Clinical trial NCT03199040
Conditions: Triple Negative Breast Cancer Triple Negative Breast Neoplasms TNBC - Triple-Negative Breast Cancer Triple-negative Breast Carcinoma
Sponsor: Washington University School of Medicine
Start date: 2019-07-23; Completion date: 2023-05-01
Enrollment: 18 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This is a single institution, open-label randomized phase 1 trial of neoantigen DNA vaccine alone vs. neoantigen DNA vaccine plus durvalumab in triple negative breast cancer (TNBC) patients following standard of care therapy. Patients with newly diagnosed clinical stage II-III TNBC are eligible for enrollment. Patients will receive standard of care therapy including chemotherapy, surgery and radiation therapy as clinically indicated. Following standard of care therapy, patients will be randomized to receive either a neoantigen DNA vaccine alone, or a neoantigen DNA vaccine + durvalumab.

Detailed description:

Arms: arm 1: * The first neoantigen DNA vaccine injection will take place following the completion of standard of care therapy. The day of the first vaccine injection will be referred to as Day 1
* The schedule of vaccination is Day 1, Day 29 ± 7, Day 57 ± 7, Day 85 ± 7, Day 113 ± 7, and Day 141 ± 7 with at least 21 days between injection days
* For patients who are randomized to the neoantigen DNA vaccine plus durvalumab arm, the neoantigen-specific T cell response will be assessed prior to Day 85. If a neoantigen-specific T cell response is present, durvalumab will be started on Day 85, and will be administered Q4W at a dose of 1500 mg over the course of 60 minutes. If a neoantigen-specific T cell response is not present, these patients will be replaced but may continue to receive the neoantigen DNA vaccine on study. They will not be transferred to the vaccine-only arm. arm 2: * The first neoantigen DNA vaccine injection will take place following the completion of standard of care therapy. The day of the first vaccine injection will be referred to as Day 1
* The schedule of vaccination is Day 1, Day 29 ± 7, Day 57 ± 7, Day 85 ± 7, Day 113 ± 7, and Day 141 ± 7 with at least 21 days between injection days
Interventions: intervention 1: Durvalumab intervention 2: Neoantigen DNA vaccine intervention 3: TDS-IM system (Inchor Medical Systems) intervention 4: Peripheral blood draw
Intervention descriptions: intervention 1: -Human monoclonal antibody intervention 2: -The vaccine will be given by the TDS-IM system intervention 3: -At each vaccination time point, patients will receive two injections at separate sites. intervention 4: -Baseline, following completion of standard of care therapy, Day 1, Day 57, Day 113, Day 159, and 1 year after initiation of neoantigen DNA vaccine therapy

Protocol documents:
Prot_SAP_000.pdf: 
Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 1 of 112 
A RANDOMIZED PHASE 1 TRIAL OF NEOANTIGEN DNA VACCINE ALONE VS. 
NEOANTIGEN VACCINE PLUS DURVALUMAB IN TRIPLE NEGATIVE BREAST 
CANCER PATIENTS FOLLOWING STANDARD OF CARE THERAPY 
HRPO #: 201710109 
Version date: 04/24/2023 
IND #: 17625 
clinicaltrials.gov #: NCT03199040 
 
INVESTIGATORS 
 
Principal Investigator: 
William E. Gillanders, M.D. 
Washington University School of Medicine 
660 South Euclid, Box 8109 
St. Louis, MO 63110 
Telephone: 314-747-0072 
E-mail: gillandersw@wustl.edu 
 
Co-Investigators: 
Robert D. Schreiber, Ph.D. 
Immunobiologist 
Washington University School of Medicine 
660 South Euclid, Box 8118 
St. Louis, MO 63110 
Telephone: 314-362-8747 
E-mail: rdschreiber@wustl.edu  
 
Foluso O. Ademuyiwa, M.D., MPH 
Medical Oncologist 
Washington University School of Medicine 
660 South Euclid, Box 8056 
St. Louis, MO 63110 
Telephone: 314-362-7201 
E-mail: bisiademuyiwa@wustl.edu  
 
Timothy Eberlein, M.D. 
Breast Surgeon 
Washington University School of Medicine 
660 South Euclid, Box 8109 
St. Louis, MO 63110 
Telephone: 314-362-8020 
E-mail: eberleint@wustl.edu 
 
Feng Gao, M.D., Ph.D. 
Biostatistician 
Washington University School of Medicine 
660 South Euclid, Box 8067 
St. Louis, MO 63110 
Telephone: 314-362-3682 
E-mail: feng@wustl.edu 
 
S. Peter Goedegebuure, Ph.D. 
Immunologist 
Washington University School of Medicine 
660 South Euclid, Box 8109 
St. Louis, MO 63110 
Telephone: 314-362-8612 
E-mail: goedegep@wustl.edu 
 
 
Rebecca L. Aft, M.D., Ph.D. 
Breast Surgeon 
Washington University School of Medicine 
660 South Euclid, Box 8109 
St. Louis, MO 63110 
Telephone: 
E-mail: aftr@wustl.edu 
 
Amy Cyr, M.D. 
Breast Surgeon 
 Washington University School of Medicine 
660 South Euclid, Box 8109 
St. Louis, MO 63110 
Telephone: 636-916-7160 
E-mail: amycyr@wustl.edu 
 
Julie A. Margenthaler, M.D. 
Breast Surgeon 
Washington University School of Medicine 
660 South Euclid, Box 8109 
St. Louis, MO 63110 
Telephone: 314-362-7534 
E-mail: jmargenthaler@wustl.edu 
 
Christopher A. Miller, Ph.D. 
Genome Scientist 
Washington University School of Medicine 
660 South Euclid, Box 8007 
St. Louis, MO 63110 
Telephone: 314-286-0263 
E-mail: c.a.miller@wustl.edu  
 
Malachi Griffith, Ph.D. 
Genome Scientist 
Washington University School of Medicine 
660 South Euclid, Box 8501 
St. Louis, MO 63110 
Telephone: 314-286-1274 
E-mail: mgriffit@wustl.edu   


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 2 of 112 
Protocol Revision History 
Initial Approval:  
09/28/17 
Amendment #1:  
03/01/18 
Amendment #2:  
08/31/18 
Amendment #3:  
06/04/19 
Amendment #4:  
08/13/19 
Amendment #5:  
02/12/20 
Amendment #6  
05/04/20 
Amendment #7  
06/11/20 
Amendment #8  
12/09/21 
Amendment #9  
04/24/23 
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 3 of 112 
SUMMARY 
Protocol: 
A RANDOMIZED PHASE 1 TRIAL OF NEOANTIGEN DNA VACCINE ALONE 
VS. NEOANTIGEN DNA VACCINE PLUS DURVALUMAB IN TRIPLE 
NEGATIVE BREAST CANCER PATIENTS FOLLOWING STANDARD OF 
CARE THERAPY 
Study Design: 
This is a single institution, open-label randomized phase 1 trial of neoantigen 
DNA vaccine alone vs. neoantigen DNA vaccine plus durvalumab in triple 
negative breast cancer (TNBC) patients who have residual disease following 
standard of care therapy. Patients with newly diagnosed clinical stage II-III TNBC 
are eligible for enrollment. Patients will receive standard of care therapy including 
chemotherapy, surgery and radiation therapy as clinically indicated. Following 
standard of care therapy, patients will be randomized to receive either a 
neoantigen DNA vaccine alone, or a neoantigen DNA vaccine + durvalumab.  
Hypotheses: 
Hypotheses tested in this study include: (1) neoantigen DNA vaccines can induce 
and/or enhance neoantigen-specific T cell responses; and (2) durvalumab will 
enhance the response to neoantigen DNA vaccines. 
Objectives: 
The primary objective is to assess the safety of neoantigen DNA vaccines given 
alone or in combination with durvalumab. Safety assessment will include both 
clinical observation and laboratory evaluation. The secondary objective is to 
measure the immune response to neoantigen DNA vaccines given alone, or in 
combination with durvalumab. The immune response will be measured in the 
peripheral blood by luminex assay, ELISPOT, and multiparametric flow 
cytometry. 
Product Description: The neoantigen DNA vaccines are composed of closed circular DNA plasmids 
that are designed to express neoantigens identified by next generation 
sequencing. The plasmids will be formulated as naked DNA plasmid vaccines, 
and supplied at a concentration of 2 mg/mL. DNA vaccines will be administered 
intramuscularly (in the deltoid or lateralis) using a TriGrid electroporation device 
(Ichor Medical Systems). Two injections (2 mg/injection, total dose 4 mg) will be 
administered at each time point. 
 
Durvalumab is a human monoclonal antibody (MAb) of the immunoglobulin G1 
kappa (IgG1κ) subclass that inhibits binding of programmed cell death ligand 1 
(PD-L1) to programmed cell death 1 (PD-1) and CD80 (B7-1). 
Subjects: 
Patients with clinical stage II-III TNBC are eligible for participation. Patients must 
provide consent for tumor/normal exome sequencing and dbGAP-based data 
sharing and provide germline and tumor DNA samples of adequate quality for 
exome and cDNA-capture sequencing. 
Study Plan: 
Twenty-four breast cancer patients will be enrolled. Tumor tissue and peripheral 
blood will be obtained prior to initiation of any treatment for sequencing and 
correlative studies. These analyses will inform the design of the neoantigen DNA 
vaccines.  
 
The neoantigen DNA vaccines will be manufactured at Siteman Cancer Center 
and then administered following standard of care therapy. It is anticipated that 
standard of care therapy in this clinical context will include surgery, adjuvant or 
neoadjuvant chemotherapy (AC + T or similar regimen), and adjuvant radiation 
therapy, taking a total of approximately 6 months to complete. 
 
Following standard of care adjuvant therapy, patients will be randomized to 
receive either a neoantigen DNA vaccine alone, or a neoantigen DNA vaccine + 
durvalumab. The vaccine will be administered by intramuscular injection on Day 
1, Day 29 ± 7, Day 57 ± 7, Day 85 ± 7, Day 113 ± 7, and Day 141 ± 7 with at 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 4 of 112 
least 21 days between injection days. Each DNA vaccination will be 4 mg 
vaccine administered intramuscularly using a TriGrid electroporation device.  
 
For patients who are randomized to neoantigen DNA vaccine + durvalumab, the 
neoantigen-specific T cell response will be assessed after two vaccinations (Day 
57). If a neoantigen-specific T cell response is present, durvalumab will be 
administered every 28 days at a dose of 1500 mg beginning on Day 85. 
Study Duration: 
Each subject will be followed for at least 12 months following the date of last 
treatment. Additional follow-up visits or telephone contact will be scheduled 
annually thereafter if the patient is alive and available for follow-up. 
Study Endpoints: 
The phenotype and function of class I and II neoantigen-specific T cell response 
will be assessed by ELISPOT, luminex, multiparametric flow cytometry, and 
CyTOF. Safety endpoints include safety of the neoantigen DNA vaccine alone, or 
neoantigen DNA vaccine plus durvalumab. Toxicity will be graded according to 
the National Cancer Institute Common Terminology Criteria for Adverse Events 
version 4.03. 
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 5 of 112 
SCHEMA 
  
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 6 of 112 
PROTOCOL SIGNATURE PAGE 
 
A RANDOMIZED PHASE 1 TRIAL OF NEOANTIGEN DNA VACCINE ALONE VS. NEOANTIGEN 
VACCINE PLUS DURVALUMAB IN TRIPLE NEGATIVE BREAST CANCER PATIENTS FOLLOWING 
STANDARD OF CARE THERAPY 
 
I have read the attached clinical protocol and agree to conduct this trial in accordance with all the 
stipulations of the protocol and in accordance with the Declaration of Helsinki/Tokyo/Venice on 
Experimentation in Humans as required by the United States Food and Drug Administration regulations, 
Code of Federal Regulations Title 21 parts 50, 56, 312, 800, Title 45 part 46 and all applicable guidelines. 
 
Name of Investigator: 
 
 
Signature 
Date 
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 7 of 112 
TABLE OF CONTENTS 
SUMMARY _________________________________________________________________ 3 
SCHEMA ___________________________________________________________________ 5 
PROTOCOL SIGNATURE PAGE ______________________________________________ 6 
1 
OBJECTIVES __________________________________________________________ 10 
1.1 
PRIMARY OBJECTIVE _______________________________________________________________ 10 
1.2 
SECONDARY OBJECTIVE _____________________________________________________________ 10 
2 
BACKGROUND ________________________________________________________ 11 
2.1 
CHECKPOINT BLOCKADE THERAPY ____________________________________________________ 11 
2.2 
NEOANTIGENS ____________________________________________________________________ 11 
2.3 
NEXT GENERATION SEQUENCING AND CANCER VACCINES ___________________________________ 12 
2.4 
SEQUENCING STRATEGIES TO IDENTIFY NEOANTIGENS _____________________________________ 13 
2.5 
PRIORITIZATION OF SEQUENCING-IDENTIFIED NEOANTIGENS _________________________________ 13 
2.6 
NEOANTIGEN DNA VACCINES ________________________________________________________ 13 
2.7 
ELECTROPORATION ________________________________________________________________ 14 
2.8 
RATIONALE FOR VACCINATION IN THE ADJUVANT SETTING __________________________________ 14 
2.9 
RATIONALE FOR TARGETING PATIENTS WITH TRIPLE NEGATIVE BREAST CANCER _________________ 15 
2.10 
DURVALUMAB (MEDI4736) _________________________________________________________ 16 
3 
PATIENT SELECTION _________________________________________________ 19 
3.1 
ELIGIBILITY CRITERIA ______________________________________________________________ 19 
3.2 
INCLUSION OF WOMEN AND MINORITIES ________________________________________________ 21 
4 
REGISTRATION PROCEDURES _________________________________________ 22 
4.1 
PRIOR TO REGISTRATION ____________________________________________________________ 22 
4.2 
REGISTRATION PROCEDURES _________________________________________________________ 22 
4.3 
CONFIRMATION OF PATIENT ELIGIBILITY ________________________________________________ 23 
4.4 
PATIENT REGISTRATION IN THE SITEMAN CANCER CENTER ONCORE DATABASE _________________ 23 
4.5 
ASSIGNMENT OF UPN ______________________________________________________________ 23 
4.6 
RANDOMIZATION __________________________________________________________________ 23 
5 
INVESTIGATIONAL AGENT: DURVALUMAB ____________________________ 24 
5.1 
DESCRIPTION _____________________________________________________________________ 24 
5.2 
PHARMACOKINETICS AND DRUG METABOLISM ___________________________________________ 24 
5.3 
SUPPLIER(S) ______________________________________________________________________ 24 
5.4 
DOSAGE FORM AND PREPARATION ____________________________________________________ 24 
5.5 
STORAGE AND STABILITY ___________________________________________________________ 24 
5.6 
ADMINISTRATION _________________________________________________________________ 25 
6 
INVESTIGATIONAL AGENT: NEOANTIGEN DNA VACCINES _____________ 26 
6.1 
CHEMICAL NAME AND STRUCTURE ____________________________________________________ 26 
6.2 
MANUFACTURING FACILITY __________________________________________________________ 27 
6.3 
MANUFACTURING PROCESS __________________________________________________________ 30 
6.4 
PRODUCT FORMULATION ____________________________________________________________ 33 
6.5 
PRODUCT RELEASE TESTS ___________________________________________________________ 34 
6.6 
STABILITY _______________________________________________________________________ 35 
6.7 
LABELS _________________________________________________________________________ 35 
6.8 
SITEMAN CANCER CENTER INVESTIGATIONAL PHARMACY __________________________________ 35 
6.9 
INVESTIGATIONAL AGENT ADMINISTRATION _____________________________________________ 36 
6.10 
INVESTIGATIONAL AGENT ACCOUNTABILITY _____________________________________________ 40 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 8 of 112 
7 
NONCLINICAL STUDIES _______________________________________________ 41 
7.1 
OVERVIEW _______________________________________________________________________ 41 
7.2 
NONCLINICAL STUDIES _____________________________________________________________ 41 
7.3 
RATIONALE FOR NO GLP SAFETY STUDIES_______________________________________________ 42 
7.4 
RATIONALE FOR NO GLP BIODISTRIBUTION STUDIES _______________________________________ 43 
7.5 
SEQUENCING PIPELINE ______________________________________________________________ 43 
7.6 
EPITOPE PREDICTION ALGORITHM _____________________________________________________ 45 
8 
TREATMENT PLAN ____________________________________________________ 46 
8.1 
NEOANTIGEN DNA VACCINE ADMINISTRATION ___________________________________________ 46 
8.2 
DURVALUMAB ADMINISTRATION ______________________________________________________ 46 
8.3 
REPLACEMENT OF PATIENTS _________________________________________________________ 46 
8.4 
GENERAL CONCOMITANT MEDICATION AND SUPPORTIVE CARE GUIDELINES ____________________ 47 
8.5 
WOMEN OF CHILDBEARING POTENTIAL _________________________________________________ 48 
8.6 
BLOOD DONATION _________________________________________________________________ 49 
8.7 
DURATION OF THERAPY _____________________________________________________________ 49 
8.8 
DURATION OF FOLLOW UP ___________________________________________________________ 49 
9 
POTENTIAL TOXICITY AND DOSE MODIFICATIONS ____________________ 50 
9.1 
POTENTIAL TOXICITY _______________________________________________________________ 50 
9.2 
TOXICITY MONITORING AND MANAGEMENT _____________________________________________ 53 
9.3 
DOSE MODIFICATIONS FOR DURVALUMAB _______________________________________________ 53 
9.4 
DOSE MODIFICATIONS FOR NEOANTIGEN DNA VACCINES ___________________________________ 79 
10 STUDY CALENDAR ____________________________________________________ 80 
11 CRITERIA FOR RESPONSE _____________________________________________ 82 
11.1 
PRIMARY OBJECTIVE: SAFETY ________________________________________________________ 82 
11.2 
SECONDARY OBJECTIVE: NEOANTIGEN-SPECIFIC T CELL RESPONSE ____________________________ 82 
11.3 
NEOANTIGEN-SPECIFIC T CELL RESPONSE _______________________________________________ 82 
12 REMOVAL OF SUBJECTS FROM PROTOCOL THERAPY _________________ 85 
12.1 
REMOVAL OF SUBJECTS FROM PROTOCOL THERAPY ________________________________________ 85 
12.2 
VOLUNTARY SUBJECT WITHDRAWAL ___________________________________________________ 85 
12.3 
PROCEDURE FOR DISCONTINUATION ___________________________________________________ 85 
13 REGULATORY AND REPORTING REQUIREMENTS ______________________ 86 
13.1 
SPONSOR-INVESTIGATOR REPORTING REQUIREMENTS _____________________________________ 86 
14 DATA AND SAFETY MONITORING _____________________________________ 91 
14.1 
PROTOCOL STOPPING CRITERIA _______________________________________________________ 91 
14.2 
DATA SAFETY MONITORING PLAN _____________________________________________________ 91 
14.3 
DEVELOPMENTAL THERAPEUTICS _____________________________________________________ 92 
14.4 
NIH RECOMBINANT DNA ADVISORY COMMITTEE ________________________________________ 92 
15 STATISTICAL CONSIDERATIONS ______________________________________ 93 
15.1 
STUDY OBJECTIVES AND ENDPOINTS ___________________________________________________ 93 
15.2 
STUDY DESIGN AND SAMPLE JUSTIFICATION _____________________________________________ 93 
15.3 
DATA ANALYSIS __________________________________________________________________ 94 
16 DATA MANAGEMENT _________________________________________________ 95 
16.1 
ADVERSE EVENT COLLECTION IN THE CASE REPORT FORMS ________________________________ 95 
17 REGULATORY AND ETHICAL OBLIGATIONS ___________________________ 97 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 9 of 112 
17.1 
INFORMED CONSENT _______________________________________________________________ 97 
17.2 
INSTITUTIONAL REVIEW BOARD ______________________________________________________ 97 
17.3 
SUBJECT CONFIDENTIALITY __________________________________________________________ 97 
18 ADMINISTRATIVE AND LEGAL OBLIGATIONS _________________________ 98 
18.1 
STUDY DOCUMENTATION AND RETENTION OF RECORDS ____________________________________ 98 
18.2 
POLICY REGARDING RESEARCH-RELATED INJURIES ________________________________________ 98 
18.3 
STUDY TERMINATION _______________________________________________________________ 98 
19 REFERENCES _________________________________________________________ 99 
20 APPENDICES _________________________________________________________ 108 
20.1 
ABBREVIATIONS _________________________________________________________________ 108 
20.2 
ECOG/ZUBROD PERFORMANCE STATUS SCALE __________________________________________ 109 
20.3 
NATIONAL CANCER INSTITUTE COMMON TERMINOLOGY CRITERIA FOR ADVERSE EVENTS ________ 109 
20.4 
DEFINITIONS FOR ADVERSE EVENT REPORTING _________________________________________ 109 
20.5 
REPORTING TIMELINES ____________________________________________________________ 111 
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 10 of 112 
1 
OBJECTIVES 
This is a single institution, open-label randomized phase 1 trial of neoantigen DNA vaccine alone vs. 
neoantigen DNA vaccine plus durvalumab in triple negative breast cancer (TNBC) patients following 
standard of care therapy. Patients with newly diagnosed clinical stage II-III TNBC are eligible for 
enrollment. Patients will receive standard of care therapy including chemotherapy, surgery and radiation 
therapy as clinically indicated. Following standard of care therapy, patients will be randomized to receive 
either a neoantigen DNA vaccine alone, or a neoantigen DNA vaccine + durvalumab. 
Hypotheses tested in this study include: (1) neoantigen DNA vaccines can induce and/or enhance 
neoantigen-specific T cell responses; and (2) durvalumab will enhance the response to neoantigen DNA 
vaccines. 
1.1 
Primary objective 
The primary objective is to assess the safety of neoantigen DNA vaccines given alone or in combination 
with durvalumab. Safety assessment will include both clinical observation and laboratory evaluation. 
Toxicity will be graded according to the National Cancer Institute Common Terminology Criteria for 
Adverse Events v4.03. 
1.2 
Secondary objective 
The secondary objective is to measure the immune response to neoantigen DNA vaccines given alone, or 
in combination with durvalumab. The immune response will be measured in the peripheral blood by 
luminexssay, ELISPOT, and multiparametric flow cytometry. 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 11 of 112 
2 
BACKGROUND 
2.1 
Checkpoint blockade therapy 
Activation of naïve T cells requires two signals: one signal is mediated through the T cell receptor when it 
interacts with a peptide-MHC complex on the antigen-presenting cell (APC) [1]. The second signal is a 
(positive) co-stimulatory signal through CD28 on the T cell and its ligand(s) B7.1/B7.2 on the APC [1, 2]. 
Physiologically-activated T cells such as those triggered by infections rapidly expand and typically 
eradicate infected cells. Several immune regulatory mechanisms exist that control the clonal expansion 
and persistence of activated T cells. CTLA4 is induced on the T cell surface upon activation; it effectively 
competes with CD28 for binding to B7 ligands, but transduces a negative co-stimulatory signal and 
abrogates T cell activation. A second regulatory pathway by which activated T cells are controlled is 
mediated through the Programmed Cell Death-1 (PD-1) molecule on T cells. PD-1 also has two ligands, 
PD-L1 and PD-L2, expressed on many cell types [1]. Since PD-1 ligation blocks signaling through the T 
cell receptor, PD-1 acts on the effector function of T cells rather than on stimulation. As such, CTLA4 and 
PD1 regulate different stages of T cell activation. These findings have been successfully exploited 
therapeutically using antibodies that block ligation of these checkpoint molecules [1-3]. The anti-CTLA4 
antibody ipilimumab received FDA approval in 2011, and two antibodies to PD-1, pembrolizumab and 
nivolumab, received approval in 2014 for the treatment of malignant melanoma. Multiple checkpoint 
inhibition clinical trials are currently ongoing in other diseases such as lung cancer, kidney cancer, 
bladder, prostate cancer, and others, and are expected to lead to approval of checkpoint therapy drugs 
for these diseases in the coming years. It is important to realize that this form of cancer therapy is unlike 
others in that the target is not the tumor but rather T cell regulatory molecules, and that checkpoint 
blockade therapy does not activate the immune system but instead neutralizes inhibitory signals that 
block tumor-specific T cells. As such, there is strong rationale to combine checkpoint blockade therapy 
with strategies that actively induce immune effector cells, such as cancer vaccine therapy.  
2.2 
Neoantigens 
Tumor antigens are often classified as shared tumor antigens and tumor-specific antigens. The majority 
of tumor-specific antigens are now believed to be the result of somatic mutations present in the tumor. 
Shared tumor antigens are expressed in multiple cancers, and are often self-differentiation antigens that 
are expressed in a limited subset of normal tissues, but overexpressed in cancers. Examples of shared 
tumor antigens include MAGE (melanoma) [4], prostatic acid phosphatase (prostate cancer) [5], and 
HER2/neu (breast cancer) [6]. 
Tumor-specific antigens are uniquely expressed in individual cancers, and are typically the result of point 
mutations or other genetic changes that are present only in the tumor (reviewed in [7, 8]). As such, tumor-
specific antigens represent the only antigens that are truly unique to the tumor and not expressed in 
normal tissues. The first human mutant tumor-specific antigen was described in 1995, resulting from a 
point mutation of cyclin-dependent kinase (CDK4) [9]. Since that time additional publications have 
described the expression of neoantigens in melanoma [10], non-small cell lung cancer [11] and other 
human cancers [12]. 
Cancer vaccine strategies targeting neoantigens have clear conceptual advantages over strategies 
targeting shared tumor antigens. Conceptual advantages include: (1) Targeting neoantigens is potentially 
safer. Neoantigens are expressed only in the tumor, decreasing the risk of autoimmunity. (2) Targeting 
neoantigens is potentially more effective. T cell responses to neoantigens are high in affinity, and are not 
limited by central mechanisms of self-tolerance. (3) Targeting neoantigens potentially limits antigen-loss, 
a common tumor escape mechanism. One of the hallmarks of cancer is genome instability, and one clear 
weakness of cancer vaccines that target a single shared tumor antigen is antigen-loss. Targeting multiple 
neoantigens may preclude antigen loss. In addition, many neoantigens play a functional role in neoplastic 
transformation (driver mutations). Immune selection resulting in loss of driver mutations may 
fundamentally alter the phenotype of targeted cancers. (4) Targeting neoantigens is likely to be 
universally applicable in solid tumors. Solid tumors appear to have a remarkable number of 
nonsynonymous mutations present (each nonsynonymous mutation is a candidate mutant tumor-specific 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 12 of 112 
antigen), suggesting that a neoantigen DNA vaccine approach could be used in most solid tumor patients, 
regardless of intrinsic subtype or HLA type.  
We have recently used next generation sequencing technologies to identify and study neoantigens in 
more detail as described in the sections below. 
2.3 
Next generation sequencing and cancer vaccines 
Cancer genome sequencing is a major focus area for Siteman Cancer Center, and for the Genome 
Institute at Washington University School of Medicine (WUSM). We recently used next-generation 
sequencing technologies to sequence and compare four DNA samples (primary tumor, brain metastasis, 
xenograft of the primary tumor, and peripheral blood) from an African-American patient with basal-like 
breast cancer. Of note, next-generation sequencing technologies are particularly well suited to breast 
cancer genome sequencing. Breast cancer is a heterogenous disease, but genome sequencing at almost 
40× haploid coverage allowed us to precisely calculate mutant allele frequencies, demonstrating genome 
remodeling, and unexpected similarities between the brain metastasis and xenograft [13]. 
Additional studies are currently ongoing, and we have successfully sequenced over 47 breast cancer 
tumor/normal genomes in postmenopausal breast cancer patients with luminal disease enrolled in the 
American College of Surgeons Oncology Group Z1031 clinical trial (neoadjuvant endocrine therapy) [14]. 
Although the main objective of this study was to define a genetic profile of susceptibility and/or resistance 
to aromatase inhibitor therapy, the results do provide important insights into the rational development of 
vaccine strategies for targeting neoantigens. 
In the 47 luminal breast cancer genomes sequenced, we identified and validated 1415 nonsynonymous 
single-nucleotide variant (SNV) mutations. This means that approximately 31 nonsynonymous SNV 
mutations are present per luminal breast cancer genome, similar to the number identified in our previous 
studies of a basal-like breast cancer [13], and similar to the number predicted by limited exome 
sequencing [15], and statistical models [16]. 79 genes contained nonsynonymous SNV mutations in more 
than one breast cancer (recurrently mutated genes). The most common recurrently mutated genes 
include PIK3CA (mutations present in 21 breast cancers), MT-ND5 (11 breast cancers), TP53 (7 breast 
cancers), SYNE1 (6 breast cancers), and TTN (5 breast cancers). Of the remaining 79 recurrently 
mutated genes, nonsynonymous SNV mutations were present in < 10% of the breast cancers studied. Of 
note, we identified 10 different PIK3CA mutations, and the most common mutation, p.H1047R, was 
present in 10 cancers. Our interpretation of these data is that a personalized vaccine approach is 
significantly more attractive than an off-the-shelf vaccine approach targeting recurrent mutations. Given 
the diversity of mutations observed, the limited number of recurrent mutations present in >10% of 
patients, and the fact that off-the-shelf vaccines would be restricted by HLA type, we estimate that < 10% 
of breast cancer patients would be eligible for an off-the-shelf vaccine targeting the most common 
PIK3CA mutation. Hence, even if multiple off-the-shelf vaccines were available for different recurrent 
mutations, only a limited number of patients would be eligible. However, given the number of mutations 
consistently observed in breast cancer, it is likely that at least a subset of these mutations could be 
successfully targeted by a personalized vaccine approach. These results suggest that a personalized 
vaccine approach is the best strategy to target neoantigens. 
There are two conceptual strategies for creating personalized cancer vaccines targeting neoantigens: a 
candidate epitope strategy, and an unbiased strategy. The candidate epitope strategy uses computer 
algorithms [17, 18] and in vitro studies to predict immunodominant epitopes, which are then integrated 
into a neoantigen vaccine. In the unbiased strategy, no attempt is made to identify the immunodominant 
epitopes, and all candidate neoantigens are integrated into a neoantigen vaccine. 
We have considered both the candidate epitope strategy and the unbiased strategy. We believe that the 
candidate epitope strategy is superior to the unbiased strategy for the following reasons. (1) Preliminary 
data from preclinical models and human correlative studies suggest that relatively few sequencing-
identified neoantigens are processed, presented and effectively recognized by the immune system. (2) 
We have now developed and validated algorithms for the prediction and prioritization of sequencing-
identified neoantigens [19, 20]. (3) Targeting a limited number of prioritized sequencing-identified 
neoantigens will facilitate vaccine design and manufacture, and streamline immune monitoring. 


