the following institution’s IRB? 
   No      Yes; Date:_____/____/_____ 
* Not Applicable should only be used if subject has not received this agent. 
Authorized Investigator Signature:   
Date:____/____/____ 
 
 
 
 
 


 
 
70 
 
 
APPENDIX IV: CORRELATIVE TISSUE FORM (FOR ALL SITES) 
 
A copy of this form should accompany the sample shipments to COH Pathology Core. 
Non-COH sites: refer to Appendix V for shipping instructions to COH Pathology Core. 
 
COH IRB number: 18069 
Shipping date (MM-DD-YYYY):   ________/________/________ 
Subject ID (issued by DCC):   
Participant Initials (F, M, L) (if applicable): 
Institution: 
Date of collection/ biopsy (MM-DD-YYYY):    ________/________/________ 
Time point:     
  Screening       
  Week 7 
Diagnosis: 
Tissue type (FFPE scrolls): 
Number of scrolls: 
Number of slides: 
 
 
 
 
 
 
CRA/Study Coordinator/Nurse Printed Name:   
CRA/Study Coordinator/Nurse Signature:   
Contact Number: 
 
 
 
 
 
 
 
 
 


 
 
71 
 
 
APPENDIX V: TISSUE SHIPPING GUIDELINES TO CITY OF HOPE PATHOLOGY CORE 
 
These guidelines apply to non-COH sites only. 
All biological material must be shipped according to applicable government and International Air 
Transport Association (IATA) regulations.  
Shipping guidelines can also be found on the FedEx website. 
1. Aim to ship samples on a Monday through Wednesday. If this is not feasible, advance 
arrangements should be made with City of Hope Pathology Core (DL-PATHCORE-
BiospecimenSupport@COH.org).   
2. Notify City of Hope Pathology Core (DL-PATHCORE-BiospecimenSupport@COH.org) of impending 
shipment.  
3. Slides/ Blocks: Batch ship at room temperature. During extreme heat, include refrigerated (not 
frozen) gel packs or gel insulators. 
It is recommended to ship samples via overnight (for a delivery by 3 pm or earlier the next day) 
or 2-day (with a morning delivery). During extreme heat, ship via overnight (for a delivery ideally 
by 10.30 am, or 3 pm the next day). 
4. Frozen samples should be batch shipped on dry ice via overnight (for a delivery by 10.30 am the 
next day). The shipment should contain enough dry ice to last at least 72 hours. 
5. On the day of shipment, email the sample shipment information to City of Hope Pathology Core 
(DL-PATHCORE-BiospecimenSupport@COH.org). 
6. Ship samples with a copy of the correlative tissue form (Appendix IV) and a copy of the pathology 
report to: 
 
Karen Miller 
COH Pathology Core 
City of Hope National Medical Center 
1500 E. Duarte Road 
Familian Science (Building 084), Room 1207 
Duarte, CA 91010 
Telephone: 626-218-8408 
Email: DL-PATHCORE-BiospecimenSupport@COH.org 
 
 
 


 
 
72 
 
 
APPENDIX VI: CORRELATIVE BLOOD COLLECTION FORM FOR NON-COH SITES ONLY 
Subject ID (issued by DCC):   
Participant Initials (F, M, L) (if applicable): 
Institution: 
 
To be used by non-COH sites for the following blood samples being sent to COH APCF: 
 
Sample # 
Time point of 
Collection * 
Tube Type Used 
# of aliquots 
Time of Collection 
Date of Collection 
Indicate which 
sample was 
collected 
1. 
 
Week 1 
Green-top - Plasma 
 
____:____ AM/ PM 
____/____/____ 
 
Green-top - PBMC 
 
____:____ AM/ PM 
____/____/____ 
 
Red-top - Serum 
 
____:____ AM/ PM 
____/____/____ 
 
2. 
 
Week 4 
Green-top - Plasma 
 
____:____ AM/ PM 
____/____/____ 
 
Green-top - PBMC 
 
____:____ AM/ PM 
____/____/____ 
 
Red-top - Serum 
 
____:____ AM/ PM 
____/____/____ 
 
3. 
 
Week 7 
Green-top - Plasma 
 
____:____ AM/ PM 
____/____/____ 
 
Green-top - PBMC 
 
____:____ AM/ PM 
____/____/____ 
 
Red-top - Serum 
 
____:____ AM/ PM 
____/____/____ 
 
4. 
 
Week 10 
Green-top - Plasma 
 
____:____ AM/ PM 
____/____/____ 
 
Green-top - PBMC 
 
____:____ AM/ PM 
____/____/____ 
 
Red-top - Serum 
 
____:____ AM/ PM 
____/____/____ 
 
5. 
 
EOT 
Green-top - Plasma 
 
____:____ AM/ PM 
____/____/____ 
 
Green-top - PBMC 
 
____:____ AM/ PM 
____/____/____ 
 
Red-top - Serum 
 
____:____ AM/ PM 
____/____/____ 
 
 
 
A copy of this form should accompany the sample shipments to COH APCF. Refer to the blood shipping guidelines 
for shipping instructions to COH APCF (Appendix VII). 
 
CRA/Study Coordinator/ Nurse:   
Contact Number: 
CRA/Study Coordinator/ Nurse Signature:   
Date: 
 
 


 
 
73 
 
 
APPENDIX VII: BLOOD SHIPPING GUIDELINES TO CITY OF HOPE APCF 
These guidelines apply to non-COH sites only. 
All biological material must be shipped according to applicable government and International Air 
Transport Association (IATA) regulations.  
Shipping guidelines can also be found on the FedEx website. 
 
1. Aim to ship samples on a Monday through Wednesday. If this is not feasible, advance 
arrangements should be made with APCF (DL-APCF@coh.org). 
1. On the day of shipment, email APCF (DL-APCF@coh.org) the Airwaybill shipment #. 
2. Ship samples with a copy of the correlative blood collection form (Appendix VI) to: 
 
Dr. Tim Synold 
Analytical Pharmacology Core Facility  
Shapiro 1042 
City of Hope National Medical Center 
1500 E. Duarte Road 
Duarte, CA 91010 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
74 
 
 
APPENDIX VIII: CORRELATIVE BLOOD COLLECTION FORM FOR NON-COH SITES ONLY 
Subject ID (issued by DCC):   
Participant Initials (F, M, L) (if applicable): 
Institution: 
 
To be used by non-COH sites for the batched shipment of the following blood samples to TGen: 
 
Sample # 
Time point of 
Collection * 
Tube Type Used 
# of aliquots 
Time of Collection 
Date of Collection 
Indicate which 
sample was 
collected 
1. 
Week 1 
Purple-top - Plasma 
 
____:____ AM/ PM 
____/____/____ 
 
2. 
Week 4 
Purple-top - Plasma 
 
____:____ AM/ PM 
____/____/____ 
 
3. 
Week 7 
Purple-top - Plasma 
 
____:____ AM/ PM 
____/____/____ 
 
4. 
Week 10 
Purple-top - Plasma 
 
____:____ AM/ PM 
____/____/____ 
 
5. 
EOT 
Purple-top - Plasma 
 
____:____ AM/ PM 
____/____/____ 
 
 
 
A copy of this form should accompany the sample shipments to TGEN.  
 
CRA/Study Coordinator/ Nurse:   
Contact Number: 
CRA/Study Coordinator/ Nurse Signature:   
Date: