A Phase Ib Study to Assess the Safety, Tolerability and Immunologic Activity of Preoperative IRX 2 In Early Stage Breast Cancer
Phase: PHASE1
Conditions: Breast Neoplasm, Breast Neoplasm, Male, Triple Negative Breast Cancer
Sponsor: Providence Health & Services

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Clinical Study Protocol 
Title: 
A Phase Ib Study to Assess the Safety, Tolerability and 
Immunologic Activity of Preoperative IRX-2 In Early Stage 
Breast Cancer Patients  
 
Protocol:  IRX-2 2016-B 
 
US BB IND #: 
132055 
Investigational Medicinal 
Product (IMP) 
IRX-2 (Leukocyte Derived Cytokine Mixture, Human) 
 
Date: 
Original Protocol:  August 23, 2016 
Amendment #1:  June 30, 2017 
Amendment #2: January 19, 2018 
Principal Investigator 
David Page, MD 
Earle A. Chiles Research Institute 
4805 NE Glisan Street, 2N87 
Portland, OR 97213 
503-215-5696 
FAX 503-215-5695 
David.Page2@Providence.org 
Sponsor: 
Providence Portland Medical Center 
4805 NE Glisan Street, 2N35 
Portland, OR 97213 

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Investigator Agreement 
I have read and approved this protocol. My signature confirms my agreement that the clinical 
study will be conducted in full compliance with the protocol and all applicable laws and 
regulations including, but not limited to, the International Conference on 
Harmonisation Guideline for Good Clinical Practice (GCP), the Code of Federal 
Regulations (CFR), the ethical principles that have their origins in the Declaration of Helsinki 
and all applicable Federal and local regulations. All required study information will be archived 
as required by regulatory authorities. 
Nothing in this document is intended to limit the authority of a physician to provide emergency 
medical care under applicable regulations. 
This document is confidential. It contains proprietary information of IRX Therapeutics, Inc. 
Any disclosure of such information that is not authorized in writing by the sponsor is strictly 
prohibited. This document must be used solely for the purpose of reviewing or performing this 
study.  
 
 
Investigator: 
 
Date:
 
 
David Page, MD 
Institution: 
Providence Portland Medical Center 
 
 
 
 
 

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Protocol Synopsis IRX-2 2016-B 
Name of Investigational drug:  IRX-2 
Title of Study:  A Phase Ib Study to Assess the Safety, Tolerability and Immunologic Activity 
of Preoperative IRX-2 in Early Stage Breast Cancer 
Study Center(s): Providence Portland Medical Center 
Study Period: 12 months 
Phase of Development: Phase Ib 
Objectives:  
Primary objective: 
 
To establish the safety and tolerability of the IRX-2 regimen when administered pre-
operatively in early stage breast cancer (ESBC) patients 
Secondary objectives: 
 
To evaluate for changes in tumor infiltrating lymphocyte (TIL) score associated with the 
IRX-2 regimen, as measured by H&E TIL count according to Salgado criteria 
 
In Cohort B: To estimate the pathologic complete response rate to neoadjuvant 
anthracycline-based and non-platinum containing chemotherapy in patients who have 
received the IRX-2 Regimen before chemotherapy 
 
Exploratory objectives:  
 
To evaluate the effect of IRX-2 on additional investigational immunologic and molecular 
biomarkers 
Study Design:  
This will be a Phase Ib study conducted to determine the safety and tolerability of an IRX-2 
regimen in early stage breast cancer, to be administered pre-operatively before standard-of-
care surgical resection and following standard-of-care diagnostic biopsy. 
Eligible subjects will have early stage breast cancer of any receptor subtype, for which 
standard-of-care surgical resection is planned.  
Cohort B will enroll subjects triple negative breast cancer (defined by ER<10%, PR<10%, and 
HER2-negative by NCCN guidelines), T1c+ tumors for which neoadjuvant anthracycline-based 
and non-platinum containing chemotherapy is planned. The IRX-2 regimen will be administered 
and completed preceding chemotherapy. Cohort B subjects must undergo post-IRX-2 Regimen 
biopsy (2-3 cores), followed by commencement of chemotherapy preferably within one week 
after biopsy. 
For all subjects, a minimum of 1 core of pre-IRX2 tumor-bearing biopsy material, either from 
diagnostic core biopsy or a planned pre-IRX2 research biopsy, must be available for research 
analysis.  
The IRX-2 regimen will be administered in all enrolled subjects according to the schema and 
dosing schedule delineated in Table 1. IRX-2 will be administered by subcutaneous injection 
into the periareolar skin of the affected breast by a nurse at Providence Medical Center.  

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Subjects will be evaluated by physical exam and blood tests at baseline and periodically during 
the study, culminating in a final study visit 30 days (±7 days) following surgical resection. Safety 
will be evaluated routinely using the NCI-CTCAE v4.03 toxicity reporting criteria. The toxicity of 
the IRX-2 has been previously evaluated in two head and neck cancer studies, and therefore 
no formalized interval safety assessments are planned. However, safety and tolerability of the 
IRX-2 regimen will be continuously evaluated by the principal investigator in collaboration with 
the IRX-2 Therapeutics, and the trial may be terminated or held at any time for unanticipated 
concerns of safety/tolerability.  
 
Therapeutic Regimen 
Days of 
administration* 
Drug 
Dose 
Route 
Comment 
1 
Cyclophosphamide
300mg/m2 
IV infusion 
Low dose does not 
require 
corticosteroids; has 
low to moderate 
emetic risk per 
NCCN Guidelines 
Any 10 days 
between days 
4-19 
IRX-2 
2 mL (230 
units of IL-2) 
on each of 
10 days 
Two 1 mL 
subcutaneous 
periareolar 
injections 
Injections will be 
administered at 
Providence Medical 
Center 
1-21 
Indomethacin 
25mg, TID 
Oral 
Divided three times 
daily x 21 days 
1-21 
Multivitamin with 
15-30 mg of 
zinc/tablet 
1 tablet 
Oral 
Once daily x 21 
days 
1-21 
Omeprazole 
1 tablet 
Oral 
Once daily x 21 
days 
Ideally 2-5 days 
after last IRX-2  
 
 
 
Clinically indicated 
breast 
surgery/biopsy 
*As necessitated by scheduling logistics, the oral medications may be discontinued after Day 
17 and surgery/biopsy performed 2-12 days later. Indomethacin must be discontinued at least 
2 days prior to scheduled surgery/biopsy. 
 
Subjects will be considered evaluable if they consent and receive any part of the 
cyclophosphamide infusion, but any who are enrolled but withdraw consent prior to beginning 
the IRX-2 Regimen will not be evaluable and will be replaced.  The rate of Grade 1-4 toxicities 
will be reported as additional, descriptive measures of tolerability. The following criteria must 
be met for that subject to be considered to have “tolerated” the therapy:  

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 
Subject receives cyclophosphamide, >7 injections of IRX-2 and >10 days of 
indomethacin, omeprazole and multivitamins with zinc and does not have Grade 3 or 4 
treatment-associated adverse events requiring discontinuation of therapy; 
 
Subject does not experience a delay in standard-of-care surgical resection related to 
treatment-associated adverse events; 
 
Subject does not have any ongoing treatment-related Grade 2-4 adverse events at the 
30-day post-operative follow-up visit; 
 
Subject does not withdraw consent after the start of the cyclophosphamide infusion. 
Because this therapy is being administered in subjects receiving therapy intended to cure their 
disease, the threshold for establishing safety/tolerability will be high, such that nearly all 
subjects must tolerate the therapy. The therapy will be deemed “safe and tolerable” if 75% or 
greater of evaluable subjects tolerate the therapy according pre-specified definition of 
tolerability (see Section 7.2 for additional detail).  
The secondary endpoint is to evaluate the effect of therapy on stromal TIL (sTIL) infiltration by 
Salgado criteria. The rationale for this endpoint is that the percentage of TILs in pre-operative 
biopsies is highly correlated with both overall survival and response to neoadjuvant therapy. If 
the IRX-2 regimen is found to increase the percentage of TILs, this would provide rationale to 
evaluate further IRX-2 in this setting. 
Exploratory endpoints will be analyzed descriptively, and will include additional peripheral blood 
and intratumoral analyses of the immunologic effects of the IRX-2 regimen, including, for 
example, some or all of the following:   
 
flow cytometric quantification and characterization of peripheral lymphocytes (including 
activated T-cells, T-regulatory cells, NK cells, and myeloid cells) 
 
TIL phenotype characterization by multispectral immunofluorescence imaging to 
assess changes in abundance of T-regulatory cells, activated T-cells, myeloid lineages 
and dendritic cells 
 
Intratumoral and T-cell clonal responses by T-cell receptor DNA deep sequencing 
 
Characterization of intratumoral immune responses by RNA expression using the 
Nanostring PanCancer Immune panel and/or Prosigna tumor recurrence score 
Based upon conservative estimates of enrollment of 2 subjects per month, the accrual time is 
estimated to be 1 year.  
Criteria for Inclusion in the Study:  
Inclusion Criteria 
1. Invasive breast cancer of any receptor subtype diagnosed by core-needle biopsy  
2. To undergo surgical resection with curative intent by partial mastectomy (lumpectomy) 
or mastectomy 
3. Tumor >5 mm in maximum diameter by ultrasound or mammography. (Subjects with 
smaller tumors may be included at the discretion of the Principal Investigator.) Cohort 
B: (defined by ER<10%, PR<10%, and HER2-negative by NCCN guidelines), T1c+ 
tumors for which neoadjuvant anthracycline-based and non-platinum containing 
chemotherapy is planned. 
4. Willing and able to provide written informed consent, including consent for use of 

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available tissue and required blood draws for research purposes 
5. Availability of at least one tumor-bearing core specimen from the breast cancer 
diagnostic biopsy 
6. Performance status of KPS 70% or greater. 
7. Female or male ≥18 years of age on day of signing informed consent. 
8. Adequate organ function, defined as 
o Absolute neutrophil count ≥1,500/μL. 
o Platelet count ≥100,000/μL. 
o Serum creatinine ≤1.5 × upper limit of normal (ULN) or CrCl ≥60 mL/min.  
o AST and ALT ≤2.5 × ULN  
o Bilirubin ≤1.5 × ULN or direct bilirubin ≤ULN. (Not applicable in patients  with 
Gilbert’s Syndrome.)  
o INR/PT and PTT ≤1.5 × ULN 
o Negative serum or urine pregnancy test if of childbearing potential  
9. Women of childbearing potential must use acceptable measures to avoid becoming 
pregnant while on treatment and for 1 year after last dose of investigational therapy. 
Exclusion Criteria 
1. Neoadjuvant systemic therapy is planned (except for Cohort B subjects) 
2. Prior surgery, radiotherapy or chemotherapy for this cancer (other than core-needle 
biopsy)  
3. Received an investigational agent within 4 weeks of the first dose of treatment. 
4. Diagnosis of immunodeficiency or has received more than replacement doses of 
corticosteroids any other immunosuppressive therapy within 4 weeks of the first dose 
of treatment 
5. Hypersensitivity to IRX-2, cyclophosphamide, indomethacin, aspirin or ciprofloxacin. 
6. Chronic anticoagulation, not including aspirin, but including heparins, warfarin, oral 
anticoagulants or other platelet function inhibitors, that cannot, in the documented 
opinion of the investigator, safely be interrupted from at least 2 days prior to the initiation 
of the study regimen until after surgical resection of the tumor.  
7. Another malignancy that required active treatment within 6 months of the first dose of 
treatment  
8. History or current evidence of any condition, therapy, or laboratory abnormality that 
might confound the results of the trial, interfere with the subject’s participation for the 
full duration of the trial, such that trial participation is not in the best interest of the 
subject, including but not limited to uncontrolled hypertension or clinically significant 
cardiovascular disease, myocardial infarction within the previous 3 months, active 
infection or pneumonitis or other pulmonary disease requiring systemic therapy, 
clinically significant gastritis or peptic ulcer disease (that would preclude the use of 
indomethacin), stroke of other symptoms of cerebral vascular insufficient within the last 
3 months, autoimmune disease that has required systemic treatment within the past 2 
years (other than hormone replacement doses), or uncontrolled psychiatric or 
substance abuse disorders. 
9. Pregnancy or lactation. 
10. Known history of Human Immunodeficiency Virus (HIV) (HIV 1/2 antibodies), active 
Hepatitis B (e.g., HBsAg reactive) or Hepatitis C (e.g., HCV RNA [qualitative] is 
detected). 
Number of Subjects: 39 anticipated 

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TABLE OF CONTENTS  
PROTOCOL SYNOPSIS IRX-2 2016-B ........................................................................................ 3 
1 
INTRODUCTION ............................................................................................................. 14 
1.1 
BACKGROUND 
14 
1.1.1 Breast Cancer .......................................................................................... 14 
1.1.2 The Role of the Immune System in Breast Cancer ................................. 14 
1.1.3 Prognostic significance of tumor infiltrating lymphocytes ........................ 15 
1.1.4 Immune Deficiency and Immunotherapy in Cancer Patients ................... 15 
1.1.5 The IRX-2 Regimen ................................................................................. 15 
1.1.6 Manufacture and Composition of IRX-2 ................................................... 15 
1.1.7 Mechanism of Action of IRX-2 ................................................................. 16 
1.1.8 Rationale for the Components of the IRX-2 Regimen ............................. 16 
1.1.9 Delivery of IRX-2...................................................................................... 17 
1.2 
Nonclinical Studies with the IRX-2 Regimen 
17 
1.3 
Clinical Experience with the IRX-2 Regimen in Subjects with HNSCC 
18 
1.3.1 Phase 1 Trial............................................................................................ 18 
1.3.2 Phase 2 Trial............................................................................................ 18 
1.3.2.1 
Study Design and Subject Demographics ............................. 18 
1.3.2.2 
Clinical Results ...................................................................... 18 
1.3.2.2.1 
Radiographic Changes: ........................................ 18 
1.3.2.2.2 
Disease-free and Overall Survival: ....................... 18 
1.3.2.3 
Safety Results ........................................................................ 18 
1.3.2.4 
Immunologic Results.............................................................. 19 
2 
STUDY OBJECTIVES ..................................................................................................... 21 
2.1 
Primary Objective 
21 
2.2 
Secondary Objective 
21 
2.3 
Exploratory Objective 
21 
3 
INVESTIGATIONAL PLAN .............................................................................................. 21 
3.1 
Study Design 
21 
3.1.1 Rationale for the Primary Objective ......................................................... 23 
3.1.2 Rationale for the Secondary Objective .................................................... 24 
3.1.3 Rationale for Exploratory Objectives ....................................................... 24 
3.1.4 Benefit/Risk Assessment ......................................................................... 24 

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3.2 
Selection of Study Population 
25 
3.2.1 Inclusion Criteria ...................................................................................... 25 
3.2.2 Exclusion Criteria ..................................................................................... 25 
3.3 
Tumor Staging 
26 
3.4 
Assessment of Surgical Resectability 
26 
3.5 
Termination of Study Treatment 
26 
3.6 
Withdrawal of Consent 
26 
4 
STUDY TREATMENTS ................................................................................................... 27 
4.1 
Study Design 
27 
4.2 
Regimen Medications 
27 
4.2.1 IRX-2 ....................................................................................................... 27 
4.2.2 IRX-2 Storage .......................................................................................... 27 
4.2.3 IRX-2 Administration ................................................................................ 27 
4.2.4 Other Medications .................................................................................... 27 
4.3 
Method of Assigning Subject Numbers 
28 
4.4 
Treatment Compliance 
28 
4.5 
Prohibited, Prior and Concomitant Treatments 
28 
5 
STUDY PROCEDURES AND SCHEDULE..................................................................... 28 
5.1 
Study Schedule 
28 
5.2 
Study Procedures 
31 
5.2.1 Screening ................................................................................................ 31 
5.2.2 Day 1 ....................................................................................................... 31 
5.2.3 Any 10 days between days 4 and 15 ....................................................... 32 
5.2.4 One to Five Days Prior to Surgery ........................................................... 32 
5.2.5 Surgery .................................................................................................... 32 
5.2.6 Follow-up Schedule ................................................................................. 33 
6 
ADVERSE EVENTS AND REPORTING REQUIREMENTS ........................................... 33 
6.1 
Adverse Event Definitions 
33 
6.1.1 Definition of Serious and Life Threatening Adverse Events .................... 34 
6.1.2 Definition of Relationship to Study Regimen and Drug ............................ 34 
6.1.3 Definition of Severity ................................................................................ 35 
6.1.4 Definition of Action Taken ........................................................................ 35 
6.1.5 Definition of Outcome to Date .................................................................. 35 

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6.2 
Notification of Serious or Unexpected Adverse Events 
36 
7 
STATISTICAL CONSIDERATIONS AND ASSESSMENT OF ENDPOINTS .................. 36 
7.1 
Sample Size Considerations 
36 
7.2 
Primary Endpoint: Safety and Tolerability of the IRX-2 Regimen 
37 
7.2.1 Laboratory values, vital signs, physical findings and other safety 
data .......................................................................................................... 38 
7.2.2 Pregnancy................................................................................................ 38 
7.3 
Secondary Endpoint:  Stromal TIL infiltration 
38 
7.4 
Exploratory Endpoints 
39 
8 
ETHICAL AND REGULATORY CONSIDERATIONS ..................................................... 40 
8.1 
Protection of Human Subjects 
40 
8.2 
Informed Consent 
40 
9 
STUDY ADMINISTRATION ............................................................................................ 40 
9.1 
Monitoring Plan 
40 
9.1.1 Site Initiation ............................................................................................ 40 
9.1.2 Interim Monitoring .................................................................................... 40 
9.1.3 Study Close-out ....................................................................................... 41 
9.1.4 Site Responsibility ................................................................................... 41 
 
9.2 
Investigational Product Accountability 
41 
9.3 
Study Documents and Access to Records 
41 
9.4 
Quality Assurance 
42 
9.5 
Confidentiality and Publication Policy 
42 
10 
REFERENCES ................................................................................................................ 43 
 

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LIST OF FIGURES  
 
Figure 1 
Overall Survival by High and Low Lymphocyte Infiltration in the Surgical 
Resection Specimen (Study IRX-2 2005-A) ........................................................ 19 
Figure 2 
Lymphocyte Infiltration (Study IRX-2 2005-A) ..................................................... 20 
Figure 3 
Overall Survival by Change in Lymphocyte Infiltration from Biopsy to Resection 
Specimen (Study IRX-2 2005-A) ......................................................................... 20 
 
LIST OF TABLES  
 
Table 1 
Therapeutic Regimen .......................................................................................... 23 
Table 2: 
Schedule of Study Events ................................................................................... 29 
LIST OF APPENDICES  
Appendix 1: 
Karnofsky Performance Status ............................................................................ 46 
Appendix 2: 
Calculation of Creatinine Clearance by Cockcroft-Gault Equation ...................... 47 

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List of Definitions and Abbreviations  
AE 
adverse event 
AJCC 
American Joint Committee on Cancer 
Akt 
protein kinase B 
ALT/SGPT 
alanine aminotransferase/serum glutamic pyruvic transaminase 
ANC 
absolute neutrophil count 
AST/SGOT 
aspartate aminotransferase/ serum glutamic oxaloacetic transaminase 
BUN 
blood urea nitrogen 
CBC 
complete blood count 
CD 
cluster of differentiation  
CFR 
Code of Federal Regulations 
CRF 
case report form 
CRO 
Contract Research Organization 
CT 
computed tomography 
DFS 
disease-free survival 
DSMB 
Data and Safety Monitoring Board 
EFS 
event-free survival 
ESBC 
early stage breast cancer 
EU 
European Union 
FDA 
Food and Drug Administration 
FDG-PET 
fluorodeoxyglucose positron emission tomography 
Fox P3 
Fox P3 protein is involved in immune system responses 
GM-CSF 
granulocyte-macrophage colony stimulating factor 
HIV 
human immunodeficiency virus 
HNSCC 
head and neck squamous cell carcinoma 
HPV 
human papillomavirus 
HR 
hazard ratio 
IB 
Investigator’s Brochure 
IEC 
Independent Ethics Committee 
IFN-α 
interferon alpha 
IFN-γ 
interferon gamma 
IL-1 
interleukin 1 beta 
IL-2 
interleukin 2 
IL-7 
interleukin 7 
IL-10 
interleukin 10 
IL-12 
interleukin 12 
IL-15 
interleukin 15 

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IRB 
Institutional Review Board 
IRX-2 
primary cell-derived biologic with multiple active cytokine components 
IRX-2 regimen 
Twenty subcutaneous injections of 115 U of IRX-2 (over 10 days) 
preceded by cyclophosphamide with a 21 day course of indomethacin 
and zinc containing multivitamins 
ITT 
Intention-To-Treat 
IV 
intravenous 
IxRS 
Interactive Recognition System 
KPS 
Karnofsky performance status 
LDH 
lactate dehydrogenase 
MedDRA 
Medical Dictionary for Regulatory Activities  
MHC 
major histocompatibility complex  
MHC class I 
major histocompatibility complex class 1 
MHC class II 
major histocompatibility complex class 2 
MRI 
magnetic resonance imaging 
NCCN 
National Comprehensive Cancer Network 
NCI-CTCAE 
National Cancer Institute Common Terminology Criteria for Adverse 
Events 
NK or NKT 
natural killer T cells 
OS 
overall survival 
PET 
positron emission tomography 
PDL1 
programmed death ligand 1  
PHA 
Phytohemagglutinin 
PI 
Principal Investigator 
PI3K 
phosphoinositide 3 kinase 
PT 
prothrombin time 
PTT 
partial thromboplastin time 
RECIST 
Response Evaluation Criteria in Solid Tumors 
SAE 
serious adverse event 
SAP 
statistical analysis plan 
SCC 
squamous cell carcinoma 
T cells 
cellular immune lymphocytes; derivation – thymus lymphocytes 
T-bet 
T box transcription factor 
TCR 
T cell receptor  
Teff 
T effector cell 
TGF-β 
Transforming growth factor beta 
Treg 
T regulatory cell; suppressor T cell 
TID 
three times a day 

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TIL 
tumor infiltrating lymphocytes 
TNF-α 
tumor necrosis factor alpha 
TNM  
tumor grading system (T-primary lesion; N-nodes; M-metastases) 
U 
unit(s) 
ULN 
upper limit of normal 
US 
United States 
 

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1 
INTRODUCTION 
1.1 
BACKGROUND 
1.1.1 
Breast Cancer 
The American Cancer Society estimates that approximately 40,000 people in the USA will die 
annually of metastatic breast cancer [American Cancer Society, 2012]. Breast cancer ranks 
second as the cause of cancer deaths in women, and while deaths rates are falling, patients 
diagnosed with advanced breast cancer remain at high risk of dying from their disease. 
Immunotherapy to facilitate immune recognition of micrometastatic disease has been of scientific 
interest, particularly in patients at high risk of tumor recurrence. There are numerous vaccination 
and immunotherapy trials listed on clinical trials.gov that are open for the adjuvant or neo-adjuvant 
treatment of patients with breast cancer. Examples of vaccines being evaluated include a peptide 
Her2/neu vaccine, vaccines against carcinoembryonic antigen (CEA) and cancer testis antigens 
(CTA). These vaccines also include various adjuvant components designed to optimize immune 
response, for example, a toll-like receptor 3 agonist, GM-CSF, or dendritic cells. In addition to 
vaccines, other immunotherapeutic approaches, for example immune checkpoint blockade with 
ipilimumab or tissue-destructive in situ vaccination approaches such as tumor cryoablation [Page, 
2014] are also being evaluated. 
Specifically for triple negative breast cancer, the standard-of-care is to consider pre-operative 
neoadjuvant chemotherapy. Benefits of this approach are to assess for complete response, which 
is prognostic for overall survival, and to potentially surgically downstage. Because immune 
infiltrates are predictive to response to chemotherapy in this population, introduction of 
immunotherapy either before or during chemotherapy is of potential therapeutic utility. 
1.1.2 
The Role of the Immune System in Breast Cancer   
The tumor microenvironment and tumor immunogenicity have recently been recognized as a 
contributing component in pathogenesis and response to therapy. Recently published studies 
suggest that distant metastatic invasion of breast cancer, specifically “triple negative breast 
cancer” (TNBC) may be induced by immune and/or inflammatory deregulation. Among a set of 
45-gene signatures that was found in distant disease recurrence, ten genes found within the 
transforming growth factor-β signaling pathway are associated with an immune-suppressed 
phenotype [Kuo, 2012]. In patients with breast cancer, either a decreased number of CD8+ 
T-cells, or an increased number of regulatory T-cells, was associated with adverse tumor 
characteristics such as lymph node metastasis, higher stage, and Ki-67 immunopositivity 
[Miyashita, 2014]. Corroborating these findings, across multiple large adjuvant and neoadjuvant 
datasets, the quantity of tumor-infiltrating lymphocytes (TILs) in early-stage TNBC has been 
associated with improved survival, and increased likelihood of response to chemotherapy 
[Miyashita, 2014; Denkert, 2010]. A study from the German Breast Group addressed the role of 
TILs in predicting response to neoadjuvant therapy with anthracycline and taxane chemotherapy 
[Denkert, 2010]. The percentages of intratumoral and stromal lymphocytes in pretreatment tumor 
biopsies were identified as significant independent predictors of pCR in both a training (n=218) 
and validation cohort (n=840) (p=0.012; -0.001). Additionally, pretreatment TILs have been linked 
to improved DFS and OS in breast cancer [Adams, 2014]. Functional assays demonstrate that 
adequate activation of TILs derived from breast cancer tissue could restore the appropriate 
antitumor immune responses [DeNardo, 2007]. This emerging data underscores the potential of 
a therapeutic strategy that increases immune-mediated antitumor activity. Therefore, a 
reasonable hypothesis to examine is that modulation of the immune system might improve 
outcomes. 

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1.1.3 
Prognostic significance of tumor infiltrating lymphocytes 
The quantity of TILs within breast cancer tumor stroma has been validated across multiple 
adjuvant studies as a strong and independent predictor of recurrence [Adams, 2014]. A 
consensus methodology of scoring stromal TILs has been developed by an international breast 
cancer TILs working group, and is being prospectively validated [Salgado, 2014]. The 
methodology involves scoring of lymphocytes on a single tumor-bearing H&E slide on a scale of 
0-100% lymphocytes. Using this methodology in a pooled analysis of 5 adjuvant triple negative 
chemotherapy trials (n=991), stromal TILs were prognostic for invasive disease free survival 
(HR=0.84, 95% CI 0.78-0.91) and overall survival [Loi, 2015]. Furthermore, stromal TILs were 
found to be independently prognostic when analyzed in conjunction with clinical and pathologic 
variables including age, tumor size, and node positivity by multivariate cox analysis. In subjects 
with matched lymph node status, increases in TILs by 20% were associated with improvements 
in 5-year distant relapse free survival rates as great as 10%.  
In light of these data, there is strong rationale to evaluate immunotherapy techniques that may 
increase stromal lymphocyte infiltration, and potentially improve outcomes, in patients with early 
stage breast cancer in pre-operative clinical trials.  
1.1.4 
Immune Deficiency and Immunotherapy in Cancer Patients 
The rationale for the pursuit of immunotherapy in patients with cancer stems from a considerable 
body of evidence, which indicates that immunologically competent cells are important host 
defense mechanisms. Many human cancers, however, are associated with cellular 
immunodeficiency. Immunotherapies now in development for the treatment of multiple cancers 
include nonspecific, systemic cell-mediated approaches, cytokine based immunotherapy 
(including delivery of granulocyte-macrophage colony stimulating factor [GM-CSF], interleukin-2 
[IL-2], interferon alpha [IFN-α], interferon gamma [IFN-γ], interleukin-12 [IL-12], and IRX-2), 
tumor-associated antigens as therapeutic targets, monoclonal antibodies and cancer vaccines, 
either peptide/protein based, dendritic-cell based or nucleic acid based, and immune checkpoint 
inhibitors. Discussion of these many approaches is beyond the scope of this protocol Introduction 
but is presented in more detail in the Investigator’s Brochure (IB) and in several reviews [ 
Petrelli F, Coinu A, Borgonovo K, Cabiddu M, Ghilardi M, Lonati V, Barni S. The value of 
platinum agents as neoadjuvant chemotherapy in triple-negative breast cancers: a systematic 
review and meta-analysis. Breast Cancer Res Treat 2014 144:223-32. 
Rapidis, 2009; Freiser, 2013; Gildener-Leapman, 2013]. 
1.1.5 
The IRX-2 Regimen 
The concept that subjects with cancer might benefit from immune stimulation led to the 
development of the IRX-2 regimen, first studied in patients with head and neck squamous cell 
cancer (HNSCC), a malignancy clearly associated with immune deficiency [Hadden, 1994]. The 
IRX-2 regimen consists of a cell-derived biologic, IRX-2, that contains multiple cytokines, 
cyclophosphamide, indomethacin, omeprazole, and multivitamins with zinc. These four 
components have been administered in patients with HNSCC as a regimen consisting of an 
intravenous (IV) infusion of low-dose cyclophosphamide on Day 1, followed by oral indomethacin, 
omeprazole and oral multivitamins with zinc daily for 21 days, and IRX-2 administered as two 
subcutaneous injections per day for ten days [Freeman, 2010; Wolf, 2011]. Omeprazole, a proton 
pump inhibitor, is given concurrently to improve tolerance of the indomethacin.  

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1.1.6 
Manufacture and Composition of IRX-2 
IRX-2 is a primary cell-derived biologic with multiple active cytokine components produced under 
pharmaceutical standards as discussed in more detail in the IB. Briefly, human leukocytes (“buffy 
coats”) pooled from multiple donors are stimulated with phytohemagglutinin and ciprofloxacin. 
Subsequently, the phytohemagglutinin, ciprofloxacin and all cellular elements are removed or 
significantly reduced, and the cell-free supernatant is filter sterilized, nanofiltered to clear viral 
particles, vialed, and frozen as IRX-2. 
IL-2 is the major cytokine in IRX-2, followed by IFN-γ, TNF-α, and interleukin 1 beta (IL-1). These 
cytokines when studied individually enhance cell-mediated immunity via several different 
mechanisms discussed below and in the IB.  
1.1.7 
Mechanism of Action of IRX-2  
Recent in vitro studies have elucidated several potential mechanisms of action of IRX-2, and 
these various mechanisms of action need not be exclusive. IRX-2 treatment of human monocyte-
derived dendritic cells results in changes consistent with the development of mature activated 
dendritic cells. Specifically, IRX-2 increased the percentage of cells expressing CD83 and CCR7, 
markers for dendritic cell maturation and migration and increased the expression of multiple 
markers that are critical mediators of T cell activation [Egan, 2007]. Similar results were obtained 
in a later study in cells obtained from patients with HNSCC [Schilling, 2013]. Also, in an in vitro 
study of peripheral blood mononuclear cells obtained from patients with HNSSC, IRX-2 up-
regulated cytotoxicity of NK cells and did so more effectively than IL-2 [Schilling, 2012]. 
IRX-2 can also protect T cells from activation induced cell death by reversing microvesicle induced 
inhibition of the PI3K/Akt pathway and correcting the imbalance of pro- versus anti-apoptotic 
proteins induced by tumor-derived microvesicles [Czystowska, 2009; Czystowska 2011]. IRX-2 
was superior to recombinant IL-7 and IL-15 in protecting T cells from tumor-induced apoptosis. 
The presence of IRX-2 in a tumor microenvironment model promoted the induction and expansion 
of IFN-γ+T-bet+ Teff and significantly decreased the induction of inducible IL-10+TGF-β+ Treg. The 
responsible mechanism involved IFN-γ+-driven T cell polarization towards Teff and suppression of 
Treg differentiation [Schilling, 2012]. In the Phase 2a study in subjects with HNSSC (described 
below in Section 1.3.1), IRX-2 mediated reductions in circulating B and NKT cell numbers, 
suggesting redistribution of these cells to tissues [Whiteside, 2012]. A decrease in naïve T cells 
was also noted, suggesting their upregulation to memory T cells, while unchanged numbers of 
Tregs (suppressor T cells) after IRX-2 therapy indicated that IRX-2 does not expand this 
compartment, potentially benefiting anti-tumor immune responses [Whiteside, 2012].  
Finally, IRX-2 has been shown to induce enhanced T cell responses when administered with 
tumor antigen vaccines, raising the possibility that IRX-2 treatment in subjects with HNSCC 
enhances endogenous antigen-specific T cell responses to the tumor [Naylor, 2010]. 
1.1.8 
Rationale for the Components of the IRX-2 Regimen 
The rationale for the components of the IRX-2 regimen is outlined below. 
Cyclophosphamide:  One mechanism for reversal of anergy and reversal of suppression of 
immune responses in subjects with malignancy by adoptive immunotherapy may be related to 
inhibition of regulatory T cell function [North, 1982; North, 1984]. Evidence indicates that 
cyclophosphamide inhibits Treg number and/or function [Emens, 2005]. Thus many clinical trials 
that involve immunotherapy or attempt to stimulate immune response to tumor antigens have 
employed low dose cyclophosphamide (300 mg/m2) as a component of the treatment regimen. 
This immunomodulatory dose is less than one-third of a typical anti-cancer dose and is intended 

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to enhance the development of cell-mediated immunity by providing contrasuppression of tumor-
associated immune suppression (to reduce the number and function of regulatory T cells, i.e. Treg) 
[Berd, 1982; Machiels, 2001]. 
Pathways of local immune tolerance, escape mechanisms active within the tumor 
microenvironment and superimposed potent systemic mechanisms of immune tolerance have 
been reviewed [Emens, 2005] and are discussed in more detail in the Investigator’s Brochure 
(Section 3.4.1). The use of cytotoxic chemotherapy in doses and schedules designed to abrogate 
specific mechanisms of immune tolerance in order to release the full potential of an antitumor 
immune response is discussed. Specifically, cyclophosphamide may be used to prime the 
immune system by promoting the differentiation of CD4+ T helper cells and by abrogating the 
suppressive influence of CD4+CD25+ T regulatory (Treg) cells. In the absence of Treg influence, 
high-avidity CD8+ T cells are recruited to an antigen-specific immune response. 
Cyclophosphamide also facilitates the establishment of memory CD8+ T cells. Thus inclusion of 
cyclophosphamide in combination with other immune-modulatory agents is supported by both 
pre-clinical and clinical data as reviewed in more detail by Emens (2005) and in the Investigator’s 
Brochure (Section 3.4.1). 
Indomethacin:  Indomethacin, a nonselective COX-1/COX-2 inhibitor, is a potent inhibitor of 
prostaglandin synthesis and may reverse the immunosuppression induced by prostaglandin 
[Lapointe, 1992; Hadden, 1994]. Numerous experimental, epidemiologic, and clinical studies 
suggest that non-steroidal anti-inflammatory drugs, including indomethacin, suppress 
cyclooxygenase and have promise as anticancer agents, particularly for chemoprevention of and 
as adjuvant therapy in patients with cancer [Thun, 2002; Investigator’s Brochure, Section 3.4.2].  
Zinc with multivitamins:  Subclinical zinc deficiency is common in subjects with HNSCC, perhaps 
related to a history of alcohol consumption [Brookes, 1981]. The importance of zinc in cellular 
immunity has been described and several reviews are available [Good, 1979; Keen, 1990; 
Blewett, 2012; Haase, 2014]. Alcohol consumption is frequently associated with nutritional 
deficiency, which can also result in impaired immune response [Słotwińska, 2014; Bianchini, 
2012; Wintergerst, 2007]. Thus, based on these observations and the lack of any contraindication 
to their use, zinc-containing multivitamins have been added to the IRX-2 regimen. 
Omeprazole:  Omeprazole, a proton pump inhibitor, is active at preventing indomethacin-induced 
gastritis. It is administered with the IRX-2 regimen to decrease the likelihood of indomethacin-
induced gastritis. 
1.1.9 
Delivery of IRX-2 
The route of administration of IRX-2 takes advantage of the normal pathways of lymph node 
activation. Normally, lymphatics drain from an area of disease, such as a tumor bed, and antigens 
and other factors associated with disease migrate in the lymphatics to the regional nodes. By 
presenting the cytokine-containing biologic in the area of the tumor-draining lymph nodes rather 
than systemically, there is an opportunity to mobilize antigen presenting cells and enhance 
dendritic cell function as well as directly activate T cells to proliferate and become cytotoxic 
lymphocytes. Additionally, subcutaneous administration has been less toxic since the systemic 
cytokine drug concentration is much lower.  
In this study, we will evaluate the feasibility of injection of the cytokine mixture, IRX-2, 
subcutaneously into the periareolar skin of the affected breast. Periareolar injection of radioactive 
colloid is an acceptable and clinically utilized technique for localizing sentinel lymph nodes in 
breast cancer [McMasters, 2011] and therefore it is assumed that periareolar injection of IRX-2 
will be a feasible, acceptable and effective method of delivering the study agent to the draining 
lymph nodes of the tumor. 

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1.2 
Nonclinical Studies with the IRX-2 Regimen 
Nonclinical studies have demonstrated an effect of IRX-2 on T cells in animal models as well as 
in cellular assays. These studies are summarized briefly in Section 1.1.7 above and in more detail 
in the IB. 
1.3 
Clinical Experience with the IRX-2 Regimen in Subjects with HNSCC 
1.3.1 
Phase 1 Trial 
IRX-2 2004-B was a multicenter, Phase 1 trial in subjects with advanced stage HSNCC who had 
progressed after surgery and/or radiation therapy that was designed to evaluate the clinical and 
laboratory safety and tolerability of the IRX-2 regimen. Results of the study are presented in the 
IB and have been published [Freeman, 2010]. The reported toxicities were acceptable overall and 
did not preclude proceeding to additional trials. 
1.3.2 
Phase 2 Trial 
1.3.2.1 Study Design and Subject Demographics 
IRX-2 2005-A was a multi-center trial entitled “A Phase 2, Open-label Trial of the Safety and 
Biological Effect of Pre-operative Subcutaneous IRX-2 (with Cyclophosphamide, Indomethacin, 
and Zinc) in Subjects with Resectable Cancer of the Head and Neck.”  Results of the study are 
presented in the IB and have been published [Wolf, 2011; Berinstein, 2012; Whiteside, 2012] and 
are also summarized here.  
 
The study objectives were to determine the safety of the IRX-2 regimen when used as 
neoadjuvant (preoperative) therapy and to evaluate clinical, pathological, and radiographic tumor 
response and disease-free survival (DFS) and overall survival (OS).  
1.3.2.2 Clinical Results 
1.3.2.2.1 
Radiographic Changes: 
Changes in tumor size between baseline and immediately pre-operative, i.e. after the IRX-2 
regimen, measurements were evaluable in 23 subjects. Based on measurement of the longest 
single tumor diameter, tumor growth or shrinkage was as follows: 4 subjects had a decrease in 
tumor size, 16 subjects had no significant change, and 3 subjects had an increase in tumor size 
[Wolf, 2011]. Review of the radiologic findings in relation to the pathology finding in the resection 
specimens, however, established that lymph node or tumor enlargement due to reactive 
hyperplasia could not be distinguished from enlargement related to tumor growth [Wolf, 2011]. 
 
One subject underwent a fluorodeoxyglucose positron emission tomography (FDG-PET) CT scan 
at baseline and at completion of the IRX-2 regimen. Elevated glycolytic activity was observed in 
2 lymph nodes and in the primary tumor on the baseline PET scan. At the completion of the IRX-2 
regimen, there was a 75% decrease in glycolytic activity in these lesions [Wolf, 2011]. 
 
1.3.2.2.2 
Disease-free and Overall Survival: 
After 5 years of follow-up, median DFS and OS had not been reached. These results for both 
DFS and OS appeared to be slightly superior to those observed in a comparable group of 81 
historical controls, treated at the University of Michigan and matched for baseline characteristics 
[G. Wolf, personal data]. 
 

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1.3.2.3 Safety Results 
The IRX-2 regimen was tolerated with minimal toxicity. Compliance was excellent; all subjects 
completed the regimen and there were no unplanned delays in surgery as a result of the 
immunotherapy regimen. There were no reports from investigators of any unanticipated or 
unusual delays or difficulties in performing the planned resections or reconstructions or during the 
postoperative recovery.  
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 [Wolf, 2011]. Note that many of these AEs, including e.g. aspiration pneumonia, 
wound infections, alcohol withdrawal, are common in HNSCC subjects during their pre- and post-
operative course. During treatment, several subjects noted decreased pain or improved 
swallowing and no significant progressive symptoms were noted.  
 
1.3.2.4 Immunologic Results 
Pretreatment tumor biopsies and the tumor surgical specimens from 25 subjects were 
characterized for lymphocyte infiltration, necrosis and fibrosis using both hematoxylin and eosin 
stains and immunohistochemistry [Berinstein, 2012]. Kaplan-Meier estimates of overall survival 
are displayed in Figure 1 as a function of high and low lymphocyte infiltration in the surgical 
specimens after the IRX-2 immunotherapy. Eighteen subjects were in the better survival group 
and 7 were in the inferior survival group; the survival curves are significantly different (p <0.05) 
[Berinstein, 2012]. 
 
Figure 1 
Overall Survival by High and Low Lymphocyte Infiltration in the Surgical 
Resection Specimen (Study IRX-2 2005-A) 
 
 

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When lymphocyte infiltration in the pretreatment biopsies was compared to that in the resected 
surgical specimen, increases in lymphocyte infiltration were seen as shown in Figure 2 (change 
in mean lymphocyte infiltration from the biopsy to the surgical specimen is shown on the y-axis) 
[Berinstein, 2012]. 
 
Figure 2 
Lymphocyte Infiltration (Study IRX-2 2005-A) 
 
 
In addition, subjects in whom the greatest increase in tumor lymphocyte infiltration from biopsy 
to surgery (n=14) was observed had a trend toward superior survival compared to subjects in 
whom no or more limited change was observed (n=11) as shown in Figure 3 (p = 0.10) (IRX 
Therapeutics, unpublished observations). 
 
Figure 3 
Overall Survival by Change in Lymphocyte Infiltration from Biopsy to 
Resection Specimen (Study IRX-2 2005-A) 

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Peripheral blood lymphocyte subsets also were monitored pre- and post-treatment with the IRX-2 
regimen to evaluate changes induced by the IRX-2 regimen (summarized in Section 1.1.7 above 
and in the IB). The IRX-2 regimen-mediated reductions in B and NKT cell numbers in the blood 
suggested a redistribution of these cells to tissues while the unchanged numbers of Tregs after 
IRX-2 therapy indicated that IRX-2 does not expand this compartment, potentially benefiting anti-
tumor immune responses [Whiteside, 2012].  
2 
STUDY OBJECTIVES  
2.1 
Primary Objective 
The primary objective is to establish the safety and tolerability of the IRX-2 regimen when 
administered pre-operatively in early stage breast cancer (ESBC) patients. 
 
2.2 
Secondary Objective 
 
To evaluate for changes in tumor infiltrating lymphocyte (TIL) score associated with the 
IRX-2 regimen, as measured by H&E TIL count according to Salgado criteria [Salgado, 
2014]. 
 
In Cohort B: To estimate the pathologic complete response rate to neoadjuvant 
anthracycline-based and non-platinum containing chemotherapy in patients who have 
received the IRX-2 Regimen before chemotherapy 
 
2.3 
Exploratory Objective 
The exploratory objective is to evaluate the effect of IRX-2 on additional investigational 
immunologic and molecular biomarkers. 

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Exploratory endpoints will be analyzed descriptively, and will include additional peripheral 
blood and intratumoral analyses of the immunologic effects of the IRX-2 regimen, including, 
for example, some or all of the following:   
 
flow cytometric quantification and characterization of peripheral lymphocytes (including 
activated T-cells, T-regulatory cells, NK cells, and myeloid cells) 
 
TIL phenotype characterization by multispectral immunofluorescence imaging to assess 
changes in abundance of T-regulatory cells, activated T-cells, myeloid lineages and 
dendritic cells 
 
Intratumoral and T-cell clonal responses by T-cell receptor DNA deep sequencing 
 
Characterization of intratumoral immune responses by RNA expression using the 
Nanostring PanCancer Immune panel and/or Prosigna tumor recurrence score 
3 
INVESTIGATIONAL PLAN 
3.1 
Study Design  
This will be a Phase Ib study conducted to determine the safety and tolerability of an IRX-2 
regimen in early stage breast cancer to be administered pre-operatively before standard-of-care 
surgical resection and following standard-of-care diagnostic biopsy, and patients with triple 
negative breast cancer who will receive the IRX-2 regimen preceding standard of care 
neoadjuvant chemotherapy. 
Eligible subjects will have early stage breast cancer of any receptor subtype, for which standard-
of care surgical resection is planned, or T1c+ triple negative breast cancer with planned standard 
of care neoadjuvant chemotherapy treatment (anthracycline-containing and non-platinum 
containing). 
To be eligible, a minimum of 1 core of tumor-bearing biopsy material must be available for 
research analysis.  
The IRX-2 regimen will be administered in all enrolled subjects according to the schema and 
dosing schedule delineated in Table 1. IRX-2 will be administered by subcutaneous injection by 
study personnel at Providence Medical Center. 
 
Subjects will be evaluated by physical exam and blood tests at baseline and periodically during 
the study, culminating in a final study visit 30 days (+/- 7 days) following surgical resection. Safety 
will be evaluated routinely using the NCI-CTCAE v4.03 toxicity reporting criteria.  
 
All subjects are to receive standard of care surgery and postoperative adjuvant radiation or 
chemo- or hormonal therapy, as appropriate, or for triple negative breast cancer patients, 
neoadjuvant treatment with chemotherapy. 
 
 
 

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Table 1 
Therapeutic Regimen 
 
Days of 
administration* 
Drug 
Dose 
Route 
Comment 
1 
Cyclophosphamide
300mg/m2 
IV infusion 
Low dose does not 
require corticosteroids; 
has low to moderate 
emetic risk per NCCN 
Guidelines 
Any 10 days 
between  
days 4-19 
IRX-2 
2 mL (230 
units of IL-2)  
on each of 10 
days 
Two 1 mL 
subcutaneous 
periareolar 
injections 
Injections will be 
administered at 
Providence Medical 
Center. 
1-21 
Indomethacin 
25mg, TID 
Oral 
Divided three times 
daily x 21 days 
1-21 
Multivitamin with 
15-30 mg of 
zinc/tablet 
1 tablet 
Oral 
Once daily x 21 days 
1-21 
Omeprazole 
1 tablet 
Oral 
Once daily x 21 days 
Ideally 2-5 days 
after last IRX-2  
 
 
 
Clinically indicated 
breast surgery, or 
post-IRX2 biopsy  
*As necessitated by scheduling logistics, the oral medications may be discontinued after Day 17 
and surgery or biopsy performed 2-12 days later. Indomethacin must be discontinued at least 2 
days prior to scheduled surgery or biopsy. 
Adverse events will be collected from Day 1 (day of cyclophosphamide) until the day of surgery. 
A final study visit 30 days (±7 days) following surgical resection any AEs felt by the investigators 
to be definitely or possibly related to the study regimen.  
Serious adverse events (not including hospitalization required for breast surgery) will be collected 
from Day 1 until 30 days after surgery. All SAEs will be tabulated regardless of relatedness to 
study drugs.  
3.1.1 
Rationale for the Primary Objective 
No significant safety concerns have been recognized in prior Phase 1 and Phase 2a clinical trials 
of the IRX-2 regimen in subjects with HNSCC. Nevertheless, both trials included only subjects 
with HNSCC, were relatively small trials, and involved injection into a different anatomical site (i.e. 
the cervical neck nodal basin). Therefore, this trial is needed to establish the safety of the IRX-2 
regimen in subjects with early stage breast cancer using an approach of periareolar sub-
cutaneous injection. The safety of IRX-2 will be determined by evaluation of AEs and assessment 
of any surgical delays associated with administration of the study regimen. 

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The rationale of cohort B is to estimate safety and immunologic effects in the pre-neoadjuvant 
chemotherapy setting.   
3.1.2 
Rationale for the Secondary Objectives 
Across multiple adjuvant clinical trials, tumor lymphocytic infiltration has been associated with 
favorable overall survival and long term outcomes. Measurement of TILs by Salgado Criteria has 
been validated retrospectively in numerous clinical trials, and the methodology has reached 
international consensus. The TILs prognostic marker is a continuous variable, with increases in 
TIL count associated with linear increases in distant disease free survival, independent of other 
prognostic markers such as lymph node status.  
The rationale for measuring TILs by Salgado criteria is to evaluate the hypothesis that IRX-2 
increases TIL count in early stage breast cancer. If so, then these data will support further study 
of the IRX-2 regimen in early stage breast cancer in a larger clinical trial with a clinical endpoint. 
In cohort B, pathologic complete response rate will be assessed preliminarily in the setting of IRX-
2 pre-treatment, on the basis that enhanced immune infiltration may mediate clearance of 
chemotherapy-resistant tumor cells. 
3.1.3 
Rationale for Exploratory Objectives 
Additional immune-based biomarkers, while less extensively validated than H&E TIL count, hold 
promise as prognostic/predictive indicators, and may also be used to further characterize the 
immunologic and tumor effects of immunotherapy [Tumeh, 2014; Feng, 2015]. It is also quite 
possible that mechanistic insights may be obtained from such analyses. For example, if IRX-2 is 
shown to increase PD-L1 expression on TILs, this would support a follow-up pre-clinical 
assessment of combination IRX-2 with PD-1/L1 blockade.  
3.1.4 
Benefit/Risk Assessment  
This trial is designed to incorporate immunotherapy into the early stage breast cancer paradigm 
while minimizing deviations from standard peri-operative care. The trial will only enroll subjects 
for whom standard neoadjuvant systemic therapy is not planned. At Providence Health Centers, 
among early stage patients not treated with neoadjuvant systemic therapy, the median time from 
diagnosis (by core biopsy) to surgical resection between 2004 and 2015 was 27 days (interquartile 
range 19-39 days, n=5165) [Page, unpublished observations]. Therefore, subjects enrolled in this 
trial are not anticipated to experience a delay in surgery beyond what is customary for the 
institution. To minimize delays in definitive surgical resection, subjects will be screened for 
eligibility and consented by the investigator as soon as possible following confirmation of invasive 
carcinoma. The study regimen calls for 21 days of pre-operative therapy, and therefore if subjects 
are consented and commence therapy within 7-10 days of diagnosis, they will be scheduled to 
undergo definitive surgical resection between days 23 and 28 after diagnosis, consistent with the 
median time from diagnosis to surgical resection among early stage subjects not treated with 
neoadjuvant systemic therapy.  
 
This regimen has not raised any significant safety concerns in several phase I/II clinical trials as 
discussed above, and therefore the risk of significant harm from adverse events is minimal. 
Furthermore, the IRX-2 regimen has been administered pre-operatively in HNSCC patients, i.e., 
in patients who undergo more extensive surgical procedures than breast cancer patients, 
including elective neck dissection and more complicated tumor excisions and reconstructive 
surgery. 
 

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One potential indirect risk is that the study drugs might influence prognostic markers obtained 
from the surgical specimen, for example RNA expression profiling for ER-positive tumors used to 
determine chemotherapy benefit. For this reason, in all subjects with ER-positive breast cancer, 
a portion of the pre-study diagnostic core biopsy will be submitted for RNA expression profiling if 
clinically indicated. 
Additional support for a low risk from the pre-operative IRX-2 regimen is that in a recent peri-
operative early stage breast cancer immunotherapy trial, subjects were treated with either 
ipilimumab single dose, tumor cryoablation, or both. The regimen was well-tolerated, with no 
sustained adverse events, no delays in pre-planned surgical resection, and no reported 
deviations/complications in post-mastectomy adjuvant chemotherapy/hormonal therapy (Page, 
2014). 
3.2 
Selection of Study Population 
3.2.1 
Inclusion Criteria 
To be enrolled in the study, subjects must meet the following inclusion criteria. 
1. Invasive breast cancer of any receptor subtype diagnosed by core-needle biopsy  
2. To undergo surgical resection with curative intent by partial mastectomy (lumpectomy) or 
mastectomy 
3. Tumor >5 mm in maximum diameter by ultrasound or mammography. (Subjects with 
smaller tumors may be included at the discretion of the Principal Investigator.) Cohort B: 
triple negative (defined by ER<10%, PR<10%, and HER2-negative by NCCN guidelines), 
T1c+ tumors for which neoadjuvant anthracycline-based and non-platinum containing 
chemotherapy is planned. 
4. Willing and able to provide written informed consent, including consent for use of available 
tissue and required blood draws for research purposes 
5. Availability of at least one tumor-bearing core specimen from the breast cancer diagnostic 
biopsy 
6. Performance status of KPS 70% or greater 
7. Female or male ≥18 years of age on day of signing informed consent 
8. Adequate organ function, defined as: 
a. Absolute neutrophil count ≥1,500/μL. 
b. Platelet count ≥100,000/μL. 
c. Serum creatinine ≤1.5 × ULN or CrCl ≥60 mL/min (calculated by by Cockcroft-
Gault Equation [Appendix 2]).  
d. AST and ALT ≤2.5 × ULN  
e. Bilirubin ≤1.5 × ULN or direct bilirubin ≤ULN. (Not applicable to patients with 
Gilbert’s Syndrome.) 
f. 
INR/PT and PTT ≤1.5 × ULN 
g. Negative serum or urine pregnancy test for women of childbearing potential  
9. Men, and women of childbearing potential must use acceptable measures to avoid 
becoming pregnant or a new father while on treatment and for 1 year after last dose of 
investigational therapy 
3.2.2 
Exclusion Criteria 
Subjects who meet any of the following criteria will be excluded from the study. 
1. Neoadjuvant systemic therapy is planned (except for Cohort B) 
2. Prior surgery, radiotherapy or chemotherapy for this cancer (other than core-needle biopsy 
or lymph node excisional biopsy)  

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3. Received an investigational agent within 4 weeks of the first dose of treatment. 
4. Diagnosis of immunodeficiency or has received more than replacement doses of 
corticosteroids any other immunosuppressive therapy within 4 weeks of the first dose of 
treatment 
5. Hypersensitivity to IRX-2, cyclophosphamide, indomethacin, omeprazole, aspirin or 
ciprofloxacin. 
6. Chronic anticoagulation, not including aspirin, but including heparins, warfarin, oral 
anticoagulants or other platelet function inhibitors , that cannot, in the documented opinion 
of the investigator, safely be interrupted from at least 2 days prior to the initiation of 
the study regimen until after surgical resection of the tumor. 
7. Another malignancy that required active treatment within 6 months of the first dose of 
treatment  
8. History or current evidence of any condition, therapy, or laboratory abnormality that might 
confound the results of the trial, interfere with the subject’s participation for the full duration 
of the trial, such that trial participation is not in the best interest of the subject, including 
but not limited to uncontrolled hypertension or clinically significant cardiovascular disease, 
myocardial infarction within the previous 3 months, active infection or pneumonitis or other 
pulmonary disease requiring systemic therapy, clinically significant gastritis or peptic ulcer 
disease (that would preclude the use of indomethacin), stroke of other symptoms of 
cerebral vascular insufficient within the last 3 months, autoimmune disease that has 
required systemic treatment within the past 2 years (other than hormone replacement 
doses), or uncontrolled psychiatric or substance abuse disorders. 
9. Pregnancy or lactation. 
10. Known history of Human Immunodeficiency Virus (HIV) (HIV 1/2 antibodies), active 
Hepatitis B (e.g., HBsAg reactive) or Hepatitis C (e.g., HCV RNA [qualitative] is detected. 
3.3 
Tumor Staging  
Initial tumor staging will be performed using the TNM staging and stage grouping is based on 
American Joint Committee on Cancer (AJCC) clinical staging criteria: 
 https://cancerstaging.org/references-tools/quickreferences/Documents/BreastMedium.pdf 
3.4 
Assessment of Surgical Resectability 
The disease must be resectable for cure, as determined by the investigating clinician (in 
consultation with a surgeon if necessary), in order for the subject to be eligible for the study.  
3.5 
Termination of Study Treatment 
The investigator may stop the study treatment regimen due to an allergic or hypersensitivity 
reaction to the study drug, an SAE or clinically significant AE or laboratory abnormality or for 
protocol noncompliance. If a subject is discontinued from the study due to an AE, the investigator 
should notify IRX Therapeutics within 24 hours of the event.  
 
NOTE: Subjects may experience changes in primary tumor or regional lymph nodes during 
treatment with the IRX-2 regimen, including inflammation, necrosis, or change in 
consistency. These changes should not be mistaken for tumor progression prior to 
surgery. 
3.6 
Withdrawal of Consent 
Any subject may terminate participation in the study at any time but every effort should be made 
to continue with study treatment and evaluations. If a subject elects to discontinue study 

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participation at any time for safety, medical or personal reasons, the investigator should make a 
reasonable effort to determine the reason for the subject’s withdrawal and document the reason 
on the CRF. 
Any clinically significant AEs or SAEs leading to premature withdrawal are to be followed until 
resolution. 
4 
STUDY TREATMENTS 
4.1 
Study Design  
Subjects who meet all eligibility criteria will be treated with the IRX-2 regimen as described in 
Table 1 above (Section 3.1). 
4.2 
Regimen Medications 
4.2.1 
IRX-2 
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, 
kit numbers, lot number, and product identity. Kit and lot numbers of IRX-2 will be recorded in the 
CRF. Study syringes for subcutaneous IRX-2 administration will be disposed of in accordance 
with biosafety procedures.  
4.2.2 
IRX-2 Storage 
IRX-2 must be stored in a secure area and maintained under labeled storage conditions at a range 
of −15oC to –25o C. 
4.2.3 
IRX-2 Administration 
IRX-2 should be administered as two subcutaneous injections of 1 mL each, in the periareolar 
region of the affected breast. Each injection should be given subcutaneously approximately 1 cm 
from the outer margin of the areola. A 4-6 mm, 28-30 gauge needle is recommended; injections 
should consistently enter the fat just underneath the skin. The first injection should be 
administered on the same axis as the tumor location as identified by physical exam, 
mammography or ultrasound. The second injection on the same day should be rotated 90 degrees 
clockwise to the first injection. On the second day of IRX-2 injections, the first injection should be 
again administered on the same axis as the tumor location, but the second injection should be 
rotated 90 degrees counter-clockwise to the first injection, i.e. to maximize the likelihood of drug 
delivery to the lymphatics, the location of the second injection each day should be alternated 
between clockwise and counter-clockwise from the primary injection site. Each injection site each 
day should be gently massaged for one minute to promote entry into lymphatics.  
 