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2.4 
Sequencing strategies to identify neoantigens 
Robust next-generation sequencing strategies for the identification of neoantigens will be required for the 
successful clinical translation of personalized cancer vaccine strategies. As such, a major focus of our 
research studies has been the development of cost-effective and accurate next-generation sequencing 
strategies to identify neoantigens and validate the expression of these antigens at the mRNA level. 
Initially a cancer genome sequencing approach was used. While cancer whole genome sequencing is 
informative and provides comprehensive information about both the coding and noncoding regions of the 
genome, this level of information may not be necessary for identifying neoantigens, or prioritizing antigens 
for immune intervention. We have now confirmed that tumor/normal exome sequencing is a robust and 
accurate strategy for the identification of neoantigens [20]. Of note, recent studies suggest that 
approximately 40% of mutations identified by cancer exome sequencing are not expressed at the mRNA 
level, so it is important to confirm expression of the mutant allele at the mRNA level. To evaluate mRNA 
expression, we have performed cDNA-capture sequencing analyses. We have confirmed that cDNA-
capture sequencing can be used to successfully confirm expression of sequencing-identified neoantigens 
at the mRNA level. This analysis also provides an estimation of how highly expressed the mutated allele 
is expressed relative to other genes in the tumor. For the phase 1 clinical trial proposed, tumor/normal 
exome sequencing analysis will be used to identify mutations (single nucleotide variants, insertions and 
deletions) present only in the tumor, and cDNA-capture sequencing will be used to confirm mutant allele 
expression and expression level in the tumor mRNA. 
2.5 
Prioritization of sequencing-identified neoantigens 
Of note, we have now developed and validated an epitope prediction algorithm for the prioritization of 
sequencing-identified neoantigens. Once somatic mutations have been identified and mutant mRNA 
expression confirmed/quantified using the sequencing strategies outlined above, neoantigens will be 
prioritized using an epitope prediction algorithm that has been designed to select and prioritize the most 
promising sequencing-identified neoantigens. Currently, the most commonly used CD8 T cell epitope 
prediction algorithm is NetMHC. However, collaborative work conducted by Robert Schreiber, Elaine 
Mardis, Max Artyomov and William Gillanders has shown that a much more accurate prediction comes 
from calculating a median affinity for each sequencing-predicted mutant epitope using multiple epitope 
prediction algorithms (NetMHC Pan; ANN; SMM; and others). We have significantly improved this epitope 
prediction algorithm by applying three filters to the initial prioritized output list: (a) elimination of 
hypothetical proteins; (b) use of an antigen processing algorithm to eliminate epitopes that are not likely 
to be proteolytically produced by constitutive proteasomes or immunoproteasomes; and (c) prioritization 
of “neo-epitopes” identified by a higher affinity binding of the mutant peptide sequence compared to the 
wildtype peptide sequence. The final output of these analyses is a rank-ordered list of the highest to 
lowest priority sequencing-identified neoantigens for each individual patient. In experiments performed 
using preclinical mouse sarcoma models, this refined prediction algorithm has successfully identified the 
major tumor rejection antigens in three out of three tumors tested to date [19, 20]. To our knowledge, this 
is the only algorithm that has been successfully applied to date to cancer vaccine development. Additional 
information about the preclinical validation of the epitope prediction algorithm is provided in Section 6.2 
Nonclinical Studies. A similar process will be used to identify and prioritize HLA class II neoantigens 
Once a rank-ordered list of the highest to lowest priority sequencing-identified mutant tumor-specific 
peptide antigens is generated, we may perform in vitro binding studies to confirm that the prioritized 
sequencing-identified mutant tumor-specific peptide antigens can bind to and stabilize the appropriate 
HLA class I allele. Up to 20 of the highest priority sequencing-identified neoantigens that are confirmed to 
bind and stabilize HLA class I or class II molecules will be targeted using the neoantigen DNA vaccine 
strategy detailed below. 
2.6 
Neoantigen DNA vaccines 
The neoantigen DNA vaccine strategy is based on the DNA vaccine platform. The observation that direct 
administration of recombinant DNA can generate potent immune responses established the field of DNA 
vaccines in the early 1990s [21-26]. Since that time, DNA vaccines have remained an area of intense 
research interest, and vaccines targeting infectious disease agents and cancers have progressed into 


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clinical trials. Advantages of the DNA vaccine platform include the remarkable safety profile of DNA 
vaccines, and the relative ease of manufacture relative to proteins and other biologics. Perhaps most 
important, however, is the molecular flexibility of the DNA vaccine platform, with the ability to genetically 
manipulate encoded antigens, and/or incorporate other genes to amplify the immune response [27, 28]. 
The molecular flexibility of the DNA vaccine platform allows us to target multiple neoantigens using a 
single polyepitope DNA vaccine. Polyepitope DNA vaccines integrate multiple epitopes in a single 
construct (Figure 1). We have optimized the polyepitope DNA vaccine platform to maximize antigen 
presentation of neoantigens by integrating a mutant ubiquitin molecule. Because MHC class I binding 
peptides are initially processed in the cytosol by the ubiquitin/proteasome pathway, we have integrated a 
mutant form of ubiquitin (UbG76V). Fusion of UbG76V to the N-terminus of the polyepitope construct 
promotes epitope generation and display. 
 
 
 
2.7 
Electroporation 
Recent research provides valuable insights into why preclinical studies of DNA vaccines have been 
successful in rodents, but less successful in larger animals. One difficulty is scaling up DNA vaccine dose 
and injection volume [29]. In rodents, hydrostatic pressure from injecting DNA in a relatively large liquid 
volume significantly improves cellular uptake of DNA and antigen expression [30]. This effect is reduced 
in larger animals as the relative volume injected and hydrostatic pressure is reduced. Electroporation 
dramatically increases DNA uptake by muscle cells, antigen expression, and immunogenicity [31-34]. Of 
particular note, electroporation has now been used successfully in non-human primates, with responses 
at levels previously not observed with other DNA vaccine approaches and similar to or superior to 
responses induced by live vectors [35-42]. The importance of electroporation to the successful clinical 
translation of DNA vaccines was recently highlighted and emphasized in a high-profile review in Nature 
Reviews [43], and in the introduction to a special issue of Vaccine [44]. We have established a 
collaboration with Ichor Medical Systems and will use the TDS-IM electroporation device in the proposed 
phase 1 clinical trial. 
2.8 
Rationale for vaccination in the adjuvant setting 
To date the majority of therapeutic cancer vaccine trials have been performed in patients with metastatic 
disease. Unfortunately, the results of cancer vaccines in patients with metastatic cancers have been 
disappointing. In a recent review of cancer vaccine trials of 440 patients at the NCI, Rosenberg et al. 
noted that the objective clinical response rate by standard RECIST criteria was only 2.6% [45]. It is now 
commonly believed that generation of an effective antitumor immune response will be difficult in patients 
Figure 1: Neoantigen DNA vaccine design. Neoantigen DNA vaccines integrate multiple epitopes in 
a single construct. We have optimized the neoantigen DNA vaccine platform to maximize antigen 
presentation of the sequencing-identified neoantigens by integrating a mutant ubiquitin molecule. 


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with metastatic cancers [46]. Metastatic breast cancer is no exception; metastatic breast cancer is 
associated with an increase in the prevalence of regulatory T cells [47, 48] and immature myeloid cells 
[49, 50] in the peripheral blood and in the tumor microenvironment, and these cells are capable of 
inhibiting endogenous or elicited antitumor immune responses.  
These issues have resulted in a fundamental reassessment of the clinical development paradigm for 
therapeutic cancer vaccines, with an emphasis on early assessment of vaccine safety and efficacy in an 
appropriate clinical context [51-53]. 
The most important consideration in the design of this clinical trial is to ensure the safe translation of the 
neoantigen DNA vaccine strategy. The Food and Drug Administration (FDA) dictates that initial studies of 
biologic therapies be performed in such a way that there is a balance between the potential risks and 
benefits in individual patients.  
2.9 
Rationale for targeting patients with triple negative breast cancer 
Triple-negative breast cancer (TNBC) lacks expression of estrogen receptor, progesterone receptor and 
HER-2. TNBC is associated with an aggressive clinical course, and there are no targeted therapies 
available. As such, development of innovative therapies for the treatment of TNBC is an important priority.  
There is strong rationale for the use of checkpoint blockade therapy in patients with TNBC.  
First, TNBC is a mutationally complex breast cancer subtype. Previous studies suggest that response to 
checkpoint blockade therapy is associated with mutational load. Several recent studies have provided 
insight into the mutational landscape in human breast cancer, including TNBC. While the average somatic 
mutational frequency across almost 30 different types of cancer is approximately one 
mutation/megabase, the frequency among all breast cancers ranges from 0.1 to 10 [54-56]. One feature 
of TNBC is the increased frequency of germline and somatic TP53 and BRCA1 mutations [57]. TP53 and 
BRCA mutations lead to defects in DNA repair mechanisms, and consequently an increased mutational 
frequency. Extensive analysis of the clonal and mutational spectrum of primary TNBC suggests that 
TNBC is characterized by a higher mutational frequency than other subtypes of breast cancer [58, 59]. 
The relative abundance of somatic mutations in TNBC compared to other breast cancer subtypes 
suggests that mutant antigens that can be targeted by the immune system are more likely to be present.  
Second, tumor infiltrating lymphocytes (TILs) are more common in TNBC, and TILs are associated with 
improved outcome in TNBC following adjuvant or neoadjuvant chemotherapy [60-62]. The association 
between TILs and improved outcome in TNBC suggests that the adaptive immune system contributes to 
the response to chemotherapy. 
Third, PD-L1 expression is higher in TNBC than other breast cancer subtypes [63, 64]. PD-L1 expression 
has been used as a biomarker for response to checkpoint blockade therapy [65]. 
We have examined the expression of PD-1 and PD-L1 in the common molecular subtypes of breast 
cancer using a well annotated human breast cancer tissue array [64, 66]. These studies demonstrate that 
expression of PD-1 on tumor-infiltrating lymphocytes and/or PD-L1 on breast cancers is more common in 
TNBC, and expression of these proteins is associated with a poor prognosis. Other investigators have 
also documented increased expression of PD-L1 in TNBC [63]. 
Fourth, a recent phase 1 clinical trial of atezolizumab in patients with metastatic TNBC demonstrates the 
safety of checkpoint blockade therapy with promising response rates. In an oral presentation at the AACR 
annual meeting in April 2015 (Abstract 2859), Emens et al. reported on 54 patients with metastatic TNBC 
enrolled in an expansion cohort. Patients received atezolizumab at 15 mg/kg, 20 mg/kg or 1200 mg flat 
dose IV q3w. AEs occurred in 67% of patients, most frequently fatigue (22%), pyrexia (15%), neutropenia 
(15%), and nausea (15%). Grade 3 AEs occurred in 11% of patients (adrenal insufficiency, neutropenia, 
nausea, vomiting, decreased WBC count). Twenty-one of the 54 patients were evaluable for efficacy, all 
of whom were PD-L1-positive (IHC 2/3). Among the 21 evaluable patients with PD-L1-expressing tumors, 
the unconfirmed RECIST ORR was 24% (95% CI, 8% to 47%) which included 3 PRs and 2 CRs. 
Adams et al reported the results of a phase 1b clinical trial of atezolizumab in combination with nab-
paclitaxel in patients with metastatic TNBC at the 2015 SABCS Meeting (Abstract P2-11-06). Twenty-four 


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patients with a minimum follow-up of ≥ 3 months were evaluable for efficacy. Across all groups the ORR 
was 71%. Based on these tests, a randomized phase 3 trial is planned (Impassion130, NCT02425891). 
2.10 Durvalumab (MEDI4736) 
The non-clinical and clinical experience is fully described in the most current version of the durvalumab 
Investigator’s Brochure (edition 10).  
Durvalumab is a human monoclonal antibody (mAb) of the immunoglobulin G (IgG) 1 kappa subclass that 
inhibits binding of PD-L1 and is being developed by AstraZeneca/MedImmune for use in the treatment of 
cancer. (MedImmune is a wholly owned subsidiary of AstraZeneca; AstraZeneca/MedImmune will be 
referred to as AstraZeneca throughout this document.) As durvalumab is an engineered mAb, it does not 
induce antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. The proposed 
mechanism of action for durvalumab is interference of the interaction of PD-L1 with PD-1 and CD80. PD-
L1 can bind to both PD-1 and CD80 on T cells. Similar to PD-L1/PD-1, the PD-L1/CD80 interaction 
delivers an inhibitory signal in T cells. Blockade of the PD-L1/CD80 interaction is expected to block the 
inhibitory signal and thereby enhance T cell responsiveness and expansion. 
To date durvalumab has been given to more than 1800 subjects as part of ongoing studies either as 
monotherapy or in combination with other anti-cancer agents. Details on the safety profile of durvalumab 
monotherapy are summarized in Section 2.10.1. Refer to the current durvalumab Investigator’s Brochure 
for a complete summary of non-clinical and clinical information including safety, efficacy and 
pharmacokinetics.  
2.10.1 Durvalumab monotherapy dose rationale 
A durvalumab dose of 20 mg/kg Q4W is supported by in-vitro data, non-clinical activity, clinical 
PK/pharmacodynamics, biomarkers, and activity data from Study 1108 in subjects with advanced solid 
tumors and from a Phase I trial performed in Japanese subjects with advanced solid tumor 
(D4190C00002). 
2.10.2 PK/Pharmacodynamic data 
Based on available PK/pharmacodynamic data from ongoing Study 1108 with doses ranging from 0.1 to 
10 mg/kg Q2W or 15 mg/kg Q3W, durvalumab exhibited non-linear (dose-dependent) PK consistent with 
target-mediated drug disposition. The PK approached linearity at ≥3 mg/kg Q2W, suggesting near 
complete target saturation (membrane-bound and sPD-L1), and further shows that the durvalumab 
dosing frequency can be adapted to a particular regimen given the linearity seen at doses higher than 3 
mg/kg. The expected half-life with doses ≥3 mg/kg Q2W is approximately 21 days. A dose-dependent 
suppression in peripheral sPD-L1 was observed over the dose range studied, consistent with 
engagement of durvalumab with PD-L1. A low level of immunogenicity has been observed. No subjects 
have experienced immune-complex disease following exposure to durvalumab (For further information on 
immunogenicity, please see the current IB).  
Data from Study D4190C00006 (Phase I trial in NSCLC subjects using the combination of durvalumab 
and tremelimumab) also show an approximately dose-proportional increase in PK exposure for 
durvalumab over the dose range of 3 to 20 mg/kg durvalumab Q4W or Q2W. (For further information on 
PK observations in Study 006, please see the current IB).  
The observed durvalumab PK data from the combination study were well in line with the predicted 
monotherapy PK data (5th median and 95th percentiles) for a Q4W regimen.  
A population PK model was developed using the data from Study 1108 (doses=0.1 to 10 mg/kg Q2W or 
15 mg/kg Q3W (Fairman et al 2014). Multiple simulations indicate that a similar overall exposure is 
expected following both 10 mg/kg Q2W and 20 mg/kg Q4W regimens, as represented by AUCss 
(4 weeks). Median Cmax,ss is expected to be higher with 20 mg/kg Q4W (~1.5 fold) and median 
Ctrough,ss is expected to be higher with 10 mg/kg Q2W (~1.25 fold). Clinical activity with the 20 mg/kg 
Q4W dosing regimen is anticipated to be consistent with 10 mg/kg Q2W with the proposed similar dose of 
20 mg/kg Q4W expected to (a) achieve complete target saturation in majority of subjects; (b) account for 
anticipated variability in PK, pharmacodynamics, and clinical activity in diverse cancer populations; 


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(c) maintain sufficient PK exposure in case of ADA impact; and (d) achieve PK exposure that yielded 
maximal antitumor activity in animal models. 
Given the similar area under the plasma drug concentration-time curve (AUC) and modest differences in 
median peak and trough levels at steady state, the observation that both regimens maintain complete 
sPD-L1 suppression at trough, and the available clinical data, the 20 mg/kg Q4W and 10 mg/kg Q2W 
regimens are expected to have similar efficacy and safety profiles, supporting further development with a 
dose of 20 mg/kg Q4W.  
2.10.3 Clinical data 
Refer to the current durvalumab Investigator’s Brochure for a complete summary of clinical information 
including safety, efficacy and pharmacokinetics at the 20mg/kg Q4W regimen. 
2.10.4 Rationale for fixed dosing 
A population PK model was developed for durvalumab using monotherapy data from a Phase I study 
(study 1108; N=292; doses= 0.1 to 10 mg/kg Q2W or 15 mg/kg Q3W; solid tumors). Population PK 
analysis indicated only minor impact of body weight (WT) on the PK of durvalumab (coefficient of ≤ 0.5). 
The impact of body WT-based (10 mg/kg Q2W) and fixed dosing (750 mg Q2W) of durvalumab was 
evaluated by comparing predicted steady state PK concentrations (5th, median and 95th percentiles) 
using the population PK model. A fixed dose of 750 mg was selected to approximate 10 mg/kg (based on 
median body WT of ~75 kg). A total of 1000 subjects were simulated using body WT distribution of 40–
120 kg. Simulation results demonstrate that body WT-based and fixed dosing regimens yield similar 
median steady state PK concentrations with slightly less overall between-subject variability with fixed 
dosing regimen.  
Similar findings have been reported by others (Ng et al 2006, Wang et al 2009, Zhang et al 2012, Narwal 
et al 2013). Wang and colleagues investigated 12 monoclonal antibodies and found that fixed and body 
size-based dosing perform similarly, with fixed dosing being better for 7 of 12 antibodies (Wang et al 
2009)]. In addition, they investigated 18 therapeutic proteins and peptides and showed that fixed dosing 
performed better for 12 of 18 in terms of reducing the between-subject variability in 
pharmacokinetic/pharmacodynamics parameters (Zhang et al 2012).  
A fixed dosing approach is preferred by the prescribing community due to ease of use and reduced 
dosing errors. Given expectation of similar pharmacokinetic exposure and variability, we considered it 
feasible to switch to fixed dosing regimens. Based on average body WT of 75 kg, a fixed dose of 1500 mg 
Q4W durvalumab (equivalent to 20 mg/kg Q4W) is included in the current study. 
2.10.5 Identified and potential risks 
Monoclonal antibodies directed against immune checkpoint proteins, such as programmed cell death 
ligand 1 (PD-L1) as well as those directed against programmed cell death-1 (PD-1) or cytotoxic T-
lymphocyte antigen-4 (CTLA-4), aim to boost endogenous immune responses directed against tumor 
cells. By stimulating the immune system however, there is the potential for adverse effects on other 
tissues. 
Most adverse drug reactions seen with the immune checkpoint inhibitor class of agents are thought to be 
due to the effects of inflammatory cells on specific tissues. Potential risks are events with a potential 
inflammatory mechanism and which may require more frequent monitoring and/or unique interventions 
such as immunosuppressants and/or endocrine replacement therapy. These risks include gastrointestinal 
AEs such as colitis and diarrhoea, pneumonitis, nephritis and acute renal failure, hepatic AEs such as 
hepatitis and liver enzyme elevations, dermatitis, and endocrinopathies such as hypo- and hyper-
thyroidism, hypophysitis and adrenal insufficiency. 
Identified risks with durvalumab are diarrhea, increases in transaminases, pneumonitis and colitis.  
Potential risks include endocrinopathies (hypo- and hyper-thyroidism, hypophysitis and adrenal 
insufficiency) hepatitis/hepatotoxicity, neurotoxicities, nephritis, pancreatitis, dermatitis, infusion-related 
reactions, anaphylaxis, hypersensitivity or allergic reactions, and immune complex disease. Further 
information on these risks can be found in the current version of the durvalumab IB. 


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In monotherapy clinical studies AEs (all grades) reported very commonly (≥ 10% of subjects) are fatigue, 
nausea, decreased appetite, dyspnea, cough, constipation, diarrhea, vomiting, back pain, pyrexia, 
abdominal pain, anemia, arthralgia, peripheral edema, headache, rash, and pruritus. Approximately 10% 
of subjects experienced an AE that resulted in permanent discontinuation of durvalumab and 
approximately 3.5% of subjects experienced an SAE that was considered to be related to durvalumab by 
the study investigator. 
The majority of treatment-related AEs were manageable with dose delays, symptomatic treatment, and in 
the case of events suspected to have an immune basis, the use of established treatment guidelines for 
immune-mediated. 
A detailed summary of durvalumab monotherapy AE data can be found in the current version of the 
durvalumab IB 
  
 


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3 
PATIENT SELECTION 
3.1 
Eligibility Criteria 
3.1.1 
Inclusion criteria 
A patient will be eligible for inclusion in this study only if ALL of the following criteria apply: 
(1) Histologically confirmed diagnosis of invasive breast cancer. 
(2) ER and PR less than Allred score of 3 or less than 1% positive staining cells in the invasive 
component of the tumor. Patients not meeting this pathology criteria, but have been clinically treated 
as having TNBC, may be enrolled at treating physician’s discretion. 
(3) HER2 negative by FISH or IHC staining 0 or 1+. 
(4) Consented for genome sequencing. 
(5) Clinical stage T1c-T4c, any N, M0 primary tumor by AJCC 7th edition clinical staging prior to 
neoadjuvant chemotherapy, with residual invasive breast cancer after neoadjuvant therapy. 
(6) At least 18 years of age. 
(7) Eastern Cooperative Oncology Group (ECOG) performance status ≤ 1. 
(8) Adequate organ and marrow function no more than 14 days prior to registration as defined below: 
absolute neutrophil count 
≥1,500/μL 
platelets 
≥100,000/μL 
hemoglobin 
≥ 9.0 g/dL 
total bilirubin 
≤1.5 X institutional upper limit of normal 
AST/ALT 
≤2.5 X institutional upper limit of normal 
serum creatinine clearance 
> 40 mL/min by the Cockcroft-Gault formula or by 24-hour urine 
collection for determination of creatinine clearance 
(9) Body weight > 30 kg. 
(10) Evidence of post-menopausal status or negative urine or serum pregnancy test for female pre-
menopausal subjects. Women will be considered post-menopausal if they have been amenorrheic for 
12 months without an alternative medical cause. The following age-specific requirements apply: 
- 
Women < 50 years of age would be considered post-menopausal if they have been amenorrheic 
for 12 months or more following cessation of exogenous hormonal treatments and if they have 
luteinizing hormone and follicle-stimulating hormone levels in the post-menopausal range for the 
institution or underwent surgical sterilization (bilateral oophorectomy or hysterectomy). 
- 
Women ≥ 50 years of age would be considered post-menopausal if they have been amenorrheic 
for 12 months or more following cessation of all exogenous hormonal treatments, had radiation-
induced menopause with last menses > 1 year ago, had chemotherapy-induced menopause with 
last menses > 1 year ago, or underwent surgical sterilization (bilateral oophorectomy, bilateral 
salpingectomy, or hysterectomy). 
(11) Able to understand and willing to sign an IRB-approved written informed consent document. 
3.1.2 
Exclusion criteria 
A patient will be ineligible for inclusion in this study if ANY of the following criteria apply: 
(1) Received chemotherapy, radiotherapy (to more than 30% of the bone marrow or with a wide field of 
radiation), or biologic therapy within the last 30 days. 
(2) Concurrent enrollment in another clinical study, unless it is an observational (non-interventional) 
clinical study or during the follow-up period of an interventional study. 


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(3) Receiving any other investigational agent(s) or has received an investigational agent within the last 
30 days. 
(4) Receipt of live attenuated vaccination within 6 months prior to study entry or within 30 days of 
receiving durvalumab. 
(5) Major surgical procedure within 28 days prior to the first dose of durvalumab. Local surgery of 
isolated lesions for palliative intent is acceptable. 
(6) Current use or prior use of immunosuppressive medication within 28 days before the first dose of 
durvalumab, with the exceptions of intranasal, inhaled, and intra-articular corticosteroids or systemic 
corticosteroids at physiological doses which are not to exceed 10 mg/day of prednisone or an 
equivalent corticosteroid. 
(7) Known metastatic disease. 
(8) Invasive cancer in the contralateral breast. 
(9) Known allergy, or history of serious adverse reaction to vaccines such as anaphylaxis, hives, or 
respiratory difficulty. 
(10) History of hypersensitivity to durvalumab or any excipient. 
(11) Mean QT interval corrected for heart rate using Fridericia’s formula (QTcF) ≥ 470 ms calculated from 
3 electrocardiograms (ECGs) (within 15 minutes at 5 minutes apart). 
(12) Any unresolved toxicity NCI CTCAE grade ≥ 2 from previous anticancer therapy with the exception of 
alopecia, vitiligo, and the laboratory values defined in the inclusion criteria. Subjects with grade ≥ 2 
neuropathy will be evaluated on a case-by-case basis after consultation with the study physician. 
Subjects with irreversible toxicity not reasonably expected to be exacerbated by treatment with 
durvalumab may be included only after consultation with the study physician. 
(13) Uncontrolled intercurrent illness including, but not limited to ongoing or active infection, symptomatic 
congestive heart failure, uncontrolled hypertension, unstable angina pectoris, cardiac arrhythmia, 
interstitial lung disease, serious chronic gastrointestinal conditions associated with diarrhea, evidence 
of any acute or chronic viral illness or disease, or psychiatric illness/social situation that would limit 
compliance with study requirements or compromise the ability of the subject to give written informed 
consent.  
(14) Active or prior documented autoimmune or inflammatory disorders (including inflammatory bowel 
disease [e.g., colitis or Crohn’s disease], diverticulitis [with the exception of diverticulosis], systemic 
lupus erythematosus, Sarcoidosis syndrome, or Wegener syndrome [granulomatosis with polyangiitis, 
Graves’ disease, rheumatoid arthritis, hypophysitis, uveitis, etc.]). The following are exceptions to this 
criterion: 
- 
Subjects with vitiligo or alopecia 
- 
Subjects with hypothyroidism (e.g., following Hashimoto syndrome) stable on hormone 
replacement 
- 
Any chronic skin condition that does not require systemic therapy 
- 
Subjects without active disease in the last 5 years may be included but only after consultation 
with the study physician 
- 
Subjects with celiac disease controlled by diet alone 
(15) History of pneumonitis or interstitial lung disease. 
(16) History of active primary immunodeficiency. 
(17) Active infection including tuberculosis (clinical evaluation that includes clinical history, physical 
examination, and radiographic findings, and TB testing in line with local practice), hepatitis B (known 
positive HBV surface antigen (HBsAg) result), hepatitis C, or human immunodeficiency virus (positive 
HIV 1/2 antibodies). Subjects with a past or resolved HBV infection (defined as the presence of 


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hepatitis B core antibody (anti-HBc) and absence of HBsAg) are eligible. Subjects positive for 
hepatitis C (HCV) antibody are eligible only if polymerase chain reaction is negative for HCV RNA. 
(18) History of allogeneic organ transplantation. 
(19) Pregnant or breastfeeding. A negative serum pregnancy test is required no more than 14 days before 
study entry. 
(20) Subjects of reproductive potential who are not willing to employ effective birth control from screening 
to 1 year after last dose of vaccine. 
(21) History of another primary malignancy except for: 
- 
Malignancy treated with curative intent and with no known active disease ≥ 5 years before the 
first dose of study treatment and low potential for risk of recurrence 
- 
Adequately treated non-melanoma skin cancer of lentigo maligna without evidence of disease 
- 
Adequately treated carcinoma in situ without evidence of disease 
(22) History of leptomeningeal carcinomatosis. 
(23) Patient must have no active major medical or psychosocial problems that could be complicated by 
study participation. 
(24) Subjects with a strong likelihood of non-adherence such as difficulties in adhering to follow-up 
schedule due to geographic distance from the Siteman Cancer Center should not knowingly be 
registered. 
(25) Individuals in whom a skinfold measurement of the cutaneous and subcutaneous tissue for eligible 
injection sites (left and right medial deltoid region) exceeds 40 mm. 
(26) Individuals in whom the ability to observe possible local reactions at the eligible injection sites (deltoid 
region) is, in the opinion of the investigator, unacceptably obscured due to a physical condition or 
permanent body art. 
(27) Therapeutic or traumatic metal implant in the skin or muscle of either deltoid region. 
(28) Acute or chronic, clinically significant hematologic, pulmonary, cardiovascular, or hepatic or renal 
functional abnormality as determined by the investigator based on medical history, physical 
examination, EKG, and/or laboratory screening test 
(29) Any chronic or active neurologic disorder, including seizures and epilepsy, excluding a single febrile 
seizure as a child, or chronic seizure disorder which is well controlled by medication with no seizures 
within the last 2 years. 
(30) Syncopal episode within 12 months of screening 
(31) Current use of any electronic stimulation device, such as cardiac demand pacemakers, automatic 
implantable cardiac defibrillator, nerve stimulators, or deep brain stimulators. 
3.2 
Inclusion of women and minorities 
Women of all races and ethnic groups are eligible for this trial. 
 


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4 
REGISTRATION PROCEDURES 
4.1 
Prior to registration 
4.1.1 
Subject recruitment 
The methods used for recruitment of subjects in the study will be devoid of any procedures that may be 
construed as coercive. The recruitment process will not involve any restrictions based on social or 
demographic factors including age, or ethnic characteristics of the subject population. However, the 
composition of the study subject population will depend on patient sources available to the investigators. 
Subjects will be identified and recruited for this study as follows: 
Patients will be recruited from Siteman Cancer Center outpatients or patient referrals by our community 
oncologists to the principal investigator and co-investigators. Patients must be willing and able to give 
their written informed consent indicating that they are aware of the investigational nature of the study. 
After a patient is deemed eligible for study, the principal investigator (or co-investigators) will discuss the 
Washington University Human Research Protection Office-approved informed consent with the patient. 
This written informed consent will be signed and dated by the patient and the principal investigator (or co-
investigators). The original consent will be placed in the patient’s permanent record and a copy will be 
given to the patient. 
Washington University School of Medicine (WUSM) has an approved Multiple Project Assurance of 
Compliance with Department of Health and Human Services Regulations for the Protection of Human 
Research Subjects on file with the Office for Human Research Protection (OHRP). The Human Research 
Protection Office Policies and Procedures for Protection of Human Research Subjects details all policies 
and procedures for the protection of human research subjects and can be obtained upon request from the 
Human Research Protection Office. 
4.1.2 
Compliance and understanding 
All patients who present with clinical stage II or III triple negative breast cancer will be screened for 
eligibility for entry into the study. As in all trials, the goal is to achieve a high level of compliance with 
protocol requirements by assuring, during the eligibility assessment, that the potential subject is fully 
informed and agrees to the protocol requirements. In addition, subjects with a strong likelihood of non-
adherence such as difficulties in adhering to follow-up schedule due to geographic distance from the 
Siteman Cancer Center, should not knowingly be registered. Adherence of the Siteman Cancer Center 
staff to careful assessment of the subject’s understanding of the trial and a clinical center environment 
which supports the continued commitment of the subjects are essential for the trial to be successfully 
completed. 
4.1.3 
Presentation of informed consent 
Consent will be obtained by either the principal investigator or by individuals approved by the principal 
investigator and whose names and copy of their curriculum vitae have been filed. The initial consent 
should be the IRB-approved version corresponding to the version of the protocol approved when the 
screening was initiated. Informed consent is to be obtained from the subject according to Section 18.1 
Informed Consent of this protocol. 
4.2 
Registration procedures 
Patients must not start any protocol intervention prior to registration through the Siteman Cancer 
Center. 
The following steps must be taken before registering patients to this study: 
(1) 
Confirmation of patient eligibility 
(2) 
Registration of patient in the Siteman Cancer Center OnCore database 
(3) 
Assignment of unique patient number (UPN) 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 23 of 112 
4.3 
Confirmation of patient eligibility 
Confirm patient eligibility by collecting the information listed below: 
(1) 
The registering MD’s name 
(2) 
Patient’s race, sex, and DOB 
(3) 
Three letters (or two letters and a dash) for the patient’s initials 
(4) 
Copy of signed consent form 
(5) 
Completed eligibility checklist, signed and dated by a member of the study team 
(6) 
Copy of appropriate source documentation confirming patient eligibility 
4.4 
Patient registration in the Siteman Cancer Center OnCore database 
All patients must be registered through the Siteman Cancer Center OnCore database. 
4.5 
Assignment of UPN 
Each patient will be identified with a unique patient number (UPN) for this study. All data will be recorded 
with this identification number on the appropriate CRFs. 
4.6 
Randomization 
Twenty-four patients will be enrolled. Patients will be randomized for participation in a 1:1 ratio to receive 
either neoantigen DNA vaccine alone or neoantigen DNA vaccine plus durvalumab. The randomization 
table will be generated using the SAS program PROC PLAN. Randomization will take place after the 
manufacture of the vaccine and after eligibility has been reconfirmed. 
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 24 of 112 
5 
INVESTIGATIONAL AGENT: DURVALUMAB 
5.1 
Description 
Durvalumab is a human monoclonal antibody (mAb) of the immunoglobulin G1 kappa (IgG1κ) subclass 
that inhibits binding of programmed cell death ligand 1 (PD-L1) (B7-H1, CD274) to programmed cell 
death 1 (PD-1; CD279) and CD80 (B7-1). Durvalumab is composed of 2 identical heavy chains and 2 
identical light chains, with an overall molecular weight of approximately 149 kDa. Durvalumab contains a 
triple mutation in the constant domain of the immunoglobulin (Ig) G1 heavy chain that reduces binding to 
complement protein C1q and the fragment crystallizable gamma (Fcγ) receptors involved in triggering 
effector function. 
5.2 
Pharmacokinetics and Drug Metabolism 
Please refer to Section 2.10.2. 
5.3 
Supplier(s) 
The Investigational Products Supply section of AstraZeneca/MedImmune will supply durvalumab 
(MEDI4736) to the investigator as a 500-mg vial solution for infusion after dilution. 
5.4 
Dosage Form and Preparation 
Durvalumab (MEDI4736) will be supplied by AstraZeneca as a 500-mg vial solution for infusion after 
dilution. The solution contains 50 mg/mL durvalumab (MEDI4736), 26 mM 
histidine/histidine-hydrochloride, 275 mM trehalose dihydrate, and 0.02% (weight/volume) polysorbate 80; 
it has a pH of 6.0. The nominal fill volume is 10 mL. Investigational product vials are stored at 2°C to 8°C 
(36°F to 46°F) and must not be frozen. Durvalumab (MEDI4736) must be used within the individually 
assigned expiry date. 
Preparation of infusion bags 
Total time from needle puncture of the durvalumab vial to the start of administration should not exceed:  
• 
24 hours at 2°C to 8°C (36°F to 46°F) 
• 
4 hours at room temperature 
If in-use storage time exceeds these limits, a new dose must be prepared from new vials. Infusion 
solutions must be allowed to equilibrate to room temperature prior to commencement of administration.  
A dose of 1500 mg (for patients >30 kg in weight) will be administered using an IV bag containing 0.9% 
(w/v) saline or 5% (w/v) dextrose, with a final durvalumab (MEDI4736) concentration ranging from 1 to 20 
mg/mL, and delivered through an IV administration set with a 0.2- or 0.22-μm in-line filter. Add 30.0 mL of 
durvalumab (MEDI4736) (ie, 1500mg of durvalumab [MEDI4736]) to the IV bag. The IV bag size should 
be selected such that the final concentration is within 1 to 20 mg/mL. Mix the bag by gently inverting to 
ensure homogeneity of the dose in the bag.  No incompatibilities between durvalumab and 
polyvinylchloride or polyolefin IV bags have been observed. Subjects whose weight drops to ≤ 30 kg 
should not receive durvalumab.  
In the event that either preparation time or infusion time (see Section 5.6) exceeds the time limits, a new 
dose must be prepared from new vials. Durvalumab does not contain preservatives, and any unused 
portion must be discarded. 
5.5 
Storage and Stability 
Total in-use storage time from needle puncture of durvalumab vial to start of administration should not 
exceed 4 hours at room temperature or 24 hours at 2-8°C (36-46°F). If in-use storage time exceeds these 
limits, a new dose must be prepared from new vials. Infusion solutions must be allowed to equilibrate to 
room temperature prior to commencement of administration. Durvalumab does not contain preservatives 
and any unused portion must be discarded. 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 25 of 112 
5.6 
Administration 
Following preparation of durvalumab, the entire contents of the IV bag should be administered as an IV 
infusion at room temperature (approximately 25°C) over approximately 60 minutes (±5 minutes), using a 
0.2-μm or 0.22-μm in-line filter.  
Standard infusion time is 1 hour. However, if there are interruptions during infusion, the total allowed time 
should not exceed 8 hours at room temperature. The IV line will be flushed with a volume of IV diluent 
equal to the priming volume of the infusion set used after the contents of the IV bag are fully 
administered, or complete the infusion according to institutional policy to ensure the full dose is 
administered and document if the line was not flushed.  
Since the compatibility of durvalumab with other IV medications and solutions, other than normal saline 
(0.9% [weight/volume] sodium chloride for injection) or 5% (weight/volume) dextrose, is not known, the 
durvalumab solution should not be infused through an IV line in which other solutions or medications are 
being administered. 
Subjects will be monitored before, during, and after the infusion with assessment of vital signs at the 
times specified in the Schedule of Assessments. Subjects are monitored (pulse rate, blood pressure) 
every 30 minutes during the infusion period (including times where infusion rate is slowed or temporarily 
stopped). 
In the event of a ≤Grade 2 infusion-related reaction, the infusion rate of study drug may be decreased by 
50% or interrupted until resolution of the event (up to 4 hours) and re-initiated at 50% of the initial rate 
until completion of the infusion. For subjects with a ≤Grade 2 infusion-related reaction, subsequent 
infusions may be administered at 50% of the initial rate. Acetaminophen and/or an antihistamine (e.g., 
diphenhydramine) or equivalent medications per institutional standard may be administered at the 
discretion of the investigator. If the infusion-related reaction is ≥Grade 3 or higher in severity, study drug 
will be discontinued. The standard infusion time is one hour, however if there are interruptions during 
infusion, the total allowed time from infusion start to completion of infusion should not exceed 8 hours at 
room temperature. For management of subjects who experience an infusion reaction, please refer to the 
toxicity and management guidelines. 
As with any antibody, allergic reactions to dose administration are possible. Appropriate drugs and 
medical equipment to treat acute anaphylactic reactions must be immediately available, and study 
personnel must be trained to recognize and treat anaphylaxis. The study site must have immediate 
access to emergency resuscitation teams and equipment in addition to the ability to admit subjects to an 
intensive care unit if necessary. 
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 26 of 112 
6 
INVESTIGATIONAL AGENT: NEOANTIGEN DNA VACCINES 
6.1 
Chemical name and structure 
The neoantigen DNA vaccines are also known as DNA plasmid vector expressing tumor-specific 
antigens. 
Neoantigen DNA vaccines will be designed in the Gillanders laboratory and manufactured at the Siteman 
Cancer Center Biological Therapy Core Facility based on the following general steps:  
(1) Breast cancer tissue and normal lymphocytes will be obtained from breast cancer patients who are 
eligible for the phase 1 clinical trial. 
(2) Tumor/normal exome sequencing and tumor cDNA-capture sequencing will be performed to identify 
candidate neoantigens. 
(3) Candidate neoantigens will be prioritized based on epitope prediction algorithms and in vitro studies. 
(4) Personalized polyepitope inserts integrating the prioritized neoantigens will be designed in the 
Gillanders laboratory and then synthesized and cloned into the pING parent vector by Blue Heron 
Biotech. 
(5) Master cell banks will be established and validated at WUSM. 
(6) The neoantigen DNA vaccines will be manufactured and vialed at WUSM. 
(7) The neoantigen DNA vaccines will undergo product release tests at WUSM and other sites prior to 
investigational use. 
The neoantigen DNA vaccines will be manufactured in the SCC Biologic Therapy Core facility. Standard 
Operating Procedures for the GMP manufacture of the neoantigen vaccines have been established and 
are in accordance with “CGMP for Phase 1 Investigational Drugs 2008,” and “Considerations for Plasmid 
DNA Vaccines for Infectious Disease Indications 2007.” 
Each neoantigen DNA vaccine drug product is composed of a deoxyribonucleic acid (DNA) plasmid 
purified from E. Coli. The pING parent vector was obtained from Alan Houghton, M.D. at Memorial Sloan 
Kettering Cancer Center (Figure 2). The pING vector has been used extensively in preclinical DNA 
vaccine studies [71, 72], and in human clinical trials, including a clinical trial at WUSM (ClinicalTrials.gov 
identifier: NCT00807781). The mammaglobin-A DNA vaccine is based on the pING vector and was 
manufactured at WUSM in the SCC Biologic Therapy Core Facility using manufacturing processes almost 
identical to the ones outlined here). 
The pING vector contains the following elements: (1) a eukaryotic promoter and enhancer from the 
Towne strain of CMV; (2) a polylinker region to facilitate cloning of a variety of DNA fragments; (3) donor 
and acceptor splice sites and a poly adenylation signal sequence derived from the bovine growth 
hormone gene; (4) the ColE1 origin of replication and (5) a gene conferring kanamycin resistance. With 
the exception of the kanamycin resistance gene, which was cloned as a PstI fragment from the plasmid 
pUC4, all other gene segments were amplified by polymerase chain reaction (PCR). 
The role of each element is as follows: (1) the CMV promoter/enhancer enables high-level expression of 
polyepitope insert in mammalian cells; (2) the polylinker region serves as a multiple cloning site for easy 
insertion of genes, in this case the polyepitope insert; (3) the polyadenylation signal sequence facilitates 
efficient transcription termination and polyadenylation of mRNA; (4) the origin of replication allows for 
high-copy number replication and growth in E. coli; (5) the kanamycin resistance gene provides for 
selection in E. coli; and (6) the prokaryotic T7 promoter of the pING plasmid produces high levels of 
polyepitope DNA transcripts in E. coli in the presence of bacterial T7 RNA polymerase. Of note, the 
kanamycin resistance gene is cloned in the opposite orientation from the polyepitope insert to limit 
transcription of kanamycin from the CMV promoter in human cells. 
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 27 of 112 
 
 
The personalized polyepitope inserts will be designed in the Gillanders Laboratory based on the results of 
next-generation sequencing and epitope prediction algorithms from each individual patient. Personalized 
polyepitope inserts will incorporate up to 20 prioritized neoantigens. The personalized polyepitope inserts 
will be synthesized by Blue Heron Technologies and cloned into the pING parent vector as detailed 
below. 
6.2 
Manufacturing facility 
The neoantigen DNA vaccines will be prepared in the Siteman Cancer Center Biologic Therapy Core 
Facility at Washington University School of Medicine. The facility is located at 500 S. Kingshighway, 
Room 719 Southwest Tower, Saint Louis, MO 63110. 
The facility adheres to cGMP practices with regard to documentation, facility maintenance, and QC/QA 
review. Within the 2,615 sq ft GMP-facility on the 7th floor of the Southwest Tower, 6 manufacturing 
rooms are available for clinical grade manufacturing of cellular therapy products, recombinant DNA or 
gene therapy products (Floor plan below).  
Figure 2: Neoantigen DNA vaccine design. Neoantigen DNA vaccines are based on the pING 
parent vector. The pING vector contains the following elements: (1) a eukaryotic promoter and 
enhancer from the Towne strain of CMV; (2) a polylinker region to facilitate cloning of a variety of DNA 
fragments; (3) donor and acceptor splice sites and a poly adenylation signal sequence derived from 
the bovine growth hormone gene; (4) the ColE1 origin of replication; (5) a gene conferring kanamycin 
resistance, and (6) a polyepitope insert integrating the prioritized neoantigens. 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 28 of 112 
 
 
In the Biologic Therapy Core facility, all manufacturing rooms are physically separated from each other 
with single pass air, and provide one-way personnel and product flow, which avoids cross-contamination 
of products. 6 different products can be worked on simultaneously without interfering with each other. 
Passthroughs are available from gowning rooms to intermediate rooms, and intermediate rooms to 
manufacturing rooms. All manufacturing rooms are completely isolated from each other by sealed walls 
and ceilings. A custom ceiling allows maintenance on the HVAC units, filters or lights without breeching 
the clean environment of the facility. 
The manufacturing laboratories are only entered, one at a time, from an intermediate room. The 
laboratories are only exited, one at a time, into another intermediate room. 
This provides easily manageable spatial and temporal segregation of products, which is a necessity to 
prevent product cross-contamination. Door interlocks, which allow for doors to be opened only one at a 
time, assure this unidirectional flow of personnel and products. 
Manufacturing rooms, intermediate rooms and gowning/degowning rooms are isolated from each other by 
air pressure gradients. The manufacturing rooms have higher air pressure in respect to the intermediate 
rooms; gowning rooms have negative air pressure towards intermediate rooms and the hallway. This also 
assures protection of the outside environment from potentially biohazardous materials produced in the 
Biologic Therapy Core facility. Air pressure gradients are maintained by individual HVAC units for each 
room. The manufacturing rooms are certified and maintained as Class 10,000, the intermediate rooms 
are certified and maintained as Class 100,000. 
Every manufacturing lab is equipped with a 6-foot biosafety cabinet and a dual chamber incubator. 
Sufficient room is available per manufacturing lab to accommodate specialized equipment (e.g. clinical 
grade magnetic bead cell separator) for different manufacturing processes. In the largest manufacturing 
Figure 3: Siteman Cancer Center Biologic Therapy Core Facility floor plan. HW: Hallway, G1: 
Gowning Room 1; G2: Gowning Room 2; IEN 1: Intermediate Entry Room 1; IEN 2: Intermediate Entry 
Room 2; M1 to M3: Manufacturing Rooms 1 to 3; M4 to M6: Manufacturing Rooms 4 to 6; IEX: 
Intermediate Exit Room; DG: Degowning Room; O: Office; A: Autoclave Room. 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 29 of 112 
room, besides the 6ft biosafety cabinet, a second 4ft biosafety cabinet is installed for work with infectious 
agents. Manufacturing room 1 can also be switched to negative air pressure and BSL-3 containment 
(vector manufacturing), and an IBC approved protocol to accomplish this task is in place. 
Quality control and monitoring plans are in effect. Daily environmental cleaning is performed by dedicated 
personnel. The facility is electronically monitored continuously, daily monitoring by core personnel 
ensures adherence to standards and is prescribed by SOPs. Environmental monitoring includes non 
viable and viable particle enumeration with limits more stringent than the standards prescribed in the 
USP. 
GMP regulations for even Phase 1 clinical trials are in place to assure a cellular or biological product to 
be safe and free of contaminants. 
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 30 of 112 
6.3 
Manufacturing process 
Overview of the neoantigen DNA vaccine manufacturing process. Schematic outline of product 
manufacture, in-process testing, and product release tests. 
 
 
Figure 4: Overview of the neoantigen DNA vaccine manufacturing process. Schematic outline of 
product manufacture, in-process testing, and product release tests. 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 31 of 112 
Design of the neoantigen DNA vaccine. Neoantigen DNA vaccines will be designed in the Gillanders 
laboratory at WUSM. Up to 20 candidate neoantigens from each patient will be integrated into the 
neoantigen DNA vaccine. Neoantigens will be identified and prioritized based on the results of next-
generation sequencing studies, epitope prediction algorithms, and in vitro studies. 
Synthesis of the polyepitope insert. A polyepitope insert integrating up to 20 of the highest priority 
neoantigens will be designed in the Gillanders laboratory. This insert will then be synthesized by Blue 
Heron Biotech. The insert will then be cloned into the pING parent vector by Blue Heron Biotech. The 
pING vector containing the polyepitope insert will be sequenced by Blue Heron Biotech. The plasmid will 
then be used to establish a master cell bank at WUSM as detailed below. 
Creation of a master cell bank. Master cell banks for each individual neoantigen DNA vaccine will be 
constructed by transformation of E. coli strain DH5 alpha with each patient’s unique pING-polyepitope 
plasmid. Briefly, 15 vials of transformed bacteria will be prepared and stored at -80°C in the Siteman 
Cancer Center Biologic Therapy Core Facility. The Johnson Controls monitoring system continuously 
monitor the freezer used for storage.  The system triggers an alarm and electronically notifies staff when 
temperature values are out of specification. In addition, daily manual monitoring is applied. For each 
bacterial culture production, a vial of the glycerol stock will be taken from the master cell bank and used 
for culture inoculation. 
Samples from each master cell bank will be characterized for host strain identity, contaminating 
organisms, viable cells, plasmid activity, and plasmid identity as detailed below in testing to be performed 
under GLP conditions at Barnes-Jewish Hospital and Washington University School of Medicine. Of note, 
in previous sequencing reactions we have identified three variations in pING vector sequence. These 
variations are in non-coding areas of the plasmid DNA, and are not expected to have any functional 
significance. They are also present in the pING parent vector obtained from MSKCC, suggesting that the 
published sequence for the pING vector may not be accurate. Please see Table 1 below. 
TABLE 1: Master cell bank validation for neoantigen DNA vaccines 
Criteria 
Method 
Specification 
Test site 
Host strain identity 
Growth on 
selective media 
Growth of E. coli 
Barnes-Jewish Hospital 
Microbiology Laboratory 
Contaminating 
organisms 
Growth on 
selective media, 
No growth 
Barnes-Jewish Hospital 
Microbiology Laboratory 
Viable cells 
Growth in LB agar 
Growth of E. coli 
Barnes-Jewish Hospital 
Microbiology Laboratory 
Plasmid activity 
Transfect cells, 
RT-PCR 
Detection of polyepitope 
construct mRNA 
Gillanders Laboratory 
Plasmid identity 
DNA sequencing 
Sequence as predicted 
Gillanders Laboratory 
Plasmid identity 
Restriction 
analysis 
As expected, no 
extraneous bands 
Gillanders Laboratory 
Plasmid DNA fermentation. The plasmid vectors will be grown in E. Coli strain DH5alpha. The host 
bacteria will be grown in 500 mL LB broth cultures in a bacterial shaker at 37°C for 16 hours. Antibiotic 
selection (kanamycin) will be applied to increase plasmid yield.  
Plasmid DNA purification. The plasmid DNA will be purified using reagents obtained from Sigma Aldrich 
(Saint Louis, MO). Distinct lots of the Genelute HP Select Plasmid Gigaprep Kit from Sigma-Aldrich (St. 
Louis, MO) will be used for the manufacture of the plasmids. The following narrative is a brief description 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 32 of 112 
of the protocol. The composition of buffers, solutions, and chromatography material is included in Table 2 
below. (Please note that the precise composition of the resin and solutions are considered by Sigma 
Aldrich to be proprietary). The harvested bacteria will be recovered by centrifugation, and then 
resuspended in a cell suspension buffer containing RNase A. Lysis buffer will be added and a 
homogenous lysate will be obtained by gentle inversion. After neutralization of the bacterial lysate, a 
lysate clearing agent will be added and allowed to stand for 5 minutes for precipitates to form. The lysate 
will be cleared by filtration, and a binding solution optimized for endotoxin-free plasmid purification will be 
added. The lysate with binding solution will then run through an equilibrated column where the plasmid 
DNA is captured onto a silica membrane in the presence of high salt, while endotoxins are prevented 
from adsorbing to the membrane. The flow-through will be discarded, and the contaminants in the column 
will be removed in three wash steps. Finally, the bound plasmid DNA will be eluted in endotoxin-free 
water. The eluted DNA will be precipitated by adding 0.1 volume of 3 M sodium acetate and 0.7 volumes 
of isopropanol. The DNA will be recovered by centrifugation at 14,000 x g in a fixed angle rotor. A wash 
and pelleting of the final DNA with 70% ethanol will be performed and the resulting purified DNA will be 
air dried in a biosafety cabinet. All vessels and buffers used for DNA preparation will be sterile for single 
use, or will be autoclaved in a monitored and quality controlled autoclave in the Biological Therapy Core 
facility. 
The only product of animal origin is the RNase solution. This is isolated from bovine pancreas and we 
have Certificates of Origin that clearly indicate that these were derived from a New Zealand herd. These 
animals passed ante and post mortem testing for viral pathogens; the samples were treated with low pH 
(1.7) for 12 hours to inactivate pathogenic viral agents; and no animal protein was fed to these animals.  
Glycerol will be used in our manufacturing process. However, all Lot #s of the glycerol utilized will be of 
plant origin. 
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 33 of 112 
TABLE 2: Composition of buffers, solutions, and chromatography material 
Solution 
Composition, Sigma # 
Wash Solution 2 
Tris-HCl (T3038), NaCl (S5150) 
Neutralization Solution 
Trizma-HCl (T5941) 
Binding Column 
Guanidine Thiocyanate (G9277), EDTA (E7889) 
Column Preparation 
Solution 
NaOH (S2770) 
Resuspension/RNase A 
Solution 
Tris-HCl (T3038), EDTA (E7889) 
Lysis Solution 
NaCl (S5150), Tris-HCl (T3038), EDTA (E7889), Ocytl-beta-
Thioglucopyranoside (O6004) 
Lysate Clearing Agent 
Guanadine HCl (G3272), Tris-HCl (T2413), EDTA (E7889), Tween20 
(P7949), Triton X-100 (T8787), Hydrochloric acid (H9892) 
Binding Solution 
Guanadine HCl (G3272), Tris Acetate (T1258), Isopropanol (I0398), 
NaOH (S2770)  
Column Prep Solution 
NaOH (S2770) 
EndoCleaning Solution 
NaOH (S2770), Tween20 (P7949) 
Wash Solution 1 
NaCl (S5150), Tris-HCl (T3038) 
Wash Solution 2 
Tris-HCl (T3038), NaCl (S5150) 
6.4 
Product formulation 
The eluted plasmid preparations will be resuspended in sterile, preservative free-PBS. A small sample 
(100 µL) will be removed aseptically and the concentration tested using a certified NanoDrop 
spectrophotometer, Qubit Fluorometer or similar device. Typically, the eluted DNA is in the range of 3-4 
mg/mL. The DNA product will be diluted to 2 mg/mL using sterile, preservative-free PBS, and aseptically 
added to 2.0 mL cryovials from Nalgene (Catalog# 5000-0020, Lot# 616412). The vials are made of 
polypropylene with a high-density polyethylene closure. They are externally-threaded for aseptic 
technique. They have a sealing ring in conjunction with specially designed threads. A white marking area, 
fill line, and graduations are printed on the vials with white paint. They are radiation-sterilized, non-
cytotoxic, non-pyrogenic, and are RNase/DNase-free. 
We plan to synthesize enough DNA for approximately 20 vials. We anticipate that 12 vials will be required 
for vaccination (2 vials per time point times 6 time points), and 5 vials will be required for product release 
tests. We are targeting 20 vials to have a reserve stock. 
Following vialing, we anticipate that 4 vials (#1, 4, 10, 19) will be used for product release testing. Vials 1 
and 19 will be used to test sterility and represent the beginning and end of the vialing procedure. Two 
additional vials will be used for the other product release tests listed in Table 3. 
If any contamination would be detected in our plasmid samples, we would initiate an alcohol precipitation 
step. This procedure is basically the final stage of the gigaprep purification, where the soluble DNA is 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 34 of 112 
precipitated using 2x volumes of cold isopropanol and the DNA is pelleted by centrifugation. The pellet is 
washed in 70% ethanol and air dried. This procedure is a standard method for eliminating any 
microbiologic contamination in DNA. 
6.5 
Product release tests 
A key issue in the quality control of plasmid DNA for human use is the development of analytical 
methodologies capable of fully characterizing the product in its final form, ensuring the production of a 
consistent product. The following list of release criteria, analytical methodologies, and specifications has 
been adapted from [73]. Neoantigen DNA vaccines will be tested for the following specifications. 
TABLE 3: Neoantigen DNA vaccine product release tests 
Criteria 
Method 
Specification 
Test Site 
Plasmid activity 
Transfect cells, RT-PCR 
Polyepitope mRNA 
present 
Gillanders Laboratory 
Residual kanamycin 
USP 29 <81> 
< 8 mcg/mL 
Alcami or equivalent 
Plasmid 
homogeneity 
Agarose gel 
electrophoresis 
≥80% closed circular 
Gillanders Laboratory 
Residual RNA 
Agarose gel 
electrophoresis 
Less than 1% 
Gillanders Laboratory 
Residual bacterial 
chromosomal DNA 
Agarose gel 
electrophoresis 
Less than 1% 
Gillanders Laboratory 
Plasmid identity 
Restriction analysis 
As expected, no 
extraneous bands 
Gillanders Laboratory 
Appearance 
Visual 
Clear and colorless, 
free of particulates 
Gillanders Laboratory 
pH 
Calibrated pH meter 
Report pH 
Biologic Therapy Core 
Facility 
DNA concentration 
Nano-drop 
spectrophotometer 
Greater than 1 
mg/mL 
Gillanders Laboratory 
Residual proteins 
BCA reagent colorimetric 
Less than 1% (< 21 
µg/mL) 
Gillanders Laboratory, 
Biologic Therapy Core 
Facility, or equivalent 
Residual 
isopropanol, EtOH 
USP <467> 
Less than 5000 ppm 
PACE Analytics or 
equivalent 
Endotoxin 
Chromogenic Limulus 
amebocyte lysate assay 
Less than 175 
EU/mL 
Biologic Therapy Core 
Facility 
Sterility 
USP <71> 
No growth 
Biologic Therapy Core 
Facility 
Alternatively, we will perform studies to demonstrate that the manufacturing process removes residual 
kanamycin, RNA, bacterial chromosomal DNA, proteins, isopropanol and EtOH below the specification 
levels, and not perform these residual studies on each lot.  To date, testing on each lot has shown that 
residual kanamycin is undetectable and residual isopropanol and EtOH is consistently below the 
specification. This testing will no longer be performed. The criteria for sterility meets specification if no 
growth after 7 days. The sterility culture will be held and monitor for 14 days as per USP <71>. 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 35 of 112 
6.6 
Stability 
The neoantigen DNA vaccines will be stored in the Biologic Therapy Core Facility in a -80°C freezer. The 
freezer used for storage is monitored 24/7 by Johnson Controls monitoring system with dedicated on-call 
personnel to address any aberrant occurrences. In addition, daily manual monitoring is applied. Overall, 
we anticipate no problems with plasmid stability as stability is considered to be one of the advantages of 
the DNA vaccine platform, and plasmid DNA formulated in PBS is considered to be very stable. 
We are currently storing similar investigational DNA vaccines in the Biologic Therapy Core Facility in a -
80°C freezer. These investigational DNA vaccines were manufactured in the Biologic Therapy Core 
Facility using an almost identical manufacturing process as the one proposed here, are based on the 
pING parental vector and are thus very similar in size and composition, and are formulated similarly in 
PBS at 2 mg/mL. These DNA vaccines have remained stable over a 3- year period.  
We do not anticipate that long-term stability testing will be required. As each lot of vaccine is personalized 
to a specific patient, we anticipate that the vaccine will be used within 6 months of completion of the 
product release tests. 
6.7 
Labels 
Each neoantigen DNA vaccine product will be made as a single lot ensuring that the labeled product is 
unique and consistent for this trial. Please see the sample label below. Prior to administration a time out 
will be performed to confirm subject identity, and the investigational agent to be administered. This will be 
performed by the clinical research associate, the oncology nurse trained to administer the vaccine by 
electroporation and the experimental pharmacist. 
SCC Biologic Therapy Core Facility 
Saint Louis, MO 
Store at -80°C 
WUSM PPDV #2 
Subject Study #, Subject Initials 
LOT # 
1 
Vial # 
003 
 
Concentration 
2mg/mL 
Volume 
1.2mL 
Date 
mm/dd/yyyy 
CAUTION Investigational drug limited by federal law to investigational use 
6.8 
Siteman Cancer Center Investigational Pharmacy 
The Investigational Pharmacy is located on the 7th floor of the Center for Advanced Medicine within the 
chemotherapy administration area of the Siteman Cancer Center. The pharmacy is locked and secured 
with entry only via an authorized swipe card entry system. Three pharmacists, including a pharmacist with 
responsibility for investigational agents, and two technicians are employed full-time. Two freezers (-70ºC 
and -20ºC) and one refrigerator have been designated for studies. Room temperature shelving extends 
over 300 feet. There are three IV admixture hoods. Two vertical flow hoods are used for preparation of 
chemotherapy agents and a laminar flow hood is used for non-chemotherapy agents. There were a total 
of 13,260 chemotherapy prescriptions filled in 2004. Pharmacy hours of operation are Monday through 
Friday from 7:00 am to 5:30 pm. The investigational pharmacist will obtain the vaccine from the TBC 
Facility and dispense the vaccine to the nurse who will be administering the agent to the patient. 


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6.9 
Investigational agent administration 
Each DNA vaccination will be 1 mL vaccine administered intramuscularly using an integrated 
electroporation administration system (TDS-IM system, Ichor Medical Systems). At each vaccination time 
point, patients will receive two injections at separate sites. 
6.9.1 
Introduction to electroporation 
Electroporation (EP) is a potent delivery technique based on the in vivo application of electrical fields that 
may improve immune responses following the administration of plasmid DNA vaccines. The EP effect is 
induced through the propagation of electrical fields of sufficient magnitude and duration within a target 
region of tissue. These electrical fields produce a transient increase in the membrane permeability of cells 
exposed to the electrical field, allowing enhanced intracellular uptake of agents distributed within the 
interstitium of the local tissue. Of note, agent delivery occurs only where the agent of interest is present 
contemporaneously with the propagation of threshold level electrical fields. Numerous non-clinical studies 
have demonstrated that the application of EP can enhance gene expression and immune responses 
following plasmid DNA vaccine delivery. 
Although DNA vaccines encoding antigens from a variety of cancers and infectious pathogens have 
exhibited considerable promise in non-clinical studies, clinical experience to date suggests that current 
administration methods may have insufficient potency to achieve the desired immune responses in 
humans. Poor delivery efficiency and low levels of antigen expression have been implicated as factors 
contributing to the sub-optimal responses observed to date [74, 75]. 
Electroporation (EP) is a potent physical delivery technique based on the in vivo application of electrical 
fields. The propagation of electrical fields of a sufficient magnitude within a target tissue induces a 
transient increase in cell membrane permeability, resulting in a significant improvement of intracellular 
uptake of exogenous substances. Shortly after pulse delivery, the cell membrane function stabilizes and 
cells within the affected tissue resume normal function. EP has been shown in non-clinical studies to be a 
potent method for delivery of DNA vaccines [40, 76-78]. 
EP has several important advantages that make it an appealing method for delivery of DNA vaccines. 
First, EP can reliably enhance delivery and expression of DNA antigens by 2-3 orders of magnitude. This 
improvement in delivery has been shown to enhance both cellular and humoral immune responses. 
Second, EP is a non-viral delivery method. As such, it has a favorable safety profile and does not elicit 
unwanted immune responses against the vector. Thus, the technique is particularly appropriate for 
indications likely to require multiple vaccine administrations to achieve response (e.g. prime / boost). It is 
also advantageous because the absence of immunogenic viral antigens minimizes potentially deleterious 
antigen competition, thereby ensuring that the immune response is concentrated on the antigen of 
interest. Lastly, unlike other non-viral approaches, EP is capable of inducing consistent responses from 
subject to subject, even at relatively low DNA doses. 
6.9.2 
The TDS-IM Electrode Array System 
The TDS-IM device is supplied by Ichor Medical Systems, Inc. The TDS-IM device utilizes the in vivo 
application of electrical fields to enhance the intracellular delivery of agents of interest in a targeted region 
of tissue (EP). The device complies with the applicable safety and electromagnetic compatibility 
requirements of International Electrotechnical Commission (IEC) 60601-1. During this study, the device 
will be operated according to the TDS-IM User Manual and applicable study-specific procedures to 
ensure consistent and safe utilization. 
Each TDS device consists of 3 parts: A pulse stimulator, an integrated applicator, and a single-use 
application cartridge (see Figure below). The components are manufactured in ISO13485-compliant, 
FDA-registered facilities including BIT MedTech, LLC. (Pulse Stimulator) and Life Science Outsourcing, 
Inc. (Integrated Applicator and Application Cartridges. 


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6.9.3 
Summary of TDS-IM Clinical Experience to Date 
To date, formal safety studies have been conducted to assess the TDS-IM as the means for delivery of 
nine DNA vaccine candidates. This includes studies of a melanoma DNA vaccine encoding a xenogeneic 
form of the tyrosinase antigen that uses the same vector backbone (pING) as the Mammaglobin-A DNA 
vaccine candidate (see Table 1). Safety studies for the various DNA vaccine candidates have included 
repeat dose studies in rabbits to assess safety and toxicology, and studies in rats to assess DNA vaccine 
biodistribution, persistence, and potential for integration into host DNA. 
In the safety and toxicology studies, notable findings associated with electroporation-mediated delivery of 
DNA vaccines were limited to localized inflammatory responses of mild to moderate severity at the site of 
administration (for an example, see Dolter, K.E., et al. Immunogenicity, safety, biodistribution and 
persistence of ADVAX, a prophylactic DNA vaccine for HIV-1, delivered by in vivo electroporation. 
Vaccine, 2011. 29(4): p. 795-803). The injection site findings were most prominent in tissue samples 
obtained 48–72 hours after administration at that site. Tissue samples obtained from administration sites 
14–43 days after administration indicate progressive resolution of the local inflammatory responses over 
time. 
The results of the biodistribution studies indicated negligible systemic uptake of vaccine DNA following 
EP-based intramuscular delivery, with no significant differences observed among the different DNA 
vaccine candidates tested to date. Persistence analysis indicated that the presence of vaccine DNA 30 to 
90 days after administration was confined to the tissues at the site of administration (muscle and skin), 
and only at very low levels (< 1,000 copies/µg host genomic DNA), suggesting minimal risk for potential 
integration of vaccine DNA into host DNA (for an example, see Dolter, 2011). 
Reports describing the results of the tyrosinase GLP safety and toxicology studies are available in BB 
IND 13275); a letter of cross reference to this IND has been provided by Ichor. 
 