All IRX-2 injections will be administered by study personnel at the study site.   
4.2.4 
Other Medications 
Commercial cyclophosphamide will be provided by the pharmacy of the study site. The relatively 
low dose of cyclophosphamide has low to moderate emetic risk per NCCN Guidelines. Use of 
corticosteroids as an antiemetic is neither required nor permitted because of its potential 
immunosuppressive effect. An antiemetic regimen of lorazepam and a serotonin (5-HT3) 
antagonist is recommended. 
IRX Therapeutics will supply indomethacin, zinc (with multivitamins) and omeprazole. 

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4.3 
Method of Assigning Subject Numbers 
After a subject has signed the informed consent form and has been deemed eligible for 
enrollment, the investigator will assign a unique subject number to that subject. Subjects who 
withdraw from the study after being assigned a subject number will retain that number. 
4.4 
Treatment Compliance 
The investigator or study personnel will administer IRX-2 injections as described in Section 4.2.4. 
Subjects must maintain a log of all oral medications taken and this must be documented in the 
clinical record. 
4.5 
Prohibited, Prior and Concomitant Treatments 
Subjects should not take aspirin (except for low-dose aspirin as prescribed for vascular disease 
or cardiac risk-reduction) or other non-prescribed, non-steroidal anti-inflammatory agents.  
All other concomitant prescribed medications, recognized over-the-counter medications, or any 
changes in medications during the study from Day 1 to the day of surgery will be collected. 
Thereafter, only medications given to treat a reported study related AE or SAE will be collected. 
5 
STUDY PROCEDURES AND SCHEDULE 
5.1 
Study Schedule 
The Schedule of Study Procedures is outlined in Table 2. 
Tests and procedures should be performed on days indicated. Variation of ±3 days for tests 
indicated in “days”, 1 week for tests indicated in “weeks” is permitted. 
 
 

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Table 2: 
Schedule of Study Events 
Study Procedure 
Screeninga
Treatment Periodb 
Follow-Up 
Post  
treatmentb
Surgery/
Biopsyb, c, 
k 
Post-
Surgery/ 
Biopsy 
Study Day 
-21 to -1 
Day 1 
Day 1-21 
Any 10 
days 
between 
Day 4-19 
~Day 
18-27 
Within 12 
days of 
last IRX-2 
~30 days 
post 
Informed consent 
X
Inclusion/Exclusion  
X
Study Drug Administration 
Cyclophosphamide  
Xd
Indomethacin, zinc with 
multivitamins  
 
 
Xe 
 
 
 
 
Omeprazole 
X
IRX-2  
Xf
Evaluations 
Complete history & physical 
examination, including ECOG 
X 
 
 
 
 
 
 
Interval history & physical 
examination of primary site and 
regional nodes  
 
 
 
 
X 
 
X 
Adverse events, concomitant 
medicationsg,h, 
 
X 
X 
X 
X 
 
X 
CBC, chemistry paneli, j 
X
X
X
X
X
PT, PTTi 
X
Pregnancy test, if applicable 
X
Core biopsy (archival or new 
biopsy) 
X 
 
 
 
 
 
 
Path specimen for research 
X
Blood and/or serum samplesj 
Xj
Xj
Xj
Xj

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Footnotes for Schedule of Events Table: 
a. Subjects may be screened for eligibility up to 21 days prior to Day 1. 
b. As necessitated by scheduling logistics, the oral medications may be discontinued after Day 15 and surgery performed 2 days 
later. 
c. Surgery (or biopsy for cohort B) is ideally to be scheduled 2-5 days following the last IRX-2 injections, but may be within 12 days 
of the last IRX-2 injection.  
d. Corticosteroids should not be needed as a prophylaxis because cyclophosphamide dose is associated with minimal nausea. 
Suggested antiemetic regimen would include a serotonin (5-HT3) antagonist and lorazepam. 
e. Indomethacin must be discontinued at least 2 days before surgery. 
f. 
Two 1 mL subcutaneous periareolar IRX-2 injections may be given on any 10 days between Days 4 and 17 (days 4-8 and 11-15 
preferred).  
g. Adverse events will be measured from Day 1 until the day of surgery. Adverse events must be recorded as observed, but at least 
on Day 1 (before cyclophosphamide), Day 4 (before and after first IRX-2 injections), and twice on Days 9-17 (after approximately 
5 and 10 days of IRX-2 injections). 
h. All concomitant medications should be documented at baseline. Thereafter, only medications given to treat a reported AE or 
SAE, or considered immunosuppressive or anti-proliferative should be documented.  
i. 
Day 1 CBC and chemistry panel will not be performed if Screening / Baseline visit is less than 7 days prior. PT/PTT at screening 
only.  
j. 
Three 10 cc CPT tubes and one 5cc Cytochex tube for research studies (including immunogenicity testing) will be collected on Day 
1 (before cyclophosphamide), ~Day 4 (before first IRX-2 injections), twice on Days 9-19 (after approximately 5 and 10 days of IRX-
2 injections), prior to surgery and at the post-operative visit. In addition, 5 mL of blood will be collected at -, on approximately Day 
17 and on the post-operative visit for serum analysis of immunogenicity testing against the active components of IRX-2. CBC/diff 
and chemistry: CMP, LDH will be drawn at the same time points. 
k. Cohort B will undergo research core biopsy during this time, and will commence neoadjuvant chemotherapy within 7 days of biopsy 
 

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5.2 
Study Procedures  
5.2.1 
Screening  
The purpose and procedures of the study will be fully explained to participants. Those wishing to 
enroll in the study will sign a written informed consent prior to initiating any protocol specific 
evaluations or procedures (unless performed within study window prior to signing consent as part 
of standard-of-care visits or procedures).. 
The following screening evaluations are to be performed within 21 days (unless otherwise noted) 
prior to Day 1: 
 
Medical history, including assessment of all entry and exclusionary criteria, demographics 
(age, sex, race, menstrual status, weight, height), history of present illness, history of 
hypersensitivity (drug allergies), general medical history and habits. 
 
Determination of ECOG performance status (Appendix 1). 
 
Concomitant medication review. 
 
Physical exam, including description of the breast and regional lymph node areas. 
 
Clinical laboratory assessments: 
o Hematology:  complete blood count (CBC), differential, platelet count, PT and PTT. 
o Serum chemistry: serum albumin, total protein, serum bilirubin, lactate dehydrogenase 
(LDH), ALT/SGPT, AST/SGOT, alkaline phosphatase, creatinine, blood urea nitrogen 
(BUN), glucose, and electrolytes. 
o Calculated creatinine clearance (if creatinine >1.5 × ULN) (by Cockcroft-Gault 
Equation [Appendix 2]). 
 
Pregnancy test (serum or urine) if appropriate. 
 
Tumor biopsy for confirmation of diagnosis and research specimen (unless diagnostic 
biopsy confirmed sufficient by pathology). 
5.2.2 
Day 1  
The following procedures will be completed at this visit: 
 
Collection of research blood and serum samples 
 
Administer the following medications: 
o 
Infusion of cyclophosphamide, 300 mg/m2. Steroids must not be administered as 
antiemetic agents since these might interfere with immunomodulation by IRX-2. 
Antiemetic regimen should include a serotonin (5-HT3) antagonist and lorazepam or 
equivalent medications. 
o 
Begin indomethacin (25 mg, TID) to be taken orally after meals from Day 1 through 
Day 21. 
o 
Begin zinc (15-30 mg, once daily) with multivitamins to be taken orally from Day 1 
through Day 21. 
o 
Begin omeprazole (20 mg, once daily) to be taken orally after meals from Day 1 
through Day 21. 

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5.2.3 
Any 10 days between days 4 and 19  
 
IRX-2 should be administered by subcutaneous injection at two sites as described in 
Section 4.2.3. 
 
Each subcutaneous injection will be approximately 1.0 mL of IRX-2 (corresponding to 115 
units of IRX-2 per injection or 2300 units during the total regimen). 
 
On the first day of IRX-2 injections, subjects will be monitored for 15 minutes after the 
IRX-2 injections for signs or symptoms of any reaction. 
 
If the nurse or other qualified medical personnel receives information about an AE, she/he will 
document the event.  
In the event of Grade 3 or higher toxicity, no further IRX-2 should be injected and indomethacin 
should be discontinued. In the event of Grade 2 toxicity, treatment should be delayed until stable 
and then resumed per protocol. Treatment should be discontinued in a subject if the treatment is 
delayed for more than 7 days, except that treatment with IRX-2 and zinc with multivitamins may 
be continued per protocol in a subject intolerant of indomethacin. 
5.2.4 
One to Five Days Prior to Surgery (or post-IRX2 biopsy) 
The following procedures will be performed at this visit: 
 
Interval history and assessment of all current symptoms including severity (AEs). 
 
Record concomitant medications. 
 
ECOG performance status. 
 
Brief physical exam.  
 
Breast and regional node exam. 
 
Indomethacin must be stopped at least 2 days before surgery/biopsy. Subjects are 
scheduled to take indomethacin through Day 21. If surgery/biopsy is to be performed 
before Day 21, then the subject should discontinue indomethacin at least 2 days before 
surgery. 
 
Clinical laboratory assessments: 
o 
Hematology: CBC, differential, platelet count. 
o 
Serum chemistry: serum albumin, total protein, serum bilirubin, LDH, ALT/SGPT, 
AST/SGOT, alkaline phosphatase, creatinine, BUN, glucose, and electrolytes. 
 
Collect blood samples for correlative studies.  
5.2.5 
Surgery (or post-IRX biopsy) 
All early stage breast cancer subjects will undergo surgical resection of their tumor, 
reconstruction, and postoperative adjuvant therapy per usual standards of care. Surgery is ideally 
to be scheduled 2-5 days following the last IRX-2 injections, but may be within 12 days of the last 
IRX-2 injections. On the rare occasion, subjects/investigators may opt for post-IRX-2 biopsy 
during this window instead of surgical resection, i.e. if original surgical date must be revised to 
accommodate unforeseen changes in surgical plan unrelated to the IRX-2 treatments. Subjects 
in Cohort B will undergo core biopsy instead of surgery, but will undergo standard-of-care surgical 
resection following completion of neoadjuvant chemotherapy, or may advance to surgery at any 
time at the discretion of the investigator. 

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 
For all subjects, surgical and pathological data must be reported as described in this 
Protocol and the CRFs. 
5.2.6 
Follow-up Schedule  
Subjects will be assessed clinically at a final study visit approximately 30 days following surgical 
resection. Efforts will be made for this visit to coincide with a standard-of-care clinical 
assessment.  
 
Subjects will be followed longitudinally, in an ongoing basis, to evaluate for post-IRX recurrence 
free survival. Because recurrences may occur many years later, no pre-defined termination date 
for follow-up is designated. 
 