TDS-IM Components
TDS-IM Integrated 
Applicator
TDS-IM Application 
Cartridge
TDS Pulse Stimulator
Syringe
TDS-IM Components
TDS-IM Integrated 
Applicator
TDS-IM Application 
Cartridge
TDS Pulse Stimulator
Syringe


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Breast Cancer Program 
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6.9.4 
Summary of TDS-IM clinical experience to date 
The TDS-IM investigational device is currently being used as the means for DNA vaccine delivery in both 
the therapeutic and prophylactic setting. Six clinical trials of the TDS-IM device have been completed as 
detailed in the Table below. 
TDS-IM Based DNA Vaccine Administration: Completed Clinical Trials 
ClinicalTrials.gov 
Study # 
Vaccine Candidate 
Subject 
Population 
NCT00545987 
Multigenic HIV-1 DNA vaccine candidate (ADVAX) 
Healthy, HIV 
uninfected adult 
volunteers 
NCT00471133 
Xenogeneic tyrosinase DNA vaccine candidate 
(pINGmuTyr) 
Patients with 
Stage IIB-IV 
melanoma 
NCT01169077 
Multi-epitope malaria DNA vaccine (EP-1300) 
Healthy adult 
volunteers 
NCT01502345 
Multi-antigen hantavirus DNA vaccine (pWRG/HTN-M(x) 
and pWRG/PUUV-M(s2)) 
Healthy adult 
volunteers 
NCT01641536 
Multi-antigen HBV DNA vaccine administered with a DNA-
based human IL-12 adjuvant (HB-110) 
HBV infected adult 
volunteers 
NCT01496989 
Multi-antigen HIV DNA vaccine (HIV-MAG) administered 
with or without a DNA-based human IL-12 adjuvant 
(GENEVAX) prior to or after administration of an 
adenovirus vector (Ad35GRIN) 
Healthy, HIV 
uninfected adult 
volunteers 
The TDS-IM device is currently being evaluated in seven ongoing clinical studies as detailed in the Table 
below. 
TDS-IM Based DNA Vaccine Administration: Ongoing Clinical Trials 
ClinicalTrials.gov 
Study # 
Vaccine Candidate 
Subject Population 
NCT01138410 
Epitope-based TRP-2 melanoma vaccine (SCIB-1) 
Patients that are HLA A1, 
A2, A24, or B35 positive 
with AJCC stage III-IV 
melanoma 
NCT01859325 
Multi-antigen HIV DNA vaccine (HIV-MAG) 
administered with or without a DNA-based human 
IL-12 adjuvant (GENEVAX) prior to administration 
of an vesicular stomatitis virus vector (rVSVgag) 
HIV infected adult 
volunteers 
NCT01578889 
Multi-antigen HIV DNA vaccine (HIV-MAG) 
administered with or without a DNA-based human 
IL-12 adjuvant (GENEVAX) prior to administration 
of an vesicular stomatitis virus vector (rVSVgag) 
Healthy adult volunteers 
NCT01266616 
Multi-antigen HIV DNA vaccine (HIV-MAG) 
administered with or without a DNA-based human 
IL-12 adjuvant (GENEVAX) 
HIV infected adult 
volunteers 
NCT01634503 
Multi-antigen HPV DNA vaccine administered with 
a DNA-based human IL-12 adjuvant (GX-188E) 
Patients with Grade 3 
Cervical Intraepithelial 
Neoplasia 
NCT01493154 
HPV DNA vaccine administered with a DNA-based 
calreticulin adjuvant (pNGVL-4a-CRT/E7 (detox)) 
HPV-associated squamous 
cell carcinoma of the head 
and neck 
NCT01984983 
VEEV DNA Vaccine Candidate 
Healthy adult volunteers 
To date, the 13 clinical trials that have been completed or are currently ongoing have enrolled over 350 
subjects in the electroporation arms of the studies (including subjects receiving either the DNA vaccine 


Washington University School of Medicine 
Protocol Date 04/24/2023 
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Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 39 of 112 
candidate or placebo). The device has been used for administration of DNA injections of up to 1.0 ml 
volume and 4.0 mg DNA dose per injection site. Subjects have received the vaccine candidate either as a 
single injection in one muscle site (total DNA dose up to 4.0 mg per administration time point) or as 2 
injections in 2 separate muscle sites (total DNA dose up to 8.0 mg per administration time point). Subjects 
given the DNA dose as a single injection have received up to 5 TDS-IM injections at up to 4.0 mg DNA, 
and subjects administered the DNA dose in 2 injections have received up to 5 administrations (i.e., 10 
total TDS-IM injections).  
Adverse responses reported in association with use of the device include localized muscle contractions 
and associated discomfort/pain during the application of EP, minor cutaneous bleeding at the site of 
injection, and transient injection site soreness of mild to moderate severity, typically resolving within 24–
72 hours following administration. Several subjects in the 2 melanoma studies have reported 
lightheadedness immediately following procedure application which, in some cases, was accompanied by 
a decrease in blood pressure. In one subject, enrolled in the xenogeneic tyrosinase study, this was 
followed by a brief syncopal episode (~30 seconds duration) shortly after procedure application. The 
subject recovered without incident. At the time of enrollment, the subject indicated a life long history of 
sinus bradycardia of unknown origin, which was confirmed by electrocardiogram (EKG) during screening. 
Multiple EKGs performed after the syncopal episode indicated no changes from pre-procedure baseline. 
Based on the judgment of the investigator, the subject was withdrawn from the study and the study 
eligibility criteria were modified to exclude subjects with sinus bradycardia. No other serious or 
unanticipated adverse events attributed to the device or administration procedure have been observed. 
The results of the completed HIV-1 ADVAX vaccine study in healthy subjects have been published [79]. 
As reported in the abstract, eight volunteers each received 0.2 mg, 1 mg, or 4 mg ADVAX or saline 
placebo via electroporation (EP), or 4 mg ADVAX via standard intramuscular injection at weeks 0 and 8. 
A third vaccination was administered to eleven volunteers at week 36. EP was safe, well-tolerated and 
considered acceptable for a prophylactic vaccine. EP delivery of ADVAX increased the magnitude of HIV-
1-specific cell mediated immunity by up to 70-fold over IM injection, as measured by gamma interferon 
ELISpot. The number of antigens to which the response was detected improved with EP and increasing 
dosage. Intracellular cytokine staining analysis of ELISpot responders revealed both CD4+ and CD8+ T 
cell responses, with co-secretion of multiple cytokines. Briefly, results from this study indicate that EP-
based delivery with the TDS-IM device at ADVAX DNA doses ranging from 0.2–4.0 mg was safe and 
effective in improving the magnitude, breadth and durability of cellular immune responses to a DNA 
vaccine candidate. Assessment of the tolerability of the EP procedure by questionnaire after each 
administration indicates that the procedure is generally acceptable for use in healthy subjects. 
6.9.5 
Preparation of the vaccine 
Doses of the neoantigen DNA vaccines will be prepared in the Siteman Cancer Center Investigational 
Pharmacy and delivered to the clinic on the day of vaccination. Vials will be thawed at room temperature 
on the day of study visits. After thawing the vaccine vial(s), the investigational pharmacist will mix the 
vial(s) completely by inverting the vial(s) at least 10 times (one inversion = one 180° turn of the wrist and 
back). The vaccine will then be withdrawn into 3.0 mL Becton Dickinson Model 309585 syringe under 
sterile conditions by the research pharmacist. For administration, a 22 gauge 1.5 inch injection needle will 
be affixed to the syringe. Once the dose is prepared, the research pharmacist will load the syringe into a 
TDS-IM Application Cartridge in a manner consistent with the instructions provided in the TDS-IM User’s 
Guide.  
6.9.6 
Vaccine administration 
Detailed instructions for procedure administration using the TDS-IM device are included in the TDS-IM 
User’s Guide. Briefly, the TDS-IM Application Cartridge is loaded into the Integrated Applicator. The Pulse 
Stimulator is connected to an appropriate power source, turned on, and then connected to the Integrated 
Applicator through the supplied cable. Prior to administration, the skin at the site of administration is 
prepared according to the standard procedures for a conventional intramuscular injection. Once the skin 
has been disinfected, the skin around the injection site is held firmly while the Application Cartridge is 
placed against the injection site at a 90° angle. An indicator light on the device notifies the user that 
sufficient pressure has been applied. The activation button is then depressed, causing the electrodes and 
injection needle to be inserted into the target tissue. An automated safety check is performed, and, if 


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Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
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passed, allows the injection of the study vaccine into the muscle. The procedure will conclude with a 
series of brief, localized muscle contractions at the administration site. The procedure will require 
approximately 7-10 seconds to complete, during which time the site is held firmly and with the device 
depressed against the skin. Once the indicator light on the device indicates that the procedure is 
complete, the device is withdrawn and site is covered with a sterile covering and pressure applied with 3 
fingers for 1 minute. 
6.10 Investigational agent accountability 
6.10.1 Documentation 
The investigational agent will be prepared by the study pharmacist at the Siteman Cancer Center 
Investigational Pharmacy. The study pharmacist will be responsible for maintaining an accurate record of 
the codes, inventory, and an accountability record of vaccine supplies for this study. Electronic 
documentation as well as paper copies will be used. 
6.10.2 Disposition 
The empty vials and the unused portion of a vial will be discarded in a biohazard containment bag and 
incinerated or autoclaved. Any unopened vials that remain at the end of the study will be returned to the 
production facility or discarded at the discretion of the principal investigator in accordance with policies 
that apply to investigational agents. Partially used vials will not be administered to other subjects or used 
for in vitro experimental studies. They will be disposed of in accordance with institutional or pharmacy 
policy. 
 
 


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Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
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7 
NONCLINICAL STUDIES 
7.1 
Overview 
The neoantigen DNA vaccine strategy is designed to target neoantigens present in the cancer, but absent 
in normal tissues. 
One of the reasons that we have pursued clinical development of a neoantigen DNA vaccine strategy 
targeting neoantigens is because we believe that this strategy has the potential to be safer than strategies 
targeting shared tumor antigens. Shared tumor antigens are typically expressed at high levels in the 
tumor, but are also typically expressed at lower levels in some normal tissues. Expression of shared 
tumor antigens in normal tissues may increase the risk of autoimmunity. Neoantigens are present only in 
the tumor. In addition, our next-generation sequencing-based epitope prediction algorithm prioritizes 
epitopes where the mutant epitope (but not the wildtype epitope) can bind to restricting HLA molecules. 
This decreases the potential that immune responses targeting neoantigens will be cross-reactive with 
wildtype antigens. 
We do not think that GLP safety and toxicology studies will provide significant insight into the safety of the 
neoantigen DNA vaccine strategy. First, it is impossible to know a priori what mutations will be present 
and/or prioritized in individual patients. We estimate that there are as many as 7 million potential 
neoantigens that could be targeted by our approach. Only a limited number of mutations could be 
targeted in GLP safety and toxicology studies. Second, to our knowledge, no mammary tumor models 
exist that would be relevant for GLP safety and toxicology studies. Third, the pING parental vector has 
proven to be safe in phase 1 clinical trials. 
We are not proposing to perform GLP biodistribution and integration studies at this stage in development. 
There is extensive GLP information available about the biodistribution and integration of DNA vaccines 
following electroporation with the TriGrid device. This includes GLP information about biodistribution and 
integration of DNA vaccines using the pING parent vector. Additional information about the biodistribution 
and integration of DNA vaccines following electroporation with the TriGrid device is summarized in the 
IND application. 
The dosing described in this study is based on previous DNA vaccine studies as well as an ongoing 
phase I clinical trial (NCT02348320). 
7.2 
Nonclinical studies 
It has long been known that there is a dynamic relationship between the immune system and cancer. This 
dynamic relationship has been studied in detail, ultimately resulting in the establishment of the cancer 
immunoediting concept [80-87].  
We have recently focused on defining the antigens recognized by the immune system during the cancer 
immunoediting process. These studies, summarized below, demonstrate that neoantigens are important 
tumor rejection antigens, and provide strong support for our personalized breast cancer vaccine strategy. 
Specifically, we have developed next-generation sequencing and epitope prediction algorithms to identify 
and prioritize neoantigens. We will use these algorithms in the proposed clinical trial. The preclinical data 
supporting the use of these algorithms are presented below. 
In initial studies we used a combination of next-generation sequencing and epitope prediction algorithms 
to identify neoantigens in the d42m1 MCA sarcoma line. These algorithms identified one particular 
mutation (an R913L mutation of SPTBN2) as a top candidate, and subsequent analyses confirmed that 
this mutant tumor-specific antigen functioned as an immunodominant tumor rejection antigen. These 
studies were published in Nature [20]. 
The d42m1 MCA sarcoma is an unedited tumor, and would therefore be expected to express strong 
tumor rejection antigens. We have since turned our attention to examining the epitope landscape in 
edited MCA sarcomas that develop in immunocompetent wildtype mice. Specifically, we have asked the 
following questions: (1) Can the next-generation sequencing and epitope prediction algorithms be used 
more broadly to identify and prioritize important neoantigens in less immunogenic tumors? (2) Can the 


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Protocol Date 04/24/2023 
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Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 42 of 112 
next-generation sequencing and epitope prediction algorithms be used to prioritize antigens for immune 
targeting and/or neoantigen vaccine therapy? 
To address these questions, we focused initial efforts on d42m1-T3. d42m1-T3 is a clone of d42m1 that 
lacks the immunodominant rejection antigen, mutant SPTBN2, and forms progressively growing tumors in 
wildtype mice. We specifically chose the d42m1-T3 clone because d42m1-T3 shares with naturally edited 
sarcomas the ability to form progressively growing tumors in wildtype mice and shows a similar sensitivity 
to checkpoint blockade.  
To identify and prioritize neoantigens from the d42m1-T3 we used optimized next-generation sequencing 
and epitope prediction algorithms. Specifically, we pipelined the candidate mutant tumor-specific antigen 
sequences into four different MHC class I epitope prediction algorithms and calculated the median 
predicted affinity for binding to the relevant class I MHC alleles. We then applied filters that account for 
proteasomal processing of the antigen and differences in MHC class I binding affinity between mutant 
and native sequences to prioritize the neoantigens. We also deprioritized hypothetical Riken proteins.  
Of the top 61 prioritized candidates, 20 were eliminated by the filtering process; including two of the top 
four candidates. Of those that remained, two [G1254V Laminin subunit α4 (mLama4) and A506T alpha-
1,3 glucosyltransferase (mAlg8)] were clearly favored above the others based on predicted binding 
affinity.  
To test whether these two “best” neoantigens were biologically relevant, we generated tumor-specific 
CD8+ T cell lines from the spleens of three independent mice that had rejected d42m1-T3 cells after anti-
PD-1 therapy and showed that each T cell line (CTL-62, CTL-73, CTL-74) displayed specificity for d42m1-
T3 but not an unrelated sarcoma, F244. To determine if the “prioritized” neoantigens were recognized by 
anti-d42m1-T3 T cell lines, we incubated 8 amino acid synthetic peptides corresponding to each of the 
top 61 initially predicted H-2Kb neoantigens with irradiated splenocytes and CTL-74 T cells and monitored 
IFN-γ production. The mLama4 and mAlg8 peptides strongly stimulated CTL-74 T cells, with mLama4 
inducing ~10x more IFN-γ  than mAlg8. No other predicted mutant epitope induced significant levels of 
IFN-γ  production in this assay. Similar results were obtained with the other two d42m1-T3 specific CD8+ 
T cell lines. Subsequent dose response experiments showed that mLama4 stimulated the tumor-specific 
T cell lines to a greater extent than mAlg8 and that the T cells reacted specifically with mutant but not 
native peptides.  
We then used four experimental systems to confirm that our optimized epitope prediction algorithms 
accurately prioritized neoantigens. First, together with the groups of Hans-Georg Rammensee in 
Tübingen and Ruedi Abersold in Zurich we detected mLama4 and mAlg8 peptides bound to H-2Kb on 
d42m1-T3 tumor cells. To our knowledge this is the first time that mutant class I epitopes have been 
detected bound to tumor cell-associated MHC class I. Second, using PE-labeled H-2Kb tetramers carrying 
mLama4 or mAlg8 peptides, CD8+ T cells with specificities for these two epitopes were found to 
accumulate in d42m1-T3 tumors in αPD-1 treated mice and reached peak values just prior to tumor 
rejection on day 12. Consistent with the results of the T cell stimulation experiments, mLama4-specific T 
cells were present in significantly higher numbers in the tumor than mAlg8-specific T cells. No mLama4- 
or mAlg8-specific T cells were observed in irrelevant, checkpoint blockade-sensitive F244 tumors. Third, 
vaccination of naïve WT mice with mutant-Lama4 or mutant-Alg8 short peptide vaccines (8mer) induced 
strong CD8+ T cell responses that were specific for the mutant, but not the WT epitope (mLama4 = 1650 
SFC/106 cells vs. wtLama4 = 75 SFC/106 cells; mAlg8 =606 SFC/106 cells vs. wtAlg8 = 50 SFC/106 cells). 
Fourth, prophylactic vaccination of mice with long peptides (~30mer) corresponding to either the mLama4 
epitope alone, or both the mLama4 and mAlg8 epitopes induced protection against subsequent challenge 
with d42m1-T3 tumor cells. The combined peptide vaccine was more protective than the vaccine 
containing the mLama4 long peptide alone. 
7.3 
Rationale for no GLP safety studies 
We do not think that GLP safety and toxicology studies will provide significant insight into the safety of the 
neoantigen DNA vaccine strategy. 
First, it is impossible to know a priori what mutations will be present and/or prioritized in patients. Next-
generation sequencing of epithelial cancers has demonstrated that there are very few recurrent mutations 


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Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
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present. Mutations can be present in any one of the approximately 20,000 protein-coding genes present 
in the human genome. Even more problematic is that the mutations targeted could be anywhere in the 
corresponding protein. It has been estimated that the average length of a protein in humans is 362 AA. 
Thus, there are potentially 7,240,000 potential neoantigens that could be targeted by our approach. This 
includes only point mutations and does not include mutations resulting from indels. If studies are 
performed in a preclinical model, only a limited number of mutations will be targeted. For example, if we 
design a neoantigen DNA vaccine specific for a murine epithelial cancer targeting 5 genes, this would 
represent only 5 of 7,240,000 potential neoantigens. Even if 1000 GLP safety and toxicology studies were 
performed, each targeting 5 neoantigens, it would still provide information on < 0.07% of potential 
neoantigens. 
Second, to our knowledge, no mammary tumor models exist that would be relevant for the proposed 
preclinical studies. In order to study neoantigens, sequencing analyses of paired tumor/normal tissues are 
required. As such, we would need to study spontaneous tumors in mice, as tumors propagated as cell 
lines do not have corresponding normal DNA to evaluate. There are very few models of spontaneous 
tumor development in wildtype mice. Spontaneous tumors do develop in genetically engineered mice, but 
these oncogene-driven tumors in genetically engineered murine models of cancer typically have a very 
limited number of mutations. We have performed extensive studies in genetically engineered mice, and 
have found that there are very few mutations present in tumors derived from these mice. For example, we 
have performed extensive studies in the p53-null transplant mammary tumor model. p53 is a tumor 
suppressor gene and plays an important role in maintaining genome stability. As such one might expect 
that there would be a significant number of mutations present in p53-null transplant mammary tumors. 
However, we have sequenced > 10 p53-null transplant mammary tumors and have found that there are 
only a limited number of mutations present in each tumor. This is not at all representative of human triple-
negative breast cancers where significantly more mutations are present. Of note, a significant number of 
mutations must be present to reliably identify neoantigens that are immunogenic, as many candidate 
neoantigens are not processed and presented by the immune system, or cannot be recognized efficiently 
by T cells. If only a limited number of mutations is present, it is possible that no neoantigens will be 
identified that are immunogenic. For meaningful GLP safety and toxicology studies of a neoantigen DNA 
cancer vaccine, one of the key considerations is to assess any potential toxicity associated with immune 
responses to the vaccine. If no neoantigens are identified that are immunogenic, the GLP safety and 
toxicology studies will have only limited value as no immune response to the mutant tumor-specific 
antigen will be generated. As such, we are not aware of any mammary tumor models in mice that would 
be appropriate for the GLP safety and toxicology studies. 
7.4 
Rationale for no GLP biodistribution studies 
We are not proposing to perform GLP biodistribution and integration studies at this stage in development. 
There is extensive GLP information available about the biodistribution and integration of DNA vaccines 
following electroporation with the TriGrid device. This includes GLP information about biodistribution and 
integration of DNA vaccines using the pING parent vector. The information available about the 
biodistribution and integration of DNA vaccines following electroporation with the TriGrid device is 
detailed below. 
7.5 
Sequencing pipeline 
Robust next-generation sequencing strategies for the identification of neoantigens will be required for the 
successful clinical translation of personalized cancer vaccine strategies. As such, a major focus of our 
research studies has been the development of cost-effective and accurate next-generation sequencing 
strategies to identify neoantigens and validate the expression of these antigens at the mRNA level.  
A key collaborator on this project is Dr. Elaine Mardis, Co-Director of the Genome Institute at Washington 
University School of Medicine. Mardis is an internationally-known expert in genomics, and cancer 
genomics. The Genome Institute has published seminal papers describing somatic mutations present in 
tumors, providing significant insights into the biology of AML [88, 89] and breast cancer [13, 14]. Mardis 
and her team have developed and optimized the sequencing and data analysis pipelines that will be used 
to identify expressed mutations in subjects’ breast cancers. 


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The first step in the sequencing pipeline is exome sequencing of breast cancer and normal DNA. Exome 
fragments are captured using Nimblegen's "VCRome" exome capture reagent. Background DNA is 
washed away while the bound exome DNA is eluted and sequenced. Separate libraries are made from 
the breast cancer and normal DNA and processed independently. Exome sequencing is performed using 
the Illumina platform. 
Exome sequences from breast cancer and normal DNA are compared separately to the human reference 
sequence and then to one another to identify somatic variation. VarScan 2 software is used to detect 
misaligned sequences and identify structural variants in the breast cancer DNA.  
The second step in the sequencing pipeline is cDNA-capture sequencing. To validate the results of the 
exome sequencing, and confirm expression of the somatic mutations in the breast cancer, cDNA-capture 
sequencing of the breast cancer RNA will be performed. cDNA-capture sequencing is very similar to RNA 
sequencing, but cDNA is captured prior to sequencing to enrich for mRNA. cDNA-capture sequencing is a 
sensitive and accurate methodology to detect expression of somatic mutations at the mRNA level in 
breast cancer. cDNA-capture sequencing is performed using the Illumina platform.  
The third step in the sequencing pipeline is data analysis to identify the expressed somatic mutations. All 
somatic mutations are subjected to a set of filters to exclude “false-positive” calls. The filters include: 
Exome data analysis:  
(1) Normal Coverage > 5x 
(2) Normal Variant Allele Frequency (VAF) < 2% 
(3) Tumor Coverage > 10x 
(4) Tumor Variant Allele Frequency > 20% 
 
Tumor cDNA Capture Data 
(5) Tumor Coverage > 10x 
(6) Tumor Variant Allele Frequency > 20% 
(7) FPKM > 1 
Breast cancer-specific mutations that meet these filters will be prioritized. We will further prioritize indels 
over missense mutations. The mutations will be further analyzed for epitopes using the epitope prediction 
algorithms detailed below (Please see Section 6.4). 
Please see below for an explanation of the terms used above. 
The term coverage is a general term to describe the fold oversampling of a DNA target by sequencing 
data. In covering a target region or genome, increasing depth of coverage leads to increased certainty of 
variant detection. Therefore, 10x “coverage” implies that the given site was independently sequenced at 
least 10 times. 
Variant allele frequency (VAF) is a metric that represents the sensitivity of identifying somatic variant over 
a range of sample purity and sequencing depths. The expected variant allele frequency acts as a 
surrogate for purity. For example, if a heterogeneous somatic variant is present in all tumor cells and the 
tumor cells represent 40% of the sample, then the observed VAF would be 20%. 
FPKM stands for Fragments Per Kilobase of transcript per Million mapped reads. This is a way to 
estimate expression of a gene. In RNA sequencing (and cDNA-capture sequencing), the relative 
expression of a transcript is proportional to the number of cDNA fragments that originate from it. Paired-
end RNA-Seq experiments produce two reads per fragment, but that doesn't necessarily mean that both 
reads will be mappable. For example, the second read is of poor quality. If we were to count reads rather 
than fragments, we might double-count some fragments but not others, leading to a skewed expression 
value. Thus, FPKM is calculated by counting fragments, not reads. If you have a gene with an FPKM of 0, 
you'll see a few reads that align to it. However, if there are 100 or more reads per sample, 1 or 2 reads is 
rather insignificant. So, while you can't really say the gene is not expressed with 100% certainty, you can 
say it was not detected.  


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 45 of 112 
7.6 
Epitope prediction algorithm 
We have developed and optimized an epitope prediction algorithm for the identification and prioritization 
of neoantigens. This optimized epitope prediction algorithm is described below and in the Gubin 2014 
manuscript [19]. We have established this algorithm is collaboration with Dr. Robert Schreiber, an 
internationally-known expert in tumor immunology [20, 80-87]. Schreiber was one of the first to use next 
generation sequencing technologies to identify neoantigens, demonstrating that these antigens are 
important tumor rejection antigens [20]. Schreiber and Gillanders have now optimized the epitope 
prediction algorithm and have demonstrated that cancer vaccines targeting neoantigens are associated 
with antitumor immunity [19].  
The goal of the optimized epitope prediction algorithm is to identify and prioritize up to 20 neoantigens. 
The algorithm uses a combination of binding algorithms, processing algorithms and in vitro binding 
assays. 
Mutations that are expressed in the breast cancer will be identified using the sequencing pipeline outlined 
in the document titled “Sequencing Pipeline.” The predicted amino acid sequences corresponding to the 
expressed mutations will be pipelined through three class I MHC epitope-binding algorithms provided by 
the Immune Epitope Database and Analysis Resource (http://www.immuneepitope.org): (i) Stabilized 
Matrix Method (SMM) [90], (ii) Artificial Neural Network (ANN) [91], and (iii) NetMHCpan [92]. 
A prioritized list of binding epitopes (i.e. IC50 < 500 nM) will be generated after calculating the median 
binding affinity value for each mutant sequence (affinity value expressed as 1/IC50 x 100).  
Filters will be applied to the list to (a) eliminate epitopes that are not processed efficiently by the 
immunoproteosome based on the NetChop algorithm [93] (peptides with a NetChop score > 0.6 will be 
prioritized), (b) deprioritize epitopes from hypothetical proteins or that form a weaker predicted binding 
epitope than that expressed by the corresponding wild type sequence, (c) prioritize mutant epitopes that 
have the highest difference in predicted binding affinity compared to their wild type counterpart, and (d) 
incorporate expression profiles of wild type genes that correspond to the mutant antigen candidates in 
normal vs tumor cells.  
To confirm the ability of prioritized neoantigens to bind to the corresponding MHC class I allele, we will 
synthesize peptides corresponding to the prioritized list of candidate epitopes. Synthetic peptides (> 95% 
purity) will be tested for binding to the respective class I MHC molecule using the T2 cell line, or one of its 
derivatives expressing common class I MHC alleles. The T2 cell line is deficient in transporters for antigen 
presentation (TAP), and as such is commonly used to assess binding of exogenously added peptides [94, 
95]. Expression of class I MHC alleles is measured by staining of T2 cells with antibody specific for the 
class I MHC allele followed by flow cytometric analysis to measure the fluorescence intensity of the class I 
MHC allele on the cell surface. Data are typically expressed as Mean Fluorescence Intensity (MFI) by 
subtracting the baseline fluorescence intensity of the class I MHC allele to that measured after addition of 
exogenous peptide. Neoantigens that do not bind to the corresponding class I MHC allele will be 
deprioritized.  
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 46 of 112 
8 
TREATMENT PLAN 
8.1 
Neoantigen DNA vaccine administration 
The first neoantigen DNA vaccine injection will take place following completion of SOC therapy. The day 
of the first vaccine injection will be referred to as Day 1. 
All subjects will be treated as outpatients in the Siteman Cancer Center. 
The schedule of vaccination is Day 1, Day 29 ± 7, Day 57 ± 7, Day 85 ± 7, Day 113 ± 7, and Day 141 ± 7 
with at least 21 days between injection days. All study injections will be given intramuscularly using an 
integrated electroporation device (TDS-IM system, Ichor Medical Systems). At each vaccination time 
point, patients will receive two injections of the neoantigen DNA vaccine, one injection into each deltoid or 
lateralis. If both injections cannot be given on the same day, the patient will be asked to return to 
complete the second injection. Standard aseptic technique and precautions will be utilized in site 
preparation, vaccine administration, and medical waste disposal to ensure maximal safety of subjects and 
study personnel. Participants’ vital signs will be monitored for 30 minutes post vaccine for safety 
purposes. 
The propagation of electroporation inducing electrical fields in the muscle will result in brief, localized 
muscle contractions at the site of administration, which are transiently painful. The neoantigen DNA 
vaccines will be administered by an experienced nurse who has completed a training seminar on the use 
of the TDS-IM device. 
The sites of immunization may be rotated for each of the immunizations. No injection will be given at a 
location in which the draining lymph nodes have been removed. 
At the discretion of the treating physician, patients may be pre-medicated with lorazepam 1mg PO (or 
similar) at least 30 minutes but no greater than 60 minutes prior to the first injection. Patients may also 
receive a second dose of lorazepam (1 mg PO) (or similar) 10 minutes prior to injection. Patients may 
also receive pain medication at treating physicians’ discretion PO at least 30 minutes but no greater than 
60 minutes prior to the first injection. 
At intervals throughout the study (both before and after vaccination) subjects will have blood drawn for 
immunologic assays. Any cells, serum or plasma not used will be stored for future immunological assays. 
Please see Section 10 Study Calendar for details on study visit procedures and monitoring. 
8.2 
Durvalumab administration 
For patients who are randomized to the neoantigen DNA vaccine plus durvalumab arm, the neoantigen-
specific T cell response will be assessed prior to Day 85. If a neoantigen-specific T cell response is 
present, durvalumab will be started on Day 85, and will be administered Q4W (± 7 days) at a dose of 
1500 mg over the course of 60 minutes. If a neoantigen-specific T cell response is not present, these 
patients will be replaced but may continue to receive the neoantigen DNA vaccine on study. They will not 
be transferred to the vaccine-only arm. 
On days when subjects receive both vaccine injections and durvalumab infusions, they will receive the 
vaccine first, followed by observation for 30 minutes, followed by administration of durvalumab.  Subjects 
will be monitored before, during and after the infusion with assessment of vital signs at the times specified 
in the Schedule of Assessment. Subjects are monitored (pulse rate, blood pressure) every 30 minutes 
during the infusion period (including times where infusion rate is slowed or temporarily stopped). Every 
effort should be made to give durvalumab and vaccine on the same day. 
If weight falls to 30 kg or less, durvalumab will no longer be administered. 
8.3 
Replacement of patients 
Patients who initially meet eligibility criteria and have a vaccine manufactured but either fail eligibility re-
check based on durvalumab-specific criteria (but are still able to receive neoantigen vaccine safely) or are 
randomized to receive durvalumab but do not experience a neoantigen-specific T cell response (and thus 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 47 of 112 
cannot receive durvalumab) may receive the neoantigen DNA vaccine but will be considered inevaluable 
and will be replaced. Safety and response data will be collected and analyzed separately. 
8.4 
General Concomitant Medication and Supportive Care Guidelines 
In the event of a ≤ grade 2 infusion-related reaction, the infusion rate of durvalumb may be decreased by 
50% or interrupted until resolution of the event and re-initiated at 50% of the initial rate until completion of 
the infusion. For subjects with a ≤ grade 2 infusion-related reaction, subsequent infusions may be 
administered at 50% of the initial rate. Acetaminophen and/or an antihistamine (e.g., diphenhydramine) or 
equivalent medications per institutional standard may be administered at the discretion of the investigator. 
If the infusion-related reaction is grade 3 or higher in severity, study drug will be discontinued. The 
standard infusion time is one hour, however if there are interruptions during infusion, the total allowed 
time from infusion start to completion of infusion should not exceed 8 hours at room temperature. For 
management of subjects who experience an infusion reaction, please refer to the toxicity and 
management guidelines. 
As with any antibody, allergic reactions to dose administration are possible. Appropriate drugs and 
medical equipment to treat acute anaphylactic reactions must be immediately available, and study 
personnel must be trained to recognize and treat anaphylaxis. The study site must have immediate 
access to emergency resuscitation teams and equipment in addition to the ability to admit subjects to an 
intensive care unit if necessary. 
8.4.1 
Permitted Concomitant Medications 
Supportive medication/class of drug: 
Usage: 
Concomitant medications or treatments 
(eg, acetaminophen or diphenhydramine) deemed 
necessary to provide adequate prophylactic or 
supportive care, except for those medications identified 
as “prohibited,” as listed above  
To be administered as prescribed by the 
Investigator 
Best supportive care (including antibiotics, nutritional 
support, correction of metabolic disorders, optimal 
symptom control, and pain management [including 
palliative radiotherapy to non-target lesions, etc]) 
Should be used, when necessary, for all 
subjects 
Inactivated viruses, such as those in the influenza 
vaccine 
Permitted 
8.4.2 
Excluded Concomitant Medications (Durvalumab Arm Only) 
Prohibited medication/class of drug: 
Usage: 
Any investigational anticancer therapy other 
than those under investigation in this study 
Should not be given concomitantly whilst the patient 
is on study treatment 
mAbs against CTLA-4, PD-1, or PD-L1 other 
than those under investigation in this study 
Should not be given concomitantly whilst the patient 
is on study treatment 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 48 of 112 
Prohibited medication/class of drug: 
Usage: 
Any concurrent chemotherapy, radiotherapy, 
immunotherapy, or biologic or hormonal 
therapy for cancer treatment other than those 
under investigation in this study 
Should not be given concomitantly whilst the patient 
is on study treatment. (Concurrent use of hormones 
for non-cancer-related conditions [eg, insulin for 
diabetes and hormone replacement therapy] is 
acceptable. Local treatment of isolated lesions, 
excluding target lesions, for palliative intent is 
acceptable [eg, by local surgery or radiotherapy]) 
Immunosuppressive medications including, but 
not limited to, systemic corticosteroids at 
doses exceeding <<10 mg/day>> of 
prednisone or equivalent, methotrexate, 
azathioprine, and tumor necrosis factor-α 
blockers 
Should not be given concomitantly. (Use of 
immunosuppressive medications for the management 
of IP-related AEs or in subjects with contrast allergies 
is acceptable). In addition, use of inhaled, topical, and 
intranasal corticosteroids is permitted. 
Drugs with laxative properties and herbal or 
natural remedies for constipation 
Should be used with caution through to 90 days after 
the last dose of durvalumab during the study 
Sunitinib 
Should not be given concomitantly or through 90 days 
after the last dose of durvalumab (acute renal failure 
has been reported with combination therapy of 
tremelimumab and sunitinib) 
EGFR TKIs 
Should not be given concomitantly. 
Should be used with caution in the 90 days post last 
dose of durvalumab. 
Increased incidences of pneumonitis (with third 
generation EGFR TKIs) and increased incidence of 
transaminase increases (with 1st generation EGFR 
TKIs) has been reported when durvalumab has been 
given concomitantly. 
Live attenuated vaccines 
Should not be given through 30 days after the last 
dose of IP (including SoC) 
8.5 
Women of Childbearing Potential 
Females of childbearing potential who are sexually active with a non-sterilized male partner must use at 
least 1 highly effective methods of contraception (see table below) from the time of screening, and must 
agree to continue using such precautions for one year after the last dose of neoantigen vaccine. 
Cessation of birth control after this point should be discussed with a responsible physician. Not engaging 
in sexual activity for the total duration of the drug treatment and the drug washout period is an acceptable 
practice; however, periodic abstinence, the rhythm method, and the withdrawal method are not 
acceptable methods of birth control. Female subjects should also refrain from breastfeeding throughout 
this period. 
Females of childbearing potential are defined as those who are not surgically sterile (i.e., bilateral tubal 
ligation, bilateral oophorectomy, or complete hysterectomy) or postmenopausal. Women will be 
considered post-menopausal if they have been amenorrheic for 12 months without an alternative medical 
cause. The following age-specific requirements apply: 
• 
Women < 50 years of age would be considered post-menopausal if they have been amenorrheic 
for 12 months or more following cessation of exogenous hormonal treatments and if they have 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 49 of 112 
luteinizing hormone and follicle-stimulating hormone levels in the post-menopausal range for the 
institution or underwent surgical sterilization (bilateral oophorectomy or hysterectomy). 
• 
Women ≥ 50 years of age would be considered post-menopausal if they have been amenorrheic 
for 12 months or more following cessation of all exogenous hormonal treatments, had radiation-
induced menopause with last menses > 1 year ago, had chemotherapy-induced menopause with 
last menses > 1 year ago, or underwent surgical sterilization (bilateral oophorectomy, bilateral 
salpingectomy, or hysterectomy). 
Highly effective methods of contraception, defined as one that results in a low failure rate (i.e., less than 
1% per year) when used consistently and correctly are described in the table below. Note that some 
contraception methods are not considered highly effective (e.g., male or female condom with or without 
spermicide; female cap, diaphragm, or sponge with or without spermicide; non-copper containing 
intrauterine device; progestogen-only oral hormonal contraceptive pills where inhibition of ovulation is not 
the primary mode of action [excluding Cerazette/desogestrel which is considered highly effective]; and 
triphasic combined oral contraceptive pills). 
Highly effective methods of contraception (< 1% failure rate) 
Barrier/Intrauterine Methods 
Hormonal Methods 
• Copper T intrauterine device 
• Levonorgestrel-releasing intrauterine 
system (e.g., Mirena ®)a 
 
• Etonogestrel implants, e.g., Implanon or Norplant 
• Intravaginal device, e.g., ethinylestradiol and 
etonogestrel 
• Medroxyprogesterone injection, e.g., Depo-Provera 
• Normal and low dose combined oral contraceptive 
pill 
• Norelgestromin/ethinylestradiol transdermal system 
• Cerazette (desogestrel) 
a This is also considered a hormonal method. 
8.6 
Blood donation 
Patients should not donate blood while participating in this study or for at least 90 days following the last 
infusion of durvalumab. 
8.7 
Duration of therapy 
In the absence of treatment delays due to adverse events, treatment may continue for 6 months (6 doses 
of neoantigen DNA vaccine).  Patients receiving durvalumab may receive up to 4 doses. Under certain 
circumstances, a subject will be terminated from participating in further injections. Subjects who are 
discontinued from additional study injections will continue to be followed according to the schedule of 
safety and immunogenicity evaluations. Please see Section 12 Removal of Patients from Protocol 
Therapy for additional details. 
8.8 
Duration of follow up 
Patients will be followed for 52 weeks or until death, whichever occurs first. Additional follow-up visits or 
telephone contact will be scheduled annually for 5 years if the patient is alive and available for follow-up. 
Patients removed from study for unacceptable adverse events will be followed until resolution or 
stabilization of the adverse event. 
 
 
 
 
 
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 50 of 112 
9 
POTENTIAL TOXICITY AND DOSE MODIFICATIONS 
9.1 
Potential toxicity 
9.1.1 
Experience with the pING parent vector 
The neoantigen DNA vaccines have not been used in humans to date. Conventional DNA cancer 
vaccines based on the pING parent vector have been used extensively in phase 1 human clinical trials. 
We have discussed this issue with Dr. Robert Jambou at the Office of Biotechnology Activities, National 
Institutes of Health. On January 12, 2009, We made a Freedom of Information Act (FOIA) request for 
copies of information on the study population, dosing regimen, and study results to include a detailed 
adverse events/safety information for six NIH OBA-registered clinical trials: 0005-394, 0105-474, 0105-
474, 0303-573, 0312-617, 0412-684 and 0412-685. Dr. Jambou sent us the relevant clinical protocols, 
Appendix M documents, safety reports and annual reports. 
In accordance with Appendix M-I-C-4 of the NIH Guidelines for Research Involving Recombinant DNA 
Molecules posted April 1, 2002 in the Federal Register, investigators who have received authorization 
from the FDA to initiate a human gene transfer protocol must report in writing any unanticipated problems 
(serious adverse events or SAEs) to the NIH Office of Biotechnology Activities (OBA). These guidelines 
state that "Principal Investigators must submit, in accordance with Appendix M-I-C-4, a written report on 
any serious adverse event that is both unexpected and associated with the use of the gene transfer 
product. Any serious adverse event that is fatal or life-threatening, that is unexpected, and associated 
with the use of the gene transfer product must be reported to the NIH OBA as soon as possible, but not 
later 24 hours after the sponsor’s initial receipt of the information. Serious adverse events that are 
unexpected and associated with the use of the gene transfer product, but are not fatal or life-threatening, 
must be reported to the NIH OBA as soon as possible, but not later than 15 calendar days after the 
sponsor’s initial receipt of the information."  
Note that on April 25, 2019, NIH revised the NIH Guidelines for Research Involving Recombinant or 
Synthetic Nucleic Acid Molecules, to streamline oversight of human gene transfer research. 
Under the amended NIH Guidelines, the NIH Office of Science Policy (OSP) will not: 
 
•           accept new human gene transfer protocols for the protocol registration process under the NIH 
Guidelines 
•           accept annual reports, safety reports, amendments or other documentation for any previously 
registered human gene transfer protocols under the NIH Guidelines (Appendix M-I-C). 
 
Additional information regarding the changes to the NIH Guidelines is available at this URL: 
https://osp.od.nih.gov/biotechnology/nih-guidelines/. 
We have used this information to prepare Table 4 below. Of note, there was only one serious adverse 
event reported. One patient with metastatic prostate cancer developed urinary retention. It is not clear if 
this was related to the DNA vaccine. Review of the available annual reports confirms minimal toxicity 
associated with DNA vaccination in these trials. Over 90% of the toxicities related to treatment were 
grade 1 and included diarrhea, dizziness, edema, fatigue, injection site reaction, nausea, vomiting, and 
rigors.  
We have also discussed these trials with Jedd Wolchock, M.D., at Memorial Sloan-Kettering Cancer 
Center, by e-mail correspondence January 15, 2009. Dr. Wolchock is the PI or co-investigator on all of 
these trials. He confirmed that there has been minimal toxicity associated with the DNA vaccines based 
on the pING parent vector.  
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 51 of 112 
Table 4 Clinical Experience with pING parent vector 
Protocol 
number  
Study 
population  
Dosing 
regimen  
Route of 
administration  
Number and 
frequency  
Safety  
0005-394  
Metastatic 
melanoma, 18 
patients total  
100, 500, or 
1500 ug 
DNA  
Intramuscularly 
using Biojector 
2000  
three 
vaccinations 
three weeks 
apart  
No SAEs 
reported  
0105-474  
Prostate 
cancer, 36 
patients  
100, 500, or 
1500 ug 
DNA  
Intramuscularly 
using Biojector 
2000  
six vaccinations 
three weeks 
apart  
One SAE 
reported, 
deemed to be 
unrelated to the 
vaccine  
0303-573  
Metastatic 
melanoma, 18 
patients total  
100, 500, or 
1500 ug 
DNA  
Intramuscularly 
using Biojector 
2000  
six vaccinations 
three weeks 
apart  
No SAEs 
reported  
0312-617  
Renal Cell 
Carcinoma, 18 
patients  
500, 1500, 
or 3000 ug 
DNA  
Intramuscularly 
using Biojector 
2000  
six vaccinations 
three weeks 
apart  
No SAEs 
reported  
0412-684  
Breast cancer, 
12 patients  
500, 1000, 
3000, or 
6000 ug 
DNA  
Intramuscularly 
using Biojector 
2000  
five 
vaccinations 
three weeks 
apart  
No SAEs 
reported  
0412-685  
Metastatic 
melanoma, 18 
patients  
500, 2000, 
or 4000 ug 
DNA  
Intramuscularly 
using Biojector 
2000  
six vaccinations 
three weeks 
apart  
No SAEs 
reported  
9.1.2 
Potential toxicity related to the neoantigen DNA vaccine 
This is the one of the first times that neoantigens identified by next generation sequencing have been 
targeted for immune therapy in humans, and one of the first times that neoantigen DNA vaccines have 
been administered to humans. However, clinical trials of similar investigational DNA vaccines suggest that 
these vaccines will be very safe. We expect that most of the toxicity to be limited to local grade 1 or 2 
reactions at the vaccination site. 
Please note that the risks detailed below are based on the risks of injections, the risks of vaccines in 
general, and the results of previous studies with investigational DNA vaccines. 
Risks associated with intramuscular injections include acute bleeding and/or bruising. Although highly 
unlikely, intramuscular injection can result in peripheral nerve damage and/or injection site infection. Due 
to the insertion and activation of multiple electrodes, use of the TDS-IM electroporation device may 
increase these risks. The propagation of electroporation inducing electrical fields in the muscle will result 
in brief, localized muscle contractions at the site of administration, which are transiently painful. As with 
any immunization, discomfort or redness at the injection site in the days following DNA vaccine 
administration may be expected. Since intramuscular DNA delivery with electroporation results in 
increased intracellular uptake of plasmid at the site of injection and electric field application, the 
procedure may increase the frequency and/or severity of local site reactions compared to conventional 
intramuscular administration of DNA vaccines. Such symptoms should not last longer than several days.  
Study subjects can receive medications such as acetaminophen, NSAIDs, or antihistamines as required. 
Steroids will not routinely be used in study subjects; if steroids are required the study subject will receive 
no further immunizations, but will continue to be monitored in follow-up visits. 


Washington University School of Medicine 
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Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
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Subjects may exhibit general signs and symptoms associated with administration of a vaccine injection, 
including fever, chills, rash, aches and pains, nausea, headache, dizziness and fatigue. These side 
effects will be monitored, but are generally short term and do not require treatment. 
The possibility of integration of the DNA plasmid vector into genomic DNA of transfected myocytes has 
been considered. Plasmid integration at a sufficiently high frequency carries the possibility of inducing 
deleterious mutations. Potential side effects could include an increased risk of malignancy arising from 
the cells harboring the mutation(s). However, current evidence from laboratory and animal studies 
indicates that the frequency of induced mutations following electroporation of DNA is conservatively 
estimated to be two to three orders of magnitude lower than that of naturally-occurring gene inactivating 
mutations in healthy humans. 
The effect of this vaccine on a fetus or nursing baby is unknown, so female subjects of childbearing 
potential will be required to agree to use birth control for sexual intercourse beginning 21 days prior to 
enrollment and continuing through the last protocol visit. Women who are pregnant or nursing will be 
excluded from the study. 
The potential discomforts of this study include having blood drawn, intramuscular injection of the vaccine, 
and possible reactions to the vaccine. Drawing blood causes transient discomfort and may cause fainting. 
Bruising at the blood draw site may occur, but can be prevented or lessened by applying pressure for 
several minutes. Injection also causes transient discomfort. Infection at the site of blood drawing or 
vaccination is extremely unlikely as alcohol swabbing and sterile equipment will be used.  
The use of plasmid DNA has the potential to cause an allergic reaction due to the presence of bacterial 
endotoxin. However, each lot of DNA will be tested for endotoxin to ensure that endotoxin content does 
not exceed USP specifications. Antibodies to DNA may potentially develop in DNA vaccine recipients. 
However, the development of such antibodies in response to DNA vaccination is rare and has not been 
associated with disease in animals or humans to date. 
9.1.3 
Potential toxicity related to the TDS-IM device 
The following are anticipated adverse reactions based on the previous clinical studies of TDS-IM based 
DNA vaccine delivery. All known risks and precautions described are explained in detail in the informed 
consent. 
Local Reactions 
Local site reactions associated with the use of the TDS-IM device in humans include acute pain 
associated with the localized muscle contractions during the application of electroporation in virtually all 
subjects. Mild, transient bleeding at the sites of electrode/needle penetration is commonly observed 
following removal of the device. Soreness, erythema, and/or induration of mild to moderate severity are 
commonly reported at the administration site. In one instance, local site soreness transiently graded as 
severe was reported in association with a device technical error related to needle deployment wherein the 
procedure was suspended prior to the application of electroporation. Other injection site findings of mild 
severity including mild bruising, hematoma, and paresthenia have been reported occasionally. All 
injection site reactions have typically resolved within 24–72 hours following administration, but, in rare 
instances, mild local tenderness has been reported to persist for up to one week. 
For the TDS-IM device there is a theoretical risk that excessive energy could be delivered to the local 
tissues of the subject, resulting in more pronounced local reactions than have been observed to date. 
However, the TDS Pulse Stimulator incorporates multiple redundant mitigations to prevent this hazard, 
including performance of a pre-pulse safety check and the use of multiple circuits that monitor total 
energy delivered and will which will terminate energy delivery. There have been no reported occurrences 
of excessive energy delivery in any of the nonclinical and clinical studies conducted with the pulse 
stimulator to date. 
Systemic Reactions 
Systemic adverse events reported during the studies utilizing the TDS-IM and judged to be possibly 
related to the study product and/or delivery device have been generally mild to moderate in severity and 
include flu-like symptoms, headache, fever, dizziness, malaise, fatigue, arthralgia, myalgia, and aphthous 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 53 of 112 
stomatitis. Transient elevation in serum creatine phosphokinase of mild to moderate severity and judged 
to be associated with procedure administration have been reported in a small minority of subjects. Two 
subjects have reported the onset of severe fatigue within 24 hours of dosing that was resolved by the 
following day and was not observed following subsequent doses. 
In approximately 1% of procedure administrations, one or more symptoms consistent with a vasovagal 
reaction (i.e., dizziness / lightheadedness, hypotension, diaphoresis, and/or skin pallor) have been 
observed immediately after procedure application. In rare instances, these reactions have progressed to 
brief syncope, particularly in subjects with pre-existing risk factors for syncope (e.g., pronounced sinus 
bradycardia). In all cases, the subjects have rapidly recovered from the syncope without incident.  
A theoretical risk associated with the use of DNA vaccines is the possibility of uptake of the vaccine 
candidate into non-target tissues and/or integration of the vaccine DNA into the genomic DNA of the test 
subject. As described in Section 8.2 below, nonclinical studies have been performed to assess the 
potential for systemic biodistribution and genomic integration following TDS based delivery multiple DNA 
vaccine candidates, including a tyrosinase melanoma DNA vaccine candidate utilizing the same vector 
backbone (pING) as the Mammaglobin-A DNA vaccine candidate. To date, there has been no evidence 
of significant systemic uptake or genomic integration events following TDS based DNA vaccine 
administration.  
9.2 
Toxicity monitoring and management 
Toxicity will be characterized according to the National Cancer Institute Common Terminology Criteria for 
Adverse Events version 4.03 (CTCAE).  
Toxicities associated or possibly associated with durvalumab treatment should be managed according to 
standard medical practice or suggested management tables provided in this protocol in Section 9.3 
below. Additional tests, such as autoimmune serology or biopsies, should be used to determine a 
possible immunogenic etiology. 
Although most immune-related adverse events observed with immunomodulatory agents have been mild 
and self-limiting, such events should be recognized early and treated promptly to avoid potential major 
complications. Discontinuation of durvalumab may not have an immediate therapeutic effect and in 
severe cases, immune-related toxicities may require acute management with topical corticosteroids, 
systemic corticosteroids, mycophenolate, or tumor necrosis factor−α inhibitors. The Investigator should 
consider the benefit-risk balance a given patient may be experiencing prior to further administration of 
durvalumab. Durvalumab should be permanently discontinued in patients with life-threatening immune-
related adverse events. 
Subjects who are immunized with the DNA vaccine will be evaluated at the time of each vaccination. 
Follow up on subject well-being will be performed by telephone on the first or second day after each 
vaccination. All information will be recorded on case report forms. Adverse events will be reported to the 
Quality Assurance and Safety Monitoring Committee of the Siteman Cancer Center, the Institutional 
Review Board, the Institutional Biosafety Committee, the Office of Biotechnology Activities and the Food 
and Drug Administration as detailed in Section 13 Adverse Event Reporting. 
Significant local inflammation will be treated with cold packs and oral analgesics as indicated. Skin 
ulceration at the vaccine site will be treated with local wound care and antibiotics as indicated. 
Autoimmune involvement of the breast will be treated conservatively with analgesics; more aggressive 
intervention (systemic corticosteroids) will be used as necessary 
9.3 
Dose modifications for durvalumab 
For adverse events (AEs) that are considered at least partly due to administration of durvalumab the 
following dose adjustment guidance may be applied: 
• 
Treat each of the toxicities with maximum supportive care (including holding the agent suspected 
of causing the toxicity where required). 
• 
If the symptoms promptly resolve with supportive care, consideration should be given to 
continuing the same dose of durvalumab along with appropriate continuing supportive care. If 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 54 of 112 
medically appropriate, dose modifications are permitted for durvalumab (see below). 
• 
All dose modifications should be documented with clear reasoning and documentation of the 
approach taken. 
All toxicities will be graded according to NCI CTCAE v4.03. 
9.3.1 
Dosing Modification and Toxicity Management Guidelines for Immune-Mediated, Infusion-
Related, and Non-Immune-Mediated Reactions 
Drug administration modifications of study drug/study regimen will be made to manage potential immune-
related AEs based on severity of treatment-emergent toxicities graded per NCI CTCAE v4.03. 
In addition to the criteria for permanent discontinuation of study drug/study regimen based on CTCAE 
grade/severity (table below), permanently discontinue study drug/study regimen for the following 
conditions: 
• 
Inability to reduce corticosteroid to a dose of ≤10 mg of prednisone per day (or equivalent) within 
12 weeks of the start of the immune-mediated adverse event (imAE) 
• 
Grade 3 recurrence of a previously experienced treatment-related imAE following resumption of 
dosing 
See tables below for dosing modifications and toxicity management guidelines for specific immune-
mediated reactions, other immune-mediated reactions, infusion-related reactions, and non-immune 
mediated reactions. 
 
 


W a s hi n gt o n U ni v er sit y S c h o ol of M e di ci n e  
Pr ot o c ol D at e 0 4/ 2 4/ 2 0 2 3  
Sit e m a n C a n c er C e nt er  
Br e a st C a n c er Pr o gr a m  
N e o a nti g e n D N A v a c ci n e +/ - d ur v al u m a b   
 
P a g e 5 5  of 1 1 2  
D o si n g M o difi c ati o n a n d T o xi cit y M a n a g e m e nt G ui d eli n e s ( T M G s) f or D ur v al u m a b  M o n ot h er a p y, D ur v al u m a b i n C o m bi n ati o n wit h ot h er Pr o d u ct s, or 
Tr e m eli m u m a b M o n ot h er a p y –   O ct o b er 2 0 2 2  
G e n er al C o n si d er ati o n s R e g ar di n g I m m u n e -M e di at e d R e a cti o n s  
T h e s e g ui d eli n e s ar e pr o vi d e d a s a r e c o m m e n d ati o n t o s u p p ort i n v e sti g at or s i n t h e m a n a g e m e nt of p ot e nti al i m m u n e -m e di at e d a d v er s e e v e nt s (i m A E s).  
 
I m m u n e-m e di at e d e v e nt s c a n o c c ur i n n e arl y a n y or g a n or ti s s u e, t h er ef or e, t h e s e g ui d eli n e s m a y n ot i n cl u d e all t h e p o s si bl e i m m u n e -m e di at e d r e a cti o n s. 
I n v e sti g at or s ar e a d vi s e d t o t a k e i nt o c o n si d er ati o n t h e a p pr o pri at e pr a cti c e g ui d eli n e s a n d ot h er s o ci et y g ui d eli n e s ( e. g., N ati o n al C o m pr e h e n si v e C a n c er N et w or k 
( N C C N), E ur o p e a n S o ci et y of M e di c al O n c ol o g y ( E S M O)) i n t h e m a n a g e m e nt of t h e s e e v e nt s. R ef er t o t h e s e cti o n of t h e t a bl e titl e d “ Oth er -I m m u n e-M e di at e d 
R e a cti o n s” f or g e n er al g ui d a n c e o n i m A E s n ot n ot e d i n t h e “ S p e cifi c I m m u n e -M e di at e d R e a cti o n s” s e cti o n.  
E arl y i d e ntifi c ati o n a n d m a n a g e m e nt of i m A E s i s e s s e nti al t o e n s ur e s af e u s e of t h e st u d y dr u g. M o nit or p ati e nt s cl o s el y f or s y m pt o m s a n d si g n s t h at m a y b e 
cli ni c al m a nif e st ati o n s of u n d erl yi n g i m A E s. P ati e nt s wit h s u s p e ct e d i m A E s s h o ul d b e t h or o u g hl y e v al u at e d t o r ul e o ut a n y alt er n ati v e eti ol o gi e s ( e. g., di s e a s e 
pr o gr e s si o n, c o n c o mit a nt m e di c ati o n s, i nf e cti o n s). I n t h e a b s e n c e  of a cl e ar alt er n ati v e eti ol o g y, all s u c h e v e nt s s h o ul d b e m a n a g e d a s if t h e y w er e i m m u n e -
m e di at e d. I n stit ut e m e di c al m a n a g e m e nt pr o m ptl y, i n cl u di n g s p e ci alt y c o n s ult ati o n a s a p pr o pri at e. I n g e n er al, wit h h ol d st u d y dr u g/ st u d y r e gi m e n f or s e v er e 
( Gr a d e 3) i m A E s. P er m a n e ntl y di s c o nti n u e st u d y dr u g/ st u d y r e gi m e n f or lif e -t hr e at e ni n g ( Gr a d e 4) i m A E s, r e c urr e nt s e v er e ( Gr a d e 3) i m A E s t h at r e q uir e s y st e mi c 
i m m u n o s u p pr e s si v e tr e at m e nt, or a n i n a bilit y t o r e d u c e c orti c o st er oi d d o s e t o 1 0 m g or l e s s of pr e d ni s o n e or e q ui v al e nt p er d a y wit hi n 1 2 w e e k s of i niti ati n g 
c orti c o st er oi d s.  
B a s e d o n t h e s e v erit y of t h e i m A E, d ur v al u m a b a n d/ or tr e m eli m u m a b s h o ul d b e wit h h el d a n d c orti c o st er oi d s a d mi ni st er e d. U p o n i m pr o v e m e nt t o Gr a d e ≤ 1, 
c orti c o st er oi d s h o ul d b e t a p er e d o v er ≥ 2 8 d a y s. M or e p ot e nt i m m u n o s u p pr e s si v e a g e nt s s h o ul d b e c o n si d er e d f or e v e nt s n ot r e s p o n di n g t o s y st e mi c st er oi d s. 
Alt er n ati v e i m m u n o s u p pr e s si v e a g e nt s n ot li st e d i n t hi s g ui d eli n e m a y b e c o n si d er e d at t h e di s cr eti o n of t h e i n v e sti g at or b a s e d o n cli ni c al  pr a cti c e a n d r el e v a nt 
g ui d eli n e s. Wit h l o n g -t er m st er oi d a n d ot h er i m m u n o s u p pr e s si v e u s e, c o n si d er t h e n e e d f or gl u c o s e m o nit ori n g.    
 
D o s e m o difi c ati o n s of st u d y dr u g/ st u d y r e gi m e n s h o ul d b e b a s e d o n s e v erit y of tr e at m e nt -e m er g e nt t o xi citi e s gr a d e d p er N CI C T C A E v er si o n i n t h e a p pli c a bl e 
st u d y pr ot o c ol.  
 
C o n si d er ati o n s f or Pr o p h yl a xi s f or L o n g T er m u s e of St er oi d s f or P ati e nt s R e c ei vi n g I m m u n e C h e c k p oi nt I n hi bit or I m m u n ot h er a p y  
 I nf e cti o n Pr o p h yl a xi s: P n e u m o c y sti s jir o v e cii p n e u m o ni a ( P J P), a ntif u n g al a n d H er p e s Z o st er r e a cti v ati o n 
 G a striti s: C o n si d er pr o p h yl a xi s f or p ati e nt s at hi g h ri s k of g a striti s ( e. g. N S AI D u s e, a nti c o a g ul ati o n)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
R el e v a nt S o ci et y G ui d eli n e s f or M a n a g e m e nt of i m A E s  


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Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
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These society guidelines are provided as references to serve in support of best clinical practice and the TMGs. Please note, these were the current 
versions of these guidelines at the time of updating TMGs. Please refer to the most up to date version of these guidelines. 
 
1. 
Brahmer JR, et al. Society for Immunotherapy of Cancer (SITC) clinical practice guideline on immune checkpoint inhibitor-related adverse events. J 
Immunother Cancer 2021;9:e002435 
2. 
Brahmer JR, et al. Management of immune-related adverse events in patients treated with immune checkpoint inhibitor therapy: American Society of 
Clinical Oncology Clinical Practice Guideline. J Clin Oncol 2018;36(17):1714-1768. 
3. 
Haanen JBAG, et al. Management of toxicities for immunotherapy: European Society for Medical Oncology (ESMO) clinical practice guidelines for 
diagnosis, treatment, and follow-up. Annals Oncol 2017;28(Suppl4):i119-i1142. 
4. 
Sangro B, et al. Diagnosis and management of toxicities of immune checkpoint inhibitors in hepatocellular carcinoma. J Hepatol 2020;72(2):320-341. 
5. 
Thompson JA, et al. National Comprehensive Cancer Network Guidelines: Management of immunotherapy-related toxicities version 1.2022. Published 
February 28, 2022. 
 
 
 
 
Event 
Grade 
Dose Modifications 
Toxicity Management 
Pneumonitis / 
Interstitial ILD 
Any Grade 
General guidance 
- Patients should be thoroughly evaluated to rule out any alternative etiology 
with similar clinical presentation (e.g. infection, progressive disease) 
- Monitor patients for signs (e.g. tachypnoea) and symptoms of pneumonitis 
or ILD (new onset or worsening shortness of breath or cough). Evaluate 
patients with imaging and pulmonary function tests including other 
diagnostic procedures as described below 
- Suspected pneumonitis should be confirmed with radiographic imaging and 
other infectious and disease-related etiologies excluded, and managed as 
described below. 
- Initial work-up may include clinical evaluation, monitoring of oxygenation 
via pulse oximetry (resting and exertion), laboratory work-up (including 
clinically relevant culture specimens to rule out infection). and high-
resolution computed tomography (CT) scan.  
- Consider Pulmonary and Infectious Diseases consults. 
Grade 1  
 
No dose modification required. 
However, consider holding study 
drug/study regimen dosing as 
clinically appropriate and during 
diagnostic 
work-up 
for 
other 
etiologies 
For Grade 1 
- Monitor and closely follow up in 2-4 days for clinical symptoms, pulse 
oximetry (resting and exertion) and laboratory work-up and then as 
clinically indicated 
 
Grade 2  
 
Hold study drug/study regimen 
dose until Grade 2 resolution to ≤ 
Grade 1. 
  
  
For Grade 2 
- Monitor symptoms daily and consider hospitalization, as clinically 
indicated. 
- Consider Pulmonary and Infectious Diseases Consults. 
- Promptly start systemic steroids (e.g., prednisone 1 to 2mg/kg/day PO or 


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Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 57 of 112 
 
IV equivalent) 
- Consider HRCT or chest CT with contrast. Repeat imaging study as 
clinically indicated. 
- If no improvement within 2 to 3 days, additional workup should be 
considered and prompt treatment with IV methylprednisolone 2 to 
4mg/kg/day started  
- If still no improvement within 2 to 3 days despite IV methylprednisone at 2 
to 4/g/kg/day, promptly start immunosuppressive therapy such as tumor 
necrosis factor (TNF) inhibitors (e.g. infliximab at 5mg/kg once, may be 
repeated at 2 and 6 weeks after initial dose at the discretion of the treating 
provider or relevant practice guidelines). Caution: It is important to rule out 
sepsis and refer to infliximab label for general guidance before using 
infliximab. 
- Consider discussing with Clinical Study Lead. 
Grade 3 or 4  
  
Permanently 
discontinue 
study 
drug/study regimen 
 
For Grade 3 or 4  
- Hospitalize the patient 
- Promptly initiate empiric IV methylprednisolone 1 to 4 mg/kg/day or 
equivalent  
- Obtain Pulmonary and Infectious Disease consults; consider, as 
necessary, discussing with Clinical Study Lead 
- Consider starting anti-infective therapy if infection is still a consideration 
on the basis of other diagnostic testing despite negative culture results.  
- Supportive Care (e.g., oxygen) 
- If no improvement within 2 days, additional workup should be considered 
and prompt treatment with additional immunosuppressive therapy such as 
TNF inhibitors (e.g. infliximab at 5mg/kg once, may be repeated at 2 and 
6 weeks after initial dose at the discretion of the treating provider or 
relevant practice guidelines). Caution: rule out sepsis and refer to 
infliximab label for general guidance before using infliximab  
Diarrhea / Colitis 
 
 
Any Grade 
General guidance 
- Patients should be thoroughly evaluated to rule out any alternative 
etiology (e.g., disease progression, other medications, or infections), 
including testing for Clostridium difficile toxin, etc. 
- Monitor for symptoms that may be related to diarrhea/enterocolitis 
(abdominal pain, cramping, or changes in bowel habits such as increased 
frequency over baseline or blood in stool) or related to bowel perforation 
(such as sepsis, peritoneal signs and ileus) 
- Consider further evaluation with imaging study with contrast. 
- Consult a gastrointestinal (GI) specialist for consideration of further 
workup. 
- WHEN SYMPTOMS OR EVALUATION INDICATE AN INTESTINAL 
PERFORATION IS SUSPECTED, CONSULT A SURGEON 
EXPERIENCED IN ABDOMINAL SURGERY IMMEDIATELY WITHOUT 
ANY DELAY. 
- PERMANENTLY DISCONTINUE STUDY DRUG FOR ANY GRADE OF 


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Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 58 of 112 
INTESTINAL PERFORATION. 
- Steroids should be considered in the absence of clear alternative etiology, 
even for low grade events, in order to prevent potential progression to 
higher grade event, including perforation 
- Use analgesics carefully; they can mask symptoms of perforation and 
peritonitis 
Grade 1 
 
No dose modifications 
For Grade 1 diarrhea:  
- Monitor closely for worsening symptoms 
- Consider 
symptomatic 
treatment 
including 
hydration, 
electrolyte 
replacement, dietary changes (e.g., American Dietetic Association colitis 
diet), loperamide, and other supportive care measures. 
- If symptoms persist, consider checking lactoferrin; if positive, treat as Grade 
2 below. If negative and no infection, continue Grade 1 management.  
Grade 2 
 
Hold study drug/study regimen 
until resolution to ≤ Grade 1 
  
For Grade 2 diarrhea:  
- Consider 
symptomatic 
treatment 
including 
hydration, 
electrolyte 
replacement, dietary changes (e.g., American Dietetic Association colitis 
diet), and loperamide and/or budesonide  
- Consider further evaluation with imaging study with contrast. 
- Consider consult of a gastrointestinal (GI) specialist for consideration of 
further workup. 
- Promptly start prednisone 1 to 2 mg/kg/day PO or IV equivalent  
- If no improvement within 3 days despite therapy with 1 to 2 mg/kg IV 
prednisone equivalent, reconsult GI specialist and, if indicated, promptly 
start immunosuppressant agent such as infliximab at 5mg/kg IV, may be 
repeated at 2 and 6 weeks after initial dose at the discretion of the treating 
provider or relevant practice guidelines. Caution: Important to rule out 
bowel perforation and refer to infliximab label for general guidance before 
using infliximab. 
-  If perforation is suspected, consult a surgeon experienced in 
abdominal surgery immediately without any delay. 
- Consider, as necessary, discussing with Clinical Study Lead if no resolution 
to ≤ Grade 1 in 3 to 4 days. 


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Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 59 of 112 
Grade 3 or 4 
 
  
Grade 3: 
• For patients treated with 
durvalumab monotherapy, hold 
study drug/study regimen until 
resolution to Grade ≤1; study 
drug/study regimen can be 
resumed after completion of 
steroid taper (<10 mg 
prednisone per day, or 
equivalent). 
• For patients treated with 
durvalumab in combination with 
other products (not 
tremelimumab), decision to be 
made at the discretion of the 
study investigator, in discussion 
with AstraZeneca Clinical Study 
Lead. 
 