5.2.7 
Cohort B 
Subjects in Cohort B (i.e. those with triple negative breast cancer receiving post-IRX-2 
neoadjuvant chemotherapy) will receive standard of care neoadjuvant anthracycline-based 
chemotherapy. Subjects for whom platinum-based neoadjuvant therapy is planned are not eligible 
to enroll. Pathologic response rate in the surgical resection specimen will be assessed following 
chemotherapy. Remaining tissue from the resection specimen, in excess of tissue needed for 
diagnostic purposes, may be used to conduct additional immunologic exploratory assessments. 
 
Subjects in cohort B will follow the same toxicity and immunologic assessments and long-term 
follow-up as cohort A, including a 30-day post-biopsy safety visit. Following the 30-day window, 
toxicity assessments pertaining to chemotherapy will not be recorded formally in the trial, but will 
be conducted per standard-of-care at the discretion of the treating physician. 
 
 
6 
ADVERSE EVENTS AND REPORTING REQUIREMENTS 
6.1 
Adverse Event Definitions 
Definitions of AEs will follow the International Conference on Harmonization E6: Good Clinical 
Practice Step 5, Consolidated Guideline 1.5.96, April 1998 edition, and are summarized below. 
For this study, the investigational product is the IRX-2 regimen, including IRX-2, 
cyclophosphamide, indomethacin, and zinc-containing multivitamins. 
An adverse drug experience (event) is any new undesirable medical occurrence or change 
(worsening) of an existing condition in a subject that occurs from Day 1 until the time of surgery 
(with the exception of an SAE, if it occurs within 30 days from the last dose of study medication), 
whether or not considered to be drug related. Therefore, AEs are treatment-emergent signs and 
symptoms. This includes those events occurring from drug overdose, whether accidental or 
intentional, from drug abuse or from drug withdrawal. In general, abnormal laboratory findings 
without clinical significance based on the investigator’s judgment are part of the database and 
should not be recorded as AEs. 
Adverse events reported by the subject or observed by the investigator will be individually listed 
on an AE CRF page. The signs and symptoms, date of onset, duration, action taken, severity, 
outcome to date, and relationship to the study drug will be recorded. The severity of AEs will be 
graded according to the National Cancer Institute Common Terminology Criteria for Adverse 
Events (NCI-CTCAE), Version 4.03 toxicity criteria. Severity will be classified as described in 
Section 6.1.3. 

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For adverse events of Grade 3 or 4, the Principal Investigator will determine whether to modify or 
discontinue study treatment. 
Adverse events will be measured from Day 1 until surgery and then 30 days post-surgery. 
Evidence of toxicity after surgery will be assessed by tabulation of AEs felt by the investigator to 
be definitely or possibly related to the study regimen.  
Serious adverse events will be measured from Day 1 until surgery and then at 30 days post-
surgery. Evidence of toxicity after surgery will be assessed by tabulation of SAEs felt by the 
investigator to be definitely or possibly related to the study regimen. Ongoing AEs at the time of 
surgery will be followed for 30 days post-surgery. Ongoing SAEs beginning from surgery will be 
reported and monitored until resolution or stability. 
At each visit, the investigator will be prompted to report AEs as “not”, “possibly,” or “definitely” 
related to the IRX-2 regimen. 
6.1.1 
Definition of Serious and Life Threatening Adverse Events 
A serious adverse drug experience (event) is any AE occurring at any dose that results in any of 
the following outcomes: 
 
Death 
 
Is life-threatening  
o A life-threatening adverse drug experience is any AE that places the subject, in the 
view of the investigator, at immediate risk of death from the reaction as it occurred. 
This does not include a reaction that, had it occurred in a more severe form, might 
have caused death. 
 
A persistent or significant disability/incapacity 
 
Inpatient hospitalization or prolongation of existing hospitalization, except that planned 
hospitalization during days in clinic and surgery or hospitalization for the social reasons or 
the convenience of the subject or physician shall not be considered an SAE  
 
A congenital anomaly/birth defect 
 
Important medical event 
o Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be serious when, based upon appropriate medical judgment, the 
event may jeopardize the subject and require medical or surgical intervention to 
prevent one of the outcomes listed above. Severe complications of standard-of-care 
surgical resection (lumpectomy or mastectomy), are to be considered “important 
medical events” and thus SAEs. 
6.1.2 
Definition of Relationship to Study Regimen and Drug 
Association or relatedness to the study regimen will be graded as either “not”, “possibly,” or 
“definitely” related to the study regimen. Determination of relatedness includes: 
 
Definitely, characterized as an AE that 
o Follows direct temporal sequence from regimen administration. 
o Abates upon discontinuation of the regimen. 

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o Cannot be explained by the known characteristics of the subject’s clinical state or by 
other modes of therapy administered to the subject.  
 
Possibly, characterized as an AE that 
o Follows a reasonable temporal sequence from regimen administration. 
o Abates upon discontinuation of the regimen. 
o Could have been produced by the subject’s clinical state or by other modes of therapy 
administered to the subject. 
 
Not Related, characterized as an AE that 
o Does not follow any temporal sequence from regimen administration.  
o Is explained by the subject’s clinical state or by other modes of therapy administered 
to the subject. 
6.1.3 
Definition of Severity 
The severity of adverse changes in physical signs or symptoms will be graded according to the 
NCI-CTCAE v4.03. For AEs not listed in the table, the severity of adverse changes in physical 
signs or symptoms will be classified as follows: 
 
Grade 1: 
Mild (transient or mild discomfort; no limitation in activity; no medical
intervention/therapy required) 
Grade 2: 
Moderate (mild to moderate limitation in activity, some assistance may be needed; 
no or minimal medical intervention/therapy required) 
Grade 3: 
Severe (marked limitation in activity, some assistance usually required; 
medical intervention/therapy required, hospitalizations possible) 
Grade 4: 
Life-threatening 
(extreme 
limitation 
in 
activity, 
significant 
assistance 
required; significant medical intervention/therapy required, hospitalization or 
hospice care probable) 
Grade 5: 
Death 
6.1.4 
Definition of Action Taken 
None: 
No action taken with regard to the study drug 
Interrupted: 
The study drug was stopped but restarted after the subject’s symptom abated. 
The subject was rechallenged with the study drug 
Discontinued: The study drug was permanently stopped 
6.1.5 
Definition of Outcome to Date 
Resolved with sequelae: 
The subject has recovered from the AE with observable  
residual effects 

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Resolved without sequelae: 
The subject has fully recovered from the AE with no  observable 
residual effects 
Unresolved: 
The AE is present and observable 
Death: 
The subject died as a result of the AE 
Lost to Follow-up: 
Source documentation confirms that repeated attempts to  
contact subject have failed 
6.2 
Notification of Serious or Unexpected Adverse Events 
The Principal Investigator is responsible for determining whether or not the suspected adverse 
reaction meets the criteria for expedited reporting in accordance with Federal Regulations 
(21 CFR §312.32). A suspected adverse reaction must be both serious and unexpected in order 
to meet the reporting requirements.  
An adverse event or suspected adverse reaction is considered “unexpected” if it is not listed in 
the investigator brochure or is not listed at the specificity or severity that has been observed. 
An adverse event or suspected adverse reaction is considered “serious” if, in the view of either 
the investigator or IRX Therapeutics, it results in any of the following outcomes: Death, a life-
threatening adverse event, inpatient hospitalization or prolongation of existing hospitalization, a 
persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions, or a congenital anomaly/birth defect. Important medical events that may not result in 
death, be life-threatening, or require hospitalization may be considered serious when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject and may require 
medical or surgical intervention to prevent one of the outcomes listed in this definition. 
The timeline for submitting an IND safety report to FDA is no later than 15 calendar days after 
the Investigator determines that the suspected adverse reaction qualifies for reporting (21 CFR 
312.32(c)(1)).  
Any unexpected fatal or life-threatening suspected adverse reaction will be reported to FDA no 
later than 7 calendar days after the Investigator’s initial receipt of the information (21 CFR 
312.32(c)(2)).  
Any relevant additional information that pertains to a previously submitted IND safety report will 
be submitted to FDA as a Follow-up IND Safety Report without delay, as soon as the information 
is available (21 CFR 312.32(d)(2)).  
Providence Portland Medical Center will also notify IRX of all SAEs in parallel with notification to 
the FDA. 
 
7 
STATISTICAL CONSIDERATIONS AND ASSESSMENT OF ENDPOINTS 
7.1 
Sample Size Considerations 
This clinical trial is a proof of concept and initial tolerability/safety study but not a “first-in-human” 
study. The number of subjects is generally consistent with similar studies of other investigation 
products at this stage of development. A sample size of 20 was selected because it provides 
adequate power to estimate the proportion of subjects who tolerate the regimen, and to 
preliminarily characterize the immunologic effects of therapy. 

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Cohort B expansion is designed similarly to evaluate feasibility and immunologic effects of pre-
neoadjuvant IRX-2 in a higher-risk triple negative breast cancer population.  Cohort B sample size 
of n=19 is also based upon the first stage of a Simon 2-stage design, to test the hypothesis that 
IRX-2 pre-treatment improves pathologic complete response rate to 50% compared to historical 
benchmark of 30% (based upon a meta-analysis of non-platinum containing chemotherapies). 
Subjects who are enrolled but withdraw consent prior to beginning the IRX-2 Regimen will not be 
evaluable and will be replaced. 
7.2 
Primary Endpoint: Safety and Tolerability of the IRX-2 Regimen (cohort A) 
 
Definitions and reporting requirements for AEs and SAEs have been presented above in Section 
6. 
Because this therapy is being administered in subjects receiving therapy intended to cure their 
disease, the threshold for establishing safety/tolerability will be high, such that nearly all subjects 
must tolerate the therapy. The therapy will be deemed “safe and tolerable” if 75% or greater of 
evaluable subjects tolerate the therapy according pre-specified definition of tolerability.  
This is based upon hypothesis testing whereby the null hypothesis is that the regimen is not 
tolerable (i.e. falls below the boundary of tolerability), and the alternative hypothesis is that the 
regimen is tolerated. The boundary of tolerability will depend on the total number of evaluable 
subjects, as depicted in the below table. 
For example, using the 75% threshold of tolerability and assuming that 20 subjects are evaluable, 
if 15 or more out of 20 patients tolerate the therapy according pre-specified definition of the 
tolerability (i.e., if the tolerability rate is 75% or greater), then the null hypothesis H0: p <= 0.58 
(i.e., the tolerability boundary) would be rejected in favor of alternative hypothesis H1: p = 0.8 with 
a type I error rate and power equal to 0.09 and 0.80, respectively. 

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# 
evaluable 
subjects 
(n) 
75% 
threshold 
(x*) 
x* / n 
Tolerability 
boundary 
under H0 
Specified 
Tolerability 
under H1
Type 1  
error 
Power 
15 
12 
0.80 
0.60 
0.85 
0.09 
0.82 
16 
12 
0.75 
0.56 
0.81 
0.10 
0.83 
17 
13 
0.76 
0.58 
0.82 
0.09 
0.82 
18 
14 
0.78 
0.60 
0.83 
0.09 
0.82 
19 
15 
0.79 
0.62 
0.84 
0.10 
0.82 
20 
15 
0.75 
0.58 
0.80 
0.09 
0.80 
 
Subjects will be considered evaluable if they consent and receive any part of the 
cyclophosphamide infusion. The rate of Grade 1-4 toxicities will be reported as additional, 
descriptive measures of tolerability. The following criteria must be met for that subject to be 
considered to have “tolerated” the therapy:  
 
Subject receives cyclophosphamide, >7 injections of IRX-2 and >10 days of indomethacin 
and multivitamins with zinc and does not have Grade 3 or 4 treatment-associated adverse 
events requiring discontinuation of therapy; 
 
Subject does not experience a delay in standard-of-care surgical resection related to 
treatment-associated adverse events; 
 
Subject does not have any ongoing treatment-related Grade 2-4 adverse events at the 30-
day post-operative follow-up visit; 
 
Subject does not withdraw consent after the start of the cyclophosphamide infusion. 
7.2.1 Laboratory values, vital signs, physical findings and other safety data 
Any laboratory results, vital signs, physical findings, or outcomes of any other investigations that 
are conducted during the course of the study that in the opinion of the investigator should be 
considered an adverse event must be documented in the appropriate section of the CRF. 
7.2.2 Pregnancy 
All pregnancies that occur during the study or for which conception is likely to have occurred within 
1 year of the last administration of IRX-2 must be reported as an adverse event, including 
pregnancies that occur in the partner of a subject.  
All pregnancies are to be monitored for the duration and outcomes reported. Congenital anomaly 
or birth defects should be reported as an SAE. 
7.3 
Secondary Endpoint:  Stromal TIL infiltration 
The secondary endpoint (cohort A) is to evaluate the effect of therapy on stromal TIL (sTIL) 
infiltration by Salgado criteria. The rationale for this endpoint is that the percentage of TILs in pre-
operative biopsies is highly correlated with both overall survival and response to neoadjuvant 
therapy. If the IRX-2 regimen is found to increase the percentage of TILs, this would provide 
rationale to evaluate further IRX-2 in this setting. 