Grade 4: 
Permanently discontinue study 
drug/study regimen 
For Grade 3 or 4: 
- Urgent GI consult and imaging/or colonoscopy as appropriate. 
- Promptly initiate empiric IV methylprednisolone 1 to 2 mg/kg/day or 
equivalent  
- Monitor stool frequency and volume and maintain hydration  
- If still no improvement within 2 days, continue steroids and promptly add 
further immunosuppressants (e.g. infliximab at 5mg/kg IV, may be 
repeated at 2 and 6 weeks after initial dose at the discretion of the treating 
provider or relevant practice guidelines). Caution: Ensure GI consult to rule 
out bowel perforation and refer to infliximab label for general guidance 
before using infliximab.  
- If perforation is suspected, consult a surgeon experienced in 
abdominal surgery immediately without any delay. 
Hepatitis  
 
Infliximab should 
not be used for 
management of 
immune-related 
hepatitis. 
 
Any Grade 
General guidance. 
For Any Grade 
- Patients should be thoroughly evaluated to rule out any alternative 
etiology (e.g., viral hepatitis, disease progression, concomitant 
medications)  
- Monitor and evaluate transaminases (aspartate aminotransferase [AST], 
alanine aminotransferase [ALT], alkaline phosphatase [ALP] and total 
bilirubin.  
ALT or AST ≤ 3 x 
ULN or total bilirubin 
≤ 1.5 x ULN 
• No dose modifications. 
• If it worsens, then consider 
holding therapy. 
- Continue transaminase and total bilirubin monitoring per protocol  
ALT or AST > 3 ≤ 5 x 
ULN or total bilirubin 
> 1.5 ≤ 3 x ULN  
 
• Hold study drug/study regimen 
dose until ALT or AST ≤ 3 X 
ULN or total bilirubin, ≤ 1.5 X 
ULN. Resume study drug/study 
regimen after completion of 
steroid taper (<10mg 
prednisone or equivalent). 
• Permanently discontinue study 
drug/study regimen for any 
case meeting Hy’s law 
laboratory criteria (AST or ALT 
≥3 xULN AND bilirubin ≥2 x 
- Regular and frequent checking of transaminases and total bilirubin (e.g. 
every 1 to 2 days) until transaminases and total bilirubin elevations 
improve or resolve.  
- 
If no resolution to ALT or AST ≤ 3 x ULN or total bilirubin 
- 
≤ 1.5 x ULN in 1-2 days, consider, discussing with Clinical Study Lead as 
needed. 
- If event is persistent (> 2 to 3 days) or worsens, promptly start prednisone 
1 to 2mg/kg/day PO or IV equivalent.  
 


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Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 60 of 112 
 
ULN without initial findings of 
cholestasis (i.e., elevated 
ALP)) and in the absence of 
any alternative cause. 
  
ALT or AST > 5- ≤ 10 
x ULN 
• 
Hold study drug/study 
regimen. Resume study 
drug/study regimen if 
elevations downgrade to 
ALT or AST ≤ 3 x ULN or 
total bilirubin ≤ 1.5 x ULN 
after completion of 
steroid taper (<10 mg 
prednisone, or 
equivalent).   
 
 
 
 
 
- Promptly initiate empiric IV methylprednisolone at 1 to 2 mg/kg/day or 
equivalent  
- Perform Hepatology Consult, abdominal workup, and imaging as 
appropriate. 
- If still no improvement within 2 to 3 days despite 1 to 2 mg/kg/day 
methylprednisolone IV or equivalent, promptly start treatment with an 
additional immunosuppressant (i.e., mycophenolate mofetil 0.5 to 1 g 
every 12 hours then taper in consultation with hepatology consult or 
relevant practice guidelines). Discuss with Clinical Study Lead if 
mycophenolate is not available. Infliximab should NOT be used. 
 
Concurrent ALT or 
AST > 3 x ULN and 
total bilirubin 
> 2 x ULNd 
 
ALT or AST 
> 10 x ULN OR total 
bilirubin > 3 x ULN 
• 
Permanently discontinue 
study drug/study regimen 
− Promptly initiate empiric IV methylprednisolone at 1 to 2 mg/kg/day or 
equivalent. 
 
If still no improvement within 2 to 3 days despite 1 to 2 mg/kg/day 
methylprednisolone IV or equivalent, promptly start treatment with an 
additional immunosuppressant.(e.g., mycophenolate mofetil 0.5 – 1 g 
every 12 hours then taper in consultation with hepatology consult or 
relevant practice guidelines). Discuss with Clinical Study Lead if 
mycophenolate is not available. Infliximab should NOT be used. 
PLEASE SEE 
shaded area 
immediately 
below this 
section to find 
guidance for 
management of 
“Hepatitis 
(elevated LFTS)” 
in 
hepatocellular 
carcinoma 
(HCC) patients 
(or 
secondary tumor 
involvement of 
the liver with 
abnormal 
baseline values 
[BLV] 
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 61 of 112 
Hepatitis  
(elevated 
transaminases 
and total 
bilirubin) 
Infliximab should 
not be used for 
management of 
immune-related 
hepatitis.  
 
 
 
See instructions 
at bottom of 
shaded area  
if transaminase 
rise is not isolated 
but (at any time) 
occurs in setting  
of either 
increasing 
bilirubin or 
signs of 
DILI/liver 
decompensation 
Any Elevations of 
AST, ALT, or T. Bili 
as Described Below 
General guidance 
For Any Elevations Described 
- Patients should be thoroughly evaluated to rule out any alternative 
etiology (e.g., viral hepatitis, disease progression, concomitant 
medications, worsening of liver cirrhosis [e.g., portal vein thrombosis]). 
- Monitor and evaluate liver function test: AST, ALT, ALP, and T. Bili. 
- For hepatitis B (HBV) + patients: evaluate quantitative HBV viral load, 
quantitative Hepatitis B surface antigen (HBsAg), or Hepatitis B envelope 
antigen (HBeAg). 
- For Hepatitis C (HCV) + patients: evaluate quantitative HCV viral load. 
- Consider consulting Hepatology or Infectious Disease specialists 
regarding changing or starting antiviral HBV medications if HBV viral load 
is >2000 IU/ml. 
- Consider consulting Hepatology or Infectious Disease specialists 
regarding changing or starting antiviral HCV medications if HCV viral load 
has increased by ≥2-fold. 
- For HCV+ with Hepatitis B core antibody (HBcAB) +: Evaluate for both 
HBV and HCV as above. 
Isolated AST or ALT 
> ULN and ≤ 2.5 X 
BLV 
  
 
 
 
 
 
 
 
 
 
• No dose modifications 
• If ALT/AST elevations 
represents significant 
worsening based on 
investigator assessment, then 
treat as described for 
elevations in the row below. 
 
For all transaminase elevations, 
see instructions at bottom of 
shaded area if transaminase rise 
is not isolated but (at any time) 
occurs in setting of either 
increasing bilirubin or signs of 
DILI/liver decompensation 
 
ALT or AST ≥ 2.5- ≤ 
5X BLV and ≤ 
20xULN 
 
 
• Hold study drug/study regimen 
dose until resolution to AST or 
ALT ≤2.5 X BLV. 
• If toxicity worsens, then treat 
as described for elevations in 
the rows below. If toxicity 
improves to AST or ALT ≤2.5 × 
BLV, resume study drug/study 
regimen after completion of 
steroid taper (<10 mg 
- Regular and frequent checking of Transaminases and total bilirubin (e.g., 
every 1 to 3 days) until elevations of these are improving or resolved. 
- Recommend consult hepatologist consider abdominal ultrasound, 
including Doppler assessment of liver perfusion. 
- Consider, as necessary, discussing with Clinical Study Lead. 
- If event is persistent (>2 to 3 days) or worsens, and investigator suspects 
toxicity to be imAE, start prednisone 1 to 2 mg/kg/day PO or IV 
equivalent. 
- If still no improvement within 2 to 3 days despite 1 to 2 mg/kg/day of 
THIS shaded 
area is 
guidance only 
for 
management 
of “Hepatitis 
(elevated 
LFTs)” in HCC 
patients  


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Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 62 of 112 
prednisone, or equivalent). 
 
prednisone PO or IV equivalent, consider additional work up. If still no 
improvement within 2 to 3 days despite 2 mg/kg/day of IV 
methylprednisolone, consider additional abdominal work up (including 
liver biopsy) and imaging (i.e., liver ultrasound), and consider starting 
additional immunosuppressants (i.e., mycophenolate mofetil 0.5 to 1 g 
every 12  hours then taper in consultation with hepatology consult or 
relevant practice guidelines). Discuss with Clinical Study Lead if 
mycophenolate mofetil is not available. Infliximab should NOT be used. 
ALT or AST  >5-7X 
BLV and ≤ 20X ULN 
OR concurrent 
2.5-5X BLV and ≤ 
20XULN AND total 
bilirubin > 1.5 - < 2 x 
ULNd 
 
 
 
 
 
 
• Hold study drug/study regimen 
dose until resolution to AST or 
ALT ≤2.5 × BLV. Resume 
study drug/study regimen if 
elevations downgrade to AST 
or ALT ≤2.2 × BLV and after 
completion of steroid taper 
(<10 mg prednisone, or 
equivalent). 
• Permanently discontinue study 
drug/study regimen if the 
elevations do not downgrade to 
AST or ALT ≤2.5 × BLV within 
14 days. 
- Regular and frequent checking of LFTs (e.g., every 1-2 days) until 
elevations of these are improving or resolved. 
- Consult hepatologist (unless investigator is hepatologist); obtain 
abdominal ultrasound, including Doppler assessment of liver perfusion; 
and consider liver biopsy. 
- Consider, discussing with Clinical Study Lead as needed. 
- If investigator suspects toxicity to be immune-mediated, promptly initiate 
empiric IV methylprednisolone at 1 to 2 mg/kg/day or equivalent. 
- If no improvement within 2 to 3 days despite 1 to 2 mg/kg/day 
methylprednisolone IV or equivalent, obtain liver biopsy (if it has not been 
done already) and promptly start treatment with an additional 
immunosuppressant (e.g., mycophenolate mofetil 0.5 to 1 g every 12 
hours then taper in consultation with a hepatologist or relevant practice 
guidelines). Discuss with Clinical Study Lead if mycophenolate is not 
available. Infliximab should NOT be used. 
ALT or AST > 7 × BLV OR >20 ULN, whichever occurs first OR bilirubin > 3ULN 
Permanently discontinue study drug/study regimen. 
- Same as above (except recommended obtaining liver biopsy early) 
 
Nephritis and/or 
Renal 
Dysfunction  
Any Grade  
General guidance 
 
 
 
- Patients should be thoroughly evaluated to rule out any alternative 
etiology (e.g., disease progression, infections, recent IV contrast, 
medications, fluid status). 
- Consider consulting a nephrologist. 
- Consider imaging studies to rule out any alternative etiology 
- Monitor for signs and symptoms that may be related to changes in renal 
function (e.g. routine urinalysis, elevated serum BUN and creatinine, 
decreased creatinine clearance, electrolyte imbalance, decrease in urine 
output, or proteinuria).  
-  Follow urine protein/creatinine ratio every 3-7 days 
Grade 1  
No dose modifications. 
For Grade 1:  
- Monitor serum creatinine weekly and any accompanying symptoms. 


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Protocol Date 04/24/2023 
Siteman Cancer Center 
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Neoantigen DNA vaccine +/- durvalumab 
 
Page 63 of 112 
• If creatinine returns to baseline, resume regular monitoring per study 
protocol. 
• If creatinine worsens, depending on the severity, treat as Grade 2, 3, or 
4. 
- Consider hydration, electrolyte replacement, and diuretics. as clinically 
indicated. 
- 
Consider nephrologist consult if not resolved within 14 days, or 
earlier as clinically indicated 
 
Grade 2  
Hold study drug/study regimen 
until resolution to Grade ≤ 1 or 
baseline.  
For Grade 2:  
- Consider including hydration, electrolyte replacement, and diuretics as 
clinically indicated.  
- Follow urine protein/creatinine ratio every 3-7 days 
- Carefully monitor serum creatinine as clinically warranted. 
- Consult nephrologist and consider renal biopsy if clinically indicated. 
- Start prednisone 0.5 – 1 mg/kg/day if other causes are ruled out 
- If event is persistent beyond 5 days or worsens, increase to prednisone 
up to 2 mg/kg/day PO or IV equivalent.  
- If event is not responsive within 5 days or worsens despite prednisone at 
1 to 2 mg/kg/day PO or IV equivalent, consider additional workup. When 
event returns to baseline, resume study drug/study regimen and routine 
serum creatinine monitoring per study protocol. 
 
Grade 3 or 4  
  
Permanently discontinue study 
drug/study regimen 
For Grade 3 or 4: 
- Carefully monitor serum creatinine daily. 
- Follow urine protein/creatinine ratio every 3-7 days 
- Consult nephrologist and consider renal biopsy if clinically indicated. 
- Promptly start prednisone 1 to 2 mg/kg/day PO or IV equivalent.  
- If event is not responsive within 3 to 5 days of steroids or worsens despite 
prednisone at 1 to 2 mg/kg/day or IV equivalent, consider additional 
workup and prompt treatment with an immunosuppressant.  
Rash or 
Dermatitis 
(including 
Pemphigoid) 
 
Any Grade  
(refer to NCI CTCAE 
v 4.03 for definition 
of severity / grade 
depending on type of 
skin rash) 
General guidance 
For Any Grade: 
- Patients should be thoroughly evaluated to rule out any alternative 
etiology.  
- Monitor for signs and symptoms of dermatitis (rash and pruritus). 
- HOLD STUDY DRUG IF GRADE 3 PEMPHIGOID OR SEVERE 
CUTANEOUS ADVERSE REACTION (SCAR)1 IS SUSPECTED. 
- PERMANENTLY DISCONTINUE STUDY DRUG IF SCAR OR GRADE 
3 PEMPIGOID IS CONFIRMED. 
 
Grade 1  
No dose modifications. 
For Grade 1: 
- Consider symptomatic treatment including oral antipruritics (e.g., 
diphenhydramine or hydroxyzine) and topical therapy (e.g., emollient, 
lotion, or institutional standard). 


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Protocol Date 04/24/2023 
Siteman Cancer Center 
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Neoantigen DNA vaccine +/- durvalumab 
 
Page 64 of 112 
Grade 2 
For persistent (> 1 week) Grade 2 
events, hold scheduled study 
drug/study regimen until resolution 
to ≤ Grade 1 or baseline  
• If toxicity improves to Grade ≤ 
1 or baseline, then resume 
drug/study regimen after 
completion of steroid taper 
(<10 mg prednisone, or 
equivalent). 
For Grade 2: 
- Consider Dermatology consult and skin biopsy, as indicated. 
- Consider symptomatic treatment including oral antipruritics (e.g., 
diphenhydramine or hydroxyzine) and topical therapy. 
- Consider moderate-strength topical steroid  
- If no improvement of rash/skin lesions occurs within 1 week or is 
worsening despite symptomatic treatment and/or use of moderate 
strength topical steroid, consider discussing with Clinical Study Lead, as 
needed, and promptly start systemic steroids prednisone 1 to 2 
mg/kg/day PO or IV equivalent.  
-  
Grade 3  
Grade 3: 
• Hold study drug/study regimen 
until resolution to Grade ≤ 1 or 
baseline 
• If toxicity improves to Grade ≤1 
or baseline, then resume study 
drug/study regimen after 
completion of steroid taper 
(<10 mg prednisone, or 
equivalent). 
 
 
For Grade 3  
- Reconsult a dermatologist. Consider skin biopsy (preferably more than 1) 
as clinically feasible.  
-  Promptly initiate empiric IV methylprednisolone 1 to 2 mg/kg/day or 
equivalent. 
- Consider hospitalization. 
- Monitor the extent of rash [Rule of Nines]. 
- Consider, as necessary, discussing with Clinical Study Lead. 
Grade 4 
For Grade 4 
 
Permanently discontinue study 
drug/study regimen 
For Grade 4 
− Reconsult a dermatologist. Consider skin biopsy (preferably more 
than 1) as clinically feasible. 
− Promptly initiate empiric IV methylprednisolone 1 to 2 mg/kg/day 
or equivalent. 
− Consider hospitalization. 
− Monitor the extent of rash [Rule of Nines]. 
Consider, as necessary, discussing with Clinical Study Lead. 
Endocrinopathy 
(e.g., 
hyperthyroidism, 
thyroiditis, 
hypothyroidism, 
Type 1 diabetes 
mellitus, 
Any Grade  
(Depending on the 
type of 
endocrinopathy, refer 
to CTCAE v 4.03 for 
definition) 
General guidance 
For Any Grade: 
- Patients should be thoroughly evaluated to rule out any alternative 
etiology (e.g., disease progression including brain metastases, or 
infections). 
- Consider consulting an endocrinologist for endocrine events. 
- Consider discussing with Clinical Study Lead, as needed. 
- Monitor patients for signs and symptoms of endocrinopathies. (Non-


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 65 of 112 
hypophysitis, 
hypopituitarism, 
and adrenal 
insufficiency) 
specific symptoms include headache, fatigue, behavior changes, mental 
status changes, photophobia, visual field cuts, vertigo, abdominal pain, 
unusual bowel habits, polydipsia, polyuria, hypotension and weakness.) 
- Depending on the suspected endocrinopathy, monitor and evaluate 
thyroid function tests: thyroid stimulating hormone (TSH), free T3 and free 
T4 and other relevant endocrine labs (e.g., blood glucose and ketone 
levels, hemoglobin A1C (HgA1c)). If a patient experiences an AE that is 
thought to be possibly of autoimmune nature (e.g., thyroiditis, 
pancreatitis, hypophysitis, diabetes insipidus), the investigator should 
send a blood sample for appropriate autoimmune antibody testing. 
- Investigators should ask subjects with endocrinopathies who may require 
prolonged or continued hormonal replacement, to consult their primary 
care physicians or endocrinologists about further monitoring and 
treatment after completion of the study. 
Grade 1 
 
No dose modifications. 
For Grade 1: 
- Monitor patient with appropriate endocrine function tests. 
- For suspected hypophysitis/hypopituitarism, consider consultating an 
endocrinologist to guide assessment of early-morning adrenocorticotropin 
hormone (ACTH), cortisol, TSH and free T4; also consider gonadotropins, 
sex hormones, and prolactin levels, as well as cosyntropin stimulation test 
(though it may not be useful in diagnosing early secondary adrenal 
insufficiency). 
- If TSH < 0.5 × LLN, or TSH > 2 × ULN or consistently out of range in 2 
subsequent measurements, include free T4 at subsequent cycles as 
clinically indicated and consider consultation of an endocrinologist. 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 66 of 112 
Grade 2, 3, or 4  
 
 
• For Grade 2-4 
endocrinopathies other than 
hypothyroidism and Type 1 
diabetes mellitus (T1DM), 
consider holding study 
drug/study regimen dose until 
acute symptoms resolve. 
• Study drug/study regimen can 
be resumed once event 
stabilizes and after completion 
of steroid taper (<10 mg 
prednisone, or equivalent). 
• Patients with endocrinopathies 
who may require prolonged or 
continued steroid replacement 
(e.g., adrenal insufficiency) can 
be retreated with study 
drug/study regimen if the 
patien is clinically stable as per 
investigator or treating 
physician’s clinical judgement. 
For Grade 2, 3, or 4: 
- Consult endocrinologist to guide evaluation of endocrine function, and as 
indicated by suspected endocrinopathy and as clinically indicated, 
consider pituitary scan. 
- For all patients with abnormal endocrine work up, except those with 
isolated hypothyroidism or Type 1 DM, and as guided by an 
endocrinologist, consider short-term, corticosteroids (e.g., 1 to 
2mg/kg/day methylprednisolone or IV equivalent) and prompt initiation of 
treatment with relevant hormone replacement. 
- Isolated hypothyroidism may be treated with replacement therapy, without 
study drug/study regimen interruption, and without corticosteroids. 
- Isolated T1DM may be treated with appropriate diabetic therapy and 
without corticosteroids. Only hold study drug/study regimen in setting 
of hyperglycemia when diagnostic workup is positive for diabetic 
ketoacidosis. 
- For patients with normal endocrine work up (laboratory assessment or 
magnetic resonance imaging (MRI) scans), repeat laboratory 
assessments/MRI as clinically indicated. 
Amylase/Lipase 
increased 
Any Grade 
General Guidance 
For Any Grade: 
- Patients should be thoroughly evaluated to rule out any alternative 
etiology (e.g. disease progression, viral infection, concomitant 
medications, substance abuse). 
- For modest asymptomatic elevations in serum amylase and lipase, 
corticosteroid treatment is not indicated as long as there are no other 
signs or symptoms of pancreatic inflammation. 
- Assess for signs/symptoms of pancreatitis. 
- Consider appropriate diagnostic testing (e.g., abdominal CT with contrast, 
MRCP if clinical suspicion of pancreatitis and no radiologic evidence on 
CT). 
- If isolated elevation of enzymes without evidence of pancreatitis, continue 
immunotherapy. Consider other causes of elevated amylase/lipase. 
- If evidence of pancreatitis, manage according to pancreatitis 
recommendations. 
Grade 1 
No dose modifications. 
Grade 2, 3, or 4 
For Grade 2, 3, or 4: 
In consultation with relevant 
gastroenterology specialist 
consider continuing study 
drug/study regimen if no 
clinical/radiologic evidence of 
pancreatitis ± improvement in 
amylase/lipase. 
Acute Pancreatitis 
Any Grade 
General Guidance 
For Any Grade: 
- Patients should be thoroughly evaluated to rule out any alternative 
etiology. 
- Consider Gastroenterology referral. 
Grade 2 
Consider holding study 
drug/regimen 
Grade 2 
- 
Consider IV hydration 
- 
Consider Gastroenterology referral 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 67 of 112 
Grade 3 or 4 
For Grade 3: 
Hold study drug/study regimen 
until resolution of elevated 
enzymes and no radiologic 
findings 
 
If no elevation in enzymes or 
return  to baseline values, then 
resume study drug/study regimen 
after completion of steroid taper 
(<10 mg prednisone, or 
equivalent). 
 
For Grade 4: 
Permanently discontinue study 
drug/study regimen. 
For Grade  3 or 4: 
- Promptly start systemic steroids prednisone 1 to 2 mg/kg/day PO or IV 
equivalent. 
- IV hydration 
Nervous System Disorders 
Aseptic Meningitis 
Any Grade  
(, Refer to NCI 
CTCAE v4.03 for 
defining the CTC 
grade/severity) 
General guidance. 
For Any Grade: 
- Consider neurology consult. 
- Consider MRI brain with and without contrast and pituitary protocol and a 
lumbar puncture for diagnosis. 
- Exclude bacterial and viral infections. (ie HSV) 
- Symptoms may include headache, photophobia, and neck stiffness, 
nausea/vomiting which may resemble an infectious meningitis. 
- Patients may be febrile. 
- Mental status should be normal 
- Consider IV acyclovir until polymerase chain reactions are available 
Any Grade  
Permanently discontinue study 
drug/study regimen 
For Any Grade 
- 
Consider neurology consult 
- 
Consider MRI brain with and without contrast with pituitary protocol 
and a lumbar puncture for diagnosis 
- 
Exclude bacterial and viral infections. (ie HSV) 
- 
Consider IV acyclovir until polymerase chain reactions are available 
- 
Consider, as necessary, discussing with Clinical Study Lead. (Last 
bullet) 
- 
Consider hospitalization 
- 
Once infection has been ruled out promptly initiate empiric IV 
methylprednisolone 1 to 2 mg/kg/day or equivalent. 


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Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 68 of 112 
Encephalitis 
Any Grade 
General Guidance 
For Any Grade 
- Consider neurology consult 
- Consider testing including MRI of the brain with and without contrast, 
lumbar puncture, electroencephalogram (EEG) to evaluate for subclinical 
seizures, ESR, CRP, antineutrophil cytoplasmic antibody (ANCA) (if 
vasculitic process suspected), thyroid panel including TPO and 
thyroglobulin and additional autoantibodies to rule out paraneoplastic 
disorders. 
- Exclude bacterial and viral infections. (ie HSV) 
- Consider IV acyclovir until polymerase chain reactions are available. 
-  Symptoms may include Confusion, altered behavior, headaches, 
seizures, short-term memory loss, depressed level of consciousness focal 
weakness, and speech abnormality. 
Grade 2 
For Grade 2 permanently 
discontinue study drug/study 
regimen 
For Grade 2 
- Consider, as necessary, discussing with the Clinical Study Lead. 
- Once infection has been ruled out methylprednisolone 1 2 mg/kg/day 
- For progressive symptoms or if oligoclonal bands are present consider 
methylprednisolone 1 g IV daily for 3-5 days plus IVIG or plasmapheresis 
Grade 3 or 4 
For Grade 3 or 4 
 
Permanently discontinue study 
drug/study regimen 
For Grade 3 or 4 
- Consider, as necessary, discussing with Clinical Study Lead. 
- Consider hospitalization. 
- Once infection is ruled out, start methylprednisolone 2 g IV daily for 3-5 
days for progressive symptoms consider adding IVIG or plasmapheresis 
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 69 of 112 
Transverse 
Myelitis 
Any Grade 
General Guidance 
- Permanently discontinue 
immunotherapy 
- Consider MRI of the spine and 
brain 
- Once imaging is complete 
consider lumbar puncture 
- 
Consider testing to rule out 
additional aetiologies: B12, 
HIV, rapid plasma regain 
(RPR), ANA, anti-Ro/La 
antibodies, aquaporin-4 IgG, 
Myelin oligodendrocyte 
glycoprotein (MOG) IgG, 
paraneoplastic panel for anti-
Hu and anti-CRMP5/CV2 
 
For Any Grade 
- 
Consider neurology consult 
- 
Impatient Care 
- 
Consider prompt initiation of high methylprednisolone pulse dosing 
- 
Strongly consider IVIG or plasmapheresis 
Peripheral 
neuropathy 
Any Grade 
(Refer to NCI 
CTCAE applicable 
version in study 
protocol for defining 
the CTCAE 
grade/severity) 
General guidance 
For Any Grade: 
- Patients should be evaluated to rule out any alternative etiology for 
neuropathy (e.g., disease progression, infections, metabolic syndromes 
and medications, etc.). It should be noted that the diagnosis of immune-
mediated peripheral neuromotor syndromes can be particularly 
challenging in patients with underlying cancer, due to the multiple 
potential confounding effects of cancer (and its treatments) throughout the 
neuraxis. Given the importance of prompt and accurate diagnosis, it is 
essential to have a low threshold to obtain a neurological consult. 
- Neurophysiologic diagnostic testing (e.g., electromyogram and nerve 
conduction investigations) are routinely indicated upon suspicion of such 
conditions and may be best facilitated by means of a neurology 
consultation. 
Grade 1 
 
No dose modifications. 
For Grade 1: 
- Consider discussing with the Clinical Study Lead, as needed. 
- Monitor symptoms for interference with ADLS, gait difficulties, imbalance, 
or autonomic dysfunction 
Grade 2 
 
Hold study drug/study regimen 
dose until resolution to Grade ≤1. 
.= 
 
For Grade 2:  
- Consult a neurologist. 
- Consider EMG/NCS  
- Consider discussing with the Clinical Study Lead, as needed.  
- Observation for additional symptoms or consider initiating prednisone 0.5-
1mg/kg orally 
- If progression initiate methylprednisolone 2-4 mg/kg/day and treat as GBS 
- Sensory neuropathy/neuropathic pain may be managed by appropriate 
medications (e.g., gabapentin or duloxetine). 
 


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Neoantigen DNA vaccine +/- durvalumab 
 
Page 70 of 112 
Grade 3 or 4 
 
For Grade 3 or 4: 
Permanently discontinue study 
drug/study regimen  
For Grade 3 or 4:  
- Consider discussing with Clinical Study Lead, as needed. 
- Recommend hospitalization. 
- Monitor symptoms and consult a neurologist. 
- Treat per Guillain-Barre Syndrome recommendations 
 
Guillain-Barre 
Syndrome (GBS) 
 
General Guidance 
− Recommend hospitalization 
− Obtain neurology consult 
− Obtain MRI of spine to rule out compression lesion 
− Obtain lumbar puncture 
− Antibody tests for GBS variants 
− Pulmonary function tests 
− Obtain electromyography (EMG) and nerve conduction studies 
− Frequently monitor pulmonary function tests and neurologic 
evaluations 
− Monitor for concurrent autonomic dysfunction 
− Initiate medication as needed for neuropathic pain 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 71 of 112 
Grade 2-4 
Grade 2-4 
Permanently discontinue 
methylprednisolone 1 gram daily for 5 days, then taper over 4 
weeks. 
Myasthenia gravis 
 
General Guidance 
− Obtain neurology consult 
− Recommend hospitalization 
− Obtain pulmonary function tests 
− Obtain labs: ESR, CRP, creatine phosphokinase (CPK), aldolase 
and anti-striational antibodies 
− Consider cardiac exam, ECG, troponin, transthoracic 
echocardiogram for possible concomitant myocarditis 
− Obtain electromyography (EMG) and nerve conduction studies 
− Consider MRI of brain/spine to rule out CNS involvement by 
disease 
− Avoid medications that might exacerbate MG (e.g. beta blockers, 
some antibiotics, IV magnesium) 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 72 of 112 
Grade 2 
Permanently discontinue 
- 
Consider pyridostigmine 30mg three times daily and gradually 
increase based on symptoms (max dose 120mg four times daily) 
− Consider starting low dose prednisone 20mg daily and increase 
every 3-5 days. (Target dose 1mg/kg/day. Max dose 100mg daily) 
Grade 3-4 
Permanently discontinue 
− Start methylprednisolone 1-2mg/kg/day. Taper steroids based on 
symptom improvement 
− Start plasmapheresis or IVIG 
− Consider rituximab if refractory to plasmapheresis or IVIG 
− Frequent PFT assessments 
− Daily neurologic evaluations 


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Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 73 of 112 
Myocarditis 
Any Grade 
 
(Refer to NCI 
CTCAE applicable 
version in study 
protocol for defining 
the CTCAE 
grade/severity) 
General guidance 
 
Discontinue drug permanently if 
biopsy-proven immune-mediated 
myocarditis. 
For Any Grade 
- Initial work-up should include clinical evaluation, B-type natriuretic peptide 
(BNP), cardiac enzymes, electrocardiogram (ECG), echocardiogram 
(ECHO), monitoring of oxygenation via pulse oximetry (resting and 
exertion), and additional laboratory work-up as indicated. Spiral CT or 
cardiac MRI can complement ECHO to assess wall motion abnormalities 
when needed. 
- Patients should be thoroughly evaluated to rule out any alternative etiology 
(e.g., disease progression, other medications, or infections) 
- The prompt diagnosis of immune-mediated myocarditis is important, 
particularly in patients with baseline cardiopulmonary disease and reduced 
cardiac function.  
- Consider discussing with the Clinical Study Lead, as needed. 
- Monitor patients for signs and symptoms of myocarditis (new onset or 
worsening chest pain, arrhythmia, shortness of breath, peripheral edema). 
As some symptoms can overlap with lung toxicities, simultaneously 
evaluate for and rule out pulmonary toxicity as well as other causes (e.g., 
pulmonary embolism, congestive heart failure, malignant pericardial 
effusion). Consult a cardiologist early, to promptly assess whether and 
when to complete a cardiac biopsy, including any other diagnostic 
procedures. 
- As indicated, Spiral CT or cardiac MRI can complement ECHO to assess 
wall motion abnormalities when needed. 
Grade 2, 3, or 4 
If Grade 2-4, permanently 
discontinue study drug/study 
regimen. 
For Grade 2-4: 
− Monitor symptoms daily, hospitalize. 
− Consider cardiology consultation and a prompt start of high-dose/pulse 
corticosteroid therapy 
− Supportive care (e.g., oxygen). 
− If no improvement consider immunosuppressive therapy such as TNF 
inhibitors (e.g., infliximab ,  IVIG or plasmapheresis or other therapies 
depending on the clinical condition of the patient, based on the discretion of 
the treating specialist consultant or relevant practice guidelines. Caution: It 
is important to rule out sepsis and refer to infliximab label for general 
guidance before using infliximab. Infliximab is contraindicated for 
patients who have heart failure.  


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Protocol Date 04/24/2023 
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Neoantigen DNA vaccine +/- durvalumab 
 
Page 74 of 112 
Myositis/ 
Polymyositis 
Any Grade 
General guidance 
For Any Grade: 
- Patients should be thoroughly evaluated to rule out any alternative 
etiology (e.g., disease progression, other medications, or infections). 
- Monitor patients for signs and symptoms of poly/myositis. Typically, 
muscle weakness/pain occurs in proximal muscles including upper arms, 
thighs, shoulders, hips, neck and back, and; also difficulty breathing 
and/or trouble swallowing can occur and progress rapidly. Increased 
general feelings of tiredness and fatigue may occur, and there can be 
new-onset falling, difficulty getting up from a fall, and trouble climbing 
stairs, standing up from a seated position, and/or reaching up. 
- If poly/myositis is suspected, a Neurology consultation should be obtained 
early, with prompt guidance on diagnostic procedures. Myocarditis may 
co-occur with poly/myositis; refer to guidance under Myocarditis. Given 
breathing complications, refer to guidance under Pneumonitis/ILD. Given 
possibility of an existent (but previously unknown) autoimmune disorder, 
consider Rheumatology consultation. 
− Consider, as necessary, discussing with the Clinical Study Lead. 
− Initial work-up should include clinical evaluation, creatine kinase, aldolase, 
lactate dehydrogenase (LDH), blood urea nitrogen (BUN)/creatinine, 
erythrocyte sedimentation rate or C-reactive protein (CRP) level, urine 
myoglobin, and additional laboratory work-up as indicated, including a 
number of possible rheumatological/antibody tests (i.e., consider whether a 
rheumatologist consultation is indicated and could guide need for 
rheumatoid factor, antinuclear antibody, anti-smooth muscle, 
antisynthetase [such as anti-Jo-1], and/or signal-recognition particle 
antibodies). Confirmatory testing may include electromyography, nerve 
conduction studies, MRI of the muscles, and/or a muscle biopsy. Consider 
Barium swallow for evaluation of dysphagia or dysphonia. 
Grade 1 
No dose modifications 
For Grade 1: 
- Monitor and closely follow up in 2 to 4 days for clinical symptoms and 
initiate evaluation as clinically indicated. 
- Consider Neurology consult. 
- Consider, as necessary, discussing with the Clinical Study Lead. 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 75 of 112 
Grade 2 
 
• Hold study drug/study regimen 
dose until resolution to Grade 
≤1. 
• Permanently discontinue study 
drug/study regimen if it does not 
resolve to Grade ≤1 within 30 
days or if there are signs of 
respiratory insufficiency. 
 
For Grade 2 
− Monitor symptoms daily and consider hospitalization. 
− Consider Rheumatology or Neurology consult, and initiate evaluation. 
− Consider, as necessary, discussing with the Clinical Study Lead. 
− If clinical course is rapidly progressive (particularly if difficulty breathing 
and/or trouble swallowing), promptly start IV methylprednisolone 2 to 4 
mg/kg/day systemic steroids along with receiving input from Neurology 
consultant  
− If clinical course is not rapidly progressive, start systemic steroids (e.g., 
prednisone 1 to 2 mg/kg/day PO or IV equivalent); if no improvement within 
2 to 3 days, continue additional work up and start treatment with IV 
methylprednisolone 2 to 4 mg/kg/day 
− If after start of IV methylprednisolone at 2 to 4 mg/kg/day there is no 
improvement within 3 days, consider additional immunosuppressive 
therapy such as TNF inhibitors (e.g., infliximab, IVIG or plasmapheresis, or 
other therapies based on the discretion of the treating specialist consultant 
or relevant practice guideline. Caution: It is important to rule out sepsis 
and refer to infliximab label for general guidance before using 
infliximab. 
Grade 3  
 
Grade 3 
• Hold study drug/study regimen 
dose until resolution to Grade 
≤1. 
• Permanently discontinue study 
drug/study regimen if Grade 3 
imAE does not resolve to Grade 
≤1 within 30 days or if there are 
signs of respiratory 
insufficiency. 
 
 
For Grade 3 or 4 
− Monitor symptoms closely; recommend hospitalization. 
− Consider Rheumatology and/or Neurology consult. 
− Consider discussing with the Clinical Study Lead, as needed. 
− Promptly start IV methylprednisolone 2 to 4 mg/kg/day systemic steroids 
along with receiving input from Neurology consultant. 
− If after start of IV methylprednisolone at 2 to 4 mg/kg/day there is no 
improvement within 2 to 3 days, consider starting another 
immunosuppressive therapy such as a TNF inhibitor (e.g., infliximab at 5 
mg/kg IV, may be repeated at 2 and 6 weeks after initial dose at the 
discretion of the treating provider or relevant practice guidelines). Caution: 
It is important to rule out sepsis and refer to infliximab label for 
general guidance before using infliximab. 
− Consider whether patient may require IV IG, plasmapheresis. 
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 76 of 112 
Grade 4 
For Grade 4 
 
Permanently discontinue study 
drug/study regimen 
Grade 4 
− Monitor symptoms closely; recommend hospitalization. 
− Consider Rheumatology and/or Neurology consult 
− Consider discussing with the Clinical Study Lead, as needed. 
− Promptly start IV methylprednisolone 2 to 4 mg/kg/day systemic 
steroids along with receiving input from Neurology consultant. 
− If after start of IV methylprednisolone at 2 to 4 mg/kg/day there is 
no improvement within 2 to 3 days, consider starting another 
immunosuppressive therapy such as a TNF inhibitor 
(e.g., infliximab at 5 mg/kg IV, may be repeated at 2 and 6 weeks 
after initial dose at the discretion of the treating provider or 
relevant practice guidelines). Caution: It is important to rule out 
sepsis and refer to infliximab label for general guidance before 
using infliximab. 
1 SCAR terms include Stevens-Johnson Syndrome (SJS), Toxic Epidermal Necrolysis (TEN), Erythema Multiforme, Acute Generalized 
Exanthematous Pustulosis, Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) and Drug-induced hypersensitivity syndrome. 
Other-Immune-Mediated Reactions 
Grade 
Dose Modifications 
Toxicity Management 
Any Grade 
Note: It is possible that events with an 
inflammatory or immune mediated mechanism 
could occur in nearly all organs, some of them are 
not noted specifically in these guidelines (e.g. 
immune thrombocytopenia, haemolytic anaemia, 
uveitis, vasculitis). 
- Patients should be thoroughly evaluated to rule 
out any alternative etiology (e.g., disease 
progression, other medications, or infections). 
- The Clinical Study Lead may be contacted for 
immune-mediated reactions not listed in the 
“specific immune-medidated reactions” section. 
- Consultation with relevant specialist 
- Treat accordingly, as per institutional standard. 
Grade 1 
No dose modifications. 
Monitor as clinical indicated. 
Grade 2 
• Hold study drug/study regimen until resolution 
to ≤Grade 1 or baseline. 
• If toxicity worsens, then treat as Grade 3 or 
Grade 4. 
• Study drug/study regimen can be resumed once 
event stabilizes to Grade ≤1 after completion of 
steroid taper. 
• Consider whether study drug/study regimen 
should be permanently discontinued in Grade 2 
events with high likelihood for morbidity and/or 
mortality when they do not rapidly improve to 
Grade <1 upon treatment with systemic steroids 
For Grade 2, 3, or 4 
Treat accordingly, as per institutional standard, 
appropriate clinical practice guidelines, and society 
guidelines. 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 77 of 112 
and following full taper. 
Grade 3 
Hold study drug/study regimen 
Grade 4 
Permanently discontinue study drug/study regimen. 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 78 of 112 
 
Infusion-Related Reactions 
Grade 
Dose Modifications 
Toxicity Management 
Any Grade 
General guidance. 
- Manage per institutional standard at the discretion of investigator 
- Monitor patients for signs and symptoms of infusion-related reactions 
(e.g., fever and/or shaking chills, flushing and/or itching, alterations in 
heart rate and blood pressure, dyspnea or chest discomfort, or skin 
rashes) and anaphylaxis (e.g., generalized urticaria, angioedema, 
wheezing, hypotension, or tachycardia). 
Grade 1 or 2 
 
For Grade 1 
The infusion rate of study drug/study regimen may be decreased 
by 50% or temporarily interrupted until resolution of the event. 
 
For Grade 2 
• The infusion rate of study drug/study regimen may be 
decreased 50% or temporarily interrupted until resolution of 
the event.  
• Subsequent infusions may be given at 50% of the initial 
infusion rate. 
For Grade 1 or Grade 2: 
- Acetaminophen and/or antihistamines may be administered per 
institutional standard at the discretion of the investigator  
- Consider premedication per institutional standard or study protocol prior to 
subsequent doses 
- Steroids should not be used for routine medication of Grade ≤ 2 infusion 
reactions. 
Grade 3 or 4 
Permanently discontinue study drug/study regimen 
For Grade 3 or 4: 
- Manage severe infusion-related reactions per institutional standard, 
appropriate clinical practice guidelines, and society guidelines. 
 
Non-immune Mediated Reactions 
Grade 
Dose Modification  
Toxicity Management  
Any Grade 
Note: dose modifications are not required for adverse events not deemed to be related to study treatment 
(i.e. events due to underlying disease) or for laboratory abnormalities not deemed to be clinically 
significant. 
Treat accordingly as per institutional standard  
 
 
 
1 
No dose modifications.  
2-3 
Hold study drug/study regimen until resolution to Grade ≤ 1 or baseline 
 
4 
Discontinue Study drug/study regimen (Note for Grade 4 labs, decision to discontinue would be based on 
accompanying clinical signs/symptoms and as per Investigator’s clinical judgment and in consultation with 
the sponsor)  
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 79 of 112 
9.4 
Dose modifications for neoantigen DNA vaccines 
No dose modifications are planned. If a subject develops an adverse event that is classified as possibly, 
probably, or definitely associated with protocol therapy, this may result in removal of the subject from 
protocol therapy as outlined in Section 12 Removal of Subjects from Protocol Therapy. Protocol 
Stopping Criteria are outlined in Section 14 Data and Safety Monitoring. 
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 80 of 112 
10 
STUDY CALENDAR 
 
Screeningj 
Eligibility Re-Checkf 
D1h 
D29 
D57 
D85 
D113 
D141 
D169n 
EOTk 
52 Weeks 
after  
completion 
of 
neoantigen 
DNA 
vaccine 
therapy 
Long-term 
 F/Ue 
Randomization 
 
Xg 
 
 
 
 
 
 
 
 
 
 
Durvalumaba,b 
 
 
 
 
 
X 
X 
X 
X 
 
 
 
Vaccined 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
Concurrent meds 
X 
 
X --------------------------------------------------------------------------- X 
 
 
Physical exam, wt, PS 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Vital signs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Medical History 
X 
X 
 
 
 
 
 
 
 
 
 
 
CBC w/diff, plts 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
CMPc 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
TSH, fT3, fT4 
X 
X 
 
 
 
Xab 
Xab 
Xab 
Xab 
 
 
 
Urinalysis 
X 
X 
 
 
 
Xab 
Xab 
Xab 
Xab 
 
 
 
ECG (in triplicate) 
X 
X 
 
 
 
As clinically indicateda 
 
 
 
PT, aPTT, INR 
X 
X 
 
 
 
As clinically indicateda 
 
 
 
Troponins I and T 
X 
X 
 
 
 
Every 4 Weeks before treatmenta 
 
 
 
AE evaluationo 
 
 
X ------------------------------------------------------------- X 
 
 
 
Tumor measurements 
X 
As clinically indicated. Documentation (radiologic) must be provided for pts removed from study for PD. 
Radiologic evaluation 
X 
Radiologic measurements will be performed as clinically indicated. 
Conf. of persistent disease 
 
X 
 
 
 
 
 
 
 
 
 
 
B-HCG 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Hepatitis serologies 
X 
 
 
 
 
 
 
 
 
 
 
 
HIV testing 
X 
 
 
 
 
 
 
 
 
 
 
 
Tissue collection 
Research bx or surgical specimen or both 
 
 
 
 
 
 
 
 
 
 
Blood for sequencing 
X 
 
 
 
 
 
 
 
 
 
 
 
Blood for immune monitoringp 
 
Xm 
X 
X 
X 
X 
X 
 
X 
 
X 
 
a: Only if randomized to receive durvalumab 
b: To begin on Cycle 4, if neoantigen-specific T cell responses are detectable only if randomized to get Durvalumab 
c: Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, potassium, total protein, SGOT [AST], SGPT [ALT], sodium 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 81 of 112 
d: +/- 7 days with at least 21 days between each injection 
e: Annually for 5 years (± 2 weeks) 
f: After completion of SOC therapy 
g: After confirmation of eligibility and manufacture of vaccine 
h: After completion of SOC therapy 
j: May take place up to 28 days prior to registration. 
k: Within 30 days of last day of study treatment 
m: After the end of SOC chemotherapy 
N: Durvalumab group only 
O: In addition to completing an AE assessment at the vaccine dosing visit, an additional follow-up between 24-48 hours after vaccination must be performed (can be performed by phone). AE assessment 
and SAE reporting continues until 90 days after the last dose of Durvalumab or until time of initiation of other anticancer therapy, whichever occurs first. 
p: ± 28 days 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 82 of 112 
11 
CRITERIA FOR RESPONSE 
11.1 Primary objective: safety  
The primary objective is to assess the safety of neoantigen DNA vaccines given alone or in combination 
with durvalumab.  
Assessment of the safety of neoantigen DNA vaccines will include both clinical observation and 
laboratory evaluation. Safety will be closely monitored after injection with eight or more clinical and 
laboratory assessments in the first 24 weeks of the trial. The following parameters will be assessed 
following vaccination: 
(1) 
Local signs and symptoms 
(2)  
Systemic signs and symptoms 
(3) 
Laboratory evaluations, including blood counts and serum chemistries 
(4) 
Adverse, and serious adverse events 
Toxicity will be graded according to the National Cancer Institute Common Terminology Criteria for 
Adverse Events v4.03. 
11.2 Secondary objective: neoantigen-specific T cell response 
The secondary objective is to measure the neoantigen-specific T cell response to neoantigen DNA 
vaccines given alone, or neoantigen DNA vaccines plus durvalumab. The immune response will be 
measured in the peripheral blood by luminex assay, ELISPOT, and multiparametric flow cytometry. 
11.3 Neoantigen-specific T cell response 
The secondary objective is to assess the neoantigen-specific T cell response to the neoantigen DNA 
vaccine. To accomplish this goal, peripheral blood will be collected at multiple time points before and after 
vaccination. After peripheral blood and primary breast cancer tissue processing and storage, various 
assays will be used to assess the neoantigen-specific T cell response. Unless otherwise noted below, 
biologic specimens will be analyzed simultaneously upon completion of the vaccination protocol in order 
to minimize assay-to-assay variation. 
11.3.1 Tumor tissue and peripheral blood processing 
Processing of tumor: The tumor will be sampled prior to initiation of therapy.  
Tumor from the research biopsy will be prioritized for tumor exome and tumor cDNA-capture sequencing 
in order to identify tumor-specific mutant antigens and produce the neoantigen DNA vaccine. The cores 
will be placed in complete RPMI or equivalent media solution on ice and transported to Dr. Gillanders’ 
laboratory (CSRB, room 6664). Upon receipt by the lab, the tissue will then be prioritized for embedding 
in optimal cutting temperature (OCT) compound. The remaining cores will be placed into formalin for 
subsequent paraffin embedding (FFPE). 
Cryomold will be filled slowly to the top with OCT compound. Tissue will be gently submerged into the 
OCT compound in the cryomold. OCT will then be hardened by placement on dry ice or in the vapor 
phase of liquid nitrogen. After hardening, the mold will be placed in a container and transferred to a -80oC 
freezer for storage. The frozen tissue will be transferred to the Laboratory of Translation Pathology (LTP, 
BJC-Institute of Health, Room 5110) or the CHiiPs Immunomonitoring Laboratory for storage. 
In addition to tumor from a research biopsy, tumor from surgical specimens may be acquired. 
Processing of peripheral blood: Blood samples will be collected at the following time points: baseline, 
following completion of standard of care therapy, and Days 1, 29, 57, 85, 113, 169 and at 52 weeks after 
completion of neoantigen DNA vaccine therapy. Seventy mL of peripheral blood will be collected in 7 
green top heparinized tubes by venipuncture (BD Vacutainer® sodium heparin, REF 367874, 
approximately 10 mL each for a total of approximately 70 mL needed total) The research patient 
coordinator will transport the blood to the laboratory of Dr. Gillanders located in the Clinical Sciences 


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Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 83 of 112 
Research Building, Room 6664. PBMC will be obtained by Ficoll-Hypaque gradient centrifugation and 
cryopreserved in 10% DMSO in fetal bovine serum according to standard procedures. Plasma will also be 
collected and stored for the various immunologic testing to be performed.  
11.3.2 Neoantigen identification 
DNA will be isolated from PBMC by the CHIIPs IML or Gillanders laboratory personnel for exome 
sequencing at The Genome Institute. To identify somatic mutations, DNA and RNA will be extracted from 
OCT-embedded tumor tissue. The OCT block will be sectioned for H&E staining by the CHIIPs IML and 
tumor-rich areas will be marked by a trained pathologist. Tumor cells will be macrodissected with or 
without laser capture, and DNA and RNA will be extracted. Tumor DNA and RNA will undergo tumor 
exome and tumor cDNA-capture sequencing, respectively, at the McDonnell Genome Institute. 
The first step in the sequencing pipeline is exome sequencing of breast cancer and normal DNA. Exome 
fragments are captured using Nimblegen's "VCRome" exome capture reagent. Background DNA is 
washed away while the bound exome DNA is eluted and sequenced. Separate libraries are made from 
the breast cancer and normal DNA and processed independently. Exome sequencing is performed using 
the Illumina platform. 
Exome sequences from breast cancer and normal DNA are compared separately to the human reference 
sequence and then to one another to identify somatic variation. VarScan 2 software is used to detect 
misaligned sequences and identify structural variants in the breast cancer DNA.  
The second step in the sequencing pipeline is cDNA-capture sequencing. To validate the results of the 
exome sequencing, and confirm expression of the somatic mutations in the breast cancer, cDNA-capture 
sequencing of the breast cancer RNA will be performed. cDNA-capture sequencing is very similar to RNA 
sequencing, but cDNA is captured prior to sequencing to enrich for mRNA. cDNA-capture sequencing is a 
sensitive and accurate methodology to detect expression of somatic mutations at the mRNA level in 
breast cancer. cDNA-capture sequencing is performed using the Illumina platform.  
We have developed and optimized an epitope prediction algorithm for the identification and prioritization 
of neoantigens. This optimized epitope prediction algorithm is described below and in our previous 
publications [19, 96]. We have established this algorithm in collaboration with Dr. Robert Schreiber, an 
internationally-known expert in tumor immunology [20, 80-87]. Schreiber and Mardis were one of the first 
to use next generation sequencing technologies to identify neoantigens, demonstrating that these 
antigens are important tumor rejection antigens [20]. Schreiber, Mardis, and Gillanders have now 
optimized the epitope prediction algorithm and have demonstrated that cancer vaccines targeting 
neoantigens are associated with antitumor immunity [19]. 
The goal of the optimized epitope prediction algorithm is to identify neoantigens and quantify the 
neoantigen load. The algorithm uses a combination of binding algorithms and processing algorithms. 
Mutations that are expressed in the breast cancer will be identified using the sequencing pipeline outlined 
above. The predicted amino acid sequences corresponding to the expressed mutations will be pipelined 
through three class I MHC epitope-binding algorithms provided by the Immune Epitope Database and 
Analysis Resource (http://www.immuneepitope.org): (i) Stabilized Matrix Method (SMM) [90], (ii) Artificial 
Neural Network (ANN) [91], and (iii) NetMHCpan [92]. 
A prioritized list of binding epitopes (i.e. IC50 < 500 nM) will be generated after calculating the median 
binding affinity value for each mutant sequence (affinity value expressed as 1/IC50 x 100). 
11.3.3 Neoantigen-specific T cell analysis 
Peripheral blood will be processed and stored as PBMC and plasma. A frozen aliquot will be used for 
DNA isolation for both exome sequencing and HLA typing (the latter to be performed by ProImmune). The 
remainder of the samples will be used to analyze the immune response to neoantigen DNA vaccine alone 
vs. neoantigen DNA vaccine + durvalumab. We hypothesize that these two therapies will generate 
durable, neoantigen-specific CD4 and CD8 T cell responses, and affect the levels of immunomodulatory 
elements within the peripheral blood. We propose the following assays. 
Multi-parametric flow cytometry will be used to measure an increase in polyclonal, activated T cells (CD3, 
CD4, CD8, HLA-DR, Ki67, PD-1, perforin, granzyme A/B), neoantigen-specific T cells (CD3, CD4, CD8, 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 84 of 112 
MHC class I tetramers, MHC class II tetramers as described above) that persist over time (CD45RA, 
CD45RO, CCR7, CD27, CD28, CD95, CD58), and immunomodulatory elements (regulatory T cells: CD3, 
CD4, Foxp3, CD25; inflammatory monocytes, granulocytes: CD45, CD11b, CD115, CCR2, CD14, CD15) 
over various time points. 
For functional analysis, Luminex and ELISPOT assays will be performed on PBMC using short peptides 
that mimic the minimal neoantigen epitope incorporated by the DNA vaccine with which the respective 
patient was vaccinated. The Luminex assay will be performed in conjunction with the Immune Monitoring 
Laboratory. ELISPOT assay will be performed as previously described [97-101] on PBMC from the 
subjects at all time points for IFN-γ. PBMC will be plated at various concentrations started at 300,000 
cells per well, in triplicate, following the protocol previously described by Dr. Mohanakumar and 
colleagues for detection of breast cancer-specific T cells. PBMC will be co-cultured with the short 
peptides. As negative controls, PBMC will be incubated in medium alone or stimulated with matching 
wild-type peptides. As a positive control, we will include a mix of viral peptides (CEF) which contains 
immunodominant epitopes for multiple common MHC class I alleles from influenza virus, cytomegalovirus, 
and Epstein-Barr virus. After 24-48 hours, the plates will be developed and the spots counted in an 
ImmunoSpot Series I analyzer (Cellular Technology). 
 
 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 85 of 112 
12 
REMOVAL OF SUBJECTS FROM PROTOCOL THERAPY 
12.1 Removal of subjects from protocol therapy 
Subjects may be removed from protocol therapy if any one or more of the following events occur: 
(1) 
Development of progressive or recurrent disease requiring systemic treatment or radiation 
therapy; 
(2) 
Intercurrent illness that prevents further administration of protocol therapy; 
(3) 
Pregnancy; 
(4) 
Type 1 hypersensitivity reaction associated with protocol therapy; 
(5) 
Grade 2 systemic or injection site adverse event classified as possibly, probably, or definitely 
associated with protocol therapy that does not resolve to at least grade 1 prior to the next 
scheduled treatment; 
(6) 
Grade 3 or 4 systemic or injection site adverse event classified as possibly, probably, or definitely 
associated with protocol therapy; 
(7) 
Grade ≥ 3 infusion reaction; 
(8) 
Any significant autoimmune disease or phenomena presumed to be related to protocol therapy; 
(9) 
Subject refusal to continue protocol therapy and/or observations; 
(10) 
Significant protocol violation or noncompliance, either on the part of the subject or investigator(s); 
(11) 
The principal investigator or study sponsor believes it is in the subject's best interest to 
discontinue participation in the study; 
(12) 
Administrative reasons, e.g., study termination by the principal investigator, Siteman Cancer 
Center, HRPO, FDA, or other group. 
Please note that even if a subject is removed from protocol therapy, they will continue to be followed for 
adverse events. 
12.2 Voluntary subject withdrawal 
The subject has the right to voluntarily withdraw from the study at any time for any reason without 
prejudice to her future medical care by the physician or at the institution. 
For any subject who withdraws consent, the date and reason for consent withdrawal should be 
documented. Subject data will be included in the analysis up to the date of the consent withdrawal. 
12.3 Procedure for discontinuation 
The procedure to be followed at the time a subject either discontinues participation or is removed from the 
study is: 
(1) 
Check for the development of adverse events. 
(2) 
Complete the End-of-Study form and include an explanation of why the subject is withdrawing or 
withdrawn. 
(3) 
Attempt to perform follow-up evaluations as outlined above. 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 86 of 112 
13 
REGULATORY AND REPORTING REQUIREMENTS 
The entities providing oversight of safety and compliance with the protocol require reporting as outlined 
below. Please refer to 20.4 for definitions and 20.5 for a grid of reporting timelines. 
Adverse events will be tracked from start of treatment through 90 days after the last dose of Durvalumab 
or until initiation of new anticancer therapy, whichever comes first. All adverse events must be recorded 
on the toxicity tracking case report form (CRF) with the exception of: 
• 
Baseline adverse events, which shall be recorded on the medical history CRF 
Refer to the data submission schedule in Section 16 for instructions on the collections of AEs in the EDC.  
Reporting requirements for Washington University study team are found in section 13.1. 
13.1 Sponsor-Investigator Reporting Requirements 
13.1.1 Reporting to the Human Research Protection Office (HRPO) at Washington University 
Reporting will be conducted in accordance with Washington University IRB Policies. 
Pre-approval of all protocol exceptions must be obtained prior to implementing the change. 
13.1.2 Reporting to the Quality Assurance and Safety Monitoring Committee (QASMC) at 
Washington University 
The Washington University Sponsor Investigator (or designee) is required to notify the QASMC of any 
unanticipated problems involving risks to participants or others occurring at WU or any BJH or SLCH 
institution that has been reported to and acknowledged by HRPO.  (Unanticipated problems reported to 
HRPO and withdrawn during the review process need not be reported to QASMC.)  
 
QASMC must be notified within 10 days of receipt of IRB acknowledgment via email to 
qasmc@wustl.edu.  Submission to QASMC must include the myIRB form and any supporting 
documentation sent with the form. 
13.1.3 Reporting to Ichor Medical Systems 
Since the TDS-IM device may be involved with the occurrence of adverse events, Ichor Medical Systems 
will be included in the Adverse Event reporting plan. Reports to Ichor for SAEs or for adverse events with 
possible relationship to the device should follow the timing for reporting to the WUSM Human Research 
Protection Office and should be directed to Drew Hannaman at dhannaman@ichorms.com.  
13.1.4 Reporting to AstraZeneca 
All SAEs will be reported, whether or not considered causally related to the investigational product, or to 
the study procedure(s). The reporting period for SAEs is the period immediately following the time that 
written informed consent is obtained through 90 days after the last dose of durvalumab or until the 
initiation of alternative anticancer therapy. The investigator and/or Sponsor are responsible for informing 
the Ethics Committee and/or the Regulatory Authority of the SAE as per local requirements. 
The investigator and/or sponsor must inform the FDA, via a MedWatch form, of any serious or 
unexpected adverse events that occur in accordance with the reporting obligations of 21 CFR 312.32, 
and will concurrently forward all such reports to AstraZeneca. A copy of the MedWatch report must be 
emailed to AstraZeneca at the time the event is reported to the FDA. It is the responsibility of the sponsor 
to compile all necessary information and ensure that the FDA receives a report according to the FDA 
reporting requirement timelines and to ensure that these reports are also submitted to AstraZeneca at the 
same time. 
* A cover page should accompany the MedWatch form indicating the following: 
• 
“Notification from an Investigator Sponsored Study” 
• 
The investigator IND number assigned by the FDA 
• 
The investigator’s name and address 
• 
The trial name/title and AstraZeneca ISS reference number (ESR-16-12613) 


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Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 87 of 112 
* Sponsor must also indicate, either in the SAE report or the cover page, the causality of events in 
relation to all study medications and if the SAE is related to disease progression, as determined by 
the principal investigator. 
* Send SAE report and accompanying cover page by way of email to AstraZeneca’s designated 
mailbox: AEMailboxClinicalTrialTCS@astrazeneca.com 
If a non-serious AE becomes serious, this and other relevant follow-up information must also be provided 
to AstraZeneca and the FDA. 
Serious adverse events that do not require expedited reporting to the FDA still need to be reported to 
AstraZeneca preferably using the MedDRA coding language for serious adverse events. This information 
should be reported on a monthly basis and under no circumstance less frequently than quarterly.  
13.1.4.1 Reporting of deaths to AstraZeneca 
All deaths that occur during the study or within the protocol-defined 90-day post-last dose of durvalumab 
safety follow-up period must be reported to AstraZeneca as follows: 
• 
Death that is clearly the result of disease progression should be documented but not reported as 
an SAE 
• 
Where death is not due (or not clearly due) to progression of the disease under study, the AE 
causing the death must be reported to AstraZeneca as an SAE within 24 hours. The report 
should contain a comment regarding the co-involvement of progression of disease, if appropriate, 
and should assign main and contributory causes of death. 
• 
Deaths with an unknown cause should always be reported as an SAE. 
Deaths that occur following the protocol-defined 90-day post-last dose of durvalumab safety follow-up 
period will be documented but will not be reported as an SAE. 
13.1.4.2 Definition of adverse events of special interest 
An adverse event of special interest (AESI) is one of scientific and medical interest specific to 
understanding of the Investigational Product and may require close monitoring and rapid communication 
by the investigator to the sponsor. An AESI may be serious or non-serious. The rapid reporting of AESIs 
allows ongoing surveillance of these events in order to characterize and understand them in association 
with the use of this investigational product. 
AESIs for durvalumab include but are not limited to events with a potential inflammatory or immune-
mediated mechanism and which may require more frequent monitoring and/or interventions such as 
steroids, immunosuppressants and/or hormone replacement therapy. These AESIs are being closely 
monitored in clinical studies with durvalumab monotherapy and combination therapy. An immune-
mediated adverse event (imAE) is defined as an adverse event that is associated with drug exposure and 
is consistent with an immune-mediated mechanism of action and where there is no clear alternate 
etiology. Serologic, immunologic, and histologic (biopsy) data, as appropriate, should be used to support 
an imAE diagnosis. Appropriate efforts should be made to rule out neoplastic, infectious, metabolic, toxin, 
or other etiologic causes of the imAE.  
If the Investigator has any questions in regards to an adverse event (AE) being an imAE, the Investigator 
should promptly contact the Clinical Study Lead. 
AESIs observed with durvalumab include: 
• 
Diarrhea / Colitis and intestinal perforation 
• 
Pneumonitis / ILD 
• 
ALT/AST increases / hepatitis / hepatotoxicity  
• 
Neuropathy / neuromuscular toxicity (i.e. Guillain-Barré and myasthenia gravis) 
• 
Endocrinopathy (i.e. events of hypophysitis, adrenal insufficiency, diabetes insipidus and hyper- 
and hypothyroidism and type I diabetes mellitus) 
• 
Rash / Dermatitis 


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 88 of 112 
• 
Nephritis / blood creatinine increases 
• 
Pancreatitis (or labs suggestive of pancreatitis - increased serum lipase, increased serum 
amylase)  
• 
Myocarditis 
• 
Myositis / Polymyositis 
• 
Other inflammatory response that are rare / less frequent with a potential immune-mediated 
aetiology include, but are not limited to, pericarditis, sarcoidosis, uveitis and other events 
involving the eyes, skin, haematological and rheumatological events, vasculitis, non-infectious 
meningitis and non-infectious encephalitis. 
 
In addition, infusion-related reactions and hypersensitivity/anaphylactic reactions with a different 
underlying pharmacological aetiology are also considered AESIs.  
Further information on these risks (e.g. presenting symptoms) can be found in the current version of the 
durvalumab Investigator Brochure. More specific guidelines for their evaluation and treatment are 
described in detail in the Dosing Modification and Toxicity Management Guidelines. These guidelines 
have been prepared to assist the Investigator in the exercise of his/her clinical judgment in treating these 
types of toxicities. These guidelines apply to AEs considered causally related to the study drug/study 
regimen by the reporting investigator. 
13.1.4.3 Hy’s Law 
Cases where a patient shows elevations in liver biochemistry may require further evaluation and 
occurrences of AST or ALT ≥ 3x ULN together with total bilirubin ≥ 2x ULN may need to be reported as 
SAEs. Please refer to the Dosing Modification and Toxicity Management Guidelines for further 
instructions on cases of increases in liver biochemistry and evaluation of Hy’s law. 
13.1.4.4 Overdose 
An overdose is defined as a subject receiving a dose of durvalumab in excess of that specified in the 
Investigator’s Brochure, unless otherwise specified in this protocol. 
Any overdose of a study subject with durvalumab, with or without associated AEs/SAEs, is required to be 
reported within 24 hours of knowledge of the event to the sponsor and AstraZeneca/MedImmune Patient 
Safety or designee using the designated Safety e-mailbox. If the overdose results in an AE, the AE must 
also be recorded as an AE. Overdose does not automatically make an AE serious, but if the 
consequences of the overdose are serious, for example death or hospitalization, the event is serious and 
must be recorded and reported as an SAE. There is currently no specific treatment in the event of an 
overdose of durvalumab.  
The investigator will use clinical judgment to treat any overdose. 
13.1.4.5 Hepatic function abnormality 
Hepatic function abnormality that fulfills the biochemical criteria of a potential Hy’s Law case in a study 
subject, with or without associated clinical manifestations, is required to be reported as “hepatic function 
abnormal” within 24 hours of knowledge of the event to the sponsor and AstraZeneca Patient Safety 
using the designated Safety e-mailbox, unless a definitive underlying diagnosis for the abnormality (e.g., 
cholelithiasis or bile duct obstruction) that is unrelated to investigational product has been confirmed. 
• 
If the definitive underlying diagnosis for the abnormality has been established and is unrelated to 
investigational product, the decision to continue dosing of the study subject will be based on the 
clinical judgment of the investigator. 
• 
If no definitive underlying diagnosis for the abnormality is established, dosing of the study subject 
must be interrupted immediately. Follow-up investigations and inquiries must be initiated by the 
investigational site without delay. 
Each reported event of hepatic function abnormality will be followed by the investigator and evaluated by 
the sponsor and AstraZeneca/MedImmune.  