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Lymphocyte infiltrates in the pre-treatment tumor biopsies and the surgical specimens will be 
determined and compared. We assume that a mean sTIL count is 10% and standard deviation is 
17% (adopted from a recent meta-analysis, Loi et al 2015). Since the data will be observed at pre 
and post times on each tumor specimen, we also assume that those pairs of pre and post 
measurements are equally correlated with a correlation coefficient of at least 0.1. A sample size 
of n=20 was determined to be sufficient to attain a power of 0.80, associated with a one-sided 
hypothesis test given type I error rate 0.1, to detect a mean increase in TIL score of 11% following 
receipt of IRX-2.  
7.4 
Exploratory Endpoints 
Exploratory endpoints will be analyzed descriptively, and will include additional peripheral blood 
and intratumoral analyses of the immunologic effects of the IRX-2 regimen, including, for example, 
some or all of the following:   
 
flow cytometric quantification and characterization of peripheral lymphocytes (including 
activated T-cells, T-regulatory cells, NK cells, and myeloid cells) 
 
TIL phenotype characterization by multispectral immunofluorescence imaging to assess 
changes in abundance of T-regulatory cells, activated T-cells, myeloid lineages and 
dendritic cells. Subsets of lymphocytes will be evaluated including total T cells and naïve, 
cytotoxic and regulatory T cells. The analyses may include quantifications of cellular 
infiltrates and degree of activation by immunohistochemistry including (but not limited to): 
CD3, CD8, CD68 (myeloid derived suppressor cells), CD45RO and Fox P3 on 
lymphocytes and MHC class I and II and PDL1 on tumor cells. Other immune subsets and 
activation markers may be evaluated. 
 
Intratumoral and T-cell clonal responses by T-cell receptor DNA deep sequencing 
 
Characterization of intratumoral immune responses by RNA expression using the 
Nanostring PanCancer Immune panel and/or Prosigna tumor recurrence score 
Exploratory endpoints will be determined in all subjects enrolled but some of these endpoints may 
be modified during the course of the study, so long as no additional blood or tissue specimens 
are required of study subjects. 
7.5 
Cohort B Statistical Considerations  
Cohort B will also test the feasibility and immune effects of IRX-2, however in a pre-neoadjuvant 
chemotherapy setting. Secondarily, the cohort is designed to be expanded (per a subsequent 
protocol revision) if a threshold pCR rate is attained, allowing for a formal comparison of pCR 
against a historical benchmark. We have selected a benchmark pCR rate of 30%, based upon a 
recent meta-analysis of five randomized trials that evaluated non-platinum containing 
neoadjuvant chemotherapy. (Petrelli F, et al).  In a minimax design, we will test the null hypothesis 
of 30% pCR rate, versus an alternative hypothesis of 50% pCR rate, with alpha of 0.05 and beta 
of 0.2. With this design, response rate of ≥7/19 will prompt consideration of an expansion to total 
39 patients. Responses in ≥17/39 would prompt the team to reject the null hypothesis, and 
consider the regimen meritorious for further study. With this design, the probability of early 
stopping is 0.67. Pathologic complete response will be assessed by an intention-to-treat principle, 
whereby all subjects receiving at least one partial dose of chemotherapy will be considered 
evaluable. Subjects lost to follow-up or not evaluable will be considered non-pCR. Pathologic 
complete response will be assessed by local pathologist, defined by the AJCC staging system 
(ypT0/Tis ypN0). Subjects who do not receive at least one partial dose of chemotherapy will be 
replaced. 

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8 
ETHICAL AND REGULATORY CONSIDERATIONS 
8.1 
Protection of Human Subjects 
This study will be conducted in accordance with the US CFR, Title 21, Parts 11, 50, 54, 56 and 
312; the Declaration of Helsinki (1964) including all amendments and revisions; the Good Clinical 
Practices: Consolidated Guideline (E6), International Conference on Harmonization, the Food 
and Drug Administration Guidance for Industry: Computerized Systems Used in Clinical Trials, 
the ethical principles that have their origins in the Declaration of Helsinki and all applicable 
country-specific and local regulations. All required study information will be archived as required 
by regulatory authorities. 
8.2 
Informed Consent 
Informed consent must be obtained and documented prior to the initiation of non-standard of care 
screening tests, study registration and study therapy. 
The investigator (or designee) will explain the study procedures, treatments, costs, potential 
benefits/risks involved, and the alternatives for care or treatment to the patient/legally authorized 
representative (hereafter referred to as “patient”). Determination of the appropriate person to give 
legal consent will be done in accordance with ICH/GCP, Providence Health & Services Policy and 
state law. All study procedures will be explained in terms the patient can understand and an 
opportunity will be given for the patient to ask questions. Special care will be given to explaining 
the use and disclosure of a patient’s protected health information and if applicable, a patient’s 
rights if the study involves genetic research. This must be done before the informed consent is 
signed. This discussion will be documented in the patient’s chart.  
A copy of the signed consent form will be provided to the patient and the original consent form 
will be kept in the patient chart at the Clinical Trials Office. 
 
9 
STUDY ADMINISTRATION 
9.1 
Monitoring Plan 
9.1.1 
Site Initiation 
The Principal Investigator and Research Nurse will conduct an implementation meeting for sub-
investigators and applicable research and clinic staff. Attendance at this meeting will be 
documented. Anyone unable to attend must complete an online study training in HealthStream 
prior to conducting any study-related activities. 
9.1.2 
Interim Monitoring 
Interim monitoring will be scheduled at regular intervals throughout the study, with the timing being 
dependent on subject enrollment. Generally, the study monitor will compare the data entered on 
the CRFs with the hospital or clinic records (primary source documents) and check for protocol 
compliance, including verification of informed consent, all subject visit dates, all AEs, all 
concomitant medications, and all key efficacy observations. In addition, the Site Regulatory Binder 
containing essential documents, study drug accountability, and supporting records will be 
reviewed. Findings from this review will be summarized in writing and presented to the 
investigator, and the dates of the monitoring will be recorded by the study monitor in a sign-in log 
to be kept in the Site Regulatory Binder.  

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9.1.3 
Study Close-out 
At the study’s conclusion close-out monitoring will be performed, including a final review and 
reconciliation of all study and regulatory documents. 
9.1.4 
Site Responsibility 
The procedures defined in the protocol and in the CRFs will be carefully reviewed by the 
investigator and his/her staff, prior to the time of study initiation, to ensure appropriate 
interpretation and implementation. No deviations from the protocol should be made. Minor 
exceptions may be approved on a case-by-case basis and must be authorized by the Principal 
Investigator and documented in writing. Significant deviations that may impact subject safety or 
study integrity will be reported to the IRB and may result in termination of study participation. Any 
changes to the protocol will originate from the Principal Investigator in the form of an amendment. 
9.2 
Investigational Product Accountability 
Drug accountability records will be maintained by the Providence Health & Services Oregon 
Region Investigational Drug Services pharmacy. Drug accountability records will be initiated with 
pharmacy receipt of drug. An Investigational Drug Accountability log will be used to account for 
all investigational drugs. At the time of dispensing, the IDS pharmacist or technician will log 
appropriate information regarding the agent being dispensed, including but not limited to: name 
of drug, strength, quantity dispensed, kit/vial/lot number, subject ID number, initials and date. At 
the time of dispensing, the DARF will be reconciled with the physical inventory. 
 
Used vials of investigational drug will be destroyed as per PH&S – Portland Service Area – EOC 
Policy on Waste Management, EC 315: 
http://in.providence.org/Documentscentral/OR/safety/Waste%20Management%20Disposal.pdf 
 
Unused investigational drug will be returned or destroyed at study close or when drug expires as 
per the referenced policy above. 
 
All investigational drug accountability records, physical inventory and other supporting documents 
(such as temperature monitoring logs) will be made available to the monitor and applicable 
regulatory agencies for auditing and monitoring purposes.  
9.3 
Study Documents and Access to Records 
It is the responsibility of the investigator and study staff to maintain a comprehensive and 
centralized filing system of all study-related documentation, which is suitable for inspection at any 
time by the study monitor, the Sponsor or its designee, the FDA and other Regulatory Agencies. 
Elements should include, but are not limited to: 
 
Study Files, containing the curriculum vitae of all investigators and his/her designees, FDA 
Form 1572, the protocol with all amendments, the IB, local lab certifications and lab normal 
ranges, and all correspondence to and from local Regulatory Authorities. 
 
Subject Files, containing the completed original supporting source documentation and the 
signed informed consent form(s). 
 
Dispensing records of test agents. 
 
In accordance with FDA regulations, the investigator shall retain records for a period of 2 
years following the date a marketing application is approved for the drug for the indication 
for which it is being investigated; or, if no application is to be filed or if the application is 

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not approved for such indication, until 2 years after the investigation is discontinued and 
FDA is notified. 
9.4 
Quality Assurance 
The Principal Investigator and all designees will ensure the integrity of all data collected and 
calculations made during the conduct of the study according to their quality assurance standards 
of operations. Edit checks will be run on the data and queries issued. All data will undergo 100% 
review. 
9.5 
Confidentiality and Publication Policy 
This trial will be listed in clinical trial databases as appropriate, e.g., www.clinicaltrials.gov. 

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 APPENDICES 
Appendix 1: ECOG Performance Status 
Score 
Performance 
100 
Normal:  no complaints, no evidence of disease. 
90 
Able to carry on normal activity; minor signs or symptoms of disease. 
80 
Normal activity with effort; some signs or symptoms of disease. 
70 
Cares for self; unable to carry on normal activity or do active work. 
60 
Requires occasional assistance from others; but able to care for most 
needs. 
50 
Requires considerable assistance from others, frequent medical care. 
40 
Disabled:  requires special care and assistance. 
30 
Severely disabled:  hospitalization indicated, death not imminent. 
20 
Very sick, hospitalization necessary, active supportive treatment necessary. 
10 
Moribund:  fatal processes are progressing rapidly. 
0 
Dead 
Source: Karnofsky D, Abelman W, Craver L, Burchenal J. The use of nitrogen mustards in the 
palliative treatment of carcinoma. Cancer 1948;1:634-56. 
 
 
 

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Appendix 2: Calculation of Creatinine Clearance by Cockcroft-Gault Equation 
 
Formula:   Creatinine Clearance  = Sex (Male = 1.0, Female = 0.85) * ((140 - Age) /  
 
 
(Serum Creatinine)) * (Weight / 72) 
 
Online calculator may be found at  
 
http://reference.medscape.com/calculator/creatinine-clearance-cockcroft-gault