Washington University School of Medicine 
Protocol Date 04/24/2023 
Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
Page 89 of 112 
13.1.4.6 Pregnancy 
If a patient becomes pregnant during the course of the study, the IPs should be discontinued immediately.  
Pregnancy itself is not regarded as an AE unless there is a suspicion that the IP under study may have 
interfered with the effectiveness of a contraceptive medication. Congenital abnormalities or birth defects 
and spontaneous miscarriages should be reported and handled as SAEs. Elective abortions without 
complications should not be handled as AEs. The outcome of all pregnancies (spontaneous miscarriage, 
elective termination, ectopic pregnancy, normal birth, or congenital abnormality) should be followed up 
and documented even if the patient was discontinued from the study.  
If any pregnancy occurs in the course of the study, then the Investigator or other site personnel should 
inform the appropriate AstraZeneca representatives within 1 day, ie, immediately, but no later than 24 
hours of when he or she becomes aware of it.  
The designated AstraZeneca representative will work with the Investigator to ensure that all relevant 
information is provided to the AstraZeneca Patient Safety data entry site within 1 to 5 calendar days for 
SAEs and within 30 days for all other pregnancies.  
The same timelines apply when outcome information is available. 
13.1.5 Reporting to the FDA 
The conduct of the study will comply with all FDA safety reporting requirements.  PLEASE NOTE THAT 
REPORTING REQUIREMENTS FOR THE FDA DIFFER FROM REPORTING REQUIREMENTS FOR 
HRPO/QASMC.  It is the responsibility of the Washington University Sponsor-Investigator to report to the 
FDA as follows: 
 
• 
Report any unexpected fatal or life-threatening suspected adverse reaction (refer to Appendix B 
for definitions) no later than 7 calendar days after initial receipt of the information.   
• 
Report a suspected adverse reaction that is both serious and unexpected (SUSAR, refer to 
Appendix B) no later than 15 calendar days after it is determined that the information qualifies for 
reporting.  Report an adverse event (refer to Appendix A) as a suspected adverse reaction only if 
there is evidence to suggest a causal relationship between the drug and the adverse event, such 
as: 
o 
A single occurrence of an event that is uncommon and known to be strongly associated 
with drug exposure 
o 
One or more occurrences of an event that is not commonly associated with drug 
exposure but is otherwise uncommon in the population exposed to the drug 
o 
An aggregate analysis of specific events observed in a clinical trial that indicates those 
events occur more frequently in the drug treatment group than in a concurrent or 
historical control group 
• 
Report any findings from epidemiological studies, pooled analysis of multiple studies, or clinical 
studies that suggest a significant risk in humans exposed to the drug no later than 15 calendar 
days after it is determined that the information qualifies for reporting. 
• 
Report any findings from animal or in vitro testing that suggest significant risk in humans exposed 
to the drug no later than 15 calendar days after it is determined that the information qualifies for 
reporting.  
• 
Report any clinically important increase in the rate of a serious suspected adverse reaction of that 
listed in the protocol or IB within 15 calendar days after it is determined that the information 
qualifies for reporting. 
 
Submit each report as an IND safety report in a narrative format or on FDA Form 3500A or in an 
electronic format that FDA can process, review, and archive.  Study teams must notify the Siteman 
Cancer Center Protocol Development team of each potentially reportable event within 1 business day 
after initial receipt of the information, and must bring the signed 1571 and FDA Form 3500A to the 
Siteman Cancer Center Protocol Development team no later than 1 business day prior to the due date for 
reporting to the FDA. 
 


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Each notification to FDA must bear prominent identification of its contents (“IND Safety Report”) and must 
be transmitted to the review division in the Center for Drug Evaluation and Research (CDER) or in the 
Center for Biologics Evaluation and Research (CBER) that has responsibility for review of the IND.  
Relevant follow-up information to an IND safety report must be submitted as soon as the information is 
available and must be identified as such (“Follow-up IND Safety Report”). 
13.1.6 Reporting to the Institutional Biosafety Committee 
In accordance with institutional policies and NIH guidelines, any unanticipated problems must be reported 
to the Institutional Biological and Chemical Safety Committee (IBC) at Washington University School of 
Medicine.   
 
The Washington University Sponsor-Investigator (or designee) must report the following events to the 
Biosafety Officer at the time of submission to HRPO: 
• 
Any overt personnel exposure to recombinant DNA-containing material, whether or not that 
exposure leads to illness 
• 
Any significant spill of recombinant DNA-containing material outside of a biological safety cabinet, 
where a significant spill is: 
o 
A spill of recombinant risk group 1 agent-containing material which requires remediation 
by EH&S or other first responders 
o 
A spill of recombinant risk group 2 agent-containing material which is greater than 1 liter 
o 
Any size spill of risk group 3 agent-containing material or material the IBC has mandated 
to be handled using BSL2+ practices and procedures 
• 
Any incident which results in the release of recombinant DNA to the environment 
 
In addition, the IBC must be informed of the following events:  
• 
Any serious adverse event which is both unexpected and associated with the use of the gene 
transfer product 
 
The Biosafety Officer may be contacted at ehsibc@wustl.edu. 
 
When submitting reports to the IBC, the FDA MedWatch form will be used.  The Washington University 
Sponsor-Investigator (or designee) will be responsible for submitting all MedWatch forms from secondary 
sites to the IBC.  
 
WUSM IBC guidelines specify that any study modifications related to the investigational agent as well as 
the IRB renewal paperwork should be sent to the IBC for approval (if a modification) or acknowledgment 
(if an annual renewal).  The IRB and IBC will review all submissions simultaneously. 
  
 
 


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Breast Cancer Program 
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14 
DATA AND SAFETY MONITORING 
14.1 Protocol stopping criteria 
The principal investigator will closely monitor and analyze study data as they become available and will 
make determinations regarding the presence and severity of adverse events. The administration of study 
injections and new enrollments will be halted and the QASMC promptly notified if any of the following 
events occurs: 
(1) 
One (or more) subject(s) experiences a Grade 3 or 4 adverse event that is classified as probably 
or definitely related to treatment with durvalumab or vaccination; 
(2) 
One (or more) subject(s) experiences an SAE related to either durvalumab or vaccine; 
(3) 
Two (or more) subjects experience the same Grade 2 or higher adverse event that is classified 
as probably or definitely related to durvalumab or vaccination: this criterion applies to fever, 
vomiting, laboratory abnormalities or other clinical adverse experiences, but does not apply to the 
subjective local or systemic symptoms of pain/tenderness, malaise, fatigue, headache, chills, 
nausea, myalgia, or arthralgia. 
(4) 
Death (other than death related to progressive disease) that occurs within 30 days after receiving 
DNA vaccination. 
(5) 
Any other observation occurs that in the opinion of the PI results in a recommendation to halt 
enrollment. 
If one of these events does occur, study injections and study enrollments would only resume if review of 
the adverse events that caused the halt resulted in a recommendation to permit further study injections 
and study enrollments. 
The QASMC, in consultation with the principal investigator, will conduct any review and make the 
decision to resume or close the study for any Grade 2 or 3 events leading to a halt in the study. 
The QASMC, with participation by the principal investigator, will consult with the FDA to conduct the 
review and make the decision to resume or close the study for all Grade 4 adverse events leading to a 
halt in the study. 
14.2 Data safety monitoring plan 
In compliance with the Washington University Institutional Data and Safety Monitoring Plan, the Principal 
Investigator will provide a Data and Safety Monitoring (DSM) report to the Washington University Quality 
Assurance and Safety Monitoring Committee (QASMC) semi-annually beginning six months after accrual 
has opened (if at least one patient has been enrolled) or one year after accrual has opened (if no patients 
have been enrolled at the six-month mark). 
The Principal Investigator will review all patient data at least every six months, and provide a semi-annual 
report to the QASMC. This report will include: 
• 
HRPO protocol number, protocol title, Principal Investigator name, data coordinator name, 
regulatory coordinator name, and statistician 
• 
Date of initial HRPO approval, date of most recent consent HRPO approval/revision, date of 
HRPO expiration, date of most recent QA audit, study status, and phase of study 
• 
History of study including summary of substantive amendments; summary of accrual suspensions 
including start/stop dates and reason; and summary of protocol exceptions, error, or breach of 
confidentiality including start/stop dates and reason 
• 
Study-wide target accrual and study-wide actual accrual 
• 
Protocol activation date 
• 
Average rate of accrual observed in year 1, year 2, and subsequent years 
• 
Expected accrual end date 
• 
Objectives of protocol with supporting data and list the number of participants who have met each 
objective 


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Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
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• 
Measures of efficacy 
• 
Early stopping rules with supporting data and list the number of participants who have met the 
early stopping rules 
• 
Summary of toxicities 
• 
Abstract submissions/publications 
• 
Summary of any recent literature that may affect the safety or ethics of the study  
The study principal investigator and Research Patient Coordinator will monitor for serious toxicities on an 
ongoing basis. Once the principal investigator or Research Patient Coordinator becomes aware of an 
adverse event, the AE will be reported to the HRPO and QASMC according to institutional guidelines. 
14.3 Developmental therapeutics 
Given the nature of this human gene transfer protocol, the principal investigator will monitor and analyze 
study data as they become available and will review this data on a monthly basis with the Developmental 
Therapeutics Group at the Siteman Cancer Center. This is an independent group that will provide more 
rigorous oversight than is routinely provided by the QASMC and the HRPO. The Director of the 
Developmental Therapeutics Group devotes 20% of their time to overseeing and managing early phase 
clinical trials. These trials consist of all phase 1 trials and selected Phase 2 trials. The Director will advise 
Dr. Gillanders in the proper conduct of this study and will review patient treatment and all toxicities in the 
weekly Phase 1 meeting. In addition, the Director will assist in the oversight of the regulatory and data 
management personnel involved in this clinical trial. 
14.4 NIH Recombinant DNA Advisory Committee 
14.4.1 Initiation of the clinical investigation 
Appendix M-I-C-1 of the NIH Guidelines for Research Involving Recombinant DNA Molecules specify: 
No later than 20 working days after enrollment (see definition of enrollment in Section I-E-7) of the 
first research participant in a human gene transfer experiment, the Principal Investigator(s) shall 
submit the following documentation to NIH OBA: (1) a copy of the informed consent document 
approved by the Institutional Review Board (IRB); (2) a copy of the protocol approved by the 
Institutional Biosafety Committee (IBC) and IRB; (3) a copy of the final IBC approval from the clinical 
trial site; (4) a copy of the final IRB approval; (5) a brief written report that includes the following 
information: (a) how the investigator(s) responded to each of the RAC’s recommendations on the 
protocol (if applicable); and (b) any modifications to the protocol as required by FDA; (6) applicable 
NIH grant number(s); (7) the FDA Investigational New Drug Application (IND) number; and (8) the 
date of the initiation of the trial. The purpose of requesting the FDA IND number is for facilitating 
interagency collaboration in the Federal oversight of human gene transfer research. 
 


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Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
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15 
STATISTICAL CONSIDERATIONS 
15.1 Study objectives and endpoints 
This is a single institution, open-label randomized phase 1 trial of neoantigen DNA vaccine alone vs. 
neoantigen DNA vaccine plus durvalumab in triple negative breast cancer (TNBC) patients following 
standard of care therapy. Patients with newly diagnosed clinical stage II-III TNBC are eligible for 
enrollment. Patients will receive standard of care therapy including chemotherapy, surgery and radiation 
therapy as clinically indicated. Following standard of care therapy, patients will be randomized to receive 
either a neoantigen DNA vaccine alone, or a neoantigen DNA vaccine + durvalumab.  
15.1.1 Primary objective 
The primary objective is to assess the safety of neoantigen DNA vaccines given alone or in combination 
with durvalumab. Safety assessment will include both clinical observation and laboratory evaluation. 
Toxicity will be graded according to the National Cancer Institute Common Terminology Criteria for 
Adverse Events v4.03. 
15.1.2 Secondary objectives.  
The secondary objective is to measure the immune response to neoantigen DNA vaccines given alone, or 
in combination with durvalumab. The immune response will be measured in the peripheral blood by 
luminex assay, ELISPOT, and multiparametric flow cytometry. 
15.2 Study design and sample justification 
This is a single institution, open-label randomized phase 1 trial of neoantigen DNA vaccine alone vs. 
neoantigen DNA vaccine plus durvalumab in triple negative breast cancer (TNBC) patients following 
standard of care therapy. Patients with newly diagnosed clinical stage II-III TNBC are eligible for 
enrollment. Patients will receive standard of care therapy including chemotherapy, surgery and radiation 
therapy as clinically indicated. Following standard of care therapy, patients will be randomized to receive 
either a neoantigen DNA vaccine alone, or a neoantigen DNA vaccine + durvalumab 
Twenty-four (24) eligible patients will be enrolled, and we expect that majority of them will receive the 
vaccine and will be eligible for evaluation. 
15.2.1 Sample Size Calculations for Immunogenicity 
A power analysis was performed using the paired t-test for over time differences (i.e., measures at 
Baseline vs. Day 1, Day 57) to test the first hypotheses that neoantigen DNA vaccines can induce and/or 
enhance neoantigen-specific T cell responses (e.g., the frequency of neoantigen-specific CD8 T cells in 
ELISPOT and multi-parameter flow cytometry). Assuming a moderate correlation between measures 
taken from the same individual, the designed sample size (n=24 patients) allows us 80% power at 1-sided 
0.05 alpha level to detect a minimum of 0.5*SD for overtime changes. A power analysis was also 
performed using the 2-sample t-test for between-arm differences (i.e., measures at Day 113, Day 169) to 
test the second hypotheses that durvalumab will enhance the response to neoantigen DNA vaccines. We 
expect that the majority of the patients enrolled will remain in the study and lead to at least 10 evaluable 
patients/arm for such comparison. The designed sample size (n=10 patients/arm) provides us 80% power 
at 1-sided 0.05 alpha level to detect a minimum of 1.2*SD for between-group differences. Preliminary 
data from our phase 1 trial for mammaglobin-A DNA vaccine, though on patients with stable metastatic 
breast cancer and targeting a tissue-specific antigen, showed that the frequency of antigen-specific CD8 
T cells can be measured with ~20% coefficient of variation (CV=SD/Mean). Therefore, the designed 
sample size allows us to detect ~10% for over-time differences and ~15% for between-group differences 
respectively. As a study to estimate preliminary information, we expect that ~20% difference will provide 
an adequate signal for immunogenicity. In addition, we anticipate that more power could be achieved 
because the actual data analysis will be performed using mixed model which uses data more efficiently 
and allows us to borrow information across multiple time points. 


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Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
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15.2.2 Sample Size Calculations for Safety 
Sample size calculations for safety are expressed in terms of the ability to detect serious adverse events. 
The ability of the study to identify serious adverse events is best expressed by the maximum true rate of 
events that would be unlikely to be observed and the minimum true rate of events that would very likely 
be observed. Based on extensive simulations regarding the sample size for translational studies, [102] 
Piantadosi recommended that a sample size of 10 to 20 patients would provide a reasonable precision for 
estimating preliminary information. For example, there is 80% chance of observing at least 1 serious 
adverse event among a sample size of n=10 patients if the "true" AE rate is at least 15%. Conversely, if 
the "true" AE rate is less than 3%, there is 3% chance to observe 2 or more serious adverse events. 
15.3 Data Analysis 
15.3.1 Safety analysis 
Safety evaluation is the primary objective of the trial. The data will be descriptive, and standard toxicity 
definitions and criteria will be used as outlined in the National Cancer Institute Common Terminology 
Criteria for Adverse Events version 4.03. 
The number and percentage of subjects experiencing each type of adverse event will be tabulated by 
severity, and relationship to treatment. If appropriate, confidence intervals will be used to characterize the 
precision of the estimate. A complete listing of adverse events will also be tabulated, and will provide 
details including severity, relationship to treatment, onset, duration, and outcome. 
Laboratory data measured on a continuous scale will be characterized by summary statistics (mean and 
standard deviation). Boxplots of laboratory data will be generated for baseline values and for values 
measured during and after protocol therapy at each specific time point. Contingency tables will also be 
used to describe the change of safe data over time as appropriate. 
15.3.2 Immune response 
Neoantigen-specific T cell response to neoantigen DNA vaccines + durvalumab is the secondary  
objective of the trial. Neoantigen specific T cell responses will be measured by Luminex, ELISPOT, 
multiparametric flow cytometry, and CyTOF. The frequency of antigen-specific CD8 T cells in each arm at 
each time will be summarized using means, standard deviations and medians. The change over time as 
well as the difference between arms will also be compared using linear mixed model for repeated 
measurement data. The immunogenicity of the neoantigen DNA vaccine will also be analyzed 
qualitatively by summarizing the phenotypic and functional characteristics of epitope-specific CD8 T cells. 
Responses will be considered positive if the number of T cells after vaccination is greater than two 
standard deviations above the mean before vaccination [101]. The frequency of positive responses at 
each time point will be assessed and binomial response rates with 95% confidence interval estimates will 
be presented. In addition to presenting the binomial response rates, graphical and tabular summaries of 
the underlying distributions will be made. 
To determine whether the observed difference is larger than might be expected by chance, a permutation 
test will be used to compare the observed test statistic to the distribution of test statistics that would be 
seen if there were no difference between the two arms. Specifically, we will randomly shuffle the data and 
calculate the test statistic from the shuffled data. This procedure will be repeated 10,000 times and the 
resultant testing statistics will provide an accurate representation of the null distribution. The observed 
test statistics of between-arm differences will be compared to the null distributions. For each outcome, the 
permuted p-value will be the fraction of permuted samples that resulted in a small statistic than the 
original sample [103]. 
 
 


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Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
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16 
DATA MANAGEMENT 
Case report forms with appropriate source documentation will be completed according to the schedule 
below. 
 
Case Report Form 
Submission Schedule 
Original Consent Form 
Prior to registration 
Demographics Form 
On-Study Form 
Prior to starting treatment 
Physical Exam Form 
Baseline 
Day 1 
Day 29 
Day 57 
Day 85 
Day 113 
Day 141 
Durvalumab Form 
Day 85 
Day 113 
Day 141 
Day 169 
Randomization Form 
After end of SOC adjuvant therapy 
Vaccine Administration Form 
Day 1 
Day 29 
Day 57 
Day 85 
Day 113 
Day 141 
Immune Monitoring Form 
Baseline 
End of SOC adjuvant therapy 
Day 1 
Day 57 
Day 113 
Day 169 
Week 52 after completion of neoantigen DNA vaccine 
therapy 
Imaging Form 
Baseline 
Toxicity Form 
Concomitant Medications Form 
Continuous  
Follow Up Form 
Week 52 after completion of neoantigen DNA vaccine 
therapy 
Year 2 
Year 3 
Year 4 
Year 5 
MedWatch Form 
See Section 13.0 for reporting requirements 
 
16.1 Adverse Event Collection in the Case Report Forms 
 
All adverse events that occur beginning with start of treatment (minus exceptions defined in Section 13) 
must be captured in the Toxicity Form.  Baseline AEs should be captured on the Medical History Form. 
 


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Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
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Participant death due to disease progression should be reported on the Toxicity Form as grade 5 disease 
progression.  If death is due to an AE (e.g. cardiac disorders: cardiac arrest), report as a grade 5 event 
under that AE.  Participant death must also be recorded on the Death Form.


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Siteman Cancer Center 
Breast Cancer Program 
Neoantigen DNA vaccine +/- durvalumab 
 
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17 
REGULATORY AND ETHICAL OBLIGATIONS 
17.1 Informed consent 
In accordance with US FDA regulations (21 CFR 50) and guidelines (Federal Register, May 9, 1997, Vol. 
62, Number 90 - ICH Good Clinical Practice Consolidated Guideline) it is the investigator’s responsibility 
to ensure that informed consent is obtained from the subject before participating in an investigational 
study, after an adequate explanation of the purpose, methods, risks, potential benefits and subject 
responsibilities of the study. Procedures that are to be performed as part of the practice of medicine and 
which would be done whether or not study entry was contemplated, such as for diagnosis or treatment of 
a disease or medical condition, may be performed and the results subsequently used for determining 
study eligibility without first obtaining consent. On the other hand, informed consent must be obtained 
prior to initiation of any screening procedures that are performed solely for the purpose of determining 
eligibility for research. 
Each subject must be given a copy of the informed consent. The original signed consent must be retained 
in the institution’s records and is subject to review by the sponsor, the HRPO and any other applicable 
regulatory agencies responsible for the conduct of the institution. All elements listed in the ICH Good 
Clinical Practice guidelines must be included in the informed consent. 
Informed consent will be obtained by either the principal investigator or by individuals approved by the 
principal investigator. Informed consent will be obtained from the subject after the details of the protocol 
have been reviewed. The individual responsible for obtaining consent will assure, prior to signing of the 
informed consent, that the subject has had all questions regarding therapy and the protocol answered. 
17.2 Institutional Review Board 
In accordance with US FDA regulations (21 CFR 56) and guidelines (Federal Register, May 9, 1997 Vol. 
62 Number 90 - ICH Good Clinical Practice Consolidated Guideline) all research involving human 
subjects must be reviewed and approved by the local IRB. All modifications to the protocol, consent 
forms, or other study documents must be reviewed and approved by the local IRB. At Washington 
University School of Medicine, the Human Research Protection Office serves as the local IRB. 
17.3 Subject confidentiality 
In order to ensure subject confidentiality, each subject will be assigned a study number. Subject samples 
and medical information will be de-identified and labeled with the study number. The link between subject 
identification and study number will be safeguarded in a secure file in a locked room, and access will be 
restricted to the principal investigator, study coordinator, and other co-investigators as necessary. 
Collected data will be recorded on case report forms. Case report forms will be safeguarded in a locked 
cabinet and/or a password-protected secure computer drive and access will be restricted to the principal 
investigator, study coordinator, and other co-investigators as necessary. Subject medical information 
related to, or obtained for the purposes of this trial are confidential, and disclosure to third parties is 
prohibited. The exception is regulatory authorities including the FDA, NIH/OBA, and the local IRB. Data 
from this study must be available for inspection on request of regulatory authorities including the FDA and 
the local IRB. 
 


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Breast Cancer Program 
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18 
ADMINISTRATIVE AND LEGAL OBLIGATIONS 
18.1 Study documentation and retention of records 
18.1.1 Study documentation 
Source documents are original documents, data, and records from which the subject’s data are obtained. 
These include but are not limited to hospital records, clinical and office charts, laboratory and pharmacy 
records, diaries, diagnostic imaging studies, and correspondence. 
The principal investigator and staff are responsible for maintaining a comprehensive file of all study-
related documents, suitable for inspection at any time by representatives from the PRMC, HRPO, FDA, 
and any other applicable regulatory agency. 
Pertinent documents in the study file include: 
(1) 
The original protocol with all amendments 
(2) 
Curriculum vitae of principal investigator and co-investigators 
(3) 
Approval notification and any other correspondence with the PRMC, HRPO, NIH RAC and FDA 
Pertinent documents in each individual subject file include: 
(1) 
Informed consent forms 
(2) 
Case report forms 
(3) 
Supporting copies of source documentation 
All original source documentation must be readily available. 
18.1.2 Retention of records 
The principal investigator must retain records related to this study including protocols; amendments; 
IRB/IBC approvals; FDA IND records and other correspondence; completed, signed and dated consent 
forms; patient medical records; case report forms; drug accountability records and any other 
correspondence related to the conduct of the study. 
U.S. FDA regulations (21 CFR 312.62[c]) require that all records pertaining to the conduct of this study, 
must be retained by the responsible investigator for a minimum of 2 years after marketing application 
approval. If no application is filed, these records must be kept 3 years after the investigation is 
discontinued and the U.S. FDA and the applicable local health authorities are notified. 
18.2 Policy regarding research-related injuries 
Washington University School of Medicine investigators and their staffs will try to reduce, control, and 
treat any complications from this research. 
Any subjects who believe that they have been injured as a result of participation in this study will be 
instructed to contact the principal investigator, William E. Gillanders, M.D. at (314) 747-0072. 
Alternatively, they can contact the Human Research Protection Office, at (800) 438-0445. 
Decisions about payment for medical treatment for research-related injuries will be made by Washington 
University School of Medicine. 
In general, Washington University School of Medicine will provide no long-term medical care or financial 
compensation for research-related injuries. 
18.3 Study termination 
The principal investigator and the Siteman Cancer Center reserve the right to terminate the study. The 
principal investigator will notify the PRMC and HRPO in writing of the study's completion or early 
termination. 


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Breast Cancer Program 
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20 
APPENDICES 
20.1 Abbreviations 
AE 
Adverse Event 
CBER 
Center for Biologics Evaluation and Research 
CFR 
Code of Federal Regulations 
CPA 
Cooperative Project Assurance 
CR 
Complete response 
CRA 
Clinical Research Associate 
CRF 
Case Report Form 
CT 
Computed Tomography 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTEP 
Cancer Therapy Evaluation Program 
DNA 
Deoxyribonucleic acid 
DCTD 
Division of Cancer Treatment and Diagnosis 
ELISPOT 
Enzyme-linked immunospot assay 
ECOG 
Eastern Cooperative Oncology Group 
FDA 
Food and Drug Administration 
FWA 
Federal-wide Assurance 
GLP 
Good Laboratory Practice 
GMP 
Good Manufacturing Practice 
GPC 
Gel permeation chromatography 
HIPAA 
Health Insurance Portability and Accountability Act 
HLA 
Human leukocyte antigen 
HRPO 
Human Research Protection Office (Institutional Review Board at WUSM) 
IBC 
Institutional Biosafety Committee 
ICH 
International Conference of Harmonization 
IDB 
Investigational Drug Branch 
IRB 
Institutional Review Board 
LD 
Longest diameter 
MPA 
Multiple Project Assurance 
MRI 
Magnetic Resonance Imaging 
NCI 
National Cancer Institute 
NIH 
National Institutes of Health 
OBA 
Office of Biotechnology Activities 
OHRP 
Office for Human Research Protection 
PBMC 
Peripheral blood mononuclear cell 
PD 
Progressive disease 
PHI 
Protected Health Information 
PR 
Partial response 
RAC 
Recombinant DNA Advisory Committee 
RECIST 
Response Evaluation Criteria in Solid Tumors 
QASMC 
Quality Assurance and Safety Monitoring Committee 
SAE 
Serious Adverse Event 
SAS 
Statistical Analysis System; Analytical software from the SAS Institute, Cary, NC 
SCC 
Siteman Cancer Center 
SCIP 
Siteman Cancer Information Portal 
SD 
Stable disease 
SD 
Standard Deviation 
THF 
Tetrahydrofuran 
UPN 
Universal Product Number 
WUSM 
Washington University School of Medicine 
WUSTL 
Washington University in Saint Louis 


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20.2 ECOG/Zubrod performance status scale 
ECOG/Zubrod Score
Performance Status
0
Asymptomatic
1
Symptomatic, fully ambulatory
2
Symptomatic, in bed < 50% of the day
3
Symptomatic, in bed > 50% of the day but not bedridden
4
Bedridden
5
Dead
 
20.3 National Cancer Institute Common Terminology Criteria for Adverse Events 
This study will collect adverse events using the NCI Common Terminology Criteria for Adverse Events 
v4.03 (CTCAE), if applicable. The CTCAE provides a descriptive terminology that is to be used for 
adverse event reporting. A grading (severity) scale is also provided in the CTCAE for each adverse event 
term. An electronic version of the CTCAE may be accessed through the web at http://ctep.cancer.gov. 
Alternatively, a full copy is available from the principal investigator.  
20.4 
Definitions for Adverse Event Reporting 
A. Adverse Events (AEs) 
 
As defined in 21 CFR 312.32: 
 
Definition: any untoward medical occurrence associated with the use of a drug in humans, whether or 
not considered drug-related. 
 
Grading: the descriptions and grading scales found in the revised NCI Common Terminology Criteria 
for Adverse Events (CTCAE) version 5.0 will be utilized for all toxicity reporting.  A copy of the 
CTCAE version 5.0 can be downloaded from the CTEP website. 
 
Attribution (relatedness), Expectedness, and Seriousness: the definitions for the terms listed that 
should be used are those provided by the Department of Health and Human Services’ Office for 
Human Research Protections (OHRP).  A copy of this guidance can be found on OHRP’s website: 
http://www.hhs.gov/ohrp/policy/advevntguid.html 
 
B. Suspected Adverse Reaction (SAR) 
 
As defined in 21 CFR 312.32: 
 
Definition: any adverse event for which there is a reasonable possibility that the drug caused the 
adverse event.  “Reasonable possibility” means there is evidence to suggest a causal relationship 
between the drug and the adverse event.  “Suspected adverse reaction” implies a lesser degree of 
certainty about causality than adverse reaction, which means any adverse event caused by a drug. 
 
C. Life-Threatening Adverse Event / Life Threatening Suspected Adverse Reaction  
 
As defined in 21 CFR 312.32: 
 
Definition: any adverse drug event or suspected adverse reaction is considered “life-threatening” if, in 
the view of the investigator, its occurrence places the patient at immediate risk of death. It does not 


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include an adverse event or suspected adverse reaction that, had it occurred in a more severe form, 
might have caused death. 
 
D. Serious Adverse Event (SAE) or Serious Suspected Adverse Reaction 
 
As defined in 21 CFR 312.32: 
 
Definition:  an adverse event or suspected adverse reaction is considered “serious” if, in the view of 
the investigator, 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 
• 
A congenital anomaly/birth defect 
• 
Any other important medical event that does not fit the criteria above but, based upon 
appropriate medical judgment, may jeopardize the subject and may require medical or 
surgical intervention to prevent one of the outcomes listed above 
 
E. Protocol Exceptions 
 
Definition: A planned change in the conduct of the research for one participant. 
 
F. Deviation 
 
Definition: Any alteration or modification to the IRB-approved research without prospective IRB 
approval.  The term “research” encompasses all IRB-approved materials and documents including 
the detailed protocol, IRB application, consent form, recruitment materials, questionnaires/data 
collection forms, and any other information relating to the research study. 
 
A minor or administrative deviation is one that does not have the potential to negatively impact the 
rights, safety, or welfare of participants or others or the scientific validity of the study. 
 
A major deviation is one that does have the potential to negatively impact the rights, safety, or welfare 
of participants or others or the scientific validity of the study. 
 
 
 


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20.5 Reporting Timelines 
Expedited Reporting Timelines 
Event 
HRPO 
QASMC 
FDA 
IBC 
Ichor Medical 
Systems 
AstraZeneca 
Serious AND 
unexpected suspected 
adverse reaction 
 
 
Report no later than 15 
calendar days after it is 
determined that the 
information qualifies for 
reporting 
Report at time of HRPO 
submission; IBC will 
retrieve information from 
myIRB and will follow up 
with study team if 
needed 
 
 
Unexpected fatal or life-
threatening suspected 
adverse reaction 
 
 
Report no later than 7 
calendar days after 
initial receipt of the 
information 
 
 
 
Unanticipated problem 
involving risk to 
participants or others 
Report within 10 working 
days.  If the event results 
in the death of a 
participant enrolled at 
WU/BJH/SLCH, report 
within 1 working day. 
Report via email 
after IRB 
acknowledgment 
 
 
If related to device, report 
within 10 working days.  If 
the event results in the 
death of a participant 
enrolled at 
WU/BJH/SLCH, report 
within 1 working day. 
 
Major deviation 
Report within 10 working 
days.  If the event results 
in the death of a 
participant enrolled at 
WU/BJH/SLCH, report 
within 1 working day. 
 
 
 
 
 
A series of minor 
deviations that are being 
reported as a continuing 
noncompliance 
Report within 10 working 
days.   
 
 
 
 
 
Protocol exception 
Approval must be 
obtained prior to 
implementing the change 
 
 
 
 
 
Clinically important 
increase in the rate of a 
serious suspected 
adverse reaction of that 
list in the protocol or IB 
 
 
Report no later than 15 
calendar days after it is 
determined that the 
information qualifies for 
reporting 
 
 
 
Complaints 
If the complaint reveals an 
unanticipated problem 
involving risks to 
participants or others OR 
 
 
 
 
 


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Expedited Reporting Timelines 
Event 
HRPO 
QASMC 
FDA 
IBC 
Ichor Medical 
Systems 
AstraZeneca 
noncompliance, report 
within 10 working days.  If 
the event results in the 
death of a participant 
enrolled at 
WU/BJH/SLCH, report 
within 1 working day.  
Otherwise, report at the 
time of continuing review. 
Breach of confidentiality 
Within 10 working days. 
 
 
 
 
 
Incarceration 
If withdrawing the 
participant poses a safety 
issue, report within 10 
working days.   
 
If withdrawing the 
participant does not 
represent a safety issue 
and the patient will be 
withdrawn, report at 
continuing review. 
 
 
 
 
 
SAEs, deaths, AESIs, 
Hy’s Law events or 
hepatic function 
abnormalities, pregnancy 
 
 
 
 
 
Reported to 
AstraZeneca 
within 24 hours 
on FDA 
MedWatch form.